Death receptor 5 signaling promotes hepatocyte lipoapoptosis.
ABSTRACT: Nonalcoholic steatohepatitis is characterized by hepatic steatosis, elevated levels of circulating free fatty acids (FFA), endoplasmic reticulum (ER) stress, and hepatocyte lipoapoptosis. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptor 5 (DR5) is significantly elevated in patients with nonalcoholic steatohepatitis, and steatotic hepatocytes demonstrate increased sensitivity to TRAIL-mediated cell death. Nonetheless, a role for TRAIL and/or DR5 in mediating lipoapoptotic pathways is unexplored. Here, we examined the contribution of DR5 death signaling to lipoapoptosis by free fatty acids. The toxic saturated free fatty acid palmitate induces an increase in DR5 mRNA and protein expression in Huh-7 human hepatoma cells leading to DR5 localization into lipid rafts, cell surface receptor clustering with subsequent recruitment of the initiator caspase-8, and ultimately cellular demise. Lipoapoptosis by palmitate was not inhibited by a soluble human recombinant DR5-Fc chimera protein suggesting that DR5 cytotoxic signaling is ligand-independent. Hepatocytes from murine TRAIL receptor knock-out mice (DR(-/-)) displayed reduced palmitate-mediated lipotoxicity. Likewise, knockdown of DR5 or caspase-8 expression by shRNA technology attenuated palmitate-induced Bax activation and apoptosis in Huh-7 cells, without altering induction of ER stress markers. Similar observations were verified in other cell models. Finally, knockdown of CHOP, an ER stress-mediated transcription factor, reduced DR5 up-regulation and DR5-mediated caspase-8 activation upon palmitate treatment. Collectively, these results suggest that ER stress-induced CHOP activation by palmitate transcriptionally up-regulates DR5, likely resulting in ligand-independent cytotoxic signaling by this death receptor.
Project description:Endoplasmic reticulum (ER) stress-mediated apoptosis is a key feature of hepatocyte cytotoxicity by saturated free fatty acids (FFA). This lipoapoptosis is dependent, in part, on the transcriptional upregulation of the BH3-only protein PUMA (p53 upregulated modulator of apoptosis). Although the activator protein (AP)-1 complex facilitates PUMA expression by saturated FFA, the transcription factor CAAT/enhancer binding homologous protein (CHOP) is also induced by ER stress and promotes apoptosis. To integrate the role of these two transcription factors in ER stress-induced apoptosis, we examined the relative contribution of CHOP and AP-1 in mediating PUMA induction by saturated FFA. Our results demonstrate that short-hairpin RNA-targeted knockdown of CHOP attenuates palmitate-induced apoptosis in Huh-7 cells. Loss of CHOP induction also reduced the increase in PUMA mRNA and protein levels as well as Bax activation by palmitate. No functional CHOP binding sites were identified in the PUMA promoter sequence. Rather, we observed that CHOP physically interacts with the AP-1 complex protein c-Jun upon palmitate treatment, and a CHOP:phosphorylated c-Jun heteromeric complex binds to the AP-1 consensus binding sequence within the PUMA promoter region. Finally, loss of function studies suggest that both transcription factors are necessary for maximal PUMA induction. Collectively, these data suggest that CHOP and AP-1 cooperatively mediate PUMA induction during hepatocyte lipoapoptosis.
Project description:Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent for esophageal squamous cell carcinoma (ESCC). Forced expression of CHOP, one of the key downstream transcription factors during endoplasmic reticulum (ER) stress, upregulates the death receptor 5 (DR5) levels and promotes oxidative stress and cell death. In this study, we show that ER stress mediated by thapsigargin promoted CHOP and DR5 synthesis thus sensitizing TRAIL treatment, which induced ESCC cells apoptosis. These effects were reversed by DR5 siRNA in vitro and CHOP siRNA both in vitro and in vivo. Besides, chemically inhibition of AMPK by Compound C and AMPK siRNA weakened the anti-cancer effect of thapsigargin and TRAIL co-treatment. Therefore, our findings suggest ER stress effectively sensitizes human ESCC to TRAIL-mediated apoptosis via the TRAIL-DR5-AMPK signaling pathway, and that activation of ER stress may be beneficial for improving the efficacy of TRAIL-based anti-cancer therapy.
Project description:Saturated free fatty acids induce hepatocyte lipoapoptosis. This lipotoxicity involves an endoplasmic reticulum stress response, activation of JNK, and altered expression and function of Bcl-2 proteins. The mono-unsaturated free fatty acid palmitoleate is an adipose-derived lipokine which suppresses free fatty acid-mediated lipotoxicity by unclear mechanisms. Herein we examined the mechanisms responsible for cytoprotection.We employed isolated human and mouse primary hepatocytes, and the Huh-7 and Hep 3B cell lines for these studies. Cells were incubated in presence and absence of palmitate (16:0), stearate (18:0), and or palmitoleate (16:1, n-7).Palmitoleate significantly reduced lipoapoptosis by palmitate or stearate in both primary cells and cell lines. Palmitoleate accentuated palmitate-induced steatosis in Huh-7 cells excluding inhibition of steatosis as a mechanism for reduced apoptosis. Palmitoleate inhibited palmitate induction of the endoplasmic reticulum stress response as demonstrated by reductions in CHOP expression, eIF2-alpha phosphorylation, XBP-1 splicing, and JNK activation. Palmitate increased expression of the BH3-only proteins PUMA and Bim, which was attenuated by palmitoleate. Consistent with its inhibition of PUMA and Bim induction, palmitoleate prevented activation of the downstream death mediator Bax.These data suggest palmitoleate inhibits lipoapoptosis by blocking endoplasmic reticulum stress-associated increases of the BH3-only proteins Bim and PUMA.
Project description:Lipoapoptosis occurring due to an excess of saturated free fatty acids such as palmitate is a key pathogenic event in the initiation of nonalcoholic fatty liver disease. Palmitate loading of cells activates the endoplasmic reticulum stress response, including induction of the proapoptotic transcription factor C/EBP homologous protein (CHOP). Furthermore, the loss of microRNAs is implicated in regulating apoptosis under conditions of endoplasmic reticulum (ER) stress. The aim of this study was to identify specific microRNAs regulating CHOP expression during palmitate-induced ER stress. Five microRNAs were repressed under palmitate-induced endoplasmic reticulum stress conditions in hepatocyte cell lines (miR-92b-3p, miR-328-3p, miR-484, miR-574-5p, and miR-615-3p). We identified miR-615-3p as a candidate microRNA which was repressed by palmitate treatment and regulated CHOP protein expression, by RNA sequencing and in silico analyses, respectively. There is a single miR-615-3p binding site in the 3'untranslated region (UTR) of the Chop transcript. We characterized this as a functional binding site using a reporter gene-based assay. Augmentation of miR-615-3p levels, using a precursor molecule, repressed CHOP expression; and under these conditions palmitate- or tunicamycin-induced cell death were significantly reduced. Our results suggest that palmitate-induced apoptosis requires maximal expression of CHOP which is achieved via the downregulation of its repressive microRNA, miR-615-3p. We speculate that enhancement of miR-615-3p levels may be of therapeutic benefit by inhibiting palmitate-induced hepatocyte lipoapoptosis.
Project description:Palmitate, a saturated fatty acid (FA), is known to induce toxicity and cell death in various types of cells. Resveratrol (RSV) is able to prevent pathogenesis and/or decelerate the progression of a variety of diseases. Several in vitro and in vivo studies have also shown a protective effect of RSV on fat accumulation induced by FAs. Additionally, endoplasmic reticulum (ER) stress has recently been linked to cellular adipogenic responses. To address the hypothesis that the RSV effect on excessive fat accumulation promoted by elevated saturated FAs could be partially mediated by a reduction of ER stress, we studied the RSV action on experimentally induced ER stress using palmitate in several cancer cell lines.We show that, unexpectedly, RSV promotes an amplification of palmitate toxicity and cell death and that this mechanism is likely due to a perturbation of palmitate accumulation in the triglyceride form and to a less important membrane fluidity variation. Additionally, RSV decreases radical oxygen species (ROS) generation in palmitate-treated cells but leads to enhanced X-box binding protein-1 (XBP1) splicing and C/EBP homologous protein (CHOP) expression. These molecular effects are induced simultaneously to caspase-3 cleavage, suggesting that RSV promotes palmitate lipoapoptosis primarily through an ER stress-dependent mechanism. Moreover, the lipotoxicity reversion induced by eicosapentaenoic acid (EPA) or by a liver X receptor (LXR) agonist reinforces the hypothesis that RSV-mediated inhibition of palmitate channeling into triglyceride pools could be a key factor in the aggravation of palmitate-induced cytotoxicity.Our results suggest that RSV exerts its cytotoxic role in cancer cells exposed to a saturated FA context primarily by triglyceride accumulation inhibition, probably leading to an intracellular palmitate accumulation that triggers a lipid-mediated cell death. Additionally, this cell death is promoted by ER stress through a CHOP-mediated apoptotic process and may represent a potential anticancer strategy.
Project description:Amiodarone is a widely used anti-arrhythmic drug that inhibits diverse ion channels, including the Na(+)/Ca(2+) exchanger (NCX), L-type Ca(2+) channels, and Na(+) channels. Here, we report that subtoxic doses of amiodarone and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) synergistically induced apoptosis of various glioma cells. Treatment of U251MG glioma cells with amiodarone increased intracellular Ca(2+) levels and enhanced the expression of the endoplasmic reticulum (ER) stress-inducible transcription factor C/EBP homologous protein (CHOP). This upregulation of CHOP was followed by marked upregulation of the TRAIL receptor, DR5. Suppression of DR5 expression by small interfering (si) RNAs almost completely blocked amiodarone/TRAIL-induced apoptosis in U251MG glioma cells, demonstrating that DR5 is critical to this cell death. siRNA-mediated CHOP suppression reduced amiodarone-induced DR5 upregulation and attenuated the cell death induced by amiodarone plus TRAIL. In addition, omitting Ca(2+) from the external medium using ethylene glycol tetraacetic acid markedly inhibited this cell death, reducing the protein levels of CHOP and DR5. These results suggest that amiodarone-induced influx of Ca(2+) plays an important role in sensitizing U251MG cells to TRAIL-mediated apoptosis through CHOP-mediated DR5 upregulation. Furthermore, subtoxic doses of bepridil and cibenzoline, two other anti-arrhythmic drugs with NCX-inhibitor activity, also sensitized glioma cells to TRAIL-mediated apoptosis, via the upregulation of both CHOP and DR5. Notably, amiodarone/TRAIL cotreatment did not induce cell death in astrocytes, nor did it affect the expression of CHOP or DR5 in these cells. These results collectively suggest that a combined regimen of amiodarone plus TRAIL may offer an effective therapeutic strategy for safely and selectively treating resistant gliomas.
Project description:<h4>Objective</h4>Cytokines contribute to beta-cell destruction in type 1 diabetes. Endoplasmic reticulum (ER) stress-mediated apoptosis has been proposed as a mechanism for beta-cell death. We tested whether ER stress was necessary for cytokine-induced beta-cell death and also whether ER stress gene activation was present in beta-cells of the NOD mouse model of type 1 diabetes.<h4>Research design and methods</h4>INS-1 beta-cells or rat islets were treated with the chemical chaperone phenyl butyric acid (PBA) and exposed or not to interleukin (IL)-1beta and gamma-interferon (IFN-gamma). Small interfering RNA (siRNA) was used to silence C/EBP homologous protein (CHOP) expression in INS-1 beta-cells. Additionally, the role of ER stress in lipid-induced cell death was assessed.<h4>Results</h4>Cytokines and palmitate triggered ER stress in beta-cells as evidenced by increased phosphorylation of PKR-like ER kinase (PERK), eukaryotic initiation factor (EIF)2alpha, and Jun NH(2)-terminal kinase (JNK) and increased expression of activating transcription factor (ATF)4 and CHOP. PBA treatment attenuated ER stress, but JNK phosphorylation was reduced only in response to palmitate, not in response to cytokines. PBA had no effect on cytokine-induced cell death but was associated with protection against palmitate-induced cell death. Similarly, siRNA-mediated reduction in CHOP expression protected against palmitate- but not against cytokine-induced cell death. In NOD islets, mRNA levels of several ER stress genes were reduced (ATF4, BiP [binding protein], GRP94 [glucose regulated protein 94], p58, and XBP-1 [X-box binding protein 1] splicing) or unchanged (CHOP and Edem1 [ER degradation enhancer, mannosidase alpha-like 1]).<h4>Conclusions</h4>While both cytokines and palmitate can induce ER stress, our results suggest that, in contrast to lipoapoptosis, the PERK-ATF4-CHOP ER stress-signaling pathway is not necessary for cytokine-induced beta-cell death.
Project description:Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has shown efficacy in a phase 2 clinical trial, development of resistance to TRAIL by tumor cells is a major roadblock. We investigated whether quercetin, a flavonoid, can sensitize human ovarian cancer cells to TRAIL. Results indicate that quercetin sensitized cancer cells to TRAIL. The quercetin induced expression of death receptor DR5 but did not affect expression of DR4 in cancer cells. The induction of DR5 was mediated through activation of JNK and through upregulation of a transcription factor CCAAT enhancer-binding protein homologous protein (CHOP); as silencing of these signaling molecules abrogated the effect of quercetin. Upregulation of DR5 was mediated through the generation of reactive oxygen species (ROS), as ROS scavengers reduced the effect of quercetin on JNK activation, CHOP upregulation, DR induction, TRAIL sensitization, downregulated the expression of cell survival proteins and upregulated the proapoptotic proteins. Furthermore, quercetin enhances TRAIL mediated inhibition of tumor growth of human SKOV-3 xenograft was associated with induction of apoptosis, activation of caspase-3, CHOP and DR5. Overall, our data suggest that quercetin enhances apoptotic death of ovarian cancer cells to TRAIL through upregulation of CHOP-induced DR5 expression following ROS mediated endoplasmic reticulum-stress.
Project description:Human glioblastoma multiforme cells demonstrate varying levels of sensitivity to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. Endoplasmic reticulum (ER) stress has been shown to trigger cell death through apoptosis. We therefore pursued a strategy of integrating clinically relevant investigational agents that cooperate mechanistically through the regulation of ER stress and apoptosis pathways. Nelfinavir belongs to the protease inhibitor class of drugs currently used to treat patients with HIV and is in clinical trials as an anti-tumor agent. We found that Nelfinavir treatment led to ER stress-induced up-regulation of the DR5 receptor. This transactivation was mediated by the transcription factor CCAAT/enhancer binding protein homologous protein (CHOP). We also determined that ER stress-induced ATF4 up-regulation was responsible for modulation of CHOP. In contrast, DR4 receptor expression was unchanged by Nelfinavir treatment. Combining Nelfinavir with TRAIL led to a significantly enhanced level of apoptosis that was abrogated by siRNA silencing of DR5. We provide evidence that Nelfinavir-induced ER stress modulates DR5 expression in human glioblastoma multiforme cells and can enhance TRAIL efficacy. These studies provide a potential mechanistic rationale for the use of the Food and Drug Administration-approved agent Nelfinavir in combination with DR5 agonists to induce apoptosis in human malignancies.
Project description:Glioblastoma multiforme (GBM) is the most aggressive malignant brain tumour in humans and is highly resistant to current treatment modalities. We have explored the combined treatment of the endoplasmic reticulum (ER) stress-inducing agent 2,5-dimethyl-celecoxib (DMC) and TNF-related apoptosis-inducing ligand (TRAIL WT) or the DR5-specific TRAIL D269H/E195R variant as a potential new strategy to eradicate GBM cells using TRAIL-resistant and -sensitive GBM cells. GBM cell lines were investigated for their sensitivity to TRAIL, DMC and combination of both agents. Cell viability was measured by MTS assay and apoptosis was assessed by Annexin V/PI and acridine orange staining. Caspase activation and protein expression levels were analysed with Western blotting. Death Receptor (DR) cell surface expression levels were quantified by flow cytometry. DR5 expression was increased in U87 cells by ectopic expression using a retroviral plasmid and survivin expression was silenced using specific siRNAs. We demonstrate that A172 expresses mainly DR5 on the cell surface and that these cells show increased sensitivity for the DR5-specific rhTRAIL D269H/E195R variant. In contrast, U87 cells show low DR cell surface levels and is insensitive via both DR4 and DR5. We determined that DMC treatment displays a dose-dependent reduction in cell viability against a number of GBM cells, associated with ER stress induction, as shown by the up-regulation of glucose-regulated protein 78 (GRP78) and CCAAT/-enhancer-binding protein homologous protein (CHOP) in A172 and U87 cells. The dramatic decrease in cell viability is not accompanied by a correspondent increase in Annexin V/PI or caspase activation typically seen in apoptotic or/and necrotic cells within 24h of treatment. Although DMC did not affect DR5 expression in the GBM cells, it increased TRAIL-induced caspase-8 activation in both TRAIL-sensitive and -resistant cells, indicating that DMC potentiates initiator caspase activation in these cells. In A172 cells, sub-toxic concentrations of DMC greatly potentiated TRAIL-induced apoptosis. Furthermore, DMC strongly reduced survivin expression in A172 and U87 cells and silencing of this anti-apoptotic protein partially sensitized cells to TRAIL-induced apoptosis. Our findings corroborate that DMC is a promising agent against GBM, and uncovers a potential synergistic cooperation with TRAIL in this highly malignant cancer.