Activation of c-fos expression by transforming Ha-ras in HC11 mouse mammary epithelial cells is PKC-dependent and mediated by the serum response element.
The mechanism by which transforming Ha-ras induces c-fos expression in HC11 mouse mammary epithelial cells was investigated with regard to controversial data concerning the role of protein kinase C (PKC) and the required promoter elements of the fos gene. HC11 cells carrying a glucocorticoid-inducible Ha-ras (val12) construct were transfected with a chloramphenicol acetyltransferase (CAT) reporter gene under the control of a human fos promoter which includes the serum response element (SRE), the adjacent c-fos AP-1 site (FAP) and the cAMP response element (CRE). Induction of the Ha-ras gene by dexamethasone lead to a transactivation of expression of the transfected fos promoter construct which was inhibited by the PKC inhibitor BM41440 and abrogated in PKC-'depleted' cells. A similar transactivation was observed when the fos promoter construct was co-transfected with a constitutively active ras expression vector. Again, this effect was depressed by the PKC inhibitor and abolished in PKC-'depleted' cells. 'PKC-depletion' was achieved by long-term exposure to 12-O-tetradecanoylphorbol-13-acetate. This procedure was shown to deplete cells of PKC alpha and to reduce significantly PKC epsilon. Long-term exposure to bryostatin 1 selectively depletes PKC alpha. Depletion of PKC alpha by bryostatin 1 does not reduce the transcriptional activation of the SRE-FAP-TK-CAT (TK: thymidine kinase) construct by Ha-ras. In order to delineate the promoter elements mediating the transcriptional activation, constructs which lack the FAP and the CRE sites but contain an intact SRE were co-transfected with the ras construct. Elimination of the FAP and CRE sequences did not affect the transcriptional activation by Ha-ras (val12). It is concluded that in HC11 cells, transforming Ha-ras activates c-fos expression in a PKC-dependent manner, presumably implying PKC epsilon, and that the SRE is sufficient to mediate transcriptional activation.
Uberall
F
F
Institute of Medical Chemistry and Biochemistry, University of Innsbruck, Austria.
Kampfer
S
S
Doppler
W
W
Grunicke
H H
HH
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D001483:Base Sequence; D002460:Cell Line; D002478:Cells, Cultured; D004268:DNA-Binding Proteins; D003907:Dexamethasone / Q000494:pharmacology; D004848:Epithelium / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation; D008321:Mammary Glands, Animal / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D011247:Pregnancy; D011493:Protein Kinase C / Q000378:metabolism; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid; D026362:Serum Response Factor; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection
D000970:Antineoplastic Agents; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D016760:Proto-Oncogene Proteins c-fos; D026362:Serum Response Factor; D014157:Transcription Factors; D003907:Dexamethasone; D011493:Protein Kinase C; D016283:Proto-Oncogene Proteins p21(ras); D013755:Tetradecanoylphorbol Acetate