Forskolin inhibits cyclin D-1 expression in cultured airway smooth-muscle cells

Accumulation of intracellular cyclic adenosine monophosphate (cAMP) has bee n shown to inhibit the growth of cultured airway smooth-muscle cells, but t he precise mechanism underlying the antimitogenic action of cAMP in these c ells is unknown. We examined the effects of forskolin, an activator of aden ylate cyclase, on DNA synthesis, cyclin D-1 expression, and cAMP response e lement-binding protein (CREB) phosphorylation and DNA binding in bovine tra cheal myocytes. DNA synthesis was assessed by measurement of [H-3]thymidine incorporation. Cyclin D-1 protein abundance and CREB phosphorylation were assessed by immunoblotting. Cyclin D-1 promoter transcriptional activation was determined by measurement of luciferase activity in cells transiently c otransfected with complementary DNAs encoding the full-length cyclin D-1 pr omoter subcloned into a luciferase reporter and beta -galactosidase (to nor malize for transfection efficiency). The binding of nuclear proteins to the cyclin D-1 promoter cAMP response element (CRE) was determined by electrop horetic mobility shift assay. We found that forskolin attenuated platelet-d erived growth factor-induced DNA synthesis in a concentration-dependent man ner. In addition, forskolin pretreatment decreased both cyclin D-1 promoter activity and protein levels. Forskolin treatment induced the phosphorylati on of CREB and increased the binding of nuclear protein to the cyclin D-1 p romoter CRE. Finally, addition of an antibody against CREB 1 induced supers hift of at least one protein-DNA complex. Together, these data suggest that cAMP suppresses cyclin D-1 gene expression via phosphorylation and transac tivation of CREB. Further studies are needed to determine whether this is t he primary mechanism of cAMP-induced growth inhibition, or whether addition al pathways are also involved.