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The Vitamin D Receptor Is Required for Mouse Hair Cycle Progression but not for Maintenance of the Epidermal Stem Cell Compartment

2008; Elsevier BV; Volume: 128; Issue: 8 Linguagem: Inglês

10.1038/jid.2008.43

1523-1747

Héctor G. Pálmer, Dolores Martínez, Geert Carmeliet, Fiona M. Watt,

Wnt/β-catenin signaling in development and cancer

dermal papilla label-retaining cell 12-O-tetradecanoylphorbol-13-acetate outer root sheath vitamin D receptor TO THE EDITOR Loss or mutational inactivation of the vitamin D receptor (VDR) is linked to post-natal hair loss in mouse and human epidermis (Hughes et al., 1991Hughes M.R. Malloy P.J. O'Malley B.W. Pike J.W. Feldman D. Genetic defects of the 1,25-dihydroxyvitamin D3 receptor.J Recept Res. 1991; 11: 699-716PubMed Google Scholar; Sakai et al., 2001Sakai Y. Kishimoto J. Demay M.B. Metabolic and cellular analysis of alopecia in vitamin D receptor knockout mice.J Clin Invest. 2001; 107: 961-966Crossref PubMed Scopus (122) Google Scholar). VDR-null mice fail to undergo the first post-natal hair growth phase (anagen), and the hair follicles convert into epidermal cysts (Sakai et al., 2001Sakai Y. Kishimoto J. Demay M.B. Metabolic and cellular analysis of alopecia in vitamin D receptor knockout mice.J Clin Invest. 2001; 107: 961-966Crossref PubMed Scopus (122) Google Scholar) (Figure 1a–d). It is reported that VDR ablation leads to a gradual decrease in hair follicle stem cells (Cianferotti et al., 2007Cianferotti L. Cox M. Skorija K. Demay M.B. Vitamin D receptor is essential for normal keratinocyte stem cell function.Proc Natl Acad Sci USA. 2007; 104: 9428-9433Crossref PubMed Scopus (137) Google Scholar). When epidermal stem cell depletion is induced by post-natal deletion of Rac1, there is progressive degeneration of hair follicles, sebaceous glands, and interfollicular epidermis, with the result that spontaneous wounds develop (Benitah et al., 2005Benitah S.A. Frye M. Glogauer M. Watt F.M. Stem cell depletion through epidermal deletion of Rac1.Science. 2005; 309: 933-935Crossref PubMed Scopus (226) Google Scholar). In contrast, VDR-/- mice have excessively thick and folded interfollicular epidermis (Figure 1c), reflecting an increase in epidermal proliferation (Figure 1e and f) (Zinser et al., 2002Zinser G.M. Sundberg J.P. Welsh J. Vitamin D(3) receptor ablation sensitizes skin to chemically induced tumorigenesis.Carcinogenesis. 2002; 23: 2103-2109Crossref PubMed Scopus (173) Google Scholar), a phenotype more consistent with a role of VDR in epidermal lineage selection. These considerations prompted us to explore the effects of VDR ablation on the epidermal stem cell compartment in more depth, using a combination of in vitro and in vivo assays. In hair follicles, the stem cells reside in a permanent area known as the bulge, which is an expansion of the outer root sheath (ORS) just below the sebaceous glands (Müller-Röver et al., 2001Müller-Röver S. Handjiski B. van der Veen C. Eichmüller S. Foitzik K. McKay I.A. et al.A comprehensive guide for the accurate classification of murine hair follicles in distinct hair cycle stages.J Invest Dermatol. 2001; 117: 3-15Crossref PubMed Google Scholar). Cianferotti et al., 2007Cianferotti L. Cox M. Skorija K. Demay M.B. Vitamin D receptor is essential for normal keratinocyte stem cell function.Proc Natl Acad Sci USA. 2007; 104: 9428-9433Crossref PubMed Scopus (137) Google Scholar reported that there is a progressive decline in the proportion of keratinocytes expressing the bulge marker CD34. CD34-positive keratinocytes also express the α6β4 integrin and therefore we performed flow cytometry of CD34/α6 double-positive cells from wild-type and VDR-negative mice. We compared back and tail epidermis of mice aged 8, 24, or 36 weeks (Figure 1i). Consistent with the findings of Cianferotti et al., 2007Cianferotti L. Cox M. Skorija K. Demay M.B. Vitamin D receptor is essential for normal keratinocyte stem cell function.Proc Natl Acad Sci USA. 2007; 104: 9428-9433Crossref PubMed Scopus (137) Google Scholar, we found that there was a reduction in the double-positive population at both body sites in VDR-null mice compared to wild-type mice. Cianferotti et al., 2007Cianferotti L. Cox M. Skorija K. Demay M.B. Vitamin D receptor is essential for normal keratinocyte stem cell function.Proc Natl Acad Sci USA. 2007; 104: 9428-9433Crossref PubMed Scopus (137) Google Scholar further reported that the clonal growth ability of keratinocytes isolated from 4-week-old VDR-null mice shows a reduced ability to form large, actively growing clones. In contrast, we saw no difference in the overall colony-forming efficiency or clone size of keratinocytes cultured from 8-week-old VDR-null and wild-type mice (Figure 1g and h). Furthermore, even though the number of CD34/α6 double-positive cells was lower in 8-week-old VDR-null mice (Figure 1i), the ability of those cells to grow at clonal density was not impaired (Figure 1j). In addition, we found that the growth rate of VDR-null immortalized keratinocytes was significantly higher than that of wild-type cells (Figure 1k). This is consistent with the observation that the proportion of proliferating Ki67-positive keratinocytes is higher in adult VDR-null than wild-type epidermis (Figure 1e and f) and fits with the increase in interfollicular epidermal thickness (Figure 1b and c). We suggest that the differences in colony-forming ability observed by Cianferotti et al., 2007Cianferotti L. Cox M. Skorija K. Demay M.B. Vitamin D receptor is essential for normal keratinocyte stem cell function.Proc Natl Acad Sci USA. 2007; 104: 9428-9433Crossref PubMed Scopus (137) Google Scholar and ourselves may be due to differences in the method used to isolate and culture the keratinocytes (Supplementary Methods). Download .pdf (.01 MB) Help with pdf files Supplementary Methods The discrepancy between the decline in CD34-positive cells and the lack of any reduction in clonal growth ability led us to examine a third marker of epidermal stem cells, DNA label retention. We injected 10-day-old mice with BrdU and followed the label-retaining cells (LRCs) in adult animals (8 weeks). Immunostaining of wild-type tail epidermis with an anti-BrdU antibody revealed a pool of LRCs in the hair follicle bulge and scattered LRCs in the sebaceous glands and interfollicular epidermis (Figure 2a). The VDR was expressed throughout the basal layer of the interfollicular epidermis, the outer root sheath of the hair follicle, and the periphery of the sebaceous gland (Figure 2b). Double staining for BrdU and VDR showed that LRCs expressed VDR, although the level of VDR in individual cell did vary (Figure 2c–f). In 8-week-old VDR-null mice, there was no obvious reduction in the number of LRCs per hair follicle. LRCs extended from below the sebaceous glands to the base of the follicle, the same location as in telogen wild-type follicles (Figure 2g and h). We observed that CD34 labeling intensity was reduced (Figure 2i and j), consistent with our own (Figure 1i and j) and previous observations (Cianferotti et al., 2007Cianferotti L. Cox M. Skorija K. Demay M.B. Vitamin D receptor is essential for normal keratinocyte stem cell function.Proc Natl Acad Sci USA. 2007; 104: 9428-9433Crossref PubMed Scopus (137) Google Scholar). CD34 staining extended the length of the degenerating follicles (Figure 2j), in agreement with the distribution of LRCs (Figure 2h). Although bulge cells in both wild-type and VDR-null follicles expressed high levels of the α6 integrin (Figure 2k and l), there was a marked difference in its subcellular localization. In wild-type mice, most bulge cells had surface α6 expression, with only a subpopulation of cells, at the boundary between the bulge and lower follicle, showing evidence of integrin internalization (Figure 2k, insets). In contrast, cells with intracellular α6 integrin were found along the length of the bulge in VDR-null follicles (Figure 2l). This is consistent with the reduction in the CD34/integrin α6 double-positive population observed by flow cytometry (Figure 1i). As LRCs and clonogenic cells were not depleted in VDR-null follicles, we considered the alternative hypothesis that follicular degeneration reflects an inability of the stem cells to respond to mitogenic stimuli in vivo. We therefore treated the tails of BrdU-labeled 8-week-old wild-type and VDR-null mice topically with 12-O-tetradecanoylphorbol-13-acetate (TPA) (Braun et al., 2003Braun K.M. Niemann C. Jensen U.B. Sundberg J.P. Silva-Vargas V. Watt F.M. Manipulation of stem cell proliferation and lineage commitment: visualisation of label-retaining cells in wholemounts of mouse epidermis.Development. 2003; 130: 5241-5255Crossref PubMed Scopus (361) Google Scholar). TPA treatment induced keratinocyte proliferation in wild-type interfollicular epidermis and sebaceous glands, and stimulated the follicles to enter anagen (Figure 2m). In VDR-null mice, proliferation was also stimulated; however, VDR-null hair follicles did not enter a normal anagen and instead there was a marked increase in the diameter of the bulge area (Figure 2n). Non-treated VDR-null mice presented a higher proportion of keratinocytes in S/G2+M phase (6.7%) than wild-type mice (1.5%) (P<0.002) (Figure 2q, lower panels), which correlated with the increased numbers of Ki67-positive cells in VDR-null skin (Figure 2g and h) (Zinser et al., 2002Zinser G.M. Sundberg J.P. Welsh J. Vitamin D(3) receptor ablation sensitizes skin to chemically induced tumorigenesis.Carcinogenesis. 2002; 23: 2103-2109Crossref PubMed Scopus (173) Google Scholar). TPA treatment of wild-type epidermis caused LRCs to enter the cell cycle, thereby diluting out the BrdU label and increasing the proportion of cells in S/G2+M of the cell cycle (Figure 2q). In VDR-null epidermis, this mobilization was more pronounced and there was a higher proportion of BrdU-labeled cells in S/M or G2 phase of the cell cycle (Figure 2q). This leads us to conclude that not only are there LRCs in VDR-null epidermis but also that they are competent to respond to an in vivo proliferative stimulus. The increased number of VDR-null cells recruited into cycle by TPA suggests an enhanced sensitivity to proliferative stimuli, most likely because of deregulated expression of cell cycle genes that are VDR targets (Deeb et al., 2007Deeb K.K. Trump D.L. Johnson C.S. Vitamin D signalling pathways in cancer: potential for anticancer therapeutics.Nat Rev. 2007; 7: 684-700Crossref Scopus (1124) Google Scholar). In humans and mice, mutations in VDR, hairless receptor, or retinoic acid receptor result in similar epidermal phenotypes characterized by atrichia with papules (Panteleyev et al., 1998Panteleyev A.A. van der Veen C. Rosenbach T. Muller-Rover S. Sokolov V.E. Paus R. Towards defining the pathogenesis of the hairless phenotype.J Invest Dermatol. 1998; 110: 902-907Crossref PubMed Scopus (79) Google Scholar; Miller et al., 2001Miller J. Djabali K. Chen T. Liu Y. Ioffreda M. Lyle S. et al.Atrichia caused by mutations in the vitamin D receptor gene is a phenocopy of generalized atrichia caused by mutations in the hairless gene.J Invest Dermatol. 2001; 117: 612-617Abstract Full Text Full Text PDF PubMed Google Scholar; Ghyselinck et al., 2002Ghyselinck N.B. Chapellier B. Calleja C. Kumar Indra A. Li M. Messaddeq N. et al.[Genetic dissection of retinoic acid function in epidermis physiology].Ann Dermatol Venereol. 2002; 129: 793-799PubMed Google Scholar; Bikle et al., 2006Bikle D.D. Elalieh H. Chang S. Xie Z. Sundberg J.P. Development and progression of alopecia in the vitamin D receptor null mouse.J Cell Physiol. 2006; 207: 340-353Crossref PubMed Scopus (70) Google Scholar) and detachment of the dermal papilla (DP) from the hair follicles (Panteleyev et al., 1998Panteleyev A.A. van der Veen C. Rosenbach T. Muller-Rover S. Sokolov V.E. Paus R. Towards defining the pathogenesis of the hairless phenotype.J Invest Dermatol. 1998; 110: 902-907Crossref PubMed Scopus (79) Google Scholar; Bikle et al., 2006Bikle D.D. Elalieh H. Chang S. Xie Z. Sundberg J.P. Development and progression of alopecia in the vitamin D receptor null mouse.J Cell Physiol. 2006; 207: 340-353Crossref PubMed Scopus (70) Google Scholar). Hairless receptor and retinoic acid receptor interact with VDR to regulate gene transcription (Hsieh et al., 2003Hsieh J.C. Sisk J.M. Jurutka P.W. Haussler C.A. Slater S.A. Haussler M.R. et al.Physical and functional interaction between the vitamin D receptor and hairless corepressor, two proteins required for hair cycling.J Biol Chem. 2003; 278: 38665-38674Crossref PubMed Scopus (190) Google Scholar; Deeb et al., 2007Deeb K.K. Trump D.L. Johnson C.S. Vitamin D signalling pathways in cancer: potential for anticancer therapeutics.Nat Rev. 2007; 7: 684-700Crossref Scopus (1124) Google Scholar). We analyzed the location of the DP by staining for tenascin C (Jiang and Chuong, 1992Jiang T.X. Chuong C.M. Mechanism of skin morphogenesis. I. Analyses with antibodies to adhesion molecules tenascin, N-CAM, and integrin.Dev Biol. 1992; 150: 82-98Crossref PubMed Scopus (91) Google Scholar) (Figure 2o and p). We observed that in wild-type skin the DP was attached to telogen hair follicles, but this was not the case in VDR-null skin (Figure 2o and p). Although, as previously suggested (Bikle et al., 2006Bikle D.D. Elalieh H. Chang S. Xie Z. Sundberg J.P. Development and progression of alopecia in the vitamin D receptor null mouse.J Cell Physiol. 2006; 207: 340-353Crossref PubMed Scopus (70) Google Scholar), the degeneration of hair follicles in the absence of VDR is likely to be due in part to the lack of DP association, an additional possibility is that it reflects an inability of keratinocytes to migrate along the follicle at the onset of anagen. This latter possibility is suggested by the altered distribution of the α6 integrin (Figure 2l). To examine this, we performed two assays of keratinocyte motility in vitro. In a wound-healing scratch assay, VDR-null keratinocytes covered a shorter distance than wild-type cells at 24 and 48 hours (Figure S1a). When the distance covered by single cells was monitored by time-lapse video microscopy, keratinocytes also migrated a shorter distance than wild-type cells (Figure S1b and c). Download .jpg (.07 MB) Help with files Figure S1VDR null primary mouse keratinocytes have reduced motility. (a) Motility of wild type and VDR null keratinoctes was assessed by a wound-healing scratch assay. Pictures were taken at the time points indicated. (b, c) Single cell motility was analyzed by tracking wild type and VDR null keratinocytes by time-lapse videomicroscopy. The movement of individual cells during 48 h is indicated by colored lines. (c) 50 individual wild type and VDR null cells were tracked. The average distance covered is shown with the correspondent standard deviation. Scale bars correspond to 2 µm in panel a and 20 mm in panel b. In conclusion, although VDR is clearly necessary to maintain hair follicles, their degeneration does not reflect a loss of follicle stem cells. We did see a reduction in cell-surface levels of CD34, but in contrast to a previous report (Cianferotti et al., 2007Cianferotti L. Cox M. Skorija K. Demay M.B. Vitamin D receptor is essential for normal keratinocyte stem cell function.Proc Natl Acad Sci USA. 2007; 104: 9428-9433Crossref PubMed Scopus (137) Google Scholar), there was no loss of clonal growth potential in culture. There was no reduction in the number of LRCs, and VDR-null LRCs were competent to enter the cell cycle in response to TPA. We propose that in addition to the previously reported DP defect (Bikle et al., 2006Bikle D.D. Elalieh H. Chang S. Xie Z. Sundberg J.P. Development and progression of alopecia in the vitamin D receptor null mouse.J Cell Physiol. 2006; 207: 340-353Crossref PubMed Scopus (70) Google Scholar), the failure of VDR-null epidermis to maintain the hair follicle may represent an inability of the cells to migrate along the follicle at the onset of anagen. This would fit with the observation that TPA treatment causes a swelling of the bulge and also with the intriguing observation that many of the outer root sheath cells have internalized α6 integrin. The authors state no conflict of interest. We thank everyone who provided us with advice and reagents, in particular George Elia and Virgilio Failla and members of the CR-UK London Research Institute FACS and Biological Resources Laboratories. HGP is the recipient of a European Union Marie Curie Fellowship. This work was supported by CR-UK. Supplementary Methods. Figure S1. VDR-null primary mouse keratinocytes have reduced motility.