GPNMB Induces BiP Expression by Enhancing Splicing of BiP Pre-mRNA during the Endoplasmic Reticulum Stress Response
Yasuhiro NodaKazuhiro TsurumaMasafumi TakataMitsue IshisakaHirotaka TanakaYusuke NakanoYuki NagaharaMasamitsu ShimazawaHideaki Hara
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Glycoprotein nonmetastatic melanoma protein B (GPNMB) has a neuroprotective effect against neuronal cell death caused by the accumulation of abnormal mutated proteins. It is known that the accumulation of pathological proteins induces endoplasmic-reticulum (ER) stress leading to cell damage. The aim of this study was to determine the role of GPNMB in the ER stress response. GPNMB was greatly up-regulated by thapsigargin-induced ER stress. Under the ER stress conditions, GPNMB relocated to the nucleus and specifically up-regulated expression of BiP at the mRNA level by promoting the BiP pre-mRNA splicing, not through the pathways initiated by the three major transducers of the unfolded protein response: IRE1, PERK, and ATF6. Furthermore, we found that the protein level of BiP and the infarction were increased and attenuated, respectively, in Gpnmb-transgenic mice after occlusion of the middle cerebral artery, in comparison with wild-type mice. Thus, our findings indicate that GPNMB enhances the BiP expression by promoting the splicing (thereby preventing cell death caused by ER stress) and could be a therapeutic target in ER stress-related disorders.Keywords:
ATF6
Thapsigargin
XBP1
Duck enteritis virus (DEV) can infect ducks, geese, and many other poultry species and leads to acute, septic and highly fatal infectious disease. Autophagy is an evolutionarily ancient pathway that plays an important role in many viral infections. We previously reported that DEV infection induces autophagy for its own benefit, but how this occurs remains unclear. In this study, endoplasmic reticulum (ER) stress was triggered by DEV infection, as demonstrated by the increased expression of the ER stress marker glucose-regulated protein 78 (GRP78) and the dilated morphology of the ER. Pathways associated with the unfolded protein response (UPR), including the PKR-like ER protein kinase (PERK) and inositol-requiring enzyme 1 (IRE1) pathways, but not the activating transcription factor 6 (ATF6) pathway, were activated in DEV-infected duck embryo fibroblast (DEF) cells. In addition, the knockdown of both PERK and IRE1 by small interfering RNAs (siRNAs) reduced the level of LC3-II and viral yields, which suggested that the PERK-eukaryotic initiation factor 2α (eIF2α) and IRE1-x-box protein1 (XBP1) pathways may contribute to DEV-induced autophagy. Collectively, these data offer new insight into the mechanisms of DEV -induced autophagy through activation of the ER stress-related UPR pathway.
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Accumulation of proteins in the endoplasmic reticulum (ER) triggers the unfolded protein response (UPR), comprising three signaling pathways initiated by Ire1, Perk and Atf6 respectively. Unfolded protein response activation was compared in chemically stressed murine wildtype melanocytes and mutant melanocytes that retain tyrosinase in the ER. Thapsigargin, an ER stressor, activated all pathways in wildtype melanocytes, triggering Caspase 12-mediated apoptosis at toxic doses. Albino melanocytes expressing mutant tyrosinase showed evidence of ER stress with increased Ire1 expression, but the downstream effector, Xbp1, was not activated even following thapsigargin treatment. Attenuation of Ire1 signaling was recapitulated in wildtype melanocytes treated with thapsigargin for 8 days, with diminished Xbp1 activation observed after 4 days. Atf6 was also activated in albino melanocytes, with no response to thapsigargin, while the Perk pathway was not activated and thapsigargin treatment elicited robust expression of the downstream effector CCAAT-enhancer-binding protein homologous protein. Thus, melanocytes adapt to ER stress by attenuating two UPR pathways.
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Diabetes is caused by loss or dysfunction of beta cells. Endoplasmic reticulum (ER) stress has been implicated in beta cell loss in both type 1 and type 2 diabetes. Hyperglycemia and increased insulin demand cause ER stress, activating the unfolded protein response (UPR). The initial beta cell response to stress increases cell number through proliferation, but prolonged stress leads to beta cell demise. Precisely characterizing the beta cell UPR could lead to a better understanding of how beta cells are lost in diabetes. In this study, mouse primary islet cells were treated with ER stressors Thapsigargin (Tg) or tunicamycin (Tm), which activate all three arms of the UPR: Atf6, Ire1 and Perk. In mouse beta cells, Tg (1uM) activated the Ire1 (tested by splicing of Xbp1) and Perk (tested by phosphorylation of eIF2-alpha) pathways within 1 hour, and induced Atf6 transcriptional targets by 4 hours. Specific roles of Atf6 in the beta cell ER stress response are not well understood, with both positive and negative roles reported in the literature. Therefore, we tested how Atf6 participates in the acute beta cell response to severe stress, including UPR pathway activation and cell death. We found that knockdown of Atf6 leads to a blunted ER stress response, with delayed activation of Atf6 target genes Grp78, Hyou1 and Pdia4, but not HerpUD1. The Ire pathway was also negatively impacted by Atf6 knockdown, with blunted activation of Xbp1 target genes Sec24D, Erdj4 and Ssr3. Perk pathway activation was not affected by knockdown of Atf6 (p-eIF2-alpha, Chop mRNA). Finally, acute knockdown of Atf6 increased beta cell death even in the absence of stress (n=4) and made beta cells more prone to stress-mediated cell death (n=2). On the other hand, overexpression of ATF6 had no impact on Tg-mediated beta cell death suggesting Atf6 was not sufficient to protect (n=2) against irreversible ER stress. In sum, our data suggest a nuanced role for Atf6 in the beta cell stress response and the cell fate choice between life and death. Disclosure R.B. Sharma: None. C.O. Darko: None. B. Gablaski: None. L.C. Alonso: None. Funding National Institutes of Health (R01DK114686); National Institute of Diabetes and Digestive and Kidney Disease (R01DK113300)
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The unfolded protein response (UPR) events triggered by the accumulation of unfolded protein in endoplasmic reticulum (ER) activate the three UPR signaling pathways mediated by IRE1, ATF6 and PERK. Spliced XBP1 mRNA induced by activated IRE1 is translated to the protein, a potent transcription factor that induces BiP expression. XBP1 is also induced by activated ATF6. It is thus thought to be an important marker reflecting both IRE1 and ATF6 signaling in response to ER stress. For quantitative measurement of XBP1 gene expression, it is important to distinguish between the spliced and non-spliced form of XBP1 mRNA. We have developed a new method to detect the spliced XBP1 mRNA by means of real-time PCR and we compared the result with measurements of the expression of the ER stress inducible gene BiP. A good correlation was found between spliced XBP1 expression and BiP expression. Thus, our method may be useful for simple and quantitative evaluation of ER stress.
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Abstract The endoplasmic reticulum (ER) responds to changes in intracellular homeostasis through activation of the unfolded protein response (UPR). UPR can facilitate the restoration of cellular homeostasis, via the concerted activation of three ER stress sensors, namely IRE1, PERK and ATF6. Global approaches in several cellular contexts have revealed that UPR regulates the expression of many miRNAs that play an important role in the regulation of life and death decisions during UPR. Here we show that expression of miR-424(322)-503 cluster is downregulated during UPR. IRE1 inhibitor (4 μ8C) and deficiency of XBP1 had no effect on downregulation of miR-424(322)-503 during UPR. Treatment of cells with CCT030312, a selective activator of EIF2AK3/PERK signalling, leads to the downregulation of miR-424(322)-503 expression. The repression of miR-424(322)-503 cluster during conditions of ER stress is compromised in PERK-deficient MEFs. miR-424 regulates the expression of ATF6 via a miR-424 binding site in its 3′ UTR and attenuates the ATF6 transcriptional activity during UPR. Further miR-424 had no effect on IRE1-XBP1 axis but enhanced the regulated IRE1-dependent decay (RIDD). Our results suggest that miR-424 constitutes an obligatory fine-tuning mechanism where PERK-mediated downregulation of miR-424(322)-503 cluster regulates optimal activation of IRE1 and ATF6 during conditions of ER stress.
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