Abstract
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.
Highlights
The binding of many hormones and growth factors to cells induces activation of adenylyl cyclase, which catalyzes synthesis of cAMP from ATP [1,2]. cAMP regulates a wide range of processes through its downstream effectors protein kinase A (PKA),2 cyclic nucleotide-gated cation channels, and a small family of guanine nucleotide exchange factors (GEFs) involved in the regulation of Ras-related proteins (Refs. 3–5 and references therein)
To analyze the role of PKA in forskolin-induced cellular proliferation and DNA synthesis, we employed a specific inhibitor of PKA, H-89, and we silenced the expression of the PKA effector CREB by RNAi
We have examined the role of two cAMP effector systems; namely, Epac- and PKA-initiated signaling cascades, on forskolininduced macrophage proliferation
Summary
Stimulation of cell growth and proliferative effects of cAMP in a PKA-independent manner involve activation of Rap via Epac. In these cases, the cell-proliferative effects of cAMP mediated by activation of PI 3-kinase/Akt signaling [6, 8, 9]. We have examined the effect of elevating intracellular cAMP levels on primary macrophage proliferation by both Epac-1-Rap and PKACREB signaling pathways. In these studies, we have used forskolin, an adenylyl cyclase activator and a prototype for elevating cAMP levels, for raising intracellular cAMP levels. Studies employing the silencing of CREB gene expression indicate that both these pathways exhibit cross-talk at the level of Akt activation
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