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.     

细胞内质网应激与糖脂代谢紊乱的机制关联

在真核细胞中,内质网是蛋白质合成、折叠、加工及其质量监控的重要场所。当内质网难以承担蛋白折叠的高负荷时则引发内质网应激(ER stress),激活细胞的未折叠蛋白响应(unfoldedprotein response,UPR)。细胞通过内质网跨膜蛋白ATF6、PERK和IRE1介导的三条极为关键的UPR信号通路,调控下游相关基因的表达,以增强内质网对蛋白折叠的处理能力。因此,UPR通路在细胞的稳态平衡中具有举足轻重的作用,而这一动态过程的调控对于维持机体的正常生理功能至关重要。近来大量研究表明,在哺乳动物中内质网应激与机体的营养感应和糖脂代谢的调控过程密切相关。在肝脏、脂肪、胰岛以及下丘脑等不同的组织器官中,内质网应激均影响代谢通路的调节机制,因此在糖脂代谢紊乱的发生发展中扮演重要的角色。综上所述,进一步深入了解内质网应激引发代谢异常的生理学机制,可以为肥胖、脂肪肝及2型糖尿病等相关代谢性疾病的防治提供新的潜在药物靶点和重要的理论线索。     

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.     

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.     

Cardiomyocyte-specific disruption of Serca2 in adult mice causes sarco(endo)plasmic reticulum stress and apoptosis

Reduced sarco(endo)plasmic reticulum (SR) Ca(2+) ATPase (SERCA2) contributes to the impaired cardiomyocyte Ca(2+) homeostasis observed in heart failure. We hypothesized that a reduction in SERCA2 also elicits myocardial ER/SR stress responses, including unfolded protein responses (UPR) and cardiomyocyte apoptosis, which may additionally contribute to the pathophysiology of this condition. Left ventricular myocardium from mice with cardiomyocyte-specific tamoxifen-inducible disruption of Serca2 (SERCA2 KO) was compared with aged-matched controls. In SERCA2 KO hearts, SERCA2 protein levels were markedly reduced to 2% of control values at 7 weeks following tamoxifen treatment. Serca2 disruption caused increased abundance of the ER stress-associated proteins CRT, GRP78, PERK, and eIF2α and increased phosphorylation of PERK and eIF2α, indicating UPR induction. Pro-apoptotic signaling was also activated in SERCA2 KO, as the abundance of CHOP, caspase 12, and Bax was increased. Indeed, TUNEL staining revealed an increased fraction of cardiomyocytes undergoing apoptosis in SERCA2 KO. ER-Tracker staining additionally revealed altered ER structure. These findings indicate that reduction in SERCA2 protein abundance is associated with marked ER/SR stress in cardiomyocytes, which induces UPR, apoptosis, and ER/SR structural alterations. This suggests that reduced SERCA2 abundance or function may contribute to the phenotype of heart failure also through induction of ER/SR stress responses.     

The protein kinase/endoribonuclease IRE1alpha that signals the unfolded protein response has a luminal N-terminal ligand-independent dimerization domain

In response to accumulation of unfolded proteins in the endoplasmic reticulum (ER), cells activate an intracellular signal transduction pathway called the unfolded protein response (UPR). IRE and PERK are the two type-I ER transmembrane protein kinase receptors that signal the UPR. The N-terminal luminal domains (NLDs) of IRE1 and PERK sense ER stress conditions by a common mechanism and transmit the signal to regulate the cytoplasmic domains of these receptors. To provide an experimental system amenable to detailed biochemical and structural analysis to elucidate the mechanism of ER-transmembrane signaling mechanism mediated by the NLD, we overexpressed the soluble luminal domain of human IRE1alpha in COS-1 cells by transient DNA transfection. Here we report the expression, purification, and characterization of the soluble NLD. The biological function of the NLD was confirmed by its ability to associate with itself and to interact with both the membrane-bound full-length IRE1alpha receptor and the ER chaperone BiP. Functional and spectral studies suggested that the highly conserved N-linked glycosylation site is not required for proper protein folding and self-association. Interestingly, we demonstrated that the NLD forms stable dimers linked by intermolecular disulfide bridges. Our data support that the luminal domain represents a novel ligand-independent dimerization domain.     

6-Shogaol induces apoptosis in human hepatocellular carcinoma cells and exhibits anti-tumor activity in vivo through endoplasmic reticulum stress

6-Shogaol is an active compound isolated from Ginger (Zingiber officinale Rosc). In this work, we demonstrated that 6-shogaol induces apoptosis in human hepatocellular carcinoma cells in relation to caspase activation and endoplasmic reticulum (ER) stress signaling. Proteomic analysis revealed that ER stress was accompanied by 6-shogaol-induced apoptosis in hepatocellular carcinoma cells. 6-shogaol affected the ER stress signaling by regulating unfolded protein response (UPR) sensor PERK and its downstream target eIF2α. However, the effect on the other two UPR sensors IRE1 and ATF6 was not obvious. In prolonged ER stress, 6-shogaol inhibited the phosphorylation of eIF2α and triggered apoptosis in SMMC-7721 cells. Salubrinal, an activator of the PERK/eIF2α pathway, strikingly enhanced the phosphorylation of eIF2α in SMMC-7721 cells with no toxicity. However, combined treatment with 6-shogaol and salubrinal resulted in significantly increase of apoptosis and dephosphorylation of eIF2α. Overexpression of eIF2α prevented 6-shogaol-mediated apoptosis in SMMC-7721 cells, whereas inhibition of eIF2α by small interfering RNA markedly enhanced 6-shogaol-mediated cell death. Furthermore, 6-shogaol-mediated inhibition of tumor growth of mouse SMMC-7721 xenograft was associated with induction of apoptosis, activation of caspase-3, and inactivation of eIF2α. Altogether our results indicate that the PERK/eIF2α pathway plays an important role in 6-shogaol-mediated ER stress and apoptosis in SMMC-7721 cells in vitro and in vivo.     

Study on the effect of IRE1α on cell growth and apoptosis via modulation PLK1 in ER stress response

The mammalian unfolded protein response (UPR) protects the cell against the stress of misfolded proteins in the endoplasmic reticulum (ER). Failure to adapt to ER stress causes the UPR to trigger apoptosis. Inositol-requiring enzyme-1a (IRE1a), as one of three unfolded protein sensors in UPR signaling pathways, senses ER unfolded proteins through an ER lumenal domain that becomes oligomerized during ER stress. It is known to be important for ER stress-mediated apoptosis and cell growth, but the exact molecular mechanism underlying these processes remains unexplored. In this study, we report that knockdown of IRE1a by an siRNA silencing approach enhanced, whereas its overexpression inhibited, cell proliferation in Hepatoma cells. Besides, overexpression of IRE1a induced, while its repression inhibited, ER stress-mediated apoptosis in Hepatomas cells. Furthermore, we found that overexpressed IRE1a can down-regulate Polo-like kinase 1(PLK1) from mRNA and protein two levels. IRE1a-mediated induction of apoptosis and inhibition of proliferation in response to ER stress is through downregulation PLK1, an early trigger for G2/M transition known to be participated in regulating cell proliferation and cell apoptosis. Collectively, these findings reveal a novel critical role of IRE1a in ER stress-mediated apoptosis and the molecular mechanisms involved. IRE1a may be a useful molecular target for the development of novel predictive and therapeutic strategies in cancer.     

Endogenous BiP reporter system for simultaneous identification of ER stress and antibody production in Chinese hamster ovary cells

As the biopharmaceutical industry expands, improving the production of therapeutic proteins using Chinese hamster ovary (CHO) cells is important. However, excessive and complicated protein production causes protein misfolding and triggers endoplasmic reticulum (ER) stress. When ER stress occurs, cells mediate the unfolded protein response (UPR) pathway to restore protein homeostasis and folding capacity of the ER. However, when the cells fail to control prolonged ER stress, UPR induces apoptosis. Therefore, monitoring the degree of UPR is required to achieve high productivity and the desired quality. In this study, we developed a fluorescence-based UPR monitoring system for CHO cells. We integrated mGFP into endogenous HSPA5 encoding BiP, a major ER chaperone and the primary ER stress activation sensor, using CRISPR/Cas9-mediated targeted integration. The mGFP expression level changed according to the ER stress induced by chemical treatment and batch culture in the engineered cell line. Using this monitoring system, we demonstrated that host cells and recombinant CHO cell lines with different mean fluorescence intensities (MFI; basal expression levels of BiP) possess a distinct capacity for stress culture conditions induced by recombinant protein production. Antibody-producing recombinant CHO cell lines were generated using site-specific integration based on host cells equipped with the BiP reporter system. Targeted integrants showed a strong correlation between productivity and MFI, reflecting the potential of this monitoring system as a screening readout for high producers. Taken together, these data demonstrate the utility of the endogenous BiP reporter system for the detection of real-time dynamic changes in endogenous UPR and its potential for applications in recombinant protein production during CHO cell line development.     

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.     

Moderate endoplasmic reticulum stress activates a PERK and p38-dependent apoptosis

The endoplasmic reticulum (ER) has the ability to signal organelle dysfunction via a complex signaling network known as the unfolded protein response (UPR). In this work, hamster fibroblast cells exhibiting moderate levels of ER stress were compared to those exhibiting severe ER stress. Inhibition of N-linked glycosylation was accomplished via a temperature-sensitive mutation in the Dad1 subunit of the oligosaccharyltransferase (OST) complex or by direct inhibition with tunicamycin (Tm). Temperature shift (TS) treatment generated weak activation of ER stress signaling when compared to doses of Tm that are typically used in ER stress studies (500–1000 nM). A dose-response analysis of key ER stress signaling mediators, inositol-requiring enzyme 1 (IRE1) and protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), revealed 20–40 nM of Tm to generate activation intensity similar to TS treatment. In parental BHK21 cells, moderate (20–40 nM) and high doses (200–1000 nM) of Tm were compared to identify physiological and signaling-based differences in stress response. Inhibition of ER Ca²⁺ release via ITPR activity with 2-aminoethoxydiphenyl borate (2-APB) or Xestospongin C (XeC) was sufficient to protect against apoptosis induced by moderate but not higher doses of Tm. Analysis of kinase activation over a range of Tm exposures revealed the p38 stress-activated protein kinase (SAPK) to display increasing activation with Tm dosage. Interestingly, Tm induced the extracellular regulated kinases (Erk1/2) only at moderate doses of Tm. Inhibition of ER transmembrane stress sensors (IRE1, PERK) or cytosolic signaling mediators (p38, Jnk1, Erk1/2) was used to evaluate pathways involved in apoptosis activation during ER stress. Inhibition of either PERK or p38 was sufficient to reduce cell death and apoptosis induced by moderate, but not high, doses of Tm. During ER stress, cells exhibited a rapid decline in anti-apoptotic Mcl-1 and survivin proteins. Inhibition of PERK was sufficient to block this affect. This work reveals moderate doses of ER stress to generate patterns of stress signaling that are distinct from higher doses and that apoptosis activation at moderate levels of stress are dependent upon PERK and p38 signaling. Studies exploring ER stress signaling should recognize that this signaling acts as a rheostat rather than a simple switch, behaving distinctively in a dose-dependent manner.