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.