Vanadate inhibits expression of the gene for phosphoenolpyruvate carboxykinase (GTP) in rat hepatoma cells.
Vanadate, at concentrations between 0.5 and 2 mM, rapidly decreased the basal level of P-enolpyruvate carboxykinase (GTP) (EC 4.1.1.32) mRNA and blocked the dibutyryl cyclic AMP (Bt2cAMP)-induced increase in enzyme mRNA in both FTO-2B and H4IIE rat hepatoma cells. The concentration of vanadate necessary to inhibit the expression of this gene was similar to that required for the vanadate-mediated activation of the insulin receptor tyrosine kinase. To determine whether vanadate could inhibit PEPCK gene transcription, a series of chimeric genes containing several deletions in the P-enolypyruvate carboxykinase promoter between -550 and -68 was linked to the structural genes for either amino-3-glycosyl phosphotransferase (neo) or chloramphenicol acetyltransferase and introduced into hepatoma cells using three methods: (a) infection with a Moloney murine leukemia virus-based retrovirus, (b) transfection and stable selection for neo expression, or (c) transient expression of chloroamphenicol acetyltransferase. In FTO-2B hepatoma cells infected with retrovirus, vanadate rapidly (within 1 h) inhibited transcription of the PEPCK-neo gene and blocked induction of gene expression caused by the addition of either Bt2cAMP or dexamethasone to the cells. Vanadate was not a general transcription inhibitor since, it like insulin, stimulated the expression of the c-fos gene. Also, the inhibitory effect of vanadate was rapidly reversible in FTO-2B cells since PEPCK gene expression could be stimulated by Bt2cAMP and dexamethasone after removal of vanadate. A series of 5' deletions in the P-enolpyruvate carboxykinase promoter (-550 to +73) was ligated to the structural gene for neo and stably transfected into hepatoma cells. Sequences responsive to vanadate were detected between -109 and -68. This result was confirmed using H4IIE hepatoma cells transiently expressing the PEPCK-CAT gene. The most likely target for vanadate in that region of the P-enolpyruvate carboxykinase promoter is cAMP regulatory element 1 which maps from -91 to -84. A comparison of the inhibitory effects of insulin and vanadate in this system indicated a major difference in the site of action of these two compounds on PEPCK gene transcription.
Bosch
F
F
Pew Center for Molecular Nutrition, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106.
Hatzoglou
M
M
Park
E A
EA
Hanson
R W
RW
D000818:Animals; D001483:Base Sequence; D003994:Bucladesine / Q000494:pharmacology; D002460:Cell Line; D002678:Chimera; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D015342:DNA Probes; D003907:Dexamethasone / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects*; D005796:Genes / Q000187:drug effects*; D008114:Liver Neoplasms, Experimental / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000235:genetics*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D015183:Restriction Mapping; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection; D014638:Vanadates / Q000494:pharmacology*
D015342:DNA Probes; D012333:RNA, Messenger; D014638:Vanadates; D003994:Bucladesine; D003907:Dexamethasone; D015500:Chloramphenicol O-Acetyltransferase; D010729:Phosphoenolpyruvate Carboxykinase (GTP)