Forskolin inhibits cyclin D1 expression in cultured airway smooth-muscle cells.

Accumulation of intracellular cyclic adenosine monophosphate (cAMP) has been shown to inhibit the growth of cultured airway smooth-muscle cells, but the precise mechanism underlying the antimitogenic action of cAMP in these cells is unknown. We examined the effects of forskolin, an activator of adenylate cyclase, on DNA synthesis, cyclin D1 expression, and cAMP response element-binding protein (CREB) phosphorylation and DNA binding in bovine tracheal myocytes. DNA synthesis was assessed by measurement of [3H]thymidine incorporation. Cyclin D1 protein abundance and CREB phosphorylation were assessed by immunoblotting. Cyclin D1 promoter transcriptional activation was determined by measurement of luciferase activity in cells transiently cotransfected with complementary DNAs encoding the full-length cyclin D1 promoter subcloned into a luciferase reporter and beta-galactosidase (to normalize for transfection efficiency). The binding of nuclear proteins to the cyclin D1 promoter cAMP response element (CRE) was determined by electrophoretic mobility shift assay. We found that forskolin attenuated platelet-derived growth factor-induced DNA synthesis in a concentration-dependent manner. In addition, forskolin pretreatment decreased both cyclin D1 promoter activity and protein levels. Forskolin treatment induced the phosphorylation of CREB and increased the binding of nuclear protein to the cyclin D1 promoter CRE. Finally, addition of an antibody against CREB1 induced supershift of at least one protein-DNA complex. Together, these data suggest that cAMP suppresses cyclin D1 gene expression via phosphorylation and transactivation of CREB. Further studies are needed to determine whether this is the primary mechanism of cAMP-induced growth inhibition, or whether additional pathways are also involved.

Catalytic activation of extracellular signal-regulated kinases induces cyclin D1 expression in primary tracheal myocytes.

We have demonstrated that extracellular signal-regulated kinases (ERKs) and cyclin D1 are required for bovine tracheal myocyte DNA synthesis. We hypothesized that catalytic activation by ERKs may regulate cyclin D1 expression in these cells. To test this hypothesis, we examined the effects of two inhibitors of ERKs and two reagents that increase the level of activated ERKs on cyclin D1 protein abundance and promoter activity. ERK activity was inhibited either by PD98059, a synthetic inhibitor of mitogen-activated protein kinase (MAPK)/ERK kinase (MEK), the upstream signaling intermediate required and sufficient for ERK activation, or by transient transfection with a dominant-negative mutant of MEK1 (MEK-2A). The level of activated ERKs was increased by transient transfection with either a constitutively active form of MEK1 (MEK-2E) or wild-type ERK2 (MAPKwt). Cyclin D1 expression was assessed either by immunoblot or cotransfection with the full-length cyclin D1 promoter subcloned into a luciferase reporter. We found that pretreatment of bovine tracheal myocytes with PD98059 significantly attenuated platelet- derived growth factor (PDGF)-induced cyclin D1 protein abundance. Furthermore, transfection with MEK-2A reduced PDGF-induced cyclin D1 promoter activity. Finally, transfection with either MEK-2E or MAPKwt induced cyclin D1 promoter activity in the absence of growth factor treatment. We conclude that catalytic activation of ERKs regulates cyclin D1 expression in airway smooth-muscle cells.