Synergism between calcium and cyclic GMP in cyclic AMP response element-dependent transcriptional regulation requires cooperation between CREB and C/EBP-beta.
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-beta but not C/EBP-alpha or -delta 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-beta 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-beta and CREB interacted directly; this interaction involved the C terminus of C/EBP-beta but occurred independently of CREB's leucine zipper domain. CREB Ser(133) phosphorylation was stimulated by calcium but not by cGMP; in cGMP-treated cells, (32)PO(4) incorporation into C/EBP-beta was decreased and C/EBP-beta/CRE complexes were increased, suggesting regulation of C/EBP-beta functions by G-kinase-dependent dephosphorylation. C/EBP-beta and CREB associated with the fos promoter in intact cells, and the amount of promoter-associated C/EBP-beta was increased by calcium and cGMP. We conclude that calcium and cGMP transcriptional synergism requires cooperation of CREB and C/EBP-beta, with calcium and cGMP modulating the phosphorylation states of CREB and C/EBP-beta, respectively.
Chen
Yongchang
Y
Department of Medicine and Cancer Center, University of California at San Diego, La Jolla, California 92093-0652, USA.
Zhuang
Shunhui
S
Cassenaer
Stijn
S
Casteel
Darren E
DE
Gudi
Tanima
T
Boss
Gerry R
GR
Pilz
Renate B
RB
D016475:3T3 Cells; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000378:metabolism*; D019556:COS Cells; D002118:Calcium / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism*; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D006152:Cyclic GMP / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D005786:Gene Expression Regulation; D005799:Genes, Dominant; D017930:Genes, Reporter; D016762:Genes, fos / Q000235:genetics; D005822:Genetic Vectors; D051379:Mice; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D020218:Response Elements; D015398:Signal Transduction; D013347:Subcellular Fractions / Q000378:metabolism; D013997:Time Factors; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured; D020798:Two-Hybrid System Techniques
D022782:CCAAT-Enhancer-Binding Protein-beta; D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D012333:RNA, Messenger; D000242:Cyclic AMP; D006152:Cyclic GMP; D002118:Calcium