The alpha and eta isoforms of protein kinase C stimulate transcription of human involucrin gene.
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.
Takahashi
H
H
Department of Dermatology, Asahikawa Medical College, Japan.
Asano
K
K
Manabe
A
A
Kinouchi
M
M
Ishida-Yamamoto
A
A
Iizuka
H
H
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
D007527:Isoenzymes; D009687:Nuclear Proteins; D011498:Protein Precursors; D018808:Transcription Factor AP-1; C032253:involucrin; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate