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• W1982681624 abstract "High throughput sequencing of a mouse keratinocyte library was used to identify an expressed sequence tag with homology to the epidermal growth factor (EGF) family of growth factors. We have named the protein encoded by this expressed sequence tag Epigen, for epithelial mitogen. Epigen encodes a protein of 152 amino acids that contains features characteristic of the EGF superfamily. Two hydrophobic regions, corresponding to a putative signal sequence and transmembrane domain, flank a core of amino acids encompassing six cysteine residues and two putativeN-linked glycosylation sites. Epigen shows 24–37% identity to members of the EGF superfamily including EGF, transforming growth factor α, and Epiregulin. Northern blotting of several adult mouse tissues indicated that Epigen was present in testis, heart, and liver. Recombinant Epigen was synthesized in Escherichia coli and refolded, and its biological activity was compared with that of EGF and transforming growth factor α in several assays. In epithelial cells, Epigen stimulated the phosphorylation of c-erbB-1 and mitogen-activated protein kinases and also activated a reporter gene containing enhancer sequences present in the c-fos promoter. Epigen also stimulated the proliferation of HaCaT cells, and this proliferation was blocked by an antibody to the extracellular domain of the receptor tyrosine kinase c-erbB-1. Thus, Epigen is the newest member of the EGF superfamily and, with its ability to promote the growth of epithelial cells, may constitute a novel molecular target for wound-healing therapy.AJ291391 High throughput sequencing of a mouse keratinocyte library was used to identify an expressed sequence tag with homology to the epidermal growth factor (EGF) family of growth factors. We have named the protein encoded by this expressed sequence tag Epigen, for epithelial mitogen. Epigen encodes a protein of 152 amino acids that contains features characteristic of the EGF superfamily. Two hydrophobic regions, corresponding to a putative signal sequence and transmembrane domain, flank a core of amino acids encompassing six cysteine residues and two putativeN-linked glycosylation sites. Epigen shows 24–37% identity to members of the EGF superfamily including EGF, transforming growth factor α, and Epiregulin. Northern blotting of several adult mouse tissues indicated that Epigen was present in testis, heart, and liver. Recombinant Epigen was synthesized in Escherichia coli and refolded, and its biological activity was compared with that of EGF and transforming growth factor α in several assays. In epithelial cells, Epigen stimulated the phosphorylation of c-erbB-1 and mitogen-activated protein kinases and also activated a reporter gene containing enhancer sequences present in the c-fos promoter. Epigen also stimulated the proliferation of HaCaT cells, and this proliferation was blocked by an antibody to the extracellular domain of the receptor tyrosine kinase c-erbB-1. Thus, Epigen is the newest member of the EGF superfamily and, with its ability to promote the growth of epithelial cells, may constitute a novel molecular target for wound-healing therapy.AJ291391 epidermal growth factor transforming growth factor α heparin-binding EGF-like growth factor amphiregulin mitogen-activated protein serum response element SDS-polyacrylamide gel electrophoresis 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid The epidermis of mammalian skin is a complex structure, the assembly and maintenance of which requires the regulation of a large number of genes (1Eckert R.L. Crish J.F. Banks E.B. Welter J.F. J. Invest. Dermatol. 1997; 109: 501-509Abstract Full Text PDF PubMed Scopus (175) Google Scholar). In particular, mRNAs encoding several members of the EGF1 superfamily of growth factors have been localized to the proliferative compartment of the epidermis, suggesting that they play an important role in the maintenance of skin structure (2Turbitt M.L. Akhurst R.J. White S.I. MacKie R.M. J. Invest. Dermatol. 1990; 95: 229-232Abstract Full Text PDF PubMed Google Scholar, 3Sakai Y. Nelson K.G. Snedeker S. Bossert N.L. Walker M.P. McLachlan J. DiAugustine R.P. Cell Growth Differ. 1994; 5: 527-535PubMed Google Scholar, 4Downing M.T. Brigstock D.R. Luquette M.H. Crissman-Combs M. Besner G.E. Histochem. J. 1997; 29: 735-744Crossref PubMed Scopus (35) Google Scholar). The EGF superfamily is an expanding group of growth factors containing several members including EGF, TGFα, Epiregulin, HB-EGF, AR, betacellulin, and the neuregulins (5Cohen S. Elliot G.A. J. Invest. Dermatol. 1963; 40: 1-5Abstract Full Text PDF PubMed Scopus (217) Google Scholar, 6de Larco J.E. Todaro G.J. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 4001-4005Crossref PubMed Scopus (1199) Google Scholar, 7Shoyab M. McDonald V.L. Bradley J.G. Todaro G.J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 6528-6532Crossref PubMed Scopus (295) Google Scholar, 8Higashiyama S. Abraham J.A. Miller J. Fiddes J.C. Klagsbrun M. Science. 1991; 251: 936-939Crossref PubMed Scopus (1044) Google Scholar, 9Holmes W.E. Sliwkowski M.X. Akita R.W. Henzel W.J. Lee J. Park J.W. Yansura D. Abadi N. Raab H. Lewis G.D. et al.Science. 1992; 256: 1205-1210Crossref PubMed Scopus (926) Google Scholar, 10Shing Y. Christofori G. Hananhan D. Ono Y. Sasada R. Igarashi K. Folkman J. Science. 1993; 259: 1604-1607Crossref PubMed Scopus (377) Google Scholar, 11Toyoda H. Komurasaki T. Ikeda Y. Yoshimoto M. Morimoto S. FEBS Lett. 1995; 377: 403-407Crossref PubMed Scopus (75) Google Scholar). These members were first identified as secreted peptides; however, subsequent cloning of their cDNA has revealed that all are derived from membrane-bound precursors that are proteolytically cleaved from the plasma membrane (9Holmes W.E. Sliwkowski M.X. Akita R.W. Henzel W.J. Lee J. Park J.W. Yansura D. Abadi N. Raab H. Lewis G.D. et al.Science. 1992; 256: 1205-1210Crossref PubMed Scopus (926) Google Scholar, 12Gray A. Dull T.J. Ullrich A. Natur e. 1983; 303: 722-725Crossref PubMed Scopus (337) Google Scholar, 13Derynck R. Roberts A.B. Winkler M.E. Chen E.Y. Goeddel D.V. Cell. 1984; 38: 287-297Abstract Full Text PDF PubMed Scopus (701) Google Scholar, 14Plowman G.D. Green J.M. McDonald V.L. Neubauer M.G. Disteche C.M. Todaro G.J. Shoyab M. Mol. Cell. Biol. 1990; 10: 1969-1981Crossref PubMed Scopus (327) Google Scholar, 15Abraham J.A. Damm D. Bajardi A. Miller J. Klagsbrun M. Ezekowitz R.A. Biochem. Biophys. Res. Commun. 1993; 190: 125-133Crossref PubMed Scopus (150) Google Scholar, 16Sasada R. Ono Y. Taniyama Y. Shing Y. Folkman J. Igarashi K. Biochem. Biophys. Res. Commun. 1993; 190: 1173-1179Crossref PubMed Scopus (119) Google Scholar). Although members of the EGF superfamily have relatively low homology with each other at the amino acid level, the presence of six conserved cysteine residues in the active peptide suggests that all have a similar tertiary structure. Indeed, the solution structure of EGF and TGFα demonstrated that identical disulfide linkages between these conserved cysteine residues enable the formation of a three-looped structure (17Campbell I.D. Cooke R.M. Baron M. Harvey T.S. Tappin M.J. Prog. Growth Factor Res. 1989; 1: 13-22Abstract Full Text PDF PubMed Scopus (65) Google Scholar). Central to the function of the EGF superfamily is a conserved domain known as the EGF motif, which is present in all EGF superfamily members identified to date. This motif encompasses three of the six conserved cysteine residues and contains additional residues important for tertiary structure stabilization and receptor binding (18Groenen L.C. Nice E.C. Burgess A.W. Growth Factors. 1994; 11: 235-257Crossref PubMed Scopus (216) Google Scholar). Just as the EGF superfamily comprises structurally similar members, so do the receptors through which the peptides signal. In mammals, the EGF receptor tyrosine kinase family includes c-erbB-1 (EGFR, HER-1), c-erbB-2 (HER-2, Neu), c-erbB-3 (HER-3), and c-erbB-4 (HER-4) (19Lin C.R. Chen W.S. Kruiger W. Stolarsky L.S. Weber W. Evans R.M. Verma I.M. Gill G.N. Rosenfield M.G. Science. 1984; 224: 843-848Crossref PubMed Scopus (279) Google Scholar, 20Coussens L. Yang-Feng T.L. Liao Y.C. Chen E. Gray A. McGrath J. Seeburg P.H. Libermann T.A. Schlessinger J. Francke U. et al.Science. 1985; 230: 1132-1139Crossref PubMed Scopus (1567) Google Scholar, 21Kraus M.H. Issing W. Miki T. Popescu N.C. Aaronson S.A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 9193-9197Crossref PubMed Scopus (674) Google Scholar, 22Plowman G.D. Culouscou J.-M. Whitney G.S. Green J.M. Carlton G.W. Foy L. Neubauer M.G. Shoyab M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1746-1750Crossref PubMed Scopus (689) Google Scholar). c-erbB receptor signal transduction begins with stabilization of a receptor homo- or heterodimer through ligand binding (23Moghal N. Sternberg P.W. Curr. Opin. Cell Biol. 1999; 11: 190-196Crossref PubMed Scopus (287) Google Scholar). Signaling is such that a single receptor, e.g. c-erbB-1, can bind to several EGF superfamily ligands (EGF, TGFα, epiregulin, AR, HB-EGF, betacellulin; see Ref. 24Riese D.J. Kim E.D. Elenius K. Buckley S. Klagsbrun M. Plowman G.D. Stern D.F. J. Biol. Chem. 1996; 271: 20047-20052Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar for review), and a single EGF superfamily ligand (e.g. epiregulin) can bind to several receptors (c-erbB-1 and B-4; Ref. 25Komurasaki T. Toyoda H. Uchida D. Morimoto S. Oncogene. 1997; 15: 2841-2848Crossref PubMed Scopus (126) Google Scholar). Tyrosine phosphorylation of C-terminal residues of c-erbB receptors activates the Ras-MAP kinase pathway, leading to regulation of c-fos expression by the binding of transcription factors to sites such as the serum inducing element and SRE within the c-fos promoter (see Ref. 26Hackel P.O. Zwick E. Prenzel N. Ullrich A. Curr. Opin. Cell Biol. 1999; 11: 184-189Crossref PubMed Scopus (546) Google Scholar for review). The Ras-MAP kinase signaling pathway is widely used by receptor tyrosine kinases to promote diverse cellular responses including cell growth, differentiation, and apoptosis (27Chin Y.E. Kitagawa M. Su W.C. You Z.H. Iwamoto Y. Fu X.Y. Science. 1996; 272: 719-722Crossref PubMed Scopus (730) Google Scholar, 28Garrington T.P. Johnson G.L. Curr. Opin. Cell Biol. 1999; 11: 211-218Crossref PubMed Scopus (1132) Google Scholar). In vitroanalysis of EGF superfamily members has revealed that all are able to stimulate or inhibit the proliferation of epithelia-derived normal and transformed cell lines, and several members are able to stimulate growth of fibroblasts, smooth muscle, and neural cell lines in culture (11Toyoda H. Komurasaki T. Ikeda Y. Yoshimoto M. Morimoto S. FEBS Lett. 1995; 377: 403-407Crossref PubMed Scopus (75) Google Scholar, 29Gill G.N. Lazar C.S. Nature. 1981; 293: 305-307Crossref PubMed Scopus (323) Google Scholar, 30Shoyab M. Plowman G.D. McDonald V.L. Bradley J.G. Todaro G.J. Science. 1989; 243: 1074-1076Crossref PubMed Scopus (488) Google Scholar, 31Traverse S. Seedorf K. Paterson H. Marshall C.J. Cohen P. Ullrich A. Curr. Biol. 1994; 4: 694-701Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar, 32Davis-Fleischer K.M. Besner G.E. Front. Biosci. 1998; 3: d288-d299Crossref PubMed Google Scholar, 33Toyoda H. Komurasaki T. Uchida D. Takayama Y. Isobe T. Okuyama T. Hanada K. J. Biol. Chem. 1995; 270: 7495-7500Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 34Watanabe T. Shintani A. Nakata M. Shing Y. Folkman J. Igarashi K. Sasada R. J. Biol. Chem. 1994; 269: 9966-9973Abstract Full Text PDF PubMed Google Scholar, 35Taylor D.S. Cheng X. Pawlowski J.E. Wallace A.R. Ferrer P. Molloy C.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1633-1638Crossref PubMed Scopus (69) Google Scholar). EGF and TGFα can promote angiogenesis in vivo and can stimulate migration of endothelial cells and keratinocytes in vitro, both features thought to be of importance in epithelial wound healing (36Gospodarowicz D. Bialecki H. Thakral T.K. Exp. Eye Res. 1979; 28: 501-514Crossref PubMed Scopus (203) Google Scholar, 37Schreiber A.B. Winkler M.E. Derynck R. Science. 1986; 232: 1250-1253Crossref PubMed Scopus (716) Google Scholar, 38Schultz G.S. White M. Mitchell R. Brown G. Lynch J. Twardzik D.R. Todaro G.J. Science. 1987; 235: 350-352Crossref PubMed Scopus (307) Google Scholar, 39Nickoloff B.J. Mitra R.S. Riser B.L. Dixit V.M. Varani J. Am. J. Pathol. 1988; 132: 543-551PubMed Google Scholar, 40Sato E. Tanaka T. Takeya T. Miyamoto H. Koide S. Endocrinology. 1991; 128: 2402-2406Crossref PubMed Scopus (25) Google Scholar, 41Nakamura M. Nishida T. Cornea. 1999; 18: 452-458Crossref PubMed Scopus (47) Google Scholar). However, aberrant activation of the EGF ligand/receptor pathway in diseases such as psoriasis and cancer suggests that disregulation of EGF superfamily members and their receptors may play a role in the progression of these and other disorders (32Davis-Fleischer K.M. Besner G.E. Front. Biosci. 1998; 3: d288-d299Crossref PubMed Google Scholar, 42Derynck R. Goeddel D.V. Ullrich A. Gutterman J.U. Williams R.D. Bringman T.S. Berger W.H. Cancer Res. 1987; 47: 707-712PubMed Google Scholar, 43Elder J.T. Fisher G.J. Lindquist P.B. Bennet G.L. Pittelkow M.R. Coffey Jr., R.J. Ellingsworth L. Derynck R. Voorhees J.J. Science. 1989; 243: 811-814Crossref PubMed Scopus (504) Google Scholar, 44Imanishi K. Yamaguchi K. Suzuki M. Honda S. Yanaihara N. Abe K. Br. J. Cancer. 1989; 59: 761-765Crossref PubMed Scopus (57) Google Scholar, 45King Jr., L.E. Gates R.E. Stoscheck C.M. Nanney L.B. J. Invest. Dermatol. 1990; 95: 10S-12SAbstract Full Text PDF PubMed Scopus (61) Google Scholar, 46Piepkorn M. Am. J. Dermatopathol. 1996; 18: 165-171Crossref PubMed Scopus (57) Google Scholar). We have identified a novel member of the EGF ligand superfamily from mouse keratinocytes, which we have named Epigen, forepithelial mitogen. mRNA encoding Epigen has a restricted tissue distribution, present in heart, liver, and testis. We have purified recombinant Epigen and compared its biological activities with that of EGF and TGFα. In epithelial cells, Epigen stimulates the phosphorylation of c-erbB-1 and MAP kinase proteins. Epigen also activates genes under the control of the SRE. In addition, Epigen is a mitogen for HaCaT cells, and this activity can be significantly reduced by a blocking antibody to the receptor c-erbB-1. HaCaT and A431 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum, 2 mml-glutamine (Sigma), 1 mm sodium pyruvate (Life Technologies, Inc.), 0.77 mml-asparagine (Sigma), 0.2 mmarginine (Sigma), 160 mm penicillin G (Sigma), 70 mm dihydrostreptomycin sulfate (Roche Molecular Biochemicals). HaCaT SRE cells were also supplemented with 0.5 mg/ml geneticin (Life Technologies, Inc.). A population of basal keratinocytes from the epidermis of neonatal mouse skin was used to generate a directionally cloned cDNA library in pBK-CMV using the Zap Express kit (Stratagene). Clones excised from the library were sequenced from the T3 primer, and homology searches were performed using the FastA and FastX algorithms. An adult mouse tissue Northern blot was purchased from CLONTECH and probed with a [32P]dATP end-labeled antisense oligonucleotide corresponding to the sequence 5′-GGTCGATATAGGAACCAGCTACCTTTGCCTGCTCTGGATCTGCTG-3′ according to the manufacturer's instructions. The blot was reprobed using the control β-actin fragment provided with the blot. For bacterial expression, the vector pET16b (Novagen) was modified to shorten the bacterial leader sequence. The vector was digested with NcoI and XhoI to remove the existing leader sequence. This was replaced by the sequence 5′-ccatgggccatcaccatcaccaccatgcgaattcgctcgag-3′, which encodes the amino acid sequence MGHHHHHHANSLE. DNA encoding residues 53–103 of Epigen was polymerase chain reaction-amplified using the primers 5′-gggaattctctgaagttctctcatc-3′ and 5′-gcggatccttaagcatacgaagttag-3′. The resulting DNA fragments were digested with EcoRI andBamHI and ligated into the modified pET16b vector. A pGL3-promoter vector (Promega) containing the neomycin resistance gene was used to develop an SRE luciferase reporter construct where the SRE was inserted 5′ of the SV40 promoter. The following primers were used to generate the enhancer region of the human c-fos promoter: SRE1, 5′-gatccgcagcccgcgagcagttcccgtcaatccctcccccctta-3′; SRE2, 5′-cacaggatgtccatattaggacatctgcgtcagcaggtttccacggcctttccctgtagcccta-3′; SRE3, 5′-cctaatatggacatcctgtgtaaggggggagggattgacgggaactgctcgcgggctgcg-3′; and SRE4, 5′-gatctagggctacagggaaaggccgtggaaacctgctgacgcagatgt-3′. Primers were annealed to prepare two double-stranded oligonucleotides: SRE1/3 and 2/4. SRE1/3 and SRE2/4 were then ligated together into theBamHI and BglII sites of pGL3 to form the SRE. A concatamer of eight SRE enhancers was prepared by subsequent rounds of digestion and SRE ligation into pGL3. Small scale expression of Epigen in pET16b was induced in BL21/DE3/Lys cells according to the manufacturer's instructions. Protein induction was monitored by analyzing whole bacterial lysates by SDS-PAGE and Western blotting using an anti-His antibody (Roche Molecular Biochemicals) according to the manufacturer's instructions. For purification, expression was scaled up, and bacterial cell pellets were resuspended in lysis buffer (20 mm Tris-HCl, pH 8.0, 10 mmβ-mercaptoethanol, 1 mm phenylmethylsulfonyl fluoride). To the lysed cells, 1% Nonidet P-40 was added, and the mixture was incubated on ice for 10 min. Lysates were further disrupted by sonication on ice at 95 watts for 4 × 15 s and then centrifuged for 15 min at 14,000 rpm to pellet the inclusion bodies. The pellet containing inclusion bodies was resuspended in lysis buffer containing 0.5% w/v CHAPS and sonicated on ice for 5–10 s. This mixture was stored on ice for 1 h and centrifuged at 14,000 rpm for 15 min at 4 °C, and the supernatant was discarded. The pellet was once more resuspended in lysis buffer containing 0.5% w/v CHAPS, sonicated, and centrifuged, and the supernatant was removed as before. The pellet was resuspended in solubilizing buffer (6 mguanidine HCl, 0.5 m NaCl, 20 mm Tris-HCl, pH 8.0), sonicated at 95 watts for 4 × 15 s, and then centrifuged for 20 min at 14,000 rpm and 4 °C to remove debris. The supernatant was stored at 4 °C until use. Recombinant Epigen was purified using a nickel-chelating Sepharose column (Amersham Pharmacia Biotech) following the manufacturer's recommended protocol. For refolding, the protein solution was added to 5 times its volume of refolding buffer (1 mm EDTA, 1.25 mm reduced glutathione, 0.25 mm oxidized glutathione, 20 mm Tris-HCl, pH 8.0) over a period of 1 h at 4 °C. The refolding buffer was stirred rapidly during this time, and stirring was continued at 4 °C overnight. The refolded proteins were then concentrated by ultrafiltration using standard protocols. For MAP kinase phosphorylation assays, HaCaT cells were seeded in 6-well dishes such that the cells reached 80% confluence after an overnight incubation. Cells were then transferred into serum-free media and incubated for a further 24 h. Recombinant Epigen, TGFα (Genzyme), or control protein (TR1002P, a secreted protein produced as for Epigen) was added to the cells at a concentration of 18 nm and incubated for up to 20 min. Media was removed after the indicated times, and cells were immediately lysed in 100 μl of radioimmune precipitation buffer (50 mm Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mmNaCl, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, leupeptin, pepstatin). MAP kinase phosphorylation was then assessed by SDS-PAGE and Western blot analysis using anti-ACTIVETM MAP kinase polyclonal antibody (Promega) following the manufacturer's instructions. Blots were stripped and reprobed using anti-ERK1/2 antibody (Santa Cruz Biotechnology) to ensure equal protein loading. For c-erbB-1 phosphorylation assays, A431 cells were substituted for HaCaT cells and treated in the same manner. c-erbB-1 phosphorylation was assessed as above, using an antibody to EGF receptor (activated form, Transduction Laboratories) following the manufacturer's instructions. Blots were stripped and reprobed using anti-EGF receptor (Transduction Laboratories) to ensure equal protein loading. HaCaT cells were stably transfected with the concatamerized SRE construct described above, using standard techniques. For the assay, 5 × 103 cells were aliquoted into wells of a 96-well plate and incubated for 24 h. Media was changed to 0.1% fetal bovine serum, and cells were incubated for an additional 24 h. Cells were incubated in the presence of recombinant Epigen, EGF (PeproTech), TGFα, or a control protein at concentrations titrating from 180 nm for 6 h, washed twice in phosphate-buffered saline, and lysed with 40 μl of lysis buffer (Promega). 10 μl was transferred to a 96-well plate, and 10 μl of luciferase substrate (Promega) was added by direct injection into each well by a Victor2 fluorometer (Wallac); the plate was shaken, and the luminescence for each well was read at 3 × 1-s intervals. -HaCaT proliferation assays were performed in 96-well flat-bottomed plates in 0.1 ml of Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 0.05% fetal calf serum. Recombinant Epigen, EGF, TGFα, or a control protein was titrated into the plates from a concentration of 12 nm, and 1 × 103 HaCaT cells were added to each well. The plates were incubated for 5 days in an atmosphere containing 10% CO2 at 37 °C. Cell growth was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye reduction. HaCaT inhibition assays were performed essentially as above except that anti-c-erbB-1 (USB) or isotype control antibodies were titrated into wells (from 1 μg/ml) containing a constant amount of Epigen (1.75 nm) or TGFα (0.1 nm). High throughput sequencing of a mouse immature keratinocyte library identified an expressed sequence tag of 1715 base pairs containing a single open reading frame of 459 nucleotides (Fig.1 A). A putative translation initiation codon was found within the sequence GAATATGG, which matched the consensus sequence established for eukaryotic translational initiation (47Kozac M. Nucleic Acids Res. 1987; 26: 8125-8148Crossref Scopus (4168) Google Scholar). Only 5 base pairs of sequence was found upstream of the ATG codon in this cDNA, whereas the 3′ untranslated region was 1.25 kilobases in length and contained a poly(A) stretch preceded by a putative polyadenylation signal (AATAAA). Conceptual translation of the 459-base pair open reading frame produced a predicted protein of 152 amino acids. Hydrophobicity analysis identified a stretch of 18 amino acids, starting from the putative initiating methionine, indicative of a signal peptide. The putative cleavage site of the signal peptide was predicted to be located between Ala18 and Ala19 (SignalP; Ref. 48Nielsen H. Engelbrecht J. Brunak S. von Heijne G. Protein Eng. 1997; 10: 1-6Crossref PubMed Scopus (4934) Google Scholar). An additional hydrophobic region was present between Ile111and Cys130, suggesting the presence of a transmembrane domain. A search against the Prosite data base (release 15.0; Ref. 49Hofmann K. Bucher P. Faiquet L. Bairoch A. Nucleic Acids Res. 1999; 27: 215-219Crossref PubMed Scopus (1005) Google Scholar) revealed that residues 83–94 (CRCFTGYTGQRC) matched the consensus pattern of EGF 1 and 2 domains. In addition, the predicted protein also contained two N-linked glycosylation sites between amino acids 36 and 39 (NWTF) and between amino acids 40 and 43 (NNTE). Alignment of the predicted protein, which we have named Epigen, with entries in the Swiss-Prot Database indicated that Epigen was similar to several members of the EGF family. Over the entire Epigen protein sequence, this homology was relatively low, displaying 26 and 29% identity to TGFα and Epiregulin, respectively. This compares favorably with the protein sequence identity between other EGF family members, which ranges from 23 to 33%. Because EGF family members exist in a functional form as small peptides, we aligned the functional peptides of the EGF family with Epigen (Fig. 1 B). This revealed that a 51-amino acid internal segment of Epigen was more than 40% identical to the active peptides of EGF, TGFα, and Epiregulin. The active peptides of the EGF family are sufficient for activity and contain several conserved residues critical for the maintenance of this activity. Epigen has also retained these residues, which include six cysteines (Cys59, Cys67, Cys72, Cys83, Cys85, Cys94), three glycines (Gly70, Gly88, Gly91), and an arginine and leucine (Arg93 and Leu99). The six cysteine residues in TGFα form three disulfide bonds that are required for optimum binding affinity to c-erbB-1 (50Tou J.S. McGrath M.F. Zupec M.E. Byatt J.C. Violand B.N. Kaempfe L.A. Vineyard B.D. Biochem. Biophys. Res. Commun. 1990; 167: 484-491Crossref PubMed Scopus (15) Google Scholar, 51Violand B.N. Tou J.S. Vineyard B.D. Seigel N.R. Smith C.E. Pyla P.D. Zobel J.F. Toren P.C. Kolodziej E.W. Int. J. Pept. Protein Res. 1991; 37: 463-467Crossref PubMed Scopus (13) Google Scholar). Mutation of Arg93 and Leu99 was found to abrogate binding of EGF and TGFα to c-erbB-1 (18Groenen L.C. Nice E.C. Burgess A.W. Growth Factors. 1994; 11: 235-257Crossref PubMed Scopus (216) Google Scholar), and Gly91 and Arg93 are highly conserved within EGF family members but not in proteins that contain EGF units without growth factor activity (52Massague J. Pandiella A. Annu. Rev. Biochem. 1993; 62: 515-541Crossref PubMed Scopus (600) Google Scholar). Together, the conserved features of Epigen with members of the EGF family at the amino acid level suggested that Epigen encoded a novel member of the EGF family. Furthermore, an increase in sequence conservation of a 51-amino acid internal peptide of Epigen with the functional peptides of the EGF family suggested that this peptide would encompass residues sufficient for its biological activity. The pattern of Epigen expression in adult mouse tissues was assessed by Northern blotting using commercial poly(A)+ blots fromCLONTECH. A [32P]dATP end-labeled synthetic oligonucleotide hybridizing to the 3′ untranslated region of mouse Epigen was used as a probe. Expression of mouse Epigen was evident in testis and liver tissues, with some expression in heart (Fig. 2). A longer exposure indicated that Epigen was also expressed in lung and kidney (data not shown). As a loading control, the blot was reprobed with a β-actin fragment. We assessed whether Epigen, like other EGF family members, could activate the Ras/MAP kinase/c-fos signal transduction pathway (26Hackel P.O. Zwick E. Prenzel N. Ullrich A. Curr. Opin. Cell Biol. 1999; 11: 184-189Crossref PubMed Scopus (546) Google Scholar). For this purpose, primers were designed to amplify cDNA that encoded Epigen from residues Leu53 to Ala103. Polymerase chain reaction products were subcloned into the bacterial expression vector pET16b, in frame with an N-terminal poly-histidine tag (Fig. 3 A). The presence of a poly-histidine tag at a similar position in TGFα was found to have no effect on its biological activity (13Derynck R. Roberts A.B. Winkler M.E. Chen E.Y. Goeddel D.V. Cell. 1984; 38: 287-297Abstract Full Text PDF PubMed Scopus (701) Google Scholar). The expression vector was transformed into Escherichia coli to prepare recombinant Epigen (Fig. 3 B). We assayed for MAP kinase phosphorylation upon stimulation of HaCaT cells by Epigen over a 20-min period. In this assay, phosphorylated MAP kinase was detected by Western blot analysis using an antibody that recognizes the phosphorylated forms of MAP kinase only (Fig. 4, A–C,panel P). As shown in Fig. 4 A, an increase in MAP kinase phosphorylation was detected following a 5-min incubation of HaCaT cells with Epigen. The level of MAP kinase phosphorylation steadily increased to a maximal level at 20 min of stimulation. TGFα also induced MAP kinase phosphorylation after 5 min of incubation; however, maximal levels were reached at 10 to 15 min post-stimulation and had begun to decrease by 20 min (Fig. 4 B). A control protein was only able to stimulate weak MAP kinase phosphorylation at 20 min when compared with Epigen and TGFα (Fig. 4 C). Reprobing each blot with an antibody recognizing total MAP kinase verified that an equal quantity of protein from the control and test samples had been analyzed (Fig. 4, A–C, panel T).Figure 4Induction of MAP kinase phosphorylation in HaCaT cells by Epigen and TGFα. HaCaT cells were cultured overnight in the absence of serum and then stimulated with 18 nm Epigen (A), TGFα (B), or control protein (C) at 37 °C for the times indicated. Cells were processed as described under “Experimental Procedures.” Lysates were analyzed by SDS-PAGE and Western blotting using antibodies to the phosphorylated form of MAP kinase protein (P) or to total MAP kinase (T).View Large Image Figure ViewerDownload Hi-res image Download (PPT) We then assessed whether Epigen activated genes under the control of the SRE. Reporter constructs containing concatamerized SRE sequences upstream of a luciferase gene were stably transfected into HaCaT cells as described under “Experimental Procedures.” Reporter activity was evaluated by measuring luciferase levels. As shown in Fig.5 A, a dose-dependant increase in luciferase level was detected when Epigen was added to the stably transfected HaCaT cell line. A 3-fold increase in luciferase levels was observed at the highest Epigen concentration used, with luciferase levels returning to base line at 1.8 nm. TGFα stimulated a 5-fold" @default.
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• W1982681624 cites W1970447875 @default.
• W1982681624 cites W1973188134 @default.
• W1982681624 cites W1973202897 @default.
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• W1982681624 cites W2017799589 @default.
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• W1982681624 cites W2020475637 @default.
• W1982681624 cites W2022916313 @default.
• W1982681624 cites W2023626809 @default.
• W1982681624 cites W2024985960 @default.
• W1982681624 cites W2027049288 @default.
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• W1982681624 cites W2031070504 @default.
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• W1982681624 cites W2034614477 @default.
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• W1982681624 cites W2038086562 @default.
• W1982681624 cites W2042390035 @default.
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• W1982681624 cites W2048905477 @default.
• W1982681624 cites W2049099038 @default.
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• W1982681624 cites W2053307807 @default.
• W1982681624 cites W2053383702 @default.
• W1982681624 cites W2054012934 @default.
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• W1982681624 cites W2071972670 @default.
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