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Gregory James Gores - One of the best experts on this subject based on the ideXlab platform.

  • Lipoapoptosis induced by saturated free fatty acids stimulates monocyte migration a novel role for pannexin1 in liver cells
    Purinergic Signalling, 2015
    Co-Authors: Feng Xiao, Steven F. Bronk, Gregory James Gores, Shar L Waldrop, Laurie S Davis, Gordan Kilic
    Abstract:

    Recruitment of monocytes in the liver is a key pathogenic feature of hepatic inflammation in nonalcoholic steatohepatitis (NASH), but the mechanisms involved are poorly understood. Here, we studied migration of human monocytes in response to supernatants obtained from liver cells after inducing Lipoapoptosis with saturated free fatty acids (FFA). Lipoapoptotic supernatants stimulated monocyte migration with the magnitude similar to a monocyte chemoattractant protein, CCL2 (MCP-1). Inhibition of c-Jun NH2-terminal kinase (JNK) in liver cells with SP600125 blocked migration of monocytes in a dose-dependent manner, indicating that JNK stimulates release of chemoattractants in Lipoapoptosis. Notably, treatment of supernatants with Apyrase to remove ATP potently inhibited migration of THP-1 monocytes and partially blocked migration of primary human monocytes. Inhibition of the CCL2 receptor (CCR2) on THP-1 monocytes with RS102895, a specific CCR2 inhibitor, did not block migration induced by lipoapoptotic supernatants. Consistent with these findings, Lipoapoptosis stimulated pathophysiological extracellular ATP (eATP) release that increased supernatant eATP concentration from 5 to ~60 nM. Importantly, inhibition of Panx1 expression in liver cells with short hairpin RNA (shRNA) decreased supernatant eATP concentration and inhibited monocyte migration, indicating that monocyte migration is mediated in part by Panx1-dependent eATP release. Moreover, JNK inhibition decreased supernatant eATP concentration and inhibited Pannexin1 activation, as determined by YoPro-1 uptake in liver cells in a dose-dependent manner. These results suggest that JNK regulates activation of Panx1 channels, and provide evidence that Pannexin1-dependent pathophysiological eATP release in Lipoapoptosis is capable of stimulating migration of human monocytes, and may participate in the recruitment of monocytes in chronic liver injury induced by saturated FFA. Electronic supplementary material The online version of this article (doi:10.1007/s11302-015-9456-5) contains supplementary material, which is available to authorized users.

  • degradation of ciaps contributes to hepatocyte Lipoapoptosis
    American Journal of Physiology-gastrointestinal and Liver Physiology, 2013
    Co-Authors: Yuko Akazawa, Nathan W Werneburg, Sophie C. Cazanave, Steven F. Bronk, Kazuhiko Nakao, Keisuke Kakisaka, Maria Eugenia Guicciardi, Gregory James Gores
    Abstract:

    Hepatocyte apoptosis is a hallmark of nonalcoholic steatohepatitis. We have previously observed that the saturated free fatty acids (FFAs) induce hepatocyte apoptosis in part via a death receptor 5 (DR5)-mediated signaling pathway. Cellular inhibitor of apoptosis protein 1 and 2 (cIAP-1 and cIAP-2) proteins are potent inhibitors of death receptor-mediated apoptosis. However, the role of the cIAPs in FFA-mediated hepatocyte apoptosis is unexplored. Our aim was to determine whether cIAPs are dysregulated during hepatocyte Lipoapoptosis. cIAP proteins underwent rapid cellular elimination following treatment with the saturated FFAs palmitate (PA) and stearate. In contrast, PA did not decrease cIAP-1 and cIAP-2 mRNA expression in the cells. Degradation of cIAPs was dependent on their E3-ligase activity, suggesting that cIAPs undergo autoubiquitination that leads to proteasomal degradation. Huh-7 cells stably expressing shRNA targeting cIAP-1, but not cIAP-2, displayed enhanced sensitivity to PA-mediated apoptosis. Incubation with the SMAC mimetic JP1584, which induces rapid degradation of cIAPs, also enhanced PA-mediated apoptosis. Hepatocytes isolated from DR5 knockout mice exhibited reduced apoptosis following treatment with PA plus JP1584, implying that degradation of cIAPs sensitizes to DR5-mediated cell death pathways. A decrease of cIAP-1 was also observed in tissue from patients with nonalcoholic steatohepatitis compared with normal obese subjects. Collectively, these results implicate proteasomal degradation of cIAPs by FFA as a mechanism contributing to hepatocyte Lipoapoptosis.

  • A Hedgehog Survival Pathway in ‘Undead’ Lipotoxic Hepatocytes
    Journal of Hepatology, 2012
    Co-Authors: Keisuke Kakisaka, Nathan W Werneburg, Sophie C. Cazanave, Steven F. Bronk, Nataliya Razumilava, Joachim C. Mertens, Gregory James Gores
    Abstract:

    Background & Aims Ballooned hepatocytes in non-alcoholic steatohepatitis (NASH) generate sonic hedgehog (SHH). This observation is consistent with a cellular phenotype in which the cell death program has been initiated but cannot be executed. Our aim was to determine whether ballooned hepatocytes have potentially disabled the cell death execution machinery, and if so, can their functional biology be modeled in vitro . Methods Immunohistochemistry was performed on human NASH specimens. In vitro studies were performed using HuH-7 cells with shRNA targeted knockdown of caspase 9 (shC9 cells) or primary hepatocytes from caspase 3 −/− mice. Results Ballooned hepatocytes in NASH display diminished expression of caspase 9. This phenotype was modeled using shC9 cells; these cells were resistant to Lipoapoptosis by palmitate (PA) or lysophosphatidylcholine (LPC) despite lipid droplet formation. During lipid loading by either PA or LPC, shC9 cells activate JNK which induces SHH expression via AP-1. An autocrine hedgehog survival signaling pathway was further delineated in both shC9 and caspase 3 −/− cells during lipotoxic stress. Conclusions Ballooned hepatocytes in NASH downregulate caspase 9, a pivotal caspase executing the mitochondrial pathway of apoptosis. Hepatocytes engineered to reduce caspase 9 expression are resistant to Lipoapoptosis, in part, due to a hedgehog autocrine survival signaling pathway.

  • mechanisms of lysophosphatidylcholine induced hepatocyte Lipoapoptosis
    American Journal of Physiology-gastrointestinal and Liver Physiology, 2012
    Co-Authors: Keisuke Kakisaka, Nathan W Werneburg, Sophie C. Cazanave, Justin L Mott, Steven F. Bronk, Christian D Fingas, Maria Eugenia Guicciardi, Gregory James Gores
    Abstract:

    Isolated hepatocytes undergo Lipoapoptosis, a feature of hepatic lipotoxicity, on treatment with saturated free fatty acids (FFA) such as palmitate (PA). However, it is unknown if palmitate is directly toxic to hepatocytes or if its toxicity is indirect via the generation of lipid metabolites such as lysophosphatidylcholine (LPC). PA-mediated hepatocyte Lipoapoptosis is associated with endoplasmic reticulum (ER) stress, c-Jun NH2-terminal kinase (JNK) activation, and a JNK-dependent upregulation of the potent proapoptotic BH3-only protein PUMA (p53 upregulated modulator of apoptosis). Our aim was to determine which of these mechanisms of lipotoxicity are activated by PA-derived LPC. We employed Huh-7 cells and isolated murine and human primary hepatocytes. Intracellular LPC concentrations increase linearly as a function of the exogenous, extracellular PA, stearate, or LPC concentration. Incubation of Huh-7 cells or primary hepatocytes with LPC induced cell death by apoptosis in a concentration-dependent manner. Substituting LPC for PA resulted in caspase-dependent cell death that was accompanied by activating phosphorylation of JNK with c-Jun phosphorylation and an increase in PUMA expression. LPC also induced ER stress as manifest by eIF2α phosphorylation and CAAT/enhancer binding homologous protein (CHOP) induction. LPC cytotoxicity was attenuated by pharmacological inhibition of JNK or glycogen synthase kinase-3 (GSK-3). Similarly, short-hairpin RNA (shRNA)-targeted knockdown of CHOP protected Huh-7 cells against LPC-induced toxicity. The LPC-induced PUMA upregulation was prevented by JNK inhibition or shRNA-targeted knockdown of CHOP. Finally, genetic deficiency of PUMA rendered murine hepatocytes resistant to LPC-induced apoptosis. We concluded that LPC-induced Lipoapoptosis is dependent on mechanisms largely indistinguishable from PA. These data suggest that FFA-mediated cytotoxicity is indirect via the generation of the toxic metabolite, LPC.

  • death receptor 5 signaling promotes hepatocyte Lipoapoptosis
    Journal of Biological Chemistry, 2011
    Co-Authors: Sophie C. Cazanave, Nathan W Werneburg, Justin L Mott, Steven F. Bronk, Scott H Kaufmann, Christian D Fingas, Wei X Meng, Niklas Finnberg, Wafik S Eldeiry, Gregory James Gores
    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.

Steven F. Bronk - One of the best experts on this subject based on the ideXlab platform.

  • Lipoapoptosis induced by saturated free fatty acids stimulates monocyte migration a novel role for pannexin1 in liver cells
    Purinergic Signalling, 2015
    Co-Authors: Feng Xiao, Steven F. Bronk, Gregory James Gores, Shar L Waldrop, Laurie S Davis, Gordan Kilic
    Abstract:

    Recruitment of monocytes in the liver is a key pathogenic feature of hepatic inflammation in nonalcoholic steatohepatitis (NASH), but the mechanisms involved are poorly understood. Here, we studied migration of human monocytes in response to supernatants obtained from liver cells after inducing Lipoapoptosis with saturated free fatty acids (FFA). Lipoapoptotic supernatants stimulated monocyte migration with the magnitude similar to a monocyte chemoattractant protein, CCL2 (MCP-1). Inhibition of c-Jun NH2-terminal kinase (JNK) in liver cells with SP600125 blocked migration of monocytes in a dose-dependent manner, indicating that JNK stimulates release of chemoattractants in Lipoapoptosis. Notably, treatment of supernatants with Apyrase to remove ATP potently inhibited migration of THP-1 monocytes and partially blocked migration of primary human monocytes. Inhibition of the CCL2 receptor (CCR2) on THP-1 monocytes with RS102895, a specific CCR2 inhibitor, did not block migration induced by lipoapoptotic supernatants. Consistent with these findings, Lipoapoptosis stimulated pathophysiological extracellular ATP (eATP) release that increased supernatant eATP concentration from 5 to ~60 nM. Importantly, inhibition of Panx1 expression in liver cells with short hairpin RNA (shRNA) decreased supernatant eATP concentration and inhibited monocyte migration, indicating that monocyte migration is mediated in part by Panx1-dependent eATP release. Moreover, JNK inhibition decreased supernatant eATP concentration and inhibited Pannexin1 activation, as determined by YoPro-1 uptake in liver cells in a dose-dependent manner. These results suggest that JNK regulates activation of Panx1 channels, and provide evidence that Pannexin1-dependent pathophysiological eATP release in Lipoapoptosis is capable of stimulating migration of human monocytes, and may participate in the recruitment of monocytes in chronic liver injury induced by saturated FFA. Electronic supplementary material The online version of this article (doi:10.1007/s11302-015-9456-5) contains supplementary material, which is available to authorized users.

  • degradation of ciaps contributes to hepatocyte Lipoapoptosis
    American Journal of Physiology-gastrointestinal and Liver Physiology, 2013
    Co-Authors: Yuko Akazawa, Nathan W Werneburg, Sophie C. Cazanave, Steven F. Bronk, Kazuhiko Nakao, Keisuke Kakisaka, Maria Eugenia Guicciardi, Gregory James Gores
    Abstract:

    Hepatocyte apoptosis is a hallmark of nonalcoholic steatohepatitis. We have previously observed that the saturated free fatty acids (FFAs) induce hepatocyte apoptosis in part via a death receptor 5 (DR5)-mediated signaling pathway. Cellular inhibitor of apoptosis protein 1 and 2 (cIAP-1 and cIAP-2) proteins are potent inhibitors of death receptor-mediated apoptosis. However, the role of the cIAPs in FFA-mediated hepatocyte apoptosis is unexplored. Our aim was to determine whether cIAPs are dysregulated during hepatocyte Lipoapoptosis. cIAP proteins underwent rapid cellular elimination following treatment with the saturated FFAs palmitate (PA) and stearate. In contrast, PA did not decrease cIAP-1 and cIAP-2 mRNA expression in the cells. Degradation of cIAPs was dependent on their E3-ligase activity, suggesting that cIAPs undergo autoubiquitination that leads to proteasomal degradation. Huh-7 cells stably expressing shRNA targeting cIAP-1, but not cIAP-2, displayed enhanced sensitivity to PA-mediated apoptosis. Incubation with the SMAC mimetic JP1584, which induces rapid degradation of cIAPs, also enhanced PA-mediated apoptosis. Hepatocytes isolated from DR5 knockout mice exhibited reduced apoptosis following treatment with PA plus JP1584, implying that degradation of cIAPs sensitizes to DR5-mediated cell death pathways. A decrease of cIAP-1 was also observed in tissue from patients with nonalcoholic steatohepatitis compared with normal obese subjects. Collectively, these results implicate proteasomal degradation of cIAPs by FFA as a mechanism contributing to hepatocyte Lipoapoptosis.

  • A Hedgehog Survival Pathway in ‘Undead’ Lipotoxic Hepatocytes
    Journal of Hepatology, 2012
    Co-Authors: Keisuke Kakisaka, Nathan W Werneburg, Sophie C. Cazanave, Steven F. Bronk, Nataliya Razumilava, Joachim C. Mertens, Gregory James Gores
    Abstract:

    Background & Aims Ballooned hepatocytes in non-alcoholic steatohepatitis (NASH) generate sonic hedgehog (SHH). This observation is consistent with a cellular phenotype in which the cell death program has been initiated but cannot be executed. Our aim was to determine whether ballooned hepatocytes have potentially disabled the cell death execution machinery, and if so, can their functional biology be modeled in vitro . Methods Immunohistochemistry was performed on human NASH specimens. In vitro studies were performed using HuH-7 cells with shRNA targeted knockdown of caspase 9 (shC9 cells) or primary hepatocytes from caspase 3 −/− mice. Results Ballooned hepatocytes in NASH display diminished expression of caspase 9. This phenotype was modeled using shC9 cells; these cells were resistant to Lipoapoptosis by palmitate (PA) or lysophosphatidylcholine (LPC) despite lipid droplet formation. During lipid loading by either PA or LPC, shC9 cells activate JNK which induces SHH expression via AP-1. An autocrine hedgehog survival signaling pathway was further delineated in both shC9 and caspase 3 −/− cells during lipotoxic stress. Conclusions Ballooned hepatocytes in NASH downregulate caspase 9, a pivotal caspase executing the mitochondrial pathway of apoptosis. Hepatocytes engineered to reduce caspase 9 expression are resistant to Lipoapoptosis, in part, due to a hedgehog autocrine survival signaling pathway.

  • mechanisms of lysophosphatidylcholine induced hepatocyte Lipoapoptosis
    American Journal of Physiology-gastrointestinal and Liver Physiology, 2012
    Co-Authors: Keisuke Kakisaka, Nathan W Werneburg, Sophie C. Cazanave, Justin L Mott, Steven F. Bronk, Christian D Fingas, Maria Eugenia Guicciardi, Gregory James Gores
    Abstract:

    Isolated hepatocytes undergo Lipoapoptosis, a feature of hepatic lipotoxicity, on treatment with saturated free fatty acids (FFA) such as palmitate (PA). However, it is unknown if palmitate is directly toxic to hepatocytes or if its toxicity is indirect via the generation of lipid metabolites such as lysophosphatidylcholine (LPC). PA-mediated hepatocyte Lipoapoptosis is associated with endoplasmic reticulum (ER) stress, c-Jun NH2-terminal kinase (JNK) activation, and a JNK-dependent upregulation of the potent proapoptotic BH3-only protein PUMA (p53 upregulated modulator of apoptosis). Our aim was to determine which of these mechanisms of lipotoxicity are activated by PA-derived LPC. We employed Huh-7 cells and isolated murine and human primary hepatocytes. Intracellular LPC concentrations increase linearly as a function of the exogenous, extracellular PA, stearate, or LPC concentration. Incubation of Huh-7 cells or primary hepatocytes with LPC induced cell death by apoptosis in a concentration-dependent manner. Substituting LPC for PA resulted in caspase-dependent cell death that was accompanied by activating phosphorylation of JNK with c-Jun phosphorylation and an increase in PUMA expression. LPC also induced ER stress as manifest by eIF2α phosphorylation and CAAT/enhancer binding homologous protein (CHOP) induction. LPC cytotoxicity was attenuated by pharmacological inhibition of JNK or glycogen synthase kinase-3 (GSK-3). Similarly, short-hairpin RNA (shRNA)-targeted knockdown of CHOP protected Huh-7 cells against LPC-induced toxicity. The LPC-induced PUMA upregulation was prevented by JNK inhibition or shRNA-targeted knockdown of CHOP. Finally, genetic deficiency of PUMA rendered murine hepatocytes resistant to LPC-induced apoptosis. We concluded that LPC-induced Lipoapoptosis is dependent on mechanisms largely indistinguishable from PA. These data suggest that FFA-mediated cytotoxicity is indirect via the generation of the toxic metabolite, LPC.

  • death receptor 5 signaling promotes hepatocyte Lipoapoptosis
    Journal of Biological Chemistry, 2011
    Co-Authors: Sophie C. Cazanave, Nathan W Werneburg, Justin L Mott, Steven F. Bronk, Scott H Kaufmann, Christian D Fingas, Wei X Meng, Niklas Finnberg, Wafik S Eldeiry, Gregory James Gores
    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.

Sophie C. Cazanave - One of the best experts on this subject based on the ideXlab platform.

  • Molecular Mechanisms of Lipotoxicity in Nonalcoholic Fatty Liver Disease
    Hepatic De Novo Lipogenesis and Regulation of Metabolism, 2015
    Co-Authors: Sophie C. Cazanave, Arun J. Sanyal
    Abstract:

    Nonalcoholic fatty liver disease (NAFLD) is a growing epidemic which affects up to 30 % of the American population. Nonalcoholic steatohepatitis (NASH), the more severe form of NAFLD, is associated with increased risk of cardiovascular morbidity and mortality, insulin resistance (IR), type 2 diabetes mellitus (T2DM), hepatic steatosis, elevated circulating levels of free fatty acids (FFAs) and hepatocyte apoptosis. Because lipid-induced toxicity, or Lipoapoptosis, represents a key pathogenic factor for NAFLD, we will focus on the evidence connecting the accumulation of certain lipid types in the liver with the progressive induction of hepatocyte injury and secondary inflammation/fibrosis during the disease. A better understanding of the molecular and cellular pathway of lipotoxicity has both diagnostic and therapeutic implication for the treatment of NAFLD.

  • saturated free fatty acids induce cholangiocyte Lipoapoptosis
    Hepatology, 2014
    Co-Authors: Sathish Kumar Natarajan, Sophie C. Cazanave, Sally A Ingham, Ashley M Mohr, Cody J Wehrkamp, Mary Anne Phillippi, Justin L Mott
    Abstract:

    Recent studies have identified a cholestatic variant of nonalcoholic fatty liver disease (NAFLD) with portal inflammation and ductular reaction. Based on reports of biliary damage, as well as increased circulating free fatty acids (FFAs) in NAFLD, we hypothesized the involvement of cholangiocyte Lipoapoptosis as a mechanism of cellular injury. Here, we demonstrate that the saturated FFAs palmitate and stearate induced robust and rapid cell death in cholangiocytes. Palmitate and stearate induced cholangiocyte Lipoapoptosis in a concentration-dependent manner in multiple cholangiocyte-derived cell lines. The mechanism of Lipoapoptosis relied on the activation of caspase 3/7 activity. There was also a significant up-regulation of the proapoptotic BH3-containing protein, PUMA. In addition, palmitate-induced cholangiocyte Lipoapoptosis involved a time-dependent increase in the nuclear localization of forkhead family of transcription factor 3 (FoxO3). We show evidence for posttranslational modification of FoxO3, including early (6 hours) deacetylation and dephosphorylation that coincide with localization of FoxO3 in the nuclear compartment. By 16 hours, nuclear FoxO3 is both phosphorylated and acetylated. Knockdown studies confirmed that FoxO3 and its downstream target, PUMA, were critical for palmitate- and stearate-induced cholangiocyte Lipoapoptosis. Interestingly, cultured cholangiocyte-derived cells did not accumulate appreciable amounts of neutral lipid upon FFA treatment. Conclusion: Our data show that the saturated FFAs palmitate and stearate induced cholangiocyte Lipoapoptosis by way of caspase activation, nuclear translocation of FoxO3, and increased proapoptotic PUMA expression. These results suggest that cholangiocyte injury may occur through Lipoapoptosis in NAFLD and nonalcoholic steatohepatitis patients. (Hepatology 2014;60:1941–1955)

  • degradation of keap1 activates bh3 only proteins bim and puma during hepatocyte Lipoapoptosis
    Cell Death & Differentiation, 2014
    Co-Authors: Sophie C. Cazanave, Xiangyang Wang, Huiping Zhou, M Rahmani, Steven Grant, D E Durrant, C D Klaassen, Masayuki Yamamoto, A J Sanyal
    Abstract:

    Non-alcoholic steatohepatitis is characterized by hepatic steatosis, elevated levels of circulating free fatty acids (FFA) and hepatocyte Lipoapoptosis. This Lipoapoptosis requires increased JNK phosphorylation and activation of the pro-apoptotic BH3-only proteins Bim and PUMA. Kelch-like ECH-associated protein (Keap)-1 is a BTB/Kelch protein that can regulate the expression of Bcl-2 protein and control apoptotic cell death. Yet, the role of Keap1 in hepatocyte lipotoxicity is unclear. Here we demonstrate that Keap1 protein was rapidly degraded in hepatocytes, through autophagy in a p62-dependent manner, in response to the toxic saturated FFA palmitate, but not following incubation with the non-toxic FFA oleic acid. Stable knockdown of Keap1 expression, using shRNA technology, in hepatocarcinoma cell lines induced spontaneous cell toxicity that was associated with JNK1-dependent upregulation of Bim and PUMA protein levels. Also, Keap1 knockdown further sensitized hepatocytes to Lipoapoptosis by palmitate. Likewise, primary hepatocytes isolated from liver-specific Keap1−/− mice displayed higher Bim and PUMA protein levels and demonstrated increased sensitivity to palmitate-induced apoptosis than wild-type mouse hepatocytes. Finally, stable knockdown of Bim or PUMA expression prevented cell toxicity induced by loss of Keap1. These results implicate p62-dependent autophagic degradation of Keap1 by palmitate as a mechanism contributing to hepatocyte Lipoapoptosis.

  • degradation of ciaps contributes to hepatocyte Lipoapoptosis
    American Journal of Physiology-gastrointestinal and Liver Physiology, 2013
    Co-Authors: Yuko Akazawa, Nathan W Werneburg, Sophie C. Cazanave, Steven F. Bronk, Kazuhiko Nakao, Keisuke Kakisaka, Maria Eugenia Guicciardi, Gregory James Gores
    Abstract:

    Hepatocyte apoptosis is a hallmark of nonalcoholic steatohepatitis. We have previously observed that the saturated free fatty acids (FFAs) induce hepatocyte apoptosis in part via a death receptor 5 (DR5)-mediated signaling pathway. Cellular inhibitor of apoptosis protein 1 and 2 (cIAP-1 and cIAP-2) proteins are potent inhibitors of death receptor-mediated apoptosis. However, the role of the cIAPs in FFA-mediated hepatocyte apoptosis is unexplored. Our aim was to determine whether cIAPs are dysregulated during hepatocyte Lipoapoptosis. cIAP proteins underwent rapid cellular elimination following treatment with the saturated FFAs palmitate (PA) and stearate. In contrast, PA did not decrease cIAP-1 and cIAP-2 mRNA expression in the cells. Degradation of cIAPs was dependent on their E3-ligase activity, suggesting that cIAPs undergo autoubiquitination that leads to proteasomal degradation. Huh-7 cells stably expressing shRNA targeting cIAP-1, but not cIAP-2, displayed enhanced sensitivity to PA-mediated apoptosis. Incubation with the SMAC mimetic JP1584, which induces rapid degradation of cIAPs, also enhanced PA-mediated apoptosis. Hepatocytes isolated from DR5 knockout mice exhibited reduced apoptosis following treatment with PA plus JP1584, implying that degradation of cIAPs sensitizes to DR5-mediated cell death pathways. A decrease of cIAP-1 was also observed in tissue from patients with nonalcoholic steatohepatitis compared with normal obese subjects. Collectively, these results implicate proteasomal degradation of cIAPs by FFA as a mechanism contributing to hepatocyte Lipoapoptosis.

  • A Hedgehog Survival Pathway in ‘Undead’ Lipotoxic Hepatocytes
    Journal of Hepatology, 2012
    Co-Authors: Keisuke Kakisaka, Nathan W Werneburg, Sophie C. Cazanave, Steven F. Bronk, Nataliya Razumilava, Joachim C. Mertens, Gregory James Gores
    Abstract:

    Background & Aims Ballooned hepatocytes in non-alcoholic steatohepatitis (NASH) generate sonic hedgehog (SHH). This observation is consistent with a cellular phenotype in which the cell death program has been initiated but cannot be executed. Our aim was to determine whether ballooned hepatocytes have potentially disabled the cell death execution machinery, and if so, can their functional biology be modeled in vitro . Methods Immunohistochemistry was performed on human NASH specimens. In vitro studies were performed using HuH-7 cells with shRNA targeted knockdown of caspase 9 (shC9 cells) or primary hepatocytes from caspase 3 −/− mice. Results Ballooned hepatocytes in NASH display diminished expression of caspase 9. This phenotype was modeled using shC9 cells; these cells were resistant to Lipoapoptosis by palmitate (PA) or lysophosphatidylcholine (LPC) despite lipid droplet formation. During lipid loading by either PA or LPC, shC9 cells activate JNK which induces SHH expression via AP-1. An autocrine hedgehog survival signaling pathway was further delineated in both shC9 and caspase 3 −/− cells during lipotoxic stress. Conclusions Ballooned hepatocytes in NASH downregulate caspase 9, a pivotal caspase executing the mitochondrial pathway of apoptosis. Hepatocytes engineered to reduce caspase 9 expression are resistant to Lipoapoptosis, in part, due to a hedgehog autocrine survival signaling pathway.

Yuko Akazawa - One of the best experts on this subject based on the ideXlab platform.

  • Toyocamycin attenuates free fatty acid-induced hepatic steatosis and apoptosis in cultured hepatocytes and ameliorates nonalcoholic fatty liver disease in mice.
    PLOS ONE, 2017
    Co-Authors: Ikuko Takahara, Ken Ohnita, Naota Taura, Yuko Akazawa, Maiko Tabuchi, Katsuya Matsuda, Hisamitsu Miyaaki, Youko Kido, Yasuko Kanda, Fuminao Takeshima
    Abstract:

    Background and aims A high serum level of saturated free fatty acids (FFAs) is associated with the development of nonalcoholic fatty liver disease (NAFLD). X-box binding protein-1 (XBP-1) is activated by FFA treatment upon splicing. XBP-1 is a transcription factor induced by the endoplasmic reticulum (ER) stress sensor endoribonuclease inositol-requiring enzyme 1 alpha (IRE1α). However, the role of XBP-1 in NAFLD remains relatively unexplored. Toyocamycin was recently reported to attenuate the activation of XBP-1, possibly by inducing a conformational change in IRE1α. In this study, we examined the effect of toyocamycin on hepatocyte Lipoapoptosis and steatosis. We also explored the effects of toyocamycin in a mouse model of NAFLD. Methods Huh-7 cells and isolated rat primary hepatocytes were treated with palmitic acid (PA), which is a saturated FFA, in the presence or absence of toyocamycin. In addition, male C57BL/6J mice were fed a diet rich in saturated fat, fructose, and cholesterol (FFC) for 4 months, after which the effect of toyocamycin was assessed. Results Toyocamycin attenuated FFA-induced steatosis. It also significantly reduced PA-induced hepatocyte Lipoapoptosis. In addition, toyocamycin reduced the expression of cytosine-cytosine-adenosine-adenosine-thymidine enhancer-binding protein homologous protein (CHOP), which is a key player in ER stress-mediated apoptosis, as well as its downstream cell death modulator, death receptor 5. In the in vivo study, toyocamycin ameliorated the liver injury caused by FFC-induced NAFLD. It also reduced hepatic steatosis and the expression of lipogenic genes. Conclusions The data we obtained suggest that toyocamycin attenuates hepatocyte lipogenesis and ameliorates NAFLD in vivo and may therefore be beneficial in the treatment of NAFLD in humans.

  • Lipotoxicity pathways intersect in hepatocytes: Endoplasmic reticulum stress, c-Jun N-terminal kinase-1, and death receptors.
    Hepatology Research, 2016
    Co-Authors: Yuko Akazawa, Kazuhiko Nakao
    Abstract:

    Non-alcoholic fatty liver disease (NAFLD) is becoming increasingly more common worldwide. Hepatocyte apoptosis caused by free fatty acids, termed hepatocyte Lipoapoptosis, is a feature of non-alcoholic steatohepatitis (NASH), an advanced form of NAFLD. As no salutary treatment for NASH exists, it is important to understand the molecular mechanisms responsible for disease development and progression. This review discusses recent developments in research on hepatocyte Lipoapoptosis, focusing on the endoplasmic reticulum stress, c-Jun N-terminal kinase-1, and death receptor-mediated pathway networks and their modulators and interactions. In addition, we describe the emerging importance of the signaling pathways that not only impact the dying hepatocytes themselves, but also influence surrounding cells and possibly promote disease progression through the release of microvesicles. Overall, a more comprehensive understanding of the molecular mediators in lipotoxicity-related pathways would likely benefit the development of mechanism-based therapies of NASH.

  • 696 XBP-1 Inhibitor Toyocamycin Attenuates Palmitate-Induced Hepatocyte Apoptosis and Steatosis
    Gastroenterology, 2014
    Co-Authors: Ikuko Takahara, Ken Ohnita, Fuminao Takeshima, Hajime Isomoto, Naota Taura, Tatsuki Ichikawa, Yuko Akazawa, Kazuhiko Nakao
    Abstract:

    Background and Aim: Free fatty acid-induced hepatocyte cytotoxicity is deeply associated withmorbidity of Non-alcoholic fatty liver disease (NAFLD) (Malhi et al, 2008, Gastroenterology). Saturated free fatty acids induce hepatocyte Lipoapoptosis via endoplasmic reticulum stress (ER) response, which leads to translational activation of DR5 and BH3 only protein Bim (Cazanave et al, JBC, 2011). We previously reported that X-box binding protein-1 (XBP-1) is activated downstream of IRE-1 arm of ER stress by free fatty acid treatment upon its splicing (Akazawa et al, 2009, J Hepatol). However, role of XBP-1 during hepatocyte Lipoapoptosis remains relatively unexplored. We examined the impact of inhibiting XBP-1 on hepatocyte Lipoapoptosis and steatosis employing Toyocamycin, which was recently reported to inhibit ER stress-mediated XBP-1 splicing (Ri M et al, Blood Cancer J. 2012). Methods: We employed hepatocellular carcinoma cell line, Huh-7 cells, for these studies. Cells were incubated with palmitate (200~800μM) in presence or absence of Toyocamycin (0.1~3μM). Apoptosis was assessed by DAPI staining and caspase 3/7 activity assay. Steatosis was assessed by Nile red staining. mRNA expression of Bim and DR5 was examined by real time PCR. Expression of Bim protein and phosphorylation of JNK was assessed by immunoblotting. Results: As expected, Toyocamycin inhibited palmitate-mediated XBP-1 splicing without affecting amount of total RNA. Toyocamycin significantly reduced palmitateinduced hepatocyte Lipoapoptosis (p

  • degradation of ciaps contributes to hepatocyte Lipoapoptosis
    American Journal of Physiology-gastrointestinal and Liver Physiology, 2013
    Co-Authors: Yuko Akazawa, Nathan W Werneburg, Sophie C. Cazanave, Steven F. Bronk, Kazuhiko Nakao, Keisuke Kakisaka, Maria Eugenia Guicciardi, Gregory James Gores
    Abstract:

    Hepatocyte apoptosis is a hallmark of nonalcoholic steatohepatitis. We have previously observed that the saturated free fatty acids (FFAs) induce hepatocyte apoptosis in part via a death receptor 5 (DR5)-mediated signaling pathway. Cellular inhibitor of apoptosis protein 1 and 2 (cIAP-1 and cIAP-2) proteins are potent inhibitors of death receptor-mediated apoptosis. However, the role of the cIAPs in FFA-mediated hepatocyte apoptosis is unexplored. Our aim was to determine whether cIAPs are dysregulated during hepatocyte Lipoapoptosis. cIAP proteins underwent rapid cellular elimination following treatment with the saturated FFAs palmitate (PA) and stearate. In contrast, PA did not decrease cIAP-1 and cIAP-2 mRNA expression in the cells. Degradation of cIAPs was dependent on their E3-ligase activity, suggesting that cIAPs undergo autoubiquitination that leads to proteasomal degradation. Huh-7 cells stably expressing shRNA targeting cIAP-1, but not cIAP-2, displayed enhanced sensitivity to PA-mediated apoptosis. Incubation with the SMAC mimetic JP1584, which induces rapid degradation of cIAPs, also enhanced PA-mediated apoptosis. Hepatocytes isolated from DR5 knockout mice exhibited reduced apoptosis following treatment with PA plus JP1584, implying that degradation of cIAPs sensitizes to DR5-mediated cell death pathways. A decrease of cIAP-1 was also observed in tissue from patients with nonalcoholic steatohepatitis compared with normal obese subjects. Collectively, these results implicate proteasomal degradation of cIAPs by FFA as a mechanism contributing to hepatocyte Lipoapoptosis.

  • Glycogen synthase kinase-3 (GSK-3) inhibition attenuates hepatocyte Lipoapoptosis.
    Journal of Hepatology, 2010
    Co-Authors: Samar C Ibrahim, Nathan W Werneburg, Sophie C. Cazanave, Nafisa A. Elmi, Steven F. Bronk, Yuko Akazawa, Daniel D. Billadeau, Gregory James Gores
    Abstract:

    Backgrounds & Aims Saturated free fatty acids induce hepatocyte Lipoapoptosis, a key pathologic feature of non-alcoholic steatohepatitis. The saturated free fatty acid palmitate induces hepatocyte Lipoapoptosis via an endoplasmic reticulum stress pathway resulting in c-Jun-N-terminal (JNK) activation. Glycogen synthase kinase (GSK)-3 is a serine/threonine kinase which may also promote JNK activation. Thus, our aim was to determine if GSK-3 inhibition suppresses palmitate induced JNK activation and Lipoapoptosis. Methods For these studies, we employed mouse primary hepatocytes, Huh-7 and Hep3B cell lines. Results Palmitate-induced GSK-3 activation was identified by phosphorylation of its substrate glycogen synthase. GSK-3 pharmacologic inhibition, by GSK-3 inhibitor IX and enzastaurin, significantly reduced PA-mediated Lipoapoptosis. More importantly, Huh-7 cells, in which either GSK-3α or GSK-3β isoforms were stably and selectively knocked down by shRNA, displayed resistance to palmitate-induced cytotoxicity. GSK-3 pharmacological inhibitors and shRNA-targeted knockdown of GSK-3α or GSK-3β also suppressed JNK activation by palmitate. JNK activation, in part, promotes lipoapotosis by inducing expression of the pro-apoptotic effector p53-upregulated modulator of apoptosis (PUMA). Consistent with this concept, GSK-3 pharmacologic inhibition also reduced PUMA cellular protein levels during exposure to palmitate. On the other hand, the GSK-3 inhibitors did not prevent PA induction of ER stress. Conclusions Our results suggest that GSK-3 activation promotes a JNK-dependent cytotoxic signaling cascade culminating in Lipoapoptosis.

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  • foxo3 increases mir 34a to cause palmitate induced cholangiocyte Lipoapoptosis
    Journal of Lipid Research, 2017
    Co-Authors: Sathish Kumar Natarajan, Ashley M Mohr, Cody J Wehrkamp, Mary Anne Phillippi, Bailey A Stringham, Sizhao Lu, Duygu Dee Harrisonfindik, Justin L Mott
    Abstract:

    Nonalcoholic steatohepatitis (NASH) patients have elevated plasma saturated free fatty acid levels. These toxic fatty acids can induce liver cell death and our recent results demonstrated that the biliary epithelium may be susceptible to lipotoxicity. Here, we explored the molecular mechanisms of cholangiocyte Lipoapoptosis in cell culture and in an animal model of NASH. Treatment of cholangiocytes with palmitate (PA) showed increased caspase 3/7 activity and increased levels of cleaved poly (ADP-ribose) polymerase and cleaved caspase 3, demonstrating cholangiocyte Lipoapoptosis. Interestingly, treatment with PA significantly increased the levels of microRNA miR-34a, a pro-apoptotic microRNA known to be elevated in NASH. PA induction of miR-34a was abolished in cholangiocytes transduced with forkhead family of transcription factor class O (FoxO)3 shRNA, demonstrating that FoxO3 activation is upstream of miR-34a and suggesting that FoxO3 is a novel transcriptional regulator of miR-34a. Further, anti-miR-34a protected cholangiocytes from PA-induced Lipoapoptosis. Direct and indirect targets of miR-34a, such as SIRT1, receptor tyrosine kinase (MET), Kruppel-like factor 4, fibroblast growth factor receptor (FGFR)1, and FGFR4, were all decreased in PA-treated cholangiocytes. SIRT1 and MET were partially rescued by a miR-34a antagonist. Cholangiocyte apoptosis and miR-34a were dramatically increased in the liver of mice with early histologic features of NASH. Our study provides evidence for the pro-apoptotic role of miR-34a in PA-induced cholangiocyte Lipoapoptosis in culture and in the liver.

  • saturated free fatty acids induce cholangiocyte Lipoapoptosis
    Hepatology, 2014
    Co-Authors: Sathish Kumar Natarajan, Sophie C. Cazanave, Sally A Ingham, Ashley M Mohr, Cody J Wehrkamp, Mary Anne Phillippi, Justin L Mott
    Abstract:

    Recent studies have identified a cholestatic variant of nonalcoholic fatty liver disease (NAFLD) with portal inflammation and ductular reaction. Based on reports of biliary damage, as well as increased circulating free fatty acids (FFAs) in NAFLD, we hypothesized the involvement of cholangiocyte Lipoapoptosis as a mechanism of cellular injury. Here, we demonstrate that the saturated FFAs palmitate and stearate induced robust and rapid cell death in cholangiocytes. Palmitate and stearate induced cholangiocyte Lipoapoptosis in a concentration-dependent manner in multiple cholangiocyte-derived cell lines. The mechanism of Lipoapoptosis relied on the activation of caspase 3/7 activity. There was also a significant up-regulation of the proapoptotic BH3-containing protein, PUMA. In addition, palmitate-induced cholangiocyte Lipoapoptosis involved a time-dependent increase in the nuclear localization of forkhead family of transcription factor 3 (FoxO3). We show evidence for posttranslational modification of FoxO3, including early (6 hours) deacetylation and dephosphorylation that coincide with localization of FoxO3 in the nuclear compartment. By 16 hours, nuclear FoxO3 is both phosphorylated and acetylated. Knockdown studies confirmed that FoxO3 and its downstream target, PUMA, were critical for palmitate- and stearate-induced cholangiocyte Lipoapoptosis. Interestingly, cultured cholangiocyte-derived cells did not accumulate appreciable amounts of neutral lipid upon FFA treatment. Conclusion: Our data show that the saturated FFAs palmitate and stearate induced cholangiocyte Lipoapoptosis by way of caspase activation, nuclear translocation of FoxO3, and increased proapoptotic PUMA expression. These results suggest that cholangiocyte injury may occur through Lipoapoptosis in NAFLD and nonalcoholic steatohepatitis patients. (Hepatology 2014;60:1941–1955)

  • mmu mir 615 3p regulates Lipoapoptosis by inhibiting c ebp homologous protein
    PLOS ONE, 2014
    Co-Authors: Yasuhiro Miyamoto, Justin L Mott, Amy S Mauer, Swarup Kumar, Harmeet Malhi
    Abstract:

    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.

  • mechanisms of lysophosphatidylcholine induced hepatocyte Lipoapoptosis
    American Journal of Physiology-gastrointestinal and Liver Physiology, 2012
    Co-Authors: Keisuke Kakisaka, Nathan W Werneburg, Sophie C. Cazanave, Justin L Mott, Steven F. Bronk, Christian D Fingas, Maria Eugenia Guicciardi, Gregory James Gores
    Abstract:

    Isolated hepatocytes undergo Lipoapoptosis, a feature of hepatic lipotoxicity, on treatment with saturated free fatty acids (FFA) such as palmitate (PA). However, it is unknown if palmitate is directly toxic to hepatocytes or if its toxicity is indirect via the generation of lipid metabolites such as lysophosphatidylcholine (LPC). PA-mediated hepatocyte Lipoapoptosis is associated with endoplasmic reticulum (ER) stress, c-Jun NH2-terminal kinase (JNK) activation, and a JNK-dependent upregulation of the potent proapoptotic BH3-only protein PUMA (p53 upregulated modulator of apoptosis). Our aim was to determine which of these mechanisms of lipotoxicity are activated by PA-derived LPC. We employed Huh-7 cells and isolated murine and human primary hepatocytes. Intracellular LPC concentrations increase linearly as a function of the exogenous, extracellular PA, stearate, or LPC concentration. Incubation of Huh-7 cells or primary hepatocytes with LPC induced cell death by apoptosis in a concentration-dependent manner. Substituting LPC for PA resulted in caspase-dependent cell death that was accompanied by activating phosphorylation of JNK with c-Jun phosphorylation and an increase in PUMA expression. LPC also induced ER stress as manifest by eIF2α phosphorylation and CAAT/enhancer binding homologous protein (CHOP) induction. LPC cytotoxicity was attenuated by pharmacological inhibition of JNK or glycogen synthase kinase-3 (GSK-3). Similarly, short-hairpin RNA (shRNA)-targeted knockdown of CHOP protected Huh-7 cells against LPC-induced toxicity. The LPC-induced PUMA upregulation was prevented by JNK inhibition or shRNA-targeted knockdown of CHOP. Finally, genetic deficiency of PUMA rendered murine hepatocytes resistant to LPC-induced apoptosis. We concluded that LPC-induced Lipoapoptosis is dependent on mechanisms largely indistinguishable from PA. These data suggest that FFA-mediated cytotoxicity is indirect via the generation of the toxic metabolite, LPC.

  • death receptor 5 signaling promotes hepatocyte Lipoapoptosis
    Journal of Biological Chemistry, 2011
    Co-Authors: Sophie C. Cazanave, Nathan W Werneburg, Justin L Mott, Steven F. Bronk, Scott H Kaufmann, Christian D Fingas, Wei X Meng, Niklas Finnberg, Wafik S Eldeiry, Gregory James Gores
    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.