Analysis of c-fos expression in the butyrate-induced F-98 glioma cell differentiation.
The functional induction of c-fos in the sodium butyrate-induced differentiation of F-98 glioma cells was studied. Fos protein level was increased by butyrate. In contrast, c-Jun protein was constitutively expressed and was not affected by butyrate. Gel-retardation assay indicates Fos as a component of the complex formed between the consensus oligonucleotide of the TPA (PMA, phorbol 12-myristate 13-acetate) response element (TRE) and nuclear extract prepared from butyrate-treated cells. Transfection studies showed that butyrate increased transcription from a multimeric TRE-driven reporter construct, and the effect was mimicked by transfecting cells with fos-expression plasmid. Furthermore, under conditions of c-fos over-expression, transactivation by butyrate was essentially abolished. These data suggest that Fos induction had a functional role in gene activation. Characterization of stable c-fos transfectants demonstrated that these cells displayed alterations in morphology, showed serum-dependent growth, had slower growth rates and grew to lower saturation densities than did untransfected F-98 cells or transfected cells that did not express c-fos. Immunofluorescent staining indicated that fos transfectants also had elevated glial fibrillary acidic protein ('GFAP') expression. Transfection of the c-fos promoter-chloramphenicol acetyltransferase fusion gene into F-98 cells revealed that activation of c-fos by butyrate was exerted at the promoter level, and sequences located within nucleotides -757 to -402 of the c-fos promoter were responsible for butyrate induction. Our data indicate that transcriptional activation of c-fos through its promoter by butyrate resulted in increased Fos protein expression. Transfection studies show that both c-fos and butyrate activate TRE-containing genes, and fos may be a downstream mediator of butyrate. Furthermore, expression of c-fos plays a major role in modulating the growth properties of F-98 cells.
Tang
S J
SJ
Institute of Marine Biotechnology, National Ocean University, Keelong, Taiwan.
Huang
Y M
YM
Wang
F F
FF
D000818:Animals; D001483:Base Sequence; D002087:Butyrates / Q000494:pharmacology*; D020148:Butyric Acid; D002454:Cell Differentiation / Q000187:drug effects*; D002455:Cell Division; D015870:Gene Expression; D016762:Genes, fos / Q000235:genetics*; D016758:Genes, jun / Q000235:genetics; D005904:Glial Fibrillary Acidic Protein / Q000235:genetics; D005910:Glioma / Q000473:pathology*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins; D012045:Regulatory Sequences, Nucleic Acid; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects; D015533:Transcriptional Activation; D014162:Transfection; D014407:Tumor Cells, Cultured
D002087:Butyrates; D005904:Glial Fibrillary Acidic Protein; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D020148:Butyric Acid; D013755:Tetradecanoylphorbol Acetate