title: Overproduction of the beta 1 form of protein kinase C enhances phorbol ester induction of glucose transporter mRNA.
abstract: Phorbol esters bind to and activate a family of Protein Kinase C (PKC) proteins, although the degree to which the various PKC forms mediate specific biological functions is unknown. We and others have previously shown that, after exposure to phorbol esters, cultured fibroblasts exhibit increased expression of the mRNA encoding the HepG2/brain glucose transporter (GT mRNA). We therefore studied phorbol ester regulation of GT mRNA in rat fibroblasts which do (R6-PKC3) or do not (R6-C1) stably overproduce a full-length cDNA encoding the PKC beta 1 isotype. When PKC beta 1 is overproduced in a cell that normally has undetectable levels, it is capable of increasing HepG2/brain GT mRNA in response to alpha-D-glucose phorbol 12-myristate 13-acetate (TPA). The level of GT mRNA is maximally induced 6 to 8-fold over basal levels 6 h after exposure to TPA (10 ng/ml) in R6-PKC3 cells that overproduce PKC beta 1, but only 2-fold over basal levels in the control R6-C1 cells. This TPA induced increase in the level of GT mRNA was observed as early as 1 h, peaked by 6-8 h and decreased markedly by 24 h in both cell types. The effect of PKC beta 1 on GT mRNA expression is probably mediated through enhancement of transcription, since the stability of GT mRNA was only minimally affected by TPA. Unlike the enhancement of TPA induced GT mRNA expression caused by overexpression of PKC beta 1, the responses of GT mRNA to calf serum, platelet-derived growth factor, epidermal growth factor, insulin or insulin-like growth factor-1 were the same in both cell types. After pretreatment with 1000 ng/ml TPA in 0.5% calf serum for 24 h, PKC activity was down-regulated and both R6-C1 and R6-PKC3 cells showed complete down-regulation of the GT mRNA responses to an additional treatment with 1000 ng/ml TPA. In contrast to the marked loss of responsiveness to TPA and PKC down-regulation, the responses of GT mRNA to serum, PDGF, EGF, insulin and IGF-1 were unaffected by down-regulation. Thus, our results provide direct evidence for both PKC-dependent and independent pathways regulating GT gene expression. Furthermore, it appears that the level of PKC beta 1 production, rather than down-stream signal transduction events, is the rate-limiting step in the pathway by which TPA induces an increase in GT mRNA.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Charles A. Dana Research Institute, Beth Israel Hospital, Boston, Massachusetts 02115.
authors.forename: K G
authors.initials: KG
authors.lastname: Mountjoy
authors.forename: G M
authors.initials: GM
authors.lastname: Housey
authors.forename: J S
authors.initials: JS
authors.lastname: Flier
mesh_terms: D000818:Animals; D005347:Fibroblasts; D005786:Gene Expression Regulation; D006133:Growth Substances / Q000097:blood / Q000494:pharmacology; D009004:Monosaccharide Transport Proteins / Q000235:genetics*; D010703:Phorbol Esters / Q000494:pharmacology*; D011493:Protein Kinase C / Q000096:biosynthesis*; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000187:drug effects; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured
chemical_list: D006133:Growth Substances; D009004:Monosaccharide Transport Proteins; D010703:Phorbol Esters; D012333:RNA, Messenger; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate