title: The alpha and eta isoforms of protein kinase C stimulate transcription of human involucrin gene.
abstract: 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: alpha, delta, epsilon, eta, and zeta. 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-alpha or PKC-eta 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-delta, -epsilon, and -zeta had no effect on the involucrin-promoter activity. Although the promoter activity was stimulated by transfection of PKC-gamma, TPA did not enhance the promoter activity in the PKC-gamma-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-alpha, or PKC-eta-transfected SVHK cells, but not of PKC-gamma-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-alpha and PKC-eta, mediates the TPA-dependent activation of involucrin gene expression of SVHK cells. PKC-gamma, which is not present in keratinocytes, also induces involucrin gene expression in a TPA-independent manner, when introduced into SVHK cells.
authors.affiliation: Department of Dermatology, Asahikawa Medical College, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Takahashi
authors.forename: K
authors.initials: K
authors.lastname: Asano
authors.forename: A
authors.initials: A
authors.lastname: Manabe
authors.forename: M
authors.initials: M
authors.lastname: Kinouchi
authors.forename: A
authors.initials: A
authors.lastname: Ishida-Yamamoto
authors.forename: H
authors.initials: H
authors.lastname: Iizuka
mesh_terms: D001483:Base Sequence; D001665:Binding Sites / Q000502:physiology; D002461:Cell Line, Transformed; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism / Q000494:pharmacology*; D015603:Keratinocytes / Q000201:enzymology; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D011493:Protein Kinase C / Q000378:metabolism / Q000494:pharmacology*; D011498:Protein Precursors / Q000235:genetics*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects*; D014162:Transfection
chemical_list: D007527:Isoenzymes; D009687:Nuclear Proteins; D011498:Protein Precursors; D018808:Transcription Factor AP-1; C032253:involucrin; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate