The regulation of p33 gene expression by insulin and calcium ionophores.

We have previously shown that insulin induces p33 transcription and mRNA levels in serum-deprived rat H4 hepatoma cells. In the current study, the effects of the calcium ionophores A23187 and ionomycin on the regulation of p33 gene expression were examined. When H4 cells were incubated with A23187 or ionomycin (1 microM) for 90-300 min, 700-900% and 400-600% increases in p33 mRNA levels were observed. The effects of ionophore and insulin together on p33 mRNA levels were not additive. Insulin-induced increases in p33 mRNA levels were diminished at low concentrations of extracellular calcium, but were unchanged by the calcium channel blocker verapamil. The chelation of intracellular calcium using 20 and 60 microM quin2-AM resulted in 50% and 90% reductions in insulin-induced p33 mRNA levels. When transcription assays were performed, A23187 treatment for 15-180 min increased p33 transcription 300-400%. The lesser effect of A23187 on transcription compared to that on mRNA levels was also true for insulin treatment. Similar results were obtained using ionomycin. Insulin- and A23187-induced p33 transcription was reduced by quin2-AM to below basal levels. These studies show that calcium ionophores stimulate p33 gene expression and suggest that changes in intracellular calcium can alter insulin's induction of this gene.