Adaptive Endoplasmic Reticulum Stress Response Research Articles

Endoplasmic reticulum oxidoreductin 1-alpha deficiency and activation of protein translation synergistically impair breast tumour resilience.

Endoplasmic reticulum (ER) stress triggers an adaptive response in tumours which fosters cell survival and resilience to stress. Activation of the ER stress response, through its PERK branch, promotes phosphorylation of the α-subunit of the translation initiation factor eIF2, thereby repressing general protein translation and augmenting the translation of ATF4 with the downstream CHOP transcription factor and the protein disulfide oxidase, ERO1-alpha EXPERIMENTAL APPROACH: Here, we show that ISRIB, a small molecule that inhibits the action of phosphorylated eIF2alpha, activating protein translation, synergistically interacts with the genetic deficiency of protein disulfide oxidase ERO1-alpha, enfeebling breast tumour growth and spread. ISRIB represses the CHOP signal, but does not inhibit ERO1. Mechanistically, ISRIB increases the ER protein load with a marked perturbing effect on ERO1-deficient triple-negative breast cancer cells, which display impaired proteostasis and have adapted to a low client protein load in hypoxia, and ERO1 deficiency impairs VEGF-dependent angiogenesis. ERO1-deficient triple-negative breast cancer xenografts have an augmented ER stress response and its PERK branch. ISRIB acts synergistically with ERO1 deficiency, inhibiting the growth of triple-negative breast cancer xenografts by impairing proliferation and angiogenesis. These results demonstrate that ISRIB together with ERO1 deficiency synergistically shatter the PERK-dependent adaptive ER stress response, by restarting protein synthesis in the setting of impaired proteostasis, finally promoting tumour cytotoxicity. Our findings suggest two surprising features in breast tumours: ERO1 is not regulated via CHOP under hypoxic conditions, and ISRIB offers a therapeutic option to efficiently inhibit tumour progression in conditions of impaired proteostasis.

XAF1 drives apoptotic switch of endoplasmic reticulum stress response through destabilization of GRP78 and CHIP

X-linked inhibitor of apoptosis-associated factor-1 (XAF1) is a stress-inducible tumor suppressor that is commonly inactivated in many human cancers. Despite accumulating evidence for the pro-apoptotic role for XAF1 under various stressful conditions, its involvement in endoplasmic reticulum (ER) stress response remains undefined. Here, we report that XAF1 increases cell sensitivity to ER stress and acts as a molecular switch in unfolded protein response (UPR)-mediated cell-fate decisions favoring apoptosis over adaptive autophagy. Mechanistically, XAF1 interacts with and destabilizes ER stress sensor GRP78 through the assembly of zinc finger protein 313 (ZNF313)-mediated destruction complex. Moreover, XAF1 expression is activated through PERK-Nrf2 signaling and destabilizes C-terminus of Hsc70-interacting protein (CHIP) ubiquitin E3 ligase, thereby blocking CHIP-mediated K63-linked ubiquitination and subsequent phosphorylation of inositol-required enzyme-1α (IRE1α) that is involved in in the adaptive ER stress response. In tumor xenograft assays, XAF1−/− tumors display substantially lower regression compared to XAF1+/+ tumors in response to cytotoxic dose of ER stress inducer. XAF1 and GRP78 expression show an inverse correlation in human cancer cell lines and primary breast carcinomas. Collectively this study uncovers an important role for XAF1 as a linchpin to govern the sensitivity to ER stress and the outcomes of UPR signaling, illuminating the mechanistic consequence of XAF1 inactivation in tumorigenesis.

Age-related neuronal damage by advanced glycation end products through altered proteostasis

Aging is a main risk factor for many diseases including neurodegenerative disorders. Numerous theories and mechanisms including accumulation of advanced glycation end products (AGEs) have been put forward in explaining brain aging. However, a focused study on the status of AGEs in the brain during progressive aging in connection with interrelated cellular processes like ubiquitin-proteasome system (UPS), unfolded protein response, autophagy-lysosome system and apoptosis is lacking. In this study, we investigated the levels of AGEs in the brain of 5-, 10-, 15- and 20-months old WNIN rats. Endoplasmic reticulum (ER) stress response, UPS components, autophagy flux, neurotrophic and presynaptic markers along with cell death markers were analyzed by immunoblotting. The neuronal architecture was analyzed by H&E and Nissl staining. The results demonstrated progressive accumulation of AGEs in the brain during aging. Adaptive ER stress response was observed by 10-months while maladaptive ER stress response was seen at 15- and 20-months of age along with impaired UPS and autophagy, and perturbations in neuronal growth factors. All these disturbances intensify with age to further exaggerate cell death mechanisms. There was a shrinkage of the cell size with aging and Congo-red staining revealed β-amyloid accumulation in higher ages. Together these results suggest that progressive accumulation of AGEs with aging in the brain may lead to neuronal damage by affecting ER homeostasis, UPS, autophagic flux, and neuronal growth factors.

Adaptive endoplasmic reticulum stress signalling via IRE1α-XBP1 preserves self-renewal of haematopoietic and pre-leukaemic stem cells.

Over their lifetime, long-term haematopoietic stem cells (HSC) are exposed to a variety of stress conditions that they must endure. Many stresses, such as infection/inflammation, reactive oxygen species, nutritional deprivation and hypoxia, activate unfolded protein response signalling, which induces either adaptive changes to resolve the stress or apoptosis to clear the damaged cell. Whether unfolded-protein-response signalling plays any role in HSC regulation remains to be established. Here, we report that the adaptive signalling of the unfolded protein response, IRE1α-XBP1, protects HSCs from endoplasmic reticulum stress-induced apoptosis. IRE1α knockout leads to reduced reconstitution of HSCs. Furthermore, we show that oncogenic N-RasG12D activates IRE1α-XBP1, through MEK-GSK3β, to promote HSC survival under endoplasmic reticulum stress. Inhibiting IRE1α-XBP1 abolished N-RasG12D-mediated survival under endoplasmic reticulum stress and diminished the competitive advantage of NrasG12D HSCs in transplant recipients. Our studies illuminate how the adaptive endoplasmic reticulum stress response is advantageous in sustaining self-renewal of HSCs and promoting pre-leukaemic clonal dominance.

Defective Flux of Thrombospondin-4 through the Secretory Pathway Impairs Cardiomyocyte Membrane Stability and Causes Cardiomyopathy.

Thrombospondins are stress-inducible secreted glycoproteins with critical functions in tissue injury and healing. Thrombospondin-4 (Thbs4) is protective in cardiac and skeletal muscle, where it activates an adaptive endoplasmic reticulum (ER) stress response, induces expansion of the ER, and enhances sarcolemmal stability. However, it is unclear if Thbs4 has these protective functions from within the cell, from the extracellular matrix, or from the secretion process itself. In this study, we generated transgenic mice with cardiac cell-specific overexpression of a secretion-defective mutant of Thbs4 to evaluate its exclusive intracellular and secretion-dependent functions. Like wild-type Thbs4, the secretion-defective mutant upregulates the adaptive ER stress response and expands the ER and intracellular vesicles in cardiomyocytes. However, only the secretion-defective Thbs4 mutant produces cardiomyopathy with sarcolemmal weakness and rupture that is associated with reduced adhesion-forming glycoproteins in the membrane. Similarly, deletion of Thbs4 in the mdx mouse model of Duchenne muscular dystrophy enhances cardiomyocyte membrane instability and cardiomyopathy. Finally, overexpression of the secretion-defective Thbs4 mutant in Drosophila, but not wild-type Thbs4, impaired muscle function and sarcomere alignment. These results suggest that transit through the secretory pathway is required for Thbs4 to augment sarcolemmal stability, while ER stress induction and vesicular expansion mediated by Thbs4 are exclusively intracellular processes.

Increased mitochondrial respiration promotes survival from endoplasmic reticulum stress.

Protein misfolding in the endoplasmic reticulum (ER) is accompanied by adaptive cellular responses to promote cell survival. We now show that activation of mitochondrial respiration is a critical component of an adaptive ER stress response, requiring the unfolded protein response (UPR) sensor Ire1, and also calcium signaling via calcineurin. In yeast and mammalian cells lacking Ire1 or calcineurin, respiratory activation is impaired in response to ER stress; accumulation of mitochondrial reactive oxygen species (ROS) triggers cell death as abrogation of ROS by antioxidants or loss of the electron transport chain (in yeast) can rescue cells from death. Significantly, cells are rescued from ER stress-induced death by mitochondrial uncoupling by CCCP to increase O2 consumption (and increase the efficiency of electron transfer). Remarkably, genetic and pharmacologic strategies to promote mitochondrial biogenesis and increase O2 consumption also alleviate ER stress-mediated ROS and death in yeast and mammalian cells. Moreover, in a yeast genetic screen, three mitochondrial proteins Mrx9, Mrm1, and Aim19 that increase mitochondrial biogenesis were identified as high copy suppressors of ER stress-mediated cell death. Our results show that enhanced mitochondrial biogenesis, linked to improved efficiency of the electron transport chain, is a powerful strategy to block ROS accumulation and promote cell survival during ER stress in eukaryotic cells.

Abstract 257: Endogenous Activating Transcription Factor 6 Preserves Heart Structure and Function in a Mouse Model of Myocardial Infarction-induced Heart Failure

Rationale: The ER stress response is activated by the accumulation of misfolded, toxic proteins in the endoplasmic reticulum (ER), and upregulates proteins that restore ER protein-folding capacity. The ER-transmembrane protein, activating transcription factor 6 (ATF6) senses ER stress and responds by transcriptionally inducing many of these genes and is thus a key component of the adaptive ER stress response. We previously showed that in the heart, ischemia activates ATF6. Furthermore, transgenic mouse hearts expressing a conditionally activated form of ATF6, and subjected to ex vivo ischemia/reperfusion, exhibited preserved heart function and smaller infarcts. Our lab also showed that by serving as a novel inducer of a global anti-oxidant gene program, endogenous ATF6 limits cardiac damage caused by reactive oxygen species during reperfusion. However, the effect of endogenous ATF6 in the failing heart is not known. Given that acute ischemia caused by occlusion of the coronary arteries is the cause of myocardial infarction (MI), we hypothesized that endogenous ATF6 limits infarct size and preserves heart function during MI. Additionally, since deleterious cardiac remodeling and heart failure can be long-term consequences of MI, we hypothesized that ATF6 can mitigate these effects. Objective/Methods: To examine the role of endogenous ATF6 in heart failure, in vivo, we used a mouse model of MI-induced heart failure in mice with a global deletion of the ATF6 gene (ATF6 KO). Infarct size was measured by TTC staining and heart function was observed via longitudinal echocardiogram. Results: We found that following infarction, ATF6 KO mouse hearts had larger infarcts compared to control. Thus, ischemic cardiac tissue in the peri-infarct region requires ATF6 to limit cardiac myocyte death. Interestingly, ejection fraction following MI decreased more over 13 weeks in ATF6 KO mice relative to control. While control and ATF6 KO mouse hearts hypertrophied to a similar degree, KO mice showed greater cardiac dilation. Conclusions: Together these findings show for the first time that endogenous ATF6 acts to preserve heart structure and function in an MI model of heart failure, suggesting that ATF6 may be a viable therapeutic target for treatment of this disease.

Abstract 467: ATF6B and ATF6A Play Complimentary Roles in Mediating Adaptive and Maladaptive Signaling in Cardiac Myocytes

Rationale: ATF6α and ATF6β are endoplasmic reticulum (ER) transmembrane proteins that sense the accumulation of toxic misfolded proteins in the ER of cardiomyocytes, which can be brought about by ER stresses as ischemia. Upon ER stress, ATF6α is proteolytically cleaved into a transcription factor that binds to ER stress response elements (ERSEs) and increases expression of cardioprotective genes that restore ER protein folding. If ER proteostasis is not restored, maladaptive signaling is initiated. ATF6β is also proteolytically cleaved during ER stress, binds to the same ERSEs as ATF6α, but does not induce transcription. Hence it is clear from the above studies done in cancer cells that there are some marked similarities and differences between ATF6α and ATF6β. However, the relative roles of ATF6α and ATF6β have not been studied in the heart, where they might work in concert to mediate the dynamic switch from adaptive to maladaptive gene programing during myocardial pathology. Methods: We used neonatal rat ventricular myocytes (NRVMs) to explore the effects of ATF6α or ATF6β loss-of-function in cells treated with the ER stressor, thapsigargin (TG), which mimics ischemic heart disease. Results: In NRVM treated with TG, knockdown of ATF6β resulted in much more pronounced cell death in isolated myocytes than knockdown of ATF6α. Consistent with this finding, transcriptome analyses showed that compared to knocking down ATF6α, knockdown of ATF6β upregulated much more maladaptive, cell death-inducing genes and downregulated more cardioprotective genes. Surprisingly, knockdown of either ATF6α or ATF6β downregulated some common adaptive ER stress response genes, such as GRP78 and Derlin while also upregulating common maladaptive ER stress response genes, such as CHOP, Bcl2, Bax. Conclusion: These data indicate that both ATF6α and ATF6 β are needed for optimal viability of NRVM subjected to ER stress. There is a common, as well as differential gene regulation program controlled by these two isoforms of ATF6. Importantly, this study demonstrates a novel mechanism by which these two isoforms of ATF6 interact to govern the progression from adaptive to maladaptive ER stress signaling during chronic misfolding of ER proteins that occurs in ischemic heart disease.

A microRNA-mediated decrease in eukaryotic initiation factor 2α promotes cell survival during PS-341 treatment.

MicroRNAs (miRs) play pivotal roles in carcinogenesis and endoplasmic reticulum (ER) that performs the folding, modification and trafficking of proteins targeted to the secretory pathway. Cancer cells often endure ER stress during tumor progression but use the adaptive ER stress response to gain survival advantage. Here we report: (i) A group of miRs, including miR-30b-5p and miR-30c-5p, are upregulated by proteasome inhibitor PS-341 treatment, in HepG2 and MDA-MB-453 cells. (ii) Two representative PS-341-induced miRs: miR-30b-5p and miR-30c-5p are found to promote cell proliferation and anti-apoptosis in both tumor cells. (iii) eIF2α is confirmed as the congenerous target of miR-30b-5p and miR-30c-5p, essential to the anti-apoptotic function of these miRs. (iv) Upregulation of miR-30b-5p or miR-30c-5p, which occurs latter than the increase of phosphorylated eIF2α (p-eIF2α) in the cell under ER stress, suppresses the p-eIF2α/ATF4/CHOP pro-apoptotic pathway. (v) Inhibition of the miR-30b-5p or miR-30c-5p sensitizes the cancer cells to the cytotoxicity of proteasome inhibition. In conclusion, we unravels a new miRs-based mechanism that helps maintain intracellular proteostasis and promote cell survival during ER stress through upregulation of miR-30b-5p and miR-30c-5p which target eIF2α and thereby inhibit the p-eIF2α/ATF4/CHOP pro-apoptotic pathway, identifying miR-30b-5p and miR-30c-5p as potentially new targets for anti-cancer therapies.

Dissection of Thrombospondin-4 Domains Involved in Intracellular Adaptive Endoplasmic Reticulum Stress-Responsive Signaling.

Thrombospondins are a family of stress-inducible secreted glycoproteins that underlie tissue remodeling. We recently reported that thrombospondin-4 (Thbs4) has a critical intracellular function, regulating the adaptive endoplasmic reticulum (ER) stress pathway through activating transcription factor 6α (Atf6α). In the present study, we dissected the domains of Thbs4 that mediate interactions with ER proteins, such as BiP (Grp78) and Atf6α, and the domains mediating activation of the ER stress response. Functionally, Thbs4 localized to the ER and post-ER vesicles and was actively secreted from cardiomyocytes, as were the type III repeat (T3R) and TSP-C domains, while the LamG domain localized to the Golgi apparatus. We also mutated the major calcium-binding motifs within the T3R domain of full-length Thbs4, causing ER retention and secretion blockade. The T3R and TSP-C domains as well as wild-type Thbs4 and the calcium-binding mutant interacted with Atf6α, induced an adaptive ER stress response, and caused expansion of intracellular vesicles. In contrast, overexpression of a related secreted oligomeric glycoprotein, Nell2, which lacks only the T3R and TSP-C domains, did not cause these effects. Finally, deletion of Atf6α abrogated Thbs4-induced vesicular expansion. Taken together, these data identify the critical intracellular functional domains of Thbs4, which was formerly thought to have only extracellular functions.

Abstract 412: Dissection of Thrombospondin-4 Domains Involved in Adaptive ER Stress Responsive Signaling and Secretory Pathway Functions

Thrombospondins are a family of stress-inducible secreted glycoproteins with well-characterized roles in the extracellular matrix and tissue remodeling. We recently reported critical intracellular functions for thrombospondin-4 (Thbs4) by activation of an adaptive endoplasmic reticulum (ER) stress pathway in cardiomyocytes, in part by promoting activation of activating transcription factor 6α (Atf6α). Here, we dissect the domains of Thbs4 that mediate interactions with ER proteins and activation of this adaptive ER stress response, and determine the domains of Thbs4 involved in secretory pathway functions. We show that the N-terminal laminin G like domain (LamG) of Thbs4 exhibits intracellular localization almost exclusively at the Golgi apparatus and is robustly secreted in cultured cardiomyocytes, indicating rapid flux through the secretory pathway. We also generated a full length Thbs4 with mutations in calcium-binding motifs within the type III repeat (T3R) domain, including mutations homologous to human disease-causing mutations identified in THBS5/COMP. While wildtype Thbs4 localized to the ER and post-ER vesicles and was actively secreted in cardiomyocytes, the Thbs4 calcium-binding mutant localized predominately to the ER and was not secreted. We identified BiP (Grp78) as a novel binding partner of Thbs4 in cardiomyocytes in vitro and in vivo, and utilized adenoviruses overexpressing Thbs4 domain mutants in cardiomyocytes to map the TSP-C domain (L-type lectin domain) of Thbs4 as the BiP-interacting motif. Additionally, the TSP-C domain of Thbs4 bound Atf6α and overexpression of the TSP-C domain alone was sufficient to induce the adaptive ER stress pathway in cardiomyocytes, activate Atf6α, and protect against ER stress-induced cell death. These studies characterize the functional domains of Thbs4 and aid in parsing out the intracellular and extracellular roles of thrombospondins. Taken together, data indicate critical functions for the TSP-C and LamG domains of Thbs4 in ER stress-responsive and secretory pathway functions, respectively.

Hrd1 and ER-Associated Protein Degradation, ERAD, are Critical Elements of the Adaptive ER Stress Response in Cardiac Myocytes.

Hydroxymethyl glutaryl-coenzyme A reductase degradation protein 1 (Hrd1) is an endoplasmic reticulum (ER)-transmembrane E3 ubiquitin ligase that has been studied in yeast, where it contributes to ER protein quality control by ER-associated degradation (ERAD) of misfolded proteins that accumulate during ER stress. Neither Hrd1 nor ERAD has been studied in the heart, or in cardiac myocytes, where protein quality control is critical for proper heart function. The objective of this study were to elucidate roles for Hrd1 in ER stress, ERAD, and viability in cultured cardiac myocytes and in the mouse heart, in vivo. The effects of small interfering RNA-mediated Hrd1 knockdown were examined in cultured neonatal rat ventricular myocytes. The effects of adeno-associated virus-mediated Hrd1 knockdown and overexpression were examined in the hearts of mice subjected to pressure overload-induced pathological cardiac hypertrophy, which challenges protein-folding capacity. In cardiac myocytes, the ER stressors, thapsigargin and tunicamycin increased ERAD, as well as adaptive ER stress proteins, and minimally affected cell death. However, when Hrd1 was knocked down, thapsigargin and tunicamycin dramatically decreased ERAD, while increasing maladaptive ER stress proteins and cell death. In vivo, Hrd1 knockdown exacerbated cardiac dysfunction and increased apoptosis and cardiac hypertrophy, whereas Hrd1 overexpression preserved cardiac function and decreased apoptosis and attenuated cardiac hypertrophy in the hearts of mice subjected to pressure overload. Hrd1 and ERAD are essential components of the adaptive ER stress response in cardiac myocytes. Hrd1 contributes to preserving heart structure and function in a mouse model of pathological cardiac hypertrophy.

Cooling-Induced ER Stress is Good for Your Brain

Cooling-Induced ER Stress is Good for Your Brain

C-Flip KO fibroblasts display lipid accumulation associated with endoplasmic reticulum stress

c-Flip proteins are well-known apoptosis modulators. They generally contribute to tissue homeostasis maintenance by inhibiting death-receptor-mediated cell death.In the present manuscript, we show that c-Flip knock-out (KO) mouse embryonic fibroblasts (MEFs) kept in culture under starvation conditions gradually modify their phenotype and accumulate vacuoles, becoming progressively larger according to the duration of starvation. Large vacuoles are present in KO MEFs though not in WT MEFs, and are Oil Red-O positive, which indicates that they represent lipid droplets. Western blot experiments reveal that, unlike WT MEFs, KO MEFs express high levels of the lipogenic transcription factor PPAR-γ. Lipid droplet accumulation was found to be associated with endoplasmic reticulum (ER) stress activation and autophagic modulation valuated by means of BIP increase, LC3 lipidation and AMP-activated protein kinase (AMPK) phosphorylation, and p62 accumulation. Interestingly, XBP-1, an ER stress-induced lipogenic transcription factor, was found to preferentially localize in the nucleus rather than in the cytoplasm of KO MEFs.These data demonstrate that, upon starvation, c-Flip affects lipid accumulation, ER stress and autophagy, thereby pointing to an important role of c-Flip in the adaptive response and ER stress response programs under both normal and pathological conditions.

Adaptive endoplasmic reticulum stress alters cellular responses to the extracellular milieu.

The ability to respond to perturbations in endoplasmic reticulum (ER) function is a critical property for all cells. In the presence of chronic ER stress, the cell must adapt so that cell survival is favored or the stress may promote apoptosis. In some pathological processes, such as neurodengeneration, persistent ER stress can be tolerated for an extended period, but eventually cell death occurs. It is not known how an adaptive response converts from survival into apoptosis. To gain a better understanding of the role of adaptive ER stress in neurodegeneration, in this study, with a neuronal cell line SH-SY5Y and primary motor neuron-glia cell mixed cultures, we induced adaptive ER stress and modified the extracellular environment with physiologically relevant changes that alone did not activate ER stress. Our data demonstrate that an adaptive ER stress favored neuronal cell survival, but when cells were exposed to additional physiological insults the level of ER stress was increased, followed by activation of the caspase pathway. Our results indicate that an adaptive ER stress response could be converted to apoptosis when the external cellular milieu changed, suggesting that the conversion from prosurvival to proapoptotic pathways can be driven by the external milieu. This conversion was due at least partially to an increased level of ER stress.

From adaption to death: endoplasmic reticulum stress as a novel target of selective neurodegeneration?

From adaption to death: endoplasmic reticulum stress as a novel target of selective neurodegeneration?

MTOR inhibition increases cell viability via autophagy induction during endoplasmic reticulum stress – An experimental and modeling study

Unfolded or misfolded proteins in the endoplasmic reticulum (ER) trigger an adaptive ER stress response known as unfolded protein response (UPR). Depending on the severity of ER stress, either autophagy-controlled survival or apoptotic cell death can be induced. The molecular mechanisms by which UPR controls multiple fate decisions have started to emerge. One such molecular mechanism involves a master regulator of cell growth, mammalian target of rapamycin (mTOR), which paradoxically is shown to have pro-apoptotic role by mutually interacting with ER stress response. How the interconnections between UPR and mTOR influence the dynamics of autophagy and apoptosis activation is still unclear. Here we make an attempt to explore this problem by using experiments and mathematical modeling. The effect of perturbed mTOR activity in ER stressed cells was studied on autophagy and cell viability by using agents causing mTOR pathway inhibition (such as rapamycin or metyrapone). We observed that mTOR inhibition led to an increase in cell viability and was accompanied by an increase in autophagic activity. It was also shown that autophagy was activated under conditions of severe ER stress but that in the latter phase of stress it was inhibited at the time of apoptosis activation. Our mathematical model shows that both the activation threshold and temporal dynamics of autophagy and apoptosis inducers are sensitive to variation in mTOR activity. These results confirm that autophagy has cytoprotective role and is activated in mutually exclusive manner with respect to ER stress levels.

The Adaptive Endoplasmic Reticulum Stress Response to Lipotoxicity in Progressive Human Nonalcoholic Fatty Liver Disease

Nonalcoholic fatty liver disease (NAFLD) may progress from simple steatosis to severe, nonalcoholic steatohepatitis (NASH) in 7%-14% of the U.S. population through a second "hit" in the form of increased oxidative stress and inflammation. Endoplasmic reticulum (ER) stress signaling and the unfolded protein response (UPR) are triggered when high levels of lipids and misfolded proteins alter ER homeostasis creating a lipotoxic environment within NAFLD livers. The objective of this study was to determine the coordinate regulation of ER stress-associated genes in the progressive stages of human NAFLD. Human liver samples categorized as normal, steatosis, NASH (Fatty), and NASH (Not Fatty) were analyzed by individual Affymetrix GeneChip Human 1.0 ST microarrays, immunoblots, and immunohistochemistry. A gene set enrichment analysis was performed on autophagy, apoptosis, lipogenesis, and ER stress/UPR gene categories. An enrichment of downregulated genes in the ER stress-associated lipogenesis and ER stress/UPR gene categories was observed in NASH. Conversely, an enrichment of upregulated ER stress-associated genes for autophagy and apoptosis gene categories was observed in NASH. Protein expression of the adaptive liver response protein STC2 and the transcription factor X-box binding protein 1 spliced (XBP-1s) were significantly elevated among NASH samples, whereas other downstream ER stress proteins including CHOP, ATF4, and phosphorylated JNK and eIF2α were not significantly changed in disease progression. Increased nuclear accumulation of total XBP-1 protein was observed in steatosis and NASH livers. The findings reveal the presence of a coordinated, adaptive transcriptional response to hepatic ER stress in human NAFLD.

Cellular Inhibitor of Apoptosis Protein-1 (cIAP1) Plays a Critical Role in β-Cell Survival under Endoplasmic Reticulum Stress: PROMOTING UBIQUITINATION AND DEGRADATION OF C/EBP HOMOLOGOUS PROTEIN (CHOP)

Lipotoxicity in pancreatic β-cells, arising from excess free fatty acid-induced endoplasmic reticulum (ER) stress response, has been recognized as a key pathogenic factor causing loss of β-cell mass and contributing to type 2 diabetes. However, how the adaptive ER stress response causes cell death remains enigmatic. We report herein a critical role of cellular inhibitor of apoptosis protein-1 (cIAP1) in controlling β-cell survival under ER stress. While both palmitate and palmitoleate induced an overt ER stress response, lipotoxicity was only observed in β-cells exposed to palmitate but not palmitoleate. Interestingly, cells treated with palmitoleate exerted a sustainable level of cIAP1, whereas the protein quickly degraded following palmitate treatment. Enforced overexpression of cIAP1 prevented palmitate-induced cell death. In contrast, siRNA-mediated knockdown of cIAP1 in β-cells or knock-out of cIap1 in mouse embryonic fibroblasts not only increased palmitate-induced apoptosis, but also committed cells to death in response to the nontoxic palmitoleate treatment. Of importance, we found that cIAP1 functions as an E3 ubiquitin ligase promoting ubiquitination and degradation of C/EBP homologous protein (CHOP), a key mediator of ER stress-induced cell death. These findings define a novel mechanism for β-cell survival under ER stress and help to identify targets for therapeutic intervention against lipotoxicity in β-cells.

A Thrombospondin-Dependent Pathway for a Protective ER Stress Response

Thrombospondin (Thbs) proteins are induced in sites of tissue damage or active remodeling. The endoplasmic reticulum (ER) stress response is also prominently induced with disease where it regulates protein production and resolution of misfolded proteins. Here we describe a function for Thbs as ER-resident effectors of an adaptive ER stress response. Thbs4 cardiac-specific transgenic mice were protected from myocardial injury, whereas Thbs4(-/-) mice were sensitized to cardiac maladaptation. Thbs induction produced a unique profile of adaptive ER stress response factors and expansion of the ER and downstream vesicles. Thbs bind the ER lumenal domain of activating transcription factor 6α (Atf6α) to promote its nuclear shuttling. Thbs4(-/-) mice showed blunted activation of Atf6α and other ER stress-response factors with injury, and Thbs4-mediated protection was lost upon Atf6α deletion. Hence, Thbs can function inside the cell during disease remodeling to augment ER function and protect through a mechanism involving regulation of Atf6α.