TSH is a novel neuroendocrine regulator of selected keratins in the human hair follicle

1 Letter to the Editor TSH is a novel neuroendocrine regulator of selected keratins in the human hair follicle Yuval Ramota,b,*, Guoyou Zhanga,c, Tamás Bíród, Erika Lisztesd, Wolfang Funke, Arieh Ingberb, Lutz Langbeinf and Ralf Pausa,g aDepartment of Dermatology, University of Lübeck, D-23538 Lübeck, Germany bDepartment of Dermatology, Hadassah - Hebrew University Medical Center, Jerusalem, Israel cDepartment of Hand and Plastic Surgery, the Second Affiliated Hospital of Wenzhou Medical College, Wenzhou, Zhejiang Province, China dDepartment of Physiology, University of Debrecen, Medical and Health Science Center, Research Center for Molecular Medicine, Debrecen, Hungary eKlinik Dr. Koslowski, Munich, Germany fDivision of Skin Carcinogenesis, Deutsches Krebsforschungszentrum, Heidelberg, Germany gSchool of Translational Medicine, University of Manchester, Manchester, UK Word count: 1099; Number of references: 14; Number of tables: 1; Number of figures: 2 This study was supported in part by a Minerva Fellowship to YR, and by grants from Manchester NIHSR Biomedical Research Center to RP and from the Wilhelm Sander Stiftung to LL (2007.133.1.). Conflict of interest: The authors have no conflict of interest to declare *Corrspending author: Department of Dermatology, Hadassah - Hebrew University Medical Center, Jerusalem, Israel, Tel.: +972 (0)2 677-7111, Fax: +972 (0)2 677-7299, e-mail y[email protected] 2 Keratins and keratin-associated proteins (KAPs) constitute the major structural protein components of the hair. Regulation of their expression is critical for proper hair follicle (HF) structure and function [1]. Therefore, it is important to fully elucidate the controls that regulate keratin expression. Although it is accepted that the expression of selected keratin genes underlies endocrine controls [1,2], our understanding of the complex regulation of keratin expression remains rather fragmentary. To better elucidate this regulation, human skin and HF organ culture offer an instructive, physiologically relevant research tool [1,3]. In particular, very little is known on the neuroendocrine controls of keratin transcription. The importance of the latter is highlighted by the recent discovery that the "pituitary" neuropeptide hormone, prolactin, which is also expressed by human HFs, potently regulates the expression of selected human keratins on the gene and protein level [3]. Moreover, microarray analyses had provided first clues that thyroid stimulating hormone (TSH) and its proximal regulator in the hypothalamic-pituitary-thyroid axis, thyrotropin-releasing hormone (TRH), might operate as previously unsuspected modulators of human hair keratin and KAP gene transcription in situ [4-6]. Also, we had recently found that TSH upregulates keratin K5 gene expression and protein synthesis as well as K14 transcription in human epidermis [4]. Therefore, we have asked whether TSH operates as a novel neuroendocrine regulator of human keratins in situ, using microdissected, organ-cultured human scalp HFs as a physiologically and clinically relevant assay systems [3-5]. This was complemented by studying the effect of TSH on keratin expression in cultured human outer root sheath (ORS) keratinocytes (KCs). Anagen VI HFs were isolated from normal frontotemporal scalp skin obtained after written informed consent from three healthy females undergoing routine face-lift surgery, as 3 previously described [4,5], adhering to Helsinki guidelines and under a licence from the ethics committee of the University of Lübeck. HF mRNA extracts from one female patient were subjected to quantitative real time PCR (qPCR) for selected hair keratin genes after 24 hrs treatment with TSH (100 mU ml-1) or vehicle. For immunohistochemical analysis, isolated HFs from additional two female patients were organ cultured for 6 days as described previously [3,4], and expression of keratins K6, K14, K17, K31, K32, K85 and MSX-2 was studied with our previously published basic immunohistology protocols [3,4] (Table 1). For epithelial keratin qPCR experiments, human ORS KCs were obtained from an additional female patient, as previously described [3]. These were treated for 24h with TSH (100 mU ml1) or vehicle. Our previous microarray results had suggested that TSH administration significantly downregulates expression of K31 [4], thus suggesting a role for TSH in the regulation of hair keratins. To further explore this microarray lead, we performed qPCR on carefully selected hair keratins that are expressed in the hair shaft-generating HF epithelium, representing three major compartments of the HF epithelium: the hair matrix/precortex and cuticle (K35), the hair cortex (K31) and the hair cuticle (K32) [2]. Particular emphasis was placed on studying KRT85 and KRT35 transcription, since both keratins are the earliest ones to be expressed in the precortical hair matrix and early cuticle [2]. These qPCR analyses demonstrated that TSH downregulated transcription of KRT31 and KRT32 genes (Fig. 1A). In these qPCR analyses, KRT35 transcription was largely unaffected by TSH. Instead, transcription of its type II counterpart keratin gene, KRT85 [2], was downregulated (Fig. 1A). 4 Immunohistochemical studies on TSH treated HFs confirmed the downregulation of the hair keratins also at the protein level (Fig. 2A-C): K31 immunoreactivity, localized to the hair cortex (Fig. 2A), K85 immunoreactivity, which is expressed in the hair cortex and in the precortical hair matrix (Fig. 2B) and K32 immunoreactivity, which serves as a marker for the hair cuticle (Fig. 2C), were significantly downregulated following TSH administration. Since MSX2 serves as a major transcription factor that regulates hair keratin expression [7], analysis of MSX2 expression was also performed. Indeed, TSH downregulated MSX2 transcription in human anagen HFs in situ (Fig. 1A). MSX2 immunoreactivity was found to correspond to the expression pattern in the mouse, the hair matrix and cortex (Fig. 2D) [8]. In line with the qPCR results, TSH downregulated MSX2 immunoreactivity (Fig. 2D). Several epithelial keratins are widely expressed in the HF ORS, where they are thought to play an important role in maintaining structural integrity and homeostasis [1]. We have previously confirmed that TSH has a regulatory effect on K5 [4], which is prominently expressed in the ORS [1]. We have therefore further explored this finding, by studying the effects of TSH on selected epithelial keratins which are constitutively expressed in the ORS [2]. HF treatment with TSH significantly downregulated K6, K14 and K17 immunoreactivity (Fig. 2E-G). In order to confirm these findings in isolated HF KCs in situ, TSH treated ORS KCs were analyzed by qPCR. This showed a significant downregulation of KRT6 and KRT17 transcription, but no effect on KRT14 transcription (Fig. 1B). Exploiting the human HF as a discovery tool for keratin research, the current pilot study identifies TSH as a novel neuroendocrine regulator of keratin expression in human skin. The data provided here also suggest that the hair shaft abnormalities seen in patients with hyperor hypothyroidism [9] may result not only from perturbations in the thyroid hormone blood 5 levels, but also from associated neuroendocrine changes in TSH levels in the blood, or even in the skin [6,10]. The regulation of hair keratin gene expression is tightly controlled by a complex mechanism involving several upstream mediators. These include members of the Wnt/β-catenin pathway and of the TGF-β family which control MSX2, FOXN1 and HOXC13 activity [1,2,7,11,12]. Since MSX2 is a major regulator of hair shaft differentiation, our observation that TSH reduces MSX2 expression on the gene and protein level encourages one to pursue the hypothesis that TSH may act at least in part via inhibiting MSX2 expression. While KRT35 transcription was not inhibited by TSH, TSH downregulated KRT85 expression. Such a unilateral keratin regulation has been documented before for the cuticle keratin K82 [13]. It is conceivable that the TSH-induced upregulation of KAPs transcription previously observed by microarray analysis [4] may compensate for the downregulation of one type of keratin, as an attempt to maintain structural stability of the hair shaft. In summary, our pilot study reveals that the expression of selected HF keratins underlies novel neuroendocrine controls, possibly as part of the unfolding hypothalamus-pituitarythyroid axis equivalent present in human skin [4-6,10]. As shown for prolactin [3] and TSH, human HF organ culture offers an excellent research tool to further dissect ”novel” neuroendocrine in situ-controls that drive keratin expression in the human system under clinically relevant conditions on the gene and protein level. 6 Acknowledgements The excellent technical assistance of A. Becker and G. Scheel with HF organ culture is gratefully appreciated. We thank E. Gáspár for her critical review of the manuscript. This study was supported in part by a Minerva Fellowship to YR, and by grants from Manchester NIHSR Biomedical Research Center to RP and from the Wilhelm Sander Stiftung to LL (2007.133.1.). References 1. Ramot Y, Paus R, Tiede S, et al. Endocrine controls of keratin expression. Bioessays 2009;31:389-99. 2. Langbein L, Schweizer J. Keratins of the human hair follicle. Int Rev Cytol 2005;243:1-78. 3. Ramot Y, Bíró T, Tiede S, et al. Prolactin - a novel neuroendocrine regulator of human keratin expression in situ. FASEB J 2010;24:1768-79. 4. Bodó E, Kromminga A, Biró T, et al. Human female hair follicles are a direct, nonclassical target for thyroid-stimulating hormone. J Invest Dermatol 2009;129:1126-1139. 5. Gáspár E, Hardenbicker C, Bodó E, et al. Thyrotropin releasing hormone (TRH): a new player in human hair-growth control. FASEB J 2010;24:393-403. 6. Bodó E, Kany B, Gáspár E, et al. Thyroid stimulating hormone (TSH), a novel, locally produced modulator of human epidermal functions, is regulated by thyrotropin releasing hormone and thyroid hormones. Endocrinology 2010;151:1633-42. 7. Cai J, Lee J, Kopan R, et al. Genetic interplays between Msx2 and Foxn1 are required for Notch1 expression and hair shaft differentiation. Dev Biol 2009;326:420-30. 8. Ma L, Liu J, Wu T, et al. ‘Cyclic alopecia’ in Msx2 mutants: defects in hair cycling and hair shaft differentiation. Development 2003;130:379-89 7 9. van Beek N, Bodó E, Kromminga A, et al. Thyroid hormones directly alter human hair follicle functions: anagen prolongation and stimulation of both hair matrix keratinocyte proliferation and hair pigmentation. J Clin Endocrinol Metab 2008;93:4381-8. 10. Paus R. Exploring the "thyroid-skin connection": concepts, questions, and clinical relevance. J Invest Dermatol 2010;130:7-10. 11. Guo J, Rahman M, Cheng L, et al. Morphogenesis and maintenance of the 3D thymic medulla and prevention of nude skin phenotype require FoxN1 in preand post-natal K14 epithelium. J Mol Med 2010 Nov 26 [Epub ahead of print] 12. Potter CS, Pruett ND, Kern MJ, et al. The nude mutant gene Foxn1 is a HOXC13 regulatory target during hair follicle and nail differentiation. J Ivest Dermatol 2010 Dec 30 [Epub ahead of print] 13. Kiso M, Tanaka S, Saba R, et al. The disruption of Sox21-mediated hair shaft cuticle differentiation causes cyclic alopecia in mice. Proc Natl Acad Sci USA 2009;106:9292-7. 14. Moll R, Divo M, Langbein L. The human keratins: biology and pathology. Histochem Cell Biol 2008;129:705-33. 8 Figure legends Fig 1. A. Relative mRNA expression of KRT35, KRT31, KRT32, KRT85 and MSX2 genes following treatment with TSH (100 mU ml-1). This dose was selected since it is within the range of customarily employed TSH concentrations in cell culture studies, and because this dose has been previously shown by microarray analysis to regulate the expression of several genes in organ-cultured human hair follicles [4]. Results represent triplicate determinations of samples. Total RNA was pooled from 20 HFs. B. Relative mRNA expression of KRT6, KRT14 and KRT17 following administration of TSH to ORS KCs in culture, extracted from HFs of an additional female patient. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001; mean ± SEM. PCR amplification was carried out by using the TaqMan primers and probes (recognizing the following human genes: Assay ID: Hs01699178_gH for KRT6, Assay ID: Hs00265033_m1 for KRT14, Assay ID: Hs00356958_m1 for KRT17, Assay ID: Hs00605539_m1 for KRT31, Assay ID: Hs00605543_g1 for KRT32, Assay ID: Hs00605557_g1 for KRT35, Assay ID: Hs00158558_m1 for KRT85, Assay ID: Hs00741177_m1 for MSX2) using the TaqMan Universal PCR Master Mix Protocol (Applied Biosystems). As internal controls, transcripts of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were determined (Assay ID: Hs99999905_m1 for human GAPDH). Fig. 2. TSH (100 mU ml-1) downregulates immunoreactivity of K31 (A), K85 (B), K32 (C), MSX-2 (D), K6 (E), K14 (F) and K17 (G) in microdissected, organ-cultured normal, human scalp skin HFs, after 6 days of administration. Densitometric measurements of staining intensities in defined reference areas (quantitative immunohistomorphometry) were performed using ImageJ software (NIH, Bethesda, MD, USA; http://rsbweb. nih.gov/ij/) as described previously [3,4]. Columns represent means±SEM ; n=15–18 HFs/group; cumulative results of two different experiments. ***P<0.001. 9 TABLE 1. Primary antibodies used Name Host Dilution Positive control Method Source Clone Keratin 6 Mouse 1:10 Suprabasal layers of the ORS; suprabasal layers of wounded skin [2,14] Indirect IF PROGEN, Heidelberg, Germany Ks6.KA12 Keratin 14 Mouse 1:50 Skin epidermis, basal layer; basal and suprabasal layers of the ORS [2,14] Indirect IF Sigma-Aldrich, Taufkirchen, Germany CKB1 Keratin 17 Mouse 1:50 Suprabasal layers of the ORS [14] Indirect IF PROGEN, Heidelberg, Germany Ks17.E3 Keratin 31 Guinea Pig 1:7000 Precortex region [2] Indirect IF Lutz Langbein, DKFZ, Heidelberg, Germany hHa1 prot.1 Keratin 32 Guinea Pig 1:2000 Hair cuticle [10,14] Indirect IF Lutz Langbein, DKFZ, Heidelberg, Germany Ha2.1 Keratin 85 Guinea Pig 1:1000 Hair matrix, cortex and cuticle [14] Indirect IF Lutz Langbein, DKFZ, Heidelberg, Germany hHb 5co.2 Msx-2 Goat 1:100 Hair matrix and cortex [8] Indirect IF Santacruz, CA, USA -