Abstract
The ability of translated cellular proteins to perform their functions requires their proper folding after synthesis. The endoplasmic reticulum (ER) is responsible for coordinating protein folding and maturation. Infections, genetic mutations, environmental factors and many other conditions can lead to challenges to the ER known as ER stress. Altering ER homeostasis results in accumulation of misfolded or unfolded proteins. To eliminate this problem, a response is initiated by the cell called the unfolded protein response (UPR), which involves multiple signaling pathways. Prolonged ER stress or a dysregulated UPR can lead to premature apoptosis and an exaggerated inflammatory response. Following these discoveries, ER stress was shown to be related to several chronic diseases, such as diabetes mellitus, neurodegenerative disorders, fatty liver disease and inflammatory bowel disease that have not yet been clearly demonstrated pathophysiologically. Here, we review the field and present up-to-date information on the relationship between biological processing, ER stress, UPR, and several chronic diseases.
Introduction
Eukaryotic cells use various response mechanisms to maintain homeostasis. Perturbations of these mechanisms induced by stressors can lead to disease. One important type of perturbation is endoplasmic reticulum (ER) stress which can lead to abnormalities in the unfolded protein response (UPR). Indeed, this may be an underlying pathophysiologic mechanism of several diseases.
The ER is the largest organelle in eukaryotic cells, and has several important functions such as synthesis, folding, trafficking and translation of proteins. For example, the ER plays a key role in lipid metabolism and also serves as an important intracellular calcium storage site.1 Hence the ER is essential to cell homeostasis and cell survival. This important organelle consists of two different structures: the nuclear envelope and a peripheral part. The rough endoplasmic reticulum (RER) is adjacent to the nucleus and has ribosomes on it. The peripheral part is the smooth endoplasmic reticulum (SER) and has no ribosomes. While the RER plays a role in protein translation, translocation and post-translational modification, the SER transfers proteins synthesized by ribosomes to other intracellular locations. The two parts of the ER are interconnected by tubular structures. These structures provide a well-controlled protective mechanism against errors in protein synthesis or folding.2
ER stress
A small amount of misfolding can occur during protein synthesis even under normal conditions. The amount of misfolded protein can increase due to the complexity of the protein structure or if proteins are produced in high volume.3 Additionally, various homeostasis-perturbing factors such as temperature changes, viral infections, and nutrient deprivation lead to disruption of ER homeostasis. This can result in decreased disulfide bond formation, reduced glycosylation, and decreased energy for chaperone activity. Owing to all of these factors, misfolded protein ratio is progressively increased in the ER lumen. ER stress is defined as altered ER homeostasis and accumulation of misfolded or unfolded proteins in the ER lumen that is due to any physiologic or pathologic reason.4–9
In one of the first studies on ER stress, glucose-regulated proteins (GRP78 and GRP94) were increased after pharmacological inhibition of protein folding in mammalian cells.10 Indeed, ER stress has become a popular topic in research using tissue/cell cultures and genetic assays. An increasing number of human studies indicate that many diseases may be induced by ER stress (figure 1). Success or failure of cellular adaptation to ER stress is a major determinant for susceptibility of these diseases. However, the relationship between ER stress and the other types of stresses such as mitochondrial, oxidative, integrated, and so on, is still not well understood.
Unfolded protein response
UPR is initiated when the transmembrane receptors of the ER sense change in the extracellular environment that disrupt cellular homeostasis. This response includes reductions in protein translation and new protein synthesis, refolding or degradation of misfolded proteins, and autophagy in cases of mild ER stress.6 11–13 In cases of severe and prolonged ER stress, premature apoptosis is triggered by the same signaling pathways.
The UPR involves multifactorial signaling and transcriptional pathways and increases ER protein folding and modification capacity. Furthermore, translation of a global mRNA can be reduced and finally, misfolded proteins can be eliminated through ER-associated degradation (ERAD) and autophagy. This UPR uses multiple signaling pathways. The first few steps of these pathways involve transmembrane receptors. There are three main transmembrane receptors: inositol-requiring enzyme 1α (IRE1α), pancreatic ER kinase (PERK) and activating transcription factor 6α (ATF6α) (figure 2).6 11–13
Inositol-requiring enzyme 1α
IRE1 is a type 1 transmembrane protein which consists of a serine/threonine kinase and an endoribonuclease (RNase) molecule. IRE1α is found in mammalian cells in two subtypes, α and β. IRE1α is essential for embryological development and cell survival. IRE1ß is expressed in all cells but is more concentrated in intestinal and respiratory epithelial cells.14–16 After dissociation of the ER chaperone, which is known as glucose regulated protein-78 or binding immunoglobulin protein (GRP78/BIP), IRE1α becomes active. Then, spliced X box protein-1 (XBP1s) is produced. It is an active transcription factor from unspliced XBP1 by extraction of a 26-base intron with RNase. After the splicing step, XBP1s controls the transcription of genes encoding proteins involved in ERAD, ER enlargement, intracellular trafficking, protein folding, quality control, and phospholipid synthesis. IRE1α also degrades particular microRNAs through regulated IRE1-dependent decay and induces inflammatory responses through activation of nuclear factor κ B (NFκβ) and Jun N-terminal kinase (JNK).11 17–20
Pancreatic ER kinase
PERK is a type 1 transmembrane protein which has cytosolic serine/threonine domains. The other names in the literature are eukaryotic translation initiation factor 2-α kinase 3 and protein kinase R-like ER kinase. When ER stress occurs, PERK becomes active and phosphorylates the eukaryotic translation initiation factor 2α (elF2α) which attenuates global protein synthesis.19 Despite phosphorylated elF2α decreasing global mRNA translation, it increases translation of specific mRNAs such as ATF4. Cytosolic ATF4 molecules enter the nucleus and encode a transcription factor controlling the transcription of genes involved in apoptosis, autophagy, amino acid metabolism, antioxidant response, ER chaperone synthesis, and ER-Golgi trafficking.11 CCAAT-enhancer-binding protein homologous protein (CHOP) which is synthesized at this stage, is involved in both apoptosis and autophagy and is also an important molecule in oxidative stress pathways.21
All UPR pathways have alternative control mechanisms. For example, P58IPK directly interacts with PERK, inhibiting its kinase activity. Under ER stress, p58IPK expression is increased by ATF6 and XBP1 and decreased by elevated BIP levels. This interaction has been explained by the integration of UPR signaling mechanisms.22 23
Most of PERK’s biological activities have been ascribed to its function as an eIF2 kinase. However, it has been shown that nuclear factor erythroid 2 related factor 2 (Nrf2) is a direct substrate of PERK and is an effector of PERK-dependent cell survival.24 Moreover, the antioxidant response is mainly mediated by Nrf2 phosphorylation.24 25
Activating transcription factor 6
Unlike other ER stress sensors, ATF6 is a type 2 membrane receptor that is dislocated from the ER membrane when activated. During ER stress, dissociation of BIP from the luminal domain allows translocating of ATF6 to the Golgi apparatus, where it is subsequently cleaved by site-1 protease (S1P) and S2P at the transmembrane site to release a cytosolic part that enters the nucleus to activate gene transcription.4 Previous studies showed that the main transcription targets in the nucleus are BIP, CHOP, and XBP1.26–28 In mammals, there are two genes, α and β, that encode the ATF6 protein. While double deletion of ATF6 is incompatible with life, normal cell functions can be achieved with a single gene deletion.
The ATF6 molecule consists of a cytosolic domain that contains a basic leucine zipper (bZip) and a stress sensing domain that is located in ER. During ER stress, cytosolic domains release from their transmembrane proteins and they enter the nucleus and regulate the gene transcription. This process is known as regulated intramembrane proteolysis (RIP).26 Different members of the ATF family with similar structures containing bZip have been identified. For example, Luman and old astrocyte specifically induced substance described in astrocytes, as well as cyclic adenosine monophosphate response element binding protein H are members of the ATF family and are shown in liver cells.29 30 According to these studies, ER stress can be effective in different tissues and cells in different RIP transcription pathways.4
ER stress and diabetes
Pancreatic ß cells abundantly synthesize, store and secrete insulin, a polypeptide hormone. Under some conditions, to maintain a stable plasma glucose level between fasting and postprandial periods, large amounts of insulin may be required. That is why pancreatic ß cells have large well-developed ERs.31 Insulin biogenesis requires complex molecular steps that are initiated from the ER of pancreatic ß cells. The precursor of insulin, preproinsulin, is cleaved in the ER lumen and converted to proinsulin. The three intramolecular disulfide bonds formed by the oxidoreductases in the ER provide a mature folding of proinsulin.32 Excessive insulin production as a response to hyperglycemia is caused by ER stress on ß cells. Then, ER stress increases the loss of ß cell mass through inflammatory transcription signals as well as via UPR-associated premature apoptosis. Thus, ER stress can create a vicious circle in the pathogenesis of diabetes. In the PERK knockout model in mice, the ß cell does not attenuate elF2α-mediated translation, so the misfolding process continues and these mice develop infantile diabetes. The most important reason for endocrine pancreatic insufficiency in PERK−/− animals is insufficient β-cell proliferation that can be caused by increasing rates of apoptosis.33 Ramalingam et al showed that angiotensin II increases the expression of CHOP and ATF4 genes and reduces insulin secretion. Additionally angiotensin receptor blockers significantly decrease ER stress level and ameliorate dysregulated insulin secretion. This study showed that these drugs used for diabetic nephropathy prophylaxis are also promising in preventing the development of diabetes.34
Microvascular complications of diabetes such as nephropathy, neuropathy and retinopathy have been found closely related to ER stress-induced apoptosis and impaired UPR.35–37 Renal tubular cells are responsible for a significant amount of renal protein production and are the first cell group in the kidney affected by ER stress.38 Suppression of ER stress can prevent tubular cell apoptosis in diabetic nephropathy.39 An in vivo diabetic retinopathy model demonstrated that hyperglycemia-induced apoptosis mainly occurs with an ER stress-dependent mechanism. Apoptosis signal regulating kinase 1 was found as a key molecule which regulates ER stress-associated apoptosis.40 Furthermore, tauro urso deoxycolic acid which is a well-known ER stress inhibitor emerged as a promising therapy for diabetic retinopathy.41 It has been shown in the Schwann cell culture that short-term fluctuations in glucose levels stimulate oxidative stress and apoptosis through ER stress response. Some of the diabetic neuropathy cases may be explained by this mechanism.42
ER stress and neurodegenerative diseases
Neurons are permanent cells that are incapable of regeneration. Additionally, they are very sensitive to environmental changes such as hypoxia, ischemia and oxidative stress. In recent studies it was found that ER stress is associated with the pathogenesis of some neurodegenerative diseases. These findings are promising for development of new treatment alternatives for some uncurable and some progressive diseases. Misfolded proteins and their aggregates accumulate in affected neurons and surrounding tissues. This is the hallmark of many neurodegenerative diseases.7 For example, misfolded amyloid ß protein was previously identified in the brain tissue of patients with Alzheimer’s disease (AD). Other examples are τ protein in progressive supranuclear palsy (PSP), Lewy bodies in Parkinson’s disease (PD), mutant superoxide dismutase in amyotrophic lateral sclerosis (ALS). Also, altered glutamate signaling has been reported in Huntington disease.7 According to studies in AD, PD and ALS, progressive neurodegeneration is associated with IRE1α UPR signaling pathways. However, in patients with PSP, this association was found with PERK/elF2/ATF4 signaling.43 The role of PERK in neurodegeneration is supported by a study that showed that the synaptic functions were improved in conditional PERK-deleted AD mice.44 Furthermore, treatment with a PERK inhibitor reduced neurodegeneration in mice transfected with prions.45 These studies are promising because they may lead to better treatments of neurodegenerative diseases.
ER stress and liver diseases
Hepatocytes play a key role in various types of metabolic activity including plasma protein synthesis and secretion, lipoprotein metabolism, cholesterol biosynthesis, xenobiotic metabolism and detoxification of drugs. In hepatocytes, ER is susceptible to stress as it synthesizes large amounts of complex proteins.46 UPR is an adaptive mechanism against ER stress. However dysregulated UPR can cause cell death and tissue damage. ER stress response mediated hepatocyte injury and dysregulation of UPR are mostly induced by oxidative stress.47 Recently, dysregulated UPR was implicated in the pathophysiology of insulin resistance, obesity, fatty liver and chronic viral hepatitis. ER stress responses are observed in many liver diseases via different pathways. Table 1 summarizes the key studies to indicate that ER stress plays important roles in development and maintenance of liver diseases.48–56 ER stress has been shown to have a role in both the development of non-alcoholic steatohepatitis (NAFLD) and its transformation into NASH accompanied by inflammation and fibrosis.57 IRE1α and PERK branches of UPR are the most active pathways during the development of fatty liver disease. Furthermore, a close relationship has been reported between impaired hepatic autophagy and ER stress.58 Although these crosstalk mechanisms are still not fully elucidated, some studies emphasized that inhibiting the ER stress and promoting autophagy can ameliorate NAFLD and saturated fatty acid-induced lipotoxitity.59 60 After understanding the importance of ER stress in NAFLD pathogenesis, various potential therapeutic approaches have been reported. Polyunsaturated fatty acids have been shown to inhibit the saturated fat-induced ER stress by reducing the expression of lectin-like oxidized low-density lipoprotein receptor 1.61 N-acetylcysteine, which is an antioxidant drug, inhibits ER stress via the PERK and ATF4 pathway and apoptosis via the caspase 3 pathway in a mouse fatty liver disease model.62 Forkhead box protein O1 (FOXO1) is a transcription factor that plays an important role in the regulation of glycogenolysis and gluconeogenesis. In high fat diet-fed mice, FOXO1 inhibition reduced ER stress-related necroptosis and significantly improved liver function.63 Slymarine is a relatively old drug and its efficacy is controversial in NAFLD treatment. However, a recent article showed that Slymarine reverses many of the detrimental changes in NAFLD mice by reducing the levels of ER stress proteins BIP and XBP-1.64
ER stress and cardiovascular diseases
Recent animal and human studies have shown that UPR and ER stress-related apoptosis is associated with many cardiovascular diseases including heart failure, atherosclerosis, plaque rupture and ischemic heart disease.65 66 In patients with heart failure, apoptosis occurs via CHOP activation instead of JNK or caspase 12.67 Another apoptotic mechanism in heart failure was described by Gao et al. A postmyocardial infarction mice model showed that rapamycin treatment inhibited mechanistic target of rapamycin (mTOR) and ER stress pathways. Due to blockage of these pathways cardiac myocyte apoptosis was inhibited. These results demonstrated that there is a significant crosstalk between the mTOR and ER stress pathways in chronic heart failure.68 Although the ejection fraction seemed within normal limits, significantly increased apoptosis was observed in the cardiac muscle tissues of patients with diabetes due to impaired mitochondrial fat metabolism and increased ER stress.69 The macrophages and smooth muscle cells present in the atherosclerotic plaque make a high amount of proteins and cytokines. That’s why these active cells are highly susceptible to ER stress. The microenvironment and the ER stress that occur during the hypoxia, ischemia and reperfusion, may lead to rupture in the vulnerable plaque by inducing apoptosis.70
A recent study showed that BIP was upregulated in hypoxic pulmonary artery smooth muscle cells. The study demonstrated that the IRE1α-XBP1 pathway is involved in the process of hypoxia-induced pulmonary vascular remodeling. The authors concluded that intervening the IRE1α-XBP1 pathway may be useful for hypoxia-induced pulmonary arterial hypertension therapy.71 In another study, hydrogen sulphate as an anti-inflammatory agent has been shown to improve ER stress and hypoxia-induced pulmonary hypertension.72
An in vivo ischemia reperfusion model showed that selective pharmacological activation of ATF6 is reducing the severity of myocardial damage and preserving cardiac functions by transcriptional reprogramming of protein homeostasis.73 In a study by Haas et al the effects of different compounds on ER stress and oxidative stress were evaluated. As a result, it was seen that most of the compounds with dual stress modifiers consist of cardioprotective drugs such as β-blockers, angiotensin receptor blockers, and statins.74 According to these studies, it can be thought that ER stress inhibitors will play an active role in the prevention and treatment of cardiovascular diseases as well as in the treatment of many diseases in the future.
ER stress and gastrointestinal disease
Intestinal epithelial cells must have a functionally intact ER in order to maintain intestinal homeostasis and correctly perform protein synthesis, folding, modification and secretion. The intestinal epithelium, the largest barrier in the human body, consists of four main types of cells: Paneth cells, goblet cells, enteroendocrine cells and absorptive enterocytes. All of these cells are prone to ER stress even under normal conditions. Paneth cells are one of the most important cells for hosting immune responses. They secrete an abundance of antimicrobial peptides that regulate the composition of the microbiota.75 Goblet cells are also responsible for mucin secretion and for the protective mucin layer structure.76 Enteroendocrine cells synthesize many peptide hormones that provide the motility of the digestive system. Eventually, absorptive enterocytes secrete cytokines and chemokines for hosting immune responses. The degree of protein production in the gastrointestinal tract is reflected in many observed clinical consequences of ER stress. If the intestinal epithelial functions are impaired, many manifestations can emerge related to irritable bowel syndrome, inflammatory bowel disease, celiac disease, and pancreatobilary disease.77
The pathogenesis of inflammatory bowel disease is still not well understood. Recent studies have shown that ER stress (and the associated UPR) has a close relationship with the factors that are suspected to underlie mechanisms in the pathogenesis of inflammatory bowel disease (IBD) such as the regulation of the mucosal barrier, innate and adaptive immune responses and modulation of the intestinal microbiota.3 11 19 78
IRE1 and IBD pathogenesis
XBP1s is a potent transcription factor that is spliced after IRE1 dimerization. This molecule is involved in protein folding, secretion, and maturation; initiation of ERAD signaling; and phospholipid synthesis.28 Spontaneous inflammation in intestinal epithelial cells and a predisposition for DSS-induced colitis occur in XBP1s knockout (KO) mice.79
The IRE1α pathway contributes to the pathogenesis of IBD through four different mechanisms. First, it binds to tumor necrosis factor α receptor-associated factor 2 and activates NFκB, which results in an inflammatory response. Second, IRE1α can initiate apoptosis by activating proapoptotic B cell lymphoma-2-associated B cell lymphoma 2 associated X protein and B cell lymphoma 2 homologous antagonist/killer proteins.80 Third, it directly stimulates p38 mitogen-activated protein kinase and extracellular signal-regulated kinase 1/2, which are stress-activated protein kinases. These molecules are part of the cellular response and ER stress-associated apoptosis.81 Fourth, IRE1α allows the destruction of some specific microRNAs that inhibit caspase 2. Thus, mitochondrial apoptosis is initiated due to activation of caspase 2.18 Mice with IRE1α deletions develop spontaneous colitis by loss of goblet cells and by impairment of the intestinal barrier integrity.82 IRE1ß is an ER stress sensor that is specifically expressed in gastrointestinal epithelium. It has a lower capacity to cleave XBP1 mRNA than IRE1α. Endonuclease activity, which allows the degradation of cytosolic ribosomal RNA is greater, but the potential role of this activity in the pathogenesis of IBD is not known.83 The genetic deletion of IRE1ß increases predisposition for dextran sulphate sodium (DSS)-induced colitis through increasing BIP levels in colonic mucosa. An impaired intestinal barrier and mucin accumulation in enterocytes have been observed in IRE1ß KO mice.14 16
PERK and IBD pathogenesis
Although PERK is structurally similar to IRE1α, it promotes the production of different transcription factors, and thus results in the production of different proteins. elF2α is the most important step in the PERK pathway that determines the fate of the cell. The cell either survives by reducing protein synthesis or dies by entering the path of apoptosis.84 Another important factor is the PERK/CHOP signaling branch. ER stress-activated CHOP can inhibit the peroxisome proliferator-activated protein pathway that degrades NFκB. Thus, NFκB can cause inflammation through increased secretion of interleukin (IL) 8 or other proinflammatory cytokines.85 Furthermore, the CHOP protein increases reactive oxygen radical concentrations and IL-1ß levels through proinflammatory cytokines, resulting in premature apoptosis and colitis.86 IL-8 and IL-23 are known to be important cytokines in the pathogenesis that induces proliferation of T helper 17 cells. Additionally, the CHOP protein enhances IL-23 production induced by toll-like receptor in ER-stressed myeloid cells.87 A meta-analysis showed that some IL-23 polymorphisms are protective for ulcerative colitis while some others are associated with increased susceptibility to ulcerative colitis.88 The studies on inflammation, ER stress and IBD will lead to promising improvements in treatment. In a recent study, it was shown that azathioprine which is a well-known IBD drug, induces autophagy via both the mTOR-1 pathway and the PERK pathway.89 Further studies involving targeted therapeutic options in ER stress and its response mechanisms are needed.
ATF6 and IBD pathogenesis
The membrane-bound transcription factor S1P (Mbtps1) gene encodes the S1P molecule which is responsible for ATF6 activation. Decreased BIP and GRP94 levels have been shown in Mbtps1 KO mice. In addition, this study showed that an incomplete UPR facilitates ER stress by ATF6 and can cause severe DSS-induced colitis.90 ER stress-induced ATF6 KO mice showed decreased BIP, GRP94 and P58 inhibitor protein kinase (p58IPK) gene expression and increased CHOP levels.91 In p58IPK KO mice, goblet cell loss increased, and these mice were more susceptible to DSS-induced colitis.91 The main outcome of all these studies is that there are increases in the susceptibility of mice to DSS-induced colitis in cases where the UPR is suppressed or altered.
Conclusions
ER stress-related disorders and UPR-induced apoptosis have become more popular research foci in the last decade and may be a source of hope for patients with chronic diseases that are difficult to diagnose and treat. However, many interesting mechanisms involving ER stress and UPR pathways are still unknown. We do know that ER stress is a very important and natural part of human life. With further investigation into UPR, the pathway to new and innovative treatments may be uncovered.
Footnotes
Contributors ARK drafted the manuscript. ARK, GNV and QZ edited the manuscript.
Funding This study was supported by the National Institute of Diabetes and Digestive and Kidney Diseases (DK099052 and DK118959) funds to GNV and QZ; and the Department of Veterans Affairs (1l01CX001477) to QZ. ARK was supported by funds received from the Akdamar Fellowship Program, Department of Gastroenterology and Hepatology, Tulane University Health Sciences Center.
Competing interests None declared.
Patient consent for publication Not required.
Provenance and peer review Not commissioned; externally peer reviewed.