The α and η Isoforms of Protein Kinase C Stimulate Transcription of Human Involucrin Gene
1998; Elsevier BV; Volume: 110; Issue: 3 Linguagem: Inglês
10.1046/j.1523-1747.1998.00110.x
ISSN1523-1747
AutoresHidetoshi Takahashi, Kazuhiro Asano, Akira Manabe, Motoshi Kinouchi, Akemi Ishida‐Yamamoto, Hajime Iizuka,
Tópico(s)Nerve injury and regeneration
ResumoInvolucrin is one of the precursor proteins of the cornified cell envelope that is formed beneath the cell membrane during terminal differentiation of keratinocytes. 12-O-tetradecanoylphorbol-13-acetate (TPA), which is a potent protein kinase C (PKC) activator, induces terminal differentiation of keratinocytes. We previously demonstrated that involucrin promoter activity is stimulated by TPA in cultured fetal rat skin keratinocytes. PKC is a large family of proteins and keratinocytes containing five PKC isozymes: α, δδ, ε, ηη, and ζζ. In order to determine the role of the PKC isozyme(s) on involucrin gene expression, we constructed the chloramphenicol acetyl transferase (CAT)-involucrin promoter expression vector by connecting the 5′-upstream region of the human involucrin gene containing the untranslated first exon to the CAT reporter gene. The CAT-involucrin promoter expression vector was transfected with various PKC isozyme expression vectors into SV40-transformed human keratinocytes (SVHK cells). Transfection of the CAT-involucrin promoter expression vector with PKC-α or PKC-ηη expression vectors resulted in a significant increase in the TPA-dependent involucrin promoter activity. The PKC inhibitor, 1-(5-isoquinoline-sulfonyl)-2-methyl piperazine dihydrochloride, inhibited the promoter activity stimulated by TPA. Transfection of PKC-δδ, -ε, and -ζζ had no effect on the involucrin-promoter activity. Although the promoter activity was stimulated by transfection of PKC-γγ, TPA did not enhance the promoter activity in the PKC-γγ-transfected SVHK cells. Previously we showed three AP-1 binding sites (AP1–1, –2, and –3) on the involucrin promoter region. Both the basal and the TPA-stimulated involucrin promoter activities were suppressed by deleting the AP1–1 site (–119 to –113) that is the most proximal to the transcription start site. The deletion of AP1–2 (–297 to –303) or AP1–3 (–447 to –453) did not affect the involucrin promoter activity. Gel retardation analyses disclosed that TPA stimulated the specific DNA binding of the nuclear protein(s) of control, PKC-α, or PKC-ηη-transfected SVHK cells, but not of PKC-γγ-transfected cells. Addition of anti-c-Jun and anti-c-Fos antibodies decreased the specific protein-DNA complex band with a concomitant appearance of supershifted bands. These results indicate that PKC, specifically PKC-α and PKC-ηη, mediates the TPA-dependent activation of involucrin gene expression of SVHK cells. PKC-γγ, which is not present in keratinocytes, also induces involucrin gene expression in a TPA-independent manner, when introduced into SVHK cells. Key word: cornified cell envelope. J Invest Dermatol 110:218–223 1998 Involucrin is one of the precursor proteins of the cornified cell envelope that is formed beneath the cell membrane during terminal differentiation of keratinocytes. 12-O-tetradecanoylphorbol-13-acetate (TPA), which is a potent protein kinase C (PKC) activator, induces terminal differentiation of keratinocytes. We previously demonstrated that involucrin promoter activity is stimulated by TPA in cultured fetal rat skin keratinocytes. PKC is a large family of proteins and keratinocytes containing five PKC isozymes: α, δδ, ε, ηη, and ζζ. In order to determine the role of the PKC isozyme(s) on involucrin gene expression, we constructed the chloramphenicol acetyl transferase (CAT)-involucrin promoter expression vector by connecting the 5′-upstream region of the human involucrin gene containing the untranslated first exon to the CAT reporter gene. The CAT-involucrin promoter expression vector was transfected with various PKC isozyme expression vectors into SV40-transformed human keratinocytes (SVHK cells). Transfection of the CAT-involucrin promoter expression vector with PKC-α or PKC-ηη expression vectors resulted in a significant increase in the TPA-dependent involucrin promoter activity. The PKC inhibitor, 1-(5-isoquinoline-sulfonyl)-2-methyl piperazine dihydrochloride, inhibited the promoter activity stimulated by TPA. Transfection of PKC-δδ, -ε, and -ζζ had no effect on the involucrin-promoter activity. Although the promoter activity was stimulated by transfection of PKC-γγ, TPA did not enhance the promoter activity in the PKC-γγ-transfected SVHK cells. Previously we showed three AP-1 binding sites (AP1–1, –2, and –3) on the involucrin promoter region. Both the basal and the TPA-stimulated involucrin promoter activities were suppressed by deleting the AP1–1 site (–119 to –113) that is the most proximal to the transcription start site. The deletion of AP1–2 (–297 to –303) or AP1–3 (–447 to –453) did not affect the involucrin promoter activity. Gel retardation analyses disclosed that TPA stimulated the specific DNA binding of the nuclear protein(s) of control, PKC-α, or PKC-ηη-transfected SVHK cells, but not of PKC-γγ-transfected cells. Addition of anti-c-Jun and anti-c-Fos antibodies decreased the specific protein-DNA complex band with a concomitant appearance of supershifted bands. These results indicate that PKC, specifically PKC-α and PKC-ηη, mediates the TPA-dependent activation of involucrin gene expression of SVHK cells. PKC-γγ, which is not present in keratinocytes, also induces involucrin gene expression in a TPA-independent manner, when introduced into SVHK cells. Key word: cornified cell envelope. J Invest Dermatol 110:218–223 1998 The cornified cell envelope (CE) is a highly insoluble structure formed beneath the plasma membrane of keratinocytes during terminal differentiation (Hohl, 1990Hohl D. Cornified envelope.Dermatologica. 1990; 180: 201-211Crossref PubMed Scopus (164) Google Scholar; Goldsmith, 1993; Eckert et al., 1993Eckert R.L. Yaffe M.B. Crish J.F. Murthy S. Rorke E.A. Welter J.F. Involucrin-structure and role in envelope assembly.J Invest Dermatol. 1993; 100: 613-617Abstract Full Text PDF PubMed Google Scholar). The CE provides a protective barrier between the environment and the living layers of the skin, and is believed to play an important role in maintaining the structural integrity of the epidermis. Transglutaminase 1 catalyzes the glutamyl-lysine cross-linking reaction of various CE precursor proteins that include involucrin (Eckert and Green, 1986Eckert R.L. Green H. Structure and evolution of the human involucrin gene.Cell. 1986; 46: 583589Abstract Full Text PDF Scopus (307) Google Scholar), loricrin (Hohl et al., 1991Hohl D. Mehrel T. Lichti U. Turner M.L. Roop D.R. Steinert P.M. Characterization of human loricrin.Structure and function of a new class of epidermal cell envelope proteins. J Biol Chem. 1991; 266: 6626-6636Google Scholar), cystatin A (keratolinin) (Takahashi et al., 1997Takahashi H. Kinouchi M. Wuepper K.D. Iizuka H. Cloning of human keratolinin cDNA. keratolinin is identical with a cysteine proteinase inhibitor, cystain A, and is regulated by Ca2 +, TPA and cAMP.J Invest Dermatol. 1997; 108: 843-847Abstract Full Text PDF PubMed Scopus (20) Google Scholar), small proline-rich protein(s) (Kartasova and van de Putte, 1988Kartasova T. van de Putte P. Isolation, characterization, and UV-stimulated expression of two families of genes encoding polypeptides of related structure in human epidermal keratinocytes.Mol Cell Biol. 1988; 8: 2195-2203Crossref PubMed Scopus (142) Google Scholar), elafin (Steinert and Marekov, 1995Steinert P.M. Marekov L.N. The protein elafin, filaggrin, keratin intermediate filaments, loricrin, and small proline-rich protein 1 and 2 are isodipeptide cross-linked components of human epidermal cornified cell envelope.J Biol Chem. 1995; 270: 17702-17711Crossref PubMed Scopus (464) Google Scholar), and envoplakin (Ruhrberg et al., 1996Ruhrberg C. Hajibagheri M.A. Simon M. Dooley T.P. Watt F.M. Envoplakin, a novel precursor of the cornified envelope that has homology to desmoplakin.J Cell Biol. 1996; 134: 715-729Crossref PubMed Scopus (146) Google Scholar). Recent evidence suggests that involucrin is an early component of CE and provides a scaffold onto which several other precursor proteins are incorporated (Steinert and Marekov, 1995Steinert P.M. Marekov L.N. The protein elafin, filaggrin, keratin intermediate filaments, loricrin, and small proline-rich protein 1 and 2 are isodipeptide cross-linked components of human epidermal cornified cell envelope.J Biol Chem. 1995; 270: 17702-17711Crossref PubMed Scopus (464) Google Scholar, Ishida-Yamamoto et al., 1997Ishida-Yamamoto A. Kartasova T. Matsuo S. Kuroki T. Iizuka H. Involucrin and SPRR are synthesized sequentially in differentiation cultured epidermal cells.J Invest Dermatol. 1997; 108: 12-16Abstract Full Text PDF PubMed Scopus (34) Google Scholar). Human involucrin gene contains a 5' exon of 43 bp and a 3' exon of 2107 bp that are separated by an intron of 1188 bp (Eckert and Green, 1986Eckert R.L. Green H. Structure and evolution of the human involucrin gene.Cell. 1986; 46: 583589Abstract Full Text PDF Scopus (307) Google Scholar). The structure is characterized by a central segment composed of 39 tandem repeats of 10 amino acids, which is flanked by N- and C-terminal segments. The repeating structure of the central portion is highly conserved in all higher primates (Eckert and Green, 1986Eckert R.L. Green H. Structure and evolution of the human involucrin gene.Cell. 1986; 46: 583589Abstract Full Text PDF Scopus (307) Google Scholar), suggesting recent evolutionary origin of involucrin. Involucrin is expressed in the squamous tissue, and is detected in the upper spinous layer and the granular layer of normal human epidermis (Banks-Schlegel and Green, 1981Banks-Schlegel S. Green H. Involucrin synthesis and tissue assembly by keratinocytes in natural and cultured epithelia.J Cell Biol. 1981; 90: 732-739Crossref PubMed Scopus (272) Google Scholar). Although considerable information is available regarding the function and the evolution of involucrin protein (Eckert et al., 1993Eckert R.L. Yaffe M.B. Crish J.F. Murthy S. Rorke E.A. Welter J.F. Involucrin-structure and role in envelope assembly.J Invest Dermatol. 1993; 100: 613-617Abstract Full Text PDF PubMed Google Scholar), less is known about the regulation of the involucrin gene expression. Transgenic animal studies have shown that 2.5 kb of the 5' upstream sequence of the involucrin gene is sufficient to confer to differentiation- dependent and tissue-specific expression of the involucrin (Crish et al., 1993Crish J.F. Howard J.M. Zaim T.M. Murthy S. Eckert R.L. Tissue-specific and differentiation- appropriate expression of the human involucrin gene in transgenic mice.an abnormal epidermal phenotype. Differentiation. 1993; 53: 191-200Google Scholar, Carrol et al., 1993Carrol J.M. Alberts K.M. Galrick L.A. Harrington R. Taichman L.B. Tissue- and stratum- specific expression of the human involucrin promoter in transgenic mice.Proc Natl Acad Sci USA. 1993; 90: 10270-10274Crossref PubMed Scopus (131) Google Scholar). 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent protein kinase C (PKC) activator, induces involucrin gene expression through a nuclear factor, AP1. The AP1, which is a complex consisting of jun and fos family proteins, binds to TPA-responsive elements (TRE) and regulates the TPA-inducible genes. The promoter of the human involucrin gene has at least five TRE (Takahashi and Iizuka, 1993Takahashi H. Iizuka H. Analysis of the 5'-upstream of human involucrin gene:activation by 12-o-tetradecanoylphorbol-13-acetate.J Invest Dermatol. 1993; 100: 10-15Abstract Full Text PDF PubMed Google Scholar, Welter et al., 1995Welter J.F. Crish J.F. Agarwal C. Eckert E.L. Fos-related antigen (Fra-1), jun B, and jun D activate human involucrin promoter transcription by binding to proximal and distal AP1 sites to mediate phorbol ester effects on promoter activity.J Biol Chem. 1995; 270: 12614-12622Crossref PubMed Scopus (172) Google Scholar, Lopez-Bayghen et al., 1996Lopez-Bayghen E. Vega A. Cadena A. Granados S.E. Jave L. Gariglio P. Alvares-Saras L.M. Transcriptional analysis of the 5'-noncoding region of the human involucrin gene.J Biol Chem. 1996; 271: 512-520Crossref PubMed Scopus (71) Google Scholar). These AP1 binding sites are designated as AP1-1 to AP1-5; AP1-1 is the most proximal to the transcription start site and AP1-5 is the most distal. We and others have also demonstrated that TEF-1, POU factors, and YY-1 decrease the transcription of the involucrin gene (Takahashi et al., 1995Takahashi H. Kobayashi H. Iizuka H. Repression of involucrin gene expression by transcriptional enhancer factor 1 (TEF-1).Arch Dermatol Res. 1995; 287: 740-746Crossref PubMed Scopus (11) Google Scholar, Welter et al., 1996Welter J.F. Gali H. Crish J.F. Eckert E.L. Regulation of human involucrin promoter activity by POU domain proteins.J Biol Chem. 1996; 271: 14727-14733Crossref PubMed Scopus (51) Google Scholar, Lopez-Bayghen et al., 1996Lopez-Bayghen E. Vega A. Cadena A. Granados S.E. Jave L. Gariglio P. Alvares-Saras L.M. Transcriptional analysis of the 5'-noncoding region of the human involucrin gene.J Biol Chem. 1996; 271: 512-520Crossref PubMed Scopus (71) Google Scholar). PKC is a large family of proteins that consists of at least 11 isozymes (Nishizuka, 1992Nishizuka Y. Intracellular signaling by hydrolysis ofphospholipids and activation of protein kinase C.Science. 1992; 258: 607-614Crossref PubMed Scopus (4158) Google Scholar). PKC-α, -βI, -βII, and -γ are the classical PKC, which depend on calcium and diacylglycerol. PKC-δ, -ε, -η, -ϴ, and -μ are the novel PKC, which do not require calcium for activation. PKC-ζ and -τ are the atypical PKC, which require neither calcium nor diacylglycerol for the activation. TPA activates classical and novel PKC but not atypical PKC. The keratinocytes contain PKC-a, -δ, -ε, -η, and -ζ (Dlugosz et al., 1992Dlugosz A.A. Mischak H. Muchinski J.F. Yuspa H.S. Transcripts encoding protein kinase C-α, δ, ε, ζ, and η are expressed in basal and differentiation mouse keratinocytes in vitro and exhibit quantitative changes in neoplastic cells.Mol Cell Biol. 1992; 12: 286-292Google Scholar). The specific function of each PKC isozyme, especially in terms of involucrin gene expression, remains unknown at present. SV40-transformed human keratinocytes (SVHK cells) are well- established immortalized cell lines sharing features of normal human keratinocytes (Steinberg and Defendi, 1983Steinberg M.L. Defendi V. Transformation and immortalization of human keratinocytes by SV40.J Invest Dermatol. 1983; 81: 131S-136SAbstract Full Text PDF PubMed Scopus (57) Google Scholar, Takahashi et al., 1990Takahashi H. Tamura T. Tsutsui M. Iizuka H. Adenylate cyclase system of FRSK cells and SV40-ransformed human keratinosytes.J Dermatol (Tokyo). 1990; 17: 457-464Crossref Scopus (16) Google Scholar). SVHK cells express relatively high amounts of involucrin compared with other cell lines, such as A431 and SCC13 (data not shown). In this study, we constructed the CAT-involucrin promoter expression (INV-CAT) vector, which was subcloned from the 5'-upstream region of the involucrin gene with the untranslated first exon and was connected to the chloramphenicol acetyl transferase (CAT) reporter gene. Using the INV-CAT vector, we investigated the effects of PKC isozymes on the involucrin promoter activity of SVHK cells. The Sca I-digested fragment from the genomic involucrin gene was inserted into the Xba I site of promoterless 0-CAT plasmid, which had been blunt-ended by Klenow fragment (INV-CAT) (Takahashi and Iizuka, 1993Takahashi H. Iizuka H. Analysis of the 5'-upstream of human involucrin gene:activation by 12-o-tetradecanoylphorbol-13-acetate.J Invest Dermatol. 1993; 100: 10-15Abstract Full Text PDF PubMed Google Scholar). The p-galactosidase expression vector was kindly supplied by Dr. T. Watanabe (Medical Institute of Bioregulation, Kyushu University, Japan). PKC expression vectors were generous gifts from Dr. S. Ohno (Department of Molecular Biology, Yokohama City University School of Medicine) (Ohno et al., 1988Ohno S. Akita Y. Konno Y. Imajoh S. Suzuki K. A novel phorbol ester receptor/protein kinase, nPKC, distantly related to the protein kinase C family.Cell. 1988; 53: 731-741Abstract Full Text PDF PubMed Scopus (293) Google Scholar). Each deleted fragment was generated by an “overlap extension” method (Ho et al., 1989Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction.Gene. 1989; 77: 51-59Crossref PubMed Scopus (6679) Google Scholar) using polymerase chain reaction (PCR) using oligonucleotides (DL—1, 5 '-CTTATGAGCATGGCATTCCTGAGAA-3'; DL-2, 5'-TTTGAAGATCTTCCCATGAAGGGGT-3'; DL-3, 5'-TCATGGGAA- GATCTTCA-3'; DL-4, 5'-TGTGACCAACTTCTGCTCTG-3'; DL-5, 5'- AGCAGAAGTTGGTCACA-3'; DL-6, 5'-ACCCCTTCCCCACAGGC- ATC-3'; DL-7, 5'-TGTCCCTGGACCCTAAAGGGTTTGC; DL-8, 5'- AGGAGTCAGGGCACACTTTATATCA). The positions of these sequences are indicated in Fig 1. In order to construct the AP1-3 deleted vector (T3), we performed two PCR amplifications using DL—1 and DL—2 oligomers or DL—3 and DL—8 oligomers using the INV-CAT vector as the template. Next we performed a PCR procedure using DL—1 and DL—8 oligomers using mixed PCR product derived from DL—1 and DL—2 oligomers or DL—3 and DL—8 oligomers. The second PCR product, which contained two Pst I sites (Fig 1, double underline), was digested by Pst I restriction enzyme and was subcloned into the Pst I sites of the deleted INV-CAT vector, which was deleted at the position of the Pst I sites. Using DL—1 and DL—8 oligomers, we performed sequence analysis and confirmed the correct promoter orientation and the deleted portion. AP1-2-deleted (T2) and AP1-1-deleted (T1) vectors were constructed using DL—1, DL—4, DL—5, and DL—8 oligomers, and DL—1, DL—6, DL—7, and DL—8 oligomers, respectively. Using T1, T2, and T3 vectors, we performed PCR with these oligomers and constructed T12 (AP1—1, —2-deleted), T13 (AP1-1, -3-deleted), T23 (AP1-2, -3-deleted), and T123 (AP1—1, -2, -3-deleted) vectors. SV40-transformed human keratinocytes were a generous gift from Dr. M.L. Steinberg (Department of Chemistry, City College of the City University of New York) (Steinberg and Defendi, 1983Steinberg M.L. Defendi V. Transformation and immortalization of human keratinocytes by SV40.J Invest Dermatol. 1983; 81: 131S-136SAbstract Full Text PDF PubMed Scopus (57) Google Scholar). Cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 5% fetal calf serum, 100 u penicillin per ml, and 100 μg streptomycin per ml at 37°C in 5% CO2 in air. Transfection of plasmid DNA into these cells was performed by the liposome method using lipofectin (Felgner et al., 1987Felgner P.L. Gadek T.R. Holm M. et al.Lipofectin: a highly efficient, lipid-mediated DNA-transfected procedure.Proc Natl Acad Sci USA. 1987; 84: 7413-7417Crossref PubMed Scopus (4185) Google Scholar). Typically, 5 μg of reporter plasmid and 2 μg of β-galactosidase plasmid were transfected into 1 X 105 SVHK cells. The β-galactosidase plasmid was used as the internal standard to normalize each transfection efficacy. After 48 h, cells were collected and CAT assay was performed (Neuman et al., 1987Neuman J.R. Morency C.A. Russian K.D. A novel rapid assay for chloramphenicol acetyltransferase gene expression.Biotechniques. 1987; 5: 441Google Scholar). The enzyme activity of β-galactosidase in the transfected cell extracts was measured spectrophotometrically (Maniatis et al., 1989Maniatis T. Fritsch E.H. Sambrook T. Molecular Cloning: A Laboratory Manual.2th edn. Cold Spring Harbor Laboratory, New York1989Google Scholar). PKC enzyme assay was performed using the protein kinase C enzyme assay kit (Amersham Japan, Tokyo, Japan). Briefly, 1 X 106 transfected SVHK cells were homogenized in the extraction buffer [50 mM Tris-HCl (pH 7.5), 5 mM ethylenediamine tetraacetic acid, 10 mM ethyleneglycol- bis(P-aminoethyl ether)-N,N,N’,N’-tetraacetic acid, 0.3% (wt/vol) β-mercapto- ethanol, 10 mM benzamidine, 50 μg phenylmethylsulfonyl flouride per ml]. The homogenates were centrifuged and the supernatants were assayed according to the protocol equipped with the assay kit. Nuclear extraction and gel retardation analyses were performed according to the method previously described (Takahashi et al., 1995Takahashi H. Kobayashi H. Iizuka H. Repression of involucrin gene expression by transcriptional enhancer factor 1 (TEF-1).Arch Dermatol Res. 1995; 287: 740-746Crossref PubMed Scopus (11) Google Scholar). The oligonucleotide probe corresponds to -125 to -104, which includes the AP1-1 site. Dulbecco’s modified Eagle’s medium was purchased from Gibco (Grand Island, NY). Penicillin and streptomycin were obtained from M.A. Bioproducts (Walkersville, MD). [3H]-Acetyl coenzyme A, [γ-32P]ATP, and [α-32P]deoxycytidine thiotriphosphate were purchased from Amersham (Tokyo, Japan). Lipofectin was obtained from BRL (Bethesda, MD). Anti-c-jun, -junB, -junD, -c-fos, -NFkB, and -fra-1 antibodies were purchased from Santa Cruz Biotechnology (Santa Crus, CA). Various isozyme-specific anti-protein kinase C antibodies were purchased from Beohringer (Penzberg, Germany). We transfected various PKC isozyme expression vectors into SVHK cells. The increased expression of PKC-a was confirmed by immunoblot analysis and was shown to be plasmid dose-dependent (Fig 2). These effects were detected by the transfection of other PKC expression vectors (data not shown). The PKC expression vector-transfected SVHK cells showed increased PKC activity by around 2-fold compared with the activity of SVHK cells without the transfection (Table I). TPA stimulated the PKC activity of the transfected SVHK cells by around 6-fold, except for PKC-ζ-transfected SVHK cells (Table I).Table IEffects of PKC expression vector on PKC enzyme activity of SVHK cellsExpression vectorPKC enzyme activity (pmoles per min per mg)aResults are the means ± SEM.MediumTPAcontrol vector58.6 ± 13.1150.3 ± 14.5PKC-α115.5 ± 22.3698.8 ± 26.7PKC-β106.5 ± 20.3647.8 ± 29.4PKC-γ125.3 ± 19.2678.3 ± 19.6PKC-δ111.6 ± 17.5688.2 ± 26.9PKC-ε121.1 ± 15.2700.9 ± 30.2PKC-ζ109.8 ± 21.1199.4 ± 28.3PKC-η105.9 ± 23.5630.4 ± 32.1a Results are the means ± SEM. Open table in a new tab To determine the role of PKC isozymes on involucrin gene expression, we performed co-transfection of the INV-CAT vector with various PKC isozyme expression vectors (Fig 3). Consistent with TPA-induced activation of endogenous PKC, involucrin promoter activity was increased by TPA in SVHK cells transfected with control vector (Fig 3, lane C). Transfection of PKC-β, -δ, -ε, -ζ had no effect on the involucrin promoter activity compared with the transfection of the control vector. Although the transfection of these vectors also increased the promoter activity in the presence of TPA, the augmentation was not significantly different from that of the control vector-transfected cells (Fig 3, lanes β, δ, ε, ζ). Involucrin promoter activity, however, was significantly stimulated by the co-transfection of PKC-α or PKC-η in a TPA-dependent manner (Fig 3, lanes a, η). The promoter activity was stimulated by the transfection of PKC-γ in the absence of TPA (Fig 3, lane g). TPA did not augment the promoter activity of PKC-γ-transfected SVHK cells. The PKC inhibitor, 1-(5-isoquinoline- sulfonyl)-2-methyl piperazine dihydrochloride (H-7), significantly inhibited the promoter activity stimulated by TPA in control, PKC-α, and -η expression vector-transfected SVHK cells (Fig 4). The addition of H-7 singly to the incubation medium significantly inhibited the promoter activity of PKC-γ-transfected SVHK cells, but had no effect on other PKC vector-transfected SVHK cells (Fig 4). These results indicate that the activation of PKC-α and -η results in stimulation of involucrin promoter activity and that PKC-γ introduced into SVHK cells is endogenously activated in the absence of TPA.Figure 4Increased promoter activity by the transfection of PKC expression vector was suppressed by PKC inhibitor H-7. Following co-transfection of INV-CAT vector and PKC isozyme (α, γ, η) expression vectors, SVHK cells were incubated with TPA (10 ng per ml) or H-7 (100 μM), or both, for 24 h. The CAT activity of cells transfected with INV-CAT alone without TPA stimulation was designated as 1.0.View Large Image Figure ViewerDownload (PPT) The 5'-upstream promoter region of the INV-CAT vector contains three AP-1 binding sequences (AP1-1, -2, and -3) (Fig 1). In order to determine the critical TRE region(s) in the INV-CAT vector, we constructed various TRE-deleted vectors. The transfection of AP1-2- or AP1-3-deleted vectors had no effect on the involucrin promoter activity that was stimulated by TPA. The transfection of AP1-1 deleted vectors (T1, T12, T13, T123), however, showed a significant decrease in the basal involucrin promoter activity and TPA-dependent stimulation was abolished (Fig 5). In addition, involucrin expression was not stimulated by TPA in PKC-α- or PKC-η-transfected SVHK cells, when AP1-1 was deleted from the INV-CAT vector (Fig 6); the deletion of AP1-2 or -3 again had no effect. The putative AP1-4 (-512 to -505), which is also present in our INV-CAT vector (Welter et al., 1995Welter J.F. Crish J.F. Agarwal C. Eckert E.L. Fos-related antigen (Fra-1), jun B, and jun D activate human involucrin promoter transcription by binding to proximal and distal AP1 sites to mediate phorbol ester effects on promoter activity.J Biol Chem. 1995; 270: 12614-12622Crossref PubMed Scopus (172) Google Scholar), was apparently not functional, because our deletion analysis showed negligible effect on the promoter activity (Figure 5, Figure 6). This is consistent with the results of Welter et al., 1995Welter J.F. Crish J.F. Agarwal C. Eckert E.L. Fos-related antigen (Fra-1), jun B, and jun D activate human involucrin promoter transcription by binding to proximal and distal AP1 sites to mediate phorbol ester effects on promoter activity.J Biol Chem. 1995; 270: 12614-12622Crossref PubMed Scopus (172) Google Scholar, indicating that within the promoter region up until -811 bp, the AP1-1 region is critical for the regulation of the basal and PKC-stimulated involucrin promoter activity in SVHK cells.Figure 6The transfection of PKC-α or PKC-ηη expression vectors stimulated AP1–1 containing INV-CAT promoter activity. Following co-transfection of TRE-deleted INV-CAT vectors with PKC-α (A) or PKC-η (B) expression vectors, SVHK cells were incubated with or without TPA (10 ng per ml) for 24 h. The activity of cells transfected with INV-CAT alone without TPA stimulation was designated as 1.0. C, INV-CAT vector; T3, AP1–3-deleted INV-CAT vector; T2, AP1–2-deleted INV-CAT vector; T1, AP1–1-deleted INV-CAT vector; T23, AP1–2- and –3-deleted INV-CAT vector; T13, AP1–1- and –3-deleted INV-CAT vector; T12, AP1–1- and –2-deleted INV-CAT vector; T123, AP1–1-, –2-, and –3-deleted INV-CAT vector.View Large Image Figure ViewerDownload (PPT) The interaction between the AP1-1 sequence and the AP-1 nuclear protein(s) was investigated by gel shift analyses (Fig 7A). Control-vector-transfected SVHK cells showed a specific band indicating the interaction of nuclear protein with AP1-1 consensus sequence, which was stimulated by the addition of TPA. The nuclear factor(s) from PKC-α- and -η-transfected SVHK cells showed a significantly increased binding to the probe, which was efficiently competed by the excess of unlabeled oligomers containing the AP1-1 binding site. As expected, PKC-γ-transfected SVHK cells did not show TPA-dependent stimulation of the specific DNA-protein binding. Anti-c-jun and antic-fos antibody decreased the specific bands, and a supershifted band appeared near the top of the lane (Fig 7B, lanes 4 and 6). No supershifted band was detected by the addition of anti-junB, -junD, or -fra-1 antibodies (Fig 7B, lanes 5, 7, and 8). These results indicate that TPA stimulates the binding of nuclear factor(s) from PKC-α- and -η-transfected SVHK cells to the AP1-1 site, and the nuclear factor is most likely composed of c-jun and c-fos. Involucrin with an extended rod-shaped structure (Yaffe et al., 1992Yaffe M.B. Beegen H. Eckert R.L. Biophysical characterization of involucrin reveals a molecule ideally suited to function as an intermolecular cross-bridge of the keratinocyte cornified envelope.J Biol Chem. 1992; 267: 12233-12238Abstract Full Text PDF PubMed Google Scholar) is assumed to be initially incorporated into CE, forming a scaffold onto which other CE precursors, such as elafin, SPRR, and loricrin, are deposited (Steinert, 1995Steinert P.M. A model for the hierarchical structure of the human epidermal cornified cell envelope.Cell Death Differ. 1995; 2: 33-40PubMed Google Scholar, Steinert and Marekov, 1995Steinert P.M. Marekov L.N. The protein elafin, filaggrin, keratin intermediate filaments, loricrin, and small proline-rich protein 1 and 2 are isodipeptide cross-linked components of human epidermal cornified cell envelope.J Biol Chem. 1995; 270: 17702-17711Crossref PubMed Scopus (464) Google Scholar). The involucrin gene expression is tissue-specific and differentiation-dependent and normally detected in the suprabasal epidermal layers (Eckert, 1989Eckert R.L. Structure, function, and differentiation of the keratinocyte.Physiol Rev. 1989; 69: 13161346Google Scholar). Although SVHK cells make up a transformed cell line, they are much more easily manipulated in transfection experiments of involucrin gene expression. Notably SVHK cells contain similar PKC isozymes (α, δ, ε, η, ζ as normal human keratinocytes and show a significant involucrin expression, which is marginally detected in A431 and SCC13 cells (data not shown). Involucrin gene is TPA-inducible and co
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