Profile of Transforming Growth Factor-β Responses During the Murine Hair Cycle
2003; Elsevier BV; Volume: 121; Issue: 5 Linguagem: Inglês
10.1046/j.1523-1747.2003.12516.x
ISSN1523-1747
AutoresTsutomu Soma, Cord Dohrmann, Toshihiko Hibino, Laurel A. Raftery,
Tópico(s)TGF-β signaling in diseases
ResumoTransforming growth factor-β (TGF-β) appears to promote the regression phase of the mammalian hair cycle, in vivo in mice and in organ culture of human hair follicles. To assess the relationship between TGF-β activity and apoptosis of epithelial cells during the murine hair cycle, we identified active TGF-β responses using phospho-Smad2/3-specific antibodies (PS2). Strong, nuclear PS2 staining was observed in the outer root sheath throughout the anagen growth phase. Some bulb matrix cells were positive for PS2 during late anagen. Extensive, but weak, staining was observed in this region at the anagen-catagen transition. We also examined expression of TGF-β-stimulated clone-22 (TSC-22), which is associated with TGF-β-induced apoptosis of some cell lines. Recombinant rat TSC-22 was used to generate a rabbit anti-TSC-22 antibody useful for immunohistochemistry. TSC-22 RNA accumulation and immunoreactivity were observed in follicles throughout the murine hair cycle, including the dermal papilla and lower epithelial strand of late-catagen hair follicles. Neither the expression pattern nor the presence of nuclear TSC-22 correlated with the sites of apoptosis, suggesting that TSC-22 is not an effector of apoptosis in mouse catagen hair follicles. These studies support a complex role for TGF-β in regulating the regression phase of the cycle, with potential for indirect promotion of apoptosis during the anagen–catagen transition. Transforming growth factor-β (TGF-β) appears to promote the regression phase of the mammalian hair cycle, in vivo in mice and in organ culture of human hair follicles. To assess the relationship between TGF-β activity and apoptosis of epithelial cells during the murine hair cycle, we identified active TGF-β responses using phospho-Smad2/3-specific antibodies (PS2). Strong, nuclear PS2 staining was observed in the outer root sheath throughout the anagen growth phase. Some bulb matrix cells were positive for PS2 during late anagen. Extensive, but weak, staining was observed in this region at the anagen-catagen transition. We also examined expression of TGF-β-stimulated clone-22 (TSC-22), which is associated with TGF-β-induced apoptosis of some cell lines. Recombinant rat TSC-22 was used to generate a rabbit anti-TSC-22 antibody useful for immunohistochemistry. TSC-22 RNA accumulation and immunoreactivity were observed in follicles throughout the murine hair cycle, including the dermal papilla and lower epithelial strand of late-catagen hair follicles. Neither the expression pattern nor the presence of nuclear TSC-22 correlated with the sites of apoptosis, suggesting that TSC-22 is not an effector of apoptosis in mouse catagen hair follicles. These studies support a complex role for TGF-β in regulating the regression phase of the cycle, with potential for indirect promotion of apoptosis during the anagen–catagen transition. dermal papilla epidermal growth factor glutathione S-transferase outer root sheath phospho-Smad2 transforming growth factor-β TGF-β-stimulated clone-22 Transforming growth factor-β (TGF-β) has potent antiproliferative effects on most cultured epithelial cells, including epidermal keratinocytes. This activity appears to be important in the mammalian hair cycle in vivo. Mature hair follicles cycle through growth (anagen), regression (catagen), and resting (telogen) phases in a species-specific pattern. In TGF-β1-null mice, initial hair follicle formation is slightly advanced (Foitzik et al., 1999Foitzik K. Paus R. Doetschman T. Dotto G.P. The TGF-beta2 isoform is both a required and sufficient inducer of murine hair follicle morphogenesis.Dev Biol. 1999; 212: 278-289https://doi.org/10.1006/dbio.1999.9325Crossref PubMed Scopus (121) Google Scholar); for mature follicles, the regression phase of the hair cycle is delayed (Foitzik et al., 2000Foitzik K. Lindner 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). TGF-β1 immunoreactivity is present in the outer root sheath (ORS) throughout the murine hair cycle (Foitzik et al., 2000Foitzik K. Lindner 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). In human hair, TGF-β2 immunoreactivity appears in the boundary area between the bulb matrix and the dermal papilla (DP) during the anagen–catagen transition (Soma et al., 2002Soma T. Tsuji Y. Hibino T. TGF-β2 is involved in human catagen hair follicles.J Invest Dermatol. 2002; 118: 993-997https://doi.org/10.1046/j.1523-1747.2002.01746.xCrossref PubMed Scopus (100) Google Scholar). In cultured human hair follicles, application of TGF-β2 promotes morphologic changes similar to catagen (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). Taken together, these data indicate that TGF-β can promote hair follicle regression, a recurring program of tissue remodeling and apoptosis in adult mammals. During catagen, massive apoptosis occurs in the epithelial components of the hair follicle (Lindner et al., 1997Lindner G. Botchkarev V.A. Botchkarev N.V. Ling G. van der Veen C. Paus R. Analysis of apoptosis during hair follicle regression (catagen).Am J Pathol. 1997; 151: 1601-1617PubMed Google Scholar;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). TGF-β2 can induce apoptosis of epithelial cells in cultured human 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). TGF-β2 is expressed during the anagen–catagen transition of the human hair cycle, and sites of TGF-β type II receptor immunoreactivity correlate with the sites of apoptosis in the regressing epithelial strand of catagen hair follicles of humans (Soma et al., 2002Soma T. Tsuji Y. Hibino T. TGF-β2 is involved in human catagen hair follicles.J Invest Dermatol. 2002; 118: 993-997https://doi.org/10.1046/j.1523-1747.2002.01746.xCrossref PubMed Scopus (100) Google Scholar) and mice (Foitzik et al., 2000Foitzik K. Lindner 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). These data suggest a simple hypothesis that apoptosis of the follicular epithelial cells in catagen hair follicles is a response to TGF-β. Such a response could be direct, mediated by TGF-β signal transducers in an immediate early response, or it could be indirect, mediated by TGF-β regulation of downstream target genes. Here we examine a direct response to TGF-β, phosphorylation of the Smad signal transducers, and a potential indirect pathway, expression of the transcriptional regulator, TGF-β-stimulated clone 22 (TSC-22;Shibanuma et al., 1992Shibanuma M. Kuroki T. Nose K. Isolation of a gene encoding a putative leucine zipper structure that is induced by transforming growth factor beta 1 and other growth factors.J Biol Chem. 1992; 267: 10219-10224Abstract Full Text PDF PubMed Google Scholar). To understand the roles of TGF-β ligands during the endogenous hair cycle, it is important to know which cells are actively responding to TGF-β signals. A universal TGF-β response is the phosphorylation of Smad proteins, which can then accumulate in the nucleus (Raftery and Sutherland, 1999Raftery L.A. Sutherland D.J. TGF-β family signal transduction in Drosophila: From Mad to Smads.Dev Biol. 1999; 210: 251-268https://doi.org/10.1006/dbio.1999.9282Crossref PubMed Scopus (266) Google Scholar). Nuclear Smad proteins interact with tissue-specific transcription factors to regulate the expression of TGF-β target genes. Because phosphorylation of Smad proteins is an immediate response to TGF-β receptor activation (reviewed inMassague, 1998Massague J. TGF-beta signal transduction.Annu Rev Biochem. 1998; 67: 753-791https://doi.org/10.1146/annurev.biochem.67.1.753Crossref PubMed Scopus (3848) Google Scholar), anti-phospho-Smad antibodies allow us to directly visualize the cells that are responding to TGF-β signals. In other developmental systems, this technological advance has provided great insight into in vivo roles of TGF-β family signals (Persson et al., 1998Persson U. Izumi H. Souchelnytskyi S. et al.The L45 loop in type I receptors for TGF-beta family members is a critical determinant in specifying Smad isoform activation.FEBS Lett. 1998; 434: 83-87https://doi.org/10.1016/S0014-5793(98)00954-5Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar;Faure et al., 2000Faure S. de Santa Barbara P. Roberts D.J. Whitman M. Endogenous patterns of BMP signaling during early chick development.Dev Biol. 2000; 244: 44-65https://doi.org/10.1006/dbio.2002.0579Crossref Scopus (131) Google Scholar). Recent studies have suggested that TGF-β can promote apoptosis by inducing expression of the TSC-22 transcriptional regulator. TSC-22 was identified as a TGF-β immediately early response gene in mouse osteoblasts (Shibanuma et al., 1992Shibanuma M. Kuroki T. Nose K. Isolation of a gene encoding a putative leucine zipper structure that is induced by transforming growth factor beta 1 and other growth factors.J Biol Chem. 1992; 267: 10219-10224Abstract Full Text PDF PubMed Google Scholar). TSC-22 is a transcription factor that has a leucine zipper similar to the bZIP family of transcription factors (Seidel et al., 1997Seidel G. Adermann K. Schindler T. Ejchart A. Jaenicke R. Forssmann W.G. Rosch P. Solution structure of porcine delta sleep-inducing peptide immunoreactive peptide A homolog of the shortsighted gene product.J Biol Chem. 1997; 272: 30918-30927https://doi.org/10.1074/jbc.272.49.30918Crossref PubMed Scopus (11) Google Scholar) but differs from this family in the DNA-binding domain (Dobens et al., 1997Dobens L.L. Hsu T. Twombly V. Gelbart W.M. Raftery L.A. The Drosophila bunched gene is a homologue of the growth factor stimulated mammalian TSC-22 sequence and is required during oogenesis.Mech Dev. 1997; 65: 197-208https://doi.org/10.1016/S0925-4773(97)00080-4Crossref PubMed Scopus (53) Google Scholar). Members of the TSC-22 family share a distinct and conserved sequence in place of the basic DNA-binding domain of bZIP proteins (Ohta et al., 1996Ohta S. Shimekake Y. Nagata K. Molecular cloning and characterization of a transcription factor for the C-type natriuretic peptide gene promoter.Eur J Biochem. 1996; 15: 460-466https://doi.org/10.1111/j.1432-1033.1996.460rr.xCrossref Scopus (59) Google Scholar;Dobens et al., 1997Dobens L.L. Hsu T. Twombly V. Gelbart W.M. Raftery L.A. The Drosophila bunched gene is a homologue of the growth factor stimulated mammalian TSC-22 sequence and is required during oogenesis.Mech Dev. 1997; 65: 197-208https://doi.org/10.1016/S0925-4773(97)00080-4Crossref PubMed Scopus (53) Google Scholar;Kester et al., 1999Kester H.A. Blanchtot C. den Hertog J. van der Saag P.T. van der Burg B. Transforming growth factor-beta-stimulated clone-22 is a member of a family of leucine zipper proteins that can homo- and heterodimerize and has transcriptional repressor activity.J Biol Chem. 1999; 24: 27439-27447https://doi.org/10.1074/jbc.274.39.27439Crossref Scopus (101) Google Scholar;Seidel et al., 1997Seidel G. Adermann K. Schindler T. Ejchart A. Jaenicke R. Forssmann W.G. Rosch P. Solution structure of porcine delta sleep-inducing peptide immunoreactive peptide A homolog of the shortsighted gene product.J Biol Chem. 1997; 272: 30918-30927https://doi.org/10.1074/jbc.272.49.30918Crossref PubMed Scopus (11) Google Scholar). TSC-22 can act as a sequence-specific DNA-binding protein (Ohta et al., 1996Ohta S. Shimekake Y. Nagata K. Molecular cloning and characterization of a transcription factor for the C-type natriuretic peptide gene promoter.Eur J Biochem. 1996; 15: 460-466https://doi.org/10.1111/j.1432-1033.1996.460rr.xCrossref Scopus (59) Google Scholar) and as a transcriptional repressor (Kester et al., 1999Kester H.A. Blanchtot C. den Hertog J. van der Saag P.T. van der Burg B. Transforming growth factor-beta-stimulated clone-22 is a member of a family of leucine zipper proteins that can homo- and heterodimerize and has transcriptional repressor activity.J Biol Chem. 1999; 24: 27439-27447https://doi.org/10.1074/jbc.274.39.27439Crossref Scopus (101) Google Scholar). Overexpression of TSC-22 induces apoptotic morphology in a human gastric carcinoma cell line under serum-free conditions (Ohta et al., 1997Ohta S. Yanagihara K. Nagata K. Mechanism of apoptotic cell death of human gastric carcinoma cells mediated by transforming growth factor beta.Biochem J. 1997; 324: 777-782Crossref PubMed Scopus (68) Google Scholar). In a salivary gland cancer cell line, overexpression of TSC-22 increased sensitivity to chemotherapeutic agents that induce cell death (Kawamata et al., 1997Kawamata H. Nakashiro K. Uchida D. Hino S. Omotehara F. Yoshida H. Induction of TSC-22 by treatment with a new anti-cancer drug, vesnarinone, in a human salivary gland cancer cell.Br J Cancer. 1997; 77: 71-78Crossref Scopus (54) Google Scholar;Omotehara et al., 2000Omotehara F. Uchida D. Hino S. Bebum N.M. Yoshida H. Sato M. Kawamata H. In vivo enhancement of chemosensitivity of human salivary gland cancer cells by overexpression of TGF-beta stimulated clone-22.Oncol Rep. 2000; 7: 737-740PubMed Google Scholar;Uchida et al., 2000Uchida D. Kawamata H. Omotehara F. et al.Over-expression of TSC-22 (TGF-beta stimulated clone-22) markedly enhances 5-fluorouracil-induced apoptosis in a human salivary gland cancer cell line.Lab Invest. 2000; 80: 955-963Crossref PubMed Scopus (43) Google Scholar). The mammalian hair cycle provides an in vivo model to examine the role of TSC-22 in apoptotic events that are promoted by TGF-β. Here, we analyzed the profile of phospho-Smad2 (PS2) antigen localization in the murine hair cycle, to determine the distribution of responses to endogenous TGF-β. To assess whether apoptosis of the follicular epithelial cells is an immediately early response to TGF-β, we compared the accumulation of PS2 with the sites of apoptotic cells during mouse catagen. To reveal whether TSC-22 is involved in apoptosis during the hair cycle, we also examined the distribution of TSC-22 immunoreactivity. These studies support a more complex role for TGF-β in the anagen–catagen transition. Anagen hair follicles were induced in the dorsal skin of 7-week-old female C57BL/6 mice (Charles River Laboratories, Wilmington, MA) by depilation as described (Paus et al., 1990Paus R. Stenn K.S. Link R.E. Telogen skin contains an inhibitor of hair growth.Br J Dermatol. 1990; 22: 777-784Crossref Scopus (208) Google Scholar). Back skin was harvested on days 0, 2, 6, 10, 12, 17, 18, and 25 after depilation. Skin samples were fixed with phosphate-buffered formalin (pH 7.2) at room temperature for 1 week or 4% paraformaldehyde in phosphate buffer (pH 7.2) at 4°C overnight and then embedded in paraffin wax. Little difference was observed between samples prepared by these fixation protocols. All animal studies had the approval of the Massachusetts General Hospital Animal Care and Use Committee. A small aliquot (100 μL) of crude anti-PS2 antiserum was affinity-purified using PS2 peptide (KKK-SSpMSp) coupled to NHS-activated Sepharose gels (Amersham, Piscataway, NJ). Both antiserum and phospho-peptide were gifts from P. ten Dyke (Persson et al., 1998Persson U. Izumi H. Souchelnytskyi S. et al.The L45 loop in type I receptors for TGF-beta family members is a critical determinant in specifying Smad isoform activation.FEBS Lett. 1998; 434: 83-87https://doi.org/10.1016/S0014-5793(98)00954-5Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar). Antibodies were eluted with 0.1 M glycine (pH 2.5) and 0.1 M ethanolamine (pH 12) as described (Harlow and Lane, 1988Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor (NY)1988: 313-315Google Scholar). High-pH eluates were routinely used for immunohistochemistry at 1:200 dilution in Tris-buffered saline (pH 7.5) containing 0.1% Triton X-100, 10% normal goat serum, and 3% bovine serum albumin. Immunoreactivity was visualized with biotinylated antirabbit IgG and peroxidase-conjugated streptavidin (Laboratory Vision) using paraffin sections. To conserve the purified antibody, the tyramide signal amplification kit (Perkin-Elmer, Boston, MA) was used after the application of peroxidase-conjugated streptavidin according to the vendor's protocol. Sections were developed with diaminobenzidine solution, followed by counterstaining with hematoxylin. Specificity of PS2 antiserum was tested on primary keratinocytes prepared from newborn Swiss mice and cultured on chambered slides in minimal essential medium with 0.05 M CaCl2, 4% chelex-treated fetal calf serum, and 10 ng per mL epidermal growth factor (EGF; Collaborative Research) as described by (Hennings et al., 1980Hennings H. Michale D. Cheng C. Steinert P. Holbrook K. Yuspa S.H. Calcium regulation of growth and differentiation of mouse epidermal cells.In Culture Cell. 1980; 19: 245-254Abstract Full Text PDF Scopus (1450) Google Scholar). TGF-β1 (R & D Systems, Minneapolis, MN) was added at 10 ng per mL for 15 or 30 min before processing for immunostaining as described above. In situ hybridization to sectioned skin samples was performed as previously described (Dohrmann et al., 1999Dohrmann C.E. Belaoussoff M. Raftery L.A. Dynamic expression of TSC-22 at sites of epithelial-mesenchymal interactions during mouse development.Mech Dev. 1999; 84: 147-151https://doi.org/10.1016/S0925-4773(99)00055-6Crossref PubMed Scopus (30) Google Scholar), using a 0.9-kb PstI–BglII fragment of the 3′-untranslated region to generate TSC-22 sense and antisense probes. Sense probes gave little or no staining in control experiments; an example is shown inDohrmann et al., 1999Dohrmann C.E. Belaoussoff M. Raftery L.A. Dynamic expression of TSC-22 at sites of epithelial-mesenchymal interactions during mouse development.Mech Dev. 1999; 84: 147-151https://doi.org/10.1016/S0925-4773(99)00055-6Crossref PubMed Scopus (30) Google Scholar. Rat TSC-22 cDNA (GenBank Accession No. L25785) was subcloned into pGEX-4T2 vector (Amersham) for bacterial production of TSC-22 fusion protein. Recombinant TSC-22 protein was purified according to the vendor's protocol. Antiserum was produced in rabbits at Poconos Farm and Research Laboratory (Canadensis, PA). One anti-TSC-22 antiserum sample was selected for use based on reactivity in immunoblotting using recombinant TSC-22 proteins as briefly described below. Recombinant TSC-22 protein was digested with thrombin, resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to Immobilon-P membrane (Millipore, Bedford, MA). After being blocked with nonfat dry skim milk, the membrane was reacted with anti-TSC-22 antisera at a 1:5000 dilution. Signal was visualized with anti-rabbit IgG antibody conjugated to alkaline phosphatase (Zymed, South San Francisco, CA) using BM-purple substrate (Hoffmann-La Roche, Basel, Switzerland). To remove anti-glutathione S-trans-ferase (GST) activity, anti-TSC-22 antiserum was passed through a HiTrap column (Amersham) coupled with recombinant GST proteins twice. Affinity purification was against recombinant TSC-22 polypeptide that was cleaved from GST protein and then bound to Affi-Gel 15 (Bio-Rad, Hercules, CA). Immunostaining with anti-TSC-22 antibodies (at 2–4 μg/mL) was performed as described above, without amplification by the tyramide signal amplification system. Sections were counterstained with methyl green. Paraffin sections were stained with a TUNEL kit (Promega, Madison, WI) by the vendor's protocol, and then sections were incubated with anti-PS2 antibodies (at 1:200 dilution) or anti-TSC-22 antibodies (at 0.1 μg/mL) overnight at 4°C. Signals by the antibodies were amplified with the tyramide signal amplification kit combined with cyanine 3 dye-conjugated streptavidin (Zymed). To determine the sites of TGF-β activity during the hair cycle, we used affinity-purified anti-PS2 antiserum to detect phospho-Smad accumulation in murine hair follicles. This antiserum detects Smad2 and Smad3 after they have been C-terminally phosphorylated by serine-threonine receptor kinases of the TGF-β receptor family (Persson et al., 1998Persson U. Izumi H. Souchelnytskyi S. et al.The L45 loop in type I receptors for TGF-beta family members is a critical determinant in specifying Smad isoform activation.FEBS Lett. 1998; 434: 83-87https://doi.org/10.1016/S0014-5793(98)00954-5Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar). To conserve the purified antibody, we used the tyramide amplification system for PS2 immunostaining. The affinity-purified anti-PS2 antibody detected nuclear antigens in mouse primary keratinocytes that were treated with 10 ng per mL TGF-β1. Incubation with untreated primary keratinocytes gave low levels of diffuse staining (data not shown). Thus, nuclear staining with affinity-purified PS2 antibodies is a specific response to TGF-β family signals. A synchronized hair cycle was artificially induced by depilation on day 0. Before depilation, telogen hair follicles had only a few cells (Figure 1a, arrows) that were positive for PS2 immunostaining. At anagen stage II (day 2 after depilation), epithelial cells in the upper follicle (Figure 1b, arrowheads) and around the DP (Figure 1b, arrows) were strongly stained by anti-PS2 antibodies. At anagen stage III (day 6), when the epithelia of the follicle become organized, the nuclei of the developing ORS (Figure 1c, arrows) and some cells of the dermal sheath (Figure 1c, arrowheads) were clearly positive for PS2 staining. In anagen hair at stage V (at day 10), we detected nuclear staining not only in the ORS (Figure 1d, arrowheads) but also in the cuticle layer of the IRS (Figure 1d, arrows). In anagen VI hair follicles (day 12), some of the bulb matrix cells (Figure 1d, arrows) adjacent to the DP were clearly positive for PS2 staining; staining continued to be present in some ORS cells and the cells of the cuticle layer of the IRS (Figure 1e). Interestingly, the intensity of staining varied from cell to cell in the ORS of late-anagen hair follicles (Figure 1d,e). The staining pattern of early-catagen hair follicles (day 17, Figure 1f) was not distinguishable from that of anagen VI hair follicles (see Figure 1e). In late-catagen hair follicles with a long epithelial strand (day 18), PS2 immunoreactivity was still detected in the nuclei of the ORS cells (Figure 1g, arrows), as well as in a few cells in the epithelial strands (Figure 1g, arrowheads). In the telogen stage after depilation (day 25), some nuclei within the regressed epithelial components (Figure 1h, arrows) were still positive for PS2 immunostaining. In sum, PS2 staining was present in epithelial components of the follicle both during anagen and catagen phases of the hair cycle. This staining was most pronounced in the ORS. Weaker levels of staining were detected in the bulb matrix as early as anagen stage V. During the anagen phase, we also detected anti-PS2 staining in occasional nuclei within the DP (Figure 1d, red arrowhead). Nevertheless, this staining was quite weak compared to the ORS or the cuticle layer of the IRS. Because the staining was nuclear, we expect this reflects a response to a TGF-β-family signal. We were interested to determine whether TSC-22 could be a mediator of TGF-β-induced apoptosis during the hair cycle. First, we examined TSC-22 RNA accumulation during the postdepilation hair cycle, using in situ hybridization to a probe from the 3′-untranslated region of the cDNA (Dohrmann et al., 1999Dohrmann C.E. Belaoussoff M. Raftery L.A. Dynamic expression of TSC-22 at sites of epithelial-mesenchymal interactions during mouse development.Mech Dev. 1999; 84: 147-151https://doi.org/10.1016/S0925-4773(99)00055-6Crossref PubMed Scopus (30) Google Scholar). TSC-22 RNA accumulation was detected in limited regions of the IRS and ORS, just above the bulb region (Figure 2). Staining in this region persisted during anagen stages III (day 5, Figure 2a), V (day 8, Figure 2b), and VI (day 12, Figure 2c). The level of RNA accumulation was very low during catagen (day 19, Figure 2d). The pattern of TSC-22 RNA accumulation was different from the pattern of TGF-β responses, indicating that TSC-22 gene expression is not strongly responsive to TGF-β signaling during the hair cycle. It has been reported that TSC-22 moves from the cytoplasm to the nucleus following DNA-damage-induced apoptosis (Hino et al., 2000Hino S. Kawamata H. Uchida D. et al.Nuclear translocation of TSC-22 (TGF-β-stimulated clone-22) concomitant with apoptosis: TSC-22 as a putative transcriptional regulator.Biochem Biophys Res Commun. 2000; 278: 659-664https://doi.org/10.1006/bbrc.2000.3840Crossref PubMed Scopus (37) Google Scholar,Hino et al., 2002Hino S. Kawamata H. Omotehara F. et al.Cytoplasmic TSC-22 (transforming growth factor-β-stimulated clone-22) markedly enhances the radiation sensitivity of salivary gland cancer cells.Biochem Biophys Res Commun. 2002; 292: 957-963https://doi.org/10.1006/bbrc.2002.6776Crossref PubMed Scopus (23) Google Scholar). We wanted to determine whether this change in subcellular localization was associated with programmed cell death during tissue remodeling in vivo. In addition, immunohistochemical staining can be a more sensitive method to detect expression. To examine TSC-22 protein localization in murine hair follicles, we generated anti-TSC-22 antiserum. Rat TSC-22 cDNA (Hamil and Hall, 1994Hamil K.G. Hall S.H. Cloning of a rat Sertoli cell follicle-stimulating hormone primary response complementary deoxyribonucleic acid: Regulation of TSC-22 gene expression.Endocrinology. 1994; 134: 1205-1212https://doi.org/10.1210/en.134.3.1205Crossref PubMed Scopus (48) Google Scholar) was used to make a bacterially expressed recombinant TSC-22 protein. This was used to generate rabbit anti-TSC-22 antiserum, which recognized TSC-22 fragments by Western blot analysis using the digested recombinant fusion proteins (Figure 3a). The specificity of anti-TSC-22 for immunostaining was confirmed in two ways. First, the crude antiserum was preadsorbed against recombinant TCS-22 protein and used for immunohistochemical staining. Skin sections incubated with this preadsorbed antiserum lacked all immunostaining (data not shown). Second, the antiserum was tested for immunoreactivity on sections of skin from mice with a disrupted TSC-22 gene (C. Dohrmann, T. Soma, J. Brissette, and L. Raftery, unpublished data); the epithelium and follicles of this tissue were negative for immunostaining. For this study, we used affinity-purified antiserum, because it gave a stronger signal. TSC-22 immunoreactivity was apparent in multiple cell types in and around mouse hair follicles (Figure 3b). Before induction of the hair cycle by depilation (day 0), occasional nuclei (Figure 3b-a, arrows) were positive for anti-TSC-22 staining near the bulge area. In early-anagen hair follicles (day 2 after depilation), anti-TSC-22 staining (Figure 3b-b, arrows) was mainly detected in the differentiating follicular keratinocytes above the DP. In anagen IV (day 6), positive signals were observed in the IRS (Figure 3b-c, arrowheads) and in the outer most cell layers of the ORS (Figure 3b-c, arrows) and also within the DP. In anagen V hair follicles (day 10), when the DP reaches its largest size, a few DP cells (Figure 3b-d, arrows) were positive for TSC-22 immunostaining, with continued positive staining in the ORS and the IRS. In anagen VI (day 12; Figure 3b-e), the DP is smaller and all DP nuclei appeared to be positive for anti-TSC-22 staining. At early catagen (day 17; Figure 3b-f), the staining pattern was not dis-tinguishable from that in late-anagen hair follicles (compare to Figure 3b-e). TSC-22 immunoreactivity (Figure 3b-g, arrows) was mainly detected in the IRS of early catagen hair follicles and in the DP. In late-catagen hair follicles (at day 18; Figure 3b-g), TSC-22 immunoreactivity was observed in the DP (Figure 3b-h, arrow) and the regressing epithelial strand (Figure 3b-h, arrowheads). In the telogen stage after depilation (day 25), the staining pattern returned to that observed before depilation. Throughout the hair cycle, TSC-22 was observed to accumulate strongly in the nuclei of some cells (Figure 3b-a, b-d, arrows) and to be predominantly cytoplasmic in others (Figure 3b-c, arrowheads). At present, the mechanisms that determine nuclear localization of TSC-22 are unclear. Nevertheless, nuclear localization of TSC-22 did not correlate with the onset of catagen, suggesting that it is not linked to programmed cell death during tissue remodeling. As observed for the pattern of RNA accumulation, the pattern of TSC-22 protein localization is distinct from that of the phospho-Smad responses, indicating that TSC-22 function is regulated by other mechanisms during the hair cycle. In anagen VI, some cells of the bulb matrix consistently exhibited a weak phospho-Smad response (Figure 1e), with an increasing proportion of cells showing a response by early catagen (Figure 1f). We directly compared the sites of PS2 accumulation with the sites of apoptosis to further assess whether apoptosis of hair follicle epithelial cells is an immediately early response to TGF-β. If so, the sites and timing of apoptosis should be very similar to the pattern of PS2 accumulation. To compare these events, we used immuno-fluorescent detection w
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