Cyclic Nucleotides Suppress Tumor Necrosis Factor α-Mediated Apoptosis by Inhibiting Caspase Activation and Cytochrome cRelease in Primary Hepatocytes via a Mechanism Independent of Akt Activation
ABSTRACT 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.
- SourceAvailable from: Leila Gobejishvili
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- "PDE4B, plays an essential role in LPS-induced TLR4 signaling and inflammatory cytokine expression by monocytes/ macrophages (Jin and Conti, 2002; Jin et al., 2005; Gobejishvili et al., 2011). In cholestatic liver injury, besides downregulating systemic and hepatic inflammatory cytokines, increases in cAMP have been shown to protect hepatocytes from apoptosis due to several related stimuli, including bile acids, LPS, Fas, and TNF-a (Fladmark et al., 1997; Li et al., 2000; Webster et al., 2002; Cullen et al., 2004; Reinehr and Haussinger, 2004). Work done with PDE inhibitors has demonstrated their beneficial effect in experimental liver injury (Fischer et al., 1993; Gantner et al., 1997; Windmeier and Gressner, 1997; Taguchi et al., 1999; Xiang et al., 1999; Matsuhashi et al., 2005; Tukov et al., 2007), but there have been no studies examining the causal role of PDEs in the pathogenesis of liver fibrosis. "
ABSTRACT: Anti-inflammatory and anti-fibrotic effects of the broad spectrum PDE inhibitor Pentoxifylline have suggested an important role for cyclic nucleotides in the pathogenesis of hepatic fibrosis; however studies examining the role of specific PDEs are lacking. Endotoxemia and Toll-like receptor 4 (TLR4) mediated inflammatory and pro-fibrotic signaling play a major role in the development of hepatic fibrosis. Since cAMPspecific PDE4 critically regulates LPS-TLR4 induced inflammatory cytokine expression, its pathogenic role in bile duct ligation induced hepatic injury and fibrogenesis in Sprague Dawley rats was examined. Initiation of cholestatic liver injury and fibrosis was accompanied by a significant induction of PDE4A, B and D expression and activity. Treatment with the PDE4-specific inhibitor, rolipram, significantly decreased liver PDE4 activity, hepatic inflammatory and pro-fibrotic cytokine expression, injury and fibrosis. At the cellular level, in relevance to endotoxemia and inflammatory cytokine production, PDE4B was observed to play a major regulatory role in the LPS-inducible TNF production by isolated Kupffer cells. Moreover, PDE4 expression was also involved in the in vitro activation and transdifferentiation of isolated hepatic stellate cells (HSCs). Particularly, PDE4A, B and D up-regulation preceded induction of the HSC activation marker α-SMA. In vitro treatment of HSCs with rolipram effectively attenuated α-SMA, collagen expression and accompanying morphological changes. Overall, these data strongly suggest that up-regulation of PDE4 expression during cholestatic liver injury plays a potential pathogenic role in the development of inflammation, injury and fibrosis.Journal of Pharmacology and Experimental Therapeutics 07/2013; 347(1). DOI:10.1124/jpet.113.204933 · 3.86 Impact Factor
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- "Therefore, improvement of cardiac contractility and allograft survival by db-cAMP in this study may be due to the suppression of NF-κB-dependent increases in inflammatory cytokine and iNOS expression. In addition, cAMP has been demonstrated to inhibit apoptosis by suppressing the activation of multiple caspases (Li et al., 2000). We showed that db-cAMP inhibited caspase-3 activation and activity. "
ABSTRACT: This study was designed to investigate the effects of cAMP on immune regulation and apoptosis during acute rat cardiac allograft rejection. We found that the production of immune markers such as inflammatory cytokines (IL-1β, IL-6, and TNF-α), iNOS expression, and nitric oxide (NO) production, was significantly increased in the blood and transplanted hearts of allograft recipients, but not of isograft controls. These increases were effectively suppressed by the administration of the membrane permeable cAMP analog dibutyryl cAMP (db-cAMP). Administration of db-cAMP reduced allograft-induced elevation of several biochemical markers, such as adhesion molecule expression, iron-nitrosyl complex formation, caspase-3 activation, and apoptotic DNA fragmentation in an animal model. Furthermore, treatment of allograft recipients with db-cAMP prolonged median graft survival to 11 days compared with a median graft survival time of 8 days in saline-treated allograft recipients. These results suggest that db-cAMP exerts a beneficial effect on murine cardiac allograft survival by modulating allogeneic immune response and cytotoxicity.Experimental and Molecular Medicine 11/2009; 42(1). DOI:10.3858/emm.2010.42.1.008 · 2.46 Impact Factor
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- "These results have been confirmed in annexin V and DNA fragmentation assays (Figs 1 and 2). PKA and its activator cAMP are established mediators of differential sensitivity to apoptosis (Van Beeren et al. 1996, Fladmark et al. 1997, Kopperud et al. 2003, Li et al. 2000 "
ABSTRACT: The present study demonstrates that 3,5,3'-tri-iodothyronine (T3) in physiological dose range inhibits tumor necrosis factor alpha(TNFalpha)/Fas-induced apoptosis in mouse hepatocytes. T3 pretreatment prevented Fas-induced early stage of apoptosis signs assessed by flow cytometry analysis of the annexin V positive cell population. T3 attenuated TNFalpha/Fas-induced cleavage of caspase-8 and DNA fragmentation. We found that T3 exerted its anti-apoptotic effects by mobilization of several non-genomic mechanisms independent of transcriptional activity. Inhibition of protein kinase A (PKA), extracellular signal-regulated kinase (ERK), and Na+/H+ exchanger blocked T3-dependent anti-apoptotic effects indicating an involvement of these intracellular targets into T3-induced signaling cascade. Furthermore, physiological concentrations of T3, but not reverse T3, caused increases in intracellular cAMP content and activated PKA. T3 markedly induced phosphorylation of ERK. We also detected T3-dependent intracellular alkalinization that abolished TNFalpha-induced acidification. PKA inhibitor KT-5720 blocked T3-induced activation of ERK and intracellular alkalinization confirming the upstream position of PKA signaling. We further detected that hepatocytes from hypothyroid mice are more sensitive to TNFalpha/Fas-induced apoptosis than euthyroid animals in vivo. Together, these findings imply that T3 triggers PKA- and ERK-regulated intracellular pathways capable of driving and ensuring hepatocytes survival in the presence of death receptor ligand-induced damage under chronic inflammatory conditions.Journal of Endocrinology 12/2006; 191(2):447-58. DOI:10.1677/joe.1.07061 · 3.59 Impact Factor