On the role of P2 purinoceptors and ecto-NTPDases in postmenopausal human osteogenesis

JOSÉ BERNARDO ALMEIDA GARRETT DE NORONHA MATOS ON THE ROLE OF P2 PURINOCEPTORS AND ECTO-NTPDASES IN POSTMENOPAUSAL HUMAN OSTEOGENESIS Tese de Candidatura ao grau de Doutor em Ciências Biomédicas, submetida ao Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto. Orientador – Professor Doutor Paulo Correia-deSá Categoria – Professor Catedrático Afiliação – Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto. Co-orientadora – Prof. Doutora Maria Adelina Costa Categoria – Professora Auxiliar Afiliação – Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto. JOSÉ BERNARDO ALMEIDA GARRETT DE NORONHA MATOS ON THE ROLE OF P2 PURINOCEPTORS AND ECTO-NTPDASES IN POSTMENOPAUSAL HUMAN OSTEOGENESIS Dissertation in fulfilment of the requirements for the PhD degree in Biomedical Sciences, submitted to Instituto de Ciências Biomédicas Abel Salazar of the University of Porto. Supervisor – Professor Paulo Correia-de-Sá Category – Full Professor Affiliation – Instituto de Ciências Biomédicas Abel Salazar of the University of Porto. Co-Supervisor – Professor Maria Adelina Costa Category – Assistant Professor Affiliation – Instituto de Ciências Biomédicas Abel Salazar of the University of Porto. This research was partially supported by Fundação para a Ciência e a Tecnologia (FCT, FEDER funding) (projects PTDC/SAU-OSM/73576/2006, REEQ/1168/SAU/2005, REEQ/1264/SAU/2005, PEst-OE/SAU/UI0215/2011 and PEst-OE/SAU/UI0215/2014) and by University of Porto / Caixa Geral de Depósitos (Investigação Científica na Pré-Graduação). The author was in receipt of a PhD Studentship from FCT (POPH – QREN/FSE funding, SRFH/BD/68584/2010). ACKNOWLEDGEMENTS My acknowledgements go to Instituto de Ciências Biomédicas Abel Salazar and to the University of Porto. I want to thank Professor Paulo Correia-de-Sá for accepting me into his group and for all he has taught me so far, for the experience of learning how to make decisions and to be more accurate and pragmatic in both science and in everything I do. More importantly, I wish to thank for his patience and friendship, and for having believed and invested in my work. My acknowledgements to Professor Adelina Costa for her assistance throughout my PhD project, in particular in the basic principles of cell cultures, and for her great friendship. I want to thank the group working at the Laboratory of Pharmacology and Neurobiology for receiving me, for all their support and care demonstrated during my entire PhD project, in particular to Dr. Mariana Certal, Doctor Ana Rita, Dr. Diogo Paramos, Dr. Bruno Bragança, Dr. Aurora Barbosa and Dr. Cátia Vieira, Professor Graça Lobo, Professor Laura Oliveira, Professor Margarida DuarteAraújo, Professor Patrícia Sousa, Professor Miguel Faria, Doctor Miguel Cordeiro and to Dr. Alexandrina Timóteo. I also thank to Mrs Belmira, Helena Costa e Silva and Suzete Liça for their friendship and technical assistance; to Dr. Isabel Silva, Dr.ª Teresa Magalhães-Cardoso, Doctor Fátima Ferreirinha, Dr. João Coimbra, Dr. Ana Sá-e-Sousa, Dr. Sónia Gomes-Guerra, Dr. Isabel Calejo, Dr. Alda Barbosa and Dr. Estrela Neto for their friendship and participation in several experiments. My acknowledgements to the orthopaedic surgeons from Centro Hospitalar de Gaia, namely to Dr. Rui Rocha, Dr. José Marinhas, Dr. David Sá, Dr. Adamir, Dr. Rolando Freitas, Dr. Joaquim Lebre and to Dr. José Neves. A special acknowledgement to all patients that allowed this work to take place. My special thanks to Professor Margarida Lima and to Professor Perpétua Pinto-do-Ó for their counselling. My special thanks to Professor Jean Sévigny for his collaboration in this work. My special thanks to Mrs Ana Paula Pereira. My acknowledgements to the Portuguese Society of Pharmacology for allowing the presentation of our work in these last annual meetings. Thank you for the precious advices and criticisms. My very special thanks to my family, in particular to my wife Luísa, our two sons Carminho and António Maria, and to my dear friends. Thank you for all the support and patience. PhD thesis – José Bernardo Noronha Matos (2011-2014) 1 TABLE OF CONTENTS ABBREVIATIONS ........................................................................................................ 3 RESUMO .................................................................................................................. 9 ABSTRACT .............................................................................................................. 14 1. INTRODUCTION ................................................................................................. 18 1.1. Stem cells and the stem cell niche ....................................................... 18 1.1.1. Adult stem cells – an introduction ..................................................... 18 1.1.2. Adult stem cells and therapy – summary of promising applications in the context of bone tissue engineering .......................................................... 20 1.1.3. The bone marrow microenvironment: a stem cell niche .................... 21 1.2. Mesenchymal stem cells and osteogenic differentiation ...................... 27 1.2.1. Mesenchymal stem cells: in vitro induction of osteogenesis ............. 27 1.2.2. Mesenchymal stem cells: osteogenesis regulation signals and gene expression ..................................................................................................... 30 1.2.3. Mesenchymal stem cells: loss of function and stem cell ageing ....... 38 1.3. Bone: a dynamic and specialized tissue .............................................. 41 1.3.1. Structure of bone: an introduction ..................................................... 41 1.3.2. Bone remodelling and metabolism .................................................... 44 1.3.3. Bone disorders and therapeutic approaches .................................... 53 1.4. Purinergic signalling and bone remodelling .......................................... 63 1.4.1. Purinergic receptors .......................................................................... 63 1.4.2. P2 receptor expression by osteoblasts ............................................. 64 1.4.3. Release of nucleotides from osteoblast and osteoblast-progenitor cells 71 1.4.4. ATP hydrolysis: ecto-nucleotidases and osteoblasts ........................ 74 1.4.5. P2 receptor expression by osteocytes and osteoclasts .................... 78 2. GOALS............................................................................................................. 82 3. ORIGINAL RESEARCH PAPERS ........................................................................... 83 Paper 1 ............................................................................................................ 84 ABSTRACT ....................................................................................................... 85 INTRODUCTION ................................................................................................. 86 PhD thesis – José Bernardo Noronha Matos (2011-2014) 2 MATERIALS AND METHODS ................................................................................ 89 RESULTS ......................................................................................................... 97 DISCUSSION................................................................................................... 115 Paper 2 .......................................................................................................... 125 ABSTRACT ..................................................................................................... 126 INTRODUCTION ............................................................................................... 127 MATERIALS AND METHODS .............................................................................. 128 RESULTS ....................................................................................................... 133 DISCUSSION................................................................................................... 148 Paper 3 .......................................................................................................... 155 ABSTRACT ..................................................................................................... 156 INTRODUCTION ............................................................................................... 157 MATERIALS AND METHODS .............................................................................. 159 RESULTS ....................................................................................................... 166 DISCUSSION................................................................................................... 184 4. DISCUSSION AND CONCLUSIONS ........................................................................ 193 5. REFERENCES ................................................................................................. 201 PhD thesis – José Bernardo Noronha Matos (2011-2014) 3 ABBREVIATIONS 3’-UTR, Three prime untranslated region 25-OH-Vitamine D, 25-OH-Cholecalciferol a.u., Arbitrary units A438079, 3-[[5-(2,3-Dichlorophenyl)-1H-tetrazol-1-yl]methyl]pyridine hydrochloride ADO, Adenosine ADPβS, Adenosine 5’-[β-thio]diphosphate ALP, Alkaline phosphatase AMP, Adenosine 5’-monophosphate AP-1, Adapter-related protein complex 1 subunit AR, Androgen receptor ARL 67156, 6-N,N-Diethyl-D-β,γ-dibromomethylene ATP trisodium salt ATP, Adenosine 5’-triphosphate ATPase, Adenosine 5'-triphosphatase BGLAP, Osteocalcin coding gene BMP, Bone morphogenic protein BMPRIA, Bone morphogenic protein receptor type IA BMSCs, Bone marrow stromal cells BRU, Bone remodelling units BTE, Bone tissue engineering BzATP, 2’(3’)-O-(4-Benzoylbenzoyl)adenosine 5’-triphosphate [Ca2+]i, Intracellular calcium cAMP, 3'-5'-Cyclic adenosine monophosphate CaSR, Calcium sensing receptors CD11b, Integrin alpha M CD14, Monocyte differentiation antigen CD14 CD19, B-lymphocyte antigen CD19 CD34, Haematopoietic progenitor cell antigen CD34 CD39, Apyrase or NTPDase1 CD44, Receptor for hyaluronic acid CD45, Protein tyrosine phosphatase, receptor type C, also known as PTPRC PhD thesis – José Bernardo Noronha Matos (2011-2014) 10 Existem evidências de que a sinalização purinérgica exerce um efeito local complexo nas células ósseas. Contudo, existe uma enorme controvérsia em torno da importância de cada recetor purinérgico e das enzimas metabolizadoras dos nucleótidos, bem como em saber de que modo a interação entre estes intervenientes promove a remodelação óssea. Infelizmente, muitos dos estudos derivam da utilização de modelos animais e do uso de células humanas imortalizadas, sendo ainda escassos os trabalhos realizados em células humanas não modificadas (osteoprogenitoras e osteoblastos). Visto que a sinalização purinérgica tem sido associada a diferentes patologias ósseas, torna-se imprescindível o seu estudo na tentativa de clarificar os mecanismos moleculares responsáveis pela atuação dos nucleótidos e nucleósidos na remodelação óssea em situação fisiológica e na doença. O sucesso deste estudo poderá resultar na descoberta de novos alvos terapêuticos para as patologias ósseas. Este projeto foi delineado para avaliar a expressão diferencial dos recetores P2 nas células humanas osteoprogenitoras / osteoblastos atendendo à idade e género dos doentes. Foi, ainda, decidido documentar as respostas funcionais destas células na presença de nucleótidos no meio extracelular e investigar o envolvimento de alguns subtipos de recetores P2 na libertação de ATP e na plasticidade membranar responsável pelos fenómenos de diferenciação/proliferação celular. Devido à sua relevância no controlo da atividade purinérgica, estudou-se a influência das NTPDases na proliferação, diferenciação e mineralização das células osteoprogenitoras. Especificamente, neste projeto investigou-se: (1) a expressão e o papel funcional dos recetores sensíveis a nucleótidos de uracilo (P2Y2, P2Y4, e P2Y6) em células osteoprogenitoras / osteoblastos, visto que o seu papel na diferenciação osteogénica era desconhecido; (2) a expressão e função dos recetores ionotrópicos P2X7 na osteogénese humana, explorando os mecanismos moleculares envolvidos no sentido de esclarecer alguma controvérsia sobre o papel destes recetores existente na literatura, no que toca às células humanas; (3) a importância das NTPDases na proliferação e diferenciação das células osteoprogenitoras da medula óssea em mulheres jovens e com idade pósmenopáusica. PhD thesis – José Bernardo Noronha Matos (2011-2014) 11 As amostras de medula óssea foram obtidas de mulheres pós-menopáusicas sujeitas a artroplastia da anca como resultado de osteoporose primária. Para comparação, foram obtidas amostras da medula óssea proveniente de mulheres jovens sujeitas a colheita de enxerto ósseo para correção cirúrgica de escoliose ou fraturas traumáticas. O isolamento das células estromais mesenquimatosas foi demonstrado por análise imunofenotípica por citometria de fluxo; estas células apresentavam marcadores encontrados caracteristicamente em células mesenquimatosas multipotentes da medula óssea, nomeadamente os marcadores CD105, CD73, CD117 e CD29, não apresentando marcação para células estaminais hematopoiéticas, nomeadamente CD14 e CD45. Relativamente ao objetivo (1), verificou-se que a incubação das células estromais mesenquimatosas da medula óssea (MSCs) provenientes de mulheres pós-menopáusicas com UTP ou UDP promove a sua diferenciação osteogénica, traduzindo-se num aumento de atividade da fosfatase alcalina (ALP) para níveis semelhantes aos observados em mulheres jovens, sem contudo se observar qualquer efeito na proliferação celular. Os nucleótidos de uracilo aumentam de forma dependente da concentração os níveis de cálcio intracelular ([Ca2+]i) em MSCs. No entanto, estes efeitos tornam-se menos evidentes com o tempo das células em cultura (7>21 dias). A ativação seletiva de recetores P2Y6 com o análogo estável do UDP, PSB 0474, mimetizou os efeitos do UTP e do UDP, enquanto o análogo estável do UTP, UTPγS, foi desprovido de efeito. O antagonista seletivo do recetor P2Y6, MRS 2578, preveniu o aumento dos níveis intraceulares de [Ca2+]i e a diferenciação osteogénica provocada pelo UDP em todos os períodos de cultura analisados. As MSCs demonstram imuno-reatividade para os recetores P2Y2, P2Y4, e P2Y6. Enquanto a expressão do recetor P2Y6 se mantém constante ao longo do período de cultura (7~21 dias), a expressão dos recetores P2Y2 e P2Y4 é mais evidente em células mais diferenciadas (7<21 dias). O catabolismo extracelular dos nucleótidos de uracilo, UTP e UDP, foi maior em células menos proliferativas e mais diferenciadas (7<21 dias), que é justificado pelo aumento da expressão das NTPDases1, -2 e -3 nas populações de células mais diferenciadas (7<21 dias). PhD thesis – José Bernardo Noronha Matos (2011-2014) 12 No que concerne ao objetivo (2), verificámos que o ATP e o agonista do recetor P2X7 aumentam os níveis intracelulares de [Ca2+]i em MSCs, paralelamente à formação de poros membranares permeáveis à sonda TO-PRO-3. A ativação dos recetores P2X7 promove a formação reversível de microvesículas na membrana plasmática e a formação de bolhas (zeiose) em MSCs. Apesar da diferenciação osteogénica promovida pelas alterações na dinâmica membranar ser independente do [Ca2+]i, ela parece envolver a atividade da PLC (fosfolipase C), da PKC (proteína cinase C) e da Rho-cinase secundárias à ativação do recetor P2X7. O agonista P2X7, BzATP, antecipa a diferenciação osteogénica (aumento da atividade ALP e da expressão dos fatores de transcrição Runx-2 e Osterix) e promove a mineralização das culturas de MSCs. Quanto ao objetivo (3), demonstrámos que o catabolismo extracelular dos nucleótidos de adenina e uracilo leva a uma perda da atividade dos recetores P2Y6 e P2X7 em MSCs de mulheres pós-menopáusicas quando comparado com as mulheres jovens. Verificou-se que a expressão da NTPDase3 aumenta com o tempo das células em cultura nas mulheres pós-menopáusicas, sendo que esta enzima está ausente em MSCs de mulheres jovens. A inibição seletiva da NTPDase3 com o fármaco, PSB 06126, aumenta 3.6 vezes os níveis endógenos de ATP em culturas de MSCs de mulheres pós-menopáusicas avaliadas ao dia 7. A inibição da NTPDase3 favorece a atividade da ALP e promove a expressão de marcadores de osteogénese, Runx-2 e Osterix, ao longo do tempo de cultura. É, ainda, de realçar que a inibição da atividade da NTPDase aumentou de forma significativa (P<0.05) a formação de nódulos de mineralização nas culturas de MSCs comparativamente as mesmas culturas na ausência dos inibidores. Este efeito positivo na osteogénese foi totalmente prevenido na presença de apirase (CD39), a enzima que converte nucleótidos de adenina (ATP) e uracilo (UTP) nos seus derivados monofosfatados, AMP e UMP respectivamente. O antagonismo seletivo de recetores P2X7 e P2Y6 atenuou (P<0.05) de igual forma os efeitos dos inibidores da NTPDase3 na osteogénese. Concluindo, neste trabalho demonstra-se que os nucleótidos de uracilo são importantes reguladores da diferenciação de células osteoprogenitoras, predominantemente por intermédio da ativação de recetores P2Y6 sensíveis ao PhD thesis – José Bernardo Noronha Matos (2011-2014) 13 UDP por um mecanismo associado ao aumento dos níveis de [Ca2+]i. A ação endógena dos nucleótidos de uracilo pode ser balanceada por NTPDases específicas que irão determinar se as células osteoprogenitoras se diferenciam ou proliferam; verificou-se, ainda, que o recetor P2X7 é um importante regulador da diferenciação osteogénica e da consequente mineralização das MSCs humanas em cultura. Os mecanismos dependentes da ativação P2X7 podem envolver oscilações do [Ca2+]i e alterações na dinâmica membranar (formação de poros e de bolhas) por mecanismos dependentes da atividade da PLC, PKC e Rhocinase; estes processos controlam a expressão de marcadores envolvidos na diferenciação osteogénica, tais como o Runx-2 e o Osterix. Demonstrou-se, ainda, que a expressão da NTPDase3 controla a atividade dos recetores P2 nas MSCs em mulheres pós-menopáusicas. A expressão da NTPDase3 parece aumentar com a idade, facto que pode contribuir para reduzir dos níveis endógenos de nucleótidos de adenina e de uracilo na medula óssea das mulheres pósmenopáusicas e, deste modo, a ativação de importantes promotores da osteogénese, nomeadamente os recetores P2X7 e P2Y6. O conhecimento de alvos reguladores da diferenciação osteogénica anteriormente ignorados permitem agora o desenvolvimento de novas estratégias terapêuticas para controlar as doenças ósseas em que a erosão se sobrepõe à formação de novo osso, tais como a osteoporose, a artrite reumatoide e osteogénese imperfecta. PhD thesis – José Bernardo Noronha Matos (2011-2014) 14 ABSTRACT Bone is a specialized connective tissue, containing cells (osteoblasts, osteoclasts and osteocytes) and inorganic mineral salts deposited within an organic collagen matrix. The continuous remodelling of the bone allows it to repair, to grow and adapt. Throughout life, many abnormalities in such balanced process may result in a huge variety of bone disorders and skeletal abnormalities (such as osteoporosis). The development of new therapeutic approaches is of major importance since some bone disorders are highly prevalent and in increasing incidence on an ageing population. Bone turnover is a complex and finely tuned process. To the importance of systemic factors that regulate bone turnover, we must include the role of local factors such as adenosine 5’-triphosphate (ATP) and its derivatives (such as adenosine). It was previously found that bone cells, such as osteoprogenitors (mesenchymal stem cells, MSCs) and osteoblasts constitutively release nucleotides (e.g. ATP) when submitted to mechanical stress or under pathological conditions, such as hypoxia and inflammation. Nucleotides may be released into the extracellular milieu through distinct mechanisms: (1) cytosolic release of ATP from sites of tissue and cell damage (including sites of bone injury), (2) exocytic release and (3) via intrinsic plasma membrane channels or pores in the absence of cytolysis, which includes hemichannels controlled release. Once released, nucleotides and their derivatives may act on purinoceptors, which are important (yet largely unknown) bone turnover regulators. The receptors for purines and pyrimidines are classified into two groups: P1 receptors (A1, A2A, A2B, A3), which are primarily activated by adenosine, and P2 receptors, which respond to adenine and uracil nucleotides (e.g. ATP, ADP, UTP, UDP). The latter may be further subdivided into P2X ligand-gated ion channels and P2Y G-protein-coupled receptors. Purinoceptors activation may be terminated by ecto-nucleotidases and other ecto-phosphatases bound to the plasma membrane, which rapidly hydrolyse extracellular nucleotides to their respective nucleoside 5’-diand monophosphates, nucleosides and free phosphates or pyrophosphates. PhD thesis – José Bernardo Noronha Matos (2011-2014) 15 Current evidence shows that purinergic signalling exerts complex local effects in bone microenvironment. However, controversy still exists around the functional relevance of each purinergic receptor and enzyme on bone cells and how they may interact to promote bone remodelling. In this regard, most of the studies derive from using animal models and immortalized cell lines and less in nonmodified human cells (osteoprogenitors / osteoblasts). Since purinergic signalling has now been implicated in many bone disorders, it is of great importance to explore these potential targets for future therapies, clarifying the molecular mechanisms operating upstream and downstream receptors activation in both health and disease conditions. To this end, this project was designed to evaluate changes in the expression of P2 receptors in human osteoprogenitors / osteoblasts taking into consideration age and gender of the patients, to document functional responses to extracellular nucleotides, to investigate the involvement of P2 receptors on ATP release and cell-membrane plasticity (required for osteoblast proliferation/differentiation), and study the impact of NTPDases in such mechanisms. Specifically, this project aimed at investigating: (1) the expression and function of uracil nucleotide-sensitive receptors (P2Y2, P2Y4, and P2Y6) in osteoprogenitors / osteoblasts, since their role in osteogenic differentiation was largely unknown; (2) the expression and function of P2X7 receptors on osteogenic differentiation of osteoprogenitors / osteoblasts in culture, exploring the molecular mechanisms involved; in literature, the role of the human P2X7 receptor is still controversial; (3) the role of the activity of NTPDases in the management of cell differentiation and/or proliferation, in both health and disease conditions; this topic is, so far, largely unknown particularly in non-modified human osteoprogenitor cells. Bone marrow specimens were obtained from postmenopausal female patients undergoing total hip arthroplasty as a result of primary osteoarthrosis. For comparison purposes, it was also used bone marrow from younger female patients requiring bone engraftment for spinal fusion to correct scoliosis or to repair traumatic bone fractures. Isolation of MSCs was proven by immunophenotypic analysis (flow cytometry), showing positivity for markers of bone marrow-derived mesenchymal stromal cells, namely CD105, CD73, CD29 and CD117, in the PhD thesis – José Bernardo Noronha Matos (2011-2014) 16 absence of the expression of haematopoietic stem cell markers, such as CD14 and CD45. Regarding goal (1), it was demonstrated that the application of UTP and UDP promotes osteogenic differentiation of postmenopausal bone marrow MSCs in culture measured as increases in alkaline phosphatase (ALP) activity to levels which are observed in younger patients, with no effects on cell proliferation. Uracil nucleotides concentration-dependently increase intracellular calcium ([Ca2+]i) in MSCs; their effects become less evident with the time (7>21 days) in culture. Selective activation of P2Y6 receptors with the stable UDP analogue, PSB 0474, mimicked the effects of both UTP and UDP, whereas UTPγS was devoid of effect. Selective blockade of P2Y6 receptors with MRS 2578 prevented [Ca2+]i rises and osteogenic differentiation caused by UDP at all culture time points. MSCs are immunoreactive against P2Y2, P2Y4, and P2Y6 receptors. While the expression of P2Y6 receptors remains fairly constant (7~21 days), P2Y2 and P2Y4 become evident only in less proliferative and more differentiated cultures (7<21 days). The rate of extracellular UTP and UDP inactivation was higher in less proliferative and more differentiated cell populations. It was also found that immunoreactivity against NTPDase1, 2, and 3 raises as cells differentiate (7<21 days). Concerning the objective (2), ATP, and the P2X7 receptor agonist, BzATP, increased [Ca2+]i in parallel to the formation of TO-PRO-3 permeable membrane pores. The two P2X7 agonists elicited reversible cell microvesiculation and plasma membrane blebbing (zeiosis). Differentiation-inducing plasma membrane dynamics was Ca2+-independent, but involved the PLC (phospholipase C), PKC (protein kinase C) and Rho-kinase pathway downstream P2X7 receptor activation. BzATP anticipated osteogenic differentiation (increases in ALP activity and in the expression of Osterix and Runx-2 transcription factors) and favoured mineralization of MSC cultures. Regarding goal (3), it was found that that enzymatic inactivation of extracellular nucleotides leads to a loss of function of P2Y6 and P2X7 purinoceptors in MSCs from postmenopausal women as compared to younger females. Interestingly, the expression of NTPDase3 increased with the culture time of MSCs from postmenopausal women; this enzyme was not expressed in the cells from younger PhD thesis – José Bernardo Noronha Matos (2011-2014) 17 female patients. Selective inhibition of NTPDase3 with PSB 06126 increased by 3.6-fold the endogenous levels of ATP in MSC cultures from postmenopausal woman at day 7. NTPDase3 inhibition also increased the ALP activity and promoted the expression of osteogenic markers, both Runx-2 and Osterix, throughout the culture period. Under these circumstances, mineralization of bonenodules increased (P<0.05) when compared to the control situation at culture day 43. The osteogenic differentiating effect of NTPDase3 inhibition was fully prevented by apyrase (CD39), the enzyme that converts directly nucleoside trisphosphates into their monophosphate derivatives. Selective blockade of P2X7 and P2Y6 receptors also attenuated (P<0.05) the osteogenic effect of NTPDase3 inhibitors. In conclusion, we demonstrated in this study that uracil nucleotides are important regulators of osteogenic differentiation of MSCs via the activation of UDP-sensitive P2Y6 receptors coupled to increases in [Ca2+]i. The endogenous actions of uracil nucleotides may be balanced through the action of specific NTPDases determining whether osteoblast progenitors are driven into proliferation or differentiation. In addition, data show that the P2X7 receptor is an important regulator of osteogenic differentiation and subsequent mineralization of human MSCs in culture. The mechanisms by which the P2X7 receptor control bone formation might involve intracellular [Ca2+]i oscillations and membrane cell dynamics (pore formation and blebbing) due to downstream activation of PLC, PKC and Rho-kinase-dependent pathways controlling osteogenic markers, like Runx-2 and Osterix. Here, we show for the first time that NTPDase3 is an important modulator of the activity of P2 purinoceptors in MSCs from postmenopausal woman. Age-related overexpression of NTPDase3 may contribute to reduce the endogenous amounts of adenine and uracil nucleotides, thus compromising the activation of important inducers of osteogenesis, such as P2X7 and P2Y6 receptors. These previously unrecognized targets for local regulation of osteogenic differentiation of bone marrow MSCs may prompt for novel therapeutic strategies to control human ossification disorders where bone destruction exceeds bone formation (e.g., osteoporosis, rheumatoid arthritis, osteogenesis imperfecta). 1.Introduction PhD thesis – José Bernardo Noronha Matos (2011-2014) 18 1. INTRODUCTION 1.1. Stem cells and the stem cell niche 1.1.1. Adult stem cells – an introduction Stem cells are the foundation cells for every organ and tissue in the body. Stem cells are cells with the ability to grow and to differentiate into more than 200 cell types. These are able to divide and to give rise to identical daughter cells (known as symmetrical division) and to differentiate into specific cells of somatic tissues (Avery et al., 2006). Stem cells may be found in: early embryos (commonly known as embryonic stem cells) and adult tissues (known as adult stem cells). Stem cells can be classified with respect to their potency, that is, the competency of each cell to differentiate into a specialized type of tissue cells of the body. There are five class of potency for stem cells: totipotent, pluripotent, multipotent, oligopotent and unipotent (see e.g., Kaveh et al., 2011). Totipotent stem cells are able to give rise to all embryonic somatic cells and germ cells; they can generate a viable embryo (including extraembryonic support tissues such as the placenta). These cells result from the fusion of an ovum and sperm cell. Cells that result from the 1st divisions of the fertilized egg are totipotent cells (Volarevic et al., 2011). Pluripotent cells descend from totipotent cells and can give rise to cells of the three germ layers: endoderm, mesoderm, and ectoderm; they have no contribution to extraembryonic membranes or the placenta. Multipotent cells give rise to cells of a particular lineage or closely related family (Behr et al., 2010), that is, they can differentiate into a number of cells but from a germ layer. Oligopotent stem cells can differentiate into a few specialized cells and unipotent cells are limited to one cell type, although with the ability of self-renewal (Mitalipov and Wolf, 2009). Stem cells are part of an individual entire lifetime. Embryonic stem cells are the cells that are derived from the inner cell mass of an early stage of the embryo known as blastocyst (which consists of 50-150 cells, after 4-5 days post fertilization). Adult stem cells reside in most mammalian tissues, and the extent to which they contribute to normal homeostasis and repair is widely variable. Stem 1.Introduction PhD thesis – José Bernardo Noronha Matos (2011-2014) 19 cells have been found in many tissues and organs including epidermis, liver and bone (Kao et al., 2008). It is believed that the main role of adult stem cells is to replace damaged and injured tissues (Volarevic et al., 2011). Among adult stem cells, we can find the haematopoietic stem cells (HSCs), epithelial stem, muscle stem, neural stem and mesenchymal stem cells (MSCs) (Kao et al., 2008; Kaveh et al., 2011). In the bone marrow reside mainly HSCs and MSCs. HSCs are the blood-forming cells, that is, they are ultimately responsible for the constant renewal of blood with the production of billions of new blood cells each day. They can also be found in cord blood, foetal liver, adult spleen, and peripheral blood. Mature haematopoietic cells are traditionally categorized into two distinct lineages: the lymphoid and the myeloid. The lymphoid lineage consists of T, B and natural killer (NK) cells, while the myeloid lineage includes a number of morphologically, phenotypically and functionally distinct cell types such as different subclasses of granulocytes (neutrophils, eosinophils and basophils), monocytes– macrophages, erythrocytes, megakaryocytes and mast cells (for a review, see Iwasaki and Akashi, 2007). HSCs usually express surface markers that can be used for their identification, namely Sca-1, CD14, CD34 and CD45. MSCs are multipotent, self-renewable cells that can be found not only in the bone marrow, but also in all postnatal organs and tissues, namely adipose tissue, umbilical cord blood and compact bone (Porada et al., 2006; Volarevic et al., 2010). These are a rare-population of non-haematopoietic stromal cells, able to differentiate into mesenchymal tissues such as bone, cartilage, adipose tissue and muscle (Augello et al., 2010). They are also able to differentiate into non-mesenchymal cells such as neurons (Kopen et al., 1999). In addition, they have a high proliferative capability, while retaining their undifferentiated state (Banfi et al., 2002; Bruder et al., 1997). MSCs show variable levels of expression of several markers, namely CD105 (SH2), CD90, CD73 (or ecto 5’-nucleotidase), stromal antigen 1, CD44, CD166 (vascular cell adhesion molecule), CD54/CD102 (intracellular adhesion molecule), CD49 and c-Kit (or CD117) (Baddoo et al., 2003; Boiret et al., 2005; Cognet and Minguell, 1999; Dennis et al., 2002; Gronthos et al., 2003; Pittenger et al., 1999; Sivasubramaniyan et al., 2012). However, some variations may be found 1.Introduction PhD thesis – José Bernardo Noronha Matos (2011-2014) 26 overexpressing the PTHR, enhances osteoblastic growth and simultaneously increases the number of HSCs in the marrow (Calvi et al., 2003). They also showed that the treatment of healthy animals with PTH similarly expands the HSC population in the marrows, since there is an expansion of osteoblast precursors. Again, administrating PTH to wild-type animals before HSC transplantation improves their survival. These results point to osteoblasts as stem cell-supportive cells in such niches. Stem cells are able to maintain their undifferentiated state within the niche (Bobis et al., 2006). In the particular case of MSCs, some findings suggest that these cells decision to differentiate or to stay quiescent is regulated by Wnt family members. Its signalling is known to prevent differentiation process by inducing high levels of oct-3/4, rex-1 and the homeodomain transcription factor Nanog, which are known as gatekeepers for embryonic stem (ES) cell pluripotency (Sato et al., 2004). On the other hand, BMP-pathways play a role in the proliferation/differentiation process of stem cells, which raises the hypothesis that these factors are important for MSCs growth in their niche. When needed, stem cells like MSCs may be recruited from the bone marrow to other tissues, circulating in blood, after particular stimuli (Bobis et al., 2006). They are kept in the cell niche since they express a number of adhesion molecules, which are also important for the niche function itself. These include N-cadherin/βcatenin, VCAM/integrin and osteopontin/β1 integrin (for a revision, see Bobis et al., 2006). Osteoblasts themselves express many adhesion molecules that may also be used during cell-cell contact with HSCs. Some of these include VLA-4 (α4β1) receptors (expressed on CD34+ cells) and the vascular cell adhesion molecule-1 (VCAM-1) (expressed by bone marrow stromal cells) (Simmons et al., 1994). Other structures are involved, namely CD34, CD44, CD164, intracellular cell adhesion molecules (ICAM-1, ICAM-3), very late antigen-4 (VLA-4), among others (for a revision, see Verfaillie, 1998). Some cell-associated or matrix-bound cytokines, like IL-3, stem cell factor (SCF) and transforming growth factor α1 (TGFα1) may themselves serve as adhesion molecules (Gordon, 1991; Hardy and Minguell, 1995; Toksoz et al., 1992). When migrating to other tissues, MSCs are 1.Introduction PhD thesis – José Bernardo Noronha Matos (2011-2014) 27 able to support the regeneration process. Such cases were documented in regenerating bone (where they differentiate into bone forming cells, the osteoblasts) (Benayahu et al., 1989), in cartilage repair (Caplan et al., 1997), muscle (De Bari et al., 2003), heart (Shake et al., 2002) and migration throughout the forebrain and cerebellum (where they differentiate into astrocytes) (Kopen et al., 1999). Homing of MSCs is possible thanks to the expression of chemokine receptors which will help them in trafficking to various tissues. An example is the CXCR4, the receptor for SDF-1, which is produced by stromal cells (Stoicov et al., 2013; Yang et al., 2013). Using such signalling mechanisms, MSCs detected in the bloodstream, are able to migrate and colonize various tissues (Gao et al., 2001). 1.2. Mesenchymal stem cells and osteogenic differentiation 1.2.1. Mesenchymal stem cells: in vitro induction of osteogenesis When cells from the whole bone marrow are plated in plastic culture dishes with 10% of foetal calf serum supplemented medium, one finds that there is a population of haematopoietic non-adherent cells together with a rare population of plastic-adherent cells (in a proportion of 1:10000 nucleated cells in the bone marrow, varying from individual to individual) (Friedenstein et al., 1970). After medium change, only the adherent cells will remain. These will start to proliferate and can differentiate into mature cells of mesenchymal lineages such as osteoblasts (Friedenstein et al., 1970; Friedenstein et al., 1976). In fact, this ability to adhere to plastic is one of the criteria for the minimal identification of mesenchymal stem cells (MSCs) by the International Society for Cell Therapy in 2006 (Dominici et al., 2006). When assessed by the FACS analysis, these cells should also have the following cell phenotype: CD73+, CD90+, CD105+, CD45-, HLA-DR-, CD14or CD11b-, CD79aor CD19- (Dominici et al., 2006). Osteoblasts are the bone forming cells and, as mentioned earlier, these derive from MSCs. In vivo, MSCs are believed to be in close relation with other cells, namely haematopoietic stem cells, forming the stem cell niche (see section 1.1.3). In order to promote the osteogenic differentiation of human MSCs in vitro, these 1.Introduction PhD thesis – José Bernardo Noronha Matos (2011-2014) 28 should be incubated with ascorbic acid, β-glycerophosphate and dexamethasone (in addition to foetal bovine serum) added to the culture medium (Jaiswal et al., 1997; Pittenger et al., 1999). Ascorbic acid is known to play a role in the collagen synthesis, since it is a cofactor for proline and lysine hydroxylases, both involved in the hydroxylation of collagen. Ascorbic acid seems to be essential for normal bone formation, since ascorbic acid was previously shown to increase alkaline phosphatase and osteocalcin mRNAs in osteoblast cultures (Franceschi and Iyer, 1992) and that this induction is blocked by inhibitors of collagen triple-helix formation. In addition, osteoblasts were shown to express a Na+-dependent transporter specific for ascorbic acid which is essential for maintenance of intracellular ascorbate concentrations (Dixon et al., 1991; Franceschi et al., 1995) which, in turn, will influence the proliferation and alkaline phosphatase expression (Franceschi and Iyer, 1992; Franceschi et al., 1994). β-glycerophosphate is a substrate for alkaline phosphatase, producing an increase in phosphate content, which may be incorporated in the bone matrix (Bellows et al., 1991) together with Ca2+. It functions as an organic phosphate donor and has been used in culture media for MSC differentiation to osteoblast-type cells (see e.g., Jaiswal et al., 1997). Besides this, the free inorganic phosphate may induce increases in mRNA and protein expression of osteogenic markers like osteopontin and regulation of Runx-2 (Beck et al., 2000; Fujita et al., 2001). Dexamethasone is a synthetic corticosteroid. It mimics the actions of several glucocorticoids located naturally in the body (cortisol, estradiol, testosterone, vitamin D3, thyroxine and retinoic acid) (Kaveh et al., 2011). Glucocorticoid-activated genes work through gene activator proteins. These, when not associated to glucocorticoids, remain in the cytosol. When present, glucocorticoids bind to their glucocorticoid receptors (nuclear receptor subfamily 3, group C, member 1 - NR3C1) and to the activator proteins, migrate to the nucleus and bind to the regulatory region of each gene regulated in this manner. Dexamethasone supports osteogenic differentiation (Liu et al., 2002) by binding to some special regulatory proteins in the cell and then activating transcription of osteoblast-specific genes, like those coding for alkaline phosphatase, which is an enzyme required for matrix mineralization, converting 1.Introduction PhD thesis – José Bernardo Noronha Matos (2011-2014) 29 pyrophosphate (PPi, a known inhibitor of mineralization) into inorganic phosphate (Cheng et al., 1994). It is worth noting that dexamethasone in vivo, in long-term intake cases, results in osteoporosis and osteopenia (bone degeneration and loss) (Baylink, 1983). This apparent contradiction is based on the fact that chronic glucocorticoid intake leads to an inhibition of MSCs proliferation and may activate the action of osteoclasts. It was shown that <10-8 M stimulated receptor activator of NF-B ligand (RANKL)-induced osteoclast formation synergistically with transforming growth factor β (Takuma et al., 2003). So, regimens of dexamethasone may limit the amount of osteoprogenitors sources (Kaveh et al., 2011), resulting in an unbalanced bone formation versus bone resorption. Besides this, glucocorticoids are known to reduce Ca2+ absorption in the intestine, leading to an increase in circulating PTH, ultimately resulting in Ca2+ mobilization from bone (Fucik et al., 1975). This topic will be further discussed in section 1.3.2 which refers to bone metabolism description. Dexamethasone treated cultures have increased ALP activity when compared to control cultures (Fernandes et al., 1997), consistent with other observations showing that long-term treatment of bone-derived cells with physiological concentrations of glucocorticoids induces their differentiation into cells with an osteoblast phenotype (Benayahu et al., 1989; Cheng et al., 1994). Fernandes and co-workers also revealed that dexamethasone treated cultures show randomly located multi-layered cell three-dimensional nodules, and that these appear to be important for in vitro mineralization of alveolar bone cells (Fernandes et al., 1997). Besides the previous indicated factors, the standard procedure for in vitro culture of MSCs is based on supplementing cell culture media with foetal bovine serum (FBS). This contains growth factors and extracellular matrix molecules that enhance cell attachment to plastic surfaces, enhancing cell proliferation and differentiation. It provides several important biological molecules such as albumin, antichymotrypsin, apolipoproteins, biotin, and growth supporting factors, which are required for optimal growth of cells. In addition to the indicated factors, 10% of FBS was shown to be the indicated amount of serum for an osteoblastic-induction medium, although some variations may be found (Tateishi et al., 2008). 1.Introduction PhD thesis – José Bernardo Noronha Matos (2011-2014) 30 Although human bone cell cultures have been regarded as a useful tool to study bone control mechanisms, it should be emphasized that the number of passages in an in vitro study is an important factor to be considered. Several authors have demonstrated that serially passaged cells keep the proliferation rate constant, but without a similar ALP activity. This enzyme activity decreases with the number of passages and the ability to form mineralized areas also decreases on serial subcultures (Fernandes et al., 1997). In fact, the loss of the osteoblast phenotype on serial passage has been reported by other authors (Coelho et al., 2000). Other interesting findings revealed that serial passage of osteoblast-like cells, MC3T3E1, alters their osteoblastic function and responsiveness to transforming growth factor-β1 (TGF-β1) and bone morphogenetic protein-2 (BMP-2). These authors showed that BMP-2 significantly enhances ALP activity and osteocalcin secretion in early passage cells while TGF-β1 has the opposite effect. Both BMP-2 and TGF-β1 effects significantly decrease on late passage cells. They also showed that ALP activity and osteocalcin secretion decreases after multiple cell passages (Chung et al., 1999). Taken all together, these evidences support the idea of a replicative senescence of in vitro cultures that may be a good starting point for cell ageing proposed in vivo. Besides this, data demonstrate that caution should be taken when considering serial passages of primary cultures, since cell phenotype and differentiation ability may be compromised. 1.2.2. Mesenchymal stem cells: osteogenesis regulation signals and gene expression As mentioned earlier, mesenchymal stem cells (MSCs) are able to differentiate into multiple cell types, including osteoblasts. The latter are the bone forming cells, and together with osteoclasts (bone-destroying cells) and osteocytes (a type of cell that derives from the osteoblast lineage), compose the three major cells involved in bone remodelling. Bone formation, which is undertaken by osteoblasts (discussed further in section 1.3.2), involves a two-step process: first, mature osteoblasts synthesize and release type I collagen which constitutes 85-90% of the organic matrix and many other non-collagenous bone matrix proteins like 1.Introduction PhD thesis – José Bernardo Noronha Matos (2011-2014) 31 alkaline phosphatase (ALP), osteocalcin, osteonectin, osteopontin and bone sialoprotein. The organic matrix, known as osteoid, is subsequently mineralized by calcium and phosphate ions to produce calcified bone tissue, a mineral that is similar to hydroxyapatite, Ca10(PO4)6(OH)2 (Orriss et al., 2010). This lineage commitment of multipotent MSCs is driven by the selective expression of one master transcriptional regulator, Runx-2 (runt-related transcription factor 2), which is necessary for the osteoblast lineage (reviewed in Jensen et al., 2010). This master regulator of osteoblastogenesis acts throughout the induction, proliferation and maturation of osteoblasts regulating the expression of many osteoblast genes, including those encoding osteocalcin, type I collagen and osteopontin (for a revision, see Franceschi and Xiao, 2003). Some of these genes are depicted in Table 1. This table summarizes some of the most important osteogenic markers, including the RUNX-2 gene (also known as CBFA1). These are commonly chosen to evaluate, in vitro, the osteogenic differentiation of MSCs. The Runx-2 activity and importance in osteoblastogenesis has been explored extensively, and studies in which mutations were induced in RUNX-2 gene showed a number of skeletal abnormalities. One example is cleidocranial dysplasia syndrome, an autosomal-dominant skeletal dysplasia that is characterized by widely patent calvarial sutures, clavicular hypoplasia, supranumerary teeth and short stature. Besides this, homozygous mutation of RUNX-2 in mice was shown to be lethal due to a complete lack of mineralized bone (Komori et al., 1997; Mundlos et al., 1997; Otto et al., 1997). The signalling pathways regulating Runx-2 activity are still under extensive investigation, but there are several examples of such dynamic control. These include binding of extracellular matrix (ECM) to cell surface integrins, fibroblast growth factor 2 (FGF2), mechanical loading, parathyroid hormone (PTH) and bone morphogenic proteins (BMPs) (for a revision, see Franceschi and Xiao, 2003). Figure 3 summarizes these signalling pathways and how they interact to promote Runx-2 activity. 1.Introduction PhD thesis – José Bernardo Noronha Matos (2011-2014) 32 Table 1. Genetic and related protein markers of osteogenesis Coding Gene Protein name Protein Function c-fos c-Fos Belongs to the AP-1 class of transcription factors. Direct gene targets include those encoding osteocalcin, collagenase III, bone sialoprotein, alkaline phosphatase promoters (Jensen et al., 2010; Owen et al., 1990). RUNX-2 Runx-2 Can directly stimulate transcription of osteoblast-related genes such as those encoding osteocalcin, type I collagen, osteopontin and collagenase III by binding to specific enhancer regions in DNA (Franceschi and Xiao, 2003). ALPL Alkaline phosphatase Ubiquitous enzymes present in many organisms from bacteria to man. The most abundantly isoform expressed in bone is TNAP. Its major role is to hydrolyse PPi to maintain a proper concentration of this mineralization inhibitor ensuring normal bone mineralization (Yegutkin, 2008). BGLAP Osteocalcin One of the most abundant matrix proteins in bones, synthetized by osteoblasts. It binds strongly to apatite and calcium, which is dependent on vitamin K (Gundberg et al., 2012). SPP1 Osteopontin One of the major phosphoproteins in bone. Osteoblast adhesion to bone matrix proteins such as osteopontin can modulate various aspects of cell behaviour, including growth, differentiation, and protein production (Liu et al., 2008a). SP7 Osterix Zinc finger-containing transcriptional activator that is distinctly expressed in all developing bones and is important for osteoblast differentiation, usually downstream of Runx-2. Osteocalcin, ALP and bone sialoprotein coding-genes are some of its targets (Sinha and Zhou, 2013). COL1A1 Type I Collagen Main constituent of the organic matrix synthesized by mature osteoblasts, which is released by exocytosis. Together with noncollagenous bone matrix proteins, constitutes the osteoid, which latter mineralizes with calcium and phosphate deposition to produce calcified bone tissue. It also induces the expression of several osteoblast-related genes through integrin mediated signal transduction (Mizuno and Kuboki, 2001). IBSP Integrin-binding sialoprotein Noncollagenous glycoprotein in mineralized tissues such as bone. May be involved in cell attachment and signalling, hydroxyapatite nucleation, and binding of type I collagen (Malaval et al., 2008). SPARC Osteonectin Glycoprotein belonging to a group of matrix associated factors that mediate cell-matrix interactions but do not serve primarily structural roles; has been shown to be a Ca2+-binding glycoprotein. Seems to have a major role in bone remodelling or repair (Brekken and Sage, 2001). These protein-coding genes are commonly used as markers of osteogenesis, in vitro, since there are extensive studies attesting their importance in this cell differentiation mechanism. See text and cited references for details. 1.Introduction PhD thesis – José Bernardo Noronha Matos (2011-2014) 33 ECM containing type I collagen is mandatory for osteoblasts to differentiate and express osteoblast-related genes such as those encoding osteocalcin, bone sialoprotein, alkaline phosphatase among others (Table 1) and, ultimately, to mineralize. Usually, the ECM signals through β1 subunit-containing integrins (like α2β1). This will promote differentiation of pre-osteoblast into mature osteoblasts. This sequence may be prevented using either blocking antibodies or peptides that BMP α2 β1 ECM Mechanical force FGF 2 I II PTH/PTHRP Smad1/5 Smad4 Gq Gs FAK RTK Runx-2 Gene PKC PKA Ras Raf MEK1/2 Erk1/2 Runx-2 Runx-2 P P Smad1/5-4 SBE OSE2 Fos/Jun AP-1 Osteoblast target gene Osx Figure 3. Signal transduction pathways modulating Runx-2 activity. This transcription factor is frequently described as the master regulator of osteoblastogenesis, since it acts throughout the induction, proliferation and maturation of osteoblasts and regulates expression of many osteoblast genes. See text for details. Based on Franceschi and Xiao, 2003. 1.Introduction PhD thesis – José Bernardo Noronha Matos (2011-2014) 34 bind to the cell-binding domain of collagen, thus preventing ECM-dependent differentiation (Danen et al., 1998). Others have shown that ECM production by murine MC3T3-E1 cells increases significantly the transcription of osteocalcin coding gene, which is Runx-2 dependent (Xiao et al., 1997). Several authors suggested that the MEK/ERK branch of the mitogen activated protein kinase pathway, or MAPK, is a plausible linkage between integrin activation and subsequent stimulation of Runx-2-dependent transcription (reviewed in Franceschi and Xiao, 2003). A previous study showed that selective inhibition of ERK1/2 phosphorylation by MEK with U0126 prevented ECM-dependent induction of the osteocalcin coding gene. The authors showed that BMP action on osteogenesis was also prevented with the same blocker (Xiao et al., 2002). Other studies showed that transfecting cells with MEK1 results in Runx-2 phosphorylation, knowing that MEK1 is involved in the MAPK pathway (Xiao et al., 2000). In conclusion, evidences demonstrate that osteoblasts differentiation requires close contact with collagen-containing ECM, interacting with it through specific β1integrins. This interaction allows the activation of the MAPK pathway, which transduce signals to the nucleus. Subsequently, Runx-2 is phosphorylated and allows the stimulation of osteogenesis by increasing transcription of osteoblast marker genes such as those depicted in Figure 3. As already mentioned, FGF2 is an important in vivo regulator of skeletal development and growth. Its administration can restore bone mass in the overiectomized female rat, which is a well-established model for postmenopausal bone loss (Liang et al., 1999). In transgenic mice, overexpression of FGF2 causes premature mineralization, achondroplasia and shortening of long bones. On the other hand, disruption of the FGF-2 gene leads to decreased bone formation and mass (Coffin et al., 1995; Montero et al., 2000). Some other interesting findings revealed that activating mutations in FGFR1 up-regulate Runx-2, enhancing differentiation of calvarial osteoblasts (Zhou et al., 2000). FGF2 was also shown to promote osteocalcin gene expression in MC3T3-E1 pre-osteoblast cells (Boudreaux and Towler, 1996). The major route of FGF receptor signalling, as shown in Figure 3, involves the activation of MEK/ERK branch of the MAPK 1.Introduction PhD thesis – José Bernardo Noronha Matos (2011-2014) 35 pathway (Nugent and Iozzo, 2000). Xiao and co-workers also showed that FGF2 could rapidly induce ERK phosphorylation and increase osteocalcin mRNA, in both MC3T3-E1 and bone marrow stromal cells (Xiao et al., 2002). Others revealed that FGF2 leads to osteocalcin production by promoting the transcription of its coding gene through an AP-1-like site that is immediately 5’ to the RUNX-2 binding site (Boudreaux and Towler, 1996) (Figure 3). AP-1 refers to a class of transcription factors that is composed of heterodimers of Fos-related factors (c-Fos, Fra1, Fra2 and FosB) and Jun proteins (c-Jun, JunB and JunD). These are highly expressed in proliferating osteoprogenitors. The c-fos gene is usually evaluated to measure the extent of osteogenesis in vitro (Table 1). Accordingly, there are a number of direct gene targets of AP-1 in osteoblasts, namely those coding for osteocalcin, collagenase-3, bone sialoprotein and alkaline phosphatase (Table 1) (Owen et al., 1990). Returning to FGF2, it was also shown that its response is synergistically stimulated by a PKA pathway activator (forskolin), which was also shown to increase the activity of AP-1 related nuclear factors (c-Fos and c-Jun). So, it seems that AP-1-like factors and Runx-2 share cooperative interactions in the promotion of osteogenesis (Franceschi and Xiao, 2003). As depicted in Figure 3, mechanical stimulation is important in the regulation of bone homeostasis. Mechanical strained human osteoblast-like cells express increased levels of coding mRNA for osteopontin, osteocalcin and collagen I and III (Carvalho et al., 1998; Harter et al., 1995). The MAPK pathway seems to be largely involved in this process, and integrins seem to be associated to these mechanotransduction signals. These proteins connect the cytoskeleton to the extracellular proteins, mediating the transduction of mechanical stimuli into biochemical signals. One particular example of this observation are the experiments undertaken by Schmidt and co-workers, who showed that MAPK activation is related to α2β1 integrins, when an osteoblastic cell line was submitted to mechanical stress, using a magnetic drag force device (Schmidt et al., 1998). Ziros and co-workers also showed that osteoblast differentiation is promoted when mechanical stretching is applied, promoting the binding of Runx-2 to OSE2 DNA in 1.Introduction PhD thesis – José Bernardo Noronha Matos (2011-2014) 42 osteoclasts and osteocytes. In order to repair, to grow and adapt, the bone needs to undergocontinuous remodelling. Throughout life, many abnormalities in such balanced process may result in a huge variety of bone disorders and skeletal abnormalities (further discussed below) (Orriss et al., 2010). Together with cartilage, bone makes the skeletal system, holding three important functions: mechanical, protective and metabolic. Mechanical, since it bears a supportive role and is a site of muscle attachment; protective, since it shields vital organs and bone marrow; metabolic, since it is a reservoir of ions for the entire organism, in particular calcium and phosphate (Adler, 2000). Its main constituents are the cells and the extracellular matrix. The extracellular matrix is composed of collagen fibres and noncollagenous proteins (see below). The matrix of bone has the unique ability to calcify (Clarke, 2008). Non-collagenous proteins together with collagen and water, are the main constituents of bone matrix. There are two main forms of extracellular matrix (ECM): the osteoid and mineralized matrix. Osteoid is an immature form of matrix excreted by osteoblasts and it’s found in areas of new bone formation. It is subsequently mineralised by calcium and phosphate ions to produce calcified bone tissue; the mineral approximates to hydroxyapatite, Ca10(PO4)6(OH)2 (Orriss et al., 2010).This mature form of matrix is largely mineral, being calcium and phosphate the main constituents. The turnover of bone minerals is highly regulated in a process known as bone remodelling. The extracellular matrix gives the bone its mechanical properties but is also important for regulation and formation of new bone (Baron, 1993). There are four major cell types within bone tissue itself: osteoclasts, osteoblasts, osteocytes, and bone lining cells. Within the cavities of the bone, there is also bone marrow, which has numerous cell types, including the progenitor cells for the haematopoietic cell lineages (Baron, 1993). The osteoblast is the cell responsible for construction of new osteoid (which eventually becomes ECM). It is also the osteocyte and the bone lining cell precursor, also involved in osteoclast regulation (Boyce, 2013). The osteoblast is derived from the mesenchymal marrow stromal cells as mentioned above. TGF-β, BMPs, PTH, and 1.Introduction PhD thesis – José Bernardo Noronha Matos (2011-2014) 43 vitamin D are all important in stimulating MSCs to become osteoblasts (Deng et al., 2008). MSCs can be found in both the bone marrow and the inner layer of the periosteum. Osteoblasts can be incorporated into the osteoid and become osteocytes, line the bone and become bone lining cells, or undergo apoptosis. They are stimulated by PTH, 1,25-hydroxyvitamin-D, and insulin-like growth factor (IGF)-I (Datta et al., 2008). The mature osteoblast is designed for protein synthesis: it has a large and efficient rough endoplasmic reticulum, Golgi apparatus, and secretory vesicles. Osteoblasts are polarized, with their synthetic functions at one end (near the cellular attachment areas) and their regulatory functions and nucleus at the other (Deng et al., 2008). Bone lining cells, which derive from osteoblasts, no longer play a role in synthesis. These flat thin cells have little metabolic activity, covering nonmetabolically active areas of the bone. However these play an important role in bone resorption: it is the peelback of the lining cells that stimulates and allows the attachment of osteoclasts to bone (Everts et al., 2002). Osteocytes are derived from the osteoblasts. During the formation of new bone, these become embedded in the bony matrix and differentiate into osteocytes. These cells form a connected cellular network that, along with the nerve fibres in bone, has a role in the response to mechanical loading (Kringelbach et al., 2014), since they sense mechanical strain and cracking, and respond by triggering bone remodelling. To balance this effect they can secrete sclerostin, which reduces bone formation (Kogawa et al., 2013). It survives in single cell-sized hole in the bone known as a lacuna. It still plays a vital role in bone homeostasis, although different from the osteoblast. 90% of all bone cells are osteocytes, and they can survive for decades. These are interconnected to one another through long cellular projections in tunnels known as canaliculi. Additionally, these tunnels serve as the source of nutrients and disposal of waste for these cells (Milovanovic et al., 2013). The osteoclast derives from the haematopoietic macrophage lineage. The osteoclast is a multinucleated giant cell that is responsible for bone resorption, usually found in contact with a calcified bone surface and within a lacuna 1.Introduction PhD thesis – José Bernardo Noronha Matos (2011-2014) 44 (Howship’s lacunae). The contact zone with the bone is characterized by the presence of a ruffled border and the attachment proteins that allow it to seal itself to the bone surface and pump carbonic acid (Baron, 1993). In addition to this acidic environment, the osteoclast synthesizes enzymes that degrade extracellular matrix proteins. Its function is intimately tied to the osteoblast cells (see below). This paired activity of bone-building and bone-absorbing cells is known as coupling and is crucial to the regulation of bone and calcium in the body (Ikeda and Takeshita, 2014) 1.3.2. Bone remodelling and metabolism As mentioned earlier in this thesis, bone has multiple functions in vertebrates, including protection of vital organs and haematopoietic marrow, structural support for muscles, and storage and release of vital ions, such as calcium, and of growth factors which are stored in the matrix. Bone remodelling is the process in which bone, in the adult skeleton, is renewed continuously in response to a variety of stimuli. The osteoclasts are involved in the removal of trenches or tunnels of bone from the surfaces of trabecular and cortical bone, respectively (Boyce, 2013). Osteoblasts subsequently fill in these trenches by laying down new bone matrix in them. Bone formation matches resorption during normal bone remodelling (Boyce and Xing, 2008). Bone remodelling involves: (1) the activity of osteoblasts and osteoclasts; (2) the actions of a variety of cytokines; (3) the turnover of bone minerals, particularly calcium and phosphate (although other minerals, such as magnesium, may also be important); (3) the actions of several hormones, such as parathyroid hormone (PTH), the vitamin D family, oestrogens, growth hormone, steroids, calcitonin and various cytokines (such as interleukins). Many other factors affect bone remodelling, such as exercise, diet and drugs (Feng and McDonald, 2011). Bone remodelling starts with recruitment of osteoclast precursors and the subsequent differentiation of these to mature multinucleated osteoclasts induced by cytokines. Osteoclast precursors (OCPs) are attracted from the bone marrow (where they are held by the stroma-derived factor-1, SDF-1) to the bloodstream by 1.Introduction PhD thesis – José Bernardo Noronha Matos (2011-2014) 45 chemokines and circulate there until they are attracted back into bones by a variety of factors released at sites undergoing resorption, called bone remodelling units (BRUs), differentiating into osteoclasts (Boyce et al., 2012). These factors include the macrophage-colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL) (Boyce et al., 2012). Osteoclasts adhere to an area of trabecular bone, developing a ruffled border at the attachment site. H+ ions are pumped through the ruffled border and, along with Cl-, form HCl, which demineralizes bone, and cathepsin K is secreted to degrade the matrix (Boyce, 2013). This process gradually releases IGF-I and TGF-β, which were trapped in the osteoid. These cytokines recruit and activate osteoblasts cells, which invade the site, synthesising and secreting the organic matrix of bone, the osteoid, and secreting IGF-I and TGF-β (see e.g. Chernausek et al., 2007, and Janssens et al., 2005) Some osteoblasts become embedded in the osteoid, differentiating into osteocytes; others interact with and activate osteoclast precursors, the process known as coupling (see above). Besides IGF-I and TGF-β, other cytokines belonging to the members of the TGF-β family, such as the bone morphogenic proteins (BMPs), a range of interleukins, various hormones and members of the tumour necrosis factor (TNF) family are involved in bone remodelling. RANK, receptor activator of nuclear factor kappa B (NFκB), is a central receptor involved in osteoclast differentiation, being NFκB the principal transcription factor involved. It is activated by RANKL, which exists as a homotrimeric protein and is typically membrane-bound on osteoblastic and activated T cells or is secreted by some cells, such as activated T cells (Kearns et al., 2008). Like other TNF receptor family members, RANK lacks intrinsic kinase activity to phosphorylate and activate downstream signalling molecules; it recruits TNF receptor-activating factors (TRAFs), particularly TRAFs 1, 2, 3, 5, and 6, which are adapter proteins that recruit protein kinases (Boyce, 2013). Briefly, this process allows the activation of NIK (NF-κB-inducing kinase), and signalling through this kinase and TRAF6 results in the activation of NF-κB and subsequent increased expression of NFATc1 (which has been called the 1.Introduction PhD thesis – José Bernardo Noronha Matos (2011-2014) 46 master regulator of osteoclastogenesis) to induce further osteoclast progenitor differentiation (Boyce, 2013). Osteoblasts also produce osteoprotegerin (OPG), a decoy ligand that inhibits osteoclast differentiation by blocking the RANK receptor. During coupling, OPG can bind to RANKL and inhibit RANKL's binding to the functional receptor, RANK, on the osteoclast precursor cell. RANKL/OPG ratio is critical in the formation and activity of osteoclasts and, thus, the optimal functioning of the RANK, RANKL and OPG system is crucial for bone remodelling (Boyce and Xing, 2008; Wright et al., 2009). Oestrogens induce osteoprotegerin synthesis, which may be one mechanism by which exogenous oestrogens inhibit osteoclast function and bone resorption in postmenopausal women (see section 1.2.3). As mentioned above, bone remodelling involves daily turnover of bone minerals, being calcium and phosphate the most important. Regarding calcium, 99% of this mineral is in bone and teeth. Normal serum calcium varies from 2.1 to 2.6 mM. Three different components may be found: ionized (approximately 50%), protein-bound (nearly 40%, mainly albumin) and complexed to anions such as phosphate and citrate (approximately 10%). It is the ionized form (approximately 1.2 mM) that exerts the biological effects, and so it is tightly regulated. Hormones regulate plasma Ca2+ by controlling Ca2+ absorption from the intestine and its excretion by the kidney. These hormones may also recruit Ca2+ from the skeletal reservoir (Rang et al., 2011). Ca2+ enters the body through the intestine by facilitated diffusion (majority of total calcium uptake) throughout the small intestine and by active transport in the proximal duodenum (regulated by vitamin D). Glucocorticoids or phenytoin depress intestinal calcium transport. There is a calcium loss due to mucosal and biliary secretions and sloughing of intestinal cells. Calcium excretion in the kidney is also highly regulated. Around 9 g of Ca2+ is filtered in the glomeruli, of which >98% is reabsorbed in the tubules. The efficiency of tubular reabsorption is regulated by PTH and influenced by filtered Na+, the presence of non–reabsorbed anions, and diuretics. Regarding phosphate, approximately 80% of its total is found in bone and around 15% in soft tissues. It is present in collagen, bone, 1.Introduction PhD thesis – José Bernardo Noronha Matos (2011-2014) 47 plasma, extracellular fluid, cell membrane phospholipids and in the intracellular fluid. Phosphorus exists in both organic and inorganic forms in the body. The former includes phospholipids and organic esters. In the extracellular fluid, phosphorus exists as inorganic phosphate (Pi) in the form of NaH2PO4 and Na2HPO4. Pi interferes with tissue concentrations of Ca2+ and plays a major role in renal acid excretion. In bone, phosphate is complexed with calcium as hydroxyapatite and calcium phosphate (see above). Phosphate uptake in the intestine is passive, although an active component may be stimulated by several factors, including vitamin D. Its excretion in the kidney is highly regulated. More than 90% of plasma phosphate is filtered at the glomeruli, and more than 80% of which is reabsorbed in the tubules. Renal phosphate reabsorption is regulated by many factors, including PTH and dietary phosphate (for a review, see Moe, 2008). The most important hormones that regulate Ca2+ and phosphate concentrations are PTH and 1,25-dihydroxyvitamin D (or calcitriol), which regulate mineral homeostasis by effects on the kidney, intestine, and bone. Besides these, other hormones are involved in bone metabolism and remodelling, such as oestrogens, calcitonin and thyroid hormone (for a review, see Raisz, 1999). PTH, parathyroid hormone, consists of a single polypeptide chain of 84 amino acids. It acts on PTH receptors in various tissues (like bone, kidney and gastrointestinal tract) and its role is to maintain a constant concentration of Ca2+ in the extracellular fluid. Parathyroid hormone is synthesised in the cells of the parathyroid glands, stored in vesicles, and its release is controlled by the concentration of ionised calcium in the plasma. Calcium acts via the calciumsensing receptor (CaSR), a GPCR that couples with Gq-PLC and Gi. Occupancy of the CaSR by Ca2+ inhibits PTH secretion (in a case of hypercalcaemia); on the other hand, hypocalcemia stimulates PTH secretion (Bisello et al., 2004; Deal, 2009). In tissues, PTH binds to the PTH type 1 receptor, a class II G protein-coupled receptor that is abundant in kidney and bone (in osteoblasts and osteocytes), and triggers classic G-protein signalling pathways, including Gs-linked cAMP production and Gq/11-dependent activation of phospholipase C (PLC) β and 1.Introduction PhD thesis – José Bernardo Noronha Matos (2011-2014) 48 subsequently calcium transients and protein kinase C (PKC) activation (Sneddon et al., 2004). In the kidney, PTH enhances the efficiency of Ca2+ reabsorption (primarily in the distal nephron through the stimulation of the epithelial Ca2+ channel TRPV5), inhibits tubular reabsorption of phosphate, and stimulates conversion of 25-OH-ergocalciferol (25-OHD) to calcitriol, since it stimulates 1αhydroxylase, enhancing the synthesis and release of calcitriol. As a result, Ca2+ plasma concentration increases, whereas phosphate is excreted and its plasma concentration falls. Calcitriol, in turn, will interact with vitamin D receptors (VDRs) in the intestine to increase the efficiency of calcium absorption, and so promotes the increase of plasma Ca2+ concentration (see e.g. Li et al., 1998). The skeletal actions of PTH are complex, since this hormone can stimulate both osteoblast-dependent bone formation and osteoclast-mediated bone resorption; PTH increases bone resorption and thereby increases Ca2+ delivery to the extracellular fluid. The major effects of PTH on differentiated osteoblasts are dependent upon binding to the transmembrane PTH/PTHrP receptor type 1 (PTHR1) and activation of transcription factors such as the activator protein-1 family, Runx-2 and cAMP response element binding protein (CREB); much of the regulation of these factors by PTH is protein kinase A (PKA)-dependent, (Swarthout et al., 2002), promoting osteoblastogenesis. On other hand, several studies suggest that continuous (but not intermittent) PTH can result in an increase in receptor activator of nuclear factor-kB ligand (RANKL) expression and consequent osteoclastogenesis in culture, with an associated inhibitory effect on osteoprotegerin expression (Locklin et al., 2003; Ma et al., 2001). Vitamin D (calciferol) consists of a group of lipophilic pre-hormones that are converted in the body into a number of biologically active metabolites that function as hormones (Reichel et al., 1989). Their main action is mediated by nuclear receptors of the steroid receptor superfamily (VDR), and consists in the keeping of plasma Ca2+ by increasing its absorption in the intestine, mobilising Ca2+ from bone and decreasing its renal excretion circulating in the blood. It also regulates the activities of various cell types, namely osteoblasts (Anderson et al., 2013). In humans, there are two sources of vitamin D, dietary ergocalciferol (D2), derived 1.Introduction PhD thesis – José Bernardo Noronha Matos (2011-2014) 49 from ergosterol in plants, and cholecalciferol (D3) generated in the skin from 7dehydrocholesterol by the action of ultraviolet irradiation (Norman, 2008). In this particular mechanism, exposure to ultraviolet light in sunlight converts 7dehydrocholesterol to cholecalciferol (vitamin D3) in the skin. In liver, hydroxylation occurs to form 25-OH-cholecalciferol or 25-OH-ergocalciferol (25-OHD). After production in the liver, 25-OHD enters the circulation, bound to vitamin D-binding protein and its final activation occurs predominantly in the kidney, where 1αhydroxylase in the proximal tubule converts 25-OHD to calcitriol. In the kidney, calcitriol stimulates Ca2+ reabsorption in the distal tubule. In the proximal duodenum, calcitriol stimulates TRPV6 Ca2+ channels and also induces the synthesis of calbindin D9K, calbindin D28K, and the serosal membrane Ca2+- ATPase, promoting calcium uptake. In the absence of calcitriol, passive diffusion via the paracellular pathway is the mechanism involved (Morris and Anderson, 2010). Regarding bone, vitamin D has a direct effect on bone cells. Osteoblasts, osteoclasts and osteocytes are capable of converting 25-OHD into calcitriol (Morris and Anderson, 2010). Human and rodent osteoblasts express the CYP27B1 enzyme (or 1α-hydroxylase) which is essential to convert 25-OHD to calcitriol and to increase expression of key genes associated with maturation and mineralization, the calcitriol responsive genes osteocalcin and osteopontin (Atkins et al., 2007). In the transgenic mouse model of osteoblast and osteocyte VDR overexpression, an increased bone volume and strength due to increased mineral apposition as well as decreased osteoclast formation was shown, proving the direct action of vitamin D on osteoblasts. However, on bone absorptive cells, the osteoclasts, it was found that these express cytoplasmic CYP27B1 and nuclear VDR proteins. In pre-osteoclasts, such as in human peripheral blood mononuclear cell preparations, CYP27B1 expression was necessary for 25D to optimise osteoclastogenesis in the presence of RANKL and M-CSF in vitro (Kogawa et al., 2010). So, vitamin D action in bone is complex and goes beyond the Ca2+ increase in the plasma. However, it is well known that clinical vitamin D deficiency (see 1.Introduction PhD thesis – José Bernardo Noronha Matos (2011-2014) 50 below), in which the mineralisation of bone is impaired administration of vitamin D restores bone formation (Norman, 2008). Calcitonin is a peptide of 32 amino acids. It is a hormone secreted by the specialised C cells found in the thyroid follicles. Its actions oppose those of PTH. Calcitonin is able to decrease blood calcium levels by direct inhibition of mediated bone resorption and by enhancing calcium excretion by the kidney (Masi and Brandi, 2007). Calcitonin secretion increases in hypercalcaemia and decreases when plasma Ca2+ is low. Circulating concentrations are normally low (<15 pg/mL in males and 10 pg/mL in females) but can be markedly elevated with C cell hyperplasia or medullary thyroid cancer (Verburg et al., 2013). Calcitonin actions are mediated by the calcitonin receptor (CR), a GPCR that couples through multiple G proteins to diverse signal transduction pathways. Different isoforms of calcitonin receptors resulting from alternative splicing of the gene have been described in various animal species with differential tissue expression transcripts and different signalling properties (Moore et al., 1995; Nussenzveig et al., 1994). One of the most important pathways is coupled to the cAMP signal transduction, but calcitonin receptors may also couple to the phospholipase C (PLC) enzyme pathway. Other authors also demonstrated that calcitonin when bound to CTRs stimulates Shc tyrosine phosphorylation of MAPK Erk1/2 (Chen et al., 1998; Pondel, 2000). Osteoclasts are the major target for the action of calcitonin. Calcitonin is able to interfere with osteoclast differentiation from precursor cells and fusion of mononucleated precursors to form multinucleated cells in bone marrow cultures (Takahashi et al., 1988). Calcitonin may also inhibit several components of the osteoclast function, such as the release of acid phosphatase and the expression of carbonic anhydrase II (Zaidi et al., 1994; Zheng et al., 1994), which is an enzyme responsible for pH regulation, CO2 and HCO3transport, and maintaining H2O and electrolyte balance (Sly and Hu, 1995). Sex steroids also play a key role in the maintenance of bone integrity. In fact, the loss of ovarian function at menopause has long been associated with bone loss (for a review, see Zallone, 2006). In males, although not so abrupt, the 1.Introduction PhD thesis – José Bernardo Noronha Matos (2011-2014) 51 decline of androgen levels has also been implicated to deleterious effects in the skeleton. In fact, it is widely accepted that androgens are locally transformed into oestrogens by aromatase on bone cells (Bilezikian, 2002). The classical receptors for oestrogens (the ERα and ERβ) or androgens (AR) are expressed in bone marrow stromal cells, osteoblasts, osteoclasts and their precursor cells (Bellido et al., 1995; Couse and Korach, 1999). This suggests that the effects of sex steroids on bone may be mediated directly on such cells (Zallone, 2006). Several studies have indicated that ER receptors are important in the regulation of cytokine production that may act in an autocrine and paracrine manner, regulating osteoclastogenesis and osteoblastogenesis. These may be produced by osteoblasts, stromal cells and lymphocytes (Manolagas and Jika, 1992). Studies have shown that after menopause, there is an increase on circulating levels of IL-1 and IL-6 (Manolagas et al., 2002). These have been associated with bone loss, and several studies have reported that osteoclastogenesis is associated with increasing levels of these interleukins. Knockout of IL-6 gene in mice prevents bone loss following a decrease of sex steroids (Bellido et al., 1995). Other studies showed that in ovariectomized women and mice there are increases of circulating levels of IL-1 and TNF-α, produced by T cells and blood mononuclear cells (Kitazawa et al., 1994; Pacifici et al., 1991). These have been associated with an increased expression of RANKL by osteoblasts, stimulating the differentiation of osteoclast precursors (Wei et al., 2005). IL-7 levels are also associated with postmenopausal bone loss. In an ovariectomy-induced bone loss model in mice, neutralization of IL-7 completely prevents bone loss and rescues bone formation (Weitzmann et al., 2002). This effect seems to be dependent on T cells, since in T cell-deficiency nude mice, injection of IL-7 failed to induce bone loss (Toraldo et al., 2003). In addition, in mice models of osteoporosis, ovariectomy causes a dramatic increase in the osteoblast and osteocyte apoptosis. Addition of both oestrogens and androgens prevents such effects, a mechanism that was shown to be mediated by a Src/Shc/ERK signalling cascade (Kousteni et al., 2001). Several 1.Introduction PhD thesis – José Bernardo Noronha Matos (2011-2014) 58 slow bone loss, decreasing bone fracture risk. It supresses osteoclast activity and bone remodelling similarly to oestrogen through high affinity interactions with ERα (Aref et al., 2013; Ettinger et al., 1999). In fact, oestrogens prevented bone loss in ovariectomized rats (Turner et al., 1993). Some authors have also hypothesized that raloxifene may also act directly on the bone matrix to improve material properties, namely affecting tissue-level biochemical properties through non-cell mediated effects on hydration (Gallant et al., 2014). As in other pharmacological approaches, raloxifene may have adverse effects, namely the increased incidence of deep venous thrombosis (Grady et al., 2004). Regarding the anabolic therapy, PTH and PTH fragments are a new line of compounds that may be used to promote bone formation. The peptide fragment (1-34) is currently in clinical use, the teriparatide. The binding of the ligand to the receptor activates adenylate cyclase and a number of phospholipases (A, C, and D) and increases intracellular levels of cAMP and calcium (Whitfield et al., 1997). Possible cellular mechanisms include the expression of skeletal IGF-I and boneforming genes. Evidence has shown that teriparatide increases osteoblast growing rate and prevents osteoblastic apoptosis. These processes increase the number of osteoblasts and the rate of new bone formation, and prolong osteoblast survival (for a review, see Misiorowski, 2011). Teriparatide is given subcutaneously once a day. Despite of well tolerated, there has been reported nausea, dizziness, headache and arthralgia. Mild hypercalcaemia, transient orthostatic hypotension and leg cramps have also been reported (Rang et al., 2011). Some agents have both anabolic and anti-resorptive actions. Such an example is strontium ranelate. This dual mode of action was demonstrated in experimental studies on bone cells and pharmacological studies in animals (for a review, see Neuprez et al., 2008). It seems that strontium decreases differentiation and resorbing activity of osteoclasts and increases osteoclast apoptosis (Marie et al., 2001). In contrast, strontium ranelate seems to enhance pre-osteoblastic cell replication and collagen synthesis in culture (for a review, see Saidak and Marie, 2012). In addition, it modulates the osteoprotegerin/receptor activator of nuclear factor kappa B ligand (OPG/RANKL) system in favour of OPG, and so preventing 1.Introduction PhD thesis – José Bernardo Noronha Matos (2011-2014) 59 osteoclast maturation (Atkins et al., 2009). Finally, strontium ranelate administration decreases bone resorption and maintains bone formation in adult ovariectomized rats, which results in the prevention of bone loss and an increase in bone strength (Morohashi et al., 1995). In addition, clinical studies have shown that strontium ranelate reduces vertebral, nonvertebral and hip fractures over 1-5 years. Its spectrum of activity covers osteopenia and osteoporosis. In elderly subjects, strontium ranelate also promotes a reduction in vertebral and nonvertebral fractures (Neuprez et al., 2008). Renal osteodystrophy refers to a varied group of metabolic bone diseases that accompany chronic kidney diseases (Feng and McDonald, 2011). Its pathophysiology is complex and reflects PTH and vitamin D importance on bone turnover and related pathological abnormalities. High-turnover renal osteodystrophy can best be categorized as a secondary hyperparathyroidism state and low-turnover disease is characterized by a relative hypoparathyroidism state. Sustained PTH levels increases bone turnover with increased osteoblasts, osteoclasts and osteocytes and a consequent disorder on remodelling leads to abnormal bone formation, increased osteoid and fibrosis. Hypocalcaemia, vitamin D deficiency, hyperphosphataemia are primary factors contributing to secondary hyperparathyroidism (Malluche et al., 2010; Moe et al., 2006). Quantitative histomorphometry in conjunction with appropriate laboratory analyses is often used to make the diagnosis (Moe et al., 2006). Abnormal mineralization reflects the process of calcification of collagen in bone, reflected histologically as abnormal osteoid. This impaired mineralization is still poorly understood, but it’s believed to be caused by vitamin D and/or mineral deficiencies, aluminium toxicity, and possibly to a prolonged acidotic state (Feng and McDonald, 2011). The most common form of osteodystrophy is characterized by an absolute or relative PTH deficiency accompanied by an extremely low rate of bone formation. Factors implicated in this disorder include increased plasma calcium, vitamin D toxicity, and altered growth factors and cytokines such as BMPs, TGFβ, IGF-I and IL-6 (Hruska et al., 2008; Malluche et al., 2010). 1.Introduction PhD thesis – José Bernardo Noronha Matos (2011-2014) 60 For high-turnover disease, the therapeutic approach is focused on phosphorous restriction or use of phosphate binders, correcting vitamin D deficiency and, occasionally, the use of calcium supplements. Calcimimetic agents are recently being used to inhibit PTH secretion. For low-turnover disease, primary methods are designed to increase PTH levels, usually reducing calcium and vitamin D as well (Feng and McDonald, 2011). Paget’s disease is a bone disorder characterized by a high bone turnover. Paget’s disease is an osteoclastic-mediated disorder of bone that results in abnormal bone resorption associated with inadequate remodelling that leads to mechanically weakened bone. It’s a disease that is more prevalent with increasing age. Most patients are asymptomatic, but many present bone pain, bone deformities, secondary arthritis, and in some instances, secondary to bone deformities, neurological problems. Although the pathogenesis of the disease is largely unknown, it is believed to be a primary disorder of increased osteoclast bone resorption with a resulting secondary increase in osteoblast activity and new bone formation. Resulting trabecular bone is abnormal with a disorganized appearance (Feng and McDonald, 2011; Singer, 2009). It seems to be a genetic cause, since it follows a familial pattern (Beauregard et al., 2013; Gruener and Camacho, 2014). One such mutation is a gene that encodes an ubiquitin-binding protein that plays a role in NF-kB signalling (Wright et al., 2013). Associated to this disease is chronic viral infection, namely with paramyxoviral and the canine distemper virus (Bianco et al., 1992; Gordon et al., 1991). The therapeutic approach is directed at inhibiting osteoclasts, usually using bisphosphonates; in alternative, calcitonin is used to both prevent osteoclast activity and as analgesic, since it helps to treat patients with substantial bone pain; additionally, therapy may include surgery in order to correct bone deformities (Siris, 1995). Osteopetrosis are that family of congenital diseases which is characterized by a strikingly radiopaque skeleton, with loss of distinction between cortex and marrow space. The hallmark of the disease is the persistence of “cartilaginous bars” deep within metaphyseal and diaphyseal bone. Such islands of devitalized cartilage surrounded by bone represent the residue of nonresorbed primary spongiosa, a 1.Introduction PhD thesis – José Bernardo Noronha Matos (2011-2014) 61 component of endochondral ossification (Teitelbaum, 1993). The forms of disease are as follows: severe and intermediate autosomal recessive types and a milder autosomal dominant subtype. There have been identified several gene mutations all which encode proteins that involve osteoclast-mediated bone resorption. The three most important mutations are related to carbonic anhydrase II, proton pump and chloride channel. The proton pump is abundantly expressed in the ruffled membrane and transports protons into the resorption lacuna to create and maintain a low pH (~4.5); this situation provides a high concentration of acid onto a strongly basic mineral to dissolve the inorganic component of bone hydroxyapatite; these protons are generated from carbon dioxide and water by carbonic anhydrase II; in the other hand, the chloride channel, also present in the ruffled membrane, transports Clinto the resorption lacuna to maintain electron neutrality. Mutations in these indicated proteins will compromise bone resorption, leading to different forms of osteopetrosis (Feng and McDonald, 2011). This disease is diagnosed through the use of a combination of clinical and radiological parameters; increased serum concentrations of creatine kinase BB isozyme and tartrate-resistance acid phosphatase (which derive from osteoclasts) are often observed (Bollerslev et al., 2000; Waguespack et al., 2002). In terms of therapy, which is only supportive, haematopoietic stem cell transplantation is usually a choice (Driessen et al., 2003); treatment regimens with interferon-γ, calcium restriction, vitamin D, steroids and PTH have all been tried, with no apparent success (Feng and McDonald, 2011). Rickets is a pathological situation in which there is a reduction of mineralisation of newly-formed bone, that is, osteoid is unmineralised (osteomalacia) and endochondral calcification at the growth plate is absent or reduced, with associated growth-plate deformity (Elder and Bishop, 2014). Vitamin D deficiency is the most common cause in most cases. It remains unclear whether an absolute threshold for vitamin D exists, below which rickets is inevitable; rickets can also occur when vitamin D is within the range associated with maximum calcium absorption, but calcium intake is low (DeLucia et al., 2003). In some cases, abnormalities that mainly affect phosphate metabolism or bone-tissue 1.Introduction PhD thesis – José Bernardo Noronha Matos (2011-2014) 62 mineralisation might be the cause. The consequences of vitamin D deficiency in bone are the direct inhibition of osteoblast progenitors and an increase in RANKL expression, with a parallel decrease in OPG, decreasing bone formation and enhancing bone turnover. Treatment is focused on fixing the vitamin D deficiency, increasing sunshine exposure and through dietary supplements (such as ergocalciferol). Some uncommon forms of rickets have been reported that could not be corrected with vitamin D therapy, which seems to be an inheritable form of the disease, eventually related to vitamin D receptor abnormalities and to vitamin D metabolism anomalies (Elder and Bishop, 2014). Osteogenesis imperfecta is an inherited connective tissue disorder of remarkable clinical variability, caused by a quantitative or qualitative defect in collagen synthesis and is characterised by bone fragility (Antoniazzi et al., 2000). It is often fatal in utero but those who survive fall into a spectrum of skeletal dysfunction ranging from little or no deformity to relentless crippling associated with hundreds of fractures. Cardinal manifestations are low-bone mass and reduced bone mineral strength, leading to bone fragility and deformity. Cortical bone is more diminished than is trabecular bone, and remodelling is typically brisk in this disease (Teitelbaum, 1993). In about 90% of individuals with the clinical diagnosis of osteogenesis imperfecta, mutations in the COL1A1 and COL1A2 genes are responsible for the disorder, which code for the α1(I) and α2(I) side chains of type I collagen (Rohrbach and Giunta, 2012). Three types of treatment are available: nonsurgical management (physical therapy, rehabilitation, bracing and splinting), surgery, and drugs to increase the strength of bone and decrease the number of fractures. These include bisphosphonates administered to children with osteogenesis imperfecta, with the rationale that bones with increased volume of osteogenesis imperfecta-quality matrix will be more fracture resistant (Rauch et al., 2006). Other approaches are being considered, as antibodies directed against RANKL, in order to shift the RANKL/OPG ratio, and decrease osteoblast signalling that normally stimulates osteoclast development (Forlino et al., 2011; Hussar and Stevenson, 2010). 1.Introduction PhD thesis – José Bernardo Noronha Matos (2011-2014) 63 In general, the development of new therapeutic approaches is of major importance since these bone disorders, like osteoporosis, are highly prevalent and in increasing incidence on an ageing population. In the next chapter, purinergic signalling, in the context of bone remodelling, will be explored, reinforcing the huge and growing potential of such area in the development of new therapeutic strategies to overcome bone deterioration in common bone-related disorders. 1.4. Purinergic signalling and bone remodelling 1.4.1. Purinergic receptors As mentioned previously, bone turnover is a complex and finely tuned process. The importance of systemic and local factors has already been stated, and to these we must include the significant role of nucleotides, such as adenosine 5’- triphosphate (ATP) and its derivatives (like adenosine). It is known that nucleotides play fundamental roles in energy metabolism, nucleic acid synthesis and enzyme regulation (Burnstock and Knight, 2004). Nucleotides, predominantly ATP, are present in intracellular concentrations between 2-5 mM, and extracellular concentrations are low, mainly due to the presence of ubiquitous ectonucleotidases and other ecto-phosphatases that rapidly hydrolyse extracellular nucleotides to their respective nucleoside 5’-diand monophosphates, nucleosides and free phosphates or pyrophosphates (see below). Nucleotides may be released from cells via three major mechanisms: 1) cytosolic ATP release from sites of tissue and cell damage (including sites of bone injury), 2) exocytic release, and 3) via intrinsic plasma membrane channels or pores in the absence of cytolysis, which includes controlled release through hemichannels (for a review, see Novak, 2003; for further details, see below). Once released, nucleotides and their derivatives may act on purinergic receptors, which are important (yet largely unknown) bone turnover regulators (Orriss et al., 2010). The receptors for purines and pyrimidines are classified into two groups: P1 receptors (A1, A2A, A2B, A3), which are primarily activated by adenosine, and P2 receptors, which respond to ATP, adenosine diphosphate (ADP), uridine 1.Introduction PhD thesis – José Bernardo Noronha Matos (2011-2014) 64 triphosphate (UTP), uridine diphosphate (UDP) and nucleotide polyphosphates. The P2 receptors may be further subdivided into P2X ligand-gated ion channels and P2Y G-protein-coupled receptors (Abbracchio and Burnstock, 1994; Burnstock and Kennedy, 1985). To date, seven P2X receptors (P2X1-7) and eight P2Y receptors (P2Y1,2,4,6,11-14) have been identified, cloned and characterized, displaying different tissue distribution and pharmacological properties (Burnstock, 2007; Orriss et al., 2010). The P2X channels are assembled (in a homoor heteromeric manner) from seven subunits. Based on agonist efficacy, elctrophysiological properties and desensitization characteristics, P2X receptors have been grouped into three distinct classes (Dubyak, 2007): 1) includes P2X1 and P2X3 receptors exhibiting high affinity for ATP (EC50 = 1 μM), which are rapidly activated and desensitized; 2) includes P2X2, P2X4, P2X5, and P2X6 receptors, which have lower affinity for ATP (EC50 = 10 μM) and show slow desensitization rate and sustained depolarizing currents; 3) is represented by the homomeric P2X7 receptor, which has very low affinity for ATP (EC50 = 300–400 μM). This receptor shows little or no desensitization and acts as a nonselective ion pore (Di Virgilio, 1995), like other P2X receptors (Williams and Jarvis, 2000). Besides acting as an ATP-gated ion channel, prolonged activation of the P2X7 receptor favour permeation of plasma membrane to high-molecular weight (up to 900 Da) hydrophilic molecules, such as ATP and glutamate. P2Y and P1 receptors are classical seven-transmembrane domain receptors couped to G-proteins and numerous intracellular second messengers, including cAMP and inositol (1,4,5)- triphosphate (IP3) cascades (Burnstock and Verkhratsky, 2010). 1.4.2. P2 receptor expression by osteoblasts Previous studies confirmed that osteoblasts and osteoblast-like cells (from human and rodent species) express P2 receptors and that extracellular nucleotides could transiently increase [Ca2+]i and induce IP3 formation (Orriss et al., 2010). Table 3 illustrates the growing body of work around P2 receptors in these cells. Data so far obtained strenghten the role of these receptors in osteoblast function. After the pioneering work of Kumagai and coworkers showing 1.Introduction PhD thesis – José Bernardo Noronha Matos (2011-2014) 65 that ATP plays a central role in bone physiology (Kumagai et al., 1991), numerous studies demonstrated that nucleotides influence osteoblast and osteoclast function through the activation of both P2X and P2Y receptors. There are, however, conflicting results in the literature regarding the predominant role of P2 receptors on bone remodelling that derive mostly from heterogeneity of P2 receptors expression among osteoprogenitors vs differentiated cell populations, cell lines vs primary cell cultures (see e.g., Wesselius et al., 2011), and the existence of striking differences on the molecular composition and activity of these receptors between human and rodent species (see e.g. Roger et al., 2010). Another confounding factor that authors often disregard, which must be considered of clinical relevance, is the influence of systemic factors, such as the hormonal condition at bone microenvironment at the time of isolation of the cells. Another constrain is on what concerns the predominant use of animal models and immortalized cell lines, and less on non-modified human cells (Table 3). To obscure the pharmacological characterization and the investigation of the relative importance of P2 purinoceptors in bone biology, most studies had to deal with a lack of commercially-available selective agonists and/or antagonists. Nevertheless, data show that P2 receptors have some role on osteoblast cells function. For instance, P2X1 and P2X3 receptors were shown to be involved in the prevention of bone nodule mineralization of osteoblast cultures from neonatal Sprague–Dawley rats, since P2X1 and P2X3 receptor agonists, α,β-meATP and β,γ-meATP, inhibited bone mineralisation without affecting collagen production (Orriss et al., 2012). The activity of the P2X2 receptor seems to be more relevant in osteoclasts, where it increased bone resorption. Resorption increased 5.6-fold when osteoclasts from neonatal rats were cultured for 26 h on ivory discs in the presence of ATP, with a maximum effect occurring at relatively low concentrations (0.2-2 µM) (Morrison et al., 1998). The osteoclast promoting action of ATP was greatly amplified when these cells were cultured in acidified media (pH 6.9-7.0) (King et al., 1997). 1.Introduction PhD thesis – José Bernardo Noronha Matos (2011-2014) 66 Table 3. P2 receptor expression by osteoblasts Receptor Species Cell type Transduction mechanisms Proposed function and signalling References P2X1 Rat Primary Cation channel (Ca2+ and Na+) Involved in bone mineralization inhibition (rat osteoblasts) Orriss et al., 2012; Orriss et al., 2013 P2X2 Rat Primary Cation channel (Ca2+) Unknown. Also expressed in osteoclasts, involved in bone resorption (rodent osteoclasts) Alqallaf et al., 2009; Hoebertz et al., 2000; Morrison et al.,1998; Nakamura et al., 2000 Human MC3T3-E1, SaOS-2 P2X3 Rat Primary Cation channel Involved in bone mineralization inhibition (rat osteoblasts) Orriss et al., 2012 P2X4 Rat Primary Cation channel (Ca2+) Involved in pore formation (human osteoblast-like cells) Alqallaf et al., 2009; Ihara et al., 2005 Human SAM-1, MG-63, SaOS-2 P2X5 Rat Primary Cation channel Involved in human osteoblast like cell’s differentiation – stimulation of the MAP kinase pathway Hoebertz et al., 2000; Ihara et al., 2005; Nakamura et al., 2000; Orriss et al., 2006 Human MC3T3-E1, SAM-1 P2X6 Rat Primary Cation channel Largely unknown; regulator of MSC commitment Ihara et al., 2005; Zippel et al., 2012 Human SAM-1 P2X7 Rat Primary Cation channel, large pore after prolonged activation Controversial: Apoptosis (human cells); induction of membrane cell blebbing and increased bone formation - LPA and PGE2 synthesis (newborn rat osteoblasts); skeletal mechanotransduction (mouse osteoblasts); also expressed in human osteoclasts, involved in cellular apoptosis and cytoskeleton rearrangements Alqallaf et al., 2009; Gartland et al., 2001; Jorgensen et al., 2002; Ke et al., 2003; Li et al., 2005; Nakamura et al., 2000; Orriss et al., 2006; Orriss et al., 2010 ; Panupinthu et al., 2007; Panupinthu et al., 2008 Human MG-63, SaOS-2, Primary Mouse Primary 1.Introduction PhD thesis – José Bernardo Noronha Matos (2011-2014) 67 Table 3. (Continued) P2 receptor expression by osteoblasts P2Y1 Rat Primary Gq/G11 PLCβ activation Modulate osteoblast responses to PTH – increased c-fos expression (human and rat osteoblasts) Bowler et al., 1999; Buckley et al., 2001; Hoebertz et al., 2000; Maier et al., 1997; Orriss et al., 2006 Human MG-63 P2Y2 Rat Primary Gq/G11, possibly Gi/G0, PLCβ activation Propagation of intracellular Ca2+ waves (human osteoblasts); inhibition of bone mineralization – inhibition of ALP (rat osteoblasts); stimulation of Erg1 and Runx-2 expression – activation of PKC and ERK pathways (osteoblast-like HOBIT cell line); sensitises mechanical stress-activated Ca2+ channels – activation of ERK, p38 MAPK and JNK1 pathways (ROS-A 17/2.8 osteoblastic cells) Bowler et al., 1995; Costessi et al., 2005; Hoebertz et al., 2000; Hoebertz et al., 2002; Jorgensen et al., 2000; Katz et al., 2006; Katz et al., 2008; Maier et al., 1997; Orriss et al., 2006; Orriss et al., 2007; Pines et al., 2003 Human MG-63, Primary, SaOS-2, Te85 P2Y4 Rat Primary Gq/G11, possibly Gi, PLCβ activation Possibly involved in the inhibition of cell mineralization (rat osteoblasts) Hoebertz et al., 2002; Maier et al., 1997; Orriss et al., 2006 Human MG-63 P2Y6 Rat Primary Gq/G11 PLCβ activation Largely unknown; also expressed in osteoclasts, involved in their survival (rat osteoclasts) Korcok et al., 2005; Maier et al., 1997; Orriss et al., 2006 Human MG-63 P2Y11 Human osteosarcoma HOS cells Gq/G11, Gs Largely unknown Liu and Chen, 2010 P2Y12 Rat Primary Gi, inhibition of adenylate cyclase Involved in bone nodule mineralization Orriss et al., 2010; Syberg et al., 2012 P2Y13 Rat Primary Gi/G0 Increased ALP activity and osteogenic differentiation (mouse bone marrow stromal cells) Biver et al., 2013; Orriss et al., 2011 P2Y14 Rat Primary Gq/G11 Unknown, although suggest to play a role in early osteogenic differentiation of human MSCs Maier et al., 1997; Orriss et al., 2006; Zippel et al., 2012 Growing number of studies indicate that extracellular nucleotides play a role in modulating osteoblast function, via P2 receptors. Alkaline phosphatase, ALP; c-jun NH2-terminal protein kinase 1, JNK1; extracellular related kinase, ERK; lysophosphatidic acid, LPA; Mesenchymal stem cells, MSCs; p38 mitogen-activated kinase, p38 MAPK; phospholipase A2, PLA2; phospholipase D, PLD; prostaglandin E2, PGE2; protein kinase C, PKC (based on indicated references; see text for details). 1.Introduction PhD thesis – José Bernardo Noronha Matos (2011-2014) 74 increased ATP release from SAOS-2 and ROS 17/2.8 osteoblasts when compared to the control stiuation (Biswas and Zanello, 2009). These authors found that ATP secretion was abolished when cells are preincubated with inhibitors of vesicular exocytosis. Similarly, siRNA VDR (vitamin D receptors) silencing prevented vitamin D3 stimulation of ATP exocytosis (Biswas and Zanello, 2009). Furthermore, bisphosphonates, which are used for the treatment of osteoporosis (see section 1.3.3), have been reported to induce ATP release, favouring P2Y receptors activation. Authors showed that risedronate promotes non-lytic ATP release leading to activation of ERKs through the involvement of P2Y receptors, namely P2Y1 and P2Y2. In fact, bisphosphonates were previously shown to exert part of their effects by using a sort of membrane-receptor triggering by means of connexin 43 direct binding, due to their charged nature (Plotkin et al., 2002). These and other studies suggest that the local ATP release in response to different stimuli may represent a local trigger in bone that may influence osteogenesis (Burnstock et al., 2013). 1.4.4. ATP hydrolysis: ecto-nucleotidases and osteoblasts Once released, nucleotides may be rapidly broken down enzymatically by a cascade of a number of ecto-nucleotidases (Burnstock et al., 2013) (Figure 4). Consequently, ectonucleotidases may command a series of physiological responses by regulating P2 receptor activation (Kukulski et al., 2005). There are four families of ecto-nucleotidases: the NTPDases (ecto-nucleoside triphosphate diphosphohydrolase), the NPPs (ecto-nucleotide pyrophosphatase/ phosphodiesterase), alkaline phosphatases and ecto-5’-nucleotidase (Zimmermann et al., 2012). Ecto-nucleotidases may have overlapping specificities. For instance, NTPDases catalyse the reactions NTP → NDP + phosphate (Pi) and NDP → NMP + (Pi), whereas NPPs hydrolyse NTP → NMP + 1.Introduction PhD thesis – José Bernardo Noronha Matos (2011-2014) 75 pyrophosphate (PPi) or NDP → NMP + Pi (Burnstock et al., 2013). Regarding the NTPDase family, these are dominantly ectonucleotidases (Zimmermann et al., 2012). Four of the eight members of this family, namely NTPDase1, NTPDase2, NTPDase3 and NTPDase8 appear to be relevant to the control of P2 receptor signalling since they are located at the outer surface of the plasma membrane and hydrolyse nucleotides in the range of concentration that activates P2 receptors (Bigonnesse et al., 2004; Lavoie et al., 2004; Mateo et al., 1999; Picher et al., 1996; Smith and Kirley, 1999). These enzymes have two plasma membrane P2X N2 ATP ADP AMP ADO N1, N3 Ecto 5’ N1, N3 Osteoblast P2Y P1 P2X P2Y Osteoclast Bone matrix: Collagen + Ca10(PO4)6(OH)2 NPP AMP + PPi Mineralization ATP Osteoclast formation/ apoptosis (?) Osteoblast proliferation/ differentiation (?) Bone resorption ATP ? Osteocytes Figure 4. Schematic overview of functional effects of ATP on bone cells. ATP release from osteoblasts, osteocytes and osteoclasts can influence their function in an autocrine or paracrine manner, influencing both bone resorption and formation. Black arrows represent P1 or P2 activation, dashed arrows represent ATP breakdown and red texts/arrows represent multiple ATP effects on bone cells. NTPDase1, N1; NTPDase2, N2; NTPDase3, N3; ecto 5’- nucleotidase, Ecto 5’; nucleotide pyrophosphatase/phosphodiesterase, NPP; largely unknown or controversial effect, ?. Figure adapted from Burnstock et al., 2013. 1.Introduction PhD thesis – José Bernardo Noronha Matos (2011-2014) 76 spanning domains with an active site facing the extracellular milieu (Zimmermann et al., 2012). In contrast, the other NTPDases, namely 4-7, are anchored to the membrane of intracellular organelles by one (NTPDases5 and -6) or two (NTPDases4 and -7) transmembrane domains and their catalytic site faces the lumen of intracellular compartments, namely the Golgi apparatus and the endoplasmatic reticulum (Kukulski et al., 2005; Trombetta and Helenius, 1999; Wang et al., 1998). Since NTPDases have different biochemical properties, they are able to regulate P2 receptor activation differently, since they have different abilities in dephosphorylating nucleoside triphosphates and diphosphates (exclusively in the presence of divalent cations, Ca2+ or Mg2+, and usually all active within a pH of 7.0-8.5) (Kukulski et al., 2005). Among the four plasma membrane bound NTPDases, NTPDase1 (also known as CD39) hydrolyses ATP and ADP with the same affinity, NTPDase2 is a preferential triphosphonucleosidase whereas NTPDase3 and 8 are functional intermediates between NTPDase1 and - 2 (Kukulski et al., 2005). NTPDase1, -2, -3 and -8 efficiently hydrolyse ATP and UTP with Km values in the micromolar range, indicating that they should terminate the effects exerted by nucleotide agonists at P2X1-7 and P2Y2,4,11. In fact, Kukulski and co-workers, using COS-7 transfected cells, showed that NTPDase1 does not allow the accumulation of ADP, suggesting that it should terminate the activation of P2Y1,12,13 receptors. In contrast, they showed that NTPDases2, -3 and -8 are expected to promote the activation of ADP receptors, because in the presence of ATP, they produce a sustained (NTPDase2) or transient (NTPDases3 and -8) accumulation of ADP (Kukulski et al., 2005). Additionally, they found that all plasma membrane NTPDases dephosphorylate UTP with a significant accumulation of UDP, favouring P2Y6 receptor activation. Furthermore, comparing Km and Vmax values obtained from combined ATP and UTP hydrolyses experiments, authors revealed that adenine nucleotides are better substrates than uracil nucleotides for human NTPDase1 and -2. Furthermore, they found that human NTPDase3 dephosphorylated ADP to AMP much faster than UDP to UMP, leading to a sustained UDP accumulation (Kukulski et al., 2005). So, differential 1.Introduction PhD thesis – José Bernardo Noronha Matos (2011-2014) 77 expression and function of these NTPDases will dictate which P2 receptors will be preferentially activated within a cell population (Figure 4). Previous studies have revealed the coexistence of both ATP consuming and ATP-generating activities (nucleoside diphosphokinase or ecto-NDPK) on the cell surface, namely in human SaOS-2 osteoblast-like cells (Buckley et al., 2003). In addition, the expression of multiple NTPDases and NPPs has been previously reported (Orriss et al., 2007; Orriss et al., 2010). Regarding NPPs, NPP1 has been found to be important in bone biology. PPi, mostly generated from ATP hydrolysis by NPP1, is a strong inhibitor of bone mineralization (for a review, see Mackenzie et al., 2012). In fact, several studies suggest that ATP is a primary source of PPi in bone, pointing to a dual inhibitory action on bone mineralization via both P2 receptors mediated signalling and direct hydrolysis to PPi (Figure 4). It is believed that PPi is important to prevent ectopic mineralization (for example in soft tissues) (see e.g. Nitschke et al., 2012). At the same time, PPi is a source of Pi which is important in hydroxyapatite formation, a conversion that is undertaken by ALP. So, the concerted action of these two enzymes, NPP1 and ALP, is an important aspect of bone mineralization process. In this regard, it was found that, in a mouse model lacking NPP1, the trabecular number, trabecular bone volume, structure model index, trabecular and cortical thickness are all significantly reduced in tibiae and femurs from NPP1 (-/-) mice when compared to control animals, showing that NPP1 (-/-) mice are characterized by severe disruption to the architecture and mineralization of long-bones, dysregulation of calcium/phosphate homeostasis, all suggesting an important role for this enzyme in bone remodelling (Mackenzie et al., 2012). Other studies suggested that generalised arterial calcification in infants and severe hypophosphataemia is associated with recessive inactivating mutations in the ENPP1 gene (see e.g. Rutsch et al., 2003). One should be aware that NPPs may be inhibited by AMP, which is one of the end products of ATP hydrolysis. Intriguingly, it binds with more affinity when compared to ATP (Landt and Butler, 1978; Stefan et al., 2005). This aspect raises the question if NPP’s action has a sustained impact on bone remodelling in vivo, particular in a circumstance in which 1.Introduction PhD thesis – José Bernardo Noronha Matos (2011-2014) 78 ecto-5’-nucleotidase activity is compromised. In such case, AMP accumulation would lead to NPP1 inhibition, reducing ATP catalysis by this enzyme. ATP breakdown throughout the enzymatic cascade leads, eventually, to adenosine production by ecto-5’-nucleotidase. Adenosine has been shown to be produced by human osteoblast cells via ATP breakdown, modulating the secretion of IL-6 and osteoblastogenesis (Costa et al., 2011; Evans et al., 2006). In fact, human bone marrow derived MSCs were shown to express ecto-5’-nucleotidase (CD73) and to express all four P1 receptors (Costa et al., 2011). Agonists for all four receptors concentration-dependently increase MSCs proliferation. In particular, authors showed that A2B activation with 5’- (Nethylcarboxamide)adenosine (NECA) facilitates osteogenic differentiation measured by increases in ALP activity; the effect of NECA was prevented with the selective A2B antagonists, PSB 603 (Costa et al., 2011). These results were strenghten by the use of A2B knockout mice, thus revealing the importance of this receptor subtype in osteogenic differentiation of mesenchymal stem cells and bone formation in vivo (Carroll et al., 2012). Regarding ecto-5’-nucleotidase (CD73) as an important enzyme on adenosine formation by osteoblasts, a recent study demonstrated that CD73 knockout mice show osteopenia, with significant decreases of osteoblast-cell markers. In the same study, in vitro experiments revealed that CD73 deficiency results in impaired osteoblast differentiation, but not in the number of osteoblast progenitors. In addition, authors demonstrated the enhanced expression of osteocalcin and bone sialoprotein in MC3T3-E1 cells overexpressing CD73 (Takedachi et al., 2012). These and other studies reinforce the role of P1 receptors on bone remodelling. 1.4.5. P2 receptor expression by osteocytes and osteoclasts So far, focus has been given to purinergic signalling in osteoblast progenitor cells, osteoblasts and, only briefly, to osteoclasts. Besides their role in bone formation, P2 receptors have been previously pointed as important modulators of bone resorption. Briefly, it was found that ATP was able to stimulate resorption by cells from human osteoclastoma (Bowler et al., 1998) and from rodent osteoclasts 1.Introduction PhD thesis – José Bernardo Noronha Matos (2011-2014) 79 (Morrison et al., 1998). It was later suggested that these pro-resorptive effects were mediated by the P2X2 receptors. ADP, via P2Y1 receptors, was also pointed as an important receptor involved in osteoclast formation and activity, since ADP and 2-methylthioADP at nanomolar to submicromolar levels caused up to four to six-fold increases in osteoclastic bone resorption (Hoebertz et al., 2001). Interestingly, P2Y1 receptor knockout animals showed reduced trabecular bone in long bones, which pointed this receptor as an important modulator of bone remodelling (Orriss et al., 2011). However, another receptor was subsequently shown to mediate ADP effects on osteoclasts. P2Y12 receptor is expressed in osteoclasts (Orriss et al., 2011), and KO of this receptor impaires ADP elicited responses by these cells (Su et al., 2012). The P2Y6 receptor seems also to be important in osteoclast survival. Its activation on cultured osteoclasts prevents TNF-α-induced apoptosis, also promoting NF-kB translocation and activation (Korcok et al., 2005). Regarding P2X7 receptor, its activity appears to be complex in osteoclasts. It was demonstrated that ATP release from osteoclasts is P2X7 mediated (BrandaoBurch et al., 2012), and that blockade of the pore-forming P2X7 receptor inhibits formation of multinucleated human osteoclasts in vitro (Gartland et al., 2003). It was further demonstrated that P2X7 activation may lead to NF-kB (Korcok et al., 2004) and PKC translocation (Armstrong et al., 2009), cytoskeletal reorganization and secretion of lytic granules into the resorption lacunae (Hazama et al., 2009). However, others have shown that ATP release in response to mechanical stimuli may act on P2X7 receptors to inhibit osteoclast resorption (Naemsch et al., 2001). Regarding P1 receptors, it was suggested that adenosine resulting from released ATP may act on P1 receptors from osteoprogenitor cells, inducing IL-6 secretion and inhibition of osteoprotegerin release, promoting osteoclastogenesis (Evans et al., 2006). The A1 receptor was pointed as a promoter of osteoclast differentiation, since blockade of this receptor resulted in disruption of the association of tumor necrosis factor receptor-associated factor 6 (TRAF6) and transforming growth factor-β-activated kinase 1 (TAK1), a signalling event that is important for NF-κB activation (He and Cronstein, 2012). A2A receptors seem to be 1.Introduction PhD thesis – José Bernardo Noronha Matos (2011-2014) 80 equally involved in osteoclast function. Studies point for a compromised osteoclast differentiation upon A2A activation (Mediero et al., 2012). Others have shown that P2X7-mediated ATP release and subsequent adenosine formation may increase osteoclast fusion, an effect that seems to be A2A mediated (Pellegatti et al., 2011). Within the osteoblast lineage, osteocytes are also important cells within the bone, since they represent 90% of all bone cells (see section 1.3.1). However, little is known about the role of purinergic signalling in their survival or function (Figure 4). Part of the difficulty to study these cells is that 3D-cultures are needed to allow the maintenance of their phenotype. As previously mentioned, they form an interconnected cell network located in the fluid-filled lacunocanaliculi system, allowing them to translate mechanical signals into biochemical signals to effector cells, facilitating bone remodelling (Burnstock et al., 2013). Using cell cultures, it was possible to realize that mechanical stimulated osteocytes could present [Ca2+]i transients that are reduced by both suramin and thapsigargin, suggesting that this is due to ATP acting via P2Y receptors (Huo et al., 2008). Osteocytes were also found to express T-type voltage-sensitive calcium channels, and its α2δ1 subunit was shown to regulate mechanical-induced release of ATP (Thompson et al., 2011). Some authors suggest that ATP release from osteocytes (Kringelbach et al., 2014) may act in vivo to reduce progressive, age-related mineral encroachment from the surrounding bone, thus preventing cell death and cell “fossilization” (Burnstock et al., 2013) (Figure 4). In fact, in MLO-Y4 osteocytes, P2X7 and P2Y2 receptors were found to be functionally expressed (Kringelbach et al., 2008). Current evidence shows that purinergic signalling exerts complex local effects on the function of bone cells (e.g. osteoblasts). The effects of this signalling system are influenced by multiple factors including the receptor subtype, the extracellular nucleotides present locally and the expression of ecto-nucleotidases which will, in turn, regulate nucleotides accumulation and activity. However, there is still a huge controversy around the importance of each of the purinergic 1.Introduction PhD thesis – José Bernardo Noronha Matos (2011-2014) 81 receptors and enzymes on bone cells and how they may interact to promote bone remodelling Since purinergic signalling has now been implicated in many bone disorders (for a review, report to Burnstock et al., 2013), it is of great importance to explore these potential targets for future therapies, clarifying the molecular mechanisms operating upstream and downstream of these receptors in both health and disease. These unexplored cell targets will certainly prove useful in the therapeutic management of bone diseases in the future. 2. Goals PhD thesis – José Bernardo Noronha Matos (2011-2014) 82 2. GOALS The specific aims of the project were to investigate: (1) The expression and function of uracil nucleotide-sensitive receptors (P2Y2, P2Y4, and P2Y6) in human MSCs, since their role in osteogenic differentiation was largely unknown; (2) The expression and function of P2X7 receptors on osteogenic differentiation of human MSCs in culture, exploring the underlying molecular mechanisms involved; this was done due to the controversy of the P2X7 receptor role on bone remodelling in human cells; (3) The relevance of NTPDases in the management of osteogenic differentiation and/or cell proliferation in younger females versus postmenopausal women; this topic is, so far, largely unknown, particularly in non-modified human cells. Hopefully, this will provide new insights for development of novel therapeutic strategies for bone disorders such as osteoporosis, in which targeting both receptors and ecto-nucleotidases may prove beneficial. 3. Original Research Papers PhD thesis – José Bernardo Noronha Matos (2011-2014) 83 3. ORIGINAL RESEARCH PAPERS The results obtained in this thesis were published / submitted for publication as original research papers, as follows:  Paper 1: Noronha-Matos JB, Costa MA, Magalhães-Cardoso T, Ferreirinha F, Freitas R, Neves JM, Sévigny J and Correia-de-Sá P. (2012). Role of ecto-NTPDases on UDP-sensitive P2Y6 receptor activation during osteogenic differentiation of primary bone marrow-derived mesenchymal stem cells from postmenopausal woman. Journal of Cellular Physiology, 227, 2694-2709. DOI: 10.1002/jcp.23014. PMID: 21898410;  Paper 2: Noronha-Matos JB, Coimbra J, Sá-e-Sousa A, Rocha R, Marinhas J, Freitas R, Gomes-Guerra S, Ferreirinha F, Costa MA and Correia-de-Sá P. (2014). P2X7-induced zeiosis promotes osteogenic differentiation and mineralization of postmenopausal bone marrow-derived mesenchymal stem cells. FASEB Journal, Epub ahead of print. DOI: 10.1096/fj.14-257923, PMID: 25169056  Paper 3: Noronha-Matos JB, Calejo I, Magalhães-Cardoso MT, Silva I, Ferreirinha F, Rocha R, Marinhas J, Freitas R, Costa MA, Pelletier J, Sévigny J and Correia-de-Sá P. (2014). Inhibition of NTPDase3 on bone marrow-derived mesenchymal stem cells may be a novel therapeutic strategy to increase bone formation in postmenopausal women. In Preparation. 3. Original Research Papers PhD thesis – José Bernardo Noronha Matos (2011-2014) 90 cells were enzymatically released with 0.04% trypsin–EDTA solution and 0.025% type I collagenase in phosphate-buffered saline (PBS), at pH 7.4 during 15–20 min. The resultant cell suspension was cultured (104 cells/cm2) (day 0) in conditions known to favour osteogenic differentiation. To this end, the standard culture medium was supplemented with 50 µg/ml ascorbic acid, 10 mM βglycerophosphate, and 10 nM dexamethasone. MSC cultures were established for 28 days in the absence (control) or in the presence of purinoceptor agonists/ antagonists that were added to the culture medium at day 1 (see e.g., Costa et al., 2011). Drugs were renewed in the culture at each medium change, i.e., twice a week. All the experiments were performed in the first subculture, since previous results showed that serial passage of bone marrow-derived MSCs result in the progressive loss of the osteoblast phenotype (Coelho et al., 2000; Fernandes et al., 1997). Cell cultures were routinely monitored by phase contrast microscopy and characterized at days 1, 4, 7, 14, 21, and 28 for cell viability/proliferation (MTT assay), alkaline phosphatase (ALP) activity, and total protein content. Cell viability/proliferation MTT assay and total protein content. Proliferation studies included MTT assay and total protein content. MTT assay consisted of the reduction of 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to a purple formazan reaction product by viable cells. In the last 4 h of each test period, cells were incubated with 0.5 mg/ml of MTT in the conditions referred above. The medium was carefully removed, decanted, and the stained product dissolved with DMSO before absorbance (A) determination at 600 nm using a microplate reader spectrometer. Results were expressed as A/cm2 (Figure 6A). Total protein content was determined by Lowry’s method, after treatment of the cell layer with 0.1 M NaOH for 1 h. Bovine serum albumin (BSA) was used as a standard, and absorbance evaluated at 750 nm. Results are expressed as µg/cm2 (Amaral et al., 2002; Costa et al., 2011). Fluorimetric DNA determination. The total cell DNA content was determined as previously described (West et al., 1985). At the end of the culture period, cells 3. Original Research Papers PhD thesis – José Bernardo Noronha Matos (2011-2014) 91 were washed twice with PBS 1X and incubated for 20 min with EDTA (10 mM, pH=12.3) at 370C. Cell plates were then kept on ice and pH adjusted to 7.0 with addition of KH2PO4 (1 M). To each sample, a solution of Hoescht 33258 (200 ng/ml in NaCl 100 mM plus Tris 10 mM buffer, pH=7.0) was added. Fluorescence was measured (Perkin Elmer, Luminescence Spectrometer LS 30, Waltham, MA) and DNA concentration determined using a DNA calibration curve (0–100 µg DNA/ml). DNA standards were prepared using salmon DNA at 1 mg/ml in Tris (10 mM) plus EDTA (1 mM) buffer (pH=8.0). Results were expressed as µg of DNA/cm2. Alkaline phosphatase (ALP) activity ALP activity was determined in cell lysates (obtained by treatment of the cell layers with 0.1% Triton X in water) and assayed for the hydrolysis (30 min at 370C) of p-nitrophenyl phosphate (25 nM) in an alkaline buffer solution (pH=10.3), followed by colorimetric determination of p-nitrophenol at 405 nm. Results were expressed in nanomoles of p-nitrophenol produced per µg of protein (nmol min-1 µg protein-1) (Amaral et al., 2002; Costa et al., 2011) (Figure 6B). ALP activity is a good indicator for osteoblast cell differentiation as previously shown (Costa et al., 2011; Hoemann et al., 2009). Histochemical staining for ALP Fixed cultures (1.5% glutaraldehyde in 0.14 M sodium cacodylate buffer, 10 min) were stained for ALP. Fixed cells were incubated for 1 h in the dark with a mixture prepared in Tris buffer (pH=10) containing 2 mg/ml of fast blue RR salt; the incubation was stopped by rinsing the samples with water. The presence of ALP was identified by a brown to black staining, according to the enzyme content (see e.g., Figure 6D). Stained culture wells were photographed using an inverted microscope (Olympus IX8, Tokyo, Japan) coupled to a high-sensitivity digital colour camera (ColorView II, Olympus, Tokyo, Japan) and analysed with the software Cell F (Olympus, Tokyo, Japan). Kinetic experiments and HPLC analysis 3. Original Research Papers PhD thesis – José Bernardo Noronha Matos (2011-2014) 92 The kinetics of inactivation of UTP and UDP in human primary MSC cultures was studied at days 7 and 21, at 370C (three replicas were performed in each individual experiment). The kinetics of extracellular ATP catabolism was also studied for comparison purposes (see e.g., Magalhães-Cardoso et al., 2003). After a 30-min equilibration period, cells were incubated with 100 µM of ATP, UTP, or UDP added to the culture medium in the conditions referred above (zero time). Samples (75 µl) were collected from each well at different times up to 30 min for high-performance liquid chromatography (HPLC, LaChrom Elite, Merck, Frankfurt, Germany) analysis of the variation of substrate disappearance and product formation. Aliquots of 20 µl of collected samples were used for nucleotide analysis. The rate of ATP disappearance and subsequent formation of ADP, AMP, and adenosine was analysed as previously described (Costa et al., 2011). Separation of UTP, UDP, UMP, and uridine was achieved by ion-pair reverse-phase HPLC, at room temperature, on a Merck Lichrospher® 100 RP-18 (5 µm) column. The composition of the mobile phase was 60 mM KH2PO4 and 5 mM tetrabutylammonium (pH=6) in methanol. Each run consisted of a linear gradient from 5 to 35% methanol (v/v) performed at 1.5 ml/min flow rate during 8 min; reequilibration of the column required an additional 7-min period. Nucleotides and nucleosides were detected by UV absorption at 262 nm. Under these experimental conditions, the retention times for uracil nucleotides and nucleosides were as follows: UTP (6.41 min), UDP (4.67 min), UMP (2.18 min), and uridine (1.49 min). The actual concentrations of UTP, UDP, UMP, and uridine were expressed in micromolar. Concentrations of the substrate and products were plotted as a function of time (progress curves). The following parameters were analysed for each progress curve: half-degradation time of the initial substrate, time of appearance of the different concentrations of the products, concentration of the substrate or any product remaining at the end of the experiment. Because enzymatic activity is usually represented as a function of the total protein content, yet in osteoblast differentiating cultures type I collagen accounts to 85–90% of the organic matrix, here we also decided to normalize the ecto-nucleotidase activity by the amount of 3. Original Research Papers PhD thesis – José Bernardo Noronha Matos (2011-2014) 93 viable cells given by the MTT assay. The spontaneous degradation of uracil nucleotides at 370C in the absence of the cells was negligible over 30 min. At the end of the experiments, the remaining incubation medium was collected and used to quantify the lactate dehydrogenase (LDH, EC 1.1.1.27) activity. The negligible activity of LDH in the samples collected at the end of the experiments is an indication of the integrity of the cells during the experimental period. Single-cell [Ca2+]i transients by confocal microscopy Human primary MSCs were seeded into 35 mm dishes at a density of 2x104 cells/ml and allowed to grow for 7 or 21 days in supplemented α-MEM medium. On the day of the experiment, cells were washed twice with PBS and incubated at 370C for 45 min with the cell-permeant fluorescent Ca2+ indicator, Fluo-4NW (2.5 µM), in PBS containing 2.5% pluronic acid in 100 µM DMSO. After removal of the fluorophore loading solution, cells were washed twice more and 150 µl of PBS was added per culture dish. Culture dishes were then mounted on a thermostatized perfusion chamber at the stage of an inverted laser-scanning confocal microscope (Olympus FV1000, Tokyo, Japan) equipped with a 20x magnification objective lens (LUCPLFL 20x PH; N.A. 0.45). From this time onwards, the chamber was perfused continuously (1 ml/min) with gassed (95% O2 plus 5% CO2) Tyrode’s solution (pH=7.4) containing (mM): NaCl 137, KCl 2.7, CaCl2 1.8, MgCl2 1, NaH2PO4 0.4, NaHCO3 11.9, and glucose 11.2, at 370C. Test drugs were delivered using a multichannel perfusion system (ValveLink 8.2, Digitimer, San Francisco, CA). Changes in fluorescence of the Fluo-4NW dye were detected in the timelapse mode with the FluoView Advanced Software (Olympus, Tokyo, Japan). Fluo4NW was excited with the 488 nm line a Multi-line Ar laser. The emitted fluorescence was detected at 510–560 nm using the scanner of the confocal microscope (Olympus FV1000, Tokyo, Japan). The fluorescence images were collected at 20 sec intervals. Intracellular Ca2+ transients induced by uracil nucleotides were calibrated to the maximal calcium load produced by ionomycin (5 µM, 100% response) (Henriksen et al., 2006; Panupinthu et al., 2007). 3. Original Research Papers PhD thesis – José Bernardo Noronha Matos (2011-2014) 94 Antibody production The development and specificity of anti-human nucleotidase antibodies has been reported previously (e.g., Dranoff et al., 2004; Munkonda et al., 2009). Hartley guinea pigs and New Zealand rabbits were obtained from Charles River Laboratories (Quebec City, Canada). Genetic immunization protocol was carried out with plasmids (pcDNA3 for human NTPDase1 and pcDNA3.1 for human ecto5’-nucleotidase) encoding each protein using New Zealand rabbits for antibodies against human NTPDase1 and Hartley guinea pigs for human ecto-5’-nucleotidase antibodies. Immunofluorescence confocal microscopy MSCs were allowed to grow in chamber slides for 7 or 21 days. At the end of each test period, cultured cells were fixed in 4% paraformaldehyde (PFA) in PBS for 10 min, washed three times in PBS (10 min each) and, subsequently, incubated with blocking buffer I (10% FBS, 1% BSA, 0.1% Triton X, 0.05% NaN3) for 1.5 h. Primary antibodies, diluted in blocking buffer II (5% FBS, 1% BSA, 0.1% Triton X, 0.05% NaN3), were applied [NTPDase1 1:150 (hN1-9LI4, rabbit), NTPDase2 1:200 (hN2-Kw3I4, rabbit), NTPDase3 1:200 (hN3-B3S, mouse), ecto5’-nucleotidase 1:300 (h5’NT-2CI4, guinea-pig), P2Y1 1:50 (goat), P2Y2 1:150 (rabbit), P2Y4 1:75 (rabbit), P2Y6 1:75 (rabbit), P2X7 1:75 (rabbit), osteocalcin 1:75 (rabbit), collagen type I 1:50 (rabbit)] and the slides incubated in the dark for 2 h. After incubation, cells were washed three times in PBS 1x (10 min each); Alexa Fluor 488 (anti-rabbit), Alexa Fluor 568 (anti-mouse), and Alexa Fluor 653 (antigoat) secondary antibodies in blocking buffer II (5% FBS, 1% BSA, 0.1% Triton X) were applied for 1 h. A last wash was performed with PBS 1x and glass slides mounted with VectaShield medium and stored at 40C. Observations were performed and analysed with a laser scanning confocal microscope (Olympus FV1000, Tokyo, Japan) (Alqallaf et al., 2009). Flow cytometry analysis 3. Original Research Papers PhD thesis – José Bernardo Noronha Matos (2011-2014) 95 Plastic-adherent human bone marrow-derived MSCs (first subculture) were allowed to grow for 7 days in conditions known to favour osteogenic differentiation. At the end of this period, the cells were enzymatically released as mentioned before. The resultant cell suspension was centrifuged at 2000 rpm (5 min); the supernatant was discarded and cells were re-suspended in 2ml of PBS 1x containing 0.2% BSA. This procedure was repeated twice. Samples (100 µl) containing 250–500x103 cells were incubated for 15 min at room temperature with 10 µl of antigen-specific anti-human (mouse) fluorochrome-conjugated monoclonal antibodies (mAb). The antibodies employed were the following: Anti-CD14 labelled with fluorescein isothiocyanate (FITC) (clone MφP9), anti-CD29 labelled with fluorescein isothiocyanate (FITC) (clone TS2/I6), anti-CD34 labelled with allophycocyanin (APC) (clone 8GI2), anti-CD45 labelled with peridinin-chlorophyll protein (PerCP) (clone 2DI), anti-CD105 labelled with phycoerythrin (PE) (clone IG2), anti-CD117 labelled with phycoerythrin (PE) (clone 95C3); in some experiments, anti-ecto-5’-nucleotidase (h5’NT-2cI4, guinea-pig) was used, combined with the secondary antibody Alexa Fluor 649 (anti-guinea-pig). Cells were first identified based on their flow cytometric characteristics—high sideward (SSC) and forward (FSC) light scatter profile. Cells were then gated on the basis of sideward scatter distribution. Once identified, cells were analysed for the expression of the surface antigens recognized by the mAb referred above. Controls were performed by incubating the cells with the conjugates or secondary antibodies alone, in order to measure unspecific fluorescence (negative controls). All experiments were done in triplicate on a FACSCalibur cytometer (BD Biosciences, San Jose, CA), using the CellQuest software version 3.1 (BD Biosciences, San Jose, CA) for sample acquisition and the Paint-a-gate Pro software (BD Biosciences, San Jose, CA) for data analysis. Reagents and antibodies Cell culture reagents were purchased from Sigma-Aldrich (St. Louis, MO). Adenosine (ADO), adenosine 5’-diphosphate sodium salt (ADP), adenosine 5’- triphosphate disodium salt (ATP), 4-[[4-formyl5-hydroxyl-6-methyl-3- 3. Original Research Papers PhD thesis – José Bernardo Noronha Matos (2011-2014) 96 [(phosphonooxy)methyl]-2-pyridinyl]azo]- 1,3-benzenedisulfonic acid tetrasodium salt (PPADS), uridine, uridine 5’-diphosphate trisodium salt (UDP), and uridine 5’- triphosphate trisodium salt (UTP) were purchased from Sigma-Aldrich (St. Louis, MO). 3-[[5-(2,3-Dichlorophenyl)- 1H-tetrazol-1-yl]methyl]pyridine hydrochloride (A438079), 2’-deoxy-N6-methyladenosine 3’,5’-bisphosphate tetrasodium salt (MRS 2179), N,N’’-1,4-butanediyl-bis-[N’-(3-isothiocyanatophenyl) thiourea (MRS 2578), 3-(2-oxo-2-phenylethyl)-uridine-5’-diphosphate disodium salt (PSB 0474), and uridine-5’-(γ-thio)-triphosphate trisodium salt (UTPγS) were obtained from Tocris Cookson Inc. (Bristol, UK). All primary antibodies used in this study have previously been validated: Anti-P2Y1 was from Santa Cruz (Santa Cruz, CA); antiP2Y2 and anti-P2Y4 were from AbCam (Cambridge, UK); anti-P2Y6 and anti-P2X7 were purchased from Alomone (Jerusalem, Israel); anti-osteocalcin and anti-Type I collagen were from AbD Serotec (Kidlington, Oxford, UK); anti-CD29-FITC (clone TS2/I6) was supplied by eBioscience (San Diego, CA); anti-CD117-PE (clone 95C3) and anti-CD105-PE (clone IG2) were supplied by Immunotech (Marseille, France); anti-CD14-FITC (clone MφP9), anti-CD45-PerCP (clone 2DI), and antiCD34-APC (clone 8GI2) were supplied by BD Biosciences, San Jose, CA. Primary antibodies anti-NTPDase1, anti-NTPDase2, anti-NTPDase3, and anti-ecto-5’- nucleotidase were developed in the Centre de Recherche en Rhumatologie et Immunologie, University Laval, Québec, Canada. Alexa Fluor 488-labeled antirabbit, Alexa Fluor 568-labeled anti-mouse, Alexa Fluor 653-labeled anti-goat and the fluorescent calcium indicator Fluo-4NW were supplied by Molecular Probes (Invitrogen, Carlsbad, CA). Dimethylsulphoxide (DMSO) was obtained from Merck (Frankfurt, Germany). Tissue culture plates: 96-well plates were purchased from Corning, Lowell, MA; FluoroDish plates for confocal microscopy were from World Precision Instruments (Hitchin, Hertfordshire, UK); chamber slides were from Nunc (Rochester, NY). Presentation of data and statistical analysis Results presented in this study are from bone marrow specimens obtained from eighteen individual female patients (68±5 years old, n=18). For each experiment 3. Original Research Papers PhD thesis – José Bernardo Noronha Matos (2011-2014) 97 and assay, 6–8 replicas were accomplished. The data are expressed as mean ± S.E.M. from an n number of patients. Data from different individuals were evaluated using one-way analysis of variance (ANOVA) and no significant differences in the pattern of cell behaviour were found. Statistical differences found between control and drug-treated cultures were determined by Bonferroni’s method. P-values <0.05 were considered to represent significant differences. RESULTS Phenotypic cells characterization by flow cytometry Primary cultures were maintained for 10–15 days until near confluence when adherent cells were enzymatically released. The resultant cell suspension was cultured in conditions that favour osteogenic differentiation. Plastic-adherent MSCs (first subculture) were allowed to grow for 7 days. All the experiments were performed in the first subculture, since previous results from our collaborators and independent groups showed that serial passage of bone marrow-MSCs result in the progressive loss of the osteoblast phenotype detected as decreases in ALP activity and mineralization potential from the 1st to the 2nd subculture (Coelho et al., 2000; Fernandes et al., 1997; Schmidt and Kulbe, 1993). MSCs are thought to be multipotent cells. These cells, which are present in the adult bone marrow, can replicate as undifferentiated cells and have the potential to differentiate to lineages of mesenchymal tissues, including bone. First passage MSCs prepared in the present experimental conditions were used for immunophenotypic analysis by flow cytometry. These cells exhibited positive immunoreactivity against several surface molecules, including CD105 (SH2), CD29 (integrin β1), and CD117 (tyrosine protein kinase Kit) (Figure 5), which have been identified as markers of bone marrow-derived mesenchymal stromal cells (Baddoo et al., 2003; Boiret et al., 2005; Cognet and Minguell, 1999; Dennis et al., 2002; Gronthos et al., 2003; Pittenger et al., 1999). Conversely, cells isolated from the human bone marrow and cultured under the experimental conditions were negative for haematopoietic surface markers, like 3. Original Research Papers PhD thesis – José Bernardo Noronha Matos (2011-2014) 98 CD14 and CD45, which has been extensively used as a good argument to distinguish bone marrow haematopoietic cells from MSCs (Baddoo et al., 2003; Pittenger et al., 1999). Flow cytometry analysis showed that a subpopulation of human bone marrow cells were moderately positive for CD34, an endothelial and haematopoietic cell marker. Overall, we consider that under the present experimental conditions first passage plastic-adherent human bone marrow cells are highly enriched in multipotent mesenchymal stromal cells. IgG PerCP CD45-PerCP IgG APC CD34-APC IgG FITC CD14-FITC IgG FITC CD29-FITC IgG PE CD105-PE IgG PE CD117-PE Mesenchymal stem cell markers Haematopoietic and endothelial cell markers Figure 5. Flow cytometric analysis of surface markers of mesenchymal stem cells (MSCs) (CD29, CD105, and CD117) and haematopoietic and endothelial cells (CD45, CD14, and CD34) in plastic-adherent MSCs (first subculture) allowed to grow for 7 days in culture. Black histograms indicate isotype controls (fluorochrome-conjugates or secondary conjugated antibodies alone), white histograms show surface antigen expression level. Staining and stem cell markers have been chosen in accordance with the minimal criteria for defining multipotent mesenchymal stromal cells by the International Society for Cellular Therapy (Dominici et al., 2006). Cells were first identified based on their flow cytometric characteristics—high sideward (SSC) and forward (FSC) light scatter profile (total cell suspension). These cells were then gated on the basis of sideward scatter distribution, and they were analyzed for the expression of certain surface antigens recognized by the antibodies employed, namely anti-CD29 labelled with fluorescein isothiocyanate (FITC) (clone TS2/I6), anti-CD105 (mouse) labeled with phycoerythrin (PE) (clone IG2), anti-CD117 (mouse) labelled with phycoerythrin (PE) (clone 95C3), anti-CD45 (mouse) labelled with peridinin-chlorophyllprotein (PerCP) (clone 2D1), antiCD34 (mouse) labelled with allophycocyanin (APC) (clone 8G12), and anti-CD14 (mouse) labelled with fluorescein isothiocyanate (FITC) (clone MwP9). Results are in mean fluorescence intensities, expressed in arbitrary relative linear units. 3. Original Research Papers PhD thesis – José Bernardo Noronha Matos (2011-2014) 99 Osteoprogenitor cells from postmenopausal women exhibit low ALP activity Human MSCs (first subculture) were allowed to grow for 28 days in an osteoblast-inducing medium. Meanwhile, cell cultures were characterized for proliferation and osteogenic differentiation events at days 1, 4, 7, 14, 21, and 28. Results regarding cell viability/proliferation were measured by the MTT assay. Cultures grown in control conditions showed a gradual increase in cell proliferation until day 28 (Figure 6A). We found no significant ( P>0.05) differences regarding the proliferation profile (MTT assay) of the cells from both age groups, i.e., reduction of the tetrazolium salt (MTT) was similar between cells obtained from bone marrow specimens of female patients whose ages were 58–83 years (postmenopausal group, n=18) and 14–40 years (control group, n=4), respectively (Figure 6A). Results concerning total protein content reflected similar information as that obtained from the MTT assay (data not shown). The enzymatic MTT assay is often considered inappropriate to compare viability/proliferation of cells that can exhibit differences in their metabolic activity, such as cells from younger versus postmenopausal groups of patients. We, therefore, decided to compare the results from the MTT assay with those where cellularity and proliferation throughout the culture period was evaluated by quantifying total DNA content per culture well. Doing this, we also found no significant differences (P>0.05) in cell growth profiles of distinct age groups (Figure 6C). As expected, significant (P<0.05) differences were observed regarding the osteogenic differentiation profile (given by ALP activity) between the two groups of patients. Cell cultures from the younger group (patients of 14–40 years old, n=4) exhibit higher ALP activity than the postmenopausal group (patients of 58–83 years old, n=18). The ALP activity in the younger group of patients reached a maximum between days 7 and 21, decreasing significantly thereafter (Figure 6B), whereas in the postmenopausal group of patients the ALP activity increased slowly, yet progressively, with the incubation time until day 28. The biochemical results were confirmed by data from histochemical ALP staining, where the activity of ALP was identified by a progressive increase in the brownish staining of the cultures from day 7 to day 21 (Figure 6D). The differences in ALP activity found between the two age groups do 3. Original Research Papers PhD thesis – José Bernardo Noronha Matos (2011-2014) 106 significantly with time, whereas the P2Y6 receptor protein expression remained fairly constant as cultures progressed. The immunostaining pattern for uracil7 days 21 days P2Y2 P2Y4 P2Y6 150µm Type I Collagen Osteo calcin Figure 9. Immunocytochemical detection of uracil-sensitive P2Y receptors (P2Y2, P2Y4, and P2Y6), type I collagen and osteocalcin on human primary bone marrow-derived mesenchymal stem cells (MSCs). Shown is the time-related immunoreactivity fluorescence detection by confocal microscopy of human MSCs (first subculture) allowed to grow for 7 and 21 days in an osteoblast-inducing medium. Cells grown in eight-well chamber slides were processed for immunocytochemistry in parallel and were visualized keeping unaltered the settings of the confocal microscope throughout the procedure. For further details on immunofluorescence labelling see Materials and Methods section. 3. Original Research Papers PhD thesis – José Bernardo Noronha Matos (2011-2014) 107 sensitive P2Y receptors with time in postmenopausal primary MSCs was slightly different from that predicted in rat primary osteoblasts (Orriss et al., 2006). These authors showed that mature rat osteoblasts preferentially express the ATPand UTP-sensitive P2Y2 receptor and to a lesser extent P2Y4 (also ATP/UTP selective) and P2Y6 (UDP-sensitive) receptors. ATP increases intracellular Ca2+ but decreases osteogenic differentiation of postmenopausal primary MSCs For comparison, we also tested the effect of ATP (100 µM) on intracellular Ca2+ accumulation and osteogenic differentiation (measured by ALP activity) throughout MSC cultures life span in similar experimental conditions as those used for uracil nucleotides (Figure 10). Contrasting with the effect of uracil nucleotides on osteogenic differentiation (see above), continuous ATP (100 µM) application progressively decreased ALP activity from the first week onwards reaching a maximum on culture day 21 (Figure 10A). Acute superfusion of 7-day MSC cultures with ATP (100 µM) elicited a fast [Ca2+]i rise, which typically peaked 40 sec after nucleotide addition and decayed back almost to baseline within 1–2 min; a second progressive, but of much lower amplitude, ATP-induced rise in intracellular Ca2+ was observed following the initial high-magnitude [Ca2+]i transient (Figure 10B). Intracellular Ca2+ responses induced by ATP (100 µM) decreased with time in culture; for example, the late response to ATP almost disappeared at culture day 21 as compared to that observed in 7-day cultures (Figure 10B). Likewise, fast [Ca2+]i transients calibrated by ionomycin (5 µM) were of smaller magnitude in 21-day cultures than in 7-day cultures. It is worth noting that ATPinduced fast [Ca2+]i rises undergo rapid desensitization, i.e., application of a second ATP (100 µM) pulse following a washout period of at least 6 min elicited a significantly (P<0.05) smaller intracellular Ca2+ response than that observed with the first pulse (Figure 10B, inserts). Consecutive UTP applications induced [Ca2+]i transients which were both of a similar magnitude, thus indicating that uracil nucleotide-sensitive P2 purinoceptors are not prone to desensitization. 3. Original Research Papers PhD thesis – José Bernardo Noronha Matos (2011-2014) 108 Recent studies have demonstrated that P2 receptor expression in osteoblasts is strongly differentiation-dependent (Orriss et al., 2006). Besides uracil activated receptors (P2Y2, P2Y4, and P2Y6) (see above), human osteoblast-like cells -0.3 -0.2 -0.1 0.0 0.1 0.2 0.3 7 14 21 ALP (nmol/min/µg protein) Drug - Ctr Days in culture ATP 100 µM n = 3 *P<0.05 * * * A ATP (100 µM, 6 min) + MRS2179 (0.3 µM) 20% Ionomycin ATP (100 µM, 6 min) + A438079 (3 µM) 20% Ionomycin ATP (100 µM, 6 min) 20% Ionomycin ATP (100 µM, 6 min) 20% Ionomycin ATP (100 µM) ATP (100 µM) 20% Ionomycin ATP (100 µM, 6 min) 20% Ionomycin ATP (100 µM) ATP (100 µM) 20% Ionomycin 7 days 21 days B P2X7 P2Y1 150µm 150µm Figure 10. Effects of ATP (100 µM) on intracellular Ca2+ accumulation and osteogenic differentiation (measured by ALP activity) of human primary bone marrow-derived mesenchymal stem cells (MSCs) kept in culture during 21 days. Panel (A), represented is the variation of alkaline phosphatase (ALP) activity of human MSCs (first subculture) exposed continuously to ATP (100 µM) during 21 days as compared to control cultures grown in its absence at the same time points (see Figure 6). Zero represents identity between the two values (drug vs. control); positive and negative values represent facilitation or inhibition of osteogenic cell differentiation relative to control data obtained at the same time points. Each column represents pooled data from an n number of individuals; 6–8 replicas were performed for each individual experiment. The vertical bars represent S.E.M. *P < 0.05 represent significant differences from control values obtained in the absence of ATP (100 µM). Panel (B), represented is the fluorescence intracellular Ca2+ oscillations in human MSCs at culture days 7 and 21 caused by ATP (100 µM) in the absence and in the presence of selective P2Y1 and P2X7 receptor antagonists, respectively MRS 2179 (0.3 µM) and A438079 (3 µM). Cells were pre-incubated with the fluorescent calcium indicator, Fluo4-NW (2.5 µM, in PBS plus 2.5% pluronic acid), for 45 min at 370C. Changes in fluorescence were detected in the timelapse mode by laser-scanning confocal microscopy (Olympus FV1000, Tokyo, Japan). Intracellular Ca2+ transients were calibrated to the maximal calcium load produced by ionomycin (5 µM, 100% response). Each point represents pooled data from: 78 cells (ATP 100 µM, three different individuals, day 7), 58 cells (ATP 100 µM, three different individuals, day 21), 64 cells (ATP 100 µM plus MRS 2179 0.3 µM, three different individuals, day 7) and 47 cells (ATP 100 µM plus A438079 3 µM, three different individuals, day 7). The vertical bars represent S.E.M. [Ca2+]i transients resulting from two consecutive ATP (100 µM, for 1 min) applications 6 min apart from each other to human MSCs cultures (7 and 21 days), are also shown for comparison purposes (figure inserts). The black bars at the bottom of each graph indicate the period of drugs exposure. Human MSCs exhibit immunoreactivity against P2Y1 and P2X7 receptors at culture day 7. Images obtained under the confocal microscope are representative of three independent experiments. Scale bar is 150 µm. ATP (100 µM, 6 min) ATP (100 µM, 6 min) ATP (100 µM, 6 min) + MRS 2179 (0.3 µM) ATP (100 µM, 6 min) + A438079 (3 µM) 3. Original Research Papers PhD thesis – José Bernardo Noronha Matos (2011-2014) 109 express P2Y1 receptors as indicated by RT-PCR (Maier et al., 1997) and immunolabeling (see Figure 10B) studies. Activation of P2Y1 receptor is thought to modulate osteoblast responses to systemic factors such as parathyroid hormone via intracellular Ca2+ mobilization (Bowler et al., 1999; Buckley et al., 2001). Controversy however exists on the role of ATP activated P2X receptors, namely the P2X7 receptor, on human osteoblasts (Orriss et al., 2010; see also for a detailed review on the P2X7 receptor, Grol et al., 2009). Early reports suggested that P2X7 receptor activation caused enhanced osteoblast apoptosis (Gartland et al., 2001). The P2X7 receptor is also thought to mediate the ERK1/2 activation caused by fluid shear stress in osteoblast-like cells, via increased [Ca2+]i and protein kinase C activation (Liu et al., 2008b). These findings prompted us to investigate the expression of P2Y1 and P2X7 receptors on postmenopausal osteoprogenitor cells in culture by immunofluorescence confocal microscopy to see whether they could be implicated in ATP-induced intracellular Ca2+ responses and decreased osteogenic cells differentiation. Figure 10B shows that postmenopausal MSCs in culture exhibit immunoreactivity against P2Y1 and P2X7 receptors. As previously demonstrated by RT-PCR analysis in rat primary osteoblasts (Orriss et al., 2006), P2Y1 and P2X7 receptors immunoreactivity was detected at early time points (culture day 7), but it declined thereafter (data not shown). These findings are in keeping with our results showing that ATP-induced intracellular Ca2+ responses decreased significantly at culture day 21 as compared to those observed in 7-day cultures (Figure 10B). Pre-incubation of 7-day cultures with the subtype selective P2Y1 receptor antagonist, MRS 2179 (0.3 µM), totally abolished the fast [Ca2+]i transient induced by ATP (100 µM), while keeping the late component of [Ca2+]i rise. Conversely, the selective P2X7 receptor antagonist, A438079 (3 µM), significantly attenuated the late [Ca2+]i response induced by ATP (100 µM) without much affecting the fast component. These results suggest that, in contrast to uracil nucleotides, ATP (100 µM) or its metabolite ADP operate biphasic [Ca2+]i responses in postmenopausal MSCs in culture, which are respectively mediated by fast desensitizing P2Y1 receptors and by slow activating (non-desensitizing) 3. Original Research Papers PhD thesis – José Bernardo Noronha Matos (2011-2014) 110 P2X7 receptors. More recent studies from Dixon’s group suggested that P2X7 stimulation leads to increased membrane blebbing and bone formation (Panupinthu et al., 2007, 2008). Thus, the potential role of the P2X7 receptor in osteoblasts deserves further clarification and so does the involvement of other P2X receptor subtypes which may be also expressed in human osteoblast-like cells. Pattern of the extracellular catabolism of ATP and uracil nucleotides (UTP and UDP) in postmenopausal primary MSC cultures by HPLC Figure 11 illustrates the time course of the extracellular catabolism of ATP and uracil nucleotides (UTP and UDP) in intact postmenopausal primary MSCs in culture. ATP (100 µM) was catabolised with a half-degradation time of 24±2 min (n=9 observations from three individuals) and 12±1 min (n=9 observations from three individuals) at culture days 7 and 21, respectively (Figure 11A, B, see also Table 4). Because enzymatic activity is usually represented as a function of the total protein content, yet in osteoblast differentiating cultures type I collagen accounts to 85–90% of the organic matrix, we decided to normalize the ectonucleotidase activity by the amount of viable cells given by the MTT assay (Table 4). Normalization of the activity of the enzyme hydrolysing ATP to the total protein content or to the amount of viable cells demonstrates that speed up of ATP catabolism as cultures progressed is due predominantly to the concomitant increase in the number of cells, since the net enzymatic activity decreased by 25– 50%. Postmenopausal primary MSCs allowed to grow for 7 days hydrolysed ATP to adenosine with a very modest appearance of ADP and AMP (Figure 11A). In contrast with the majority of tissue systems, MSCs exhibit low level of ectoadenosine deaminase activity (Costa et al., 2011), so that adenosine progressively accumulates in 7-day cultures to a maximum of 38.3±3.2 µM 30 min after ATP (100 µM) application with only negligible (<1 µM) inosine and hypoxanthine formation. At 21-day cell cultures, ADP accumulation in the medium transiently 3. Original Research Papers PhD thesis – José Bernardo Noronha Matos (2011-2014) 111 0 20 40 60 80 100 010 20 30 Concentration (µM) Time (min) UTP (100 µM, 21 days) (n=3, triplicates) UTP UDP UMP Uridine 0 6 12 18 24 30 010 20 30 Concentration (µM) Time (min) ATP (100 µM, 21 days) (n=3, triplicates) ATP ADP AMP ADO 100 80 60 40 20 0 20 40 60 80 100 010 20 30 Concentration (µM) Time (min) UDP (100 µM, 21 days) (n=3, triplicates) UDP UMP Uridine 0 20 40 60 80 100 010 20 30 Concentration (µM) Time (min) UDP (100 µM, 7 days) (n=3, triplicates) UDP UMP Uridine 0 20 40 60 80 100 010 20 30 Concentration (µM) Time (min) UTP (100 µM, 7 days) (n=3, triplicates) UTP UDP UMP Uridine 0 6 12 18 24 30 010 20 30 Concentration (µM) Time (min) ATP (100 µM, 7 days) (n=3, triplicates) ATP ADP AMP ADO 100 80 60 40 20 A B C D E F Figure 11. Time course of extracellular ATP (A and B), UTP (C and D), and UDP (E and F) metabolism in human primary bone marrow-derived mesenchymal stem cells (MSCs) in culture at days 7 (A, C, and E) and 21 (B, D, and F). Nucleotides (100 µM) were added to the culture medium at time zero. Samples (75 µl) were collected from each well at indicated times in the abscissa. Each collected sample was analysed by HPLC to separate and quantify ATP or UTP (filled circles), ADP or UDP (open circles), AMP or UMP (filled squares), and adenosine (ADO) or uridine (open squares). Each point represents pooled data from three individuals; three replicas were performed in each individual experiment. The vertical bars represent S.E.M. and are shown when they exceed the symbols in size. 3. Original Research Papers PhD thesis – José Bernardo Noronha Matos (2011-2014) 112 increased to a maximum of 16.0±0.5 µM 10 min after ATP (100 µM) application (Figure 11B). Formation of adenosine from dephosphorylation of AMP by ecto-5’- nucleotidase (CD73 membrane-cell maker, EC 3.1.3.5) was moderately delayed as compared to 7-day cultures; adenosine reached a maximal concentration of 36.0±5.6 µM 30 min after ATP (100 µM) application to human MSCs cultured for 21 days. Given the linearity of the semi-logarithmic progress curves obtained by polynomial fitting of the catabolism of ATP and AMP, the analysis of the corresponding half-degradation time values suggests that the extracellular catabolism of ATP into AMP through ecto-nucleotidases is the rate-limiting step to generate adenosine from exogenously added adenine nucleotides in MSC cultures (cf. Costa et al., 2011). Extracellular UTP (100 µM) was catabolised with a half degradation time of 143±29 min (n=9 observations from three individuals) and 12±1 min (n=9 observations from three individuals) at culture days 7 and 21, respectively (Figure 11C, D). The UTP metabolites detected in the bath were UDP, UMP, and uridine, whose concentrations increased with time in human MSC cultures (Figure 11C, D). UDP transiently accumulated to maximal concentrations of 14.7±1.3 µM and 11.1±1.2 µM 5 min after UTP (100 µM) application to 7and 21-day cultures, respectively. Uridine progressively accumulated in human MSC cultures to maximal concentrations of 21.1±2.3 µM and 62.2±2.8 µM 30 min after UTP (100 µM) application at days 7 and 21, respectively. The net enzymatic inactivation of UTP normalized to the total protein content or to the amount of viable cells increased by 6–10-fold as cultures progressed from day 7 to 21, respectively (Table 4). Given that the number of viable cells increased only by about twofold in the same period of time in culture, the results suggest that more differentiated human MSCs exhibit higher ecto-nucleotidase activity implicated in the inactivation of extracellular UTP. In these circumstances, the kinetics of inactivation of UTP and ATP become comparable yet differences may be noted in the profile of products formation (Figure 11). The progress curves of UDP (100 µM) disappearance in postmenopausal primary MSC cultures at days 7 and 21 are represented in Figure 11 E, F, 3. Original Research Papers PhD thesis – José Bernardo Noronha Matos (2011-2014) 113 respectively. The results show that extracellular UDP (100 µM) is slowly inactivated in less differentiated cultures; the rate UDP catabolism speeds up (2– 3-fold) significantly as cultures progressed to more differentiate status. That is, UDP (100 µM) was catabolised with a half-degradation time of 69±16 min (n=9 observations from three individuals) and 6±1 min (n=9 observations from three individuals) at culture days 7 and 21, respectively (Figure 11E, F). The UDP metabolites detected in the bath were UMP and uridine. Thirty minutes after UDP (100 µM) application, the concentration of uridine in the culture medium was about 25.8±6.0 µM and 85.5±3.2 µM in samples collected from cell cultures at days 7 and 21, respectively. At culture day 21, the enzymatic inactivation rate of UDP normalized to the total protein content or to the amount of viable cells was the strongest of all nucleotides tested, whereas it had only intermediate values in 7day cultures (Table 4). This might explain why UDP does not easily accumulate in Table 1: Ectonucleotidase activity and half-degradation time values of ATP, UTP and UDP on human primary bone marrow stromal cells during osteogenic differentiation. Day 7 Day 21 t½ (min) pmol/µg prot. /15 min pmol/MTT abs. /15 min t½ (min) pmol/µg prot. /15 min pmol/MTT abs. /15 min ATP 24±2 173±32 6488±1203 12±1* 82±15* 4871±899 UTP 143±29 16±14 576±492 12±1** 85±5** 5042±254** UDP 69±16 60±14 2255±492 6±1** 114±2** 6783±122** ATP and ADP (100 µM) were added to culture media of human primary osteoblast cells at days 7 and 21. Samples (75 µl) were collected from each well at times 1, 5, 10, 15 and 30 min after application of the substrate. Each collected sample was analyzed by HPLC to separate and quantify ATP and/or UTP, ADP and/or UDP, AMP and/or UMP, and adenosine and/or uridine (see Figure 5). Enzymatic activity is usually presented as a function of the total protein content (day 7: 17.5±2.6 µg/cm2 vs day 21: 52.5±10.6 µg/cm2, n=14). Yet, in osteoblast differentiating cultures type I collagen accounts to 8590% of the organic matrix, thus normalization of the ectonucleotidase activity was also done by the amount of viable cells given by the MTT assay (day 7: 0.480±0.024 A/cm2 vs day 21: 0.892±0.049 A/cm2, n=20). Values are means ± SEM from 3 individuals; 3 replicas were performed in each individual experiment. *P<0.05 and **P<0.01 represent significant differences when compared with day 7. ATP, UTP and UDP (100 µM) were added to culture media of human primary MSCs at days 7 and 21. Samples (75 µl) were collected from each well at times 1, 5, 10, 15, and 30 min after application of the substrate. Each collected sample was analysed by HPLC to separate and quantify ATP and/or UTP, ADP and/or UDP, AMP and/or UMP, and adenosine and/or uridine (see Figure 11). Enzymatic activity is usually presented as a function of the total protein content (day 7: 17.5±2.6 µg/cm2 vs. day 21: 52.5±10.6 µg/cm2, n=14). Yet, in osteoblast differentiating cultures type I collagen accounts to 85–90% of the organic matrix, thus normalization of the ectonucleotidase activity was also done by the amount of viable cells given by the MTT assay (day 7: 0.480±0.024 A/cm2 vs. day 21: 0.892±0.049 A/cm2, n=20). Values are means SEM from three individuals; three replicas were performed in each individual experiment. *P<0.05 represents significant differences when compared with day 7. **P<0.01 represents significant differences when compared with day 7. Table 4. Ectonucleotidase activity and half-degradation time values of ATP, UTP and UDP on human primary bone marrow-derived mesenchymal stem cells (MSCs) during osteogenic differentiation. 3. Original Research Papers PhD thesis – José Bernardo Noronha Matos (2011-2014) 114 more mature MSC cultures when UTP (100 µM) was used as a substrate (Figure 11D). The presence of a saturating concentration (10 mM) of β-glycerophosphate in the culture medium did not alter the degradation kinetics of ATP and uracil nucleotides (UTP and UDP), suggesting that the contribution of non-specific phosphatases (e.g., ALP, EC 3.1.3.1) to the extracellular catabolism of nucleotides applied in a 100-µM concentration is negligible (data not shown). Detection of NTPDase1, -2, -3, and ecto-5’-nucleotidase in postmenopausal primary MSCs by immunofluorescence Comparing the kinetics of adenine and uracil nucleotides hydrolysis suggests that the former are much better substrates than uracil nucleotides for enzymes of the ecto-nucleotidase family putatively expressed in less differentiated (7-day) human MSC cultures. Reversion of this enzymatic pattern that plays a major role in triand diphosphonucleoside hydrolysis may be a hallmark of osteogenic differentiation of human MSCs in culture and has the potential to fine-tuning regulate P2 receptors signalling in the human bone. Human primary MSCs were allowed to grow in chamber slides for 7 and 21 days in supplemented α-MEM before immunolabeling with specific primary antibodies directed against human NTPDase1, -2 and -3, and ecto-5´- nucleotidase. Fluorescence immunoreactivity against ecto-NTPDase1, -2 and -3, and ecto-5’-nucleotidase in human primary MSCs shows a cytoplasmic/membrane-staining pattern typical for ectoenzymes, which increased in intensity with time (7<21 days) in culture (Figure 12). This is particularly evident for NTPDase1 and -3 (Figure 12A, C). Interestingly, immunoreactivity against NTPDase2 was almost unapparent at 7-day cultures (Figure 12Bi) and became stronger in more differentiated (21-day) cell cultures (Figure 12Bii). Human primary MSCs also exhibited positive immunoreactivity against CD73 (ecto-5’- nucleotidase) that is found to be expressed exclusively (>95%) in osteoprogenitors (see e.g., Liu et al., 2009). Immunolabeling of CD73 molecules in postmenopausal 3. Original Research Papers PhD thesis – José Bernardo Noronha Matos (2011-2014) 115 primary MSCs was demonstrated by both immunocytochemistry (Figure 12) and flow cytometry (data not shown). DISCUSSION The actions of extracellular adenine nucleotides on the basic functions of bone cells appear to be relatively clear-cut. ATP and ADP exert striking in vitro actions at concentrations in the low micromolar range to stimulate the formation and resorptive activity of osteoclasts, whereas ATP decrease ALP activity (see e.g., Figure 10A) and inhibit mineralized bone formation by osteoblasts (Hoebertz et al., 2003). Identification of uracil nucleotides-sensitive P2Y2, P2Y4, and P2Y6 receptors suggests that extracellular UTP/UDP have also important roles in regulating cellular function in a variety of tissues and organs. Few reports have, however, questioned the action of these compounds as autocrine/paracrine mediators in human bone physiology. In this study, we show that uracil 7 days 21 days NTPDase 1 NTPDase 2 NTPDase 3 5’-NTase Ai Aii Bi Bii Ci Cii Di Dii Figure 12. Immunocytochemical detection of NTPDase1, -2, and -3 and ecto-5’-nucleotidase on human primary bone marrow-derived mesenchymal stem cells (MSCs). Shown is the timerelated immunoreactivity fluorescence detection by confocal microscopy of human MSCs (first subculture) allowed to grow for 7 (Ai–Di) and 21 (Aii–Dii) days in an osteoblast-inducing medium. Cells grown in eight-well chamber slides were processed for immunocytochemistry in parallel and were visualized keeping unaltered the settings of the confocal microscope throughout the procedure. For further details on immunofluorescence labelling see Materials and Methods section. Scale bar is 150 µm. 3. Original Research Papers PhD thesis – José Bernardo Noronha Matos (2011-2014) 122 or fracture, micromolar concentrations of purine or pyrimidine nucleotides will be present to activate P2 receptors. Nevertheless, it is obvious that nucleotides must exist transiently in the bone microenvironment without cell damage to be physiologically relevant regulators of bone remodelling. There are two distinct nonlytic mechanisms by which nucleotides may be constitutively released from cells: (a) Exocytic release specifically concentrated within secretory granules or vesicles; and (b) release of cytosolic nucleotides via intrinsic plasma membrane channels or pores, which includes ABC transporters and connexin hemichannels controlled release (Bodin and Burnstock, 2001; Novak, 2003). Various agonists to P2 receptors can also initiate nucleotides release; in a positive feedback loop, UTP may act through P2Y2 receptors to upregulate ATP release from primary human osteoblasts (Bowler et al., 1998). In addition, bone cells constitutively release ATP (and possibly UTP) upon mechanical stimulation, hypoxia, and hypo/hypertonic stress (reviewed by Bowler et al., 2001). The ability of various cell types (e.g., murine airway epithelial cells, human cardiomyocytes) to release UTP has been directly confirmed (Lazarowski et al., 1997, 2003). It is important to stress that the extracellular accumulation of nucleotides such as UTP, UDP, and ADP may not necessarily involve a requirement for their selective release from intracellular pools. Rather, these nucleotides may accumulate as a secondary consequence of extracellular metabolism (degradation or synthesis) of nucleotide precursors that are directly released in various physiological or pathological conditions (Burrell et al., 2005; Buxton et al., 2001; Joseph et al., 2004; Yegutkin et al., 2001, 2002). Thus, ADP or UDP may either be released directly or be generated via the extracellular hydrolysis of directly released ATP or UTP. It is also possible that extracellular UTP may be secondarily generated via an ectonucleoside diphosphokinase (NDPK)-mediated transphosphorylation of directly released UDP by ATP that is co-released from the same cell or released coincidently from adjacent cells (Donaldson et al., 2000; Lazarowski et al., 1997, 2000; Lazarowski and Harden, 1999). The human osteoblastic cell line SaOS-2 expresses a strong ecto-NDPK activity that acts to generate additional extracellular ATP in the presence of a γ-phosphate donor that might be UTP or 3. Original Research Papers PhD thesis – José Bernardo Noronha Matos (2011-2014) 123 GTP (Buckley et al., 2003). Unfortunately, we were unable to detect the coexistence of UTP-consuming and UTP-generating enzymes, like the ectoNDPK, in the kinetic studies performed in this study (Figure 11). Membrane compartmentalization of ecto-enzymes and other components of purinergic signalling cascade have been demonstrated (reviewed by Yegutkin, 2008). According to that model, nucleotide-inactivating ecto-enzymes are located in close proximity with each other and next to nucleotides permeation sites, releasable stores and purinoceptors, presumably associated with lipid rafts and caveolae. Based on the compartmentalization model it seems reasonable to accept that released extracellular nucleotides and their derivatives are mainly concentrated on the cell surface, where they are subsequently ‘‘hand-to-hand’’ delivered for the succeeding phosphatase reactions. This important feature permits divergent cellular functions to take place at specific microdomains on the cell surface. Altogether, these multiple mechanisms for extracellular accumulation of particular P2 nucleotide agonists underscore why the characterization of potential nucleotide release mechanisms in a particular tissue or cell model should usually involve a corresponding analysis of extracellular nucleotide metabolism in that cell system (for a review, see Dubyak, 2007). In keeping with this model, our data emphasizes the value of a concerted action of UTP-consuming enzymes yielding to UDP generation and subsequent P2Y6 receptors activation in determining whether osteoblast progenitors are driven into proliferation or differentiation in postmenopausal human bone. These previously unrecognized targets for local regulation of osteogenic differentiation of bone marrow-derived MSCs may prompt for novel therapeutic strategies to control human diseases where bone destruction exceeds bone formation (e.g., osteoporosis, rheumatoid arthritis, osteogenesis imperfecta, fracture malunion). This research was partially supported by Fundação para a Ciência e a Tecnologia (FCT, FEDER funding) (PTDC/SAUOSM/73576/2006, REEQ/1168/SAU/2005, REEQ/1264/SAU/2005 and UMIB-215/94), by University of Porto/Caixa Geral de Depósitos (Investigação Científica na Pré-Graduação) 3. Original Research Papers PhD thesis – José Bernardo Noronha Matos (2011-2014) 124 projects (to P.C.S.), and by grants from the Canadian Institutes of Health Research (CIHR) and from The Arthritis Society (to J.S.). J.B.N.M. was in receipt of BIC Scholarship from FCT (PTDC/SAU-OSM/73576/2006) and J.S. was a recipient of a Junior 2 Scholarship from the Fonds de la Recherche en Santé du Québec (FRSQ). The authors acknowledge João Coimbra (MSc student) for his collaboration in cell cultures and Joanna Lecka for her assistance in the generation of NTPDase antibodies. Professor Margarida Lima (MD, PhD) was their guide throughout flow cytometry assays due to her vast experience in human haematological and lymphoproliferative diseases characterization. The authors also thank Mrs. M. Helena Costa e Silva and Belmira Silva for their technical assistance. 3. Original Research Papers PhD thesis – José Bernardo Noronha Matos (2011-2014) 125 Paper 2 FASEB Journal. 2014. Epub ahead of print DOI: 10.1096/fj.14-257923, PMID: 25169056 3. Original Research Papers PhD thesis – José Bernardo Noronha Matos (2011-2014) 126 P2X7-induced zeiosis promotes osteogenic differentiation and mineralization of postmenopausal bone marrow-derived mesenchymal stem cells J.B. NORONHA MATOS1,3, J. COIMBRA1,3, A. SÁ-E-SOUSA1,3, R. ROCHA4, J. MARINHAS4, R. FREITAS4, S. GUERRA-GOMES1,3, F. FERREIRINHA1,3, M.A. COSTA1,2,3, AND P. CORREIA-DE-SÁ1,3 1Laboratório de Farmacologia e Neurobiologia and 2Departamento de Química, Unit for Multidisciplinary Research in Biomedicine (UMIB), and 3Center for Drug Discovery and Innovative Medicines, Instituto de Ciências Biomédicas Abel Salazar–Universidade do Porto (ICBAS-UP), Porto, Portugal; and 4Serviço de Ortopedia e Traumatologia, Centro Hospitalar de Vila Nova de Gaia-Espinho, Portugal ABSTRACT Polymorphisms of the P2X7 receptor have been associated with increased risk of fractures in postmenopausal women. Although both osteoblasts and osteoclasts express P2X7 receptors, their function in osteogenesis remains controversial. Here, we investigated the role of the P2X7 receptor on osteogenic differentiation and mineralization of bone marrow mesenchymal stem cell (MSC) cultures from postmenopausal women (age 71±3 yr, n=18). We focused on the mechanisms related to intracellular [Ca2+]i oscillations and plasma membrane-dynamics. ATP, and the P2X7 agonist BzATP (100 µM), increased [Ca2+]i in parallel to the formation of membrane pores permeable to TO-PRO-3 dye uptake. ATP and BzATP elicited reversible membrane blebs (zeiosis) in 38±1 and 70±1% of the cells, respectively. P2X7-induced zeiosis was Ca2+ independent, but involved phospholipase C, protein kinase C, and Rho-kinase activation. BzATP (100 µM) progressively increased the expression of Runx-2 and Osterix transcription factors by 452 and 226% (at day 21), respectively, alkaline phosphatase activity by 88% (at day 28), and mineralization by 329% (at day 43) of bone marrow MSC cultures in a Rho-kinase-dependent manner. In summary, reversible plasma membrane zeiosis involving cytoskeleton rearrangements due to activation of the P2X7-Rhokinase axis promotes osteogenic differentiation and mineralization of bone marrow MSCs, thus providing new therapeutic targets for postmenopausal bone loss. 3. Original Research Papers PhD thesis – José Bernardo Noronha Matos (2011-2014) 127 INTRODUCTION Mesenchymal stem cells (MSCs) are a rare population of non-haematopoietic stromal cells present in the adult bone marrow, which exhibit extensive proliferative ability in uncommitted state while retaining great potential to differentiate into osteoblasts under appropriate conditions (Bobis et al., 2006; Pittenger et al., 1999). These unique properties make MSCs attractive candidates for bone growth and repair during aging, fracture healing, and diseases accompanied by excessive bone loss. Extracellular adenine nucleotides acting via several P2 purinoceptors play important roles in the regulation of bone formation as they are constitutively released into the bone microenvironment, the levels of which significantly increase during bone injury (reviewed in Burnstock et al., 2013). Adenine nucleotides stimulate bone formation by increasing the expression of osteoblast-related genes, such as RUNX-2 (Ciciarello et al., 2013). Released ATP also inhibits osteoclastinduced bone resorption by altering cytoskeletal structure (Miyazaki et al., 2012) and NF-kB translocation to the nucleus (Korcok et al., 2004). Communication between osteoblasts and osteoclasts involves intercellular Ca2+ signalling, which requires ATP release and activation of P2X7 receptors in osteoclasts (Jorgensen et al., 2002). Expression of the P2X7 receptor was also shown in a subpopulation of human differentiated osteoblasts (Gartland et al., 2001; Henriksen et al., 2006), as well as in several human osteoblast like cell lines (e.g., MG-63, SaOS-2, Te85; Nakamura et al., 2000), but not in others (reviewed in Wesselius et al., 2011). These differences, together with heterogeneity on the molecular composition and activity of P2X7 receptors among human and rodent species (e.g., see Roger et al., 2010), may explain the controversy in the literature regarding the predominant role of the P2X7 receptor in bone remodelling (reviewed in Burnstock et al., 2013). Bone turnover is coordinated in adulthood. Yet later in life, especially in women after menopause, osteoclast function is increased relative to osteoblast activity, and this unbalanced cellular activity causes increased bone resorption. Epidemiological studies indicate that single nucleotide polymorphisms in the P2X7 3. Original Research Papers PhD thesis – José Bernardo Noronha Matos (2011-2014) 128 receptor gene may favor the loss of bone mineral density and the risk of fracture in postmenopausal women (e.g., see Ohlendorff et al., 2007 but also Jorgensen et al., 2012). Although the high incidence of vertebral fractures may result from increased number of osteoclasts, we hypothesized that heterogeneity of the P2X7 receptor expression among osteoprogenitors and differentiated osteoblast-like cell populations might also play a role (Burnstock et al., 2013). Therefore, this study was designed to investigate the mechanisms underlying participation of the P2X7 receptor in osteogenic differentiation and mineralization of MSCs in postmenopausal women. The P2X7 receptor channel possesses unique characteristics among the P2X family by their much longer intracellular C-terminal tail, slow desensitization rate, and ability to switch between two open states depending on activation conditions. Basal P2X7 receptor activation leads to opening of membrane channels permeable to small cations (e.g., Ca2+, K+, Na+; Alloisio et al., 2010; Carrasquero et al., 2009; Orriss et al., 2006). During mechanical stress, tissue trauma and/or inflammation, prolonged exposure to high concentrations of ATP (>100 µM) leads to a leftward shift in agonist affinity and to opening of dilated plasma membrane pores permeable to molecules up to 900 Da (Panupinthu et al., 2007). This renders cells more prone to reversible morphological changes (e.g., microvesiculation and blebbing), as observed in mouse osteoblasts (Li et al., 2005) and/or susceptible to apoptosis, as occurs in osteoclasts (North, 2002). Given the osteogenic potential of MSCs for bone growth and repair in adulthood, and the controversy regarding the participation of the P2X7 receptor in human bone turnover, we focused our attention on the mechanisms underlying P2X7-induced membrane blebbing, pore formation, osteogenic differentiation, and mineralization of bone marrow-derived MSCs isolated from postmenopausal women as compared to younger females. MATERIALS AND METHODS Reagents and antibodies 3. Original Research Papers PhD thesis – José Bernardo Noronha Matos (2011-2014) 129 ATP, ADP, adenosine 5′-[β-thio]diphosphate (ADPβS), and 2’,3’-O-(benzoyl-4benzoyl)-adenosine 5′-triphosphate (BzATP) were from Sigma-Aldrich (St. Louis, MO, USA). We obtained 3-[[5-(2,3-dichlorophenyl)-1H-tetrazol-1-yl]methyl]pyridine (A438079), chelerythrine (CHL), (S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl) sulfonyl]-hexahydro-1H-1,4-diazepine (H1152), 2'-deoxy-N6-methyladenosine 3',5'- bisphosphate (MRS 2179), phorbol 12-myristate 13-acetate (PMA), thapsigargin and 1-[6-[[(17β)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5dione (U73122) from Tocris Cookson Inc. (Bristol, UK). Primary antibodies used were anti-P2Y1, anti-Runx-2 (M-70), anti-Osterix (M-15) (Santa Cruz Biotechnology, Santa Cruz, CA, USA); and anti-P2X7 (Alomone, Jerusalem, Israel). Anti-β-actin and horseradish-peroxidase-conjugated secondary antibodies were from AbCam (Cambridge, UK); Alexa Fluor 488-labelled anti-rabbit, Alexa Fluor 653-labelled anti-goat, Fluo-4NW and quinolinium 4-[3-(3-methyl-2(3H)- benzothiazolylidene)-1-propenyl]-1-[3-(trimethylammonio)propyl]-diiodide (TOPRO-3) were from Molecular Probes (Invitrogen, Carlsbad, USA). Cell cultures and phenotypic characterization Human bone marrow samples were obtained from the neck of the femur of postmenopausal women (age 71±3 yr, n=18) undergoing total hip arthroplasty as a result of primary osteoarthrosis. For comparison purposes, we also used bone marrow specimens from the sacrum of three younger female patients (age 14 yr) requiring bone engraftment for spinal fusion to correct scoliosis. Handling of bone marrow samples and culture of adherent cells was performed during 10–15 days (until near confluence), as described previously (Noronha-Matos et al., 2012). First subcultures were maintained for 43 days in standard culture medium [α-minimal essential medium (α-MEM) plus 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2.5 µg/ml amphotericin B] supplemented with 50 µg/ml ascorbic acid, 10 mM β-glycerophosphate, and 10 nM dexamethasone to promote osteogenic differentiation. Phenotypic characterization of the cells (first subculture) was performed by flow cytometry (Noronha-Matos et al., 2012). These cells exhibited positive immunoreactivity against CD105 (SH2), CD29 (integrin β1) and 3. Original Research Papers PhD thesis – José Bernardo Noronha Matos (2011-2014) 130 CD117 (tyrosine-protein kinase Kit), which have been identified as surface markers of bone marrow-derived MSCs (Bobis et al., 2006; Pittenger et al., 1999). Conversely, the cells were negative for haematopoietic surface markers, like CD14 and CD45, which have been extensively used as a good argument to distinguish bone marrow haematopoietic cells from MSCs (Baddoo et al., 2003; Pittenger et al., 1999). Thus, first passage plastic-adherent human bone marrow cells obtained under the present experimental conditions are highly enriched in multipotent MSCs. Viability/proliferation and osteogenic differentiation of bone marrow MSCs Cell viability/proliferation was evaluated by the 3-[4,5-dimethylthiazol2-yl]-2,5diphenyltetrasodium bromide (MTT) assay (Costa et al., 2011; Noronha-Matos et al., 2012). Data from the MTT assay correlates positively with the results measuring cell proliferation from total DNA quantification per culture well (e.g., see Noronha-Matos et al., 2012). Osteogenic differentiation of bone marrow-derived MSCs was inferred from increases in alkaline phosphatase (ALP) activity and from the expression of osteogenic transcription factors, namely Runx-2 and Osterix. ALP activity was determined in cell lysates by colorimetric determination of pnitrophenol (PNP) hydrolysis, as described previously; obtained values were expressed in nanomoles of PNP per minute normalized by the MTT absorbance (nmol·min-1·MTT-1; Noronha-Matos et al., 2012). Levels of Runx-2 and Osterix proteins were determined by Western blot analysis at culture day 7 and 21. Equal protein amounts (10 µg) loaded into SDS-PAGE (10%) gels were transferred onto a polyvinyl identifluoride membrane using a Mini-Protean Tetra Cell coupled to a Mini-Trans-Blot module (Bio-Rad, Hercules, CA, USA). Blocked membranes were incubated with anti-human primary antibodies (1:200): anti-Runx-2 (M-70, rabbit), anti-Osterix (M-15, goat). β-Actin (rabbit) was used as control. The peroxidase detection system (1.25 mM luminol; 0.2 mM coumaric acid; 0.1 M Tris, pH 8.5; and 0.032% hydrogen peroxide) was used for visualization of the immunoreactivity. Gels were analyzed using a gel blot imaging system (ChemiDoc MP; Bio-Rad). At culture day 43, calcium deposition in mineralized nodules was revealed by alizarin 3. Original Research Papers PhD thesis – José Bernardo Noronha Matos (2011-2014) 131 red staining and photographed using a microscope (Zeiss Axiophot; Carl Zeiss, Oberkochen, Germany) equipped with a CCD camera (CoolSnap HQ; Ropers Inc., Tucson, AZ, USA) running MetaFluor 6.3 image acquisition software (Photometrics, Tuczon, AZ, USA) (23). Images were exported to ImageJ 1.37c (U.S. National Institutes of Health, Bethesda, MD, USA) for quantification of the area of bone nodules. Single-cell intracellular calcium ([Ca2+]i) transients and morphological changes by confocal microscopy [Ca2+]i oscillations were evaluated in cells loaded with the fluorescent Ca2+ indicator, Fluo-4NW (2.5 µM), as described previously (Noronha-Matos et al., 2012). Briefly, 7-day culture dishes were mounted on the stage of a laser-scanning confocal microscope (Olympus FV1000; Olympus, Tokyo, Japan) and perfused continuously (1 ml/min) with gassed (95% O2 and 5% CO2) Tyrode’s solution (pH 7.4) containing (mM): 137 NaCl, 2.7 KCl, 1.8 CaCl2, 1 MgCl2, 0.4 NaH2PO4, 11.9 NaHCO3, and 11.2 glucose, at 37ºC (e.g., see Noronha-Matos et al., 2012). In some experiments, CaCl2 was omitted, and the extracellular Ca2+ chelator, EGTA (1 mM), was added. [Ca2+]i transients were calibrated to the maximal calcium load produced by the Ca2+ ionophore, ionomycin (5 µM, 100% response; NoronhaMatos et al., 2012). The percentage of cells in each microscopic field (LUCPLFL 20xPH; NA 0.45) exhibiting microvesiculation and blebbing (zeiosis) before and during addition of test drugs was also determined. For cell area quantification, the morphology of the cells was compared before and after the drug exposure (6 min). The area (µm2) of loaded cells with Fluo-4NW (2.5 µM) was measured using the FluoView Advanced software package (Olympus); images were exported to Image J 1.37c for mathematical analysis. TO-PRO-3 dye uptake and pore formation Membrane pore formation was monitored by measuring TO-PRO-3 dye uptake synchronously to Ca2+ signals in cells previously loaded with the fluorescent Ca2+ indicator, Fluo-4NW (2.5 µM; see above for details). After mounting culture dishes 3. Original Research Papers PhD thesis – José Bernardo Noronha Matos (2011-2014) 138 BzATP (100 µM), whereas MRS 2179 (0.3 µM) was ineffective. These results suggest that despite the role the P2Y1 receptor might play in [Ca2+]i mobilization caused by BzATP, this receptor lacks effect on membrane cell dynamics, which may justify the lack of action of the P2Y1 receptor ligands, ADP (100 µM) and ADPβS (100 µM), on membrane zeiosis (see Figure 13B, C). P2X7-induced plasma membrane blebbing is independent of Ca2+, but involves activation of phospholipase C (PLC), protein kinase C (PKC), and Rho-associated kinase in MSCs from postmenopausal women Although the pharmacology and channel properties of the P2X7 receptor have been studied extensively, signal transduction pathways are relatively unknown. It has been demonstrated that P2X7 receptors signal through phospholipase D (PLD) and phospholipase A2 (PLA2) in osteoblasts from newborn rats (Panupinthu et al., 2007). These researchers showed that lysophosphatidic acid (LPA) resulting from PLD activity acts on specific receptors on osteoblasts to cause membrane cell blebbing via a pathway dependent on Rho-associated kinase (Panupinthu et al., 2007). The interplay between PKC and Rho-kinase activation has been hypothesized, although this interaction has not been experimentally proven. Here, we examined whether inhibition of Rho-associated kinase using H1152 (3 µM; Sasaki et al., 2002; Tamura et al., 2005) or inhibition of PKC by CHL (5 µM; Herbert et al., 1990) affected membrane cell blebbing in cultured postmenopausal MSCs challenged with BzATP (100 µM). Figure 16Ai shows that BzATP (100 µM) - induced plasma membrane blebbing was prevented by both H1152 (3 µM, Figure 16Aii) and CHL (5 µM, Figure 16Aiii); statistical analysis of these experiments is shown in Figure 16B. Despite their effect on membrane cell blebbing, the two Figure 15. (Continued) Cells were challenged with BzATP (100 µM, for 6 min) in the absence and in the presence of A438079 (3 µM, a selective P2X7 receptor antagonist) or MRS 2179 (0.3 µM, a selective P2Y1 receptor antagonist). (B) Each point represents pooled data from 3–5 individuals for a total of 32 cells (BzATP), 80 cells (BzATP plus A438079), and 50 cells (BzATP plus MRS 2179). [Ca2+]i transients were calibrated to the maximal calcium load produced by ionomycin (5 µM, 100% response). (C) Bars represent pooled data from 3 individuals for n cells. Values are in arbitrary fluorescence units. *P<0.05 vs. BzATP alone. (D) Percentage of cells exhibiting plasma membrane blebs within each cell populations. Bars represent pooled data from n cells. Error bars = S.E.M. *P<0.05 vs. control (before addition of drugs); **P<0.05 vs. BzATP alone. 3. Original Research Papers PhD thesis – José Bernardo Noronha Matos (2011-2014) 139 enzyme inhibitors differently affected BzATP (100 µM)-induced [Ca2+]i response. Figure 16. P2X7-induced membrane cell dynamics require activation of PLC, PKC and Rhoassociated kinase in MSCs from postmenopausal women. [Ca2+]i oscillations and membrane blebs were monitored by confocal microscopy in the time-lapse mode in MSCs loaded with Fluo-4 NW, at culture day 7. (A) Left panels: [Ca2+]i transients calibrated to the maximal calcium load produced by ionomycin (5 μM, 100% response). 2+ Ca  M  + Thapsigargin 2 * ** * P M A 1 0 M + C H L 5 M * ** Before M  + CHL 5 M  + U73122 3 M  + H1152 3 0 20 40 60 80 100 n= 53-140 *,**P< 0.05 * ** ** ** Blebbing cells (%) A 20 % ionomycin BzATP (100 µM, 6 min) Before Treated (i) Scale bars: 50 μmScale bars: 50 μm Before Treated BzATP (100 µM, 6 min) + U73122 (3 µM) (v) Scale bars: 50 μm Before Treated PMA (10 µM, 6 min) (iv) Scale bars: 50 μm BzATP (100 µM, 6 min) + CHL (5 µM) (iii)Before Treated Scale bars: 50 μm B (ii) BzATP (100 µM, 6 min) + H1152 (3 µM) Before Treated Before Treated BzATP 100 µM 3. Original Research Papers PhD thesis – José Bernardo Noronha Matos (2011-2014) 140 Inhibition of Rho-associated kinase by H1152 (3 µM) globally depressed [Ca2+]i transients caused by BzATP (100 µM; Figure 16Aii). Conversely, CHL (5 µM) prevented the sustained component of BzATP (100 µM)-induced [Ca2+]i response, while keeping almost unaltered the initial [Ca2+]i rise (Figure 16Aiii). To confirm the PKC involvement in zeiosis, we tested the effect of a phorbol ester activator of PKC, PMA (5 µM; Castagna et al., 1982). PMA mimicked the effect of BzATP on the total number of cells exhibiting membrane blebbing, while producing a smaller [Ca2+]i response than the nucleotide (Figure 16Aiv, B). Data suggest that PKC and Rho-associated kinase effectively mediate membrane cell blebbing downstream P2X7 receptor activation. As mentioned above, P2X7 activation may lead to the production of LPA and subsequent activation of the Gq-coupled LPA1 receptor, which may mediate blebbing in osteoblasts and, thus, osteogenic differentiation during skeletal development in rats (Panupinthu et al., 2007). Stimulation of Gq-coupled receptors leads, most commonly, to PLC activation and increases in plasma membrane diacylglycerol (DAG) and cytosolic IP3 levels (Bleasdale et al., 1990). DAG would otherwise activate PKC. Pretreatment of the cells with U73122 (3 µM) significantly reduced BzATP-induced [Ca2+]i response, as well as membrane cell blebbing; under these conditions, only 35±1% of the cells exposed to the nucleotide showed blebbing (Figure 16Av, B). Overall, these results suggest that P2X7-induced plasma membrane blebbing involves activation of PLC, PKC, and Rho-associated kinase in MSCs from postmenopausal women. Figure 16. (Continued) [Ca2+]i oscillations and membrane blebs were monitored by confocal microscopy in the time-lapse mode in MSCs loaded with Fluo-4NW, at culture day 7. (A) Left panels: [Ca2+]i transients calibrated to the maximal calcium load produced by ionomycin (5 μM, 100% response). Each point represents pooled data from 3-5 different individuals for a total of 30 cells (BzATP 100 μM, Ai), 61 cells (BzATP 100 μM plus H1152 3 μM, Aii), 80 cells (BzATP 100 μM plus CHL 5 μM, Aiii), 36 cells (PMA 10 μM, Aiv) and 65 cells (BzATP 100 μM plus U73122 3 μM, Av). Error bars = S.E.M. Right panels: pseudocolor micrographs obtained before and during drug treatment. Arrows indicate membrane blebs formed during perfusion with BzATP (100 μM) and PMA (10 μM). Scale bars = 50 μm. (B) Percentage of cells exhibiting membrane blebs within each situation, along with the effect of BzATP (100 μM) in a Ca2+-free solution (CaØ + 1mM EGTA) obtained in the absence or in the presence of thapsigargin (2 μM), used to deplete intracellular Ca2+ reservoirs. Bars represente pooled data from n cells (3-5 individuals). Error bars = S.E.M. *P<0.05 vs. control (before addition of drugs); **P<0.05 vs. BzATP or PMA alone. 3. Original Research Papers PhD thesis – José Bernardo Noronha Matos (2011-2014) 141 Taking into consideration that BzATP (100 µM) elicits plasma membrane blebbing irrespective of the pattern obtained for the [Ca2+]i response, we decided to perform similar experiments in the absence of external Ca2+ (plus EGTA, 1 mM) and after depleting the intracellular Ca2+ stores with thapsigargin (2 µM). BzATP (100 µM)-induced membrane cell blebbing was still observed in 57±1% of the cells tested in Ca2+-free Tyrode’s solution supplemented with the Ca2+ chelator, EGTA (1 mM; Figure 16B). BzATP (100 µM) accelerated Ca2+ influx from the extracellular milieu on readmission of CaCl2 to control levels (1.8 mM) in the Tyrode’s solution. Under control conditions, [Ca2+]i peaked to 74±3% (n=48 cells) of the ionomycin response in 200 sec, while the maximal [Ca2+]i load (76±2% of the ionomycin response, n=44 cells) was anticipated by 80 sec (P<0.05) when the cells were preincubated with the P2X7 receptor agonist. The specific inhibitor of endoplasmic reticulum Ca2+-ATPase, thapsigargin (2 µM; Thastrup et al., 1990), failed to affect the formation of membrane cell blebs caused by BzATP (100 µM), which were observed in 65±1% of tested cells (Figure 16B). This reinforces the idea that P2X7-induced membrane cell blebbing is a Ca2+-independent phenomenon in MSCs from postmenopausal women. P2X7-induced changes in the morphology of MSCs from postmenopausal women depend on PLC, PKC, and Rho-associated kinase The mechanism underlying membrane cell blebbing is thought to involve actomyosin contraction, but the initiating events are poorly understood. Interestingly, cell shape and cytoskeletal tension generated by actomyosin have been shown to regulate osteogenic differentiation of human MSCs (McBeath et al., 2004). In the same set of previous experiments, we were able to measure the changes in cell area on application of BzATP (100 µM) in the absence and in the presence of the selective P2X7 antagonist A438079 (3 µM), the PLC inhibitor U73122 (3 µM), the PKC inhibitor CHL (5 µM), and the Rho-associated kinase inhibitor H1152 (3 µM). Figure 17A shows a representative image of a cell loaded with the Ca2+ indicator, Fluo-4NW, and the corresponding differential interference contrast (DIC) image, just before and during BzATP (100 µM) application. One can 3. Original Research Papers PhD thesis – José Bernardo Noronha Matos (2011-2014) 142 appreciate a clear retraction of the cell synchronous to application of the nucleotide along with the formation of plasma membrane blebs (Figure 17A, magnified inset). On average, the total area of the cells shrank by 316±93 µm2 (n=67) after exposure to BzATP (100 µM) for 6 min. Contraction of the cells caused by BzATP (100 µM) was significantly attenuated in the presence of A438079 (3 µM), H1152 (3 µM), chelerythrine (5 µM), and U73122 (3 µM) (Figure 17B). These results suggest that P2X7-induced changes in the morphology of MSCs from postmenopausal women depend on PLC, PKC, and Rho-associated kinase. The involvement of PKC in cell shrinkage was confirmed using PMA (10 µM). Like that observed for membrane cell blebbing, PMA (10 µM) also mimicked the effect of BzATP (100 µM) on cell retraction. A reduction of the total cell area by 425±133 µm2 (n=32) was observed in the presence of PMA (10 µM), an effect that was completely reverted by CHL (5 µM, n=41), strengthening the involvement of PKC in cell shape changes (Figure 17B). P2X7-induced morphological changes in MSCs from postmenopausal women are mostly Ca2+-independent, because the cell shrinkage effect of BzATP (100 µM) was enhanced, rather than reduced, to 511±56 µm2 (n=97) in the absence of extracellular Ca2+ (plus 1 mM EGTA) and depletion of internal Ca2+ stores with thapsigargin (2 µM) had no significant effect (316±74 µm2, n=79). Osteogenic commitment of MSCs from postmenopausal women is impaired as compared to younger females Cell viability/proliferation (given by the MTT assay) of postmenopausal MSCs grown in osteogenesis-inducing conditions gradually increase until day 28, a situation that was not different from that observed with the cells isolated from younger females (e.g., see Noronha-Matos et al., 2012). Significant (P<0.05) differences were, however, observed regarding the osteogenic differentiation profile between the two groups (Figure 18). Cell cultures from the younger group (age 14 yr, n=3) exhibited higher ALP activity normalized by the number of viable cells (MTT value) than the postmenopausal group (age 71±3 yr, n=18). ALP activity in the younger group of patients reached a maximum at day 14 (Figure 3. Original Research Papers PhD thesis – José Bernardo Noronha Matos (2011-2014) 143 Figure 17. PLC, PKC and Rho-associated kinase are involved in cell retraction induced by activation of the P2X7 receptor in MSCs from postmenopausal women. (A) Representative confocal micrographs of postmenopausal MSCs loaded with Fluo-4NW, obtained at culture day 7 just before and during perfusion with BzATP (100 μM). Arrows indicate high magnification plasma membrane blebs. Scale bars = 50 μm. Pseudocolor images represent [Ca2+]i levels; cell boundaries are best appreciated by differential interference contrast (DIC) microscopy. (B) Variation of cell area (μm2) after application of drugs as compared to the control situation (negative values indicate cell retraction). Bars represents pooled data from 3-5 individuals for a total of 67 cells (BzATP 100 μM), 57 cells (BzATP 100 μM plus A438079 3 μM), 59 cells (BzATP 100 μM plus CHL 5 μM), 42 cells (BzATP 100 μM plus U73122 3 μM), 29 cells (BzATP 100 μM plus H1152 3 μM), 32 cells (PMA 10 μM), and 41 cells (PMA 10 μM plus CHL 5 μM). Error bars = S.E.M. *P<0.05 vs. control (before addition of drugs); **P<0.05 vs. BzATP or PMA alone. A Before Treated BzATP (100 μM) Scale bars: 50 μm Fluo4-NW DIC B x4 x1 -600 -500 -400 -300 -200 -100 0 100 200 300 *,**P< 0.05 * ** + A438079 3 M (n= 57) + H1152 3 M (n= 29) BzATP 100 M (n= 67) + CHL 5 M (n = 59) ** ** + U73122 3 M (n = 42) ** Cell area / m2(Drug-Ctr) *,**P< 0.05 * + CHL 5 M (n = 41) PMA 10 M (n = 32) ** 3. Original Research Papers PhD thesis – José Bernardo Noronha Matos (2011-2014) 144 18A), whereas in the postmenopausal group, ALP activity increased slowly, yet progressively, with incubation time until day 28. Activation of the P2X7 receptor on postmenopausal MSCs resumes the osteogenic differentiation profile detected in younger females The effects of the P2X7 receptor activation on osteoblast cell cultures have been addressed in several studies, often with conflicting results (for a review, see Burnstock et al., 2013). For instance, the P2X7 receptor has been implicated in osteogenesis promotion in newborn rats (Panupinthu et al., 2007) and mediates shockwave-induced osteogenic differentiation of MSCs from healthy human volunteers (Sun et al., 2013). Conversely, other researchers observed reductions in ALP activity and bone mineralization in osteoblast cultures isolated from 2-daysold neonatal rats (Orriss et al., 2012). In SaOS-2 cells, a human osteosarcoma cell line, BzATP favoured osteoblast apoptosis (Gartland et al., 2001). This dispute ALP activity (14 yr) 4 7 14 21 28 0 10 20 30 40 50 60 BzATP (100 M) Control A438079 (3 M) H1152 (3 M) n= 8 - 16 *P<0.05 ** * * * ***** Days in culture nmol / minute / MTT ALP activity (71  3) 4 7 14 21 28 0 10 20 30 40 50 60 BzATP 100 M Control A438079 (3 M) H1152 (3 M) n= 16 - 42 *P<0.05 ** ** * ** * * Days in culture nmol / minute / MTT A B Figure 18. Activity of ALP in cell lysates of bone marrow-derived MSCs from young females (A) and postmenopausal women (B) exposed to BzATP (100 μM), A438079 (3 μM) and H1152 (3 μM) for 28 days. Ordinates are nanomoles of p-nitrophenol (pNP; at 405 nm) produced per minute from p-nitrophenylphosphate (pNPP) catalysed by ALP normalized by MTT values at given time points. Bone marrow samples were obtained from 3 young female patients (age 14 yr) and 5 postmenopausal women (age 71±3 yr); 3-8 replicas were performed in each individual experiment. Error bars = S.E.M. *P<0.05 vs. control. 3. Original Research Papers PhD thesis – José Bernardo Noronha Matos (2011-2014) 145 prompted us to investigate the role of the P2X7 receptor on osteogenic differentiation of MSCs from postmenopausal women, given that the gain of function of this receptor has been associated with increased bone mass (Husted et al., 2013). Treatment of MSCs with BzATP (100 µM) significantly increased the ALP activity above the control level. This was verified in MSC cultures from postmenopausal women (Figure 18B), but not in those from younger females (Figure 18A). On the contrary, the P2X7 receptor antagonist, A438079, failed to modify the ALP activity in MSC cultures from postmenopausal women (Figure 18B), when it was applied in a concentration (3 µM) that almost prevented osteogenic differentiation in cells from younger female patients (Figure 18A). Inhibition of Rho-kinase with H1152 (3 µM) significantly decreased the ALP activity measured in MSC cultures from both age groups (Figure 18A, B). The enhancement of ALP activity caused by BzATP (100 µM) in postmenopausal MSCs was significantly attenuated by blocking the P2X7 receptor with A438079 (3 µM). Coapplication of BzATP (100 µM) with the Rho-associated kinase inhibitor, H1152 (3 µM), abolished the increase in ALP activity observed with the nucleotide alone in cells from both age groups (Figure 18A, B). These findings suggest that P2X7 receptor activation promotes osteogenic differentiation in MSCs from postmenopausal women through a mechanism that involves stimulation of Rho associated kinase. Data also indicate that P2X7 receptors are tonically activated by endogenous ATP in cells from young females, but the osteogenic differentiating tone of the P2X7 receptor seems to be impaired in MSC cultures from postmenopausal women, probably because the nucleotide does not reach high enough concentrations in the cell microenvironment to activate this receptor. Runx-2 and Osterix are important transcription factors involved in bone formation. Runx-2 is involved in osteoblastic differentiation and skeletal morphogenesis; it is essential for the maturation of osteoblasts and ossification, both intramembranous and endochondral. It binds to a number of enhancers and promoters, including osteocalcin, osteopontin, bone sialoprotein and type I collagen (Ducy et al., 1997; Kern et al., 2001). Osterix acts downstream of Runx-2 3. Original Research Papers PhD thesis – José Bernardo Noronha Matos (2011-2014) 146 and is a zinc-finger-containing transcription factor, which is essential for embryonic osteoblast differentiation and bone formation (Nakashima et al., 2002). In this study, we collected total cell lysates for Runx-2 and osterix protein quantification by Western blot analysis. Western blot gels (Figure 19A, B) show that postmenopausal MSCs at culture day 7 and 21 express the two osteogenic differentiation markers, Runx-2 and Osterix. BzATP (100 µM) increased the expression of Runx-2 and osterix proteins from day 7 to 21 by ~452 and 226%, respectively (Figure 19C). In addition to the increments in ALP activity observed with BzATP, the ATP analogue increased the expression of important bone transcription factors, Runx-2 and Osterix, indicating that P2X7 receptor promotes osteogenic commitment of MSCs from postmenopausal women. P2X7 receptor activation promotes mineralization of postmenopausal MSC cultures The ultimate goal of bone-forming osteoprogenitors is the ability to mineralize the extracellular matrix via ALP activity. In this context, we performed histochemical assays using the Alizarin red staining at culture day 43 in order to identify extracellular calcium deposits corresponding to areas of mineralization of the cultures. We compared data obtained in the absence and in the presence of BzATP (100 µM) in MSCs from postmenopausal women and younger females. In control conditions, the total mineralized area (Figure 20A) and the total number of bone nodules formed per culture well (Figure 20B) were significantly higher in the young female group as compared to MSC cultures from postmenopausal women. Continuous application of BzATP (100 µM) significantly increased the total mineralized area and the total number of bone nodules formed per culture well in both age groups (Figure 20). The mineralization effect of the P2X7 receptor agonist was more notorious in the young female group, but BzATP (100 µM) was still capable of increasing by ~4-fold mineralization of MSCs cultures from postmenopausal women as compared to the control level. It is also worth noting that both total mineralized area and bone nodule formation in postmenopausal MSC cultures treated with BzATP (100 µM) overcame mineralization parameters 3. Original Research Papers PhD thesis – José Bernardo Noronha Matos (2011-2014) 147 determined in control conditions (no drugs added) for the young female group (Figure 20). BzATP (100 µM)-induced mineralization of MSCs cultures was fully prevented in the presence of the selective P2X7 antagonist, A438079 (3 µM). Likewise, inhibition of the Rho-associated kinase with H1152 (3 µM) also abolished mineralization of the cultures caused by BzATP (100 µM). Interestingly, mineralization of the cultures was slightly reduced when A438079 (3 µM) and H1152 (3 µM) were applied alone (Figure 20); differences reached statistical O steogen ic m arkers (75  8 yr) + B zA T P (100 M ) 7 2 1 0 200 400 600 800 1000 R un x-2 O sterix D ays in cu lture R elative expression (% of C tr) n = 3 *P< 0 .0 5 * A C Ctr 7 21 21 7 Days Osterix 58 KDa β-Actin 45 KDa +BzATP (100 µM) - - + + B 7 21 21 7 Days Runx-2 59 KDa β-Actin 45 KDa +BzATP (100 µM) - - + + Figure 19. (A), (B) Western blot analysis of Osterix (58 KDa; A) and Runx-2 (59 KDa; B) transcription factors in MSCs from 3 postmenopausal women (age 75±8 yr) at culture day 7 and 21: influence of BzATP (100 μM). β-Actin (45 KDa) was used as control. (C) Relative expression of Osterix and Runx-2 in postmenopausal MSCs as a percentage of control values detected without adding BzATP (dashed horizontal line). Error bars = S.E.M. *P<0.05 vs. control values obtained in the absence of BzATP.