Cyclic nucleotides suppress tumor necrosis factor alpha-mediated apoptosis by 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 processes in many cell types; however, the mechanisms by which cyclic nucleotides regulate apoptosis are unclear. In this study, we demonstrated that cAMP as well as cGMP analogs suppressed tumor necrosis factor alpha (TNFalpha) plus actinomycin D (ActD)-induced apoptosis in a dose-dependent manner in cultured primary hepatocytes. Furthermore, forskolin, which increases intracellular cAMP levels, also effectively suppressed TNFalpha+ActD-induced apoptosis. Activation of multiple caspases was suppressed in cells exposed to TNFalpha+ActD in the presence of cAMP or cGMP analogs. TNFalpha+ActD-induced cytochrome c release from mitochondria was also inhibited by cAMP or cGMP, reinforcing our conclusion that cyclic nucleotides interfere with the early signaling events of TNFalpha-mediated apoptosis. We evaluated the possibility that cAMP and cGMP inhibit apoptosis by activating the serine/threonine kinase Akt, which is known to promote cell survival. Both cAMP- and cGMP-elevating agents led to marked increases in Akt activation that was inhibited by the phosphatidylinositol 3'-kinase inhibitors, LY294002 and wortmannin. However, complete inhibition of cyclic nucleotide-induced Akt activation had little effect on cyclic nucleotide-mediated cell survival, indicating the existence of other survival pathways. Interestingly, the specific inhibitor 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-mediated suppression of apoptosis signaling. Our data suggest that multiple survival signaling pathways coexist in cells and that cyclic nucleotides delay apoptosis by interfering with apoptosis signaling by both PKA-dependent and -independent mechanisms.
Li
J
J
Department of Surgery, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA. jianrong@pitt.edu
Yang
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Billiar
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D000590:Amino Acid Chloromethyl Ketones / Q000378:metabolism; D000818:Animals; D017209:Apoptosis; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D002470:Cell Survival; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism; D003574:Cytochrome c Group / Q000037:antagonists & inhibitors*; D053938:DNA Fragmentation; D003609:Dactinomycin / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D007328:Insulin / Q000494:pharmacology; D008099:Liver / Q000201:enzymology*; D008297:Male; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
D000590:Amino Acid Chloromethyl Ketones; D061945:Caspase Inhibitors; D003574:Cytochrome c Group; D007328:Insulin; D019384:Nucleic Acid Synthesis Inhibitors; D011518:Proto-Oncogene Proteins; D014409:Tumor Necrosis Factor-alpha; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D003609:Dactinomycin; D000242:Cyclic AMP; C494920:Akt1 protein, rat; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D017868:Cyclic AMP-Dependent Protein Kinases; C505410:Casp3 protein, rat; C505528:Casp8 protein, rat; C506435:Casp9 protein, rat; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; D006152:Cyclic GMP