Synergism between Calcium and Cyclic GMP in Cyclic AMP Response Element-Dependent Transcriptional Regulation Requires Cooperation between CREB and C/EBP-β
Authors:
Open Access
- 1 June 2003
- journal article
- Published by Taylor & Francis in Molecular and Cellular Biology
- Vol. 23 (12) , 4066-4082
Abstract
Calcium induces transcriptional activation of the fos promoter by activation of the cyclic AMP response element (CRE)-binding protein (CREB), and in some cells its effect is enhanced synergistically by cyclic GMP (cGMP) through an unknown mechanism. We observed calcium-cGMP synergism in neuronal and osteogenic cells which express type II cGMP-dependent protein kinase (G-kinase); the effect on the fos promoter was mediated by the CRE and proportional to G-kinase activity. Dominant negative transcription factors showed involvement of CREB- and C/EBP-related proteins but not of AP-1. Expression of C/EBP-β but not C/EBP-α or -δ enhanced the effects of calcium and cGMP on a CRE-dependent reporter gene. The transactivation potential of full-length CREB fused to the DNA-binding domain of Gal4 was increased synergistically by calcium and cGMP, and overexpression of C/EBP-β enhanced the effect, while a dominant negative C/EBP inhibited it. With a mammalian two-hybrid system, coimmunoprecipitation experiments, and in vitro binding studies, we demonstrated that C/EBP-β and CREB interacted directly; this interaction involved the C terminus of C/EBP-β but occurred independently of CREB's leucine zipper domain. CREB Ser133 phosphorylation was stimulated by calcium but not by cGMP; in cGMP-treated cells, 32PO4 incorporation into C/EBP-β was decreased and C/EBP-β/CRE complexes were increased, suggesting regulation of C/EBP-β functions by G-kinase-dependent dephosphorylation. C/EBP-β and CREB associated with the fos promoter in intact cells, and the amount of promoter-associated C/EBP-β was increased by calcium and cGMP. We conclude that calcium and cGMP transcriptional synergism requires cooperation of CREB and C/EBP-β, with calcium and cGMP modulating the phosphorylation states of CREB and C/EBP-β, respectively.Keywords
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