Cyclic nucleotides suppress tumor necrosis factor alpha-mediated apoptosisby inhibiting caspase activation and cytochrome c release in primary hepatocytes via a mechanism independent of Akt activation

Cyclic nucleotides have been previously shown to modulate cell death proces ses in many cell types; however, the mechanisms by which cyclic nucleotides regulate apoptosis are unclear. In this study, we demonstrated that cAMP a s well as cGMP analogs suppressed tumor necrosis factor alpha (TNF alpha) p lus actinomycin D (ActD)-induced apoptosis in a dose-dependent manner in cu ltured primary hepatocytes. Furthermore, forskolin, which increases intrace llular cAMP levels, also effectively suppressed TNF alpha+ActD-induced apop tosis. Activation of multiple caspases was suppressed in cells exposed to T NF alpha+ActD in the presence of cAMP or cGMP analogs. TNF alpha+ActD-induc ed cytochrome c release from mitochondria was also inhibited by cAMP or cGM P, reinforcing our conclusion that cyclic nucleotides interfere with the ea rly signaling events of TNF alpha-mediated apoptosis, We evaluated the poss ibility that cAMP and cGMP inhibit apoptosis by activating the serine/threo nine kinase Akt, which is known to promote cell survival. Both cAMP- and cG MP-elevating agents led to marked increases in Akt activation that was inhi bited by the phosphatidylinositol 3'-kinase inhibitors, LY294002 and wortma nnin. However, complete inhibition of cyclic nucleotide-induced Akt activat ion had little effect on cyclic nucleotide-mediated cell survival, indicati ng the existence of other survival pathways. Interestingly, the specific in hibitor of protein kinase A (PKA), KT5720, blocked cGMP-mediated protection but only partially prevented the anti-apoptotic effect of cAMP, indicating that both PKA-dependent and -independent mechanisms are involved in cAMP-m ediated suppression of apoptosis signaling. Our data suggest that multiple survival signaling pathways coexist in cells and that cyclic nucleotides de lay apoptosis by interfering with apoptosis signaling by both PKA-dependent and -independent mechanisms.