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Showing papers on "Transcription Factor CHOP published in 2010"


Journal ArticleDOI
TL;DR: It is found that ER stress triggers PUMA induction and cell death through a p53-independent mechanism mediated by the ER-stress-inducible transcription factor ATF4 (activating transcription factor 4), and it is proposed that this pathway may be an important therapeutic target relevant to a number of neurodegenerative conditions.
Abstract: An increasing body of evidence points to a key role of endoplasmic reticulum (ER) stress in acute and chronic neurodegenerative conditions. Extensive ER stress can trigger neuronal apoptosis, but the signaling pathways that regulate this cell death remain unclear. In the present study, we demonstrate that PUMA, a Bcl-2 homology 3 (BH3)-only member of the Bcl-2 family, is transcriptionally activated in cortical neurons by ER stress and is essential for ER-stress-induced cell death. PUMA is known to be a key transcriptional target of p53, but we have found that ER stress triggers PUMA induction and cell death through a p53-independent mechanism mediated by the ER-stress-inducible transcription factor ATF4 (activating transcription factor 4). Specifically, we demonstrate that ectopic expression of ATF4 sensitizes mouse cortical neurons to ER-stress-induced apoptosis and that ATF4-deficient neurons exhibit markedly reduced levels of PUMA expression and cell death. However, chromatin immunoprecipitation experiments suggest that ATF4 does not directly regulate the PUMA promoter. Rather, we found that ATF4 induces expression of the transcription factor CHOP (C/EBP homologous protein) and that CHOP in turn activates PUMA induction. Specifically, we demonstrate that CHOP binds to the PUMA promoter during ER stress and that CHOP knockdown attenuates PUMA induction and neuronal apoptosis. In summary, we have identified a key signaling pathway in ER-stress-induced neuronal death involving ATF4–CHOP-mediated transactivation of the proapoptotic Bcl-2 family member PUMA. We propose that this pathway may be an important therapeutic target relevant to a number of neurodegenerative conditions.

300 citations


Journal ArticleDOI
TL;DR: Investigation of proinflammatory cytokine expression by monocyte-derived dendritic cells is affected by the induction of ER stress found the IL-23 gene was found to be a target of the ER stress-induced transcription factor C/EBP homologous protein (CHOP), which exhibited enhanced binding in the context of both ER stress and TLR stimulation.
Abstract: The endoplasmic reticulum (ER) stress response detects malfunctions in cellular physiology, and microbial pattern recognition receptors recognize external threats posed by infectious agents. This study has investigated whether proinflammatory cytokine expression by monocyte-derived dendritic cells is affected by the induction of ER stress. Activation of ER stress, in combination with Toll-like receptor (TLR) agonists, markedly enhanced expression of mRNA of the unique p19 subunit of IL-23, and also significantly augmented secretion of IL-23 protein. These effects were not seen for IL-12 secretion. The IL-23 gene was found to be a target of the ER stress-induced transcription factor C/EBP homologous protein (CHOP), which exhibited enhanced binding in the context of both ER stress and TLR stimulation. Knockdown of CHOP in U937 cells significantly reduced the synergistic effects of TLR and ER stress on IL-23p19 expression, but did not affect expression of other LPS-responsive genes. The integration of ER stress signals and the requirement for CHOP in the induction of IL-23 responses was also investigated in a physiological setting: infection of myeloid cells with Chlamydia trachomatis resulted in the expression of CHOP mRNA and induced the binding of CHOP to the IL-23 promoter. Furthermore, knockdown of CHOP significantly reduced the expression of IL-23 in response to this intracellular bacterium. Therefore, the effects of pathogens and other environmental factors on ER stress can profoundly affect the nature of innate and adaptive immune responses.

277 citations


Journal ArticleDOI
TL;DR: The preservation of photoreceptor function resulting from elevated levels of BiP is due to suppression of apoptosis rather than to a promotion of rhodopsin folding, and the preservation of vision in ADRP rats is preserved.
Abstract: The P23H mutation within the rhodopsin gene (RHO) causes rhodopsin misfolding, endoplasmic reticulum (ER) stress, and activates the unfolded protein response (UPR), leading to rod photoreceptor degeneration and autosomal dominant retinitis pigmentosa (ADRP). Grp78/BiP is an ER-localized chaperone that is induced by UPR signaling in response to ER stress. We have previously demonstrated that BiP mRNA levels are selectively reduced in animal models of ADRP arising from P23H rhodopsin expression at ages that precede photoreceptor degeneration. We have now overexpressed BiP to test the hypothesis that this chaperone promotes the trafficking of P23H rhodopsin to the cell membrane, reprograms the UPR favoring the survival of photoreceptors, blocks apoptosis, and, ultimately, preserves vision in ADRP rats. In cell culture, increasing levels of BiP had no impact on the localization of P23H rhodopsin. However, BiP overexpression alleviated ER stress by reducing levels of cleaved pATF6 protein, phosphorylated eIF2α and the proapoptotic protein CHOP. In P23H rats, photoreceptor levels of cleaved ATF6, pEIF2α, CHOP, and caspase-7 were much higher than those of wild-type rats. Subretinal delivery of AAV5 expressing BiP to transgenic rats led to reduction in CHOP and photoreceptor apoptosis and to a sustained increase in electroretinogram amplitudes. We detected complexes between BiP, caspase-12, and the BH3-only protein BiK that may contribute to the antiapoptotic activity of BiP. Thus, the preservation of photoreceptor function resulting from elevated levels of BiP is due to suppression of apoptosis rather than to a promotion of rhodopsin folding.

263 citations


Journal ArticleDOI
TL;DR: Data reveal an unexpected and novel prosur‐vival role of CerS6/C16‐ceramide involved in the protection against ER‐stress‐induced apoptosis and induction of HNSCC tumor growth.
Abstract: Emerging results suggest that ceramides with different fatty acid chain lengths might play distinct functions in the regulation of tumor growth and therapy. Here we report that de novo-generated C(18)- and C(16)-ceramides by ceramide synthases 1 and 6 (CerS1 and CerS6) play opposing proapoptotic and prosurvival roles, respectively, in human head and neck squamous cell carcinomas (HNSCCs). Unexpectedly, knockdown of CerS6/C(16)-ceramide using small interfering RNA induced endoplasmic reticulum (ER)-stress-mediated apoptosis. Reconstitution of C(16)-ceramide generation by induced expression of wild-type CerS6, but not its catalytically inactive mutant, protected cells from cell death induced by knockdown of CerS6. Moreover, using molecular tools coupled with analysis of sphingolipid metabolism showed that generation of C(16)-ceramide, and not dihydro-C(16)-ceramide, by induced expression of CerS6 rescued cells from ER stress and apoptosis. Mechanistically, regulation of ER-stress-induced apoptosis by CerS6/C(16)-ceramide was linked to the activation of a specific arm, ATF6/CHOP, of the unfolded protein response pathway. Notably, while expression of CerS1/C(18)-ceramide inhibited HNSCC xenograft growth, CerS6/C(16)-ceramide significantly protected ER stress, leading to enhanced tumor development and growth in vivo, consistent with their pro- and antiapoptotic roles, respectively. Thus, these data reveal an unexpected and novel prosurvival role of CerS6/C(16)-ceramide involved in the protection against ER-stress-induced apoptosis and induction of HNSCC tumor growth.

224 citations


Journal ArticleDOI
TL;DR: This study addressed the underlying mechanism for variable expression of ATF4 in response to eIF2∼P during different stress conditions and the biological significance of omission of enhanced ATF4 function, and showed that in addition to translational control, ATF4 expression is subject to transcriptional regulation.

202 citations


Journal ArticleDOI
TL;DR: The results demonstrate that short-hairpin RNA-targeted knockdown of CHOP attenuates palmitate-induced apoptosis in Huh-7 cells and suggest that CHOP and AP-1 cooperatively mediate PUMA induction during hepatocyte lipoapoptosis, and loss of function studies suggest that both transcription factors are necessary for maximalPUMA induction.
Abstract: Endoplasmic reticulum (ER) stress-mediated apoptosis is a key feature of hepatocyte cytotoxicity by saturated free fatty acids (FFA). This lipoapoptosis is dependent, in part, on the transcriptional upregulation of the BH3-only protein PUMA (p53 upregulated modulator of apoptosis). Although the activator protein (AP)-1 complex facilitates PUMA expression by saturated FFA, the transcription factor CAAT/enhancer binding homologous protein (CHOP) is also induced by ER stress and promotes apoptosis. To integrate the role of these two transcription factors in ER stress-induced apoptosis, we examined the relative contribution of CHOP and AP-1 in mediating PUMA induction by saturated FFA. Our results demonstrate that short-hairpin RNA-targeted knockdown of CHOP attenuates palmitate-induced apoptosis in Huh-7 cells. Loss of CHOP induction also reduced the increase in PUMA mRNA and protein levels as well as Bax activation by palmitate. No functional CHOP binding sites were identified in the PUMA promoter sequence. Rather, we observed that CHOP physically interacts with the AP-1 complex protein c-Jun upon palmitate treatment, and a CHOP:phosphorylated c-Jun heteromeric complex binds to the AP-1 consensus binding sequence within the PUMA promoter region. Finally, loss of function studies suggest that both transcription factors are necessary for maximal PUMA induction. Collectively, these data suggest that CHOP and AP-1 cooperatively mediate PUMA induction during hepatocyte lipoapoptosis.

171 citations


Journal ArticleDOI
TL;DR: The results suggest a novel, post-transcriptional mechanism whereby Th17 cytokines are inhibited by VDR, which may underscore future therapeutic usage of vitamin D in treatment of autoimmune diseases.

170 citations


Journal ArticleDOI
TL;DR: It is shown that palmitic acid increases podocyte cell death, both apoptosis and necrosis of podocytes, in a dose and time-dependent fashion and offers a rationale for interventional studies aimed at testing whether dietary shifting of the FFA balance toward unsaturated FFAs can delay the progression of DN.
Abstract: Apoptosis of podocytes is considered critical in the pathogenesis of diabetic nephropathy (DN). Free fatty acids (FFAs) are critically involved in the pathogenesis of diabetes mellitus type 2, in particular the regulation of pancreatic β cell survival. The objectives of this study were to elucidate the role of palmitic acid, palmitoleic, and oleic acid in the regulation of podocyte cell death and endoplasmic reticulum (ER) stress. We show that palmitic acid increases podocyte cell death, both apoptosis and necrosis of podocytes, in a dose and time-dependent fashion. Palmitic acid induces podocyte ER stress, leading to an unfolded protein response as reflected by the induction of the ER chaperone immunoglobulin heavy chain binding protein (BiP) and proapoptotic C/EBP homologous protein (CHOP) transcription factor. Of note, the monounsaturated palmitoleic and oleic acid can attenuate the palmitic acid-induced upregulation of CHOP, thereby preventing cell death. Similarly, gene silencing of CHOP protects against palmitic acid-induced podocyte apoptosis. Our results offer a rationale for interventional studies aimed at testing whether dietary shifting of the FFA balance toward unsaturated FFAs can delay the progression of DN.

164 citations


Journal ArticleDOI
TL;DR: The findings suggest an unprecedented link by which protein synthesis and/or misfolding in the ER causes oxidative stress and should encourage the development of novel strategies to treat diabetes.
Abstract: Diabetes is an epidemic of worldwide proportions caused by β-cell failure. Nutrient fluctuations and insulin resistance drive β-cells to synthesize insulin beyond their capacity for protein folding and secretion and thereby activate the unfolded protein response (UPR), an adaptive signalling pathway to promote cell survival upon accumulation of unfolded protein in the endoplasmic reticulum (ER). Protein kinase-like endoplasmic reticulum kinase (PERK) signals one component of the UPR through phosphorylation of eukaryotic initiation factor 2 on the α-subunit (eIF2α) to attenuate protein synthesis, thereby reducing the biosynthetic burden. β-Cells uniquely require PERK-mediated phosphorylation of eIF2α to preserve cell function. Unabated protein synthesis in β-cells is sufficient to initiate a cascade of events, including oxidative stress, that are characteristic of β-cell failure observed in type 2 diabetes. In contrast to acute adaptive UPR activation, chronic activation increases expression of the proapoptotic transcription factor CAAT/enhancer-binding protein homologous protein (CHOP). Chop deletion in insulin-resistant mice profoundly increases β-cell mass and prevents β-cell failure to forestall the progression of diabetes. The findings suggest an unprecedented link by which protein synthesis and/or misfolding in the ER causes oxidative stress and should encourage the development of novel strategies to treat diabetes.

119 citations


Journal ArticleDOI
TL;DR: It is suggested that baicalein could protect HT22 neuronal cells against ER stress-induced apoptosis by reducing CHOP induction as well as ROS accumulation and mitochondrial damage.
Abstract: Baicalein is one of the major flavonoids in Scutellaria baicalensis Georgi and possesses various effects, including cytoprotection and anti-inflammation. Because endoplasmic reticulum (ER) stress has been implicated in neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and cerebral ischemia, we investigated the effects of baicalein on apoptotic death of HT22 mouse hippocampal neuronal cells induced by thapsigargin (TG) and brefeldin A (BFA), two representative ER stress inducers. Apoptosis, reactive oxygen species (ROS) production, and mitochondrial membrane potential (MMP) were measured by flow cytometry. Expression level and phosphorylation status of ER stress-associated proteins and activation and cleavage of apoptosis-associated proteins were analyzed by Western blot. Baicalein reduced TG- and BFA-induced apoptosis of HT22 cells and activation and cleavage of apoptosis-associated proteins, such as caspase-12 and -3 and poly(ADP-ribose) polymerase. Baicalein also reduced the TG- and BFA-induced expression of ER stress-associated proteins, including C/EBP homologous protein (CHOP) and glucose-regulated protein 78, the cleavage of X-box binding protein-1 and activating transcription factor 6α, and the phosphorylation of eukaryotic initiation factor-2α and mitogen-activated protein kinases, such as p38, JNK, and ERK. Knock-down of CHOP expression by siRNA transfection and specific inhibitors of p38 (SB203580), JNK (SP600125), and ERK (PD98059) as well as anti-oxidant (N-acetylcysteine) reduced TG- or BFA-induced cell death. Baicalein also reduced TG- and BFA-induced ROS accumulation and MMP reduction. Taken together, these results suggest that baicalein could protect HT22 neuronal cells against ER stress-induced apoptosis by reducing CHOP induction as well as ROS accumulation and mitochondrial damage.

105 citations


Journal ArticleDOI
TL;DR: It is shown that the bZIP protein C/EBPβ isoform LIP is required for nuclear translocation of CHOP during ER stress and the LIP-CHOP interaction has a stabilizing role for LIP, and this functional cooperativity as molecular symbiosis between proteins is proposed.
Abstract: Induction of the transcription factor CHOP (CCAAT-binding homologous protein; GADD 153) is a critical cellular response for the transcriptional control of endoplasmic reticulum (ER) stress-induced apoptosis. Upon nuclear translocation, CHOP upregulates the transcription of proapoptotic factors and downregulates antiapoptotic genes. Transcriptional activation by CHOP involves heterodimerization with other members of the basic leucine zipper transcription factor (bZIP) family. We show that the bZIP protein C/EBP beta isoform LIP is required for nuclear translocation of CHOP during ER stress. In early ER stress, LIP undergoes proteasomal degradation in the cytoplasmic compartment. During later ER stress, LIP binds CHOP in both cytoplasmic and nuclear compartments and contributes to its nuclear import. By using CHOP-deficient cells and transfections of LIP-expressing vectors in C/EBP beta(-/-) mouse embryonic fibroblasts (MEFs), we show that the LIP-CHOP interaction has a stabilizing role for LIP. At the same time, CHOP uses LIP as a vehicle for nuclear import. LIP-expressing C/EBP beta(-/-) MEFs showed enhanced ER stress-induced apoptosis compared to C/EBP beta-null cells, a finding in agreement with the decreased levels of Bcl-2, a known transcriptional control target of CHOP. In view of the positive effect of CHOP-LIP interaction in mediating their proapoptotic functions, we propose this functional cooperativity as molecular symbiosis between proteins.

Journal ArticleDOI
16 Dec 2010-PLOS ONE
TL;DR: Results suggest that inhibition of de novo MUFA synthesis by Scd1 extinction could be a promising anti-cancer target by inducing cell death through UPR and CHOP activation.
Abstract: Background Cancer cells present a sustained de novo fatty acid synthesis with an increase of saturated and monounsaturated fatty acid (MUFA) production. This change in fatty acid metabolism is associated with overexpression of stearoyl-CoA desaturase 1 (Scd1), which catalyses the transformation of saturated fatty acids into monounsaturated fatty acids (e.g., oleic acid). Several reports demonstrated that inhibition of Scd1 led to the blocking of proliferation and induction of apoptosis in cancer cells. Nevertheless, mechanisms of cell death activation remain to be better understood. Principal Findings In this study, we demonstrated that Scd1 extinction by siRNA triggered abolition of de novo MUFA synthesis in cancer and non-cancer cells. Scd1 inhibition-activated cell death was only observed in cancer cells with induction of caspase 3 activity and PARP-cleavage. Exogenous supplementation with oleic acid did not reverse the Scd1 ablation-mediated cell death. In addition, Scd1 depletion induced unfolded protein response (UPR) hallmarks such as Xbp1 mRNA splicing, phosphorylation of eIF2α and increase of CHOP expression. However, the chaperone GRP78 expression, another UPR hallmark, was not affected by Scd1 knockdown in these cancer cells indicating a peculiar UPR activation. Finally, we showed that CHOP induction participated to cell death activation by Scd1 extinction. Indeed, overexpression of dominant negative CHOP construct and extinction of CHOP partially restored viability in Scd1-depleted cancer cells. Conclusion These results suggest that inhibition of de novo MUFA synthesis by Scd1 extinction could be a promising anti-cancer target by inducing cell death through UPR and CHOP activation.

Journal ArticleDOI
TL;DR: The data suggest that CVB3 infection activates UPR pathways and induces ER stress-mediated apoptosis through the suppression of P58IPK and induction/activation of CHOP, SREBP1, and caspase-12.
Abstract: Coxsackievirus B3 (CVB3) is a single-stranded positive-sense RNA virus of the genus Enterovirus in the family Picornaviridae and can cause acute or chronic viral myocarditis. Epidemiological studies reveal that viral myocarditis is one of the major heart diseases worldwide, particularly in infants, children, and adolescents (17). Further, CVB3-induced myocarditis can result in dilated cardiomyopathy, a condition for which the only treatment is heart transplantation (12). CVB3 infection has been studied for decades in various systems, but the mechanisms of pathogenesis underlying CVB3-induced myocarditis in humans remain poorly defined. Cumulative evidence suggests that both direct viral injury and subsequent inflammatory responses contribute to the damage of cardiac myocytes, and that the extent of such damage determines the severity of late-stage heart dysfunction (10, 35). Previous studies have documented that apoptosis in cardiomyocytes can result in damaged myocardial tissue and is a hallmark of CVB3-induced myocarditis (1, 45). Although it has been shown that apoptosis facilitates the release of viral progeny during CVB3 infection (9, 48), the molecular events leading to apoptosis in CVB3-infected cells have not been well characterized. The endoplasmic reticulum (ER) system, a primary site for protein synthesis and folding, is a major site of signal initiation and transduction in response to a variety of stimuli, including virus infections (24, 66). Endogenous imbalances in cells, such as the overproduction of proteins, the accumulation of mutant proteins, or the loss of calcium homeostasis, can cause a malfunction of cellular processes and stress to the ER system (26). In response to ER stress, a coordinated adaptive program called the unfolded protein response (UPR) is activated and serves to minimize the accumulation and aggregation of misfolded proteins by increasing the capacity of the ER machinery to fold proteins correctly and activate the degradation of aberrant proteins. The UPR program represents a network of signal transduction from the ER to various locations within the cytoplasm and the nucleus, resulting in either the enhancement of cell survival or the induction of apoptosis (5). Glucose-regulated protein 78 (GRP78) functions as a master regulator of the UPR, and its upregulation indicates the activation of the UPR program (18, 27, 44). Under normal conditions, GRP78 is associated with stress sensor proteins in the ER luminal domain. Under stress conditions, GRP78 is released and binds to misfolded proteins, resulting in the activation of stress sensors (22). The UPR network of interactions is considered to have three major arms, each activated by a characteristic sensor, PERK (PKR-like ER protein kinase), IRE1 (inositol-requiring enzyme 1), and ATF6a (activating transcription factor 6a) (59). The activation of the PERK pathway results in the phosphorylation of the eukaryotic translation initiation factor 2α (eIF2α) subunit, leading to translation attenuation (18). PERK also activates the expression of ATF4, a transcription factor, leading to an upregulation of the proapoptotic genes CHOP (c/EBP homologous protein) and GADD34 (growth arrest and DNA damage-inducible protein-34) (38). IRE1 is a bifunctional ER transmembrane protein with both serine-threonine kinase and RNase activities (52, 56). Upon activation, IRE1 can remove a 26-nucleotide (nt) intron from unspliced X box binding protein 1 (XBP1) mRNA (XBP1u) by RNase activity, resulting in a translational frameshift. The spliced form of XBP1 mRNA (XBP1s) encodes a protein with a novel C terminus and acts as a potent transcriptional activator of many genes involved in the UPR (42). ATF6a is an ER transmembrane protein residing in the cytosol under normal physiological conditions. Upon the accumulation of misfolded proteins in the ER, ATF6a migrates to the Golgi apparatus, where it is cleaved by S1P and S2P proteases, releasing a soluble fragment that enters the nucleus and activates the transcription of ER chaperones and other genes responsible for correct protein folding (20). A number of viruses have been shown to trigger ER stress upon infection. However, the pattern of molecular interactions that occurs within the UPR program differs depending on virus identity and type of host cell. Many viruses apparently activate only one or a subset of UPR pathways, and interestingly, some viral infections activate one pathway yet suppress others. For example, the expression of hepatitis C virus (HCV) proteins activates the PERK- and ATF6a-initiated pathways (4, 8, 40) yet suppresses the IRE1-XBP1 pathway (51). Similarly, human cytomegalovirus (CMV) activates PERK and IRE1-XBP1 but suppresses the ATF6a pathway (23, 53). In this study, with the use of mouse cardiomyocytes and unmodified HeLa cells, as well as HeLa cell lines engineered to inducibly express genes integral to the UPR, we focus on the mechanisms of linkage between the ER stress response to CVB3 infection and the induction of apoptosis. We found that CVB3 infection activates ER stress effectors and differentially regulates the three arms of the UPR. CVB3 infection produced a downregulation of p58IPK and associated enhancement of PKR (PERK) phosphorylation activity, and these alterations affected the other two arms of the UPR. Subsequently, the proapoptotic proteins CHOP, SREBP1 (sterol regulatory element binding protein 1), and caspase-12 can become induced. Taken together, these activities appear to participate in a coordinated shifting of the ER stress response to an apoptotic program in CVB3-infected cells.

Journal ArticleDOI
TL;DR: It is hypothesized that ASFV has other mechanisms to prevent the eIF2α phosphorylation and the subsequent protein synthesis inhibition, and it is demonstrated that DP71L causes dephosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2 α) in resting cells and during chemical-induced endoplasmic reticulum stress and acts to enhance expression of cotransfected reporter genes.
Abstract: The African swine fever virus (ASFV) DP71L protein is present in all isolates as either a short form of 70 to 72 amino acids or a long form of about 184 amino acids, and both of these share sequence similarity to the C-terminal domain of the herpes simplex virus ICP34.5 protein and cellular protein GADD34. In the present study we expressed DP71L in different mammalian cells and demonstrated that DP71L causes dephosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α) in resting cells and during chemical-induced endoplasmic reticulum stress and acts to enhance expression of cotransfected reporter genes. We showed that DP71L binds to all the three isoforms (α, β, and γ) of the protein phosphatase 1 catalytic subunit (PP1c) and acts by recruiting PP1c to eIF2α. We also showed that DP71L inhibits the induction of ATF4 and its downstream target, CHOP. We investigated the eIF2α phosphorylation status and induction of CHOP in porcine macrophages infected by two ASFV field isolates, Malawi Lil20/1 and Benin 97/1, and two DP71L deletion mutants, MalawiΔNL and E70ΔNL. Our results showed that deletion of the DP71L gene did not cause an increase in the level of eIF2α phosphorylation or induction of CHOP, indicating that DP71L is not the only factor required by the virus to control the phosphorylation level of eIF2α during infection. We therefore hypothesize that ASFV has other mechanisms to prevent the eIF2α phosphorylation and the subsequent protein synthesis inhibition.

Journal ArticleDOI
TL;DR: It is demonstrated that TRB3 can inhibit basal and stimulated podocyte production of MCP-1, a chemokine that contributes to the inflammatory injury associated with diabetic nephropathy.
Abstract: The prevalence of diabetic nephropathy continues to rise, highlighting the importance of investigating and discovering novel treatment strategies. TRB3 is a kinase-like molecule that modifies cellular survival and metabolism and interferes with signal transduction pathways. Herein, we report that TRB3 expression is increased in the kidneys of type 1 and type 2 diabetic mice. TRB3 is expressed in conditionally immortalized podocytes; however, it is not stimulated by elevated glucose. The diabetic milieu is associated with increased oxidative stress and circulating free fatty acids (FFA). We show that reactive oxygen species (ROS) such as H(2)O(2) and superoxide anion (via the xanthine/xanthine oxidase reaction) as well as the FFA palmitate augment TRB3 expression in podocytes. C/EBP homologous protein (CHOP) is a transcription factor that is associated with the endoplasmic reticulum stress response. CHOP expression increases in diabetic mouse kidneys and in podocytes treated with ROS and FFA. In podocytes, transfection of CHOP increases TRB3 expression, and ROS augment recruitment of CHOP to the proximal TRB3 promoter. MCP-1/CCL2 is a chemokine that contributes to the inflammatory injury associated with diabetic nephropathy. In these studies, we demonstrate that TRB3 can inhibit basal and stimulated podocyte production of MCP-1. In summary, enhanced ROS and/or FFA associated with the diabetic milieu induce podocyte CHOP and TRB3 expression. Because TRB3 inhibits MCP-1, manipulation of TRB3 expression could provide a novel therapeutic approach in diabetic kidney disease.

Journal ArticleDOI
TL;DR: The collective results support the view that balances between CHOP and PPARγ are crucial for epithelial homeostasis, and disruption of these balances in mucosal ER stress can etiologically affect the progress of human inflammatory bowel diseases.

Journal ArticleDOI
29 Jul 2010-PLOS ONE
TL;DR: ER stress plays an important role in α-TEA induced apoptosis by enhancing DR5/caspase-8 pro-apoptotic signaling and suppressing anti-ap optotic factors c-FLIP and Bcl-2 via ER stress mediated JNK/CHOP/DR5/ caspase -8 signaling.
Abstract: Background: a-TEA (RRR-a-tocopherol ether-linked acetic acid analog), a derivative of RRR-a-tocopherol (vitamin E) exhibits anticancer actions in vitro and in vivo in variety of cancer types. The objective of this study was to obtain additional insights into the mechanisms involved in a-TEA induced apoptosis in human breast cancer cells. Methodology/Principal Findings: a-TEA induces endoplasmic reticulum (ER) stress as indicated by increased expression of CCAAT/enhancer binding protein homologous protein (CHOP) as well as by enhanced expression or activation of specific markers of ER stress such as glucose regulated protein (GRP78), phosphorylated alpha subunit of eukaryotic initiation factor 2 (peIF-2a), and spliced XBP-1 mRNA. Knockdown studies using siRNAs to TRAIL, DR5, JNK and CHOP as well as chemical inhibitors of ER stress and caspase-8 showed that: i) a-TEA activation of DR5/caspase-8 induces an ER stress mediated JNK/ CHOP/DR5 positive amplification loop; ii) a-TEA downregulation of c-FLIP (L) protein levels is mediated by JNK/CHOP/DR5 loop via a JNK dependent Itch E3 ligase ubiquitination that further serves to enhance the JNK/CHOP/DR5 amplification loop by preventing c-FLIP’s inhibition of caspase-8; and (iii) a-TEA downregulation of Bcl-2 is mediated by the ER stress dependent JNK/CHOP/DR5 signaling. Conclusion: Taken together, ER stress plays an important role in a-TEA induced apoptosis by enhancing DR5/caspase-8 proapoptotic signaling and suppressing anti-apoptotic factors c-FLIP and Bcl-2 via ER stress mediated JNK/CHOP/DR5/caspase8 signaling.

Journal ArticleDOI
TL;DR: It is shown that anisomycin-induced CHOP expression depends on phosphorylated eIF4E/S209 and eIF2α/S51 and the significance of this cis-regulatory element in conferring on the transcript a stress-responsive translational inducibility is pinpointed.
Abstract: Cells respond to environmental stress by inducing translation of a subset of mRNAs important for survival or apoptosis. CHOP, a downstream transcriptional target of stress-induced ATF4, is also regulated translationally in a uORF-dependent manner under stress. Low concentration of anisomycin induces CHOP expression at both transcriptional and translational levels. To study specifically the translational aspect of CHOP expression, and further clarify the regulatory mechanisms underlying stress-induced translation initiation, we developed a CMV promoter-regulated, uORFchop-driven reporter platform. Here we show that anisomycin-induced CHOP expression depends on phosphorylated eIF4E/S209 and eIF2α/S51. Contrary to phospho-eIF2α/S51, phospho-eIF4E/S209 is not involved in thapsigargin-induced CHOP expression. Studies using various kinase inhibitors and mutants uncovered that both the p38MAPK-Mnk and mTOR signaling pathways contribute to stress-responsive reporter and CHOP expression. We also demonstrated that anisomycin-induced translation is tightly regulated by partner binding preference of eIF4E. Furthermore, mutating the uORF sequence abolished the anisomycin-induced association of chop mRNA with phospho-eIF4E and polysomes, thus demonstrating the significance of this cis-regulatory element in conferring on the transcript a stress-responsive translational inducibility. Strikingly, although insulin treatment activated ERK-Mnk and mTOR pathways, and consequently eIF4E/S209 phosphorylation, it failed to induce phospho-eIF2α/S51 and reporter translation, thus pinpointing a crucial determinant in stress-responsive translation.

Journal ArticleDOI
TL;DR: The role of C/EBP homologous protein (CHOP), a transcription factor mediating apoptosis in many cell types that experience high levels of ER stress, is analyzed, implying tissue-specific roles for CHOP in differentiation and stress.

Journal ArticleDOI
TL;DR: It is shown that tunicamycin, an antibiotic promoting ER stress, suppresses the expression of p21, a tumor suppressor that induces cell cycle arrest and inhibits apoptosis and is consistent with a CHOP-dependent role for p21 in the shift from the pro-survival to thePro-apoptotic function of UPR.
Abstract: The transcription factor CHOP/GADD153 is induced during the unfolded protein response (UPR) and is associated to the induction of ER stress-related apoptosis. However, how the transition between the pro-survival and the pro-apoptotic role of ER stress is being orchestrated remains poorly understood. Here we show that tunicamycin, an antibiotic promoting ER stress, suppresses the expression of p21, a tumor suppressor that induces cell cycle arrest and inhibits apoptosis. This suppression of p21 levels was independent of p53 that is the major transcriptional regulator of p21, but could be reproduced by forced expression of CHOP. Consistently with these findings, siRNA-mediated inhibition of p21 levels restored the sensitivity of CHOP-deficient cells to tunicamycin. Our findings are consistent with a CHOP-dependent role for p21 in the shift from the pro-survival to the pro-apoptotic function of UPR.

Journal ArticleDOI
TL;DR: In this paper, the authors investigated the involvement of the transcription factors belonging to the CCAAT/enhancer binding protein (C/EBP) family that regulate expression of many of the pro-inflammatory genes which show increased expression following injury to the aged brain.

Journal ArticleDOI
TL;DR: It is suggested that increased UPR and induction of CHOP are involved in enhanced anti-MCL activity of the combination of panobinostat and bortezomib.
Abstract: Purpose: Bortezomib induces unfolded protein response (UPR) and endoplasmic reticulum stress, as well as exhibits clinical activity in patients with relapsed and refractory mantle cell lymphoma (MCL). Here, we determined the molecular basis of the improved in vitro and in vivo activity of the combination of the pan-histone deacetylase inhibitor panobinostat and bortezomib against human, cultured, and primary MCL cells. Experimental Design: Immunoblot analyses, reverse transcription-PCR, and immunofluorescent and electron microscopy were used to determine the effects of panobinostat on bortezomib-induced aggresome formation and endoplasmic reticulum stress in MCL cells. Results: Treatment with panobinostat induced heat shock protein 90 acetylation; depleted the levels of heat shock protein 90 client proteins, cyclin-dependent kinase 4, c-RAF, and AKT; and abrogated bortezomib-induced aggresome formation in MCL cells. Panobinostat also induced lethal UPR, associated with induction of CAAT/enhancer binding protein homologous protein (CHOP). Conversely, knockdown of CHOP attenuated panobinostat-induced cell death of MCL cells. Compared with each agent alone, cotreatment with panobinostat increased bortezomib-induced expression of CHOP and NOXA, as well as increased bortezomib-induced UPR and apoptosis of cultured and primary MCL cells. Cotreatment with panobinostat also increased bortezomib-mediated in vivo tumor growth inhibition and improved survival of mice bearing human Z138C MCL cell xenograft. Conclusion: These findings suggest that increased UPR and induction of CHOP are involved in enhanced anti-MCL activity of the combination of panobinostat and bortezomib. Clin Cancer Res; 16(19); 4742–54. ©2010 AACR.

Journal ArticleDOI
TL;DR: It is concluded that hypusination by DHS is required for the ongoing production of proteins, particularly CHOP, in response to ER stress in the β cell.

Journal ArticleDOI
TL;DR: The data suggest that activation of the apoptotic arm of the UPR and its downstream effector CHOP/GADD153 is involved, at least in part, in RES-induced apoptosis in Burkitt's lymphoma cells.
Abstract: Resveratrol (RES), a natural phytoalexin found at high levels in grapes and red wine, has been shown to induce anti-proliferation and apoptosis of human cancer cell lines. However, the underlying molecular mechanisms are at present only partially understood. The effects of RES on activation of unfolded protein responses (UPR) were evaluated using Western blotting, semi-quantitative and real-time RT-PCR. Cell death was evaluated using Annexin V/PI staining and subsequent FACS. Similar as tunicamycin, treatment with RES lead to the activation of all 3 branches of the UPR, with early splicing of XBP-1 indicative of IRE1 activation, phosphorylation of eIF2α consistent with ER resident kinase (PERK) activation, activating transcription factor 6 (ATF6) splicing, and increase in expression levels of the downstream molecules GRP78/BiP, GRP94 and CHOP/GADD153 in human Burkitt's lymphoma Raji and Daudi cell lines. RES was shown to induce cell death, which could be attenuated by thwarting upregulation of CHOP. Our data suggest that activation of the apoptotic arm of the UPR and its downstream effector CHOP/GADD153 is involved, at least in part, in RES-induced apoptosis in Burkitt's lymphoma cells.

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TL;DR: Silencing using siRNA of both bax and bak genes, but not bax, bak, or bim alone, resulted in reduction of cytochrome c release, caspase-3 activation, DNA ladder formation and cytotoxicity, indicating that Bax and Bak were involved in apoptosis.

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TL;DR: Taurine statistically significantly attenuated lung injury following LIR, as shown by reduced malondialdehyde content, reduced cell apoptosis, and expression of activating transcription factor 4, X-box binding protein 1 (XBP1), and transcriptional activators of the CHOP gene.
Abstract: Background and purpose CHOP is a C/EBP family transcription factor involved in endoplasmic reticulum (ER) stress-mediated apoptosis. Several studies have demonstrated that ischemia reperfusion resu...

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TL;DR: It is suggested that ER stress plays a critical role in palmitate-induced lipotoxicity both in vitro and in vivo.

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TL;DR: It is shown that nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways regulate the expression of CCAAT/enhancer-binding protein homologous protein (CHOP), which mediates endoplasmic reticulum stress-induced apoptosis.

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Toru Hosoi1, Ayaka Kume1, Kayo Otani1, Tatsuya Oba1, Koichiro Ozawa1 
TL;DR: This study found that amiloride, a potassium‐sparing diuretic, has unique properties relating to such stress as well as pharmacological modulators of these ER‐regulated stress responses.
Abstract: Background and purpose: Stress on the endoplasmic reticulum (ER) can trigger rescuer responses such as the unfolded protein response (UPR). However, pharmacological modulators of these ER-regulated stress responses are not well understood. In the present study, we found that amiloride, a potassium-sparing diuretic, has unique properties relating to such stress. Experimental approach: We treated mouse primary cultured glial cells with amiloride, in the absence and presence of the ER stress-inducing reagents tunicamycin (Tm) or dithiothreitol, and measured UPR and ER stress-induced cell death. IRE1α phosphorylation, eIF2α phosphorylation, X-box binding protein 1 (XBP1) splicing, glucose regulated protein 78 (GRP78) and CCAAT/enhancer-binding protein homologous protein (CHOP) expression by reverse transcription-polymerase chain reaction and Western blotting were used to assess UPR and lactate dehydrogenase activity was determined to measure ER stress-induced cell death. Key results: Amiloride completely inhibited ER stress-induced activation of IRE1α, an ER-localized stress sensor protein, splicing of XBP1, and subsequent expression of GRP78 at the mRNA and protein levels. ER stress induces the phosphorylation of eIF2α, leading to the expression of CHOP or an attenuation of translation in cells. Surprisingly, treatment with amiloride alone markedly promoted the phosphorylation but actually inhibited ER stress-induced CHOP expression. Finally, we found that amiloride (200 µM) synergistically enhanced ER stress-induced cell death, which was mediated through caspases. On the other hand, a low dose of amiloride (20 µM) significantly prevented Tm-induced cell death. Conclusions and implications: These results suggest that amiloride can modulate UPR. They also suggest amiloride to be an important pharmacological agent and provide basic information for understanding and preventing ER stress-related diseases.

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TL;DR: It is demonstrated that elevated concentrations of UCB (50 μM) are able to activate select components of the ER stress pathway in Hepa 1c1c7 cells, which may contribute to UCB‐mediated apoptosis.
Abstract: Elevated concentrations of unconjugated bilirubin (UCB) are responsible for neonatal jaundice and can eventually lead to kernicterus or death. The molecular mechanism of UCB toxicity is incompletely elucidated. The purpose of this study was to analyze changes in gene regulation mediated by UCB to determine novel pathways that contribute to UCB-mediated toxicity. We employed microarray analysis to determine changes in gene regulation mediated by UCB at both pro- (50 μM) and antioxidant (70 nM) concentrations in Hepa 1c1c7 cells at 1 and 6 h. The changes observed in select genes were validated with qPCR. Using immunoblot analysis, we validated these changes at the protein level for select genes and documented the activation of two proteins involved in the endoplasmic reticulum (ER) stress pathway, eIF2α and PERK. Following treatment with 50 μM UCB, microarray analysis revealed the upregulation of many genes involved in ER stress (ATF3, BiP, CHOP, Dnajb1, and Herp). We demonstrate that upregulation of the proapoptotic transcription factor CHOP results in increased intracellular protein content. It was determined that activation of proteins involved in ER stress was an early event in UCB toxicity as eIF2α and PERK were both phosphorylated and activated by 1 h posttreatment. We also demonstrate that procaspase-12 content, a proposed initiator caspase in ER stress-mediated apoptosis, is decreased by 4 h posttreatment. In conclusion, this study demonstrates that elevated concentrations of UCB (50 μM) are able to activate select components of the ER stress pathway in Hepa 1c1c7 cells, which may contribute to UCB-mediated apoptosis. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:73–88, 2010; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20313