Synergistic activation of cAMP and calcium on cAMP-response-element-mediated gene expression in GH3 pituitary tumor cells.

Signals responsible for expression of the vasoactive intestinal peptide (VIP)-stimulated prolactin gene in GH3 pituitary tumor cells were examined. Transfection with a deoxyribonucleic acid (DNA) construct containing the chloramphenicol acetyltransferase (CAT) gene fused to the 2.5-kb prolactin 5'-upstream regulatory sequence indicated that VIP stimulated CAT expression. However, this effect could not be mimicked by 8-bromo-cyclic adenosine monophosphate (8-Br-cAMP), and was inhibited by the L-type Ca(2+)-channel blocker verapamil. While KCl had little effect on CAT activity, combined treatment with KCl and 8-Br-cAMP synergistically activated CAT expression. Potentiation between KCl and 8-Br-c-AMP was also seen with c-fos messenger ribonucleic acid (mRNA) expression. In addition, KCl and 8-Br-cAMP synergistically activated cAMP response element (CRE)-mediated CAT expression, and the synergism was abolished by verapamil. In the presence of okadaic acid, cAMP had no significant activation on CRE-driven CAT expression, whereas KCl-stimulated CAT expression was greatly potentiated. These results indicate that cAMP and Ca2+ synergistically activated CRE-driven gene expression through non-overlapping phosphorylation events in GH3 cells.