Dataset Information

HCV induces oxidative and ER stress, and sensitizes infected cells to apoptosis in SCID/Alb-uPA mice.

ABSTRACT: Hepatitis C virus (HCV) is a blood-borne pathogen and a major cause of liver disease worldwide. Gene expression profiling was used to characterize the transcriptional response to HCV H77c infection. Evidence is presented for activation of innate antiviral signaling pathways as well as induction of lipid metabolism genes, which may contribute to oxidative stress. We also found that infection of chimeric SCID/Alb-uPA mice by HCV led to signs of hepatocyte damage and apoptosis, which in patients plays a role in activation of stellate cells, recruitment of macrophages, and the subsequent development of fibrosis. Infection of chimeric mice with HCV H77c also led an inflammatory response characterized by infiltration of monocytes and macrophages. There was increased apoptosis in HCV-infected human hepatocytes in H77c-infected mice but not in mice inoculated with a replication incompetent H77c mutant. Moreover, TUNEL reactivity was restricted to HCV-infected hepatocytes, but an increase in FAS expression was not. To gain insight into the factors contributing specific apoptosis of HCV infected cells, immunohistological and confocal microscopy using antibodies for key apoptotic mediators was done. We found that the ER chaperone BiP/GRP78 was increased in HCV-infected cells as was activated BAX, but the activator of ER stress-mediated apoptosis CHOP was not. We found that overall levels of NF-kappaB and BCL-xL were increased by infection; however, within an infected liver, comparison of infected cells to uninfected cells indicated both NF-kappaB and BCL-xL were decreased in HCV-infected cells. We conclude that HCV contributes to hepatocyte damage and apoptosis by inducing stress and pro-apoptotic BAX while preventing the induction of anti-apoptotic NF-kappaB and BCL-xL, thus sensitizing hepatocytes to apoptosis.

SUBMITTER: Joyce MA

PROVIDER: S-EPMC2647842 | biostudies-literature | 2009 Feb

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

HCV induces oxidative and ER stress, and sensitizes infected cells to apoptosis in SCID/Alb-uPA mice.

Joyce Michael A MA Walters Kathie-Anne KA Lamb Sue-Ellen SE Yeh Mathew M MM Zhu Lin-Fu LF Kneteman Norman N Doyle Jason S JS Katze Michael G MG Tyrrell D Lorne DL

PLoS pathogens 20090206 2

Hepatitis C virus (HCV) is a blood-borne pathogen and a major cause of liver disease worldwide. Gene expression profiling was used to characterize the transcriptional response to HCV H77c infection. Evidence is presented for activation of innate antiviral signaling pathways as well as induction of lipid metabolism genes, which may contribute to oxidative stress. We also found that infection of chimeric SCID/Alb-uPA mice by HCV led to signs of hepatocyte damage and apoptosis, which in patients pl ...[more]

PMID: 19242562

Similar Datasets

Project description:Pathologic alterations in podocytes lead to failure of an essential component of the glomerular filtration barrier and proteinuria in chronic kidney diseases. Elevated levels of saturated free fatty acid (FFA) are harmful to various tissues, implemented in the progression of diabetes and its complications such as proteinuria in diabetic nephropathy. Here, we investigated the molecular mechanism of palmitate cytotoxicity in cultured mouse podocytes. Incubation with palmitate dose-dependently increased cytosolic and mitochondrial reactive oxygen species, depolarized the mitochondrial membrane potential, impaired ATP synthesis and elicited apoptotic cell death. Palmitate not only evoked mitochondrial fragmentation but also caused marked dilation of the endoplasmic reticulum (ER). Consistently, palmitate upregulated ER stress proteins, oligomerized stromal interaction molecule 1 (STIM1) in the subplasmalemmal ER membrane, abolished the cyclopiazonic acid-induced cytosolic Ca(2+) increase due to depletion of luminal ER Ca(2+). Palmitate-induced ER Ca(2+) depletion and cytotoxicity were blocked by a selective inhibitor of the fatty-acid transporter FAT/CD36. Loss of the ER Ca(2+) pool induced by palmitate was reverted by the phospholipase C (PLC) inhibitor edelfosine. Palmitate-dependent activation of PLC was further demonstrated by following cytosolic translocation of the pleckstrin homology domain of PLC in palmitate-treated podocytes. An inhibitor of diacylglycerol (DAG) kinase, which elevates cytosolic DAG, strongly promoted ER Ca(2+) depletion by low-dose palmitate. GF109203X, a PKC inhibitor, partially prevented palmitate-induced ER Ca(2+) loss. Remarkably, the mitochondrial antioxidant mitoTEMPO inhibited palmitate-induced PLC activation, ER Ca(2+) depletion and cytotoxicity. Palmitate elicited cytoskeletal changes in podocytes and increased albumin permeability, which was also blocked by mitoTEMPO. These data suggest that oxidative stress caused by saturated FFA leads to mitochondrial dysfunction and ER Ca(2+) depletion through FAT/CD36 and PLC signaling, possibly contributing to podocyte injury.

Project description:Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with poor survival rates. The presence of cancer stem-like cells (CSCs) is believed to be among the underlying reasons for the aggressiveness of PDAC, which contributes to chemoresistance and recurrence. However, the mechanisms that induce chemoresistance and inhibit apoptosis remain largely unknown.We used serum-free medium to enrich CSCs from panc-1 human pancreatic cancer cells and performed sphere formation testing, flow cytometry, quantitative reverse transcription polymerase chain reaction (RT-qPCR) and semi-quantitative western blotting to confirm the stemness of panc-1 CSCs. Hallmarks of endoplasmic reticulum (ER) stress, including IRE1, PERK, ATF4, ATF6?, GRP78 and uPA expression, were detected after gemcitabine treatment. Effects of gemcitabine-induced uPA expression on cell invasion, sphere formation, colony formation and gemcitabine sensitivity were detected. Electrophoretic mobility shift assays (EMSAs) and RNA-immunoprecipitation (RIP) were performed to detect interaction between the uPA mRNA 3'-UTR and mutant p53-R273H expressed by panc-1 CSCs. The effects of upregulated uPA by gemcitabine on apoptosis were detected by Annexin V-FITC/PI staining, and the impact of uPA on small molecule CP-31398-restored mutant p53 transcriptional activity was measured by a luciferase reporter assay.Enriched panc-1 CSCs expressing high levels of CD44 and CD133 also produced significantly higher amounts of Oct4 and Nanog. Compared with panc-1 cells, panc-1 CSCs presented chemoresistance to gemcitabine. ER stress gene detections demonstrated effects of gemcitabine-induced ER stress on both the pro-apoptotic and pro-survival branches. ER stress-induced ATF6? upregulated level of uPA by transcriptionally activating GRP78. Gemcitabine-induced uPA promoted invasion, sphere formation and colony formation and attenuated apoptosis induced by gemcitabine in panc-1 CSCs, depending on interaction with mutant p53-R273H. Upregulation of uPA abolished CP-31398-mediated restoration of mutant p53 transcriptional activity in panc-1 CSCs.Gemcitabine treatment induced ER stress and promoted mutant p53-R273H stabilization via transcriptionally activated uPA which may contribute to chemoresistance to gemcitabine. Notably, upregulation of uPA by gemcitabine treatment may lead to the failure of CP-31398; thus, a novel strategy for modulating mutant p53 function needs to be developed.

Project description:We have used homozygous albumin enhancer/promoter-driven urokinase-type plasminogen activator/severe combined immunodeficient (uPA/SCID) mice as hosts for chimeric mice with humanized livers. However, uPA/SCID mice show four disadvantages: the human hepatocytes (h-heps) replacement index in mouse liver is decreased due to deletion of uPA transgene by homologous recombination, kidney disorders are likely to develop, body size is small, and hemizygotes cannot be used as hosts as more frequent homologous recombination than homozygotes. To solve these disadvantages, we have established a novel host strain that has a transgene containing albumin promoter/enhancer and urokinase-type plasminogen activator cDNA and has a SCID background (cDNA-uPA/SCID). We applied the embryonic stem cell technique to simultaneously generate a number of transgenic lines, and found the line with the most appropriate levels of uPA expression-not detrimental but with a sufficiently damaged liver. We transplanted h-heps into homozygous and hemizygous cDNA-uPA/SCID mice via the spleen, and monitored their human albumin (h-alb) levels and body weight. Blood h-alb levels and body weight gradually increased in the hemizygous cDNA-uPA/SCID mice and were maintained until they were approximately 30 weeks old. By contrast, blood h-alb levels and body weight in uPA/SCID chimeric mice decreased from 16 weeks of age onwards. A similar decrease in body weight was observed in the homozygous cDNA-uPA/SCID genotype, but h-alb levels were maintained until they were approximately 30 weeks old. Microarray analyses revealed identical h-heps gene expression profiles in homozygous and hemizygous cDNA-uPA/SCID mice were identical to that observed in the uPA/SCID mice. Furthermore, like uPA/SCID chimeric mice, homozygous and hemizygous cDNA-uPA/SCID chimeric mice were successfully infected with hepatitis B virus and C virus. These results indicate that hemizygous cDNA-uPA/SCID mice may be novel and useful hosts for producing chimeric mice for use in future long-term studies, including hepatitis virus infection analysis or drug toxicity studies.

Dataset Information

HCV induces oxidative and ER stress, and sensitizes infected cells to apoptosis in SCID/Alb-uPA mice.

Publications

HCV induces oxidative and ER stress, and sensitizes infected cells to apoptosis in SCID/Alb-uPA mice.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets