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Hair Cycle Control by Vanilloid Receptor-1 (TRPV1): Evidence from TRPV1 Knockout Mice

2006; Elsevier BV; Volume: 126; Issue: 8 Linguagem: Inglês

10.1038/sj.jid.5700321

1523-1747

Tamás Bı́ró, Enikö Bodó, Andrea Telek, Tamás Géczy, Birte Tychsen, László Kovács, Ralf Paus,

Dermatology and Skin Diseases

immunoreactivity receptor potential-1 The vanilloid receptor-1 (or transient receptor potential-1, TRPV1) is a Ca2+-permeable cation channel that be stimulated by capsaicin, the pungent ingredient of chili peppers (Caterina et al., 1997Caterina M.J. Schumacher M.A. Tominaga M. Rosen T.A. Levine J.D. Julius D. The capsaicin receptor: a heat-activated ion channel in the pain pathway.Nature. 1997; 389: 816-824Crossref PubMed Scopus (6541) Google Scholar; Szallasi and Blumberg, 1999Szallasi A. Blumberg P.M. Vanilloid (Capsaicin) receptors and mechanisms.Pharmacol Rev. 1999; 51: 159-212PubMed Google Scholar). TRPV1 was first described on capsaicin-sensitive nociceptive neurons that respond to various painful stimuli (Di Marzo et al., 2002Di Marzo V. Blumberg P.M. Szallasi A. Endovanilloid signaling in pain.Curr Opin Neurobiol. 2002; 12: 372-379Crossref PubMed Scopus (237) Google Scholar). Therefore, TRPV1 is recognized as a central integrator of noxious stimuli (Tominaga et al., 1998Tominaga M. Caterina M.J. Malmberg A.B. Rosen T.A. Gilbert H. Skinner K. et al.The cloned capsaicin receptor integrates multiple pain-producing stimuli.Neuron. 1998; 21: 531-543Abstract Full Text Full Text PDF PubMed Scopus (2443) Google Scholar). There is increasing appreciation, however, that functions of TRPV1 signaling extend far beyond the sensory nervous system (Bíró et al., 1998aBíró T. Brodie C. Modarres S. Lewin N.E. Ács P. Blumberg P.M. Specific vanilloid responses in C6 rat glioma cells.Mol Brain Res. 1998; 56: 89-98Crossref PubMed Scopus (60) Google Scholar, Bíró et al., 1998bBíró T. Maurer M. Modarres S. Lewin N.E. Brodie C. Ács G. et al.Characterization of functional vanilloid receptors expressed by mast cells.Blood. 1998; 91: 1332-1340PubMed Google Scholar; Veronesi et al., 1999Veronesi B. Oortgiesen M. Carter J.D. Devlin R.B. Particulate matter initiates inflammatory cytokine release by activation of capsaicin and acid receptors in a human bronchial epithelial cell line.Toxicol Appl Pharmacol. 1999; 154: 106-115Crossref PubMed Scopus (113) Google Scholar; Birder et al., 2001Birder L.A. Kanai A.J. de Groat W.C. Kiss S. Nealen M.L. Berke N.E. et al.Vanilloid receptor expression suggests a sensory role for urinary bladder epithelial cells.Proc Natl Acad Sci USA. 2001; 98: 1339-13401Crossref Scopus (445) Google Scholar). In the skin, human epidermal and hair follicle keratinocytes, mast cells, and Langerhans cells are prominently positive for TRPV1 (Denda et al., 2001Denda M. Fuziwara S. Inoue K. Denda S. Akamatsu H. Tomitaka A. et al.Immunreactivity of VR1 on epidermal keratinocyte of human skin.Biochem Biophys Res Commun. 2001; 285: 1250-1252Crossref PubMed Scopus (181) Google Scholar; Inoue et al., 2002Inoue K. Koizumi S. Fuziwara S. Denda S. Inoue K. Denda M. Functional vanilloid receptors in cultured normal human epidermal keratinocytes.Biochem Biophys Res Commun. 2002; 291: 124-129Crossref PubMed Scopus (231) Google Scholar; Ständer et al., 2004Ständer S. Moormann C. Schumacher M. Buddenkotte J. Artuc M. Shpacovitch V. et al.Expression of vanilloid receptor subtype 1 in cutaneous sensory fibers, mast cells, and epithelial cells of appendage structures.Exp Dermatol. 2004; 13: 129-139Crossref PubMed Scopus (316) Google Scholar; Bodó et al., 2004Bodó E. Kovács I. Telek A. Varga A. Paus R. Kovács L. et al.Vanilloid receptor-1 is widely expressed on various epithelial and mesenchymal cell types of human skin.J Invest Dermatol. 2004; 123: 410-413Crossref PubMed Scopus (95) Google Scholar, Bodó et al., 2005Bodó E. Bíró T. Telek A. Czifra G. Telek A. Tóth B.I. et al.A hot new twist to hair biology – Involvement of vanilloid receptor-1 (VR1/TRPV1) signalling in human hair growth control.Am J Pathol. 2005; 166: 985-998Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar) and TRPV1 agonists have been shown to modulate mast cell (Bíró et al., 1998bBíró T. Maurer M. Modarres S. Lewin N.E. Brodie C. Ács G. et al.Characterization of functional vanilloid receptors expressed by mast cells.Blood. 1998; 91: 1332-1340PubMed Google Scholar) and keratinocyte functions (Inoue et al., 2002Inoue K. Koizumi S. Fuziwara S. Denda S. Inoue K. Denda M. Functional vanilloid receptors in cultured normal human epidermal keratinocytes.Biochem Biophys Res Commun. 2002; 291: 124-129Crossref PubMed Scopus (231) Google Scholar; Southall et al., 2003Southall M.D. Li T. Gharibova L.S. Pei Y. Nicol G.D. Travers J.B. Activation of epidermal vanilloid receptor-1 induces release of proinflammatory mediators in human keratinocytes.J Pharmacol Exp Ther. 2003; 304: 217-222Crossref PubMed Scopus (249) Google Scholar). In addition, we have recently provided the first evidence that TRPV1 signaling is indeed physiologically important in normal human skin in situ, by presenting that TRPV1 activation promotes hair follicle regression (catagen) and hair matrix keratinocyte apoptosis, whereas it inhibits hair matrix keratinocyte proliferation and retards hair shaft elongation in vitro (Bodó et al., 2005Bodó E. Bíró T. Telek A. Czifra G. Telek A. Tóth B.I. et al.A hot new twist to hair biology – Involvement of vanilloid receptor-1 (VR1/TRPV1) signalling in human hair growth control.Am J Pathol. 2005; 166: 985-998Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Given the unsurpassed instructiveness of mouse models for hair research (Nakamura et al., 2001Nakamura M. Sundberg J.P. Paus R. Mutant laboratory mice with abnormalities in hair follicle morphogenesis, cycling, and/or structure: annotated tables.Exp Dermatol. 2001; 10: 369-390Crossref PubMed Scopus (76) Google Scholar; Stenn and Paus, 2001Stenn K.S. Paus R. Controls of hair follicle cycling.Phys Rev. 2001; 81: 449-494PubMed Google Scholar), we now wish to examine whether (1) the expression of TRPV1 changes during the murine hair cycle and (2) the deletion of functional TRPV1 has any effect on hair follicle cycling in vivo. A tissue bank was prepared from adolescent back skin of female C57BL/6 mice in which hair follicle cycling had been induced by depilation (Paus et al., 1994Paus R. Hoffmann U. Eichmüller S. Czarnetzki B.M. Distribution and changing density of gamma-delta T cells in murine skin during the induced hair cycle.Br J Dermatol. 1994; 130: 281-289Crossref PubMed Scopus (78) Google Scholar, Maurer et al., 1997Maurer M. Fischer E. Handjinski B. Von Stebut E. Algermissen B. Bavandi A. et al.Activated skin mast cells are involved in murine hair follicle regression (catagen).Lab Invest. 1997; 77: 319-332PubMed Google Scholar; 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). This was used for immunohistological detection of hair cycle-associated differences in TRPV1 expression. The functional role of TRPV1 signaling was addressed by quantitative histomorphometry of spontaneous, experimentally unmanipulated hair follicle cycling during the first murine hair cycle (P19–P45), comparing TRPV1 knockout B6.129S4-Trpv1 mice (Jackson Laboratory, Bar Harbor, MA) and their age-matched littermates. Cryostat sections of back skin (at least three animals each per time point) processed for histology; hematoxylin–eosin-stained sections were counted and hair follicles were morphologically assigned to their respective hair cycle stages. For the detection of TRPV1 immunoreactivity, the tyramide-amplification (TSA, Ito et al., 2004Ito T. Ito N. Bettermann A. Tokura Y. Takigawa M. Paus R. Collapse and restoration of MHC class-I dependent immune privilege: exploiting the human hair follicle as a model.Am J Pathol. 2004; 164: 623-634Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar) and a peroxidase-based ABC technique (Paus et al., 1998Paus R. van der Veen C. Eichmüller S. Kopp T. Hagen E. Müller-Röver S. et al.Generation and cyclic remodeling of the hair follicle immune system in mice.J Invest Dermatol. 1998; 111: 7-18Crossref PubMed Scopus (110) Google Scholar) were performed. Sections were first incubated with a primary rabbit anti-TRPV1 antibody (H-150, sc-20813; Santa Cruz BT, Santa Cruz, CA) (1:500 in TNB buffer for TSA, Perkin Elmer, Boston, MA and 1:50 in TBS for ABC), with biotinylated multilink swine anti-goat/mouse/rabbit IgG (DAKO, Glostrup, Denmark, 1:200 in TNB), and then by a streptavidin–horseradish peroxidase (1:100 in TNB for TSA; avidin–biotin peroxidase for ABC, Linaris, Wertheim, Germany). Finally, we applied fluorescein isothiocyanate-tyramide (1:50 in Amplification Diluent, TSA kit), or diamino-benzidine (Linaris), respectively and then sections were counterstained. The employed positive (mouse spinal cord) and negative controls (the primary antibody was omitted or sections were preincubated with a specific blocking peptide; spinal cord and skin of Trpv-1−/− mice) unambiguously argued the specificity and sensitivity of the immunoreactivity patterns. (Note that the TRPV1 positivity on sebaceous glands is a false-positive result as negative controls as well as sebaceous gland of Trpv-1−/− mice skin showed immunosignals.) The study was approved by the Institutional Research Ethics Committee. Similar to human epidermis, adolescent wild-type C57BL/6 mouse skin showed strong TRPV1 immunreactivity (IR) on (mostly basal) epidermal keratinocytes (Figure 1a). In addition, also similarly to our previous human data, in the hair follicle, TRPV1-IR was exclusively restricted to the epithelial compartments (note the TRPV1-negativity of the dermal papilla during all hair cycle phases). Analysis of depilation-induced hair follicle cycling in these mice, however, revealed discrete, but important and statistically significant changes in the observed specific IR patterns corresponding to TRPV1 protein expression (Figure 1a–q). Intriguingly, the strongest IR signal was detected on keratinocytes of the epithelial strand of the regressing catagen follicle (Figure 1o–p) and of the secondary hair germ of telogen hair follicles (Figure 1c, d, m and n). With the exception of an asymmetric, disc-like region in the anagen VI hair matrix (Figure 1i and k), the most highly proliferating cell populations in the hair follicle epithelium showed a slightly reduced intensity of TRPV1-IR (Figure 1i-l). The inner root sheath and the distal, precortical hair matrix also showed only strongly reduced TRPV1-IR (Figure 1b, e–l). As TRPV1 activation by capsaicin caused hair follicle regression (anagen–catagen transition) in human hair follicle organ culture (Bodó et al., 2005Bodó E. Bíró T. Telek A. Czifra G. Telek A. Tóth B.I. et al.A hot new twist to hair biology – Involvement of vanilloid receptor-1 (VR1/TRPV1) signalling in human hair growth control.Am J Pathol. 2005; 166: 985-998Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar), spontaneous hair follicle-cycling was compared between age-matched TRPV1 wild-type and knockout mice by quantitative histomorphometry. The skin of Trpv-1−/− mice showed no obvious macroscopic or microscopic abnormalities compared to age-matched wild-type control. However, on day 19, Trpv-1−/− mice exhibited a significant delay in the first spontaneous transition of their hair follicles from morphogenesis stage 8 (which is often confused with "the first anagen", see Paus et al., 1999Paus R. Müller-Röver S. Van der Veen C. Maurer M. Eichmüller S. Ling G. et al.A comprehensive guide for the recognition and classification of distinct stages of hair follicle morphogenesis.J Invest Dermatol. 1999; 113: 523-532Crossref PubMed Scopus (421) Google Scholar) compared to wild-type littermates (Figure 2). This catagen retardation was independently confirmed by cumulative hair cycle score (Maurer et al., 1997Maurer M. Fischer E. Handjinski B. Von Stebut E. Algermissen B. Bavandi A. et al.Activated skin mast cells are involved in murine hair follicle regression (catagen).Lab Invest. 1997; 77: 319-332PubMed Google Scholar; Peters et al., 2004Peters E.M.J. Handjiski B. Kuhlmei A. Hagen E. Bielas H. Braun A. et al.Neurogenic inflammation in stress-induced termination of murine hair growth is promoted by nerve growth factor.Am J Pathol. 2004; 165: 259-271Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) (383±16 vs 267±26 in wild-type and Trpv-1−/− mice, respectively; mean±SEM, P<0.05). Likewise, subsequent telogen development (P25) was slightly but significantly retarded (hair scores of 449±9 vs 412±3 in wild-type and Trpv-1−/− mice, respectively; mean±SEM, P<0.05) in the absence of functional TRPV1 signaling (Figure 2). Instead, the first spontaneous anagen development (Figure 2, P32) and subsequent hair follicle cycling (Figure 2, P45) were not significantly different between Trpv-1-competent and -deficient mice. This suggests that, in murine back skin pelage hair follicles, TRPV1-mediated signaling is important for modulating the transition from the final stages of hair follicle morphogenesis to that of cycling skin appendage, whereas signaling via this receptor looses functional importance once hair follicle cycling has been initiated. In summary, we present here the first evidence that, very similar to human skin, murine skin expresses TRPV1 well outside of sensory nerves, namely in defined epithelial regions of the epidermis and hair follicle. The reported murine hair cycle analyses not only reveal hair cycle-dependent differences in the expression of TRPV1, but – by showing that the absence of TRPV1 is associated with a subtle yet significant delay in the spontaneous involution of hair follicles (catagen-telogen transition) – might also argue for that these receptors are indeed functional. Although one can also assume that the lack of TRPV1 in other cell types (i.e., besides keratinocytes) might also contribute to the observed hair cycle changes, the presented novel results (besides supporting our previous findings in human hair follicles, Bodó et al., 2005Bodó E. Bíró T. Telek A. Czifra G. Telek A. Tóth B.I. et al.A hot new twist to hair biology – Involvement of vanilloid receptor-1 (VR1/TRPV1) signalling in human hair growth control.Am J Pathol. 2005; 166: 985-998Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar) suggest that TRPV1 exerts much more widespread functions in mammalian skin and hair follicle biology than previously thought, which extend across species barriers and may include the inhibition of hair follicle keratinocyte proliferation. The authors state no conflict of interest. This work was supported in part by a grant from DFG to RP (Pa 345/6-4) and by Hungarian research grants (NKFP 1A/008/04, OTKA T049231, OTKA K63153, RET 06/2004, ETT 365/2003) to TB. EB was recipient of an Erasmus student fellowship. The authors are grateful to S. Wegerich and G. Pilnitz-Stolze for excellent technical assistance and Dr Franziska Conrad for helpful advice.