Endoplasmic Reticulum Stress and Related Pathological Processes.

NIH Public Access Author Manuscript J Pharmacol Biomed Anal. Author manuscript; available in PMC 2014 March 05.

NIH-PA Author Manuscript

Published in final edited form as: J Pharmacol Biomed Anal. ; 1(2): 1000107–.

Endoplasmic Reticulum Stress and Related Pathological Processes Yu Mei1, Melissa D Thompson1, Richard A Cohen1, and XiaoYong Tong1,* 1Vascular Biology Section, Boston University School of Medicine, Boston, Massachusetts 02118, USA

Abstract


The endoplasmic reticulum (ER) plays a pivotal role in lipid and protein biosynthesis as well as calcium store regulation, which determines its essential role in cell function. Hypoxia, nutrient deprivation, perturbation of redox status and aberrant calcium regulation can all trigger the ER stress response, which is mediated through three main sensors, namely inositol requiring element-1 (IRE-1), protein kinase-like ER kinase (PERK) and activating transcription factor 6 (ATF6). This review explores the interaction of ER stress and ER stress-associated pathological processes, including inflammation, apoptosis, aberrant autophagy, mitochondrial dysfunction and hypoxic responses. In addition, the correlation of ER stress with lipid and calcium homeostasis and dysregulation, and its role in disease development is also presented. Improved understanding of ER stress and its cofactors in pathological processes may provide new perspective on disease development and control.

Keywords ER stress; Inflammation; Autophagy; Mitochondrial dysfunction; Hypoxia; Calcium; Lipids; Apoptosis

Introduction


The ER is an important organelle of eukaryotic cells that functions in a variety of biological processes, including essential roles in lipid and protein biosynthesis [1,2]. In addition to serving as the site of protein synthesis, folding and post-tranlational modification, the ER is the major intracellular storage location for Ca2+, and maintains Ca2+ homeostasis through multiple integrated systems [3]. When ER homeostasis is disrupted, ER stress ensues, and an adaptive process called the unfolded protein response (UPR) is initiated [4]. ER stress can be triggered by hypoxia, nutrient deprivation, perturbation of redox status, aberrant Ca2+ regulation, viral infection, failure of posttranslational modifications, and increased protein synthesis and/or accumulation of unfolded or misfolded proteins in the ER [3,5-8]. A threepronged signal-transduction cascade is activated to resolve ER stress, including IRE-1, PERK and ATF6. Under unstressed conditions, the luminal domains of these sensors are bound to the ER chaperone binding immunoglobulin protein (BiP), which maintains them in the inactive state [9]. When unfolded or misfolded proteins accumulate, BiP instead preferentially binds to these abnormal proteins, releasing its inhibitory hold on PERK, ATF6, and IRE1. Upon dissociation from BiP and activation, these sensors initiate three

Copyright © 2013, SciTechnol, All Rights Reserved. * Corresponding author: XiaoYong Tong, Vascular Biology Section, Boston University, School of Medicine, 650 Albany Street, X729, Boston, MA, 02118, USA, [email protected].

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distinct but complementary signal transduction pathways: The PERK pathway induces global translational attenuation to avoid further accumulation of misfolded proteins in the ER, while promoting selective translation of proteins involved in the resolution of ER stress; ATF6 translocates to the Golgi apparatus where it is cleaved by site 1 and 2 proteases before a second translocation into the nucleus where it activates transcription of chaperones and other genes involved in ER quality control; IRE1 activates ER-associated degradation in an attempt to rectify the accumulation of misfolded protein. In the past decade, ER stress has drawn much attention due to its potential roles in disease development, such as cardiovascular disease [1], insulin resistance [10,11], cancer development [12] and neurological disease [13,14]. In addition, the impact of ER stress is not limited to the ER. ER stress has been confirmed to be involved in multiple pathological processes, including inflammation, impaired autophagy, mitochondrial dysfunction and hypoxic responses. Furthermore, as the major site of intracellular Ca2+ storage, the ER has the capacity to regulate Ca2+ homeostasis and Ca2+-related biological processes, and it has been shown that ER stress-associated Ca2+ depletion mediates apoptosis and disease development. This review will present recent research findings on ER stress and its related pathological processes. ER Function


The ER is an interconnected network of tubules, vesicles and flattened sacs, and is classified as either rough endoplasmic reticulum (RER) or smooth endoplasmic reticulum (SER) [1,2]. The cytosolic surface of RER is studded with ribosomes that form granules to give it a rough appearance. RER is localized in the perinuclear region. The membrane on the nuclear side of RER is a continuation of the outer nuclear membrane. mRNA exported from the nucleus is assembled on ribosomes and labeled with specific amino acid sequences which allow ribosomal recognition and binding to RER, which enables insertion of the new protein into the ER where it obtains its tertiary structure. After folding and maturation, one part of the RER membrane breaks off to form a vesicle which shuttles the protein to the Golgi apparatus or cell membrane. Unlike RER, SER has a tubular structure that lacks ribosome attachments. Its major functions are synthesis of lipids and membrane proteins, regulation of Ca2+ stores, and detoxification of drugs and alcohols. SER also contains glucose-6 phosphatase and is involved in gluconeogenesis. Smooth ER is found in both smooth and striated muscle, where it is called sarcoplasmic reticulum (SR). SR in muscle is a dynamic regulator of Ca2+, balancing Ca2+ storage, release, and reuptake through luminal Ca2+binding proteins, SR Ca2+ release channels and SR Ca2+-ATPase (SERCA) pumps respectively. ER Stress


ER stress induction—Maintenance of ER homeostasis is critical to cell survival. When ER homeostasis is disrupted, ER stress ensues, and an adaptive process called the unfolded protein response (UPR) is initiated. The accumulation of unfolded proteins in the ER causes ER stress, which can be triggered by hypoxia, nutrient deprivation, perturbation of redox regulation, aberrant Ca2+ regulation, viral infection, failure of posttranslational modifications, and increased protein synthesis [3,5-8]. As a consequence, cell signaling cascades are activated to resolve this stress by reducing protein synthesis and increasing the capacity for protein folding. Three sensors transduce ER stress signals across the ER membrane, namely IRE-1, PERK and ATF6 [1,4]. These sensors remain inactive while bound to the ER chaperone BiP. Upon accumulation of unfolded or misfolded proteins in the ER, BiP dissociates from PERK, ATF6, and IRE1 allowing for their activation (Figure 1). BiP—BiP is also known as 78 kDa glucose-regulated protein (GRP78); BiP/GRP78 binds transiently to newly synthesized proteins, and more permanently to underglycosylated or J Pharmacol Biomed Anal. Author manuscript; available in PMC 2014 March 05.

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misfolded proteins in the ER. It has a wide range of functions within the ER, including inhibition of specific transmembrane receptor proteins (ATF6, IRE1 and PERK) by binding to their luminal domains. However, when the ER is overloaded with misfolded proteins, BiP/GRP78 preferentially binds to the exposed hydrophobic regions of the misfolded proteins, causing it to dissociate from these receptors. Dissociation from BiP allows these ER stress transducers to become active and initiate subsequent signaling. Thus, BiP is an essential regulator of ER homeostasis, and its expression is currently viewed as a marker of ER stress due to its key regulation of ER stress sensor activation and ER stress initiation [1,2].


IRE1—BiP controls IRE1 activity through association and dissociation under unstressed and stressed conditions, respectively. It has been identified as a negative regulator of IRE1, as overexpression of BiP attenuates the UPR [15]. Importantly, rather than serving as a simple on or off switch for IRE1 activity, BiP is an adjustor for sensitivity to various stresses [16]. BiP modulates the sensitivity and dynamics of IRE1 activity to restore protein folding homeostasis in the ER [17]. IRE1 possesses serine/threonine kinase activity in its cytoplasmic domain, and interestingly the only known substrate is itself. During ER stress, both kinase-kinase and nuclease-nuclease interactions mediate the formation of an IRE1 homodimer. ER stress-induced dimerization of the luminal domain facilitates the proximal position of cytosolic domains for trans-autophosphorylation of the kinase activation loop. Subsequently the trans-autophosphorylation of IRE1 activates transcription factors that induce various genes required for protein refolding, secretion, and degradation of misfolded proteins [18,19]. In addition, ER stress-induced homodimerization and transautophosphorylation cause IRE1 to gain endoribonuclease activity, thus allowing it to excise a 26-nucleotide intron from the mRNA of X-box binding protein (XBP) 1 to generate a spliced XBP1s. Spliced XBP1 serves as a potent transcriptional transactivator to upregulate many UPR target genes such as the chaperones BiP/GRP78 and XBP1-U, and XBP1-U, in turn, augments the protective response by providing more substrate for expression of the XBP1s transcription factor [20,21]. Importantly, IRE1 signaling attenuates after persistent ER stress, and this involves dissociation of IRE1 clusters, IRE1 dephosphorylation, and reduction of endoribonuclease activity, which indicates that the UPR will switch from the protective phase to apoptotic phase. However, enhanced cell survival is observed with sustained IRE1 activity maintained artificially, suggesting a correlation between UPR duration and cell fate after ER stress [22,23].


PERK—Similar to IRE1, PERK is the second major branch of the ER stress response. PERK is a type I transmembrane serine/threonine kinase which is activated by transautophosphorylation and homodimerization under conditions of ER stress. Activated PERK phosphorylates eukaryotic translation initiation factor 2 α (eIF2α), which inhibits the assembly of the 80-S ribosome and thereby attenuates protein synthesis and alleviates ER protein overload [24,25]. However, while causing global translational attenuation, eIF2α phosphorylation simultaneously promotes selective translation mRNAs that contain small open reading frames in their 5’ untranslated regions, thereby increasing the translation of proteins important in the resolution of ER stress, such as activating transcription factor-4 (ATF4) [26]. In addition to regulating protein synthesis, PERK/eIF2α also regulate the cell cycle by inhibiting cyclinD1 expression during the UPR, leading to cell cycle arrest [27]. Furthermore, elevated ATF4 signaling enhances transcription of C/EBP-homologous protein (CHOP), which induces apoptosis in response to ER stress [28]. It has recently been reported that poly (ADP-ribose) polymerase 16 enzyme activity is upregulated during the UPR, which, as a consequence, increases PERK and IRE1 kinase activity by ADPribosylation [29]. It has also been shown that PERK induces miR-211, which targets the proximal CHOP promoter to increase histone methylation and repress CHOP expression,

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suggesting a role for PERK-dependent miR-211 induction in preventing CHOP accumulation and thereby re-establishing ER homeostasis [30].

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ATF6—The third major sensor of ER stress is ATF6. Upon the accumulation of misfolded proteins in the ER, ATF6 dissociates from BiP and translocates to the Golgi apparatus where it is cleaved by site 1 and site 2 proteases [31], resulting in liberation of the ATF6 cytoplasmic domain from its membrane anchor. Cleaved ATF6 then translocates to the nucleus and binds to the ER stress response element CCAAT (N) 9CCACG to induce the transcription of ER chaperones such as BiP and GRP94. ATF6 also regulates other UPR target genes encoding ER chaperones and folding enzymes such as XBP1, CHOP, hyperhomocysteinemia-induced ER stress responsive protein and protein disulfide isomerase (PDI) to aid protein folding, secretion, and degradation [21,32]. Recent findings indicate that thrombospondin can promote ATF6 nuclear shuttling by binding to its ER luminal domain [33], and that ATF6 exerts its protective effects during ER stress, in part, through downregulation of miRNA455 [34]. In the later phase of the UPR, XBP1U, the negative regulator of XBP1s, accelerates proteasomal degradation of ATF6 to stop UPR target gene expression. In one clinically important disease, Wolfman syndrome 1, ubiquitination and degradation of ATF6 is enhanced through recruitment of ATF6 to the E3 ligase upon ER stress [35]. Activation of the three distinct but complementary signal transduction pathways described above (IRE1, PERK and ATF6) facilitates cellular adaptation to and resolution of ER stress. However, as described previously, prolonged ER stress will lead to cell death or apoptosis. ER Stress Related Pathological Processes


ER stress and inflammation—ER stress and the three branches of the UPR have been linked to inflammatory signaling through several mechanisms [2,36]. For example, PERK activation results in nuclear factor kappa B (NF-κB) translocation to the nucleus and induction of inflammatory gene expression, such as interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α). Activation of IRE1α leads to recruitment of TNF-α-receptor-associated factor 2 (TRAF2), and this complex interacts with and activates c-Jun N-terminal kinase (JNK) and IκB kinase (IKK), which phosphorylate and activate downstream inflammatory mediators. The ATF6 pathway also activates NF-κB. Additionally XBP1s and ATF4, stimulate IL-8, IL-6, and monocyte chemoattractant protein 1 (MCP1) in human endothelial cells and IFN-α production in dendritic cells. The eIF2α kinase PKR (double-stranded RNA-activated protein kinase) interacts directly with inflammatory kinases such as IKK and JNK [2]. ER stress-mediated inflammation has been found to be associated with obesity, type 2 diabetes, intestinal disease and cancer. For example, inflammatory cytokine production is associated with expression of ER stress markers in the adipose tissue of mice fed a high fat diet, and the chemical chaperones 4-phenylbutyric acid and tauroursodeoxycholic acid suppress inflammatory cytokine production in adipose tissue by alleviating ER stress [37]. ER stress also mediates intestinal inflammation. Inflammatory bowel disease is defined as a group of chronic, inflammatory disorders of the colon and/or small intestine. The ATF6 and IRE1 pathways are activated in inflammatory bowel disease, as determined by increased gene transcription and corresponding protein production in inflamed samples from patients with ulcerative colitis and colonic Crohn's disease [38]. Inflammatory responses also contribute to tumor initiation, progression, and metastasis [39]. For instance, NF-κB activation, a major pro-inflammatory signal, has been linked to the transformation of premalignant cells into malignant cells [40]. Interestingly, ER stress in tumor cells was transmissible to macrophages and directed them toward a proinflammatory phenotype and inflammatory cytokine release, thus facilitating tumor progression [41].


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ER stress and apoptosis—The UPR is an adaptive response to misfolded proteins in the ER which initiates cytoprotective mechanisms and protects cells from death. Failure to adapt to and resolve ER stress triggers cell death, typically through apoptosis [3,42,43]. Caspases are major mediators of apoptosis, and a novel finding is that ER stress triggers the caspase cascade, which includes caspase-4, -8 and -3. Certain members of the caspase family are now shown to be required in ER stress, and the caspase cascade plays an important role in ER stress-induced apoptosis [44]. During ER stress, disruption of ER Ca2+ homeostasis and accumulation of misfolded proteins activate caspase-12, which mediates an ER-specific apoptosis pathway, as evidenced by defective apoptotic responses in caspase-12 deficient mice under ER stress [45]. Also during ER stress, the recruitment of the apoptosis signalregulating kinase 1 (ASK1) to oligomerized IRE1 complexes activates this kinase and leads to downstream activation of JNK. JNK activates the proapoptotic protein Bim by phosphorylation, while inhibiting the antiapoptotic protein Bcl-2 [46]. CHOP is a well recognized regulator of apoptosis during ER stress, and overexpression of CHOP induces apoptosis through the Bcl-2 pathway. CHOP activity is enhanced at a posttranscriptional level by cross-talk between the PERK/eIF2α pathway and the IRE1/TRAF2/ASK1 pathway. Phosphorylation of CHOP at serine 78 and serine 81 increases its transcriptional and apoptotic activity [47]. Additionally, translocon is an ER protein complex which is associated with protein translation. Recently, it has been found that translocon activates ER Ca2+ leak channels to trigger ER stress and apoptosis, and that this process is modulated by the ER stress marker BiP/GRP78 [48]. ER stress and autophagy—The three major ER stress pathways are involved in activation of autophagy [46,49,50]. Autophagy (self-eating) is a multistep process of degradation and recycling of cellular components to adapt cells to adverse conditions such as nutrient deprivation, hypoxia and oxidative stress. It is characterized by microtubuleassociated protein 1 light chain 3 (LC3) conversion from LC3-I to LC3-II and fusion of autophagosomes with lysosomes to form autophagolysosomes. Dysregulation of autophagy is associated with aging, neurodegeneration, inflammation, cardiovascular diseases, and cancer. As a UPR regulator and ER chaperone, BiP is required for ER integrity and stressinduced autophagy in mammalian cells [51]. The PERK/eIF2alpha pathway upregulates autophagy-related protein 12 expression, and promotes conversion of LC3-I to LC3-II and subsequent autophagosome formation [52]. The IRE1-TRAF2-JNK pathway is also essential for the induction of autophagy, and mediates LC3 translocation in mouse embryonic fibroblasts challenged with ER stressors [1]. ATF6 is required for the IFN-γ induced autophagy response [53].


Depending on the context, autophagy activation enhances cell survival or directs the cell to undergo non-apoptotic death under conditions of ER stress. For example, ER stress-induced autophagy has cardioprotective effects. Low dose tunicamycin or thapsigargin elicits ER stress, which in turn potentiates autophagy in the heart in the face of myocardial ischemia and reperfusion. As a result of increased autophagy, the ischemic heart is preconditioned and protected, as demonstrated by improved left ventricular function and reduced myocardial infarct size and cardiomyocyte apoptosis [54]. Additionally, autophagy plays a role in cancer development. Cyclosporine A (an immunophilin/calcineurin inhibitor) induces autophagy in malignant glioma cells via induction of ER stress, which exerts a cytoprotective effect by suppressing caspase activation and poly ADP ribose polymerase degradation [55]. c-Myc and N-Myc stimulate the UPR through activation of the PERK/ eIF2α/ATF4 branch, which leads to cytoprotective autophagy to promote Myc-dependent tumor growth [12]. Additionally, autophagy is recognized to play a role in drug resistance in cancer therapy [56]. Finally, autophagy also participates in angiogenesis. MCP-induced protein-(MCPIP) mediated oxidative and nitrosative stress leads to ER stress, which promotes autophagy and angiogenesis [57]. J Pharmacol Biomed Anal. Author manuscript; available in PMC 2014 March 05.

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ER stress and mitochondrial dysfunction—Mitochondrial dysfunction and ER stress interact to perpetuate one another and facilitate disease development [58]. The mitochondria-associated ER membrane (MAM) plays critical roles in cellular physiology and homeostasis, including lipid transport, Ca2+ signaling, and apoptosis. A number of proteins bound to the mitochondria or ER serve as important mediators of communication between the two organelles at the MAM [59]. ER stress can induce mitochondrial dysfunction through regulation of Ca2+ signaling and ROS production. Prolonged ER stress leads to release of Ca2+ at the MAM and increased Ca2+ uptake into the mitochondrial matrix, which induces Ca2+-dependent mitochondrial outer membrane permeabilization and apoptosis [60]. Concomitantly, ROS are produced by proteins in the ER oxidoreductin 1 (ERO1) family [61]. ER stress activates ERO1 and leads to excessive production of ROS [62], which, in turn, inactivates SERCA and activates inositol-1,4,5-trisphosphate receptors (IP3R) via oxidation, resulting in elevated levels of cytosolic Ca2+, increased mitochondrial uptake of Ca2+, and ultimately mitochondrial dysfunction [63,64]. The ER stress sensor PERK is a fundamental component of the MAM owing to its essential roles in maintaining ER-mitochondria juxtaposition and driving ROS-mediated mitochondrial apoptosis [65]. In addition, PERK/ATF3 and IRE1/JNK signaling are also involved in palmitate-induced pancreatic β-cell dysfunction and apoptosis through crosstalk with the intrinsic mitochondrial pathway [66]. ER stress also conveys apoptotic command to the mitochondrial executive machinery through cleavage of the proto-oncoprotein CRK [67].


Just as ER stress can lead to mitochondrial dysfunction, mitochondrial dysfunction also induces ER Stress. For example, nitric oxide disrupts the mitochondrial respiratory chain and causes changes in mitochondrial Ca2+ flux which induce ER stress and subsequently increase GRP78 to exert cytoprotective effects against thapsigargin [68]. In addition, mitochondrial dysfunction downregulates adiponectin synthesis in adipose tissue through activation of JNK-and ATF3-mediated ER stresses signaling pathways [69].


ER stress and hypoxia—Hypoxia or ischemia has been observed to induce ER stress, which plays dual roles in cell function. On one side, ER stress initiates protective signals to support cell survival. However, an uncontrolled, aberrantly regulated or unresolved ER stress response participates in disease development. Sustained ER stress leads to inflammation, apoptosis and promotes disease development. Severe hypoxia triggers an unresolved UPR, and subsequently increases the release of tumor necrosis factor receptor 1 (TNFR1) and inflammation [70]. In retinal cells, hypoxia upregulates ATF4, which, in turn, promotes diabetic macular edema by inducing inflammatory cytokine release [71]. Furthermore, ischemic stroke-mediated neuronal ER stress hinders vascular regeneration [72]. Superoxide produced during ischemia/reperfusion injury triggers ER stress-mediated CHOP induction, which exacerbates myocardial ischemia/reperfusion injury by facilitating cardiomyocyte apoptosis and myocardial inflammation [73]. Notably, hypoxia also induces very low density lipoprotein receptor (VLDLR) expression due to the interaction of hypoxia inducible factor with a hypoxia-responsive element in the VLDLR promoter. In addition to mediating endocytosis of lipoproteins, VLDLR promotes ER stress and apoptosis, suggesting that VLDLR-induced lipid accumulation worsens survival of ischemic cardiomyocytes by increasing ER stress and apoptosis [74]. Although the ER stress response is associated with many pathological processes, it is important to note that ER stress initiates critical protective signals to support cell survival. The oxygen sensor prolyl-4-hydroxylase domain 3 interacts with the zipper II domain of ATF4, thus reducing ATF4 under normoxic conditions. However, under anoxic conditions, ATF4 is induced due to increased protein stability [75]. ATF4 can also promote autophagy through upregulation of LC3B by directly binding to a cyclic AMP response element binding site in the LC3B promoter. Therefore, severe hypoxia can not only stabilize ATF4, J Pharmacol Biomed Anal. Author manuscript; available in PMC 2014 March 05.

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but also induce ATF4-dependent autophagy through LC3 as a survival mechanism [76]. Endogenous ATF6 plays a crucial role in ischemia-mediated ER stress activation and cell survival. Ischemia reduces ER-associated ATF6 expression and increases ATF6 nuclear localization, which, in turn, induces GRP78 gene expression and promotes cell survival [77]. Another role of activated ATF6 in the alleviation of ischemia-induced ER stress damage is to robustly induce Derlin-3 which enhances ER-associated degradation and protects cardiomyocytes from ischemia–induced cell death [78]. In addition, ATF6 induces protein disulfide isomerase associated 6 (PDIA6) gene expression by binding to an ER stress response element in the PDIA6 promoter in cardiac myocytes. PDIA6 catalyzes protein disulfide bond formation, enhances ER protein folding and protects cardiac myocytes against ischemia/reperfusion-induced death [79].


ER stress and lipid disorders—It is well-established that the ER plays a critical role in lipid metabolism and homeostasis. ER-stress-dependent dysregulation of lipid metabolism may lead to dyslipidemia, insulin resistance, cardiovascular disease, type 2 diabetes, and obesity. All three arms of the UPR and their downstream signaling molecules are involved in the regulation of lipid metabolism [80,81]. GRP78 expression reduces hepatic steatosis via inhibiting ER stress-induced sterol-regulatory-element-binding protein-1c activation [82]. PERK-dependent signaling contributes to lipogenic differentiation by maintaining sustained expression of lipogenic enzymes [83]. ATF4 is also required for lipogenic gene expression in white adipose tissue, as evidenced by ATF4 knockout mice having less white adipose tissue [84]. Hepatocyte-specific IRE1α-null mice exhibit altered lipid metabolism when challenged with the ER stress inducing agent tunicamycin [85]. IRE1β-null mice fed a high-fat and high-cholesterol diet develop hyperlipidemia, indicating its important role in lipid metabolism [86]. As a downstream transcription factor of IRE1, XBP1 is required for de novo lipid synthesis in the liver, as evidenced by XBP1 deletion in the liver resulting in reduced expression of genes encoding lipogenic enzymes, and diminished hepatic triglyceride secretion and lipid synthesis [80]. Similar to hepatocyte-specific IRE1α-null mice, tunicamycin triggered hepatic steatosis in ATF6α knockout mice by inducing lipid droplet formation, which is the outcome of reduced β-oxidation of fatty acids and attenuated VLDL formation [87].


Conversely, lipids can induce ER stress. Excess cholesterol induces acute ER stress by perturbing SERCA structure and SERCA-mediated Ca2+ homeostasis in the ER [88]. In addition, increased lipid synthesis in the obese liver results in an increased ratio of phosphatidylcholine to phosphatidylethanolamine, which inhibits SERCA activity. Impaired SERCA activity leads to ER Ca2+ depletion and ER stress [89]. Finally, burn injuries can induce liver dysfunction and hyperglycemia, which is associated with hepatic ER stress, because these injuries lead to hepatocyte ER Ca2+ depletion, ER stress, mitochondrial dysfunction and apoptosis [90,91]. ER stress and ER calcium homeostasis—As the principal intracellular Ca2+ storage site, the ER contains Ca2+ at a concentration thousands of times greater than that in the cytosol [3]. ER Ca2+ levels are modulated by ER-located Ca2+ release channels, including IP3R and ryanodine receptors, and Ca2+ uptake is mediated by SERCA (Figure 2). Moreover, Ca2+ uptake by the MAM triggers ATP production that provides energy to enable SERCA to reload the ER for continued Ca2+ efflux at the MAM, which subsequently stimulates the mitochondria to produce more ROS. ROS can further direct the ER Ca2+ channels and protein folding enzymes to promote ER Ca2+ release and ER stress. Three major proteins involved in ER stress through their regulation of ER Ca2+ levels, are SERCA, ERO1 and calreticulin. SERCA plays a critical role in maintaining ER Ca2+ homeostasis and normal ER function. Decreased SERCA expression is associated with depletion of ER Ca2+ stores and ER stress-associated apoptosis [92]. Conversely, SERCA over-expression J Pharmacol Biomed Anal. Author manuscript; available in PMC 2014 March 05.

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alleviates ER stress [89]. ER redox status also determines Ca2+ release and reuptake. ERO1 is recognized as a dynamic regulator of the redox environment in the ER. ERO1 maintained in the reduced state is associated with decreased ERO1 activity and Ca2+ efflux. ERO1 also mediates CHOP-induced apoptosis by facilitating IP3R-induced Ca2+ release during ER stress [64]. Calreticulin is a multifunctional protein that binds Ca2+ with low affinity and high capacity. During ER stress, calreticulin and another chaperone calnexin can recognize and bind to misfolded proteins, and ultimately promote protein degradation. Based on the important role of Ca2+ in ER stress, new therapeutic strategies targeting ER stress-related diseases may act via regulation of intracellular Ca2+ homeostasis. For example, sorcin (a Ca2+ binding protein) enhances Ca2+ accumulation in the ER by interacting with ryanodine receptor 2, which, in turn, prevents ER stress. Therefore, overexpressing sorcin could serve as an adaptive strategy to overcome ER stress and its associated apoptosis [93]. In addition, the ER chaperone protein σ1 receptor regulates ER/ mitochondrial Ca2+ mobilization through IP3R. Overexpression of σ1 receptor facilitates mitochondrial elongation, and increases ER-mitochondrial surface contact and the efficiency of mitochondrial Ca2+ uptake, thus enhancing ATP production and promoting cell survival in the presence of ER stress [94].

Conclusion NIH-PA Author Manuscript

The present review details recent evidence of the integration of ER stress, inflammation, apoptosis, mitochondrial dysfunction, autophagy and hypoxia in disease development. In view of the prominent effects of ER stress and other related pathologic processes in disease development, new therapeutics targeting ER stress may become a promising approach for disease control and prevention. Given the complexity of the ER stress signaling network, targeting one signal in this network may not be sufficient to control disease pathogenesis. Therefore, a combination of treatments targeting different arms of the stress response and components of disease would likely provide the most effective and efficient results.

Acknowledgments Sources of Funding This study was supported by grants from the National Institutes of Health (HL031607, HL068758, HL104017, HL105287, R.A.C.) and American Diabetes Association award (7-09-JF-69, X.Y.T.).

Abbreviations NIH-PA Author Manuscript

ASK1

Apoptosis Signal-regulating Kinase 1

ATF4

Activating Transcription Factor 4

ATF6

Activating Transcription Factor 6

BiP/GRP78

Binding Immunoglobulin Protein/Glucose-Regulated Protein

CHOP

C/EBP-Homologous Protein

CRAC

Ca2+ Release Activated Ca2+

eIF2α

Eukaryotic Translation Initiation Factor 2α

ER

Endoplasmic Reticulum

ERO1

ER Oxidoreductin 1

IKK

IκB Kinase


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IL-1

Interleukin-1

IFN

Interferon

IP3R

Inositol-1,4,5-trisphosphate Receptor

IRE-1

Inositol Requiring Element-1

JNK

c-Jun N-terminal Kinase

LC3

Light Chain 3

MAM

Mitochondria-Associated ER Membrane

MCP1

Monocyte Chemoattractant Protein 1

MCPIP

MCP-Induced Protein

NF-kb

Nuclear Factor Kappa B

PERK

Protein Kinase-like ER Kinase

PDI

Protein Disulfide Isomerase

PDIA6

Nuclear Protein Disulfide Isomerase Associated 6

RER

Rough Endoplasmic Reticulum

ROS

Reactive Oxygen Species

SER

Smooth Endoplasmic Reticulum

SERCA

Sarco/Endoplasmic Reticulum Ca2+-ATPase

SOCE

Store-Operated Ca2+ Entry

STIM1 and STIM2

Stromal Interaction Molecule 1 and 2

TGF-β

Transforming Growth Factor β

TNF-α

Tumor Necrosis Factor-α

TNFR1

Tumor Necrosis Factor Receptor 1 (TNFR1)

TRAF2

TNF-α-Receptor-Associated Factor 2

UPR

Unfolded Protein Response

VLDLR

Very-Low-Density Lipoprotein Receptor

XBP-1

X-box Binding Protein-1


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Figure 1.


ER stress signaling pathway. Upon induction of ER stress, the ER chaperone BiP binds to unfolded proteins and dissociates from three ER stress sensors, namely PERK, ATF6 and IRE1. Activated PERK attenuates protein translation by phosphorylating eIF2α. Phosphorylated eIF2α also enhances transcription of ATF4 and subsequently increases cytoprotective gene transcription. In addition, phosphorylated eIF2α induces apoptosis through upregulation of CHOP. ATF6 becomes an active transcription factor by translocating to the Golgi apparatus where it is cleaved by proteases. Activated ATF6 stimulates the expression of ER chaperones. ER stress also triggers IRE1 autophosphorylation to activate its endoribonuclease activity, which results in the production of spliced and active XBP-1s, leading to the expression and production of ER chaperones and ER associated degradation proteins. Eukaryotic translation initiation factor 2α (eIF2α); C/EBP-homologous protein (CHOP); activating transcription factor-6 (ATF6); X-box binding protein-1 (XBP-1).


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Figure 2.

Calcium homeostasis in a single cell. In mammalian cells, extracellular calcium fluxes into the cell through plasma membrane (PM) calcium channels and leaves the cell through the plasma membrane (Ca2+-Mg2+)-ATPase (PMCA) and Na+/ Ca2+ exchangers. As the major intracellular Ca2+ storage site, the ER contains Ca2+ at a concentration thousands of times greater than that in the cytosol. The Ca2+ levels inside the ER are modulated by the ERlocated Ca2+ release channels, including inositol-1,4,5-trisphosphate receptor (IP3R) and the ryanodine receptor, and Ca2+ uptake is mediated by the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA). ER-resident stromal interaction molecule (STIM1) can sense changes in ER Ca2+ concentration and activate store-operated Ca2+ entry (SOCE) by interacting with ORAI or TRPC channels in the plasma membrane.

NIH-PA Author Manuscript J Pharmacol Biomed Anal. Author manuscript; available in PMC 2014 March 05.

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