Physiological Unfolded Protein Response Research Articles

CELSR2 deficiency suppresses lipid accumulation in hepatocyte by impairing the UPR and elevating ROS level.

Cadherin EGF LAG seven-pass G-type receptor 2 (CELSR2), a mammalian orthologue of drosophila flamingo, belongs to the cadherin subfamily. CELSR2 mainly function in neural development and cilium polarity. Recent studies showed that the CELSR2 gene is related to many human diseases, including coronary artery disease, idiopathic scoliosis, and cancer. Genome-Wide Association Studies data showed that SNP in the CELSR2-PSRC1-SORT1 gene loci has a strong association with circulating lipid levels and coronary artery disease. However, the function and underlying mechanism of CELSR2 in hepatic lipid metabolism remain unknown. Here, we found that CELSR2 expression is decreased in the liver of NAFLD/NASH patients and db/db mice. Depletion of CELSR2 significantly decreased the lipid accumulation in hepatocytes by suppressing the expression of lipid synthesis enzymes. Moreover, CELSR2 deficiency impaired the physiological unfolded protein response (UPR), which damages the ER homeostasis, and elevates the reactive oxygen species (ROS) level by decreasing the antioxidant expression. Scavenging of ROS by N-acetylcysteine treatment could restore the decreased lipid accumulation of CELSR2 knockdown cells. Furthermore, CELSR2 loss impaired cell survival by suppressing cell proliferation and promoting apoptosis. Our results uncovered a new role of CELSR2 in regulating lipid homeostasis and UPR, suggesting CELSR2 may be a new therapeutic target for non-alcoholic fatty liver disease.

Planarian stem cells specify fate yet retain potency during the cell cycle.

Planarian whole-body regeneration is enabled by stem cells called neoblasts. At least some neoblasts are individually pluripotent. Neoblasts are also heterogeneous, with subpopulations of specialized neoblasts having different specified fates. Fate specification in neoblasts is regulated by fate-specific transcription factor (FSTF) expression. Here, we find that FSTF expression is common in neoblast S/G2/M cell-cycle phases but less common in G1. We find that specialized neoblasts can divide to produce progeny with asymmetric cell fates, suggesting that they could retain pluripotency. Furthermore, no known neoblast class was present in all neoblast colonies, suggesting that pluripotency is not the exclusive property of any known class. We tested this possibility with single-cell transplantations, which indicate that at least some specialized neoblasts are likely clonogenic. On the basis of these findings, we propose a model for neoblast pluripotency in which neoblasts can undergo specialization during the cell cycle without loss of potency.

PERK-mediated translational control is required for collagen secretion in chondrocytes

As chondrocytes are highly secretory and they experience a variety of stresses, physiological unfolded protein response (UPR) signalling is essential for extracellular matrix (ECM) secretion and chondrogenesis. In the three branches of the UPR pathway, PERK governs the translational attenuation and transcriptional upregulation of amino acid and redox metabolism and induction of apoptosis. It was previously demonstrated that a defect of the PERK branch of the UPR signalling pathway causes the accumulation of unfolded proteins, leading to cell death without perturbing endoplasmic reticulum (ER)-to-Golgi transport in pancreatic β cells. However, little is known about the role of PERK in chondrocytes. In this study, we found that PERK signalling is activated in chondrocytes, and inhibition of PERK reduces collagen secretion despite causing excessive collagen synthesis in the ER. Perk−/− mice displayed reduced collagen in articular cartilage but no differences in chondrocyte proliferation or apoptosis compared to the findings in wild-type mice. PERK inhibition increases misfolded protein levels in the ER, which largely hinder ER-to-Golgi transport. These results suggest that the translational control mediated by PERK is a critical determinant of ECM secretion in chondrocytes.

Therapeutics for Microcephaly - The Three “M”s

Event Abstract Back to Event Therapeutics for Microcephaly - The Three “M”s Ivan GLADWYN-NG1*, Ivan Gladwyn-Ng1*, Christian Alfano1, Giovanni Morelli2, Michelle America3, Celine D'Alessandro1 and Laurent Nguyen1 1 GIGA-Neurosciences, University of Liège, Belgium 2 Biomedical Research Institute, Hasselt University, Belgium 3 Department of Biology, KU Leuven, Belgium Neurodegenerative and neurodevelopmental disorders are increasingly prevalent diseases (1; 2; 3) with significant socio-economic costs (4; 5; 6). We present a conceptual work plan for investigating neurodevelopmental causes of microcephaly towards identifying potential drug candidates based on our recent publication (7) and EU patent application (8). Emerging epidemiological and clinical articles report the causal link between Zika virus (ZIKV) infection during gestation and congenital microcephaly. Presently, we highlight results from combinatorial analyses of ZIKV-infected human foetuses and mouse embryos to elucidate underlying cellular and molecular mechanisms pf ZIKV-induced MIC. Firstly, we demonstrate that ZIKV triggers endoplasmic reticulum (ER) stress and an unfolded protein response (UPR) in the cerebral cortex of infected post-mortem human foetuses. Secondly, we designed an in vivo mouse model - via intracerebral (ICV) and intraplacental (IPL) infection - that recapitulates the clinical phenotype. In combination, the ER stress in human foetuses and embryonic mouse brains perturbs a physiological UPR within cortical progenitors that controls neurogenesis. Thirdly, ZIKV-infected progenitors generate fewer projection neurons that eventually settle in the cerebral cortex, whereupon sustained ER stress subsequently leads to apoptosis. Lastly, we demonstrate that administration of pharmacological inhibitors of the UPR counteracts these pathophysiological mechanisms and abrogates ZIKV-induced MIC in murine embryos and pups. In conclusion, we demonstrate a conceptual work plan to elucidate pathophysiological mechanisms underlying genetic and acquired causes of MIC, as well as to investigate putative compounds that potentially ameliorate these neurodevelopmental disorders. Figure 1 Figure 2 Figure 3 Acknowledgements CORDON-BARRIS, Lluis; CREEPE, Catherine; COUDERC, Thérèse; THELEN, Nicolas; BESSIÈRES, Bettina; ENCHA-RAZAVI, Férechté; BONNIÈRE Maryse; SUZUKI, Ikuo K.; FLAMAND, Marie; VANDERHAEGHEN, Pierre; THIRY, Marc; LECUIT, Marc.

Stress-induced unfolded protein response contributes to Zika virus-associated microcephaly.

Accumulating evidence support a causal link between Zika virus (ZIKV) infection during gestation and congenital microcephaly. However, the mechanism of ZIKV-associated microcephaly remains unclear. We combined analyses of ZIKV-infected human fetuses, cultured human neural stem cells and mouse embryos to understand how ZIKV induces microcephaly. We show that ZIKV triggers endoplasmic reticulum stress and unfolded protein response in the cerebral cortex of infected postmortem human fetuses as well as in cultured human neural stem cells. After intracerebral and intraplacental inoculation of ZIKV in mouse embryos, we show that it triggers endoplasmic reticulum stress in embryonic brains in vivo. This perturbs a physiological unfolded protein response within cortical progenitors that controls neurogenesis. Thus, ZIKV-infected progenitors generate fewer projection neurons that eventually settle in the cerebral cortex, whereupon sustained endoplasmic reticulum stress leads to apoptosis. Furthermore, we demonstrate that administration of pharmacological inhibitors of unfolded protein response counteracts these pathophysiological mechanisms and prevents microcephaly in ZIKV-infected mouse embryos. Such defects are specific to ZIKV, as they are not observed upon intraplacental injection of other related flaviviruses in mice.

Retinoic Acid-induced differentiation sensitizes myeloid progenitors cells to ER stress

The clonal expansion of hematopoietic myeloid precursors blocked at different stages of differentiation characterizes the acute myeloid leukemia (AML) phenotype. A subtype of AML, acute promyelocytic leukemia (APL), characterized by the chimeric protein PML-RARα is considered a paradigm of differentiation therapy. In this leukemia subtype the all-trans-retinoic acid (RA)-based treatments are able to induce PML-RARα degradation and leukemic blast terminal differentiation [1-2]. Granulocytic differentiation of APL cells driven by RA triggers a physiological Unfolded Protein Response (UPR), a series of pathways emanating from the ER in case of ER stress, which ensues when higher protein folding activity is required as during differentiation. We show here that, although mild, the ER stress induced by RA is sufficient to render human APL cell lines and primary blasts very sensitive to low doses of Tunicamycin (Tm), an ER stress inducing drug, at doses that are not toxic in the absence of RA. Importantly only human progenitors cells derived from APL patients resulted sensitive to the combined treatment with RA and Tm whereas those obtained from healthy donors were not affected. We also show that the UPR pathway downstream of PERK plays a major protective role against ER stress in differentiating cells and, by using a specific PERK inhibitor, we potentiated the toxic effect of the combination of RA and Tm. In conclusion, our findings identify the ER stress-related pathways as potential targets in the search for novel therapeutic strategies in AML.

The unfolded protein response is required for dendrite morphogenesis.

Precise patterning of dendritic fields is essential for the formation and function of neuronal circuits. During development, dendrites acquire their morphology by exuberant branching. How neurons cope with the increased load of protein production required for this rapid growth is poorly understood. Here we show that the physiological unfolded protein response (UPR) is induced in the highly branched Caenorhabditis elegans sensory neuron PVD during dendrite morphogenesis. Perturbation of the IRE1 arm of the UPR pathway causes loss of dendritic branches, a phenotype that can be rescued by overexpression of the ER chaperone HSP-4 (a homolog of mammalian BiP/grp78). Surprisingly, a single transmembrane leucine-rich repeat protein, DMA-1, plays a major role in the induction of the UPR and the dendritic phenotype in the UPR mutants. These findings reveal a significant role for the physiological UPR in the maintenance of ER homeostasis during morphogenesis of large dendritic arbors.

IRE1 phosphatase PP2Ce regulates adaptive ER stress response in the postpartum mammary gland.

We recently reported that the PPM1l gene encodes an endoplasmic reticulum (ER) membrane targeted protein phosphatase (named PP2Ce) with highly specific activity towards Inositol-requiring protein-1 (IRE1) and regulates the functional outcome of ER stress. In the present report, we found that the PP2Ce protein is highly expressed in lactating epithelium of the mammary gland. Loss of PP2Ce in vivo impairs physiological unfolded protein response (UPR) and induces stress kinase activation, resulting in loss of milk production and induction of epithelial apoptosis in the lactating mammary gland. This study provides the first in vivo evidence that PP2Ce is an essential regulator of normal lactation, possibly involving IRE1 signaling and ER stress regulation in mammary epithelium.

Bcl-xL Protein Protects from C/EBP Homologous Protein (CHOP)-dependent Apoptosis during Plasma Cell Differentiation

Although it is known that the unfolded protein response (UPR) plays a significant role in the process of plasma cell differentiation, the contribution of the individual sensors of the UPR to this process remains unclear. In this study we examine the death signals and compensatory survival signals activated during B cell activation and the first stages of plasma cell differentiation. During in vitro differentiation of both primary murine B cells and the Bcl1 cell line, we demonstrate that in addition to activation of the physiological UPR, changes in the expression of several Bcl-2 proteins occur, which are consistent with a lowering of the apoptotic threshold of the cell. Specifically, we observed decreased expression of Bcl-2 and Mcl-1 and increased expression of the proapoptotic protein Bim. However, these changes were countered by Bcl-xL induction, which is necessary to protect differentiating cells both from ER stress-induced death by tunicamycin and from the death signals inherent in differentiation. Consistent with differentiating cells becoming dependent on Bcl-xL for survival, the addition of ABT-737 resulted in apoptosis in differentiating cells through the inhibition of sequestration of Bim. Confirming this result, differentiation in the context of RNAi-mediated Bcl-xL knockdown also induced apoptosis. This cell death is C/EBP homologous protein (CHOP)-dependent, connecting these events to the UPR. Thus plasma cell differentiation proceeds through a Bcl-xL-dependent intermediate.

Endoplasmic Reticulum Stress in Cardiometabolic Disorders

When endoplasmic reticulum (ER) homeostasis is disrupted, an adaptive signaling pathway, called the unfolded protein response (UPR) is activated to help ER cope with the stress. The UPR is an important signal transduction pathway, crucial for the survival and function of all cells. Recently, there has been a substantial progress made in understanding the molecular mechanisms of physiological UPR regulation and its role in the pathogenesis of many diseases including metabolic diseases. Studies using mouse models lacking or overexpressing the factors involved in ER stress signaling as well as work performed on humans have revealed the contribution of UPR to disease progression. This review focuses on the regulation of UPR signaling and its relevance in pathogenesis of metabolic diseases.

Crosstalk between Advanced Glycation and Endoplasmic Reticulum Stress: Emerging Therapeutic Targeting for Metabolic Diseases

Advanced glycation, the major posttranslational modification of proteins, DNA, and lipids, is accelerated under conditions of increased oxidative stress, hyperglycemia, and hypoxia contributing to a variety of metabolic diseases such as diabetes mellitus, obesity, inflammation, polycystic ovarian syndrome, ischemic cardiovascular disease, and neurodegenerative disorders. The potential role of advanced glycation in endoplasmic reticulum (ER) homeostasis is largely unknown. Basic and clinical peer-reviewed articles on advanced glycation and ER stress related to metabolic regulation were searched in PubMed from 2000-2011. The resulting articles as well as relevant cited references were reviewed. Recent evidence indicates that hyperglycemia, hypoxia, and oxidative stress, apart of triggering advanced glycation, can also adversely affect ER function, leading to pathogenic ER stress, followed by the unfolded protein response. The concomitant presence of advanced glycation in the same conditions with ER stress suggests their crosstalk in the progression of diseases associated with hypoxic and oxidative stress. Current data support the direct or indirect induction of ER stress response by advanced glycation end products or advanced glycation end product precursors in the pathogenesis of metabolic diseases. Inhibitors of advanced glycation acting as potent ER stress modulators with beneficial effects in restoring ER homeostasis and adjusting physiological unfolded protein response level present an emerging therapeutic approach with significant applications, especially in the context of metabolic dysfunction.

Physiological unfolded protein response regulated by OASIS family members, transmembrane bZIP transcription factors

The endoplasmic reticulum (ER) plays role in the maintenance of numerous aspects of cellular and organismal homeostasis by folding, modifying, and exporting nascent secretory and transmembrane proteins. Failure of the ER's adaptive capacity results in accumulation of unfolded or malfolded proteins in the ER lumen (ER stress). To avoid cellular damage, mammalian cells activate the specific signals from the ER to the cytosol or nucleus to enhance the capacity for protein folding, attenuate the synthesis of proteins, and degrade unfolded proteins. These signaling pathways are collectively known as the unfolded protein response (UPR). The canonical branches of the UPR are mediated by three ER membrane-bound proteins, PERK, IRE1, and ATF6. These ER stress transducers basically play important roles in cell survival after ER stress. Recently, novel types of ER stress transducers, OASIS family members that share a region of high sequence similarity with ATF6 have been identified. They have a transmembrane domain, which allows them to associate with the ER, and possess a transcription-activation domain and a bZIP domain. OASIS family proteins include OASIS, BBF2H7, CREBH, AIbZIP, and Luman. Despite the structural similarities among OASIS family proteins and ATF6, differences in activating stimuli, tissue distribution, and response element binding indicate specialized functions of each member on regulating the UPR in the specific organs and tissues. Here, we summarize our current understanding of biochemical characteristics and in vivo functions of OASIS family proteins, particularly focusing on OASIS and BBF2H7. A growing body of new works suggests that the UPR branches regulated by OASIS family members play essential roles in cell differentiation and maturation or maintenance of basal cellular homeostasis in mammals.

A Phos-Tag-Based Approach Reveals the Extent of Physiological Endoplasmic Reticulum Stress

Cellular response to endoplasmic reticulum (ER) stress or unfolded protein response (UPR) is a key defense mechanism associated with many human diseases. Despite its basic and clinical importance, the extent of ER stress inflicted by physiological and pathophysiological conditions remains difficult to quantitate, posing a huge obstacle that has hindered our further understanding of physiological UPR and its future therapeutic potential. Here we have optimized a Phos-tag-based system to detect the activation status of two proximal UPR sensors at the ER membrane. This method allowed for a quantitative assessment of the level of stress in the ER. Our data revealed quantitatively the extent of tissue-specific basal ER stress as well as ER stress caused by the accumulation of misfolded proteins and the fasting-refeeding cycle. Our study may pave the foundation for future studies on physiological UPR, aid in the diagnosis of ER-associated diseases and improve and facilitate therapeutic strategies targeting UPR in vivo.

The Role for Endoplasmic Reticulum Stress in Diabetes Mellitus

Accumulating evidence suggests that endoplasmic reticulum (ER) stress plays a role in the pathogenesis of diabetes, contributing to pancreatic beta-cell loss and insulin resistance. Components of the unfolded protein response (UPR) play a dual role in beta-cells, acting as beneficial regulators under physiological conditions or as triggers of beta-cell dysfunction and apoptosis under situations of chronic stress. Novel findings suggest that "what makes a beta-cell a beta-cell", i.e., its enormous capacity to synthesize and secrete insulin, is also its Achilles heel, rendering it vulnerable to chronic high glucose and fatty acid exposure, agents that contribute to beta-cell failure in type 2 diabetes. In this review, we address the transition from physiology to pathology, namely how and why the physiological UPR evolves to a proapoptotic ER stress response and which defenses are triggered by beta-cells against these challenges. ER stress may also link obesity and insulin resistance in type 2 diabetes. High fat feeding and obesity induce ER stress in liver, which suppresses insulin signaling via c-Jun N-terminal kinase activation. In vitro data suggest that ER stress may also contribute to cytokine-induced beta-cell death. Thus, the cytokines IL-1beta and interferon-gamma, putative mediators of beta-cell loss in type 1 diabetes, induce severe ER stress through, respectively, NO-mediated depletion of ER calcium and inhibition of ER chaperones, thus hampering beta-cell defenses and amplifying the proapoptotic pathways. A better understanding of the pathways regulating ER stress in beta-cells may be instrumental for the design of novel therapies to prevent beta-cell loss in diabetes.

Targeting UPR Sensors: A Novel Approach To Promote Death of Multiple Myeloma Cells.

Multiple Myeloma (MM) is a plasma cell (PC) malignancy producing large amounts of monoclonal Igs requiring a highly developed endoplasmic reticulum (ER). A coordinated adaptative program called the unfolded protein response (UPR) allows cells to survive the stress consecutive to a transient protein overload of the ER. This process is referred to as "physiological UPR". If the stress is excessive or sustained, the UPR leads to cell death ("terminal UPR"). We have recently documented that the spontaneous death of non transformed human PC is initiated by an ER stress. Moreover the group of R. Sitia has established that the proteasomal activity decreases in the late phases of PC differentiation. These findings suggest that the imbalance between the high-rate of Igs secretion and the impaired proteolytic machinery predisposes PC to apoptosis. We have postulated that a dysregulated adaptative UPR contributes to the increased survival capacity of MM cells. We have undertaken a study aimed at promoting death of MM cells by disruption of ER homeostasis. Our approach has consisted in using RNA interference to knock-down the UPR sensors PERK, ATF6 and IRE1, in two MM cell lines (U 266 and NCI-H929). We show that the anti-UPR siRNAs induce a 70 to 80% reduction of the levels of expression of PERK, ATF6 and IRE1 and promote cell death of these two human MM cell lines. The PERK-targeted siRNA was consistently found to be the best apoptosis inducer. We found that invalidation of PERK leads to induction of a terminal UPR as revealed by the upregulation of GADD153/CHOP and of the spliced form of XBP-1. The death elicited by invalidation of the UPR sensors was accompanied by phosphatidylserine (PS) exposure and early loss of the mitochondrial transmembrane potential. By contrast, we found no evidence of DNA fragmentation and electronic microscopy revealed only a partial nuclear chromatin condensation. Cell death induced by UPR-targeted siRNAs operated in a caspase -independent fashion since it was not inhibited by the pan-caspase inhibitor z-VAD-fmk. Accordingly, the activated forms of caspase-3 and-4 were undetectable. However, calpain inhibitors and calcium chelators prevented the loss of the mitochondrial transmembrane potential and partially inhibited cell death. This suggests that the death signal initiated at the level of the ER is transmitted to the mitochondria via the calcium cysteine protease calpain. Transmission electron microscopy revealed that the PERK siRNA induces formation of numerous autophagic vacuoles, a result which was confirmed by the positive staining with monodansylcadaverine (MDC). Appearance of mature autophagic vacuoles preceeded acquisition of death morphology which was characterized by partial nuclear condensation and protein accumulation. The anti-UPR siRNAs induced the presence of immature and mature autophagic vacuoles but, in contrast, when acquiring death morphology, cells were devoid of autophagic vacuoles although they showed partial nuclear condensation. The autophagic specific inhibitor 3 methyladenine reduced both PS exposure and MDC staining induced by PERK siRNA. This suggests that UPR-targeted siRNAs induce death of MM cells by autophagy. Altogether, our data demonstrate that disruption of ER homeostasis by extinction of UPR sensors induce death of MM cells. They also reveal that a connection exists between ER stress and autophagy in MM cells.

The physiological unfolded protein response in the thyroid epithelial cells

The perturbation of the endoplasmic reticulum (ER) homeostasis by the pharmacological agents or mutant secretory proteins invokes the adaptive cellular reaction, unfolded protein response (UPR). Here we show the activation of UPR under the physiological conditions of the hormone-stimulated expression of thyroglobulin (Tg), the major secretory product of thyroid cells. The early increase in the Tg synthesis within 1h after stimulation apparently triggers UPR as it is evidenced by the activation of ATF6- and PERK-dependent pathways of UPR and subsequent expression of the UPR target genes, ER molecular chaperones. However, little or no activation of the other UPR sensor, IRE1, was observed, as judged by the absence of the spliced mRNA of its downstream effector, transcription factor XBP1. We conclude that the physiological UPR in differentiating thyrocytes differs substantially from the drug-induced stress by its weaker manifestation and distinct pattern of the activation of UPR sensors.