Seiichi Oyadomari

Roles of CHOP/GADD153 in endoplasmic reticulum stress

AbstractEndoplasmic reticulum (ER) is the site of synthesis and folding of secretory proteins. Perturbations of ER homeostasis affect protein folding and cause ER stress. ER can sense the stress and respond to it through translational attenuation, upregulation of the genes for ER chaperones and related proteins, and degradation of unfolded proteins by a quality-control system. However, when the ER function is severely impaired, the organelle elicits apoptotic signals. ER stress has been implicated in a variety of common diseases such as diabetes, ischemia and neurodegenerative disorders. One of the components of the ER stress-mediated apoptosis pathway is C/EBP homologous protein (CHOP), also known as growth arrest- and DNA damage-inducible gene 153 (GADD153). Here, we summarize the current understanding of the roles of CHOP/GADD153 in ER stress-mediated apoptosis and in diseases including diabetes, brain ischemia and neurodegenerative disease.

Obesity-induced gut microbial metabolite promotes liver cancer through senescence secretome

Obesity has become more prevalent in most developed countries over the past few decades, and is increasingly recognized as a major risk factor for several common types of cancer. As the worldwide obesity epidemic has shown no signs of abating, better understanding of the mechanisms underlying obesity-associated cancer is urgently needed. Although several events were proposed to be involved in obesity-associated cancer, the exact molecular mechanisms that integrate these events have remained largely unclear. Here we show that senescence-associated secretory phenotype (SASP) has crucial roles in promoting obesity-associated hepatocellular carcinoma (HCC) development in mice. Dietary or genetic obesity induces alterations of gut microbiota, thereby increasing the levels of deoxycholic acid (DCA), a gut bacterial metabolite known to cause DNA damage. The enterohepatic circulation of DCA provokes SASP phenotype in hepatic stellate cells (HSCs), which in turn secretes various inflammatory and tumour-promoting factors in the liver, thus facilitating HCC development in mice after exposure to chemical carcinogen. Notably, blocking DCA production or reducing gut bacteria efficiently prevents HCC development in obese mice. Similar results were also observed in mice lacking an SASP inducer or depleted of senescent HSCs, indicating that the DCA–SASP axis in HSCs has key roles in obesity-associated HCC development. Moreover, signs of SASP were also observed in the HSCs in the area of HCC arising in patients with non-alcoholic steatohepatitis, indicating that a similar pathway may contribute to at least certain aspects of obesity-associated HCC development in humans as well. These findings provide valuable new insights into the development of obesity-associated cancer and open up new possibilities for its control.

Targeted disruption of the Chop gene delays endoplasmic reticulum stress–mediated diabetes

Overload of pancreatic beta cells in conditions such as hyperglycemia, obesity, and long-term treatment with sulfonylureas leads to beta cell exhaustion and type 2 diabetes. Because beta cell mass declines under these conditions, apparently as a result of apoptosis, we speculated that overload kills beta cells as a result of endoplasmic reticulum (ER) stress. The Akita mouse, which carries a conformation-altering missense mutation (Cys96Tyr) in Insulin 2, likewise exhibits hyperglycemia and a reduced beta cell mass. In the development of diabetes in Akita mice, mRNAs for the ER chaperone Bip and the ER stress-associated apoptosis factor Chop were induced in the pancreas. Overexpression of the mutant insulin in mouse MIN6 beta cells induced Chop expression and led to apoptosis. Targeted disruption of the Chop gene delayed the onset of diabetes in heterozygous Akita mice by 8-10 weeks. We conclude that ER overload in beta cells causes ER stress and leads to apoptosis via Chop induction. Our findings suggest a new therapeutic approach for preventing the onset of diabetes by inhibiting Chop induction or by increasing chaperone capacity in the ER.

Nitric oxide-induced apoptosis in pancreatic β cells is mediated by the endoplasmic reticulum stress pathway

Excessive nitric oxide (NO) production in cytokine-activated β cells has been implicated in β cell disruption in type 1 diabetes. β cells are very vulnerable to NO-induced apoptosis. However, the mechanism underlying this phenomenon is unclear. Low concentrations of NO that lead to apoptosis apparently do not cause severe DNA damage in mouse MIN6 β cells. CHOP, a C/EBP homologous protein that is induced by endoplasmic reticulum (ER) stress and plays a role in growth arrest and cell death, was induced by a NO donor, S-nitroso-N-acetyl-d,l-penicillamine (SNAP). SNAP increased cytosolic Ca2+, and only agents depleting ER Ca2+ induced CHOP expression and led to apoptosis, suggesting that NO depletes ER Ca2+. Overexpression of calreticulin increased the Ca2+ content of ER and afforded protection to cells against NO-mediated apoptosis. Furthermore, pancreatic islets from CHOP knockout mice showed resistance to NO. We conclude that NO depletes ER Ca2+, causes ER stress, and leads to apoptosis. Thus, ER Ca2+ stores are a new target of NO, and the ER stress pathway is a major mechanism of NO-mediated β cell apoptosis.

Endoplasmic reticulum stress-mediated apoptosis in pancreatic β-cells

Apoptotic cell death in pancreatic β-cells is involved in the pathogenesis of diabetes. Signals from death receptors and DNA damage have been widely accepted as being triggers of apoptosis in β-cells. Recent studies indicated that the endoplasmic reticulum (ER) can sense and transduce apoptotic signals. Various genetic and environmental stresses interfere with protein folding in the ER and induce ER stress. In mammals, ER stress transducer proteins IRE1, PERK and ATF6 activate both survival and apoptotic pathways. The former includes transcriptional induction of ER chaperones, translational attenuation, and ER-associated degradation (ERAD) while the latter includes transcriptional induction of CHOP/GADD153, the activation of cJUN NH2-terminal kinase, and the activation of caspase-12. A characteristic feature of β-cells is the highly developed ER apparently due to a heavy engagement in insulin secretion. β-cells are most susceptible to ER stress. The recent studies reviewed in this article revealed that ER stress-mediated apoptosis in β-cells plays an important role in the development of diabetes.

Ischemia-induced neuronal cell death is mediated by the endoplasmic reticulum stress pathway involving CHOP

AbstractBrain ischemia induces apoptosis in neuronal cells, but the mechanism is not well understood. When wild-type mice were subjected to bilateral common carotid arteries occlusion (BCCAO) for 15 min, apoptosis-associated morphological changes and appearance of TUNEL-positive cells were observed in the striatum and in the hippocampus at 48 h after occlusion. RT-PCR analysis revealed that mRNAs for ER stress-associated proapoptotic factor CHOP and an ER chaperone BiP are markedly induced at 12 h after BCCAO. Immunohistochemical analysis showed that CHOP protein is induced in nuclei of damaged neurons at 24 h after occlusion. In contrast, ischemia-associated apoptotic loss of neurons was decreased in CHOP−/− mice. Primary hippocampal neurons from CHOP−/− mice were more resistant to hypoxia-reoxygenation-induced apoptosis than those from wild-type animals. These results indicate that ischemia-induced neuronal cell death is mediated by the ER stress pathway involving CHOP induction.

Dephosphorylation of Translation Initiation Factor 2α Enhances Glucose Tolerance and Attenuates Hepatosteatosis in Mice

The molecular mechanisms linking the stress of unfolded proteins in the endoplasmic reticulum (ER stress) to glucose intolerance in obese animals are poorly understood. In this study, enforced expression of a translation initiation factor 2alpha (eIF2alpha)-specific phosphatase, GADD34, was used to selectively compromise signaling in the eIF2(alphaP)-dependent arm of the ER unfolded protein response in liver of transgenic mice. The transgene resulted in lower liver glycogen levels and susceptibility to fasting hypoglycemia in lean mice and glucose tolerance and diminished hepatosteatosis in animals fed a high-fat diet. Attenuated eIF2(alphaP) correlated with lower expression of the adipogenic nuclear receptor PPARgamma and its upstream regulators, the transcription factors C/EBPalpha and C/EBPbeta, in transgenic mouse liver, whereas eIF2alpha phosphorylation promoted C/EBP translation in cultured cells and primary hepatocytes. These observations suggest that eIF2(alphaP)-mediated translation of key hepatic transcriptional regulators of intermediary metabolism contributes to the detrimental consequences of nutrient excess.

hsp70-DnaJ chaperone pair prevents nitric oxide- and CHOP-induced apoptosis by inhibiting translocation of Bax to mitochondria.

AbstractWe reported that the endoplasmic reticulum (ER) stress pathway involving CHOP, a member of the C/EBP transcription factor family, plays a key role in nitric oxide (NO)-mediated apoptosis of macrophages and pancreatic β cells. We also showed that the cytosolic chaperone pair of hsp70 and dj1 (hsp40/hdj-1) or dj2 (HSDJ/hdj-2) prevents NO-mediated apoptosis upstream of cytochrome c release from mitochondria. To analyze roles of the chaperone pair in preventing apoptosis, RAW 264.7 macrophages stably expressing hsp70 and dj1 or dj2 were established. The chaperone pair prevented LPS/IFN-γ-induced and NO-mediated apoptosis downstream of CHOP induction. hsp70 mutant protein lacking the ATPase domain or the C-terminal EEVD sequence were not effective in preventing CHOP-induced apoptosis. A mutant dj2 lacking the C-terminal prenylation CaaX motif, was also not effective. When wild-type RAW 264.7 cells were treated with LPS/IFN-γ, NO-mediated apoptosis was induced, and proapoptotic Bcl-2 family protein Bax was translocated from cytosol to mitochondria. This translocation was prevented in cells stably expressing hsp70/dj2, and in CHOP knockout cells. Overexpression of CHOP in wild-type cells also induced translocation of Bax and this translocation was prevented in cells expressing hsp70/dj2. CHOP-induced apoptosis was prevented by Bax knock-down. Coimmunoprecipitation experiments showed that Bax interacts with both hsp70 and dj1/dj2. ATPase domain of hsp70 was necessary for the binding with Bax. These findings indicate that CHOP-induced apoptosis is mediated by translocation of Bax from the cytosol to the mitochondria, and hsp70/dj1 or dj2 chaperone pair prevents apoptosis by interacting with Bax and preventing translocation to the mitochondria.

Nitric Oxide-induced Apoptosis in RAW 264.7 Macrophages Is Mediated by Endoplasmic Reticulum Stress Pathway Involving ATF6 and CHOP*

Excess nitric oxide (NO) induces apoptosis in some cell types including macrophages; however, the cascade of NO-mediated apoptosis is not fully understood. We investigated the initial steps of NO-mediated apoptosis in mouse macrophage-like RAW 264.7 cells. When cells were treated with bacterial lipopolysaccharide (LPS) plus interferon-γ (IFN-γ), NO-mediated apoptosis occurred. Under these conditions, p53 accumulation was not observed, indicating that DNA damage is not the main trigger of NO-mediated apoptosis. On the other hand, mRNA and protein for CHOP, a transcription factor known to be induced by endoplasmic reticulum (ER) stress, were induced. The CHOP induction by LPS/IFN-γ treatment preceded cytochrome c release from mitochondria. In addition, p90ATF6, an ER membrane-bound transcription factor involved in ER stress response, was cleaved to its active soluble form p50ATF6, which was transported to nucleus and bound to the ER stress response element of the CHOP gene. In the luciferase reporter assay, both the CHOP-binding element of the Rous sarcoma virus long terminal repeat and ER stress response element of theCHOP gene were activated by LPS/IFN-γ treatment. When RAW 264.7 cells or COS-7 cells were transfected with expression plasmids for CHOP, p90ATF6, or p50ATF6, cell death was observed. In addition, apoptosis induced by p50ATF6 was prevented by a CHOP dominant negative form as well as by an ATF6 dominant negative form, and LPS/IFN-γ-induced apoptosis was prevented by the CHOP dominant negative form. Peritoneal macrophages from CHOP knockout mice showed resistance to NO-induced apoptosis. These results indicate that the ER stress pathway involving ATF6 and CHOP plays a key role in NO-mediated apoptosis in macrophages.

Cotranslocational Degradation Protects the Stressed Endoplasmic Reticulum from Protein Overload

Summary The ERs capacity to process proteins is limited, and stress caused by accumulation of unfolded and misfolded proteins (ER stress) contributes to human disease. ER stress elicits the unfolded protein response (UPR), whose components attenuate protein synthesis, increase folding capacity, and enhance misfolded protein degradation. Here, we report that P58 IPK /DNAJC3 , a UPR-responsive gene previously implicated in translational control, encodes a cytosolic cochaperone that associates with the ER protein translocation channel Sec61. P58 IPK recruits HSP70 chaperones to the cytosolic face of Sec61 and can be crosslinked to proteins entering the ER that are delayed at the translocon. Proteasome-mediated cytosolic degradation of translocating proteins delayed at Sec61 is cochaperone dependent. In P58 IPK−/− mice, cells with a high secretory burden are markedly compromised in their ability to cope with ER stress. Thus, P58 IPK is a key mediator of cotranslocational ER protein degradation, and this process likely contributes to ER homeostasis in stressed cells.