Onset Of Endoplasmic Reticulum Stress Research Articles

MTORC1 inhibition impairs activation of the unfolded protein response and induces cell death during ER stress in cardiomyocytes.

The mechanistic target of rapamycin complex 1 (mTORC1) is a central regulator of protein synthesis that senses and responds to a variety of stimuli to coordinate cellular metabolism with environmental conditions. To ensure that protein synthesis is inhibited during unfavorable conditions, translation is directly coupled to the sensing of cellular protein homeostasis. Thus, translation is attenuated during endoplasmic reticulum (ER) stress by direct inhibition of the mTORC1 pathway. However, residual mTORC1 activity is maintained during prolonged ER stress which is thought to be involved in translational reprogramming and adaption to ER stress. By analyzing the dynamics of mTORC1 regulation during ER stress, we unexpectedly found that mTORC1 is transiently activated in cardiomyocytes within minutes at the onset of ER stress before being inhibited during chronic ER stress. This dynamic regulation of mTORC1 appears to be mediated, at least in part, by ATF6, as its activation was sufficient to induce the biphasic control of mTORC1. We further showed that protein synthesis remains dependent on mTORC1 throughout the ER stress response and that mTORC1 activity is essential for posttranscriptional induction of several unfolded protein response elements. Pharmacological inhibition of mTORC1 increased cell death during ER stress, indicating that the mTORC1 pathway serves adaptive functions during ER stress in cardiomyocytes potentially by controlling the expression of the protective unfolded protein response.

Intracellular Ca2+ signaling and ORAI calcium release-activated calcium modulator 1 are associated with hepatic lipidosis in dairy cattle.

Fatty liver is a common metabolic disorder afflicting dairy cows during the periparturient period and is closely associated with endoplasmic reticulum (ER) stress. The onset of ER stress in humans and mice alters hepatic lipid metabolism, but it is unknown if such event contributes to fatty liver in dairy cows soon after parturition. ORAI calcium release-activated calcium modulator 1 (ORAI1) is a key component of the store-operated Ca2+ entry mechanism regulating cellular Ca2+ balance. The purpose of this study was to investigate the role of ORAI1 on hepatic lipidosis via ER stress in dairy cows. Liver tissue biopsies were collected from Holstein cows diagnosed as healthy (n = 6) or with hepatic lipidosis (n = 6). Protein and mRNA abundance of ER stress-related targets, lipogenic targets, or the transcription regulator SREBP1 and ORAI1 were greater in cows with lipidosis. In vitro, hepatocytes were isolated from four healthy female calves and used for culture with a 1.2 mM mixture of fatty acids (oleic, linoleic, palmitic, stearic, and palmitoleic acid) for various times (0, 3, 6, 9, or 12 h). As incubation time progressed, increases in concentration of Ca2+ and abundance of protein kinase RNA-like ER kinase (PERK), inositol-requiring protein 1α (IRE1α), and activating transcription factor-6 (ATF6) protein in response to exogenous fatty acids underscored a mechanistic link among Ca2+, fatty acids, and ER stress. In a subsequent study, hepatocytes were transfected with small interfering RNA (siORAI1) or the ORAI1 inhibitor BTP2 for 48 h or 2 h followed by a challenge with the 1.2 mM mixture of fatty acids for 6 h. Compared with control group, silencing or inhibition of ORAI1 led to decreased abundance of fatty acid synthesis (FASN, SREBP1, and ACACA) and ER stress-related proteins in bovine hepatocytes. Overall, data suggested that NEFA through ORAI1 regulate intracellular Ca2+ signaling, induce ER stress, and lead to lipidosis in isolated hepatocytes.

ER stress causes widespread protein aggregation and prion formation.

Disturbances in endoplasmic reticulum (ER) homeostasis create a condition termed ER stress. This activates the unfolded protein response (UPR), which alters the expression of many genes involved in ER quality control. We show here that ER stress causes the aggregation of proteins, most of which are not ER or secretory pathway proteins. Proteomic analysis of the aggregated proteins revealed enrichment for intrinsically aggregation-prone proteins rather than proteins which are affected in a stress-specific manner. Aggregation does not arise because of overwhelming proteasome-mediated degradation but because of a general disruption of cellular protein homeostasis. We further show that overexpression of certain chaperones abrogates protein aggregation and protects a UPR mutant against ER stress conditions. The onset of ER stress is known to correlate with various disease processes, and our data indicate that widespread amorphous and amyloid protein aggregation is an unanticipated outcome of such stress.

ER Stress-Induced Clearance of Misfolded GPI-Anchored Proteins via the Secretory Pathway

SummaryProteins destined for the cell surface are first assessed in the endoplasmic reticulum (ER) for proper folding before release into the secretory pathway. This ensures that defective proteins are normally prevented from entering the extracellular environment, where they could be disruptive. Here, we report that, when ER folding capacity is saturated during stress, misfolded glycosylphosphatidylinositol-anchored proteins dissociate from resident ER chaperones, engage export receptors, and quantitatively leave the ER via vesicular transport to the Golgi. Clearance from the ER commences within minutes of acute ER stress, before the transcriptional component of the unfolded protein response is activated. These aberrant proteins then access the cell surface transiently before destruction in lysosomes. Inhibiting this stress-induced pathway by depleting the ER-export receptors leads to aggregation of the ER-retained misfolded protein. Thus, this rapid response alleviates the elevated burden of misfolded proteins in the ER at the onset of ER stress, promoting protein homeostasis in the ER.

Endoplasmic reticulum: ER stress regulates mitochondrial bioenergetics

Endoplasmic reticulum (ER) stress activates an adaptive unfolded protein response (UPR) that facilitates cellular repair, however, under prolonged ER stress, the UPR can ultimately trigger apoptosis thereby terminating damaged cells. The molecular mechanisms responsible for execution of the cell death program are relatively well characterized, but the metabolic events taking place during the adaptive phase of ER stress remain largely undefined. Here we discuss emerging evidence regarding the metabolic changes that occur during the onset of ER stress and how ER influences mitochondrial function through mechanisms involving calcium transfer, thereby facilitating cellular adaptation. Finally, we highlight how dysregulation of ER–mitochondrial calcium homeostasis during prolonged ER stress is emerging as a novel mechanism implicated in the onset of metabolic disorders.

Increased ER-mitochondrial coupling promotes mitochondrial respiration and bioenergetics during early phases of ER stress.

Increasing evidence indicates that endoplasmic reticulum (ER) stress activates the adaptive unfolded protein response (UPR), but that beyond a certain degree of ER damage, this response triggers apoptotic pathways. The general mechanisms of the UPR and its apoptotic pathways are well characterized. However, the metabolic events that occur during the adaptive phase of ER stress, before the cell death response, remain unknown. Here, we show that, during the onset of ER stress, the reticular and mitochondrial networks are redistributed towards the perinuclear area and their points of connection are increased in a microtubule-dependent fashion. A localized increase in mitochondrial transmembrane potential is observed only in redistributed mitochondria, whereas mitochondria that remain in other subcellular zones display no significant changes. Spatial re-organization of these organelles correlates with an increase in ATP levels, oxygen consumption, reductive power and increased mitochondrial Ca²⁺ uptake. Accordingly, uncoupling of the organelles or blocking Ca²⁺ transfer impaired the metabolic response, rendering cells more vulnerable to ER stress. Overall, these data indicate that ER stress induces an early increase in mitochondrial metabolism that depends crucially upon organelle coupling and Ca²⁺ transfer, which, by enhancing cellular bioenergetics, establishes the metabolic basis for the adaptation to this response.

Dual involvement of caspase-4 in inflammatory and ER stress-induced apoptotic responses in human retinal pigment epithelial cells.

To investigate the functional involvement of caspase-4 in human retinal pigment epithelial (hRPE) cells. Expression and activation of caspase-4 in hRPE cells were measured after stimulation with proinflammatory agents IL-1beta (2 ng/mL), TNF-alpha (20 ng/mL), lipopolysaccharide (1000 ng/mL), interferon-gamma (500 U/mL), or monocyte coculture in the absence or presence of immunomodulating agent cyclosporine (3 or 30 ng/mL), dexamethasone (10 microM), or IL-10 (100 U/mL) and endoplasmic reticulum (ER) stress inducer thapsigargin (25 nM) or tunicamycin (3 or 10 microM). The onset of ER stress was determined by expression of GRP78. The involvement of caspase-4 in inflammation and apoptosis was further examined by treating the cells with caspase-4 inhibitor Z-LEVD-fmk, caspase-1 and -4 inhibitor Z-YVAD-fmk, and pan-caspase inhibitor Z-VAD-fmk. Caspase-4 mRNA expression and protein activation were induced by all the proinflammatory agents and ER stress inducers tested in this study. Caspase-4 activation was blocked or reduced by dexamethasone and IL-10. Elevated ER stress by proinflammatory agents and ER stress inducers was shown by increased expression of the ER stress marker GRP78. The induced caspase-4 and caspase-3 activities by tunicamycin and the stimulated IL-8 protein expression by IL-1beta were markedly reduced by caspase-4 inhibitor Z-LEVD-fmk. Although caspase-4 inhibitor Z-LEVD-fmk and caspase-1 and -4 inhibitor Z-YVAD-fmk reduced tunicamycin-induced hRPE apoptotic cell death by 59% and 86%, respectively, pan-caspase inhibitor Z-VAD-fmk completely abolished the induced apoptosis. Caspase-4 is dually involved in hRPE proinflammatory and proapoptotic responses. Various proinflammatory stimuli and ER stress induce hRPE caspase-4 mRNA synthesis and protein activation. ER stress-induced hRPE cell death is caspase and, in part, caspase-4 dependent.

Endoplasmic Reticulum (ER) Chaperone Regulation and Survival of Cells Compensating for Deficiency in the ER Stress Response Kinase, PERK

The activity of PERK, an endoplasmic reticulum (ER) transmembrane protein kinase, assists in an ER stress response designed to inhibit general protein synthesis while allowing upregulated synthesis of selective proteins such as the ATF4 transcription factor. PERK null mice exhibit phenotypes that especially affect secretory cell types. Although embryonic fibroblasts from these mice are difficult to transfect with high efficiency, we have generated 293 cells stably expressing the PERK-K618A dominant negative mutant. 293/PERK-K618A cells, in response to ER stress: (a) do not properly inhibit general protein synthesis, (b) exhibit defective/delayed induction of ATF4 and BiP, and (c) exhibit exuberant splice activation of XBP1 and robust cleavage activation of ATF6, with abnormal regulation of calreticulin levels. The data suggest compensatory mechanisms allowing for cell survival in the absence of functional PERK. Interestingly, although induction of CHOP (a transcription factor implicated in apoptosis) is notably delayed after onset of ER stress, 293/PERK-K618A cells eventually produce CHOP at normal or even supranormal levels and exhibit increased apoptosis either in response to general ER stress or, more importantly, to specific misfolded secretory proteins.

In Vivo Regulation of Grp78/BiP Transcription in the Embryonic Heart: ROLE OF THE ENDOPLASMIC RETICULUM STRESS RESPONSE ELEMENT AND GATA-4

The transcriptional activation of GRP78, which controls multiple signaling pathways of the unfolded protein response, has been used extensively as an indicator for the onset of endoplasmic reticulum stress in tissue culture systems. Here we investigate the mechanism of Grp78 induction during mouse embryonic development. Our results reveal that in transgenic mouse models, reporter gene activity driven by the Grp78 promoter is strongly activated during early embryonic heart development but subsides in later stages. This activation is strictly dependent on a 100-base pair region of the Grp78 promoter containing the endoplasmic reticulum stress response elements (ERSEs). Previous studies establish that endoplasmic reticulum stress induces in vivo binding of YY1 and the nuclear form of ATF6 to the ERSE. Since the expression of YY1 as well as ATF6 is ubiquitous in the mouse embryo, activation of the Grp78 promoter in the early embryonic heart may involve a specific mechanism. Here we report that GATA-4, a transcription factor essential for heart development, binds to the Grp78 promoter in vivo and activates the ERSE, which does not contain a consensus GATA binding site. GATA-4 cooperatively activates the Grp78 promoter with YY1, and the DNA binding domain of YY1 is necessary and sufficient for this cooperation. In addition, GATA-4 activation of the Grp78 promoter is enhanced by the nuclear form of ATF6, and this synergy is further potentiated by YY1. These results suggest that during early heart organogenesis, Grp78 can be activated through cooperation between the cell type-specific transcription factors and ERSE-binding factors.