cAMP prevents TNF-induced apoptosis through inhibiting DISC complex formation in rat hepatocytes
Introduction
Tumor necrosis factor alpha (TNF) is a pleiotropic proinflammatory cytokine that mediates the inflammatory response and immune functions and triggers the apoptosis of certain tumor cells [1], [2]. TNF is implicated in the excessive hepatocyte apoptosis seen in many conditions associated with damage in the liver [3], [4]. Excessive production of TNF or the sustained activation of TNF signaling has been implicated in the pathogenesis of a wide spectrum of conditions in humans, including sepsis, cerebral malaria, diabetes, cancer, osteoporosis, allograft rejection, and autoimmune diseases [5], [6], [7], [8]. The pleiotropic nature of TNF is due to the formation of different signaling complexes upon the binding of TNF to one of its receptors, TNF-receptor type 1 (TNFR1). TNFR1 is responsible for the pro-death signaling induced by TNF.
TNF signaling involves the binding of the TNF trimer to the extracellular domain of TNFR1 and the subsequent release of the inhibitory protein – silencer of death domains (SODD) – from the intracellular death domain (DD) of TNFR1. Once TNFR1 is engaged and activated, its DD rapidly binds to the adapter protein TNFR-associated death domain (TRADD), which subsequently recruits other signaling proteins such as TNFR-associated protein 2 (TRAF2) [9], receptor-associated protein kinase (RIPK)1, and cellular inhibitor of apoptosis proteins (cIAP)1 and cIAP2. The resulting complex then recruits the IκB kinase (IKK) complex through the K63-specific polyubiquitin chain on RIPK1 and consequently triggers an NF-κB response but does not lead to apoptosis. NF-κB induces the activation of several anti-apoptotic proteins, including cIAP1, cIAP2 and c-FLIP that prevent apoptosis [10]. If the initially formed complex fails to initiate the expression of the anti-apoptotic factor c-FLIP through NF-κB activation, then a second complex comprising FADD and procaspase-8 subsequently forms in the cytoplasm that leads to the formation of a death-inducing signaling complex (DISC) [11]. The activity of the second complex is inhibited by c-FLIP, a caspase-8 homologue that competes with caspase-8 for binding with FADD. The absence of c-FLIP induces FADD-mediated caspase-8 activation and apoptosis in the DISC of breast cancer cells [12]. Caspase-8-mediated activation of the mitochondrial death pathway through the cleavage of Bid plays a critical role in Aloe-emodin induced apoptosis of human nasopharyngeal carcinoma cells [13].
TNF-induced apoptosis in hepatocytes requires the addition of a sensitizing agent such as actinomycin D (Act D) or cytochrome p450. Eum et al. showed that the sensitizing agent ActD both accelerated and amplified the appearance of the DISC components in both cytosol and the mitochondria in TNF-treated hepatocytes [14]. Nevertheless, the molecular mechanisms of the shift toward apoptosis following sensitization are still not clear. It is known that cAMP prevents TNF + ActD-induced apoptosis in hepatocytes and that this occurs in association with a near complete inhibition of the upregulation of FADD via a PKA-dependent mechanism [15], but the effect of cAMP on DISC formation in hepatocytes is not known.
Cyclic adenosine monophosphate (cAMP) is an intracellular second messenger formed in response to diverse extracellular stimuli, including hormones or neurotransmitters, and its maintargets of cAMP are protein kinase A (PKA) [16]. cAMP regulates numerous cellular processes, including gene expression, cell differentiation, cell cycle progression and apoptosis, both in a PKA-dependent and PKA-independent manner [17], [18], [19]. Some studies indicate that analogues of cyclic nucleotides cause apoptosis in renal mesangial cells, multiple myeloma cells and human cancer cellsandactivation of cAMP signaling enhances Fas-mediated apoptosis and activation-induced cell death through potentiation of caspase-8 activation [20], [21], [22], [23]. In contrast, there have also been studies that have shown that cAMP analogues inhibit apoptosis induced by different stimuli in hepatocytes, neutrophils, smooth muscle cells, andpre-B acute lymphoblastic leukemia NALM-6 cells [15], [24], [25]. Previous studies have reported that cell permeable cyclic nucleotides inhibit apoptosis by modulating caspase activation, cytochromec release, and cIAP expression, and inducing a heat shock protein. A previously study from our laboratory has also shown that cAMP prevents the elevations in FADD levels in response to TNF + ActD in hepatocytes [15]. FADD is elevated early following TNF + ActD exposure in hepatocytes suggesting that the effect of cAMP occurs at a proximal step in TNF-induced signaling. Here we assessed that the effect of cAMP on TNF + ActD-induced DISC formation in cultured hepatocytes.
Section snippets
Materials
William’s medium E, penicillin, streptomycin, l-glutamine and HEPES were purchased from Life Technologies Inc. Mouse recombinant TNF was obtained from R&D systems (Minneapolis, MN). Insulin was purchased from Eli Lilly (Indianapolis, IN). Benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (Z-VAD-fmk), Sp-isomer ((Sp)-cAMPS), and KT5720 were from Alexis Corp (San Diego, CA). Actinomycin D, dibutyryl cyclic adenosine monophosphate (db-cAMP), and were obtained from Sigma (St. Louis, MO). Antibodies
cAMP inhibits cell death in rat hepatocytes
We first investigated the timing and extent of TNF + ActD-induced hepatocyte death in the absence and presence of db-cAMP. Cell viability was measured by crystal violet staining at time points between 0 h (control) and 18 h following treatment. No significant cell death occurred in hepatocytes treated with 200 μM db-cAMP alone at any time point (Fig. 1A). TNF + ActD alone caused a significant degree of cell death by 8 h (Fig. 1A). However, the co-treatment of TNF + ActD with db-cAMP inhibited cell death
Discussion
Cyclic nucleotides, including cAMP and cGMP inhibit TNF + ActD-induced apoptosis in hepatocytes. We have previously shown that cyclic nucleotides block caspase activation, cytochrome c release and signaling downstream of the initiator caspases [26], and that the inhibition of apoptosis is mediated by PKA [15]. Here we extend these previous observations to show that cAMP-mediated inhibition of TNF signaling occurs at a proximal step in DISC formation. cAMP prevented TNF-induced NF-κB activation
Conflict of Interest
Authors do not have any financial relationship with the granting authority. The authors declare that they have no conflict of interest.
Acknowledgment
This work has been supported by the National Institutes of Health Research Grant R37 GM44100.
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These authors contributed equally to this work.