Cyclic-adenosine 3',5'-monophosphate-stimulated c-fos gene transcription involves distinct calcium pathways in single beta-cells.

In beta-cells activation of the cyclic AMP (cAMP)-signaling cascade stimulates c-fos mRNA expression, which involves cAMP- and Ca(2+)-mediated mechanisms. To delineate potential crosstalk between both pathways at the transcriptional level we simultaneously measured c-fos promoter-driven enhanced green fluorescent protein (EGFP) expression and cytosolic free calcium (Ca(2+)) in single beta-cells (HIT-T15). Forskolin stimulated a rapid rise in cellular cAMP and in Ca(2+) through activation of voltage-sensitive Ca(2+)-influx and enhanced wild-type c-fos promoter-driven EGFP (pF711d2EGFP) expression about 4-fold after 6 h. The voltage-sensitive Ca(2+) channel (VSCC)-blocker nifedipine, which completely blocked the forskolin-induced rise in Ca(2+), partially inhibited the forskolin-induced increase in pF711d2EGFP expression, while it was completely abolished in Ca(2+)-free medium. VSCC-dependent Ca(2+)-influx per se when stimulated by K(+) (45 mM) increased pF711d2EGFP expression only minimally. No correlations could be delineated between the forskolin-induced amplitude of the Ca(2+) signal and the expression of pF711d2EGFP at the single cell level, which may indicate that small rises in Ca(2+) are sufficient to fully activate the Ca(2+)-dependent pathways required for cAMP-dependent c-fos promoter regulation. In experiments with various deletion constructs of the c-fos promoter, it could be shown that cAMP-mediated activation of the c-fos promoter involves both the cAMP-responsive element (CRE) and the serum-responsive element (SRE). While nifedipine completely abrogated the cAMP-dependent activation of c-fos transcription via the SRE, the CRE-mediated effect of cAMP on the c-fos promoter remained unaffected by nifedipine. Thus, cAMP and Ca(2+) are required for full c-fos promoter activation by the cAMP-signaling pathway in beta-cells. cAMP-dependent Ca(2+)-influx through VSCC is crucial for c-fos gene transcription via the SRE, whereas cAMP-mediated activation of the CRE demands Ca(2+)-influx, which is distinct from voltage-sensitive Ca(2+)-influx. This indicates a complex interplay between cAMP and Ca(2+) in controlling c-fos gene transcription and suggests that the mode of Ca(2+) entry may differentially act on signaling pathways leading to gene transcription in beta-cells.