The endoplasmic reticulum (ER) is the site of synthesis and folding of membrane and secretory proteins, which, collectively, represent a large fraction of the total protein output of a mammalian cell. Therefore, the protein flux through the ER must be carefully monitored for abnormalities, including the buildup of misfolded proteins. Mammalian cells have evolved an intricate set of signaling pathways from the ER to the cytosol and nucleus, to allow the cell to respond to the presence of misfolded proteins within the ER. These pathways, known collectively as the unfolded protein response, are important for normal cellular homeostasis and organismal development and may also play key roles in the pathogenesis of many diseases. This review provides background information on the unfolded protein response and discusses a selection of diseases whose pathogenesis involves ER stress.
Knockout studies have shown that the transcription factor Nrf1 is essential for embryonic development. Nrf1 has been implicated to play a role in mediating activation of oxidative stress response genes through the antioxidant response element (ARE). Because of embryonic lethality in knockout mice, analysis of this function in the adult knockout mouse was not possible. We report here that mice with somatic inactivation of nrf1 in the liver developed hepatic cancer. Before cancer development, mutant livers exhibited steatosis, apoptosis, necrosis, inflammation, and fibrosis. In addition, hepatocytes lacking Nrf1 showed oxidative stress, and gene expression analysis showed decreased expression of various ARE-containing genes, and up-regulation of CYP4A genes. These results suggest that reactive oxygen species generated from CYP4A-mediated fatty acid oxidation work synergistically with diminished expression of ARE-responsive genes to cause oxidative stress in mutant hepatocytes. Thus, Nrf1 has a protective function against oxidative stress and, potentially, a function in lipid homeostasis in the liver. Because the phenotype is similar to nonalcoholic steatohepatitis, these animals may prove useful as a model for investigating molecular mechanisms of nonalcoholic steatohepatitis and liver cancer. hepatocellular carcinoma ͉ oxidative stress ͉ knockout mouse T ranscription of many cytoprotective genes and phase-2 xenobiotic metabolizing genes is regulated through cis-active sequences known as antioxidant response elements (ARE) (1, 2). Regulation of ARE function is mediated by various basic leucine zipper (bZIP) transcription factors including members of the ''cap n collar'' (CNC)-bZIP and small-Maf family of proteins. Nrf1 and Nrf2 are CNC-bZIP proteins, and they function as obligate heterodimers by complexing with small-Maf and other bZIP proteins (3). An important role for Nrf2 in xenobiotic metabolism and oxidative stress response had been identified through knockout studies in mice (4-10). These and other studies indicate that Nrf2 is an important activator of AREs.In contrast, the function of Nrf1 is not fully understood. Mice deficient in Nrf1 function die during development (11). Analysis of nrf1 and nrf1::nrf2 mutant cells suggests that Nrf1 is also involved in the oxidative stress response (12, 13). However, the importance of Nrf1 in this function in an intact animal is not certain because early lethality precludes analysis of Nrf1-deficient animals beyond embryonic development. Chimeric mice generated with Nrf1-deficient embryonic stem cells showed widespread apoptosis in fetal livers at late gestation, demonstrating a cell autonomous role of Nrf1 in the survival of hepatocytes (14). This finding suggests that Nrf1 is required for normal function of hepatocytes. Based on these findings, we hypothesize that Nrf1 is critical to the oxidative stress response in the adult liver, and that it plays an important role in oxidative stress-induced liver disease. To bypass embryonic lethality, we used a Cre-lox system ...
The CNC-basic leucine zipper (CNC-bZIP) family is a subfamily of bZIP proteins identified from independent searches for factors that bind the AP-1-like cis-elements in the β-globin locus control region. Three members, p45-Nf-e2, Nrf-1 and Nrf-2 have been identified in mammals. Expression of p45-Nf-e2 is largely restricted to hematopoietic cells while Nrf-1 and Nrf-2 are expressed in a wide range of tissues. To determine the function of Nrf-1, targeted disruption of the Nrf-1 gene was carried out. Homozygous Nrf-1 mutant mice are anemic due to a non-cell autonomous defect in definitive erythropoiesis and die in utero.
The basal core promoter (BCP) of hepatitis B virus (HBV) controls the transcription of both the precore RNA and the core RNA. The precore RNA codes for the secreted e antigen, while the core RNA codes for the major core protein and the DNA polymerase and also is the pregenomic RNA. The double mutation of nucleotides 1762 and 1764 in the BCP from A and G to T and A, respectively, is frequently observed in HBV sequences isolated from chronic patients. Several papers have reported conflicting results regarding whether this double mutation is important for e antigen expression. In order to address this issue, we have introduced this double mutation into the HBV genome and studied its effects on HBV gene expression and replication. Our results indicate that the mutated BCP can no longer bind a liver-enriched transcription factor(s) and that the transcription of only precore RNA and, consequently, the expression of e antigen were reduced. The reduction of precore gene expression was accompanied by an increase in progeny virus production. This increase was found to occur at or immediately prior to the encapsidation of the pregenomic RNA. Thus, the results of our in vitro study resolve the discrepancy of previous clinical observations and indicate that this double mutation suppresses but does not abolish the e antigen phenotype. The implications of these findings in the pathogenesis of HBV are discussed.
Proteins that directly regulate TNFR signaling play critical roles in regulating cellular activation and survival. A20 Binding and Inhibitor of NFkB (ABIN-1) is a novel protein that is thought to inhibit NFkB signaling (1, 2). Here we show that mice deficient for ABIN-1 die during embryogenesis with fetal liver apoptosis, anemia and hypoplasia. ABIN-1 deficient cells are hypersensitive to TNF-induced PCD, and TNF deficiency rescues ABIN-1 deficient embryos. ABIN-1 inhibits caspase 8 recruitment to FADD in TNF-induced signaling complexes, preventing caspase 8 cleavage and PCD. Moreover, ABIN-1 directly binds polyubiquitin chains and this ubiquitin sensing activity is required for ABIN-1’s anti-apoptotic activity. These studies provide new insights into how ubiquitination and ubiquitin sensing proteins regulate cellular and organismal survival.
Hepatitis C virus (HCV) is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Studies of HCV replication and pathogenesis have so far been hampered by the lack of an efficient tissue culture system for propagating HCV in vitro. Although HCV is primarily a hepatotropic virus, an increasing body of evidence suggests that HCV also replicates in extrahepatic tissues in natural infection. In this study, we established a B-cell line (SB) from an HCV-infected non-Hodgkin's B-cell lymphoma. HCV RNA and proteins were detectable by RNase protection assay and immunoblotting. The cell line continuously produces infectious HCV virions in culture. The virus particles produced from the culture had a buoyant density of 1.13 to 1.15 g/ml in sucrose and could infect primary human hepatocytes, peripheral blood mononuclear cells Hepatitis C virus (HCV) has been the major etiological agent of posttransfusion non-A, non-B hepatitis and currently afflicts Ͼ100 million people worldwide. Acute HCV infection is usually subclinical without obvious symptoms. About 15 to 20% of patients can mount a successful immune response to clear the virus in the acute phase; however, 80 to 85% of patients become chronic carriers, and these patients are at high risk of developing liver cirrhosis and/or hepatocellular carcinoma.Besides causing liver pathology, HCV infection is frequently associated with mixed cryoglobulinemia, non-Hodgkin's B-cell lymphoma, and Sjögren's syndrome, all of which involve B-cell proliferation (8, 10, 27, 37, 49; P. Pioltelli, G. Zehender, G. Minti, A. Monteverde, and M. Galli, Letter, Lancet 347:624-625, 1996), suggesting that HCV may infect B cells or affect B-cell functions in natural infection. Negative-strand HCV RNA has been detected by reverse transcriptase (RT) PCR in the peripheral lymphocytes, bone marrow, lymph nodes, and central nervous system of some HCV patients (23,30,34). Analysis of positive-strand HCV RNA sequences and quasispecies patterns suggested that HCV RNAs in these cells are different from those in the serum (22). However, the possibility that HCV replicates in extrahepatic cells remains controversial because of the lack of isolation and characterization of viruses from the infected cells. Further, the use of RT-PCR for detection of viral RNA in these studies could not rigorously rule out possible contamination by the virus from the serum. Several laboratories have also shown that HCV can infect B-cell (30), T-cell (18, 32, 39), and hepatoma cell (14, 41) lines in culture, but the infection is usually transient and inefficient. Nevertheless, these studies suggested that B or T cells could support HCV replication, albeit inefficiently, at least in vitro.The molecular cloning of the HCV genome has made possible the delineation of the gene functions and the potential mechanism of pathogenesis of this virus. Recently, establishment of self-replicating HCV subgenomic (2, 25) and genomic (13, 33) replicons in Huh-7 cells has also provided an important new tool for the study...
As the human tetraspanin CD81 binds hepatitis C virus (HCV) envelope glycoprotein E2, we addressed the role CD81 may play in cellular trafficking of HCV envelope proteins. Studies on HCV life cycle are complicated by the lack of a robust cell culture system; we therefore transfected mammalian cells with HCV E1-E2 cDNA, with or without human CD81 (huCD81) cDNA. In the absence of huCD81, HCV envelope proteins are almost completely retained in the endoplasmic reticulum. Instead, when huCD81 is present, a fraction of HCV envelope proteins passes through the Golgi apparatus, matures acquiring complex sugars and is found extracellularly associated with exosomes. These are 60-100-nm membrane vesicles enriched in tetraspanins, released into the extracellular milieu by many cell types and having fusogenic activity. We also report that human plasma contains exosomes and that in HCV patients, viral RNA is associated with these circulating vesicles. We propose that the HCV-CD81 complex leaves cells in the form of exosomes, circulates in this form and exploits the fusogenic capabilities of these vesicles to infect cells even in the presence of neutralizing antibodies.
The hepatitis B virus (HBV) X gene product (pX) could be important in disease pathogenesis because it is known to transactivate transcription from many viral and cellular gene promoters, including the HBV core gene promoter, the human immunodeficiency virus (HIV-1) long terminal repeat, and the c-myc promoter. We have previously shown that only a subset of the promoters that can be transactivated by pX is transactivated in any particular cell line, and have proposed that pX acts through multiple, cell type-specific transcription factors. We show here that pX acts through both AP-1 and AP-2 sites, and that pX has a transcription activation domain. We conclude that transactivation by pX depends on at least two distinct cellular DNA-binding transcription factors and we present a model for the action of pX.
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