Cyclosporin A Promotes Hair Epithelial Cell Proliferation and Modulates Protein Kinase C Expression and Translocation in Hair Epithelial Cells
2001; Elsevier BV; Volume: 117; Issue: 3 Linguagem: Inglês
10.1046/j.0022-202x.2001.01452.x
ISSN1523-1747
AutoresTomoya Takahashi, Ayako Kamimura,
Tópico(s)NF-κB Signaling Pathways
ResumoCyclosporin A is an immunosuppressive agent known to cause hirsutism. The mechanisms of action that cause hirsutism have not been fully elucidated, however. We have previously reported that several selective protein kinase C inhibitors promote the growth of murine hair epithelial cells and stimulate anagen induction. In this paper, we report on an investigation of the mechanisms of action of hair-growing activity possessed by cyclosporin A from the viewpoint of whether it promotes hair epithelial cell growth or whether it modulates the expression or translocation of protein kinase C isozymes in hair epithelial cells. Our results indicate that cyclosporin A (over a wide dosage range of 1–1000 ng per ml) stimulates cultured murine hair epithelial cell growth to about 150%-160% relative to controls. We also observed growth-promoting effects on murine epidermal keratinocytes (about 140%) at the dose range of 1–100 ng per ml. At high dose ranges above 3 µg per ml, the growth of both cells was inhibited. On the other hand, we found that cyclosporin A reduces the overall expression of protein kinase C α, βI, and βII in cultured murine hair epithelial cells, and reduces the levels of protein kinase C α, βI, βII, and η in the particulate fraction from cultured murine hair epithelial cells. From these results, we speculate that the hair-growing activity of cyclosporin A is at least partially attributable to its growth-promoting influence on hair epithelial cells sequential to its downregulation of some protein kinase C isozymes in hair epithelial cells or inhibition of translocation of some protein kinase C isozymes to the membrane or cytoskeleton of hair epithelial cells. Cyclosporin A is an immunosuppressive agent known to cause hirsutism. The mechanisms of action that cause hirsutism have not been fully elucidated, however. We have previously reported that several selective protein kinase C inhibitors promote the growth of murine hair epithelial cells and stimulate anagen induction. In this paper, we report on an investigation of the mechanisms of action of hair-growing activity possessed by cyclosporin A from the viewpoint of whether it promotes hair epithelial cell growth or whether it modulates the expression or translocation of protein kinase C isozymes in hair epithelial cells. Our results indicate that cyclosporin A (over a wide dosage range of 1–1000 ng per ml) stimulates cultured murine hair epithelial cell growth to about 150%-160% relative to controls. We also observed growth-promoting effects on murine epidermal keratinocytes (about 140%) at the dose range of 1–100 ng per ml. At high dose ranges above 3 µg per ml, the growth of both cells was inhibited. On the other hand, we found that cyclosporin A reduces the overall expression of protein kinase C α, βI, and βII in cultured murine hair epithelial cells, and reduces the levels of protein kinase C α, βI, βII, and η in the particulate fraction from cultured murine hair epithelial cells. From these results, we speculate that the hair-growing activity of cyclosporin A is at least partially attributable to its growth-promoting influence on hair epithelial cells sequential to its downregulation of some protein kinase C isozymes in hair epithelial cells or inhibition of translocation of some protein kinase C isozymes to the membrane or cytoskeleton of hair epithelial cells. Dulbecco's phosphate-buffered calcium- and magnesium-free saline Cyclosporin A is a highly lipophilic cyclic undecapeptide, produced by the fungus Trichoderma polysporum (Link ex Pers.) Rifai, Cylindrocarpon lucidum Booth, and Tolypocladium inflatum Gams (Borel et al., 1976Borel J.F. Feurer C. Gubler H.U. Stähelin H. Biological effects of cyclosporin A, a new antilymphocytic agent.Agents Actions. 1976; 6: 468-475Crossref PubMed Scopus (1171) Google Scholar;Dreyfuss et al., 1976Dreyfuss M. Härri E. Hofmann H. Kobel H. Pache W. Tscherter H. Cyclosporin A and C: new metabolites from Trichoderma polysporum (Link ex Pers.) Rifai.European J Appl Microbiol. 1976; 3: 125-133Crossref Scopus (290) Google Scholar;Stähelin, 1986Stähelin H. Cyclosporin A: historical background.Prog Allergy. 1986; 38: 19-27PubMed Google Scholar). It is known to possess a variety of biologic and physiologic actions such as antiparasitic, fungicidal, anti-inflammatory effects, and immunosuppressive properties. Up to now, it has been used in medical treatment as an immunosuppressant after organ transplantation and as a cure for several autoimmune disorders such as psoriasis, atopic dermatitis, Behçet's disease, and myasthenia gravis. In addition, cyclosporin A is known to cause hirsutism as a side-effect (Laupacis, 1983Laupacis A. Complications of cyclosporine therapy: a comparison to azathioprine.Transplant Proc The. 1983; 15: 2748-2753Google Scholar;Harper et al., 1984Harper J.I. Kendra J.R. Desai S. Staughton R.C.D. Barrett A.J. Hobbs J.R. Dermatological aspects of the use of cyclosporin A for prophylaxis of graft-versus-host disease.Br J Dermatol. 1984; 110: 469-474Crossref PubMed Scopus (49) Google Scholar;Wysocki and Daley, 1987Wysocki G.P. Daley T.D. Hypertrichosis in patients receiving cyclosporine therapy.Clin Exp Dermatol. 1987; 12: 191-196Crossref PubMed Scopus (61) Google Scholar). Its hair-growing effects have been examined by in vitro and in vivo experiments by several researchers. It has been shown that topical and intraperitoneal cyclosporin A administration induces anagen hair formation in C57BL-6 mice (Paus et al., 1989Paus R. Stenn K.S. Link R.E. The induction of anagen hair growth in telogen mouse skin by cyclosporine A administration.Lab Invest. 1989; 60: 365-369PubMed Google Scholar;Paus et al., 1996Paus R. Bottge J.-A. Henz B.M. Maurer M. Hair growth control by immunosuppression.Arch Dermatol Res. 1996; 288: 408-410https://doi.org/10.1007/s004030050072Crossref PubMed Scopus (30) Google Scholar) and it has also been shown that oral (Sawada et al., 1987Sawada M. Terada N. Taniguchi H. Tateishi R. Mori Y. Cyclosporin A stimulates hair growth in nude mice.Lab Invest. 1987; 56: 684-686PubMed Google Scholar;Buhl et al., 1990Buhl A.E. Waldon D.J. Miller B.F. Brunden M.N. Differences in activity of minoxidil and cyclosporin A on hair growth in nude and normal mice.Lab Invest. 1990; 62: 104-107PubMed Google Scholar), topical, and subcutaneous (Watanabe et al., 1991Watanabe S. Mochizuki A. Wagatsuma K. Kobayashi M. Kawa Y. Takahashi H. Hair growth on nude mice due to cyclosporin A.J Dermatol. 1991; 18: 714-719Crossref PubMed Scopus (27) Google Scholar) administration of cyclosporin A causes hair growth on nude mice. Moreover, its effects on male pattern baldness in humans have been investigated by several researchers (Picascia and Roenigk, 1987Picascia D.D. Roenigk Jr, H.H. Cyclosporine and male-pattern alopecia.Arch Dermatol. 1987; 123: 1432Crossref PubMed Scopus (17) Google Scholar;Picascia and Roenigk, 1988Picascia D.D. Roenigk Jr, H.H. Effects of oral and topical cyclosporine in male pattern alopecia.Transplant Proc The. 1988; 20: 109-111PubMed Google Scholar;Gilhar et al., 1990Gilhar A. Pillar T. Etzioni A. Topical cyclosporine in male pattern alopecia.J Am Acad Dermatol. 1990; 22: 251-253Abstract Full Text PDF PubMed Scopus (26) Google Scholar;Lutz, 1994Lutz G. Effects of cyclosporin A on hair.Skin Pharmacol. 1994; 7: 101-104Crossref PubMed Scopus (20) Google Scholar). The mechanisms of action of its hair-growing activity have not yet been fully described, however (Paus et al., 1996Paus R. Bottge J.-A. Henz B.M. Maurer M. Hair growth control by immunosuppression.Arch Dermatol Res. 1996; 288: 408-410https://doi.org/10.1007/s004030050072Crossref PubMed Scopus (30) Google Scholar). There are at present no data to support the hypothesis that cyclosporin A can directly stimulate hair epithelial cell proliferation. Only one piece of data exists that implies that cyclosporin A prolongs the anagen phase of the hair cycle, assumed from the fact that cyclosporin A prolongs the term of hair shaft growth in the hair follicle tissue organ culture system (Taylor et al., 1993Taylor M. Ashcroft A.T.T. Messenger A.G. Cyclosporin A prolongs human hair growth in vitro.J Invest Dermatol. 1993; 100: 237-239Abstract Full Text PDF PubMed Google Scholar). On the other hand, it is reported that cyclosporin A has cytostatic effects on epidermal keratinocytes (Furue et al., 1988Furue M. Gaspari A.A. Katz S.I. The effect of cyclosporin A on epidermal cells. II. Cyclosporin A inhibits proliferation of normal and transformed keratinocytes.J Invest Dermatol. 1988; 90: 796-800Abstract Full Text PDF PubMed Google Scholar;Nickoloff et al., 1988Nickoloff B.J. Fisher G.J. Mitra R.S. Voorhees J.J. Additive and synergistic antiproliferative effects of cyclosporin A and gamma interferon on cultured human keratinocytes.Am J Pathol. 1988; 131: 12-18PubMed Google Scholar). In this report, we investigated the effect of cyclosporin A on cultured hair epithelial cell growth. It has been reported that protein kinase C (PKC) acts as a negative hair-growing factor (Harmon et al., 1995Harmon C.S. Nevins T.D. Bollag W.B. Protein kinase C inhibits human hair follicle growth and hair fibre production in organ culture.Br J Dermatol. 1995; 133: 686-693Crossref PubMed Scopus (29) Google Scholar;Harmon et al., 1997Harmon C.S. Nevins T.D. Ducote J. Lutz D. Bisindolylmaleimide protein-kinase-C inhibitors delay the decline in DNA synthesis in mouse hair follicle organ cultures.Skin Pharmacol. 1997; 10: 71-78Crossref PubMed Scopus (10) Google Scholar;Xiong and Harmon, 1997Xiong Y. Harmon C.S. Interleukin-1β is differentially expressed by human dermal papilla cells in response to PKC activation and is a potent inhibitor of human hair follicle growth in organ culture.J Interferon Cytokine Res. 1997; 17: 151-157Crossref PubMed Scopus (32) Google Scholar;Takahashi et al., 2000Takahashi T. Kamimura A. Shirai A. Yokoo Y. Several selective protein kinase C inhibitors including procyanidins promote hair growth.Skin Pharmacol Appl Skin Physiol. 2000; 13: 133-142Crossref PubMed Scopus (27) Google Scholar). In this report, we examine whether cyclosporin A, which possesses hair-growing effects, has an influence on PKC expression or translocation in hair epithelial cells. PKC is a major signal transduction pathway in many tissues and cells, and is known to play a key role in cell differentiation and proliferation (Blobe et al., 1996Blobe G.C. Stribling S. Obeid L.M. Hannun Y.A. Protein kinase C isoenzymes: regulation and function.Cancer Surv. 1996; 27: 213-248PubMed Google Scholar). The role of PKC in hair follicle tissue has not been elucidated, however. PKC was first identified and characterized by Nishizuka in 1977 as a serine threonine kinase (Nishizuka et al., 1978Nishizuka Y. Takai Y. Kishimoto A. et al.A role of calcium in the activation of a new protein kinase system.Adv Cyclic Nucleotide Res. 1978; 9: 209-220PubMed Google Scholar;Nishizuka, 1989Nishizuka Y. Studies and prospectives of the protein kinase C family for cellular regulation.Cancer. 1989; 63: 1892-1903Crossref PubMed Scopus (267) Google Scholar). Up to now, at least 12 isozymes have been isolated. PKC is now classified into three major subgroups. The first group is conventional PKC (α, βI, βII, and γ), which is calcium- and diacylglycerol-dependent. The second group is novel PKC (δ, ε, η, and θ) whose activity is calcium-independent but diacylglycerol-dependent. The third group is atypical PKC (ζ, λ, I, and µ) whose activity is calcium- and diacylglycerol-independent (Quest, 1996Quest A.F.G. Regulation of protein kinase C. a tale of lipids and proteins.Enzyme Protein. 1996; 49: 231-261Crossref PubMed Scopus (64) Google Scholar). There is as yet limited information, however, on PKC isozyme expression in hair follicles. In human hair follicles, expression of PKC-α, PKC-β, PKC-δ, and PKC-ζ has been confirmed in cultured outer root sheath keratinocytes (Hoffmann et al., 1996Hoffmann R. Schwende H. Happle R. Distribution of protein kinase C isoenzymes in different cells of the human hair follicle.Eur J Dermatol. 1996; 6: 295-296Google Scholar), and expression of PKC-α in mice (Wang and Smart, 1999Wang H.Q. Smart R.C. Overexpression of protein kinase C-α in the epidermis of transgenic mice results in striking alterations in phorbol ester-induced inflammation and COX-2, MIP-2 and TNF-α expression but not tumor promotion.J Cell Sci. 1999; 112: 3497-3506PubMed Google Scholar) and PKC-η in humans (Koizumi et al., 1993Koizumi H. Kohno Y. Osada S. Ohno S. Ohkawara A. Kuroki T. Differentiation-associated localization of nPKCη, a Ca++-independent protein kinase C, in normal human skin and skin diseases.J Invest Dermatol. 1993; 101: 858-863Abstract Full Text PDF PubMed Google Scholar;Wollina et al., 1997Wollina U. Histochemistry of the human hair follicle.in: Jollès P. Zahn H. Höcker H. Formation and Structure of Human Hair. Birkhäuser Verlag, Basel1997: 31-58Crossref Google Scholar) has been confirmed in outer root sheaths in immunohistochemical studies. We examined the expression and translocation of PKC-α, PKC-βI, PKC-βII, and PKC-η in cultured murine hair epithelial cells. The mechanism of action by which cyclosporin A causes its immunosuppressive effects has been a major focus of research interest. Cyclosporin A is known to block activation of T cells by blocking interleukin-2 (IL-2) production (Buurman et al., 1986Buurman W.A. Ruers T.J.M. Daemen I.A.J.J.M. van der Linden C.J. Groenewegen G. Cyclosporin A inhibits IL-2-driven proliferation of human alloactivated T cells.J Immunol. 1986; 136: 4035-4039PubMed Google Scholar). Up to now, as the targets of cyclosporin A, at least two routes have been proposed that lead to inhibition of IL-2 production. One is the cyclophilin-calcineurin-NFAT (nuclear factor of activated T cells) route. It has been elucidated that cyclosporin A binds to cyclophilin (cis-trans peptidyl-prolyl isomerase) (Fischer et al., 1989Fischer G. Wittmann-Liebold B. Lang K. Kiefhaber T. Schmid F.X. Cyclophilin and peptidyl-prolyl cis-trans isomerase are probably identical proteins.Nature. 1989; 337: 476-478Crossref PubMed Scopus (1172) Google Scholar;Takahashi et al., 1989Takahashi N. Hayano T. Suzuki M. Peptidyl-prolyl cis-trans isomerase is the cyclosporin A-binding protein cyclophilin.Nature. 1989; 337: 473-475Crossref PubMed Scopus (913) Google Scholar;Friedman and Weissman, 1991Friedman J. Weissman I. Two cytoplasmic candidates for immunophilin action are revealed by affinity for a new cyclophilin: one in the presence and one in the absence of CsA.Cell. 1991; 66: 799-806Abstract Full Text PDF PubMed Scopus (356) Google Scholar), an intracellular receptor, and this complex binds to and inhibits the calcium- and calmodulin-dependent phosphatase calcineurin (Liu et al., 1991Liu J. Farmer Jr, J.D. Lane W.S. Friedman J. Weissman I. Schreiber S.L. Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506 complexes.Cell. 1991; 66: 807-815Abstract Full Text PDF PubMed Scopus (3466) Google Scholar), which is known to regulate the translocation of the transcription factor NFAT to the nucleus (Flanagan et al., 1991Flanagan W.M. Corthésy B. Bram R.J. Crabtree G.R. Nuclear association of a T-cell transcription factor blocked by FK-506 and cyclosporin A.Nature. 1991; 352: 803-807Crossref PubMed Scopus (924) Google Scholar), resulting in prevention of IL-2 gene transcription (Elliott et al., 1984Elliott J.F. Lin Y. Mizel S.B. Bleackley R.C. Harnish D.G. Paetkau V. Induction of interleukin 2 messenger RNA inhibited by cyclosporin A.Science. 1984; 226: 1439-1441Crossref PubMed Scopus (334) Google Scholar;Granelli-Piperno et al., 1984Granelli-Piperno A. Inaba K. Steinman R.M. Stimulation of lymphokine release from T lymphoblasts.J Exp Med. 1984; 160: 1792-1802Crossref PubMed Scopus (147) Google Scholar;Krönke et al., 1984Krönke M. Leonard W.J. Depper J.M. et al.Cyclosporin A inhibits T-cell growth factor gene expression at the level of mRNA transcription.Proc Natl Acad Sci USA. 1984; 81: 5214-5218Crossref PubMed Scopus (553) Google Scholar). Another hypothesis involves the phospholipid metabolism PKC route. Cyclosporin A has been shown to be associated with PKC isozyme induction in lymphocytes (Kimball et al., 1993Kimball P.M. Kerman R.K. van Buren C.T. Lewis R.M. Katz S. Kahan B.D. Cyclosporine and rapamycin affect protein kinase C induction of the intracellular activation signal, activator of DNA replication.Transplantation. 1993; 55: 1128-1132Crossref PubMed Scopus (12) Google Scholar). Prevention of the activation of some PKC isozymes such as PKC-β is hypothesized as the mechanism by which cyclosporin A inhibits IL-2 synthesis and proliferation in stimulated human lymphocytes (Szamel et al., 1993Szamel M. Bartels F. Resch K. Cyclosporin A inhibits T cell receptor-induced interleukin-2 synthesis of human T lymphocytes by selectively preventing a transmembrane signal transduction pathway leading to sustained activation of a protein kinase C isoenzyme, protein kinase C-β.Eur J Immunol. 1993; 23: 3072-3081Crossref PubMed Scopus (45) Google Scholar). We investigated the effect of cyclosporin A on the expression and translocation of PKC isozymes in hair epithelial cells. In this report, we discuss the hypothesis that the hair-growing activity of cyclosporin A is related to its downregulation or inhibition of translocation of some PKC isozymes in hair epithelial cells. Cyclosporin A was purchased from Sigma (St. Louis, MO). Antihuman rabbit polyclonal antibodies against PKC-α, PKC-βI, PKC-βII, and PKC-η were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The second antibody used was biotinylated goat antirabbit immunoglobulins purchased from DAKO (Glostrup, Denmark). Streptavidin-horseradish peroxidase conjugate was purchased from Amersham Pharmacia Biotech (Little Chalfont, Buckinghamshire, U.K.). Murine hair epithelial cells were isolated and cultured according to the method reported byTanigaki et al., 1990Tanigaki N. Ando H. Ito M. Hashimoto A. Kitano Y. Electron microscopic study of cultured cells from the murine hair tissues: cell growth and differentiation.Arch Dermatol Res. 1990; 282: 402-407Crossref PubMed Scopus (19) Google Scholar with suitable modifications. The dorsal skin was peeled from 4-d-old C3H/HeNCrj mice (Charles River Japan, Kanagawa, Japan), cut into approximately 5 mm widths, washed three times with Eagle's minimum essential medium (MEM) containing 60 mg per liter of kanamycin and 10% fetal bovine serum (FBS), and then dipped into MEM containing 750 IU per ml of dispase (from Bacillus polymyxa; Godo Shusei, Tokyo, Japan), 60 mg per liter of kanamycin, and 10% FBS at 4°C for 20 h. After washing with Dulbecco's phosphate-buffered calcium- and magnesium-free saline (PBS) containing 50,000 U per liter of penicillin and 50 mg per liter of streptomycin (PBS-PS), the epidermis was peeled off and the remaining dermis layer was washed three times with PBS-PS and dispersed in Dulbecco's modified Eagle's medium (DMEM) containing 0.25% collagenase (from Streptomyces parvulus; Nitta Gelatin, Osaka, Japan), 50,000 U per liter of penicillin, 50 mg per liter of streptomycin, 0.5% bovine serum albumin, and 20% FBS at 37°C for 1 h, stirring occasionally. This dermis suspension was filtered through a 212 µm nylon mesh, and the filtrate was centrifuged at 1400 rpm for 7 min. The pellet was then resuspended in PBS-PS. The suspension was left to stand for 15 min, allowing the hair follicle tissue to precipitate due to the difference in specific gravity, after which the supernatant was removed using an aspirator. The hair follicle tissue was resuspended in PBS-PS and then precipitated. This precipitation process was repeated three times. Finally, the hair follicle tissue was incubated in 0.05% ethylenediamine tetraacetic acid (EDTA)-0.25% trypsin in Hanks' balanced calcium- and magnesium-free salt solution (HBSS) (Life Technologies, MD) at 37°C for 5 min. The hair follicle cells were suspended in DMEM supplemented with 50,000 U per liter of penicillin, 50 mg per liter of streptomycin, and 10% FBS at a density of 2 × 105 cells per ml after filtration via a 212 µm nylon mesh. This hair follicle cell suspension was pipetted into a 24-well type I collagen-coated plate (2 cm2 per well; Iwaki Glass, Chiba, Japan) at a rate of 1 ml per well (1 × 105 cells per cm2) and incubated in a humidified atmosphere containing 5% CO2 at 37°C for 24 h. After 24 h incubation, the medium was exchanged with MCDB153 (Sigma) (Boyce and Ham, 1983Boyce S.T. Ham R.G. Calcium-regulated differentiation of normal human epidermal keratinocytes in chemically defined clonal culture and serum-free serial culture.J Invest Dermatol. 1983; 81: 33S-40SCrossref PubMed Scopus (917) Google Scholar) containing 5 mg per liter of bovine insulin, 5 µg per liter of mouse epidermal growth factor, 40 mg per liter of bovine pituitary extract, 10 mg per liter of human transferrin, 0.4 mg per liter of hydrocortisone, 0.63 µg per liter of progesterone, 14 mg per liter of O-phosphorylethanolamine, 6.1 mg per liter of ethanolamine, 50,000 U per liter of penicillin, and 50 mg per liter of streptomycin after washing with PBS. It was then further incubated in a humidified atmosphere containing 5% CO2 at 37°C for 5 d. During incubation, the medium was removed and replaced with fresh medium every other day. The dorsal skin was peeled from 4-d-old C3H/HeNCrj mice (Charles River Japan), cut into approximately 5 mm widths, washed three times with MEM containing 60 mg per liter of kanamycin and 10% FBS, and then dipped into MEM containing 750 IU per ml of dispase (from Bacillus polymyxa; Godo Shusei), 60 mg per liter of kanamycin, and 10% FBS at 4°C for 20 h. After washing with PBS-PS, the epidermis was peeled off, washed five times with PBS-PS, and then immersed in 0.05% EDTA-0.25% trypsin in HBSS at 37°C for 10 min, stirring occasionally. After 10 min, DMEM (supplemented with 50,000 U per liter of penicillin, 50 mg per liter of streptomycin, and 10% FBS) was added, and the suspension was filtered through a 212 µm nylon mesh, followed by centrifugation at 1500 rpm for 5 min. The keratinocytes thus obtained were suspended in DMEM supplemented with 50,000 U per liter of penicillin, 50 mg per liter of streptomycin, and 10% FBS, pipetted into a 24-well type I collagen-coated plate (2 cm2 per well; Iwaki Glass) at an initial cell density of 1 × 105 cells per cm2, and incubated in a humidified atmosphere containing 5% CO2 at 37°C for 24 h. After 24 h incubation, the medium was exchanged with MCDB153 containing the same additives as those for the hair epithelial cells after washing with PBS. It was then further incubated in a humidified atmosphere containing 5% CO2 at 37°C for 5 d. During incubation, the medium was removed and replaced with fresh medium every other day. The degree of cell growth was determined by means of an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay (Carmichael et al., 1987Carmichael J. DeGraff W.G. Gazdar A.F. Minna J.D. Mitchell J.B. Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing.Cancer Res. 1987; 47: 936-942PubMed Google Scholar). MTT reagent (Sigma) was dissolved in PBS at a concentration of 5 mg per ml, filtered through a 0.45 µm membrane filter (cellulose acetate, DISMIC-13 cp; Advantec, Tokyo, Japan), and added 10% (vol/vol) to the culture medium. The culture plate was further incubated in a humidified atmosphere containing 5% CO2 at 37°C for 4 h. After removing the medium, the formed dye was extracted with acidic isopropanol containing 0.04 N HCl (adding 1.0 ml per 2 cm2 well), and the absorbance was measured at 570 nm relative to 640 nm. Cultured hair epithelial cells were washed twice with PBS, treated for a few minutes with 0.05% trypsin (Sigma) dissolved in PBS, and, after addition of an equal amount of 0.06% trypsin inhibitor dissolved in PBS (from soybean, inhibiting activity of 1.4 mg of trypsin per 1.0 mg; Sigma), scraped, and centrifuged (1500 rpm, 5 min). The resultant cell pellet was sonicated in five 10 s bursts in Buffer A [20 mM Tris(hydroxymethyl)aminomethane (Tris) HCl (pH 7.5), 2 mM EDTA, 10 mM ethyleneglycol-bis(β-aminoethyl ether)-N, N, N′,N′-tetraacetic acid, 0.25 M sucrose, 2 mM phenylmethylsulfonyl fluoride, 10 µg per ml leupeptin, and 10 mM 2-mercaptoethanol, final concentrations] and centrifuged at 100,000 × g for 60 min (4°C). The supernatants were concentrated to 1/10 volume using an ultrafilter (M.W. 30,000 cutting, UFP2 TTK; Millipore, MA) and stored as the cytosol fraction at -80°C. The pellets were then dissolved in Buffer B [Buffer A + 0.5% (wt/vol) polyoxyethylene (10) octylphenyl ether (Triton X-100)], sonicated in five 10 s bursts, and centrifuged at 100,000 × g for 60 min (4°C). The supernatants were concentrated to 1/10 volume using an ultrafilter (M.W. 30,000 cutting, UFP2 TTK; Millipore) and stored as the particulate fraction at -80°C. Protein concentrations were determined spectrophotometrically using a DC-Protein Assay kit (Bio-Rad, CA). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to the method ofLaemmli, 1970Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature. 1970; 227: 680-685Crossref PubMed Scopus (202762) Google Scholar. Aliquots (10 µg of protein per lane) of the cytosol or particulate fractions were mixed with sample buffer (0.125 M Tris-HCl (pH 6.8), 2% (wt/vol) SDS, 10% (wt/vol) glycerol, and 5% (vol/vol) 2-mercaptoethanol, final concentrations), heated at 98°C for 3 min, and subjected to SDS-PAGE using 7.5% polyacrylamide gel (Pagel NPU-7.5; Atto, Tokyo, Japan). Protein standards for molecular weight were as follows: phosphorylase B, 97.4 kDa; bovine serum albumin, 68.0 kDa; ovalbumin, 46.0 kDa; carbonic anhydrase, 31.0 kDa; trypsin inhibitor, 20.1 kDa; lysozyme, 14.4 kDa (ECL protein molecular weight markers, Amersham Pharmacia Biotech). The proteins were electro-blotted onto a nitrocellulose membrane (Schleicher & Schuell, Keene, NH) using a submarine transfer apparatus (Trans-Blot Cell, Bio-Rad) for 3 h at 60 V/320 cm2 (Towbin et al., 1979Towbin H. Staehelin T. Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.Proc Natl Acad Sci USA. 1979; 76: 4350-4354Crossref PubMed Scopus (44209) Google Scholar). The transfer buffer conditions were as follows: 25 mM Tris-HCl (pH 8.3), 192 mM glycine, and 20% (vol/vol) methanol. The blocking was performed on a rocking platform in a solution containing 5% bovine serum albumin and 0.1% (wt/vol) polyoxyethylene (20) sorbitan monolaurate (Tween 20) in PBS (PBS-T) for 1 h at room temperature, and the membranes were then washed with PBS-T for 5 min (three times). Next, the membranes were incubated overnight at 4°C with diluted polyclonal antibodies (antihuman rabbit antibodies, ×500 dilution by the blocking solution) against PKC isozymes (α, βI, βII, and η). The membranes were then washed with PBS-T, incubated with biotinylated goat antirabbit immunoglobulins (×3000 dilution by PBS-T) for 1 h at room temperature, and washed with PBS-T. Next, the membranes were incubated with streptavidin-horseradish peroxidase conjugate (×1000 dilution with PBS-T) for 1 h at room temperature and then washed with PBS-T. Detection of immunoreactive protein was achieved by chemiluminescence using the ECL Western blotting detection system (Amersham Pharmacia Biotech) and exposed to X-ray film (RX-U, Fuji Photo Film, Tokyo, Japan). Protein bands were identified as PKC by their molecular weight, comigration with their standard proteins (PKC-α, PKC-βI, PKC-βII, and PKC-η; human recombinant; Calbiochem-Novabiochem, CA), and lack of staining by the secondary antibody when the primary antibody was omitted. Quantitative analysis of PKC isozyme expression was performed by densitometry (CS-9000; Shimadzu, Kyoto, Japan). It is reported that cyclosporin A inhibits the growth of epidermal keratinocytes (Furue et al., 1988Furue M. Gaspari A.A. Katz S.I. The effect of cyclosporin A on epidermal cells. II. Cyclosporin A inhibits proliferation of normal and transformed keratinocytes.J Invest Dermatol. 1988; 90: 796-800Abstract Full Text PDF PubMed Google Scholar;Nickoloff et al., 1988Nickoloff B.J. Fisher G.J. Mitra R.S. Voorhees J.J. Additive and synergistic antiproliferative effects of cyclosporin A and gamma interferon on cultured human keratinocytes.Am J Pathol. 1988; 131: 12-18PubMed Google Scholar). We compared the effects of cyclosporin A on epidermal keratinocyte and hair epithelial cell growth. Our results confirmed that cyclosporin A promotes cultured murine hair epithelial cell growth at about 150%-160% relative to controls over the wide concentration range of 1–1000 ng per ml. At a high dose of 3 µg per ml of cyclosporin A, hair epithelial cell growth was partially inhibited; and hair epithelial cell growth was completely inhibited above a cyclosporin A concentration of 10 µg per ml Figure 1. We observed morphologic changes, such as elongation and vacuolization, in hair epithelial cells incubated in 3 µg per ml of cyclosporin A Figure 2c. We also confirmed that cyclosporin A promotes murine epidermal keratinocyte growth at about 140% relative to controls over the wide concentration range of 1–100 ng per ml and partially inhibits the growth of epidermal keratinocytes at concentrations of 1–3 µg per ml. Epidermal keratinocyte growth was completely inhibited above a cyclosporin A concentration of 10 µg per ml Figure 3. We observed morphologic changes, such as enlargement and vacuolization, in epidermal keratinocytes incubated with 3 µg per ml of cyclosporin A Figure 2e.Figure 2Micrographs of cultured hair epithe
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