Cytosolic free Ca2+ concentration exhibits a characteristic temporal pattern during in vitro cartilage differentiation: a possible regulatory role of calcineurin in Ca-signalling of chondrogenic cells

UNCORRECTED PROOF YCECA 972 1—14 Please ci e his a icle in p ess as: C. Ma a, e al., Cy osolic ee Ca2+ concen a ion exhibi s a cha ac e is ic empo al pa e n du ing in i o ca ilage di e en ia ion: A possible egula o y ole o calcineu in in Ca-signalling o chond ogenic cells, Cell Calcium (2008), doi:10.1016/j.ceca.2007.12.010 ARTICLE IN PRESS +Model YCECA 972 1—14 Cell Calcium (2008) xxx, xxx—xxx 1 jou nal homepage: www.else ie .com/loca e/ceca Cy osolic ee Ca2+ concen a ion exhibi s a Q1 cha ac e is ic empo al pa e n du ing in i o ca ilage di e en ia ion: A possible egula o y ole o calcineu in in Ca-signalling o chond ogenic cells 2 3 4 5 Csaba Ma aa,1,J ´ anos Fodo b,1, Zsol Sz ´ ıjgy´ a ´ oc,Tam ´ as Juh´ asza, P´ al Ge gelyc,L ´ aszl´ o Cse nochb,R ´ oza Z´ ak´ anya,∗ 6 7 aDepa men o Ana omy, His ology and Emb yology, Medical and Heal h Science Cen e, Uni e si y o Deb ecen, Q2 Nagye dei k . 98, H-4032 Deb ecen, Hunga y 8 9 bDepa men o Physiology, Medical and Heal h Science Cen e, Uni e si y o Deb ecen, Hunga y10 cCell Biology and Signalling Resea ch G oup o he Hunga ian Academy o Sciences, Depa men o Medical Chemis y, Resea ch Cen e o Molecula Medicine, Uni e si y o Deb ecen, Hunga y 11 12 Recei ed 24 July 2007; ecei ed in e ised o m 15 No embe 2007; accep ed 29 Decembe 200713 KEYWORDS Chond ogenesis; High-densi y cul u e; In acellula Ca2+ concen a ion; Fu a-2; Cyclospo ine A; Sox9 Summa y We measu ed changes o cy osolic Ca2+ concen a ion du ing chond ogenesis, which occu s in high-densi y cul u es (HDC) o chond i ying chicken mesenchymal cells. A significan , ansien ele a ion was de ec ed in Fu a-2-loaded cells on day 3 o cul u ing, when majo i y o chond ogenic cells o HDC become di e en ia ed. This 140 nM peak o cy osolic Ca2+ concen- a ion is a esul o inc eased Ca-influx and is indispensable o p ope chond ogenesis, because addi ion o 0.8 mM EGTA o cul u e medium on day 2 o 3 significan ly dec eased he in acel- lula Ca2+ concen a ion abolishing he Ca2+-peak o day 3 and inhibi ed ca ilage o ma ion. Uncon olled Ca2+ influx e oked by a Ca2+ ionopho e exe ed dual e ec s on chond ogenesis in a 14 15 16 17 18 19 20 21 Abb e ia ions: AAS, a omic abso p ion spec ome y; AM, ace oxy-me hyles e ; BMP, bone mo phogenic p o ein; CMF-PBS, calcium and magnesium ee phospha e bu e ed saline; CPA, cyclopiazonic acid; CsA, cyclospo ine A; DMMB, dime hylme hylene blue; DMSO, dime hyl sul oxide; dNTP, deoxy ibonucleo ide iphospha e; DTT, di hio h ei ol; ECM, ex acellula ma ix; EGTA, e hylene glycol e aace ic acid; ER, endoplasmic e iculum; FACS, fluo escence ac i a ed cell so e ; FCS, oe al cal se um; FGF, fib oblas g ow h ac o ; GAPDH, glyce aldehide-3-phospha e dehyd ogenase; HDC, high-densi y cell cul u e; IGF, insulin-like g ow h ac o ; IP3, inosi ol-1,4,5- isphospha e; MAPK, mi ogen-ac i a ed p o ein kinase; MTT, 3-(4,5-dime hyl hiazolyl-2)-2,5-diphenyl e azolium b omide; N-CAM, neu al cell adhesion molecule; OA, okadaic acid; PBS, phospha e bu e ed saline; PBST, phospha e bu e ed saline wi h 0.1% Tween 20; PKC, p o ein kinase C; PMCA, plasma memb ane Ca2+ ATPase; PMSF, phenylme hylsulphonyl; RT-PCR, e e se ansc ip ion and polyme ase chain eac ion; RyR, yanodine ecep o ; SDS-PAGE, sodium dodecyl sulpha e polyac ilamide gel elec opho esis; Se , se ine; SERCA, sa co(endo)plasmic e iculum Ca2+ ATPase; SOCE, s o e-ope a ed calcium en y; Th , h eonine; TRPV, ansien ecep o po en ial ion channel. ∗Co esponding au ho . Tel.: +36 52 416 392; ax: +36 52 432 290. Q3 E-mail add ess: [email p o ec ed] (R. Z´ ak´ any). 1These wo au ho s con ibu ed equally o his wo k. 0143-4160/$ — see on ma e © 2008 Else ie L d. All igh s ese ed. doi:10.1016/j.ceca.2007.12.010 UNCORRECTED PROOF YCECA 972 1—14 Please ci e his a icle in p ess as: C. Ma a, e al., Cy osolic ee Ca2+ concen a ion exhibi s a cha ac e is ic empo al pa e n du ing in i o ca ilage di e en ia ion: A possible egula o y ole o calcineu in in Ca-signalling o chond ogenic cells, Cell Calcium (2008), doi:10.1016/j.ceca.2007.12.010 ARTICLE IN PRESS +Model YCECA 972 1—14 2 C. Ma a e al. concen a ion-dependen manne ; 0.1 mg/L A23187 inc eased, whe eas 5 mg/L A23187 almos o ally blocked ca ilage o ma ion. In acellula Ca-s o es seemed no o ha e any significan pa icipa ion in he egula ion o changes o cy osolic Ca2+ concen a ion o chond i ying cells. Ac i i y o Ca—calmodulin-dependen p o ein phospha ase, calcineu in esponded o changes o in acellula Ca2+ concen a ion induced by EGTA o A23187 in a di e en ia ion s age-dependen manne . Since inhibi ion o calcineu in wi h cyclospo ine A elimina ed he peak in he cy osolic Ca2+ concen a ion, an ac i e egula o y ole o calcineu in on Ca2+ influx o chond i ying cells can be supposed. 22 23 24 25 26 27 28 29 © 2008 Else ie L d. All igh s ese ed.30 In oduc ion31 Hyaline ca ilage is an impo an elemen o he e e-32 b a e skele al sys em. I p o ides p imo dia o bones o med33 by endochond al ossifica ion and emains he majo shock-34 abso bing s uc u e o he a icula su aces o join s.35 Chond ogenic mesenchymal cells can be de i ed om di -36 e en emb yonic s uc u es: he c anial pa o neu al37 c es is he sou ce o ca ilage p imo dia o se e al c an-38 io acial bones; scle o ome o somi es di e en ia es in o39 e eb ae; appendicula bones de i e om mesenchymal40 cells o soma opleu a [1]. 41 High-densi y cell cul u e sys em (HDC) es ablished om42 chond ogenic mesenchymal cells isola ed om limb buds 43 o 4-day-old chicken emb yos is a well-known model o in44 i o ca ilage di e en ia ion [2—4]. This simple model can45 p o ide in o ma ion on he molecula s eps leading o di - 46 e en ia ion o chond op ogeni o cells o chond oblas s.47 In HDC, o ma ion o ca ilage s a s wi h he ec ui - 48 men o chond op ogeni o mesenchymal p ogeni o cells49 ha a e condensa ion and nodule o ma ion, di e en i-50 a e in o chond oblas s and chond ocy es. Condensa ion and51 nodule o ma ion ake place on he fi s day o cul u ing 52 and a e pa ly egula ed by ansien appea ance o Ca2+-53 dependen in e cellula junc ions like N-CAM (neu al cell54 adhesion molecule) and N-cadhe in [5]. Chond op ogeni o 55 cells di e en ia e in o chond oblas s on he second and56 hi d day o cul u ing [4,6], con olled by nume ous g ow h57 ac o s and o he signal molecules, e.g. FGF, BMP, Wn , IGF 58 and membe s o Hedgehog and Sox ansc ip ion ac o ami-59 lies [7]. In pa allel o he in acellula changes, ex acellula 60 ma ix (ECM) su ounding he di e en ia ing chond ogenic 61 cells is also subjec o p o ound changes: di e en ia ing62 cells s a o sec e e ca ilage-specific ma ix componen s,63 such as collagen ype II and agg ecan on he hi d day o cul- 64 u ing pe iod [8]. The unique composi ion and o ganiza ion 65 o ECM is c ucial o main enance o he p ope mo phol-66 ogy and unc ion o hese cells [9]. Exp ession o collagen67 ype II and co e p o ein o agg ecan is con olled by Sox9, a68 high-mobili y-g oup domain con aining ansc ip ion ac o 69 [10—12]. De ec ion o he exp ession le el and he phospho-70 yla ion s a us o Sox9, as well as moni o ing he exp ession 71 o he co e p o ein o agg ecan a e a eliable ma ke s o 72 chond ogenesis.73 Calcium ion is a ubiqui ous cellula signal. The concen-74 a ion o in acellula ee Ca2+ (∼10−7M) is 104 imes75 lowe han ha o he ex acellula fluid. This dis ibu- 76 ion p o ides he po en ial o he influx o Ca2+ in o cells,77 whe e i can ac as a second messenge . Va ious s imuli78 p omo e he mo emen o Ca2+ ei he om he ex acel-79 lula space o om in acellula s o es in o he cy osol. 80 The ele a ed le el o cy osolic ee Ca2+ exe s a a ie y 81 o specific changes in cellula unc ion, such as ac i a- 82 ion o p o ein kinases and p o ein phospha ases, which, 83 in u n, egula e o he p ocesses, like p oli e a ion o di - 84 e en ia ion [13]. The molecula s eps leading o ca ilage 85 di e en ia ion, among o he ac o s a e egula ed by Ca2+ 86 sensi i e enzymes like one o he Se /Th specific p o- 87 ein kinases, PKCalpha [14] o he Se /Th -specific p o ein 88 phospha ase calcineu in [15,16], ha is unique among phos- 89 pha ases o i s abili y o sense changes o in acellula 90 Ca2+ concen a ion h ough i s ac i a ion by i s calcium 91 binding subuni and calmodulin. Calcineu in is bes known 92 as a egula o o T-lymphocy e ac i a ion, since i s pha - 93 macological inhibi o s, cyclospo ine A (CsA), ac olimus, 94 pimec olimus and apamycin a e all used in he clinical p ac- 95 ice as immunosupp essan s [17]. Calcineu in is also known 96 o pa icipa e in se e al di e en ia ion p ocesses, such as 97 de elopmen o di e en muscle issues and he ne ous 98 sys em [18].99 In his s udy we measu ed he cy osolic ee Ca2+ concen- 100 a ion du ing ca ilage di e en ia ion in he chond ogenic 101 cells o HDC. A cha ac e is ic empo al pa e n in he 102 changes o cy osolic Ca2+ concen a ion could be obse ed; 103 he e was a significan and ansien ele a ion on he hi d 104 cul u ing day, he c ucial day o chond ocy e di e en ia- 105 ion. Mo eo e , beside he changes o he basal cy osolic 106 Ca2+ le el, cells o chond i ying mic omass cul u es also 107 exhibi spon aneous calcium e en s, a phenomenon cha - 108 ac e is ic o se e al o he p ima y cell cul u es [19,20].We 109 p o ide e idence ha he empo al pa e n o he changes 110 o cy osolic ee Ca2+ concen a ion in chond i ying cells is 111 indispensible o p ope ca ilage o ma ion and depends on 112 ex acellula Ca2+ a he han he a ailabili y o in acellu- 113 la Ca-s o es. We also demons a e ha calcineu in can play 114 a dual ole in Ca-signalling o chond ogenic cells: i s ac i i y 115 is modula ed by cy osolic Ca2+ concen a ion and he inhibi- 116 ion o calcineu in wi h CsA elimina es he Ca2+ peak o HDC 117 esul ing in a p onounced dec ease in ca ilage o ma ion. 118 This second obse a ion aises he possibili y o he ac i e 119 egula o y e ec o his enzyme on he enhancemen o Ca2+ 120 influx o chond i ying cells. 121 Ma e ials and me hods 122 Cell cul u e 123 Dis al pa s o he limb buds o 4-day-old Ross hyb id 124 chicken emb yos (Hambu ge —Hamil on s ages 22—24 [21])125 UNCORRECTED PROOF YCECA 972 1—14 Please ci e his a icle in p ess as: C. Ma a, e al., Cy osolic ee Ca2+ concen a ion exhibi s a cha ac e is ic empo al pa e n du ing in i o ca ilage di e en ia ion: A possible egula o y ole o calcineu in in Ca-signalling o chond ogenic cells, Cell Calcium (2008), doi:10.1016/j.ceca.2007.12.010 ARTICLE IN PRESS +Model YCECA 972 1—14 Possible egula o y ole o calcineu in in Ca-signalling o chond ogenic cells 3 we e emo ed and p ima y mic omass cul u es o chon-126 d i ying mesenchymal cells we e es ablished om a cell127 suspension wi h a densi y o 1.5 ×107cells/mL. Fi een128 mic oli e s d ople s o he suspension we e inocula ed129 on ound co e glasses (diame e : 30 mm; Menzel-Gl¨ ase ,130 Menzel GmbH, B aunschweig, Ge many) placed in o plas-131 ic Pe i dishes (Nunc, Nape ille, IL, USA). Cells we e132 allowed o a ach o he su ace o 2 h a 37 ◦C. Day133 o inocula ion is conside ed as day 0. Colonies we e 134 g own in Ham’s F12 medium (Sigma, Budapes , Hun-135 ga y) supplemen ed wi h 10% oe al cal se um (Gibco,136 Gai he sbu g, MD, USA) and we e kep a 37 ◦C in an a mo-137 sphe e o 95% ai and 5% CO2and 80% humidi y. The138 medium was changed on e e y second day o a e ea -139 men s.140 De e mina ion o cy osolic ee Ca2+ concen a ion141 Measu emen s we e pe o med on di e en days o cul u ing142 using he calcium-dependen fluo escen dye Fu a-2. Cul-143 u es we e ans e ed o 2 mL esh Ham’s F12 medium 144 con aining 10 ␮L Fu a-2-AM (10 ␮M) and 4 ␮L neos igmin145 (0.3 nM), in o de o inhibi ex acellula choline es e ases. 146 A e 60 min o incuba ion a 37 ◦CinaCO 2incuba o ,147 cul u es we e washed wice in Ty ode’s solu ion con- 148 aining 137 mM NaCl, 5.4 mM KCl, 0.5 mM MgCl2, 1.8 mM149 CaCl2, 11.8 mM Hepes—NaOH, 1 g/L glucose, pH 7.4, in150 o de o emo e he Fu a-2-AM a ached o he ex a-151 cellula ma ix. Fu a-2-loaded cells we e placed on he152 s age o an in e ed fluo escen mic oscope (Diapho , Nikon, 153 Kowasaki, Japan) and iewed using a 40×oil imme -154 sion objec i e. Measu emen s we e ca ied ou in he 155 same sal solu ion in a pe usion chambe using a dual156 wa eleng h monoch oma o (Del aScan, Pho on Technolo-157 gies In e na ional, Law ence ille, KY, USA) equipmen .158 All measu emen s we e pe o med a oom empe a u e. 159 Fluo escence o Fu a-2-loaded cells was measu ed using 160 exci a ion wa eleng hs o 340 and 380 nm and an emis-161 sion wa eleng h o 510 nm. In acellula Ca2+ concen a ions162 we e calcula ed om he a ios o in ensi ies (R=F340/F380)163 as desc ibed by G ynkiewicz e al. [22]. In acellula Ca2+ 164 le els o HDC we e measu ed 2 h a e inocula ion on165 ound co e glasses hen on cul u ing days 1—6 a he same 166 pe iod o each day. In acellula Ca2+ le els o un ea ed167 con ol cul u es and cul u es ea ed wi h EGTA, calcium168 ionopho e A23187, cyclopiazonic acid (CPA) o CsA we e169 assayed in 5 independen expe imen s measu ing 30 cells170 in each case. All measu emen s we e ca ied ou di ec ly 171 a e ea men s wi h EGTA, calcium ionopho e A23187, 172 CPA o CsA. Da a we e s a is ically analyzed by S uden ’s173 - es .174 T ea men s wi h cyclopiazonic acid and ca eine 175 To de e mine he ole o in acellula Ca-s o es in he176 changes o he cy osolic Ca2+ concen a ion, he in acel-177 lula Ca2+ pump inhibi o CPA was adminis e ed o HDC.178 Fo single cell measu emen s, CPA was dissol ed in DMSO179 and dilu ed in a modified, Ca2+- ee T yode’s solu ion180 (con aining 5 mM EGTA, wi hou CaCl2) o a final concen-181 a ion o 10 ␮M. The inhibi o was di ec ly pe used in 182 he close p oximi y (app oxima ely 50 ␮m) o cells. P io 183 o adminis a ion o CPA, cells we e washed wi h Ca2+-184 ee Ty ode’s solu ion o emo e all aces o ee Ca2+ 185 om he medium. To in es iga e p olonged e ec o deple- 186 ion o in acellula Ca2+ sou ce, CPA was adminis e ed 187 di ec ly in o he cul u e medium o 12 h on cul u ing 188 day 2 o 3 a a final concen a ion o 10 ␮M. Ca eine 189 (15 mM, dilu ed in Ty ode’s solu ion), an agonis o yan- 190 odine ecep o (RyR) was adminis e ed a close p oximi y 191 (abou 50 ␮m) o cells o 100 s du ing single cell measu e- 192 men s. 193 Measu emen and analysis o spon aneous calcium 194 ansien s 195 Spon aneous calcium ansien s we e moni o ed using LSM 196 510 META Lase Scanning Con ocal Mic oscope (Zeiss, 197 Obe kochen, Ge many). Cells o high-densi y mic omass 198 cell cul u es we e incuba ed o 1 h a 37 ◦C wi h 10 ␮M199 Fluo-4-AM in Ham’s F12 medium. Calcium imaging was pe - 200 o med in no mal Ty ode’s solu ion (see abo e). x—yanalysis 201 and line scan images we e aken o moni o he fluo es- 202 cence in ensi y du ing spon aneous ac i i ies. Fi y images 203 we e eco ded du ing a 48.4 s in e al o pe o m he x—y204 analysis. Line scan images we e eco ded a 1.54 ms/line, 205 512 pixels/line and 4096 lines using a 63×wa e imme sion 206 objec i e. Fluo-4-loaded cells we e exci ed wi h a 488 nm 207 a gon ion lase and emi ed fluo escence was collec ed 208 a 500—570 nm. Images we e analyzed using an au oma ic 209 e en de ec ion p og am de eloped in he Depa men o 210 Physiology. 211 De e mina ion o Ca concen a ion o he cul u e 212 medium 213 Ca concen a ion o he cul u e medium (F12 supplemen ed 214 wi h 10% FCS) was de e mined wi h a omic abso p ion 215 spec ome y (AAS) on Philips PU9200X equipmen in ai - 216 ace ylene flame. Measu emen s we e pe o med a he 217 wa eleng h o 422.7 nm; each sample was measu ed h ee 218 imes o 4 s. De e mina ion o Ca concen a ion was ca ied 219 ou wi h s anda d addi ion me hod; samples we e dilu ed in 220 0.1 M ni ic acid [23].221 T ea men s wi h EGTA, A23187 calcium ionopho e 222 and cyclospo ine A 223 In o de o examine he e ec s o emo al o Ca2+ om 224 he ex acellula fluid, cell cul u es we e ed a cul u e 225 medium con aining 0.8 mM EGTA (Am esco, Solon, OH, USA; 226 pH adjus ed o 7.4) o 12 h on day 2 o 3 o cul u ing. 227 To assess he e ec o ele a ed in acellula Ca2+, cul- 228 u es we e ed wi h a cul u e medium con aining calcium 229 ionopho e A23187 (Sigma, Budapes , Hunga y) a concen- 230 a ions o 0.1 and 5 mg/L o 1 h on cul u ing days 2 and 231 3. Calcium ionopho e A23187 was dissol ed in DMSO hen 232 dilu ed in cul u e medium. Ac i i y o calcineu in was inhib- 233 i ed wi h he con inuous applica ion o 2 ␮M cyclospo ine A 234 (Sigma, Budapes , Hunga y) s a ed on day 1. UNCORRECTED PROOF YCECA 972 1—14 Please ci e his a icle in p ess as: C. Ma a, e al., Cy osolic ee Ca2+ concen a ion exhibi s a cha ac e is ic empo al pa e n du ing in i o ca ilage di e en ia ion: A possible egula o y ole o calcineu in in Ca-signalling o chond ogenic cells, Cell Calcium (2008), doi:10.1016/j.ceca.2007.12.010 ARTICLE IN PRESS +Model YCECA 972 1—14 4 C. Ma a e al. Ligh mic oscopical analysis o ca ilage235 di e en ia ion236 Ca ilage ma ix was isualized by s aining wi h dime hyl- 237 me hylene blue (DMMB, Ald ich, Ge many) as desc ibed238 p e iously [2]. The amoun o sulpha ed ma ix compo-239 nen s was de e mined wi h a semi-quan i a i e me hod,240 by measu ing he op ical densi y o ex ac ed oluidine241 blue (Reanal, Budapes , Hunga y) bound o glycosamino-242 glycans in ma u e HDC. Six-day-old cell cul u es we e243 fixed in a solu ion con aining 28% e hanol, 4% o ma-244 lin and 2% ace ic acid, s ained wi h 0.1% oluidine blue 245 dissol ed in glycine—HCl bu e (pH 1.8) o 15 min, he 246 unbound oluidine blue was washed in glycine—HCl bu e 247 o 1 h. The dye bound o highly sulpha ed p o eogly- 248 cans and glycosaminoglycans was ex ac ed in 8% HCl249 dissol ed in absolu e e hanol. Abso bance o samples con-250 aining ex ac ed oluidine blue was measu ed a he251 wa eleng h o 625 nm on a mic opla e eade (Chameleon,252 Hidex, Tu ku, Finland). Samples om 10 cul u es o each253 expe imen al g oup we e de e mined in 5 independen 254 expe imen s. Da a we e s a is ically analyzed wi h S uden ’s255 - es .256 Measu emen o cell p oli e a ion wi h257 3H- hymidine labelling and mi ochond ial ac i i y258 wi h MTT assay 259 Fo measu emen o p oli e a ion a e o cells in HDC260 15 ␮L d ople s o cell suspension we e inocula ed in o261 wells o special, opaque 96-well mic o i e pla es (Wal-262 lac, Pe kinElme Li e and Analy ical Sciences, Shel on, CT,263 USA). Ham’s F12 medium con aining 1 ␮Ci/mL (185 GBq/mM)264 3H- hymidine (dilu ed om me hyl-3H- hymidine solu ion,265 Ame sham Biosciences, Budapes , Hunga y) was added o266 he wells o 16 h on di e en days o cul u ing. A e 267 washing wice wi h PBS (phospha e bu e ed saline), p o-268 eins we e p ecipi a ed wi h ice-cold 5% ichlo oace ic269 acid, washed wi h PBS again, and placed in an exsicca-270 o con aining phospho ous pen oxide in o de o abso b271 mois u e. P io o measu emen s, 50 ␮L scin illa ion solu-272 ion (MaxiLigh ; Hidex, Finland) was added o each well,273 and adioac i i y was coun ed by a liquid scin illa ion274 coun e (Chameleon, Hidex, Tu ku, Finland). Ten sam-275 ples o each expe imen al g oup om fi e independen 276 expe imen s we e s a is ically analyzed wi h S uden ’s -277 es . 278 Cellula me abolic ac i i y was de e mined by MTT assay,279 a means o measu ing he ac i i y o li ing cells ia mi o-280 chond ial dehyd ogenases. Cells cul u ed in wells o 96-well281 mic o i e pla es we e used and 10 ␮L MTT eagen [3- 282 (4,5-dime hyl hiazolyl-2)-2,5-diphenyl e azolium b omide;283 5 mg MTT/1 mL PBS] was added in o each well. Cells we e 284 incuba ed o 2 h a 37 ◦C in MTT-con aining Ham’s F12285 medium. Following addi ion o 100 ␮L MTT solubilizing solu-286 ion (10% T i on X-100 and 0.1 M HCl dissol ed in anhyd ous287 isop opanol) op ical densi y was measu ed a 570 nm on 288 a mic opla e eade (Chameleon, Hidex, Tu ku, Finland). 289 Ten samples o each expe imen al g oup om ou sepa- 290 a e expe imen s we e s a is ically analyzed wi h S uden ’s291 - es .292 De e mina ion o cell su i al by FACS analysis 293 One hund ed mic oli e s d ople s o cell suspension we e 294 inocula ed in o plas ic Pe i dishes. Following ea men s 295 o ei he A23187 o EGTA as gi en in he ‘‘T ea men s 296 wi h EGTA, A23187 calcium ionopho e and cyclospo ine A’’ 297 sec ion, cul u es we e washed wice wi h CMF-PBS (cal- 298 cium and magnesium ee PBS), incuba ed wi h 250 ␮L299 annexin-V DY647 eagen (Cen al Eu opean Biosys ems, 300 Budapes , Hunga y) and/o 10 ␮g/mL p opidium-iodide o 301 10 min a oom empe a u e, washed again wi h CMF-PBS, 302 and ypsinized o 15 min o ob ain cell suspension. Cells 303 we e collec ed by cen i uga ion a 800 ×g o 10 min, esus- 304 pended in 1 mL FACS bu e (PBS supplemen ed wi h 1% 305 BSA and 0.05% NaN3) and a e o cell su i al was de e - 306 mined using a CyFlow®space Flow Cy ome e (Pa ec GmbH, 307 M¨ uns e , Ge many). Annexin-V DY647 was moni o ed a 308 670 nm, p opidium-iodide was measu ed a 620 nm. Mea- 309 su emen lowe h eshold we e se on cell-size pa icles. 310 Analysis was pe o med wi h WinMDI 2.8 So wa e. 311 RT-PCR analysis 312 To al RNA was isola ed om cells using RNA Isola ion Ki 313 acco ding o he manu ac u e ’s ins uc ion (Gen a Sys- 314 ems Inc., Minneapolis, MN, USA). The assay mix u e o 315 e e se ansc ip ase eac ion con ained 2 ␮g RNA, 0.112 ␮M316 oligo(dT), 0.5 mM dNTP, 200 uni s M-MLV RT in 1 ×RT bu e . 317 The sequences o p ime pai s used o PCR eac ions we e 318 as ollows: o chicken agg ecan 5-CAA TGC AGA GTA CAG 319 AGA-3and 5-TCT GTC TCA CGG ACA CCG-3, o chicken 320 Sox9 5-CCC CAA CGC CAT CTT CAA-3and 5-CTG CTG ATG 321 CCG TAG GTA-3, o chicken calcineu in 5-CTG CTC TGA 322 TGA ACC AAC AGT T-3and 5-ACG GCA AGG ACC AGG TAA 323 ACA-3, o chicken GAPDH 5-GAG AAC GGG AAA CTT GTC 324 AT-3and 5-GGC AGG TCA GGT CAA CAA-3, o chicken 325 inosi ol-1,4,5- isphospha e ecep o (IP3R) ype-1 5-CGG 326 CTG TGG TCT GAG ATA C-3and 5-GGT AAT AGG GAA GAT 327 GGT AGT G-3, o chicken IP3R ype-2 5-AAG CCT ACC 328 TTA TGA CCT CC-3and 5-CAT TGT TTC CTC CAT CCT G- 329 3, and o chicken IP3R ype-3 5-TGT GGG TGG ACA AGA 330 AAG G-3and 5-GCA GGA ACT GAT GGG TGA A-3. Ampli- 331 fica ions we e pe o med in a he mocycle (PCR Exp ess 332 Tempe a u e Cycling Sys em, Hybaid, UK) as ollows: 94 ◦C, 333 1 min, ollowed by 30 cycles (94 ◦C, 30 s, 54 ◦C, 30 s, 72 ◦C, 334 30 s) and hen 72 ◦C, 5 min. Thi y-fi e cycles we e used 335 a 57 ◦C o IP3- ecep o s. PCR p oduc s we e analyzed by 336 elec opho esis in 1.2% aga ose gel con aining e hidium b o- 337 mide. 338 Wes e n-blo analysis 339 To al cell lysa es o endoplasmic e iculum (ER) ac ion o 340 HDC we e examined by Wes e n blo . Cell cul u es we e 341 ha es ed immedia ely a e ea men s on espec i e days 342 o cul u ing. Cell pelle s we e suspended in 100 ␮Lo 343 homogeniza ion bu e [con aining 50 mM T is—HCl bu e 344 (pH 7.0), 0.5 mM di hio h ei ol, 10 ␮g/mL Go dox, 10 ␮g/mL 345 leupep in, 1 mM phenylme hylsulphonyl (PMSF), 5 mM ben- 346 zamidine and 10 ␮g/mL ypsin inhibi o ]. A e s o ing hem 347 a −70 ◦C, suspensions we e sonica ed by pulsing bu s o 348 UNCORRECTED PROOF YCECA 972 1—14 Please ci e his a icle in p ess as: C. Ma a, e al., Cy osolic ee Ca2+ concen a ion exhibi s a cha ac e is ic empo al pa e n du ing in i o ca ilage di e en ia ion: A possible egula o y ole o calcineu in in Ca-signalling o chond ogenic cells, Cell Calcium (2008), doi:10.1016/j.ceca.2007.12.010 ARTICLE IN PRESS +Model YCECA 972 1—14 Possible egula o y ole o calcineu in in Ca-signalling o chond ogenic cells 5 ou imes 30 s by 50 cycles (B anson Sonifie , Danbu y,349 USA). ER ac ion o HDC was p epa ed om 3-day-old cul-350 u es g own in Pe i dishes. Cells we e homogenized using351 a Dounce homogenise in a bu e con aining 5 mM HEPES,352 320 mM suc ose and p o ease inhibi o s [3.4 ␮g/mL Go dox,353 3.4 ␮g/mL leupep in, 1 mM phenylme hylsulphonyl (PMSF),354 1.6 mM benzamidine and 3.4 ␮g/mL ypsin inhibi o , pH355 7.4]. A e cen i uga ion a 4500 ×g o 20 min, supe -356 na an was collec ed and cen i uged a 10,000 ×g o 357 15 min. Supe na an was cen i uged a 150,000 ×g o 358 120 min. Pelle con aining endoplasmic e iculum esicles359 (mic osome ac ion) was collec ed in lysis bu e (50 mM360 T is—HCl, pH 7.2 con aining p o ease inhibi o s, see abo e),361 snap- ozen in liquid ni ogen and s o ed a −70 ◦C. Sam-362 ples o SDS-PAGE we e p epa ed by he addi ion o 1/5363 olume o fi e old concen a ed elec opho esis sample364 bu e (310 mM T is—HCl, pH 6.8; 10% SDS, 50% glyc- 365 e ol, 100 mM DTT, 0.01% b omophenol blue) o cell lysa es366 and boiled o 10 min. Abou 40 ␮g o p o ein was sepa-367 a ed by 7.5% SDS-PAGE gel o immunological de ec ion368 o Sox9, phospho-Sox9, RyR, IP3- ecep o and calcineu in.369 P o eins we e ans e ed elec opho e ically o ni ocel-370 lulose memb ane. A e blocking in 5% non- a d y milk371 in PBS, memb anes we e incuba ed wi h he ollowing 372 p ima y an ibodies o e nigh a 4 ◦C: polyclonal an i-Sox9373 an ibody (Abcam L d., Camb idge, UK) in 1:200 dilu ion, 374 polyclonal an i-phospho-Sox9 an ibody (Sigma, Budapes , 375 Hunga y) in 1:200 dilu ion, monoclonal an i-RyR an ibody376 (A fini y BioReagen s, Golden, CO, USA) in 1:1000 dilu ion,377 polyclonal an i-IP3R ype 1 an ibody (Sigma, Budapes , Hun-378 ga y) in 1:250 dilu ion and polyclonal an i-calcineu in (␣379 subuni ) an ibody (Ups a e, Dundee, Sco land, UK) in 1:200380 dilu ion. A e washing h ee imes o 10 min wi h PBST (PBS 381 supplemen ed wi h 0.1% Tween 20), memb anes we e incu- 382 ba ed wi h a seconda y an ibody, an i-mouse IgG (Sigma,383 Budapes , Hunga y) in 1:1000 dilu ion o RyR and an i-384 abbi IgG (Sigma, Budapes , Hunga y) in 1:1000 dilu ion 385 o de ec ion o IP3 ecep o , Sox9, phospho-Sox9 and cal-386 cineu in in PBS con aining 5% non- a d y milk o 1 h. Signals 387 we e de ec ed by enhanced chemiluminescence (Ame sham388 Biosciences, Budapes , Hunga y).389 Enzyme ac i i y assay o calcineu in390 Ac i i y o calcineu in was measu ed by he elease391 o 32Pi om 32P-labelled p o ein phospha ase inhibi o - 392 1 (780 cpm/pmol) as desc ibed by Yang e al. [24] 393 wi h some modifica ions [25]. Thi y mic oli e s o he 394 assay mix u e (50 mM T is—HCl bu e pH 7.0) con ain-395 ing 0.16 mM di hio h ei ol, 3.4 ␮g/mL Go dox, 3.4 ␮g/mL396 leupep in, 1 mM phenylme hylsulphonyl (PMSF), 1.6 mM ben-397 zamidine, 3.4 ␮g/mL ypsin inhibi o , 40 ␮g/mL calmodulin, 398 0.2 mM CaCl2, 100 nM okadaic acid (OA), 2 nM p o ein399 phospha ase inhibi o -2, ex ac con aining abou 80 ␮g400 p o ein and 32P-labelled p o ein phospha ase inhibi o -1401 (20—30,000 cpm/ eac ion mix u e) was incuba ed a 30 ◦C 402 o 20 min. The eac ion was e mina ed by he addi ion o 403 100 ␮L o 20% ichlo oace ic acid and 100 ␮L o 6 mg/mL 404 bo ine se um albumin. A e cen i uga ion, 32Pi-con en o 405 180 ␮L o he supe na an ac ion was de e mined in a liq-406 uid scin illa ion coun e .407 Resul s 408 Cy osolic ee Ca2+ concen a ion o un ea ed cell 409 cul u es shows a cha ac e is ic age-dependen 410 pa e n 411 Cy osolic ee Ca2+ concen a ion was de e mined in Fu a- 412 2-loaded cells on di e en days o cul u ing. Basal le el o 413 in acellula Ca2+ concen a ion o chond oblas s was ound 414 o ha e an age-dependen pa e n (Fig. 1A). Ini ially, Ca2+ 415 le el is low, wi h a s a ing concen a ion o abou 75 nM on 416 day 0, hen i sligh ly inc eases in pa allel wi h he p og es- 417 sion o di e en ia ion. A 140 nM peak o he cy osolic ee 418 Ca2+ concen a ion was obse ed on day 3 o cul u ing in 419 cells o un ea ed con ol cul u es. I should be no ed ha 420 chond ogenic cells o HDC di e en ia e in o chond oblas s 421 on his day o cul u ing [3]. F om day 4, Ca2+ le el d ops, 422 howe e , i e ains a sligh ly ele a ed concen a ion (abou 423 100 nM) as compa ed o days 0—2. 424 Di e en ia ing chicken chond op ogeni o cells exhibi 425 pe iodical inc eases in cy osolic ee Ca2+ (Fig. 1B—D). These 426 oscilla ions we e de ec able mos ly on cul u ing day 3. F e- 427 quencies o oscilla ions we e simila in all he cells obse ed: 428 he pe iod was 4 ±1.2 s (mean ±s anda d e o o he mean; 429 n= 20), maximum ampli udes we e 15—20% highe han he 430 mean basal fluo escence in ensi y. 431 Changes o cy osolic Ca2+ concen a ion o HDC 432 ha dly depend on in acellula sou ces 433 We aimed o de e mine whe he he ex acellula Ca2+ o 434 he in acellula Ca-s o es could be he sou ce o ele a ed 435 cy osolic Ca2+ le els. The Ca concen a ion o F12 medium 436 con aining 10% FCS p o ed o be app oxima ely 0.78 mM 437 acco ding o AAS measu emen s (da a no shown). In o de 438 o educe he concen a ion o ee Ca2+ in he cul u e 439 medium, EGTA was applied in equimola (0.8 mM) concen a- 440 ion. EGTA ea men significan ly dec eased he cy osolic 441 Ca2+ le el o app oxima ely 60% o ha o un ea ed con ol 442 cells (Fig. 2A). Twel e hou s o EGTA ea men p o ed o be 443 e ec i e; when main ained in 0.8 mM EGTA h oughou he 444 cul u ing pe iod, cul u es de ached om he glass o plas- 445 ic su ace and died. To in es iga e he ole o in acellula 446 Ca-s o es, cyclopiazonic acid (CPA), an inhibi o o he Ca2+ 447 pump o smoo h endoplasmic e iculum was adminis e ed a 448 a concen a ion o 10 ␮M o 8 min. To emo e ee Ca2+ om 449 he medium, cells we e washed wi h Ca2+- ee Ty ode’s p io 450 o adminis a ion o CPA. A e addi ion o Ca2+- ee Ty ode’s 451 he basal cy osolic Ca2+ le el dec eased om 140 o 120 nM 452 showing he dependence o his pa ame e on he ex a- 453 cellula Ca2+ concen a ion. App oxima ely 60 s a e he 454 adminis a ion o 10 ␮M CPA, cy osolic Ca2+ le el s a ed o 455 inc ease e y slowly (Fig. 2B). The sligh ele a ion in cy oso- 456 lic Ca2+ clea ly shows ha he in acellula Ca2+-s o es a e 457 no emp y, howe e , he amoun o s o ed Ca2+ is ei he low 458 o he a e o leak is small. When adminis a ion o bo h CPA 459 and Ca- ee Ty ode’s ceased and we e washed ou wi h no - 460 mal Ca2+ con aining solu ion, a well-defined peak in cy osolic 461 Ca2+ le el could be obse ed owing o he en y o ex acel- 462 lula Ca2+ in o he cy osol, eflec ing on a possible ac i a ion 463 UNCORRECTED PROOF YCECA 972 1—14 Please ci e his a icle in p ess as: C. Ma a, e al., Cy osolic ee Ca2+ concen a ion exhibi s a cha ac e is ic empo al pa e n du ing in i o ca ilage di e en ia ion: A possible egula o y ole o calcineu in in Ca-signalling o chond ogenic cells, Cell Calcium (2008), doi:10.1016/j.ceca.2007.12.010 ARTICLE IN PRESS +Model YCECA 972 1—14 6 C. Ma a e al. Figu e 1 Day-by-day a ia ion o basal in acellula Ca2+ le els in chond i ying cells o un ea ed con ol HDC (A). Ca2+ concen- a ions we e de e mined in Fu a-2-loaded cells as desc ibed in he ‘‘Ma e ials and me hods’’ sec ion. Rep esen a i e da a o fi e independen expe imen s showing mean alues o basal in acellula Ca2+ le els o 30 cells ±s anda d e o o he mean. S a is ical analysis by S uden ’s - es compa ing he espec i e da a o he p e ious cul u ing day, *P< 0.01. Spon aneous calcium e en s in cells o chond i ying high-densi y mic omass cell cul u es on cul u ing day 3 (B—D). Calcium ansien s we e de e mined in Fluo-4-AM loaded cells as gi en in he ‘‘Ma e ials and me hods’’ sec ion. Changes o fluo escence in ensi y we e eco ded in a 50 s in e al. x—yanalysis o a ep esen a i e cell is shown in panel B, ime cou se o mean in ensi y is shown in panel C. A ep esen a i e calcium e en measu emen pe o med by line scan imaging is shown in panel D. UNCORRECTED PROOF YCECA 972 1—14 Please ci e his a icle in p ess as: C. Ma a, e al., Cy osolic ee Ca2+ concen a ion exhibi s a cha ac e is ic empo al pa e n du ing in i o ca ilage di e en ia ion: A possible egula o y ole o calcineu in in Ca-signalling o chond ogenic cells, Cell Calcium (2008), doi:10.1016/j.ceca.2007.12.010 ARTICLE IN PRESS +Model YCECA 972 1—14 Possible egula o y ole o calcineu in in Ca-signalling o chond ogenic cells 7 Figu e 2 Basal in acellula Ca2+ le els o 0.8 mM EGTA- ea ed cul u es on days 2 and 3 (A). Measu emen s we e ca ied ou ollowing 12 h o EGTA ea men s. Da a ep esen mean ±s anda d e o o he mean o in acellula Ca2+ le els o 30 cells measu ed in 5 ep esen a i e expe imen s. E ec o cyclopiazonic acid (CPA) on he elease o Ca2+ om in acellula s o es (B). Rep esen a i e eco d ou o 30 cells in 5 independen expe imen s. As e isks indica e significan (*P< 0.01) dec ease in basal Ca2+ concen a ion as compa ed o he espec i e con ol. o s o e-ope a ed Ca2+ en y (SOCE) channels [26]. The exac 464 na u e o his phenomenon, howe e , emains o be u he 465 in es iga ed.466 To in es iga e he possible ole o in e nal Ca-s o es in467 he egula ion o Ca-homeos asis o di e en ia ing chon-468 d op ogeni o cells, u he expe imen s we e pe o med.469 Ten mic omola CPA was adminis e ed o he cul u e medium 470 o HDC on day 2 o 3 o 12 h. This p olonged inhibi ion o 471 he Ca-pump o smoo h endoplasmic e iculum mus ha e472 esul ed in a comple e abolishmen o in acellula s o es, 473 howe e , no de imen al e ec on chond ogenesis could be474 obse ed (Fig. 3A). CPA- ea men sligh ly ele a ed cy oso-475 lic Ca2+ le el (Fig. 3B), which could be ela ed o he unc ion476 o SOCE channels. Combined ea men s wi h EGTA and CPA477 o 12 h (i.e., inhibi ion o Ca2+ en y om bo h ex acellu-478 la and in acellula sou ces) esul ed in a comple e loss o 479 me ach oma ically s ained ca ilage ma ix demons a ing 480 he Ca2+ dependence o in i o chond ocy e di e en ia-481 ion (da a no shown). This phenomenon may implica e he482 insu ficien capaci y o in acellula Ca-s o es o eplenish483 he unc ion o Ca2+ en y pa hways in chond ogenic cells.484 The impo ance o Ca2+-influx om ex acellula space485 is u he suppo ed by in es iga ing he endoplasmic486 e iculum yanodine ecep o (RyR) and he inosi ol-1,4,5-487 isphospha e (IP3) ecep o s. RyR was no de ec able by488 Wes e n blo analyses pe o med on o al cell lysa es o HDC 489 and only a weak band was obse ed in samples con aining 490 sepa a ed endoplasmic e iculum ac ion o HDC (Fig. 4A). 491 Fu he mo e, no esponse was de ec ed when ca eine, an 492 agonis o RyR was adminis e ed du ing single cell measu e- 493 men s (Fig. 4B). These esul s indica e ha hough p esen , 494 he low amoun o RyR loca ed in he endoplasmic e icu- 495 lum o cells o HDC may no be unc ioning and p obably 496 does no significan ly con ibu e o he ele a ion o basal 497 cy osolic Ca2+. Amplifica ion o IP3 ecep o iso o ms by RT- 498 PCR shows ha only he mRNA o IP3R ype 1 is exp essed 499 by cells o HDC (Fig. 4C). The IP3 ecep o p o ein could be 500 ha dly de ec ed in samples p epa ed om he endoplasmic 501 e iculum ac ion o chond ogenic cells by Wes e n blo ing 502 (Fig. 4D). 503 Dec eased ex acellula Ca2+ concen a ion inhibi s 504 ca ilage o ma ion and educes he exp ession o 505 chond ogenic mas e ansc ip ion ac o Sox9 506 Cul u es ea ed wi h EGTA o 12 h on day 2 o 3 o cul u - 507 ing showed a p o ound dec ease in me ach oma ic s aining 508 pe o med on day 6 o cul u ing (9% o 17% o un ea ed 509 con ol cul u es, espec i ely, Fig. 5A), demons a ing a sig- 510 Figu e 3 E ec o 10 ␮M CPA on ca ilage o ma ion in chond i ying mic omass cul u es (A). Me ach oma ic ca ilage a eas in 6-day-old high-densi y colonies isualized wi h DMMB dissol ed in 3% ace ic acid. Op ical densi y (OD625) o samples con aining oluidine blue ex ac ed wi h 8% HCl dissol ed in absolu e e hanol. Da a a e mean alues ±s anda d e o o he mean (±7%) o each expe imen al g oup ou o 10 measu emen s. Basal in acellula Ca2+ le els o 10 ␮M CPA- ea ed cul u es on days 2 and 3 (B). Measu emen s we e ca ied ou di ec ly a e he 12-h ea men wi h CPA. Da a ep esen mean ±s anda d e o o he mean o in acellula Ca2+ le els o 30 cells measu ed in 5 independen expe imen s. UNCORRECTED PROOF YCECA 972 1—14 Please ci e his a icle in p ess as: C. Ma a, e al., Cy osolic ee Ca2+ concen a ion exhibi s a cha ac e is ic empo al pa e n du ing in i o ca ilage di e en ia ion: A possible egula o y ole o calcineu in in Ca-signalling o chond ogenic cells, Cell Calcium (2008), doi:10.1016/j.ceca.2007.12.010 ARTICLE IN PRESS +Model YCECA 972 1—14 8 C. Ma a e al. Figu e 4 De ec ion o he exp ession and unc ion o RyR and IP3R in HDC. Wes e n blo analysis o RyR in ER ac ion o cells o HDC on cul u ing day 3 (A). Rep esen a i e da a o fi e independen expe imen s. E ec o he RyR-agonis ca eine (15 mM) on basal cy osolic Ca2+ le el o cells o HDC on cul u ing day 3 (B). Rep esen a i e eco d ou o 30 cells in 5 independen expe imen s. Amplifica ion o chicken IP3R ype 1 ecep o (C). Rep esen a i e esul ou o h ee independen expe imen s. Wes e n blo analysis o IP3R in ER ac ion o cells in HDC (D). Rep esen a i e analysis ou o h ee independen expe imen s. nifican educ ion o ca ilage o ma ion. Adminis a ion o 511 EGTA a e day 5 did no ha e any significan e ec on ma ix512 p oduc ion (da a no shown).513 Inhibi ion o chond ogenesis was moni o ed by de ec ion514 o mRNAs o agg ecan co e p o ein and Sox9, he majo 515 ca ilage-specific ansc ip ion ac o . A significan dec ease516 in he mRNA le els o bo h agg ecan co e p o ein and Sox9 517 was obse ed unde he e ec o EGTA on each day o ea - 518 men s, demons a ing ha educed in acellula Ca2+ le el519 dec eases ca ilage o ma ion, a leas pa ly, ia inhibi ion 520 o ca ilage di e en ia ion (Fig. 5B). These findings we e 521 u he suppo ed by Wes e n blo analyses showing ha 522 ea men wi h EGTA sligh ly educed he p o ein le el o 523 Sox9 on day 2, and a significan dec ease was obse ed in524 he phospho yla ion le el o Sox9 on day 3 (Fig. 5C). 525 Ca2+ ionopho e has dual concen a ion-dependen 526 e ec s on ca ilage o ma ion 527 Abou 0.1 mg/L concen a ion o he Ca2+ ionopho e A23187 528 aised he in acellula Ca2+ le els o app oxima ely 125% o 529 un ea ed con ol cells, and 5 mg/L concen a ion o Ca2+ 530 ionopho e esul ed in an e en highe inc ease (abou 150%) 531 in cy osolic Ca2+ le els (Fig. 6A). On he o he hand, he wo 532 concen a ions o ionopho e applied had opposi e e ec s 533 on ca ilage o ma ion. Following ea men wi h he lowe 534 concen a ion (0.1 mg/L) o A23187 on bo h days 2 and 3 o 535 one hou , an ex ensi e ca ilage o ma ion occu ed by day 536 6; when ea men was pe o med on day 3 only, he amoun 537 o me ach oma ically s ained ca ilage ma ix inc eased o 538 153% o con ol cul u es (Fig. 6B). On he con a y, highe 539 Figu e 5 E ec o 0.8 mM EGTA on ca ilage de elopmen o chond i ying mic omass cul u es (A). Me ach oma ic ca ilage a eas in 6-day-old high-densi y colonies isualized wi h DMMB dissol ed in 3% ace ic acid. Op ical densi y (OD625) o samples con aining oluidine blue ex ac ed wi h 8% HCl dissol ed in absolu e e hanol. Da a a e mean alues ±s anda d e o o he mean (±5%) o each expe imen al g oup ou o 10 measu emen s. E ec o EGTA on he exp ession o agg ecan and he exp ession and phospho yla ion o Sox9 ansc ip ion ac o in HDC o a ious ages (B and C). Fo RT-PCR eac ions GAPDH was used as a con ol. Rep esen a i e da a o fi e independen expe imen s. As e isks indica e significan (*P< 0.01) dec ease in op ical densi y o ex ac ed oluidine blue as compa ed o he espec i e con ol. UNCORRECTED PROOF YCECA 972 1—14 Please ci e his a icle in p ess as: C. Ma a, e al., Cy osolic ee Ca2+ concen a ion exhibi s a cha ac e is ic empo al pa e n du ing in i o ca ilage di e en ia ion: A possible egula o y ole o calcineu in in Ca-signalling o chond ogenic cells, Cell Calcium (2008), doi:10.1016/j.ceca.2007.12.010 ARTICLE IN PRESS +Model YCECA 972 1—14 Possible egula o y ole o calcineu in in Ca-signalling o chond ogenic cells 9 Figu e 6 E ec s o he Ca2+ ionopho e A23187 on ca ilage de elopmen o chond i ying mic omass cul u es. Basal in acellula Ca2+ le els o 0.1 o 5 mg/L A23187- ea ed cul u es on days 2 and 3 (A). Measu emen s we e ca ied ou di ec ly a e he 30-min ea men wi h A23187. Da a ep esen mean ±s anda d e o o he mean o in acellula Ca2+ le els o 30 cells measu ed in 5 independen expe imen s. Me ach oma ic ca ilage a eas in 6-day-old high-densi y colonies isualized wi h DMMB dissol ed in 3% ace ic acid (B). Op ical densi y (OD625) o samples con aining oluidine blue ex ac ed wi h 8% HCl dissol ed in absolu e e hanol. Da a shown a e mean alues ±s anda d e o o he mean (±6%) o each expe imen al g oup ou o 10 measu emen s. E ec o A23187 on he mRNA exp ession o agg ecan and Sox9 ansc ip ion ac o (C) and p o ein exp ession and phospho yla ion o Sox9 (D) in HDC o a ious ages. Fo RT-PCR eac ions GAPDH was used as a con ol. Rep esen a i e da a o fi e independen expe imen s. As e isks indica e significan (*P< 0.01) inc ease in basal Ca2+ concen a ions o significan (*P< 0.01) change in op ical densi y o ex ac ed oluidine blue as compa ed o he espec i e con ol. concen a ion (5 mg/L) o A23187 caused a ma ked inhibi- 540 ion o ca ilage o ma ion, especially when adminis e ed541 on day 2, educing he amoun o me ach oma ically s ained542 ca ilage ma ix o app oxima ely 36% o un ea ed con ol543 cul u es (Fig. 6B). Since mRNA le els o ca ilage di e en-544 ia ion ma ke s Sox9 and agg ecan co e p o ein, and bo h 545 p o ein le el and phospho yla ion o Sox9 showed a sligh 546 inc ease a he han inhibi ion unde he e ec o 5 mg/L547 ionopho e (Fig. 6C and D), he mechanism o he dec ease o 548 ca ilage o ma ion should be u he in es iga ed. Admin-549 is a ion o A23187 a e day 5 did no ha e any significan 550 e ec on ma ix p oduc ion (da a no shown). 551 E ec s o EGTA, A23187 Ca2+ ionopho e and CPA552 on a e o p oli e a ion, mi ochond ial ac i i y and 553 cellula iabili y o cells o HDC554 Cell p oli e a ion, mi ochond ial ac i i y and cellula iabil-555 i y assays we e pe o med ollowing ea men s wi h EGTA,556 he Ca2+ ionopho e A23187 and CPA, since dec eased ca -557 ilage ma ix p oduc ion could be accoun ed o ei he by 558 he inhibi ion o he di e en ia ion o chond ogenic mes-559 enchymal cells o chond oblas s o by he dec ease in cell560 numbe s. Al hough he p oli e a ion a e o cells in mic o- 561 mass cul u es was significan ly educed unde he e ec o 562 0.8 mM EGTA (Fig. 7A), he mi ochond ial ac i i y o cells 563 was no a ec ed (Fig. 7B) as e ealed by 3H- hymidine 564 inco po a ion and MTT assay, espec i ely. Cell p oli e a- 565 ion was sligh ly s imula ed by low concen a ion o he Ca2+ 566 ionopho e A23187 on day 3 (Fig. 7A). High concen a ion 567 (5 mg/L) o A23187 educed he a e o cell p oli e a ion 568 on bo h days, bu i did no cause any significan dec ease 569 in mi ochond ial ac i i y (Fig. 7B). T ea men wi h CPA did 570 no influence he p oli e a ion a e o cells (Fig. 7A) and 571 i did no p o e o be cy o oxic acco ding o MTT assays 572 (Fig. 7B). 573 Cellula iabili y ollowing ea men s wi h EGTA o he 574 Ca2+ ionopho e A23187 was also analyzed by FACS (Fig. 7C575 and D). The ionopho e did no influence he dis ibu ion o 576 cells compa ed o he con ol. The pe cen age inc ease o 577 dead cells we e as ollows: 3.2% and 0.7% (in he p esence o 578 0.1 mg/L) and 1.8% and 3.5% (in he p esence o 5 mg/L) on 579 days 2 and 3, espec i ely. T ea men wi h 0.8 mM EGTA led 580 o a highe a e o cell dea h, hus he pe cen age inc ease 581 o dead cells p o ed o be 10% and 9.1% on days 2 and 3, 582 espec i ely. Apop osis was no de ec ed in he ionopho e- 583 ea ed HDC (da a no shown).