Coordinate regulation of forskolin-induced cellular proliferation in macrophages by protein kinase A/cAMP-response element-binding protein (CREB) and Epac1-Rap1 signaling: effects of silencing CREB gene expression on Akt activation.
In this study, we have examined the role of two cAMP downstream effectors protein kinase A (PKA) and Epac, in forskolin-induced macrophage proliferation. Treatment of macrophages with forskolin enhanced [(3)H]thymidine uptake and increased cell number, and both were profoundly reduced by prior treatment of cells with H-89, a specific PKA inhibitor. Incubation of macrophages with forskolin triggered the activation of Akt, predominantly by phosphorylation of Ser-473, as measured by Western blotting and assay of its kinase activity. Akt activation was significantly inhibited by LY294002 and wortmannin, specific inhibitors of phosphatidylinositol 3-kinase, but not by H-89. Incubation of macrophages with forskolin also increased Epac1 and Rap1.GTP. Immunoprecipitation of Epac1 in forskolin-stimulated cells co-immunoprecipitated Rap1, p-Akt(Thr-308), and p-Akt(Ser-473). Silencing of CREB gene expression by RNA interference prior to forskolin treatment not only decreased CREB protein and its phosphorylation at Ser-133, but also phosphorylation of Akt at Ser-473, and Thr-308. Concomitantly, this treatment inhibited [(3)H]thymidine uptake and reduced forskolin-induced proliferation of macrophages. Forskolin treatment also inhibited activation of the apoptotic mechanism while promoting up-regulation of the anti-apoptotic pathway. We conclude that forskolin mediates cellular proliferation via cAMP-dependent activation of both PKA and Epac.
Misra
Uma K
UK
Department of Pathology, Duke University, Medical Center, Durham, North Carolina 27710, USA.
Pizzo
Salvatore V
SV
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002452:Cell Count; D049109:Cell Proliferation; D002867:Chromones / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology*; D000242:Cyclic AMP / Q000378:metabolism; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D019938:Cyclin D1 / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors / Q000378:metabolism; D005786:Gene Expression Regulation; D020868:Gene Silencing; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000737:chemistry; D047468:Immunoprecipitation; D007546:Isoquinolines / Q000494:pharmacology; D008264:Macrophages / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D009025:Morpholines / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000737:chemistry; D012694:Serine / Q000737:chemistry; D015398:Signal Transduction; D013449:Sulfonamides / Q000494:pharmacology; D013912:Threonine / Q000737:chemistry; D013936:Thymidine / Q000378:metabolism; D013997:Time Factors; D015854:Up-Regulation; D000077191:Wortmannin; D051636:X-Linked Inhibitor of Apoptosis Protein / Q000378:metabolism; D051025:bcl-Associated Death Protein / Q000378:metabolism; D020827:rap1 GTP-Binding Proteins / Q000378:metabolism*
D000730:Androstadienes; D002867:Chromones; D017362:Cyclic AMP Response Element-Binding Protein; D004791:Enzyme Inhibitors; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors; C467200:Foxo1 protein, mouse; D020662:Guanine Nucleotide Exchange Factors; D007546:Isoquinolines; D009025:Morpholines; D019253:Proto-Oncogene Proteins c-bcl-2; C116122:RAPGEF3 protein, human; D012330:RNA, Double-Stranded; D013449:Sulfonamides; D051636:X-Linked Inhibitor of Apoptosis Protein; D051025:bcl-Associated Death Protein; D019938:Cyclin D1; D005576:Colforsin; D013912:Threonine; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D012694:Serine; D006160:Guanosine Triphosphate; D000242:Cyclic AMP; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D017868:Cyclic AMP-Dependent Protein Kinases; D020827:rap1 GTP-Binding Proteins; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; D013936:Thymidine; D000077191:Wortmannin