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• W2007153143 abstract "The epidermal growth factor receptor and its ligands initiate a major signaling pathway that regulates keratinocyte growth in an autocrine manner. It is well known that high doses of epidermal growth factor receptor ligands inhibit keratinocyte growth. Recently, signal transducers and activators of transcription 1-dependent p21Waf1/Cip1 induction were reported to be involved in high-dose epidermal growth factor-dependent cell growth arrest in the A431 squamous cell carcinoma cell line; however, transfection of dominant-negative signal transducers and activators of transcription 1 adenovirus vector did not block epidermal growth factor-induced growth inhibition in normal human keratinocytes. As transforming growth factor β is a potent inhibitor of keratinocyte proliferation, we hypothesized that transforming growth factor β contributes to epidermal growth factor-mediated keratinocyte growth inhibition. Epidermal growth factor concentrations of 10 ng per ml enhanced transforming growth factor β1 mRNA expression from 3 to 6 h poststimulation. Enzyme-linked immunosorbent assay analysis detected 150 pg per ml of transforming growth factor β1 in the culture medium of keratinocytes incubated with 10 and 100 ng per ml epidermal growth factor, whereas 0.1 and 1.0 ng per ml epidermal growth factor slightly enhance transforming growth factor β1 production. Epidermal growth factor (100 ng per ml) upregulated luciferase activity of p3TP-lux, which contains three tandem transforming growth factor β-Smad signaling responsive elements, 6-fold compared with unstimulated cells. The epidermal growth factor-dependent induction of p3TP-lux luciferase activity was disrupted by transfection of the dominant negative form of transforming growth factor β type I receptor adenovirus vector (AxdnALK5), which suggests that epidermal growth factor-induced transforming growth factor β acts in an autocrine manner in keratinocytes. Moreover, transfection of AxdnALK5 completely blocked the growth inhibition induced by 100 ng per ml of epidermal growth factor in normal keratinocytes. These data demonstrate that an autocrine transforming growth factor β1-ALK5 pathway is a negative feedback mechanism for epidermal growth factor-induced normal human keratinocyte growth. The epidermal growth factor receptor and its ligands initiate a major signaling pathway that regulates keratinocyte growth in an autocrine manner. It is well known that high doses of epidermal growth factor receptor ligands inhibit keratinocyte growth. Recently, signal transducers and activators of transcription 1-dependent p21Waf1/Cip1 induction were reported to be involved in high-dose epidermal growth factor-dependent cell growth arrest in the A431 squamous cell carcinoma cell line; however, transfection of dominant-negative signal transducers and activators of transcription 1 adenovirus vector did not block epidermal growth factor-induced growth inhibition in normal human keratinocytes. As transforming growth factor β is a potent inhibitor of keratinocyte proliferation, we hypothesized that transforming growth factor β contributes to epidermal growth factor-mediated keratinocyte growth inhibition. Epidermal growth factor concentrations of 10 ng per ml enhanced transforming growth factor β1 mRNA expression from 3 to 6 h poststimulation. Enzyme-linked immunosorbent assay analysis detected 150 pg per ml of transforming growth factor β1 in the culture medium of keratinocytes incubated with 10 and 100 ng per ml epidermal growth factor, whereas 0.1 and 1.0 ng per ml epidermal growth factor slightly enhance transforming growth factor β1 production. Epidermal growth factor (100 ng per ml) upregulated luciferase activity of p3TP-lux, which contains three tandem transforming growth factor β-Smad signaling responsive elements, 6-fold compared with unstimulated cells. The epidermal growth factor-dependent induction of p3TP-lux luciferase activity was disrupted by transfection of the dominant negative form of transforming growth factor β type I receptor adenovirus vector (AxdnALK5), which suggests that epidermal growth factor-induced transforming growth factor β acts in an autocrine manner in keratinocytes. Moreover, transfection of AxdnALK5 completely blocked the growth inhibition induced by 100 ng per ml of epidermal growth factor in normal keratinocytes. These data demonstrate that an autocrine transforming growth factor β1-ALK5 pathway is a negative feedback mechanism for epidermal growth factor-induced normal human keratinocyte growth. epidermal growth factor receptor signal transducers and activators of transcription activin receptor-like kinase The binding of epidermal growth factor (EGF) family ligands to EGF receptors (EGFR) initiates a major signaling pathway that regulates epithelial cell growth (Gusterson et al., 1984Gusterson B. Cowley G. Smith J.A. Ozanne B. Cellular localisation of human epidermal growth factor receptor.Cell Biol Int Rep. 1984; 8: 649-658Crossref PubMed Scopus (151) Google Scholar;Johnson et al., 1993Johnson G.R. Kannan B. Shoyab M. Stromberg K. Amphiregulin induces tyrosine phosphorylation of the epidermal growth factor receptor and p185erbB2. Evidence that amphiregulin acts exclusively through the epidermal growth factor receptor at the surface of human epithelial cells.J Biol Chem. 1993; 268: 2924-2931PubMed Google Scholar;Alison and Sarraf, 1994Alison M.R. Sarraf C.E. The role of growth factors in gastrointestinal cell proliferation.Cell Biol Int. 1994; 18: 1-10Crossref PubMed Scopus (76) Google Scholar;Filipe et al., 1995Filipe M.I. Osborn M. Linehan J. Sanidas E. Brito M.J. Jankowski J. Expression of transforming growth factor alpha, epidermal growth factor receptor and epidermal growth factor in precursor lesions to gastric carcinoma.Br J Cancer. 1995; 71: 30-36Crossref PubMed Scopus (49) Google Scholar;Rajagopal et al., 1995Rajagopal S. Huang S. Moskal T.L. Lee B.N. el-Naggar A.K. Chakrabarty S. Epidermal growth factor expression in human colon and colon carcinomas: anti-sense epidermal growth factor receptor RNA down-regulates the proliferation of human colon cancer cells.Int J Cancer. 1995; 62: 661-667Crossref PubMed Scopus (37) Google Scholar). Some types of breast cancer cells have abundant EGFR, and EGFR plays a crucial part in breast cancer tumor progression (Battaglia et al., 1988Battaglia F. Polizzi G. Scambia G. et al.Receptors for epidermal growth factor and steroid hormones in human breast cancer.Oncology. 1988; 45: 424-427Crossref PubMed Scopus (23) Google Scholar;Bevilacqua et al., 1990Bevilacqua P. Gasparini G. Dal Fior S. Corradi G. Immunocytochemical determination of epidermal growth factor receptor with monoclonal EGFR1 antibody in primary breast cancer patients.Oncology. 1990; 47: 313-317Crossref PubMed Scopus (26) Google Scholar;Toi et al., 1990Toi M. Nakamura T. Mukaida H. et al.Relationship between epidermal growth factor receptor status and various prognostic factors in human breast cancer.Cancer. 1990; 65: 1980-1984Crossref PubMed Scopus (89) Google Scholar). Although the A431 squamous carcinoma cell line has abundant EGFR, high doses of EGF inhibit A431 cell growth (Gill and Lazar, 1981Gill G.N. Lazar C.S. Increased phosphotyrosine content and inhibition of proliferation in EGF-treated A431 cells.Nature. 1981; 293: 305-307Crossref PubMed Scopus (323) Google Scholar;Barnes, 1982Barnes D.W. Epidermal growth factor inhibits growth of A431 human epidermoid carcinoma in serum-free cell culture.J Cell Biol. 1982; 93: 1-4Crossref PubMed Scopus (235) Google Scholar). The EGF family, which includes heparin-binding epidermal growth factor-like growth factor, transforming growth factor (TGF)-α, and amphiregulin, is well known as an autocrine growth factor for normal human keratinocytes (Coffey et al., 1987Coffey R.J.J. Derynck R. Wilcox J.N. Bringman T.S. Goustin A.S. Moses H.L. Pittelkow M.R. Production and autoinduction of transforming growth factor-α in human keratinocytes.Nature. 1987; 328: 817-820Crossref PubMed Scopus (691) Google Scholar;Cook et al., 1991Cook P.W. Mattox P.A. Keeble W.W. et al.A heparin sulfate-regulated human keratinocytes autocrine factor is similar or identical to amphiregulin.Mol Cell Biol. 1991; 11: 2547-2557Crossref PubMed Scopus (208) Google Scholar;Hashimoto et al., 1994Hashimoto K. Higashiyama S. Asada H. et al.Heparin-binding epidermal growth factor-like growth factor is an autocrine growth factor of human keratinocytes.J Biol Chem. 1994; 269: 20060-20066PubMed Google Scholar;Shirakata et al., 2000Shirakata Y. Komurasaki T. Toyoda H. et al.Epiregulin, a novel member of the epidermal growth factor family, is an autocrine growth factor in normal human keratinocytes.J Biol Chem. 2000; 275: 5748-5753Crossref PubMed Scopus (144) Google Scholar). Similar to A431 cells, low doses of EGF, heparin-binding-EGF, and epiregulin stimulation promote keratinocyte growth, whereas high doses of 10–100 ng per ml inhibit proliferation (Hashimoto et al., 1994Hashimoto K. Higashiyama S. Asada H. et al.Heparin-binding epidermal growth factor-like growth factor is an autocrine growth factor of human keratinocytes.J Biol Chem. 1994; 269: 20060-20066PubMed Google Scholar;Chen et al., 1995Chen T.C. Persons K. Liu W.W. Chen M.L. Holick M.F. The antiproliferative and differentiative activities of 1,25-dihydroxyvitamin D3 are potentiated by epidermal growth factor and attenuated by insulin in cultured human keratinocytes.J Invest Dermatol. 1995; 104: 113-117Abstract Full Text PDF PubMed Scopus (59) Google Scholar;Shirakata et al., 2000Shirakata Y. Komurasaki T. Toyoda H. et al.Epiregulin, a novel member of the epidermal growth factor family, is an autocrine growth factor in normal human keratinocytes.J Biol Chem. 2000; 275: 5748-5753Crossref PubMed Scopus (144) Google Scholar). EGFR internalization occurs after the binding of EGFR ligands (Gusterson et al., 1984Gusterson B. Cowley G. Smith J.A. Ozanne B. Cellular localisation of human epidermal growth factor receptor.Cell Biol Int Rep. 1984; 8: 649-658Crossref PubMed Scopus (151) Google Scholar;Lehtola et al., 1989Lehtola L. Lehvaslaiho H. Sistonen L. Beguinot L. Alitalo K. Receptor downregulation and DNA synthesis are modulated by EGF and TPA in cells expressing an EGFR/neu chimera.Growth Factors. 1989; 1: 323-334Crossref PubMed Scopus (7) Google Scholar;Bevilacqua et al., 1990Bevilacqua P. Gasparini G. Dal Fior S. Corradi G. Immunocytochemical determination of epidermal growth factor receptor with monoclonal EGFR1 antibody in primary breast cancer patients.Oncology. 1990; 47: 313-317Crossref PubMed Scopus (26) Google Scholar). This downregulation is one of the EGFR signal regulatory mechanisms (Masui et al., 1993Masui H. Castro L. Mendelsohn J. Consumption of EGF by A431 cells: evidence for receptor recycling.J Cell Biol. 1993; 120: 85-93Crossref PubMed Scopus (68) Google Scholar;Skarpen et al., 1998Skarpen E. Johannessen L.E. Bjerk K. et al.Endocytosed epidermal growth factor (EGF) receptors contribute to the EGF-mediated growth arrest in A431 cells by inducing a sustained increase in p21/CIP1.Exp Cell Res. 1998; 243: 161-172Crossref PubMed Scopus (43) Google Scholar); however, the mechanism of EGF-dependent growth inhibition has yet to be fully assessed in keratinocytes. Recently, some groups reported that in A431 cells, the cyclin-dependent kinase (CDK) inhibitor p21 is involved in EGF-dependent cell growth arrest (Jakus and Yeudall, 1996Jakus J. Yeudall W.A. Growth inhibitory concentrations of EGF induce p21 (WAF1/Cip1) and alter cell cycle control in squamous carcinoma cells.Oncogene. 1996; 12: 2369-2376PubMed Google Scholar;Ohtsubo et al., 1998Ohtsubo M. Gamou S. Shimizu N. Anti-sense oligonucleotide of WAF1 gene prevents EGF-induced cell-cycle arrest in A431 cells.Oncogene. 1998; 16: 797-802Crossref PubMed Scopus (25) Google Scholar;Skarpen et al., 1998Skarpen E. Johannessen L.E. Bjerk K. et al.Endocytosed epidermal growth factor (EGF) receptors contribute to the EGF-mediated growth arrest in A431 cells by inducing a sustained increase in p21/CIP1.Exp Cell Res. 1998; 243: 161-172Crossref PubMed Scopus (43) Google Scholar), and that EGF-dependent STAT (signal transducers and activators of transcription) 1 activation induces p21 (Bromberg et al., 1998Bromberg J.F. Fan Z. Brown C. Mendelsohn J. Darnell Jr, J.E. Epidermal growth factor-induced growth inhibition requires Stat1 activation.Cell Growth Differ. 1998; 9: 505-512PubMed Google Scholar;Ohtsubo et al., 2000Ohtsubo M. Takayanagi A. Gamou S. Shimizu N. Interruption of NFkappaB-STAT1 signaling mediates EGF-induced cell-cycle arrest.J Cell Physiol. 2000; 184: 131-137Crossref PubMed Scopus (23) Google Scholar); however, our preliminary experiment shows that transfection of dominant-negative STAT1 adenovirus vector did not block the growth inhibition dependent on high-dose EGF in human primary keratinocytes. These data suggest that another molecule is involved in EGF-induced growth inhibition in these cells. Interferon (IFN)-γ and TGF-β are potent inhibitors of keratinocyte proliferation (Nickoloff et al., 1984Nickoloff B.J. Basham T.Y. Merigan T.C. Morhenn V.B. Antiproliferative effects of recombinant alpha- and gamma-interferons on cultured human keratinocytes.Lab Invest. 1984; 51: 697-701PubMed Google Scholar;Matsumoto et al., 1990Matsumoto K. Hashimoto K. Hashiro M. Yoshimasa H. Yoshikawa K. Modulation of growth and differentiation in normal human keratinocytes by transforming growth factor-beta.J Cell Physiol. 1990; 145: 95-101Crossref PubMed Scopus (80) Google Scholar). In keratinocytes, IFN-γ stimulation activates STAT1 and induces transcription of the CDK inhibitor p21. TGF-β binds to TGF-β type II receptor, which then phosphorylates the TGF-β type I receptor (activin receptor-like kinase 5; ALK5). Phosphorylated ALK5 recruits and activates the signal transduction molecules Smad2 and Smad3 (Attisano and Wrana, 2000Attisano L. Wrana J.L. Smads as transcriptional co-modulators.Curr Opin Cell Biol. 2000; 12: 235-243Crossref PubMed Scopus (476) Google Scholar;Datto and Wang, 2000Datto M. Wang X.F. The Smads: transcriptional regulation and mouse models.Cytokine Growth Factor Rev. 2000; 11: 37-48Crossref PubMed Scopus (49) Google Scholar;Massague and Wotton, 2000Massague J. Wotton D. Transcriptional control by the TGF-beta/Smad signaling system.EMBO J. 2000; 19: 1745-1754Crossref PubMed Google Scholar;Miyazono, 2000Miyazono K. TGF-beta signaling by Smad proteins.Cytokine Growth Factor Rev. 2000; 11: 15-22Crossref PubMed Scopus (233) Google Scholar). TGF-β induced G0/G1 phase cell cycle arrest of various cells (Longstreet et al., 1992Longstreet M. Miller B. Howe P.H. Loss of transforming growth factor beta 1 (TGF-beta 1)-induced growth arrest and p34cdc2 regulation in ras-transfected epithelial cells.Oncogene. 1992; 7: 1549-1556PubMed Google Scholar;Reddy et al., 1994Reddy K.B. Hocevar B.A. Howe P.H. Inhibition of G1 phase cyclin dependent kinases by transforming growth factor beta 1.J Cell Biochem. 1994; 56: 418-425Crossref PubMed Scopus (34) Google Scholar), and TGF-β induces the CDK inhibitors p27 and p16 in keratinocytes and inhibits cell proliferation (Hannon and Beach, 1994Hannon G.J. Beach D. p15INK4B is a potential effector of TGF-beta-induced cell cycle arrest.Nature. 1994; 371: 257-261Crossref PubMed Scopus (1885) Google Scholar;Ewen, 1996Ewen M.E. p53-dependent repression of cdk4 synthesis in transforming growth factor-beta-induced G1 cell cycle arrest.J Lab Clin Med. 1996; 128: 355-360Abstract Full Text PDF PubMed Scopus (24) Google Scholar). Keratinocyte IFN-γ production is species dependent; there are reports that human keratinocytes produce IFN-γ, whereas murine keratinocytes do not (Howie et al., 1996Howie S.E. Aldridge R.D. McVittie E. Forsey R.J. Sands C. Hunter J.A. Epidermal keratinocyte production of interferon-gamma immunoreactive protein and mRNA is an early event in allergic contact dermatitis.J Invest Dermatol. 1996; 106: 1218-1223Abstract Full Text PDF PubMed Scopus (31) Google Scholar;Akiba et al., 2001Akiba H. Ducluzeau M.T. Nicolas J.F. Interferon-gamma production in skin during contact hypersensitivity. No contribution from keratinocytes.J Invest Dermatol. 2001; 117: 163Abstract Full Text Full Text PDF PubMed Google Scholar). On the other hand, both human and murine keratinocytes produce TGF-β1 and TGF-β2 (Bascom et al., 1989Bascom C.C. Wolfshohl J.R. Coffey Jr, R.J. et al.Complex regulation of transforming growth factor beta 1, beta 2, and beta 3 mRNA expression in mouse fibroblasts and keratinocytes by transforming growth factors beta 1 and beta 2.Mol Cell Biol. 1989; 9: 5508-5515Crossref PubMed Google Scholar;Glick et al., 1990Glick A.B. Danielpour D. Morgan D. Sporn M.B. Yuspa S.H. Induction and autocrine receptor binding of transforming growth factor-beta 2 during terminal differentiation of primary mouse keratinocytes.Mol Endocrinol. 1990; 4: 46-52Crossref PubMed Scopus (61) Google Scholar;Keski-Oja and Koli, 1992Keski-Oja J. Koli K. Enhanced production of plasminogen activator activity in human and murine keratinocytes by transforming growth factor-beta 1.J Invest Dermatol. 1992; 99: 193-200Abstract Full Text PDF PubMed Scopus (26) Google Scholar). The activator protein-1 site in the TGF-β promoter region is stimulated by growth factors through mitogen-activated protein kinase activation (Salbert et al., 1993Salbert G. Fanjul A. Piedrafita F.J. Lu X.P. Kim S.J. Tran P. Pfahl M. Retinoic acid receptors and retinoid X receptor-alpha down-regulate the transforming growth factor-beta 1 promoter by antagonizing AP-1 activity.Mol Endocrinol. 1993; 7: 1347-1356Crossref PubMed Google Scholar;Rak et al., 1995Rak J. Filmus J. Finkenzeller G. Grugel S. Marme D. Kerbel R.S. Oncogenes as inducers of tumor angiogenesis.Cancer Metastasis Rev. 1995; 14: 263-277Crossref PubMed Scopus (213) Google Scholar). The EGF family phosphorylates EGFR, and EGFR activates mitogen-activated protein kinases, phosphatidylinositol 3 kinase, STAT, and G proteins (Nakamura et al., 1996Nakamura N. Chin H. Miyasaka N. Miura O. An epidermal growth factor receptor/Jak2 tyrosine kinase domain chimera induces tyrosine phosphorylation of Stat5 and transduces a growth signal in hematopoietic cells.J Biol Chem. 1996; 271: 19483-19488Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar;Grandis et al., 1998Grandis J.R. Drenning S.D. Chakraborty A. Zhou M.Y. Zeng Q. Pitt A.S. Tweardy D.J. Requirement of Stat3 but not Stat1 activation for epidermal growth factor receptor-mediated cell growth In vitro.J Clin Invest. 1998; 102: 1385-1392Crossref PubMed Google Scholar;Toyoda et al., 1998Toyoda M. Gotoh N. Handa H. Shibuya M. Involvement of MAP kinase-independent protein kinase C signaling pathway in the EGF-induced p21 (WAF1/Cip1) expression and growth inhibition of A431 cells.Biochem Biophys Res Commun. 1998; 250: 430-435Crossref PubMed Scopus (25) Google Scholar;Hackel et al., 1999Hackel P.O. Zwick E. Prenzel N. Ullrich A. Epidermal growth factor receptors: critical mediators of multiple receptor pathways.Curr Opin Cell Biol. 1999; 11: 184-189Crossref PubMed Scopus (546) Google Scholar;Liebmann, 2001Liebmann C. Regulation of MAP kinase activity by peptide receptor signalling pathway: paradigms of multiplicity.Cell Signal. 2001; 13: 777-785Crossref PubMed Scopus (250) Google Scholar). Therefore, we hypothesize the involvement of TGF-β in high-dose EGF-dependent keratinocyte growth inhibition. This study demonstrates that inhibiting TGF-β receptor signaling results in the suppression of high-dose EGF-induced growth inhibition in keratinocytes. Normal human skin obtained from plastic surgery was cut into 3–5 mm strips and incubated with 250 U per ml of dispase in Dulbecco's modified Eagle's medium overnight at 4°C. The epidermis was separated from the dermis by forceps, and the epidermal sheets were rinsed with phosphate-buffered saline (–), incubated in a 0.25% trypsin solution for 10 min at 37°C, and teased with forceps. Epidermal cells were rinsed, collected by centrifugation, and resuspended in MCDB153 medium supplemented with insulin (1 μg per ml), hydrocortisone (0.5 μg per ml), ethanolamine (0.1 mM), phosphoethanolamine (0.1 mM), CaCl2 (0.03 mM), and bovine hypothalamic extract (BHE) (50 μg per ml). Second or third passage cells were used for all experiments. All procedure involving human subjects received prior approval from the ethical committee of the Ehime University School of Medicine, and all subjects provided written informed consent. Recombinant IFN-γ and EGF were generous gifts from Ohtsuka Pharmaceutical Co. Ltd (Tokyo, Japan). Recombinant TGF-β1, anti-TGF-β1 monoclonal neutralizing antibody, and normal mouse IgG were obtained from R&D Systems (Minneapolis, MN). The cosmid cassette pAxCAw (Miyake et al., 1996Miyake S. Makimura M. Kanegae Y. et al.Efficient generation of recombinant adenoviruses using adenovirus DNA-terminal protein complex and a cosmid bearing the full-length virus genome.Proc Natl Acad Sci USA. 1996; 93: 1320-1324Crossref PubMed Scopus (786) Google Scholar), control adenovirus AxCALacZ, and the parent virus Ad5-dLX (Miyake et al., 1996Miyake S. Makimura M. Kanegae Y. et al.Efficient generation of recombinant adenoviruses using adenovirus DNA-terminal protein complex and a cosmid bearing the full-length virus genome.Proc Natl Acad Sci USA. 1996; 93: 1320-1324Crossref PubMed Scopus (786) Google Scholar) were all kind gifts from Dr Izumi Saito (Tokyo University, Japan). Fragments of Stat1F were subcloned into the adenovirus cosmid cassette pAxCAw. STAT1F codes a dominant-negative mutant, which has the tyrosine phosphorylation site substituted with phenylalanine. Adenovirus containing the CA promoter and STAT1F (AxSTAT1F) was generated by the COS-TPC method (Miyake et al., 1996Miyake S. Makimura M. Kanegae Y. et al.Efficient generation of recombinant adenoviruses using adenovirus DNA-terminal protein complex and a cosmid bearing the full-length virus genome.Proc Natl Acad Sci USA. 1996; 93: 1320-1324Crossref PubMed Scopus (786) Google Scholar). The cosmid DNA was mixed with the EcoT22I-digested DNA-terminal protein complex of Ad5-dLX, and used to cotransfect 293 cells. Recombinant viruses were generated by homologous recombination in 293 cells. Virus stocks were prepared by a standard procedure (Miyake et al., 1996Miyake S. Makimura M. Kanegae Y. et al.Efficient generation of recombinant adenoviruses using adenovirus DNA-terminal protein complex and a cosmid bearing the full-length virus genome.Proc Natl Acad Sci USA. 1996; 93: 1320-1324Crossref PubMed Scopus (786) Google Scholar). Concentrated, purified virus stocks were prepared by CsCl gradient, and the virus titer was verified using a plaque-formation assay. Construction of the adenovirus vector expressing dominant-negative TGF-β type I receptor, ALK5 (AxdnALK5) has been described previously (Fujii et al., 1999Fujii M. Takeda K. Imamura T. et al.Roles of bone morphogenetic protein type I receptors and Smad proteins in osteoblast and chondroblast differentiation.Mol Biol Cell. 1999; 10: 3801-3813Crossref PubMed Scopus (369) Google Scholar). Normal human keratinocytes were infected with adenovirus vectors at a multiplicity of infection (m.o.i.) of 5. AxCALacZ was transfected as a control vector. Keratinocytes were seeded at 4.0×104 cells per well in six-well collagen-coated dishes (Iwaki Glass, Tokyo, Japan) and cultured in 3 ml of MCDB153 medium with BHE. Twenty-four hours later, the medium was replaced with fresh MCDB153 without BHE, and adenovirus vectors (m.o.i.=5) were added. The cells were incubated for 24 h for infection and gene expression. Then, various concentrations of cytokines were added and the cells were incubated for a further 96 h. For experiments with anti-TGF-β1 monoclonal neutralizing antibody, antibody, or normal mouse IgG (1 ng per ml) was added at the time of EGF stimulation. Cells were trypsinized, harvested, and the total cell number was counted using a Coulter™ Z1 (Coulter, Tokyo, Japan). The proliferation rates were calibrated by comparing the total number of stimulated and unstimulated cells. The mean and SD of relative values obtained from three independent experiments were plotted on graphs. Keratinocytes were seeded at 5.0×103 cells per well in 96-well collagen-coated dishes (Iwaki Glass) and cultured in 0.1 ml of MCDB153 medium with BHE. Adenovirus vector and cytokines were added as described for the cell counts. Ninety-six hours after stimulation, cell proliferation was quantitated using a MTT-based assay (CellTiter96™ Non-Radioactive Cell Proliferation Assay, Promega, Madison, WI). The optical density at 570 nm was measured using a plate reader (Immunomini 2100, Nalge Nunc International K.K, Tokyo, Japan) and the value obtained was subtracted from the optical density at 630 nm. The values indicate proliferation relative to the respective unstimulated control. The mean and SD obtained from three independent experiments were plotted on graphs. Total cellular RNA was isolated from cultured human keratinocytes with Isogen® (Nippon Gene, Toyama, Japan). Single-stranded anti-sense riboprobes were prepared by in vitro transcription of human cDNA fragments using a RiboQuant® In Vitro Transcription kit (Pharmingen, San Diego, CA) in the presence of [α-32P]uridine triphosphate. An hCK-3 probe set (Pharmingen) was used as a template for in vitro transcription. Samples of total RNA (10 μg each) were hybridized with 32P-labeled riboprobe and digested with RNase using a RiboQuant® RPA kit (Pharmingen) according to the manufacturer's instructions. Hybridization products were separated on a 5% polyacrylamide–8 M urea gel. The gel was absorbed on to filter paper, dried, and then exposed to Kodak Biomax MS® film (Kodak, Japan) at – 70°C. Band density was analyzed using the Diversity Database™ (PDI Inc., New York). The relative quantities of TGF-β1 and TGF-β2 mRNA were estimated using GAPDH as an internal reference, and normalized against the respective relative values at 0 h post-TGF-β2 stimulation. The relative values of TGF-β1 and TGF-β2 mRNA were plotted on graphs. Keratinocytes were seeded at 5.0×105 cells per dish in 10 cm diameter collagen-coated dishes (Iwaki Glass), and cultured in 10 ml of MCDB153 medium with BHE. The medium was replaced after 48 h with fresh MCDB153 without BHE (10 ml). Various concentrations of EGF were added and the cells were cultured for a further 3 d. Culture media were collected and stored at –20°C for analysis. TGF-β1 and TGF-β2 were measured in the culture media using QuantikineTM (R&D Systems) according to the manufacturer's instructions. The optical density at 450 nm was determined using a plate reader (Immunomini 2100, Nalge Nunc International K.K) and subtracted from the optical density at 540 nm. The mean and SD obtained from three independent experiments were plotted on graphs. Keratinocytes were seeded at 4.0×104 cells per well in 12-well collagen-coated dishes (Iwaki Glass) and cultured in 1 ml of MCDB153 medium with BHE. The medium was replaced 48 h later with 1 ml fresh MCDB153 without BHE, and AxdnALK5 or AxCALacZ were transfected (m.o.i.=5). Twenty-four hours after adenovirus transfection, 0.5 μg of p3TP-Lux (provided by J.L. Wrana), and 10 ng of pRL-CMV (Promega) were transfected using DOTAP™ (Roche Diagnostic, Tokyo, Japan) according to the manufacturer's instructions. p3TP-Lux contains three tandem repeats of a TGF-β-signal responsive element, which is activated by TGF-β stimulation (Hayashi et al., 1997Hayashi H. Abdollah S. Qiu Y. et al.The MAD-related protein Smad7 associates with the TGF-beta receptor and functions as an antagonist of TGF-beta signaling.Cell. 1997; 89: 1165-1173Abstract Full Text Full Text PDF PubMed Scopus (1159) Google Scholar). After a 10 h transfection of the reporter gene, the medium was replaced with 1 ml fresh MCDB153 without BHE, various concentrations of EGF, or 0.1 ng per ml of TGF-β1, was added. The cells were harvested 72 h later, and luciferase activity was measured using a Dual-Luciferase™ Reporter Assay System (Promega) according to the manufacturer's instructions. Relative values of p3TP-Lux luciferase activity were estimated using pRL-CMV luciferase activity as an internal reference, and normalized against the respective relative values at nonstimulation. The mean and SD of relative values obtained from three independent experiments were plotted on graphs. STAT1 is involved in EGF-dependent growth inhibition in A431 squamous carcinoma cells (Bromberg et al., 1998Bromberg J.F. Fan Z. Brown C. Mendelsohn J. Darnell Jr, J.E. Epidermal growth factor-induced growth inhibition requires Stat1 activation.Cell Growth Differ. 1998; 9: 505-512PubMed Google Scholar;Ohtsubo et al., 2000Ohtsubo M. Takayanagi A. Gamou S. Shimizu N. Interruption of NFkappaB-STAT1 signaling mediates EGF-induced cell-cycle arrest.J Cell Physiol. 2000; 184: 131-137Crossref PubMed Scopus (23) Google Scholar). We generated adenovirus vector expressing dominant-negative STAT1 (AxSTAT1F), and examined the involvement of STAT1 in primary keratinocyte proliferation. IFN-γ is well known as a potent inhibitor of primary keratinocyte growth (Nickoloff et al., 1984Nickoloff B.J. Basham T.Y. Merigan T.C. Morhenn V.B. Antiproliferative effects of recombinant alpha- and gamma-interferons on cultured human keratinocytes.Lab Invest. 1984; 51: 697-701PubMed Google Scholar), and it phosphorylates and activates STAT1 in keratinocytes (Jiang et al., 1994Jiang C.K. Flanagan S. Ohtsuki M. Shuai K. Freedberg I.M. Blumenberg M. Disease-activated transcription factor. allergic reactions in human skin cause nuclear translocation of STAT-91 and induce synthesis of keratin K17.Mol Cell Biol. 1994; 14: 4759-4769Crossref PubMed Scopus (69) Google Scholar;Aragane et al., 1997Aragane Y. Kulms D. Luger T.A. Schwarz T." @default.
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• W2007153143 title "Keratinocyte Growth Inhibition by High-Dose Epidermal Growth Factor Is Mediated by Transforming Growth Factor β Autoinduction: A Negative Feedback Mechanism for Keratinocyte Growth" @default.
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