Cyclic-adenosine 3′,5′-monophosphate-stimulated c-fos gene transcription involves distinct calcium pathways in single β-cells

Abstract

In β-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+] i ) in single β-cells (HIT-T15). Forskolin stimulated a rapid rise in cellular cAMP and in [Ca 2+] i 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+] i , 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+] i 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 β-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 β-cells.