The cell biology of the unfolded protein response

The unfolded protein response (UPR) is an ensemble of signal transduction pathways that respond to perturbations in the oxidative, pro-folding environment of the endoplasmic reticulum. During the past decade, ongoing research implicated these pathways in maintaining homeostasis of cells and organisms exposed to various stresses. Herein, we highlight recent findings regarding the functional role of the UPR in both normal and pathophysiologic processes.     

Cellular response to cigarette smoke and oxidants: adapting to survive

The gaseous and soluble phases of cigarette smoke are sources of oxidants that contribute to the pathogenesis of chronic obstructive pulmonary disease (COPD). Chronic oxidative stress of cigarette smoking induces mucus secretion and inhibits cystic fibrosis transmembrane conductance regulator function. The increased mucus viscosity renders the airways susceptible to bacterial infections, a hallmark of chronic bronchitis. Furthermore, lungs chronically exposed to the toxic mixture of oxidants in cigarette smoke show signs of endoplasmic reticulum stress, unfolded protein response, altered ceramide metabolism, and apoptosis. Fortunately, the respiratory tract has developed effective adaptive cellular mechanisms to limit oxidant damage. Numerous antioxidant enzymes and glutathione-dependent detoxification systems are increased in healthy smokers. The regulation of the antioxidant response is largely dependent on the nuclear factor erythroid 2-related factor-2 (Nrf2) pathway. However, patients with COPD have defective Nrf2 responses. Novel therapies such as 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) to correct defective Nrf2-dependent cellular response may hold promise for patients with COPD.     

IL-8 production in response to cigarette smoke is decreased in epithelial cells from COPD patients

Cigarette smoke is the principal cause of chronic obstructive pulmonary disease (COPD), a disorder characterized by airway inflammation. As epithelial cells are the first line of defense against foreign material, the response of normal epithelial cells to smoke has been extensively studied. However, little is known about how epithelial cells derived from COPD patients respond to ongoing smoke exposure. This study was aimed at comparing the intracellular response of normal human bronchial/tracheal epithelial cells (NHBE) and COPD-diseased human bronchial/tracheal epithelial cells (DHBE) to cigarette smoke.NHBE and DHBE cells were treated with cigarette smoke condensate (CSC) for 24 h. IL-8 production was measured by ELISA and western blot was used to measure TLR4 expression. Cells were pretreated with CLI-095, a TLR4 inhibitor, or the signaling pathway inhibitors PD184352, Helenalin, or PI-103, which inhibit the ERK1/2, NF-κB and PI3K pathways, respectively.NHBE cells increased IL-8 production in a dose-dependent manner in response to CSC while DHBE cells did not show any significant difference and had a much lower production of IL-8 in response to CSC compared to NHBE cells. There was no change in TLR4 expression with CSC exposure. CLI-095 and PD184352 attenuated IL-8 secretion, indicating that CSC-induced inflammation is both TLR4- and ERK1/2-dependent.These results demonstrate that NHBE and DHBE cells differentially respond to cigarette smoke. DHBE cells exhibit a dampened IL-8 release, indicating that COPD is associated with a reduced capacity of airway epithelial cells to respond to foreign material.     

The unfolded protein response affects neuronal cell cycle protein expression: implications for Alzheimer's disease pathogenesis

Alzheimer's disease (AD) is characterized by the accumulation and aggregation of misfolded proteins. The presence of misfolded proteins in the endoplasmic reticulum (ER) triggers a cellular stress response called the unfolded protein response (UPR). Previously, we have shown that the UPR is activated in AD neurons. In actively dividing cells, activation of the UPR is accompanied by decreased cell cycle protein expression and an arrest in the G1 phase of the cell cycle. Aberrant expression of cell cycle proteins has been observed in post mitotic neurons in AD and is suggested to be involved in neurodegeneration. In this study we show that the protein levels of BiP/GRP78, an ER-stress marker, is increased in Braak stages B and C for amyloid deposits. This is in contrast to the levels of cell cycle markers cyclin D1, cyclin E and phosphorylated retinoblastoma protein (ppRb) which are decreased in Braak stage C compared to Braak stage A for amyloid deposits. In addition, we report a negative correlation between neuronal expression of ppRb and expression levels of BiP/GRP78 in control and AD cases. Activation of the UPR in neuronal cells induces changes in cell cycle protein expression similar to these observed in AD brain. ER stress inducers tunicamycin and thapsigargin down-regulate cell cycle proteins ppRb and cyclin D1 in differentiated neuroblastoma cells. In contrast, protein levels of p27, a cyclin dependent kinase inhibitor, are increased after induction of ER-stress using tunicamycin. These data suggest that activation of the UPR affects cell cycle protein expression in neurons during neurodegeneration in AD.     

The unfolded protein response in lung disease

The early steps in the biogenesis of secreted and membrane proteins occur in the lumen of the endoplasmic reticulum (ER), where resident proteins that make up the ER machinery assist in their folding, maturation, and complex assembly. Variation in the load of ER client proteins and in the function of the organelle's aforementioned machinery for coping with that load can lead to an imbalance between the two that is referred to as ER stress. This triggers a cellular response, mediated by highly conserved signaling pathways that collectively restore equilibrium to the protein-folding environment in the organelle by increasing the expression of genes that enhance nearly all aspects of ER function, and by transiently repressing the biosynthesis of new client proteins. Evidence accrued over the past 10 years suggests that ER stress and response to it influence the fate of mutant proteins that fold inefficiently, impact on the functionality of cells and tissues that cope with unusual loads of ER client proteins, and intersect with signaling pathways that influence inflammation and cancer biology. Here, we review some of the basic workings of unfolded protein response and relate them to processes that are of potential relevance to pulmonary disease.     

Acute cigarette smoke exposure in dogs: the inflammatory response

Acute cigarette smoke causes polymorphonuclear leukocyte (neutrophil, PMN) recruitment to the lung followed by loss of elastase from the recruited cells. Dogs were exposed to cigarette smoke with different oxidant content, bronchoalveolar lavage (BAL) was performed, and the cell distribution in the recovered alveolar lining fluid was analyzed. Exposures were 1, 3, or 6 cigarettes on one or multiple days with a maximum dose of 42 cigarettes. The mean percent PMN present in control lavage was 2.01%, while the mean percent PMN recovered in BAL after a dose of 42 1R1 cigarettes was 13.05%. Recoverable PMN, after a single exposure to three 1R1 cigarettes, also increased from 1.7 to 10.4% by 15 h after cessation of smoke exposure. The cell response for multiple (2 and 7) day exposures was similar. The elastase content per BAL neutrophil decreased relative to peripheral blood PMN from the same animals. No free elastolytic activity was found in BAL, but PMN elastase antigen was present. Increased frequency of cigarette smoke exposure delayed the return to homeostatic cell conditions. The increased PMN accumulation observed may result in an increased proteolytic load in the pulmonary interstitium and contribute to the pathogenesis of emphysema.     

Prenatal cigarette smoke exposure and postnatal respiratory responses to hypoxia and hypercapnia

Prenatal cigarette smoke (CS) exposure, in combination with hypoxia and/or hyperthermia can lead to gasping and attenuated recovery from hypoxia in 7 days old rat pups. We studied 95 unanesthetized spontaneously breathing 14 days old rat pups to investigate if the destabilizing effects of increased ambient temperature and prenatal CS exposure on respiratory control observed in 7 days old rats were still evident at day 14. This postnatal age was selected as it is beyond the analogous risk period for SIDS in human. Furthermore, we investigated if the breathing responses to hypercapnia are affected by prenatal CS exposure. Since high ambient (HA) temperature can lead to gasping and aberrant respiratory control, we recorded respiratory patterns at low (24-25°C) and high (29-30°C) ambient temperatures, and under hypoxic or hypercapnic states. No gasping was observed in 14 days old rat pups. During hypoxia, breathing frequency increased in the CS-exposed group under low and HA temperatures. Rectal temperature decreased only in the sham group in response to low ambient temperature hypoxia. At HA temperature, breathing frequency increased in both sham and CS-exposed groups during hypercapnia, however, it remained elevated during washout period only in the sham group. We demonstrate that prenatal CS exposure continues to have profound effects on respiratory and thermoregulatory responses to hypoxia and hypercapnia at day 14. The attenuated respiratory and thermoregulatory responses to acute hypoxia and hypercapnia on day 14 demonstrate a strong interaction between CS exposure, respiratory control, and thermoregulation during postnatal maturation.     

The unfolded protein response and translation attenuation: a modelling approach

Unfolded protein response (UPR) is a stress response to increased levels of unfolded proteins in the endoplasmic reticulum (ER). To deal with this stress, all eukaryotic cells share a well-conserved strategy--the upregulation of chaperons and proteases to facilitate protein folding and to degrade the misfolded proteins. For metazoans, however, an additional and seemingly redundant strategy has been evolved--translation attenuation (TA) of proteins targeted to the ER via the protein kinase PERK pathway. PERK is essential in secretory cells, such as the pancreatic β-cells, but not in non-secretory cell types. We have recently developed a mathematical model of UPR, focusing on the interplay and synergy between the TA arm and the conserved Ire1 arm of the UPR. The model showed that the TA mechanism is beneficial in highly fluctuating environment, for example, in the case where the ER stress changes frequently. Under highly variable levels of ER stress, tight regulation of the ER load by TA avoids excess amount of chaperons and proteases being produced. The model also showed that TA is of greater importance when there is a large flux of proteins through the ER. In this study, we further expand our model to investigate different types of ER stress and different temporal profiles of the stress. We found that TA is more desirable in dealing with the translation stress, for example, prolonged stimulation of proinsulin biosynthesis, than the chemical stress.     

Acinar cell injury induced by inadequate unfolded protein response in acute pancreatitis

Acute pancreatitis (AP) is an inflammatory disorder of pancreatic tissue initiated in injured acinar cells. Severe AP remains a significant challenge due to the lack of effective treatment. The widely-accepted autodigestion theory of AP is now facing challenges, since inhibiting protease activation has negligible effectiveness for AP treatment despite numerous efforts. Furthermore, accumulating evidence supports a new concept that malfunction of a self-protective mechanism, the unfolded protein response(UPR), is the driving force behind the pathogenesis of AP. The UPR is induced by endoplasmic reticulum(ER) stress, a disturbance frequently found in acinar cells, to prevent the aggravation of ER stress that can otherwise lead to cell injury. In addition, the UPR's signaling pathways control NFκB activation and autophagy flux, and these dysregulations cause acinar cell inflammatory injury in AP, but with poorly understood mechanisms. We therefore summarize the protective role of the UPR in AP, propose mechanistic models of how inadequate UPR could promote NFκB's pro-inflammatory activity and impair autophagy's protective function in acinar cells, and discuss its relevance to current AP treatment. We hope that insight provided in this review will help facilitate the research and management of AP.     

Different lung responses to cigarette smoke in two strains of mice sensitive to oxidants.

The development of cigarette smoke-induced pulmonary changes in C57 Bl/6J and DBA/2 mice was investigated. Both strains are sensitive to oxidants and C57Bl/6J mice are moderately deficient in serum alpha1-proteinase inhibitor. Following chronic exposure to cigarette smoke, patchy emphysema was present in mice of both strains, but developed faster in DBA/2 mice. A positive reaction for mouse neutrophil elastase was seen on the septa of both strains. Additionally, the DBA/2 mice developed a uniform parenchymal dilation that was preceded by the appearance of apoptotic cells in areas with a low signal for vascular endothelial growth factor-receptor 2. Fibrotic areas scattered throughout the parenchyma, coupled with a positive immunohistochemical reaction for transforming growth factor-beta was seen only in DBA/2 mice. Both DBA/2 and C57Bl/6J strains showed epithelial cell injury and areas of deciliation in their airways. However, the appearance of goblet cell metaplasia was common in C57Bl/6J mice but rare in DBA/2 mice. A positive immunohistochemical reaction for interleukin (IL)-4, IL-13 and MUC5AC was seen only in the airways of C57Bl/6J mice. Strain characteristics (alpha1-proteinase inhibitor levels, sensitivity to oxidants, and constitutive levels of vascular endothelial growth factor-receptor 2) and phenotypical responses (apoptosis and cytokine distribution) may condition parenchymal and airway changes to cigarette smoke.     

The role of the unfolded protein response in axial spondyloarthritis

Susceptibility to ankylosing spondylitis is highly genetic, with a heritability greater than 90 %. Presence of the HLA-B27 MHC class I allele remains the greatest genetic risk factor identified to date. Beyond its nominal role in antigen presentation, HLA-B27 displays interesting and possibly unique biochemical characteristics which may contribute to disease pathogenesis. During its biosynthesis in the endoplasmic reticulum (ER), HLA-B27 folds very slowly and misfolds, inducing ER stress. Herein, we describe a major outcome of ER stress, the unfolded protein response (UPR), as well as consequences of the UPR for inflammation and autophagy. The ability of the UPR to augment inflammatory cytokine production is particularly intriguing given the centrality of cytokines in spondyloarthritis. Evidence for the relevance of an HLA-B27-related UPR to spondyloarthritis pathogenesis in animal models and human subjects will be reviewed. As greater pharmacologic capacity to modulate ER stress becomes available, improved understanding of the role of the UPR in spondyloarthritis may yield new therapeutic targets.     

The role of the unfolded protein response in the heart

The misfolding of nascent proteins, or the unfolding of proteins after synthesis is complete, can occur in response to numerous environmental stresses, or as a result of mutations that de-stabilize protein structure. Cells have developed elaborate protein quality control systems that recognize improperly folded proteins and either refold them or facilitate their degradation. One such quality control system is the unfolded protein response, or the UPR. The UPR is a highly conserved signal transduction system that is activated when cells are subjected to conditions that alter the endoplasmic reticulum (ER) in ways that impair the folding of nascent proteins in this organelle. Recent observations indicate that in the heart, the UPR is activated during acute stresses, including ischemia/reperfusion, as well as upon longer term stresses that lead to cardiac hypertrophy and heart failure. Moreover, certain aspects of the UPR are activated during, and are required for proper heart development. This review summarizes recent studies of the UPR in the heart, focusing on the possible roles of the UPR in contributing to, or protecting from ischemia/reperfusion damage.     

香烟烟雾提取物对动物气道上皮细胞E钙粘着蛋白表达的影响

目的观察香烟烟雾提取物（ＣＳＥ）对气道上皮细胞（ＡＥＣ）、上皮钙粘附素（ＥＣＤ）表达的影响,探讨其表达变化在吸烟致ＡＥＣ损伤中的作用。方法体外培养小鼠气管环及猪ＡＥＣ,加入一定浓度的ＣＳＥ,应用免疫组化染色和图像分析法检测ＡＥＣ中ＥＣＤ表达的变化。结果正常ＡＥＣ中ＥＣＤ表达于相邻上皮细胞连接处的胞膜上,２０％ＣＳＥ作用１２和２４小时后,膜上ＥＣＤ表达减少,胞浆内表达量分别为正常对照组的１６１和１３２倍（Ｐ均＜００１）,且２４小时表达量为１２小时的１２１倍（Ｐ＜００１）。结论ＣＳＥ使ＡＥＣ中ＥＣＤ膜上表达下调,胞浆内表达增高,该表达变化可能介导了吸烟对ＡＥＣ的损伤作用。