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Involvement of Transforming Growth Factor-β2 in Catagen Induction During the Human Hair Cycle

2002; Elsevier BV; Volume: 118; Issue: 6 Linguagem: Inglês

10.1046/j.1523-1747.2002.01746.x

1523-1747

Tsutomu Soma, Yumiko Tsuji, Toshihiko Hibino,

TGF-β signaling in diseases

The involvement of transforming growth factor-β isoforms in the induction of the regressing phase (catagen) of human hair follicles were examined in vivo. In the growing phase (anagen), transforming growth factor-β1 was detected at the hair cuticle and connective tissue sheath. Transforming growth factor-β2 was restricted to the outermost cell layer of the outer root sheath. Transforming growth factor-β3 was observed in the precortical hair matrix of anagen hair follicles. During the anagen–catagen transition phase, strong transforming growth factor-β2 immunoreactivity appeared in the lower bulb matrix cells adjacent to the dermal papilla. In addition, transforming growth factor-β2 and transforming growth factor-β type II receptor were colocalized in the regressing epithelial strands, where terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling-positive apoptotic cells were also found. Transforming growth factor-β1 and transforming growth factor-β3 were mostly negative in the strand. Using an organ culture system, we investigated whether transforming growth factor-β2 and its antagonists affected the transition process. Elongation of hair was significantly suppressed by transforming growth factor-β2. Next, a neutralizing antibody and fetuin, a potent transforming growth factor-β antagonist was tested. In the presence of the antibody as well as fetuin, hair follicles were markedly elongated in a concentration-dependent manner. These results strongly suggest that transforming growth factor-β2 plays an essential part in the induction of the catagen phase of the human hair cycle. The involvement of transforming growth factor-β isoforms in the induction of the regressing phase (catagen) of human hair follicles were examined in vivo. In the growing phase (anagen), transforming growth factor-β1 was detected at the hair cuticle and connective tissue sheath. Transforming growth factor-β2 was restricted to the outermost cell layer of the outer root sheath. Transforming growth factor-β3 was observed in the precortical hair matrix of anagen hair follicles. During the anagen–catagen transition phase, strong transforming growth factor-β2 immunoreactivity appeared in the lower bulb matrix cells adjacent to the dermal papilla. In addition, transforming growth factor-β2 and transforming growth factor-β type II receptor were colocalized in the regressing epithelial strands, where terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling-positive apoptotic cells were also found. Transforming growth factor-β1 and transforming growth factor-β3 were mostly negative in the strand. Using an organ culture system, we investigated whether transforming growth factor-β2 and its antagonists affected the transition process. Elongation of hair was significantly suppressed by transforming growth factor-β2. Next, a neutralizing antibody and fetuin, a potent transforming growth factor-β antagonist was tested. In the presence of the antibody as well as fetuin, hair follicles were markedly elongated in a concentration-dependent manner. These results strongly suggest that transforming growth factor-β2 plays an essential part in the induction of the catagen phase of the human hair cycle. Transforming growth factor (TGF)-β is a family of multifunctional cytokines. Three isoforms, designated as TGF-β1, TGF-β2, and TGF-β3, are present in mammals (Roberts and Sporn, 1990Roberts A.B. Sporn M.B. The transforming growth factor-βs.in: Sporn M.B. Robert A.B. Peptide Growth Factors and Their Receptors—Handbook of Experimental Pharmacology. Springer-Verlag, Heidelberg1990: 419-472Crossref Google Scholar). The signals of all three TGF-β are mediated through a heteromeric complex of type I and type II TGF-β receptors, which have serine/threonine kinase activities (Massague, 1998Massague J. TGF-β signal transduction.Annu Rev Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3848) Google Scholar). Recently, it was revealed that the signal transduction downstream of the TGF-β receptors involves the Smad family (Hoodless and Wrana, 1998Hoodless P.A. Wrana J.L. Mechanism and function of signaling by the TGF-β superfamily.Curr Top Microbiol Immunol. 1998; 228: 235-272Crossref PubMed Google Scholar). TGF-β regulate various physiologic reactions, such as cellular growth and differentiation, morphogenesis, angiogenesis, adhesion and chemotaxis, extracellular matrix formation (Massague et al., 1992Massague J. Cheifetz S. Laiho M. Ralph D.A. Weis F.M. Zentella A. Transforming growth factor-beta.Cancer Surv. 1992; 12: 81-103PubMed Google Scholar), and apoptotic cell death (Oberhammer et al., 1992Oberhammer F.A. Pavelka M. Sharman S. Tiefenbacher R. Purchio A. Bursch W. Schulte-Hermann R. Induction of apoptosis in cultured hepatocytes and in regressing liver by transforming growth factor-β1.Proc Natl Acad Sci USA. 1992; 89: 5408-5412Crossref PubMed Scopus (673) Google Scholar;Ohta et al., 1994Ohta S. Yanagihara K. Nagata Y. Mechanism of apoptotic cell death of human gastric carcinoma cells mediated by transforming growth factor β.Biochem J. 1994; 324: 777-782Crossref Scopus (68) Google Scholar). The three TGF-β isoforms are differentially expressed in many tissues, including whisker follicles during mouse embryogenesis (Schmid et al., 1991Schmid P. Cox D. Bilbe G. Maier R. McMaster G.K. Differential expression of TGF-β1, β2 and β3 genes during mouse embryogenesis.Development. 1991; 111: 117-130PubMed Google Scholar). Three null mutation studies revealed characteristic and only partially overlapping phenotypes (Letterio et al., 1994Letterio J.J. Geiser A.G. Kulkarni A.B. Roche N.S. Sporn M.B. Roberts A.B. Maternal rescue of transforming growth factor-β1 null mice.Science. 1994; 264: 1936-1938Crossref PubMed Scopus (429) Google Scholar;Dickson et al., 1995Dickson M.C. Martin J.S. Cousins F.M. Kulkarin A.B. Karlsson S.K. Akhurst R.J. Defective haematopoiesis and vasculogenesis in TGF-β1 knock out mice.Development. 1995; 121: 1845-1854Crossref PubMed Google Scholar;Kaartinen et al., 1995Kaartinen V. Voncken J.W. Shuler C. Warburton D. Bu D. Heisterkamp N. Groffen J. Abnormal lung development and cleft palate in mice lacking TGF-β3 indicates defects of epithelial–mesenchymal interaction.Nature Genet. 1995; 11: 415-421Crossref PubMed Scopus (843) Google Scholar;Snaford et al., 1997Snaford L.P. Ormsby I. Gittenburger-de Groot A.C. et al.TGF-β2 knockout mice have multiple developmental defects that are no overlapping with other TGF-β knockout phenotype.Development. 1997; 124: 2659-2670PubMed Google Scholar), suggesting that these isoforms have distinct functions, depending on their expression sites, in embryogenesis, morphogenesis, and various biologic reactions. The hair cycle is a highly regulated process. Three phases have been defined for the mammalian hair cycle: anagen (growing phase), catagen (regressing phase), and telogen (resting phase) (Kligman, 1959Kligman A.M. The human hair cycle.J Invest Dermatol. 1959; 33: 307-316Crossref PubMed Scopus (219) Google Scholar). It is important to understand the mechanism of hair cycle regulation in order to prevent hair loss. Recent investigations have revealed involvement of the Shh and Wnt signal pathways in hair morphogenesis, as well as in the hair induction process (St-Jacques et al., 1998St-Jacques B. Dassule H.R. Karavanova I. et al.Sonic hedgehog signaling is essential for hair development.Curr Biol. 1998; 8: 1058-1068Abstract Full Text Full Text PDF PubMed Google Scholar;Chiang et al., 1999Chiang C. Swan R.Z. Grachtchouk M. et al.Essential role for sonic hedgehog during hair follicle morphogenesis.Dev Biol. 1999; 205: 1-9Crossref PubMed Scopus (403) Google Scholar;Millar et al., 1999Millar S.E. Willert K. Salinas P.C. Roelink H. Nusse R. Suuman D.J. Barsh G.S. WNT signaling in the control of hair growth and structure.Dev Biol. 1999; 207: 133-149Crossref PubMed Scopus (223) Google Scholar). A previous study using a single hair follicle organ culture clearly demonstrated that TGF-β could induce morphologic changes and apoptotic cell death indistinguishable from those seen in human catagen hair follicles (Soma et al., 1998Soma T. Ogo M. Suzuki J. Takahasi T. Hibino T. Analysis of apoptotic cell death in human hair follicles.J Invest Dermatol. 1998; 112: 518-526Google Scholar). In this study, we report that among TGF-β isoforms, TGF-β2 is the key molecule that modulates catagen entry in the human hair cycle in vivo and in vitro. Furthermore, it is shown that TGF-β antagonists suppress catagen-like morphologic changes, resulting in the elongation of hair follicles. Williams E medium, Dulbecco's modified Eagle's medium, fetal bovine serum, penicillin, streptomycin, and fungizone were supplied by Life Technologies (Rockville, MD). All other tissue culture reagents, including bovine fetuin, were purchased from Sigma (St Louis, MO). Human scalp skin specimens were obtained from plastic surgery. Scalp skin pieces and isolated hair follicles washed with ice-cold phosphate-buffered saline (PBS) were fixed with 4% paraformaldehyde in phosphate buffer (pH 7.4) at 4°C for 4 h, and embedded in paraffin wax. Serial sections of 3–5 μm were cut and mounted on slides precoated with silane (Matsunami, Tokyo, Japan). For double staining of TGF-β receptor type II and apoptotic cells, frozen sections with 10 μm were used. All antibodies were subjected to keratin treatment in order to reduce nonspecific binding of antibodies to epidermal and follicular keratins. Human cornified cell extract was washed three times with Tris-buffered (pH 7.6) saline, once with acetone, and then air-dried. Antibodies diluted to the working concentration were incubated with 100 mg per ml keratin powder in PBS containing 3% bovine serum albumin with vigorous shaking at 4°C overnight. After centrifugation, supernatants were collected for immunohistochemistry. Rabbit polyclonal antibodies specific to human TGF-β1 (SC-146), TGF-β2 (SC-90), TGF-β3 (SC-82), and TGF-β receptor type II (SC-220) were purchased from Santa Cruz (Santa Cruz Biotechnology, Santa Cruz, CA). Anti-TGF-β1 antibody was used at a dilution of 1:20, and anti-TGF-β2, anti-TGF-β3, and TGF-β type II receptor antibodies were used at 1:100. In order to test the specificity of antibodies, each antibody was incubated with 100 μg per ml of an appropriate immunogen peptide for 1 h at room temperature. After centrifugation, the supernatant was adjusted to the same concentration as the original antibody, and used in immunohistochemistry as a negative control. Peptides used for immunoadsorption were SC-146P, SC-90P, SC-82P, and SC-220P (Santa Cruz) for anti-TGF-β1, anti-TGF-β2, anti-TGF-β3, and anti-TGF-β, type II receptor antibodies, respectively. Paraffin-embedded tissue sections were deparaffinized, rehydrated, and equilibrated in PBS for 10 min at room temperature. After blocking with 10% normal goat serum at room temperature for 20 min, sections were incubated with each antibody treated with keratin powder at 4°C overnight. For TGF-β1 immunostaining, but not others, tissue sections were moderately digested with 10 μg per ml proteinase K (Nakalai Tesque, Tokyo, Japan) in PBS at 37°C for 30 min before the blocking procedure. A biotinylated rabbit anti-mouse IgG (Nichirei, Tokyo, Japan) was used as a secondary antibody followed by reaction with peroxidase-conjugated streptavidin (Nichirei). Tetramethylbenzidine (Kirkegaard & Perry Laboratories Inc., Gaithersburg, MD) was used as a color-developing reagent in Tris-buffer (pH 7.6) containing 0.01% H2O2. The sections were counterstained with nuclear fast red (Sigma). In the case of anagen hairs, more than 60 follicles from six patients (minimum 10 follicles from each patient) were used for each antibody staining. For catagen hairs, 20 follicles (two to four catagen follicles per patients) were used for the staining. For visualization of apoptotic cells and TGF-β receptor type II, cryostat sections were fixed with 4% paraformaldehyde in phosphate buffer (pH 7.4) at room temperature for 20 min and subjected to immunohistochemical staining using Texas Red® dye-conjugated anti-rabbit IgG (donkey) as a secondary antibody. TUNEL reaction was performed using a fluorescein in situ cell death detection kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's instructions. Human hair follicles were isolated and cultured as previously described (Soma et al., 1998Soma T. Ogo M. Suzuki J. Takahasi T. Hibino T. Analysis of apoptotic cell death in human hair follicles.J Invest Dermatol. 1998; 112: 518-526Google Scholar). Isolated anagen follicles were maintained in 1 ml of Williams E medium containing 100 U penicillin per ml, 10 μg streptomycin per ml, and 2.5 μg fungizone per ml (basal medium) at 37°C in 5% CO2 and 95% air. To evaluate the effects of TGF-β antagonistic molecules, hair follicles were incubated with 10 or 20 μg per ml of anti-TGF-β neutralizing antibody (Genzyme, Cambridge, MA) or 20 μg per ml of normal mouse IgG1 (Chemicon, Temecula CA). In separate experiments, fetal bovine serum fetuin or bovine serum albumin was added to the basal medium. Fetuin is a potent TGF-β antagonist, having a homologous domain to TGF-β type II receptor binding site (Demetrious et al., 1996Demetrious M. Binker C. Sukhu B. Tenenbaum H.C. Dennis J.W. Fetuin/ae2-HS glycoprotein is a TGF-β-type II receptor mimic and cytokine antagonist.J Biol Chem. 1996; 271: 12755-12761Crossref PubMed Scopus (235) Google Scholar). Culture medium was replaced every 3 d unless otherwise mentioned. Tissue sections of hair follicles were prepared as described above. Ten hair follicles were used for each sample concentration. The same experiment was repeated three times using hair follicles obtained from three different patients. In late anagen hair follicles, TGF-β1 immunoreactivity was detected in the hair cuticle and the connective tissue sheath cells (Figure 1a). The hair cuticle was also strongly positive for anti-TGF-β1 antibody. TGF-β2 was detected at the outermost layer of outer root sheath cells, which showed an elongated and palisade structure (Figure 1b). The lower bulb portion of anagen hair follicles was negative for TGF-β2. Anti-TGF-β3 antibody showed strong staining at the hair cortex and the hair cuticle in the keratogenous zone of the upper hair bulb (Figure 1c). Hair medulla was not stained (data not shown). Treatment of each antibody with the appropriate blocking peptide completely abolished the positive staining for TGF-β1, TGF-β2, or TGF-β3, on hair follicles, confirming the specificity of each antibody used in this study. We also confirmed loss of the positive staining when the primary antibodies were omitted (data not shown). In late catagen hair follicles, only a few cells in the connective tissue sheath were positive for TGF-β1 (Figure 2a). Regressing hair follicles were not stained by anti-TGF-β1 antibody. Strong TGF-β2 immunoreactivity was detected in the regressing epithelial strand (Figure 2b), indicating an important role of TGF-β2 in the catagen progression. Specific staining for TGF-β3 seen in the keratogenous zone in the anagen phase was no longer observed in catagen hair follicles (Figure 2c). Weak staining was still observed in the inner cell layers of the outer root sheath. In order to analyze involvement of the TGF-β signal pathway in apoptotic processes, double staining for TGF-β type II receptor and TUNEL-positive cells was carried out. In late catagen hair follicles, TGF-β type II receptor was found in a relatively wide range of the epithelial component, including the edge of club hair and regressing epithelial strand (Figure 3a). TUNEL-positive apoptotic cells were also detected in this area, although their localization was more restricted (Figure 3b). The superimposed image showed that TUNEL-positive cells were found in the TGF-β type II receptor-positive cells (Figure 3c). In order to determine whether TGF-β2 plays a part in the early catagen phase, hair follicles at the transitional stages were isolated from human scalp skin by microdissection based on the early morphologic changes of hair bulbs. They were easily detectable as upward removal of the hair shaft from the dermal papilla cells (DP), decrease of hair color due to the downregulation of melanogenesis and increased thickness of the connective tissue sheath. Hair follicles were immediately fixed and stained for TGF-β2. Figure 4 shows an example of a series of isolated hair follicles exhibiting anagen–catagen transition (Figure 4a–d). In the late anagen hair follicle, a faint staining for TGF-β2 could be detected around the germinative matrix cells (Figure 4e). In the very early catagen hair follicles, which are characterized by the removal of melanocytes above the DP (Figure 4b, c), strong TGF-β2 deposition was demonstrated in the lower part of the boundary area between the DP and the germinative matrix cells (Figure 4f, g). With the progression of the catagen phase, the intercellular space of DP became positive and the basal plate was mostly positive for TGF-β (Figure 4h). In the organ culture system, we confirmed essentially the same TGF-β2 staining pattern, starting as early as at 3 d of culture (data not shown). We then tested the effect of TGF-β2 on the elongation of hair follicles cultured in vitro. Hair elongation was suppressed in the presence of TGF-β2, especially at 50 ng per ml concentration (p < 0.01) (Figure 5a). Overall elongation of control hair follicles reached 1.5 mm from the starting point, whereas that of TGF-β2-treated follicles was approximately 1.2 mm during the 5 d culture period. The anti-TGF-β neutralizing antibody (Genzyme), which inhibits TGF-β action, can neutralize all three TGF-β isoforms in cell culture (Dasch et al., 1989Dasch J.R. Pace D.R. Waegell W. Inenaga D. Ellingsworth L. Monoclonal antibodies recognizing TGF-β. Bioactivity neutralization and TGF-β2 affinity purification.J Immunol. 1989; 142: 1536-1541PubMed Google Scholar). To block endogenous TGF-β2 seen around the germinative matrix cells, we used this antibody in our organ culture system. Hair growth was stimulated in the presence of the neutralizing antibody and showed about 7% (p < 0.1) and 11% increases (p < 0.01) at the concentrations of 10 μg per ml and 20 μg per ml of the antibody, respectively (Figure 5b). A recent study revealed that fetuin and some synthetic peptides derived from both fetuin and TGF-β type II receptor could work as antagonists for TGF-β activity (Demetrious et al., 1996Demetrious M. Binker C. Sukhu B. Tenenbaum H.C. Dennis J.W. Fetuin/ae2-HS glycoprotein is a TGF-β-type II receptor mimic and cytokine antagonist.J Biol Chem. 1996; 271: 12755-12761Crossref PubMed Scopus (235) Google Scholar). We also examined whether fetuin could have similar effect, as seen by the neutralizing antibody. In the presence of fetuin, hair growth was markedly and significantly stimulated at day 8 in a concentration-dependent manner, compared with the effect of bovine serum albumin (Figure 5c). We examined the regressing phase of the human hair cycle to identify the key molecule responsible for catagen induction. Our results revealed that TGF-β2 plays an essential part in the induction of catagen. TGF-β is not only a potent growth inhibitor (Glick et al., 1991Glick A.B. Sporn M.B. Yuspa S.H. Altered regulation of TGF-β and TGF-α in primary keratinocytes and papillomas expressing v-Ha-ras.Mol Carcinog. 1991; 4: 210-219Crossref PubMed Scopus (81) Google Scholar;Alexandrow and Moses, 1995Alexandrow M.G. Moses H.L. Transforming growth factor β1 inhibits mouse keratinocytes late in G1 independent of effects on gene transcription.Cancer Res. 1995; 55: 3928-3932PubMed Google Scholar) but also an apoptosis inducer (Oberhammer et al., 1992Oberhammer F.A. Pavelka M. Sharman S. Tiefenbacher R. Purchio A. Bursch W. Schulte-Hermann R. Induction of apoptosis in cultured hepatocytes and in regressing liver by transforming growth factor-β1.Proc Natl Acad Sci USA. 1992; 89: 5408-5412Crossref PubMed Scopus (673) Google Scholar;Ohta et al., 1994Ohta S. Yanagihara K. Nagata Y. Mechanism of apoptotic cell death of human gastric carcinoma cells mediated by transforming growth factor β.Biochem J. 1994; 324: 777-782Crossref Scopus (68) Google Scholar) of epithelial cells, including interfollicular and follicular epithelium. Recently, we demonstrated that TGF-β2 suppressed the proliferation of hair follicle cells and accelerated the catagen-like morphologic changes associated with increased apoptosis of these cells (Soma et al., 1998Soma T. Ogo M. Suzuki J. Takahasi T. Hibino T. Analysis of apoptotic cell death in human hair follicles.J Invest Dermatol. 1998; 112: 518-526Google Scholar). The most striking finding of our study was the strong deposition of TGF-β2 at the boundary area between the germinative matrix and the DP during the anagen–catagen transition (Figure 3). The germinative matrix cells cease to grow as soon as the hair follicle enters the catagen phase. In this study, it was also demonstrated that TGF-β2 markedly inhibited hair elongation in the organ culture system. Furthermore, as the catagen phase proceeds, TGF-β2- and TUNEL-positive cells were found in similar areas of the regressing epithelial strand (Figure 2b and Figure 3b), where the TGF-β type II receptor was strongly positive (Figure 3a). Taking all these results into account, induction of catagen is suggested to take place as follows. In late anagen, deposition of TGF-β2 occurs around the germinative matrix cells, where proliferating cells reside. High levels of TGF-β2 result in growth inhibition and further lead to the apoptosis of these cells. An inductive role of TGF-β2 is also supported by the inhibition studies. We tried to suppress TGF-β action by using two antagonistic molecules, a neutralizing antibody and fetuin. In the culture system, we found that the catagen-like morphologic changes well reflected the in vivo condition, with the upregulation of TGF-β2 and the same deposition pattern of TGF-β2 in the lower part of the hair bulb. Both antagonistic molecules promoted remarkable hair follicle elongation, suggesting suppression of the catagen entry process by TGF-β2. These lines of evidence strongly indicate that TGF-β2 plays an essential part in catagen induction of human hair follicles. Furthermore, inhibition of TGF-β action can prolong the anagen phase in human hair. TGF-β may also be involved in the early catagen signaling in the mouse hair cycle. TGF-β2 was expressed immediately before catagen (Seiberg et al., 1995Seiberg M. Marthinuss J. Stenn K.S. Changes in expression of apoptosis-associated genes in skin mark early catagen.J Invest Dermatol. 1995; 104: 78-82Crossref PubMed Scopus (85) Google Scholar). Moreover, TGF-β1 injection in mice resulted in the inhibition of anagen and caused premature catagen induction (Foitzik et al., 2000Foitzik K. Lndner G. Mueller-Roever S. et al.Control of murine hair cycle regression (catagen) by TGF-β1 in vivo.FASEB J. 2000; 14: 752-760Crossref PubMed Scopus (239) Google Scholar). Maximal immunoreactivity as well as mRNA levels of TGF-β type II receptor was observed in the anagen VI to early catagen transformation in the mouse hair cycle (Paus et al., 1997Paus R. Foitzik K. Werker P. Bulfone-Paus S. Eichmuller S. Transforming growth factor-β receptor type I and type II expression during hair development and cycling.J Invest Dermatol. 1997; 109: 518-526Crossref PubMed Scopus (106) Google Scholar). In summary, this study has elucidated the spatiotemporal localization of TGF-β isoforms during the human hair cycle. Upregulation and specific localization of TGF-β2 in the anagen–catagen transition phase would be the key to initiate the process of catagen. The effect of antagonistic molecules to TGF-β strongly supports this hypothesis, providing the basis for a fuller understanding of hair cycle regulation. The authors would like to thank Dr Tetsuo Ezaki for his cooperation in obtaining materials and Miss Hitomi Uegaki for her assistance in immunohistochemical analysis.