Synergism between Calcium and Cyclic GMP in Cyclic AMP Response Element-Dependent Transcriptional Regulation Requires Cooperation between CREB and C/EBP-β

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.

ACKNOWLEDGMENTS

We are grateful to M. Ellisman, P. F. Johnson, N. C. Jones, S. Lohmann, R. A. Maurer, M. Montmini, E. J. Murray, L. Sealy, and C. Vinson for providing cell lines and plasmids.

This work was supported by Public Health Service grants 1R01 GM-55586 (to R.B.P.) and 1R01 CA-90932 (to G.R.B.).