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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.

ELSEVIER, Nakamura, S; Takahashi, H; Kinouchi, M; Manabe, A; Ishida-Yamamoto, A; Hashimoto, Y; Iizuka, H, JOURNAL OF DERMATOLOGICAL SCIENCE, 25(2), 139-149, 2001. authorSV-40 transformed human keratinocytes (SVHK cells) were stimulated with epidermal growth factor (EGF)2 and ultraviolet B (UVB) irradiation. Following the stimulation, cell growth, apoptosis, and the activities of mitogen-activated protein (MAP) kinase families were analyzed. EGF (100ng/ml) increased SVHK cell number compared with control cells cultured in serum-free DMEM medium. The EGF-stimulated cells did not show DNA fragmentation. In contrast, UVB irradiation (40mJ/cm2) markedly decreased viable cell number, that was accompanied with DNA fragmentation. EGF stimulated extracellular signal-regulated kinase (ERK) and stress-activated protein kinase/c-Jun N-terminal kinase (JNK). Following the EGF stimulation, phosphorylated ERK and JNK were detected by phospho-p42/44 MAP kinase antibody and phospho-SAPK/JNK antibody, respectively. On the other hand, UVB irradiation stimulated the phosphorylation of p38 and JNK but not of ERK. The stimulation of ERK and JNK induced by EGF was observed earlier than the stimulation of p38 and JNK induced by UVB. PD98059, a specific MAP kinase kinase (MAPKK) 1 (also referred to as MEK1) inhibitor, inhibited EGF-dependent cell proliferation, that was associated with the inhibition of ERK and JNK phosphorylation. In contrast, UVB-induced overall cell death was not significantly affected by PD98059, that inhibited phosphorylation of JNK but not of p38. PD98059, however, significantly augmented UVB-induced cell death earlier time points (30min - 2 h). These results indicate that ERK and JNK are activated following EGF stimulation, that might be associated with cell proliferation. On the other hand, UVB-induced apoptosis seems to be mostly associated with the activation of p38. JNK stimulation might provide an anti-apoptotic tonus during the UVB-induced, p38-associated SVHK cell death

A cDNA encoding M2-type pyruvate kinase from mouse was cloned and its nucleotide sequence was determined. The cDNA encoded a protein containing 531 amino acids, the nucleotide and amino acid sequences of which were 93.2% and 98.1%, respectively, homologous to those of rat M2 pyruvate kinase. Expression of the pyruvate kinase in mouse embryonal carcinoma P19 cells altered according to differentiation stages; high at undifferentiated and low at differentiated stages.

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