4-苯基丁酸钠通过抑制内质网应激减轻皮层神经元氧糖剥夺/再灌注损伤

4-苯基丁酸钠（4-phenylbutyric acid,4-PBA）是协助内质网中蛋白质转录后修饰和折叠的分子伴侣,故可减轻非折叠蛋白反应（unfolded protein response,UPR）及其介导的细胞凋亡。既往研究表明,4-PBA可以减轻脑组织的缺血性损伤,但采用原代皮层神经元构建氧糖剥夺/再灌注（oxygen glucose deprivation/reoxygenation, OGD/R）损伤模型,来研究4-PBA对神经元损伤的保护作用及其机制尚未见报道。本文采用原代培养的皮层神经元OGD/R损伤模型,同时给予4-PBA处理,探讨4-PBA对OGD/R诱导的神经元内质网应激（endoplasmic reticulum stress,ERS）的作用及其机制。分别采用MTT、LDH和Hoechst 33342染色法检测神经元存活率、细胞膜完整性和细胞凋亡情况。Western印迹检测ERS标志物葡萄糖调节蛋白78 (glucose regulated protein 78,GRP78),以及肌醇必需酶1（inositol requiring enzyme 1, IRE1）通路相关蛋白质的表达。Western印迹结果显示,在OGD/R后0～48 h,GRP78的表达较对照组明显升高。MTT、LDH漏出率和Hoechst 33342染色法检测显示,4-PBA显著改善OGD/R所导致的神经元存活率下降、LDH漏出率升高和细胞凋亡增加,且具有明显的剂量依赖性。通过Western印迹检测发现,4-PBA显著逆转OGD/R所致GRP78蛋白表达水平的上调。此外,对肌醇必需酶1通路相关蛋白质的检测显示,4-PBA下调氧糖剥夺/再灌注组神经元p IRE1和p JNK的表达,增加抗凋亡蛋白Bcl 2表达。上述研究结果表明,4-PBA在氧糖剥夺/再灌注情况下对神经元具有保护作用,该保护作用可能是通过抑制肌醇必需酶1信号通路介导的非折叠蛋白反应和内质网应激实现的。     

Geniposide inhibits glucolipotoxicity and cooperates with Txnip knockdown to potentiate cell adaption to endoplasmic reticulum stress in pancreatic beta cells

Thioredoxin‐interacting protein (Txnip), a negative regulator of thioredoxin, has become an attractive therapeutic target to alleviate metabolic diseases. Our previous data demonstrated that geniposide improved glucose‐stimulated insulin secretion by accelerating Txnip degradation and prevented the early‐stage apoptosis of pancreatic β cells induced by palmitate, but the underlying mechanisms are still unclear. The objective of this study is to identify the role of Txnip in geniposide preventing the apoptosis of pancreatic β cells induced by high glucose and palmitate (HG/PA). The results revealed that geniposide attenuated HG/PA‐induced cell apoptosis and the expression of Bax and caspase‐3, while increasing mitochondrial membrane potential and the anti‐apoptotic protein levels of heme‐oxygenase‐1 (HO‐1) and Bcl‐2 in INS‐1 rat pancreatic β cells. Knockdown of the Txnip gene raised the levels of anti‐apoptotic proteins HO‐1 and Bcl‐2 and geniposide potentiated the effect of Txnip when the INS‐1 cells were challenged by HG/PA. Furthermore, geniposide enhanced the adoptive unfolded protein response by increasing the phosphorylation of PERK/eIF2α and IRE1α in HG/PA‐treated INS‐1 cells. The results together suggest that geniposide might be useful to antagonize glucolipotoxicity and Txnip might be a pleiotropic cellular factor in pancreatic β cells.     

Regulation of insulin biosynthesis in pancreatic beta cells by an endoplasmic reticulum-resident protein kinase IRE1

In pancreatic beta cells, the endoplasmic reticulum (ER) is an important site for insulin biosynthesis and the folding of newly synthesized proinsulin. Here, we show that IRE1alpha, an ER-resident protein kinase, has a crucial function in insulin biosynthesis. IRE1alpha phosphorylation is coupled to insulin biosynthesis in response to transient exposure to high glucose; inactivation of IRE1alpha signaling by siRNA or inhibition of IRE1alpha phosphorylation hinders insulin biosynthesis. IRE1 activation by high glucose does not accompany XBP-1 splicing and BiP dissociation but upregulates its target genes such as WFS1. Thus, IRE1 signaling activated by transient exposure to high glucose uses a unique subset of downstream components and has a beneficial effect on pancreatic beta cells. In contrast, chronic exposure of beta cells to high glucose causes ER stress and hyperactivation of IRE1, leading to the suppression of insulin gene expression. IRE1 signaling is therefore a potential target for therapeutic regulation of insulin biosynthesis.     

PERK-dependent compartmentalization of ERAD and unfolded protein response machineries during ER stress

Accumulation of misfolded proteins in the endoplasmic reticulum (ER) activates the ER membrane kinases PERK and IRE1 leading to the unfolded protein response (UPR). We show here that UPR activation triggers PERK and IRE1 segregation from BiP and their sorting with misfolded proteins to the ER-derived quality control compartment (ERQC), a pericentriolar compartment that we had identified previously. PERK phosphorylates translation factor eIF2alpha, which then accumulates on the cytosolic side of the ERQC. Dominant negative PERK or eIF2alpha(S51A) mutants prevent the compartmentalization, whereas eIF2alpha(S51D) mutant, which mimics constitutive phosphorylation, promotes it. This suggests a feedback loop where eIF2alpha phosphorylation causes pericentriolar concentration at the ERQC, which in turn amplifies the UPR. ER-associated degradation (ERAD) is an UPR-dependent process; we also find that ERAD components (Sec61beta, HRD1, p97/VCP, ubiquitin) are recruited to the ERQC, making it a likely site for retrotranslocation. In addition, we show that autophagy, suggested to play a role in elimination of aggregated proteins, is unrelated to protein accumulation in the ERQC.     

ER signaling in unfolded protein response

Abnormally folded proteins are susceptible to aggregation and accumulation in cells, ultimately leading to cell death. To protect cells against such dangers, expression of various genes including molecular chaperones can be induced and ER-associated protein degradation (ERAD) activated in response to the accumulation of unfolded protein in the endoplasmic reticulum (ER). This is known as the unfolded protein response (UPR). ERAD requires retrograde transport of unfolded proteins from the ER back to the cytosol via the translocon for degradation by the ubiquitin-proteasome system. Hrd1p is a UPR-induced ER membrane protein that acts as a ubiquitin ligase (E3) in the ERAD system. Hrd3p interacts with and stabilizes Hrd1p. We have isolated and identified human homologs (HRD1 and SEL1/HRD3) of Saccharomyces cerevisiae Hrd1p and Hrd3p. Human HRD1 and SEL1 were up-regulated in response to ER stress and overexpression of human IRE1 and ATF6, which are ER stress-sensor molecules in the ER. HEK293T cells overexpressing HRD1 showed resistance to ER stress-induced cell death. These results suggest that HRD1 and SEL1 are up-regulated by the UPR and contribute to protection against the ER stress-induced cell death by degrading unfolded proteins accumulated in the ER.     

Cytomegalovirus Downregulates IRE1 to Repress the Unfolded Protein Response

During viral infection, a massive demand for viral glycoproteins can overwhelm the capacity of the protein folding and quality control machinery, leading to an accumulation of unfolded proteins in the endoplasmic reticulum (ER). To restore ER homeostasis, cells initiate the unfolded protein response (UPR) by activating three ER-to-nucleus signaling pathways, of which the inositol-requiring enzyme 1 (IRE1)-dependent pathway is the most conserved. To reduce ER stress, the UPR decreases protein synthesis, increases degradation of unfolded proteins, and upregulates chaperone expression to enhance protein folding. Cytomegaloviruses, as other viral pathogens, modulate the UPR to their own advantage. However, the molecular mechanisms and the viral proteins responsible for UPR modulation remained to be identified. In this study, we investigated the modulation of IRE1 signaling by murine cytomegalovirus (MCMV) and found that IRE1-mediated mRNA splicing and expression of the X-box binding protein 1 (XBP1) is repressed in infected cells. By affinity purification, we identified the viral M50 protein as an IRE1-interacting protein. M50 expression in transfected or MCMV-infected cells induced a substantial downregulation of IRE1 protein levels. The N-terminal conserved region of M50 was found to be required for interaction with and downregulation of IRE1. Moreover, UL50, the human cytomegalovirus (HCMV) homolog of M50, affected IRE1 in the same way. Thus we concluded that IRE1 downregulation represents a previously undescribed viral strategy to curb the UPR.     

Tauroursodeoxycholic acid attenuates cyclosporine-induced renal fibrogenesis in the mouse model

Chronic exposure to cyclosporine causes nephrotoxicity and organ damage. Here we show that cyclosporine nephrotoxicity in vivo is associated with the activation of the unfolded protein response (UPR) pathway to initiate tissue fibrosis. We demonstrate that cyclosporine therapy activated the IRE1α branch of the unfolded protein response (UPR) and stimulated the TGFβ1 signaling pathway in the kidneys of male mice. Co-administration of the proteostasis promoter tauroursodeoxycholic acid (TUDCA) with cyclosporine inhibited the UPR pathway in the kidneys of treated male mice as well as decreased the development of renal fibrogenesis.     

Endoplasmic Reticulum Stress Impairs Insulin Receptor Signaling in the Brains of Obese Rats

The incidence of obesity is increasing worldwide. It was reported that endoplasmic reticulum stress (ERS) could inhibit insulin receptor signaling by activating c-Jun N-terminal kinase (JNK) in the liver. However, the relationship between ERS and insulin receptor signaling in the brain during obesity remains unclear. The aim of the current study was to assess whether ERS alters insulin receptor signaling through the hyper-activation of JNK in the hippocampus and frontal cortex in the brains of obese rats. Obesity was induced using a high fat diet (HFD). The Morris water maze test was then performed to evaluate decreases in cognitive function, and western blot was used to verify whether abnormal insulin receptor signaling was induced by ERS in HFD rats exhibiting cognitive decline. In addition, to determine whether ERS activated JNK and consequently impaired insulin receptor signaling, SH-SY5Y cells were treated with the JNK inhibitor, SP600125, followed by tunicamycin or thapsigargin, and primary rat hippocampal and cortical neurons were transfected with siRNA against IRE1α and JNK. We found that the expression of phosphorylation of PKR-like kinase (PERK), phosphorylation of α subunit of translation initiation factor 2 (eIF2α), and phosphorylation of inositol-requiring kinase-1α (IRE-1α) were increased in the brains of rats with HFD when compared with control rats. The level of serine phosphorylation of insulin receptor substrate-1 (IRS-1) was also increased, while protein kinase B (PKB/Akt) was reduced. ERS was also found to inhibit insulin receptor signaling via the activation of JNK in SH-SY5Y cells, primary rat hippocampal, and cortical neurons. These results indicate that ERS was increased, thereby resulting in impaired insulin receptor signaling in the hippocampus and frontal cortex of obese rats.     

Protective role of Gipie, a Girdin family protein, in endoplasmic reticulum stress responses in endothelial cells

Continued exposure of endothelial cells to mechanical/shear stress elicits the unfolded protein response (UPR), which enhances intracellular homeostasis and protect cells against the accumulation of improperly folded proteins. Cells commit to apoptosis when subjected to continuous and high endoplasmic reticulum (ER) stress unless homeostasis is maintained. It is unknown how endothelial cells differentially regulate the UPR. Here we show that a novel Girdin family protein, Gipie (78 kDa glucose-regulated protein [GRP78]-interacting protein induced by ER stress), is expressed in endothelial cells, where it interacts with GRP78, a master regulator of the UPR. Gipie stabilizes the interaction between GRP78 and the ER stress sensor inositol-requiring protein 1 (IRE1) at the ER, leading to the attenuation of IRE1-induced c-Jun N-terminal kinase (JNK) activation. Gipie expression is induced upon ER stress and suppresses the IRE1-JNK pathway and ER stress-induced apoptosis. Furthermore we found that Gipie expression is up-regulated in the neointima of carotid arteries after balloon injury in a rat model that is known to result in the induction of the UPR. Thus our data indicate that Gipie/GRP78 interaction controls the IRE1-JNK signaling pathway. That interaction appears to protect endothelial cells against ER stress-induced apoptosis in pathological contexts such as atherosclerosis and vascular endothelial dysfunction.     

Intrinsic capacities of molecular sensors of the unfolded protein response to sense alternate forms of endoplasmic reticulum stress

The unfolded protein response (UPR) regulates the protein-folding capacity of the endoplasmic reticulum (ER) according to cellular demand. In mammalian cells, three ER transmembrane components, IRE1, PERK, and ATF6, initiate distinct UPR signaling branches. We show that these UPR components display distinct sensitivities toward different forms of ER stress. ER stress induced by ER Ca2+ release in particular revealed fundamental differences in the properties of UPR signaling branches. Compared with the rapid response of both IRE1 and PERK to ER stress induced by thapsigargin, an ER Ca2+ ATPase inhibitor, the response of ATF6 was markedly delayed. These studies are the first side-by-side comparisons of UPR signaling branch activation and reveal intrinsic features of UPR stress sensor activation in response to alternate forms of ER stress. As such, they provide initial groundwork toward understanding how ER stress sensors can confer different responses and how optimal UPR responses are achieved in physiological settings.     

The Role of IRE1α in the Degradation of Insulin mRNA in Pancreatic β-Cells

Background

The endoplasmic reticulum (ER) is a cellular compartment for the biosynthesis and folding of newly synthesized secretory proteins such as insulin. Perturbations to ER homeostasis cause ER stress and subsequently activate cell signaling pathways, collectively known as the Unfolded Protein Response (UPR). IRE1α is a central component of the UPR. In pancreatic β-cells, IRE1α also functions in the regulation of insulin biosynthesis.

Principal Findings

Here we report that hyperactivation of IRE1α caused by chronic high glucose treatment or IRE1α overexpression leads to insulin mRNA degradation in pancreatic β-cells. Inhibition of IRE1α signaling using its dominant negative form prevents insulin mRNA degradation. Islets from mice heterozygous for IRE1α retain expression of more insulin mRNA after chronic high glucose treatment than do their wild-type littermates.

Conclusions/Significance

These results reveal a role of IRE1α in insulin mRNA expression under ER stress conditions caused by chronic high glucose. The rapid degradation of insulin mRNA could provide immediate relief for the ER and free up the translocation machinery. Thus, this mechanism would preserve ER homeostasis and help ensure that the insulin already inside the ER can be properly folded and secreted. This adaptation may be crucial for the maintenance of β-cell homeostasis and may explain why the β-cells of type 2 diabetic patients with chronic hyperglycemia stop producing insulin in the absence of apoptosis. This mechanism may also be involved in suppression of the autoimmune type 1 diabetes by reducing the amount of misfolded insulin, which could be a source of “neo-autoantigens.”     

Sustained IRE1 and ATF6 signaling is important for survival of melanoma cells undergoing ER stress

Apoptosis triggered by endoplasmic reticulum (ER) stress is associated with rapid attenuation of the IRE1α and ATF6 pathways but persistent activation of the PERK branch of the unfolded protein response (UPR) in cells. However, melanoma cells are largely resistant to ER stress-induced apoptosis, suggesting that the kinetics and durations of activation of the UPR pathways are deregulated in melanoma cells undergoing ER stress. We show here that the IRE1α and ATF6 pathways are sustained along with the PERK signaling in melanoma cells subjected to pharmacological ER stress, and that this is, at least in part, due to increased activation of the MEK/ERK pathway. In contrast to an initial increase followed by rapid reduction in activation of IRE1α and ATF6 signaling in control cells that were relatively sensitive to ER stress-induced apoptosis, activation of IRE1α and ATF6 by the pharmacological ER stress inducer tunicamycin (TM) or thapsigargin (TG) persisted in melanoma cells. On the other hand, the increase in PERK signaling lasted similarly in both types of cells. Sustained activation of IRE1α and ATF6 signaling played an important role in protecting melanoma cells from ER stress-induced apoptosis, as interruption of IRE1α or ATF6 rendered melanoma cells sensitive to apoptosis induced by TM or TG. Inhibition of MEK partially blocked IRE1α and ATF6 activation, suggesting that MEK/ERK signaling contributed to sustained activation of IRE1α and ATF6. Taken together, these results identify sustained activation of the IRE1α and ATF6 pathways of the UPR driven by the MEK/ERK pathway as an important protective mechanism against ER stress-induced apoptosis in melanoma cells.     

Divergent Effects of PERK and IRE1 Signaling on Cell Viability

Protein misfolding in the endoplasmic reticulum (ER) activates a set of intracellular signaling pathways, collectively termed the Unfolded Protein Response (UPR). UPR signaling promotes cell survival by reducing misfolded protein levels. If homeostasis cannot be restored, UPR signaling promotes cell death. The molecular basis for the switch between prosurvival and proapoptotic UPR function is poorly understood. The ER-resident proteins, PERK and IRE1, control two key UPR signaling pathways. Protein misfolding concomitantly activates PERK and IRE1 and has clouded insight into their contributions toward life or death cell fates. Here, we employed chemical-genetic strategies to activate individually PERK or IRE1 uncoupled from protein misfolding. We found that sustained PERK signaling impaired cell proliferation and promoted apoptosis. By contrast, equivalent durations of IRE1 signaling enhanced cell proliferation without promoting cell death. These results demonstrate that extended PERK and IRE1 signaling have opposite effects on cell viability. Differential activation of PERK and IRE1 may determine life or death decisions after ER protein misfolding.     

A ubiquitin ligase HRD1 promotes the degradation of Pael receptor, a substrate of Parkin

It has been proposed that in autosomal recessive juvenile parkinsonism (AR-JP), a ubiquitin ligase (E3) Parkin, which is involved in endoplasmic reticulum-associated degradation (ERAD), lacks E3 activity. The resulting accumulation of Parkin-associated endothelin receptor-like receptor (Pael-R), a substrate of Parkin, leads to endoplasmic reticulum stress, causing neuronal death. We previously reported that human E3 HRD1 in the endoplasmic reticulum protects against endoplasmic reticulum stress-induced apoptosis. This study shows that HRD1 was expressed in substantia nigra pars compacta (SNC) dopaminergic neurons and interacted with Pael-R through the HRD1 proline-rich region, promoting the ubiquitylation and degradation of Pael-R. Furthermore, the disruption of endogenous HRD1 by small interfering RNA (siRNA) induced Pael-R accumulation and caspase-3 activation. We also found that ATF6 overexpression, which induced HRD1, accelerated and caused Pael-R degradation; the suppression of HRD1 expression by siRNA partially prevents this degradation. These results suggest that in addition to Parkin, HRD1 is also involved in the degradation of Pael-R.