Association of protein kinase C activation with induction of ornithine decarboxylase in murine but not human keratinocyte cultures.
The goal of this study was to compare the response of mouse epidermal keratinocytes (MEKs) and human epidermal keratinocytes (HEKs) to 12-O-tetradecanoylphorbol-13-acetate (TPA) with respect to the activation and downregulation of protein kinase C (PKC), the expression of c-jun and c-fos, and the expression and induction of ornithine decarboxylase (ODC) activity. Keratinocytes from adult CD-1 mice and from discarded adult human skin were grown in primary culture in a high-calcium serum-free medium that supported proliferation and differentiation. Immunoblotting of freshly isolated and cultured MEKs and HEKs for isozymes of protein kinase C revealed that fresh HEKs contained PKC alpha, PKC beta, and PKC delta; no PKC gamma, PKC epsilon, or PKC zeta were detected. In fresh MEKs, PKC alpha, PKC beta, PKC delta, and PKC zeta were observed, but not PKC gamma or PKC epsilon. After 2 wk in culture, the isozyme profiles of MEKs and HEKs were similar except that PKC gamma was noticeably present in HEK cultures. Activation of partially purified total PKC by TPA was similar in freshly isolated and cultured MEKs and HEKs, indicating that the two species were similar in this regard and that 2 wk of culture did not alter this characteristic. When MEK and HEK cultures were treated with TPA for 3 h, less than 30% of the control level of PKC activity was detected, indicating that TPA-induced downregulation of PKC was similar in MEKs and HEKs. After treatment with TPA, MEK cultures produced a large induction of both c-jun and c-fos mRNA by 60 min, as determined by northern blot analysis, and a large induction of ODC mRNA and enzyme activity by 6 h. TPA treatment of cultured HEKs, however, did not induce ODC activity; in fact, less activity, compared with that of control cultures, was observed. Northern blot analysis also revealed no increase in c-jun, c-fos, and ODC mRNA in HEKs. However, c-jun and c-fos mRNA and both ODC mRNA and enzyme activity were induced in HEKs fed growth factors after several days of deprivation. This suggests that the lack of ODC induction by TPA in HEKs is probably due to species differences in downstream steps in PKC signal transduction.
Fischer
S M
SM
University of Texas M. D. Anderson Cancer Center, Science Park-Research Division, Smithville 78957.
Lee
M L
ML
Maldve
R E
RE
Morris
R J
RJ
Trono
D
D
Burow
D L
DL
Butler
A P
AP
Pavone
A
A
Warren
B
B
D000328:Adult; D000818:Animals; D002478:Cells, Cultured; D004789:Enzyme Activation; D004790:Enzyme Induction; D015870:Gene Expression / Q000187:drug effects; D016762:Genes, fos / Q000187:drug effects; D016758:Genes, jun / Q000187:drug effects; D006133:Growth Substances / Q000494:pharmacology; D006801:Humans; D015603:Keratinocytes / Q000187:drug effects / Q000201:enzymology*; D007700:Kinetics; D051379:Mice; D008815:Mice, Inbred Strains; D008875:Middle Aged; D009955:Ornithine Decarboxylase / Q000096:biosynthesis*; D011493:Protein Kinase C / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D012867:Skin / Q000166:cytology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D006133:Growth Substances; D012333:RNA, Messenger; D011493:Protein Kinase C; D009955:Ornithine Decarboxylase; D013755:Tetradecanoylphorbol Acetate