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

Am J Respir Cell Mol Biol. 1999 Feb;20(2):352-8. doi: 10.1165/ajrcmb.20.2.3160.


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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Cattle
  • Colforsin / pharmacology*
  • Cyclic AMP / pharmacology
  • Cyclic AMP Response Element-Binding Protein / metabolism
  • Cyclin D1 / genetics
  • Cyclin D1 / metabolism*
  • DNA Replication / drug effects
  • Muscle, Smooth / cytology
  • Muscle, Smooth / drug effects*
  • Muscle, Smooth / metabolism
  • Nuclear Proteins / metabolism
  • Phosphorylation
  • Platelet-Derived Growth Factor / pharmacology
  • Promoter Regions, Genetic
  • Protein Binding
  • Trachea / cytology
  • Trachea / drug effects*
  • Trachea / metabolism
  • Transcriptional Activation / drug effects


  • Cyclic AMP Response Element-Binding Protein
  • Nuclear Proteins
  • Platelet-Derived Growth Factor
  • Cyclin D1
  • Colforsin
  • Cyclic AMP