HCV causes chronic endoplasmic reticulum stress leading to adaptation and interference with the unfolded protein response

Background

The endoplasmic reticulum (ER) is the cellular site for protein folding. ER stress occurs when protein folding capacity is exceeded. This stress induces a cyto-protective signaling cascades termed the unfolded protein response (UPR) aimed at restoring homeostasis. While acute ER stress is lethal, chronic sub-lethal ER stress causes cells to adapt by attenuation of UPR activation. Hepatitis C virus (HCV), a major human pathogen, was shown to cause ER stress, however it is unclear whether HCV induces chronic ER stress, and if so whether adaptation mechanisms are initiated. We wanted to characterize the kinetics of HCV-induced ER stress during infection and assess adaptation mechanisms and their significance.

Methods and Findings

The HuH7.5.1 cellular system and HCV-transgenic (HCV-Tg) mice were used to characterize HCV-induced ER stress/UPR pathway activation and adaptation. HCV induced a wave of acute ER stress peaking 2–5 days post-infection, which rapidly subsided thereafter. UPR pathways were activated including IRE1 and EIF2α phosphorylation, ATF6 cleavage and XBP-1 splicing. Downstream target genes including GADD34, ERdj4, p58ipk, ATF3 and ATF4 were upregulated. CHOP, a UPR regulated protein was activated and translocated to the nucleus. Remarkably, UPR activity did not return to baseline but remained elevated for up to 14 days post infection suggesting that chronic ER stress is induced. At this time, cells adapted to ER stress and were less responsive to further drug-induced ER stress. Similar results were obtained in HCV-Tg mice. Suppression of HCV by Interferon-α 2a treatment, restored UPR responsiveness to ER stress tolerant cells.

Conclusions

Our study shows, for the first time, that HCV induces adaptation to chronic ER stress which was reversed upon viral suppression. These finding represent a novel viral mechanism to manipulate cellular response pathways.     

Compromising the unfolded protein response induces autophagy-mediated cell death in multiple myeloma cells

Objective

To determine whether the Unfolded Protein Response (UPR) sensors (PERK, ATF6 and IRE-1) can be targeted to promote death of Multiple Myeloma (MM) cells.

Methods

We have knocked-down separately each UPR stress sensor in human MM cell lines using RNA interference and followed MM cell death by monitoring the membrane, mitochondrial and nuclear alterations. Involvement of caspases in MM cell death consecutive to UPR sensor knock-down was analyzed by western blotting, measurement of their enzymatic activity using fluorigenic substrates and susceptibility to a pan-caspase inhibitor. Activation of the autophagic process was measured directly by detection of autophagosomes (electronic microscopy), monodansylcadaverine staining, production of the cleaved form of the microtubule-associated protein 1A/1B light chain 3 (LC3) and indirectly by analyzing the impact of pharmacological inhibitors of autophagy such as 3MA and bafilomycin A1.

Results

We show that extinction of a single UPR stress sensor (PERK) induces a non-apoptotic form of cell death in MM cells that requires autophagy for its execution. We also show that this cytotoxic autophagic process represses the apoptosis program by reducing the cytosolic release of the apoptogenic factors Smac/DIABLO and cytochrome c.

Interpretation

Altogether our findings suggest that autophagy can contribute to execution of death in mammalian cells that are exposed to mild ER stress. They also suggest that the autophagic process can regulate the intrinsic apoptotic pathway by inhibiting production of death effectors by the mitochondria, thus preventing formation of a functional apoptosome. Altogether these findings give credit to the idea that UPR sensors can be envisaged as therapeutic targets for the treatment of MM.     

The Role of IRE1α in the Degradation of Insulin mRNA in Pancreatic β-Cells

Background

The endoplasmic reticulum (ER) is a cellular compartment for the biosynthesis and folding of newly synthesized secretory proteins such as insulin. Perturbations to ER homeostasis cause ER stress and subsequently activate cell signaling pathways, collectively known as the Unfolded Protein Response (UPR). IRE1α is a central component of the UPR. In pancreatic β-cells, IRE1α also functions in the regulation of insulin biosynthesis.

Principal Findings

Here we report that hyperactivation of IRE1α caused by chronic high glucose treatment or IRE1α overexpression leads to insulin mRNA degradation in pancreatic β-cells. Inhibition of IRE1α signaling using its dominant negative form prevents insulin mRNA degradation. Islets from mice heterozygous for IRE1α retain expression of more insulin mRNA after chronic high glucose treatment than do their wild-type littermates.

Conclusions/Significance

These results reveal a role of IRE1α in insulin mRNA expression under ER stress conditions caused by chronic high glucose. The rapid degradation of insulin mRNA could provide immediate relief for the ER and free up the translocation machinery. Thus, this mechanism would preserve ER homeostasis and help ensure that the insulin already inside the ER can be properly folded and secreted. This adaptation may be crucial for the maintenance of β-cell homeostasis and may explain why the β-cells of type 2 diabetic patients with chronic hyperglycemia stop producing insulin in the absence of apoptosis. This mechanism may also be involved in suppression of the autoimmune type 1 diabetes by reducing the amount of misfolded insulin, which could be a source of “neo-autoantigens.”     

Protective Unfolded Protein Response in Human Pancreatic Beta Cells Transplanted into Mice

Background

There is great interest about the possible contribution of ER stress to the apoptosis of pancreatic beta cells in the diabetic state and with islet transplantation.

Methods and Findings

Expression of genes involved in ER stress were examined in beta cell enriched tissue obtained with laser capture microdissection (LCM) from frozen sections of pancreases obtained from non-diabetic subjects at surgery and from human islets transplanted into ICR-SCID mice for 4 wk. Because mice have higher glucose levels than humans, the transplanted beta cells were exposed to mild hyperglycemia and the abnormal environment of the transplant site. RNA was extracted from the LCM specimens, amplified and then subjected to microarray analysis. The transplanted beta cells showed an unfolded protein response (UPR). There was activation of many genes of the IRE-1 pathway that provide protection against the deleterious effects of ER stress, increased expression of ER chaperones and ERAD (ER-associated protein degradation) proteins. The other two arms of ER stress, PERK and ATF-6, had many down regulated genes. Downregulation of EIF2A could protect by inhibiting protein synthesis. Two genes known to contribute to apoptosis, CHOP and JNK, were downregulated.

Conclusions

Human beta cells in a transplant site had UPR changes in gene expression that protect against the proapoptotic effects of unfolded proteins.     

Suppression of p75 neurotrophin receptor surface expression with intrabodies influences Bcl-xL mRNA expression and neurite outgrowth in PC12 cells

Background

Although p75 neurotrophin receptor (p75NTR) is the first neurotrophin receptor isolated, its diverse physiological functions and signaling have remained elusive for many years. Loss-of-function phenotypic analyses for p75NTR were mainly focused at the genetic level; however these approaches were impacted by off-target effect, insufficient stability, unspecific stress response or alternative active splicing products. In this study, p75NTR surface expression was suppressed for the first time at the protein level by endoplasmic reticulum (ER) retained intrabodies.

Results

Three monoclonal recombinant antibody fragments (scFv) with affinities in the low nanomolar range to murine p75NTR were isolated by antibody phage display. To suppress p75NTR cell surface expression, the encoding genes of these scFvs extended by the ER retention peptide KDEL were transiently transfected into the neuron-like rat pheochromocytoma cell line PC12 and the mouse neuroblastoma x mouse spinal cord hybrid cell line NSC19. The ER retained intrabody construct, SH325-G7-KDEL, mediated a downregulation of p75NTR cell surface expression as shown by flow cytometry. This effect was maintained over a period of at least eight days without activating an unfolded protein response (UPR). Moreover, the ER retention of p75NTR resulted in downregulation of mRNA levels of the anti-apoptotic protein Bcl-xL as well as in strong inhibition of NGF-induced neurite outgrowth in PC12 cells.

Conclusion

The ER retained intrabody SH325-G7-KDEL not only induces phenotypic knockdown of this p75NTR but also p75NTR-associated cellular responses in PC12 cells.     

Intestinal Epithelial-Derived TAK1 Signaling Is Essential for Cytoprotection against Chemical-Induced Colitis

Background

We have previously reported that intestinal epithelium-specific TAK1 deleted mice exhibit severe inflammation and mortality at postnatal day 1 due to TNF-induced epithelial cell death. Although deletion of TNF receptor 1 (TNFR1) can largely rescue those neonatal phenotypes, mice harboring double deletion of TNF receptor 1 (TNFR1) and intestinal epithelium-specific deletion of TAK1 (TNFR1KO/TAK1^IEKO) still occasionally show increased inflammation. This indicates that TAK1 is important for TNF-independent regulation of intestinal integrity.

Methodology/Principal Findings

In this study, we investigated the TNF-independent role of TAK1 in the intestinal epithelium. Because the inflammatory conditions were sporadically developed in the double mutant TNFR1KO/TAK1^IEKO mice, we hypothesize that epithelial TAK1 signaling is important for preventing stress-induced barrier dysfunction. To test this hypothesis, the TNFR1KO/TAK1^IEKO mice were subjected to acute colitis by administration of dextran sulfate sodium (DSS). We found that loss of TAK1 significantly augments DSS-induced experimental colitis. DSS induced weight loss, intestinal damages and inflammatory markers in TNFR1KO/TAK1^IEKO mice at higher levels compared to the TNFR1KO control mice. Apoptosis was strongly induced and epithelial cell proliferation was decreased in the TAK1-deficient intestinal epithelium upon DSS exposure. These suggest that epithelial-derived TAK1 signaling is important for cytoprotection and repair against injury. Finally, we showed that TAK1 is essential for interleukin 1- and bacterial components-induced expression of cytoprotective factors such as interleukin 6 and cycloxygenase 2.

Conclusions

Homeostatic cytokines and microbes-induced intestinal epithelial TAK1 signaling regulates cytoprotective factors and cell proliferation, which is pivotal for protecting the intestinal epithelium against injury.     

Fibroblast Growth Factor Receptor 2c Signaling Is Required for Intestinal Cell Differentiation in Zebrafish

Background

There are four cell lineages derived from intestinal stem cells that are located at the crypt and villus in the mammalian intestine the non-secretory absorptive enterocytes, and the secretory cells, which include mucous-secreting goblet cells, regulatory peptide-secreting enteroendocrine cells and antimicrobial peptide-secreting Paneth cells. Although fibroblast growth factor (Fgf) signaling is important for cell proliferation and differentiation in various tissues, its role in intestinal differentiation is less well understood.

Methodology/Principal Findings

We used a loss of function approach to investigate the importance of Fgf signaling in intestinal cell differentiation in zebrafish; abnormal differentiation of goblet cells was observed when Fgf signaling was inhibited using SU5402 or in the Tg(hsp70ldnfgfr1-EGFP) transgenic line. We identified Fgfr2c as an important receptor for cell differentiation. The number of goblet cells and enteroendocrine cells was reduced in fgfr2c morphants. In addition to secretory cells, enterocyte differentiation was also disrupted in fgfr2c morphants. Furthermore, proliferating cells were increased in the morphants. Interestingly, the loss of fgfr2c expression repressed secretory cell differentiation and increased cell proliferation in the mib^ta52b mutant that had defective Notch signaling.

Conclusions/Significance

In conclusion, we found that Fgfr2c signaling derived from mesenchymal cells is important for regulating the differentiation of zebrafish intestine epithelial cells by promoting cell cycle exit. The results of Fgfr2c knockdown in mib^ta52b mutants indicated that Fgfr2c signaling is required for intestinal cell differentiation. These findings provide new evidences that Fgf signaling is required for the differentiation of intestinal cells in the zebrafish developing gut.     

Sulfur Metabolism Actively Promotes Initiation of Cell Division in Yeast

Background

Sulfur metabolism is required for initiation of cell division, but whether or not it can actively promote cell division remains unknown.

Methodology/Principal Findings

Here we show that yeast cells with more mtDNA have an expanded reductive phase of their metabolic cycle and an increased sulfur metabolic flux. We also show that in wild type cells manipulations of sulfur metabolic flux phenocopy the enhanced growth rate of cells with more mtDNA. Furthermore, introduction of a hyperactive cystathionine-β-synthase (CBS) allele in wild type cells accelerates initiation of DNA replication.

Conclusions/Significance

Our results reveal a novel connection between a key sulfur metabolic enzyme, CBS, and the cell cycle. Since the analogous hyperactive CBS allele in human CBS suppresses other disease-causing CBS mutations, our findings may be relevant for human pathology. Taken together, our results demonstrate the importance of sulfur metabolism in actively promoting initiation of cell division.     

Protein C mutation (A267T) results in ER retention and unfolded protein response activation

Background

Protein C (PC) deficiency is associated with a high risk of venous thrombosis. Recently, we identified the PC-A267T mutation in a patient with PC deficiency and revealed by in vitro studies decreased intracellular and secreted levels of the mutant. The aim of the present study was to characterize the underlying mechanism(s).

Methodology/Principal Findings

CHO-K1 cells stably expressing the wild-type (PC-wt) or the PC mutant were generated. In order to examine whether the PC mutant was subjected to increased intracellular degradation, the cells were treated with several inhibitors of various degradation pathways and pulse-chase experiments were performed. Protein-chaperone complexes were analyzed by treating the cells with a cross-linker followed by Western blotting (WB). Expression levels of the immunoglobulin-binding protein (BiP) and the phosphorylated eukaryotic initiation factor 2α (P-eIF2α), both common ER stress markers, were determined by WB to examine if the mutation induced ER stress and unfolded protein response (UPR) activation. We found no major differences in the intracellular degradation between the PC variants. The PC mutant was retained in the endoplasmic reticulum (ER) and had increased association with the Grp-94 and calreticulin chaperones. Retention of the PC-A267T in ER resulted in UPR activation demonstrated by increased expression levels of the ER stress markers BiP and P-eIF2α and caused also increased apoptotic activity in CHO-K1 cells as evidenced by elevated levels of DNA fragmentation.

Conclusions/Significance

The reduced intracellular level and impaired secretion of the PC mutant were due to retention in ER. In contrast to other PC mutations, retention of the PC-A267T in ER resulted in minor increased proteasomal degradation, rather it induced ER stress, UPR activation and apoptosis.     

The Jagged-2/Notch-1/Hes-1 Pathway Is Involved in Intestinal Epithelium Regeneration after Intestinal Ischemia-Reperfusion Injury

Background

Notch signaling plays a critical role in the maintenance of intestinal crypt epithelial cell proliferation. The aim of this study was to investigate the role of Notch signaling in the proliferation and regeneration of intestinal epithelium after intestinal ischemia reperfusion (I/R) injury.

Methods

Male Sprague-Dawley rats were subjected to sham operation or I/R by occlusion of the superior mesenteric artery (SMA) for 20 min. Intestinal tissue samples were collected at 0, 1, 2, 4, and 6 h after reperfusion. Proliferation of the intestinal epithelium was evaluated by immunohistochemical staining of proliferating nuclear antigen (PCNA). The mRNA and protein expression levels of Notch signaling components were examined using Real-time PCR and Western blot analyses. Immunofluorescence was also performed to detect the expression and location of Jagged-2, cleaved Notch-1, and Hes-1 in the intestine. Finally, the γ-secretase inhibitor DAPT and the siRNA for Jagged-2 and Hes-1 were applied to investigate the functional role of Notch signaling in the proliferation of intestinal epithelial cells in an in vitro IEC-6 culture system.

Results

I/R injury caused increased intestinal crypt epithelial cell proliferation and increased mRNA and protein expression of Jagged-2, Notch-1, and Hes-1. The immunofluorescence results further confirmed increased protein expression of Jagged-2, cleaved Notch-1, and Hes-1 in the intestinal crypts. The inhibition of Notch signaling with DAPT and the suppression of Jagged-2 and Hes-1 expression using siRNA both significantly inhibited the proliferation of IEC-6 cells.

Conclusion

The Jagged-2/Notch-1/Hes-1 signaling pathway is involved in intestinal epithelium regeneration early after I/R injury by increasing crypt epithelial cell proliferation.     

2-Hydroxyoleic Acid Induces ER Stress and Autophagy in Various Human Glioma Cell Lines

Background

2-Hydroxyoleic acid is a synthetic fatty acid with potent anti-cancer activity which does not induce undesired side effects. However, the molecular and cellular mechanisms by which this compound selectively kills human glioma cancer cells without killing normal cells is not fully understood. The present study was designed to determine the molecular bases underlying the potency against 1321N1, SF-767 and U118 human glioma cell lines growth without affecting non cancer MRC-5 cells.

Methodology/Principal Findings

The cellular levels of endoplasmic reticulum (ER) stress, unfolded protein response (UPR) and autophagy markers were determined by quantitative RT-PCR and immunoblotting on 1321N1, SF-767 and U118 human glioma cells and non-tumor MRC-5 cells incubated in the presence or absence of 2OHOA or the ER stress/autophagy inducer, palmitate. The cellular response to these agents was evaluated by fluorescence microscopy, electron microscopy and flow cytometry. We have observed that 2OHOA treatments induced augments in the expression of important ER stress/UPR markers, such as phosphorylated eIF2α, IRE1α, CHOP, ATF4 and the spliced form of XBP1 in human glioma cells. Concomitantly, 2OHOA led to the arrest of 1321N1 cells in the G₂/M phase of the cell cycle, with down-regulation of cyclin B1 and Cdk1/Cdc2 proteins in the three glioma cell lines studied. Finally, 2OHOA induced autophagy in 1321N1, SF-767 and U118 cells, with the appearance of autophagic vesicles and the up-regulation of LC3BI, LC3BII and ATG7 in 1321N1 cells, increases of LC3BI, LC3BII and ATG5 in SF-767 cells and up-regulation of LC3BI and LC3BII in U118 cells. Importantly, 2OHOA failed to induce such changes in non-tumor MRC-5 cells.

Conclusion/Significance

The present results demonstrate that 2OHOA induces ER stress/UPR and autophagy in human glioma (1321N1, SF-767 and U118 cell lines) but not normal (MRC-5) cells, unraveling the molecular bases underlying the efficacy and lack of toxicity of this compound.     

Involvement of Endoplasmic Reticulum Stress in Inflammatory Bowel Disease: A Different Implication for Colonic and Ileal Disease?

Background

Endoplasmic reticulum (ER) stress has been suggested to play a role in inflammatory bowel disease (IBD). The three branches (ATF6, IRE1 and PERK) of the unfolded protein response (UPR) have different roles and are not necessarily activated simultaneously.

Methodology/Principal Findings

Expression of UPR-related genes was investigated in colonic and ileal biopsies of 23 controls, 15 ulcerative colitis (UC) and 54 Crohn''s disease (CD) patients. This expression was confirmed at protein level in colonic and ileal samples of five controls, UC and CD patients. HSPA5, PDIA4 and XBP1s were significantly increased in colonic IBD at mRNA and/or protein levels, indicating activation of the ATF6 and IRE1 branch. Colonic IBD was associated with increased phosphorylation of EIF2A suggesting the activation of the PERK branch, but subsequent induction of GADD34 was not observed. In ileal CD, no differential expression of the UPR-related genes was observed, but our data suggested a higher basal activation of the UPR in the ileal mucosa of controls. This was confirmed by the increased expression of 16 UPR-related genes as 12 of them were significantly more expressed in ileal controls compared to colonic controls. Tunicamycin stimulation of colonic and ileal samples of healthy individuals revealed that although the ileal mucosa is exhibiting this higher basal UPR activation, it is still responsive to ER stress, even more than colonic mucosa.

Conclusions/Significance

Activation of the three UPR-related arms is seen in colonic IBD-associated inflammation. However, despite EIF2A activation, inflamed colonic tissue did not increase GADD34 expression, which is usually involved in re-establishment of ER homeostasis. This study also implies the presence of a constitutive UPR activation in healthy ileal mucosa, with no further activation during inflammation. Therefore, engagement of the UPR differs between colon and ileum and this could be a factor in the development of ileal or colonic disease.     

Evaluating the Potential Bioactivity of a Novel Compound ER1626

Background

ER1626, a novel compound, is a derivate of indeno-isoquinoline ketone. This study was designed to evaluate the biological activity and potential anti-tumor mechanism of ER1626.

Method

MTT assay, scratch assay and flow cytometry were used to determine cell proliferation, cell migration and cell cycle distribution as well as cell apoptosis on human breast cancer MCF-7 cells and endometrial cancer Ishikawa cells. We also explored the antiangiogenic effect of ER1626 on HUVEC cells and chicken embryos. The expression of estrogen receptor protein was investigated with western-blot analysis.

Results

ER1626 down-regulated the expression of estrogen receptor α protein and up-regulated β protein in MCF-7 and Ishikawa cells. The value of IC₅₀ of ER1626 on MCF-7 and Ishikawa cells were respectively 8.52 and 3.08 µmol/L. Meanwhile, ER1626 decreased VEGF secretion of MCF-7 and Ishikawa cells, disturbed the formation of VEGF-stimulated tubular structure in HUVEC cells, and inhibited the angiogenesis on the chicken chorioallantoic membrane. Scratch assay revealed that ER1626 suppressed the migration of MCF-7, Ishikawa and HUVEC cells. In addition to induction tumor cell apoptosis, ER1626 arrested cell cycle in G1/G0 phase in MCF-7 cells and G2/M phase in Ishikawa cells.

Conclusion

In conclusion, our results demonstrated that ER1626 has favorable bioactivities to be a potential candidate against breast cancer and angiogenesis.     

Yokukansan inhibits neuronal death during ER stress by regulating the unfolded protein response

Background

Recently, several studies have reported Yokukansan (Tsumura TJ-54), a traditional Japanese medicine, as a potential new drug for the treatment of Alzheimer''s disease (AD). Endoplasmic reticulum (ER) stress is known to play an important role in the pathogenesis of AD, particularly in neuronal death. Therefore, we examined the effect of Yokukansan on ER stress-induced neurotoxicity and on familial AD-linked presenilin-1 mutation-associated cell death.

Methods

We employed the WST-1 assay and monitored morphological changes to evaluate cell viability following Yokukansan treatment or treatment with its components. Western blotting and PCR were used to observe the expression levels of GRP78/BiP, caspase-4 and C/EBP homologous protein.

Results

Yokukansan inhibited neuronal death during ER stress, with Cnidii Rhizoma (Senkyu), a component of Yokukansan, being particularly effective. We also showed that Yokukansan and Senkyu affect the unfolded protein response following ER stress and that these drugs inhibit the activation of caspase-4, resulting in the inhibition of ER stress-induced neuronal death. Furthermore, we found that the protective effect of Yokukansan and Senkyu against ER stress could be attributed to the ferulic acid content of these two drugs.

Conclusions

Our results indicate that Yokukansan, Senkyu and ferulic acid are protective against ER stress-induced neuronal cell death and may provide a possible new treatment for AD.     

Endoplasmic Reticulum Stress,Unfolded Protein Response and Altered T Cell Differentiation in Necrotizing Enterocolitis

Background

Endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) play important roles in chronic intestinal inflammation. Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency in preterm infants and is characterized by acute intestinal inflammation and necrosis. The objective of the study is to investigate the role of ER stress and the UPR in NEC patients.

Methods

Ileal tissues from NEC and control patients were obtained during surgical resection and/or at stoma closure. Splicing of XBP1 was detected using PCR, and gene expression was quantified using qPCR and Western blot.

Results

Splicing of XBP1 was only detected in a subset of acute NEC (A-NEC) patients, and not in NEC patients who had undergone reanastomosis (R-NEC). The other ER stress and the UPR pathways, PERK and ATF6, were not activated in NEC patients. A-NEC patients showing XBP1 splicing (A-NEC-XBP1s) had increased mucosal expression of GRP78, CHOP, IL6 and IL8. Similar results were obtained by inducing ER stress and the UPR in vitro. A-NEC-XBP1s patients showed altered T cell differentiation indicated by decreased mucosal expression of RORC, IL17A and FOXP3. A-NEC-XBP1s patients additionally showed more severe morphological damage and a worse surgical outcome. Compared with A-NEC patients, R-NEC patients showed lower mucosal IL6 and IL8 expression and higher mucosal FOXP3 expression.

Conclusions

XBP1 splicing, ER stress and the UPR in NEC are associated with increased IL6 and IL8 expression levels, altered T cell differentiation and severe epithelial injury.