Decreased protein and mRNA expression of ER stress proteins GRP78 and GRP94 in HepG2 cells over-expressing CYP2E1

Department of Pharmacology and Biological Chemistry, Mount Sinai School of Medicine, New York, NY 10029, USA.
Archives of Biochemistry and Biophysics (Impact Factor: 3.02). 04/2006; 447(2):155-66. DOI: 10.1016/
Source: PubMed


CYP2E1 causes oxidative stress mediated cell death; the latter is one mechanism for endoplasmic reticulum (ER) stress in the cell. Unfolded proteins accumulate during ER stress and ER resident proteins GRP78 and GRP94 protect cells against ER dysfunction. We examined the possible role of GRP78 and GRP94 as protective factors against CYP2E1-mediated toxicity in HepG2 cells expressing CYP2E1 (E47 cells). E47 cells expressed high levels of CYP2E1 protein and catalytic activity which is associated with increased ROS generation, lipid peroxidation and the elevated presence of ubiquinated and aggregated proteins as compared to control HepG2 C34 cells which do not express CYP2E1. The mRNA and protein expression of GRP78 and GRP94 were decreased in E47 cells compared to the C34 cells, which may explain the accumulation of ubiquinated and aggregated proteins. Expression of these GRP proteins was induced with the ER stress agent thapsigargin in E47 cells, and E47 cells were more resistant to the toxicity caused by thapsigargin and calcimycin, possibly due to this upregulation and also because of the high expression of GSH and antioxidant enzymes in E47 cells. Antioxidants such as trolox and N-acetylcysteine increased GRP78 and GRP94 levels in the E47 cells, suggesting that CYP2E1- derived oxidant stress was responsible for down regulation of these GRPs in the E47 cells. Thapsigargin mediated toxicity was decreased in cells treated with the antioxidant trolox indicating a role for oxidative stress in this toxicity. These results suggest that CYP2E1 mediated oxidative stress downregulates the expression of GRP proteins in HepG2 cells and oxidative stress is an important mechanism in causing ER dysfunction in these cells.

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    • "Chaperones have been proposed to facilitate correct protein folding in the ER and they also prevent protein unfolding, which causes the aggregation of proteins in the ER and induces ER stress (Lambert and Prange, 2003). Two well-characterized ER chaperone proteins are the 78- kDa glucose-regulated protein (GRP78 or BiP) and GRP94, which are known to be induced by ER stress (Boelens et al., 2013; Dey et al., 2006; Eletto et al., 2010; Kaufman, 1999; Lee, 2005; Patil and Walter, 2001) and they are involved with the ER stress signaling pathway. GRP78 was also demonstrated to function during HBV viral morphogenesis where it interacts with the large surface protein of HBV and regulates its posttranscriptional topological reorientation (Cho et al., 2003; Lambert and Prange, 2003). "
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    ABSTRACT: The compound p-hydroxyacetophenone (PHAP) isolated from Artemisia morrisonensis was found to have potential anti-HBV effects in HepG2 2.2.15 cells. We clarified its antiviral mode further and HBV-transfected Huh7 cells were used as the platform. During viral gene expression, treatment with PHAP had no apparent effects on the viral precore/pregenomic RNA. However, the 2.4-kb preS RNA of viral surface gene increased significantly relative to the 2.1-kb S RNA with PHAP. Promoter activity analysis demonstrated that PHAP had a potent effect on augmenting the viral preS promoter activity. The subsequent increase in the large surface protein and induce endoplasmic reticular (ER) stress has been reported previously. Interestingly, PHAP specifically reduced ER stress related GRP78 RNA/protein levels, but not those of GRP94, in treated Huh7 cells while PHAP also led to the significant intracellular accumulation of virus. Moreover, treatment with the ER chaperone inducer thapsigargin relieved the inhibitory effect of PHAP based on the supernatant HBV DNA levels of HBV-expressed cells. In conclusion, this study suggests that the mechanism of HBV inhibition by PHAP might involve the regulation of viral surface gene expression and block virion secretion by interference with the ER stress signaling pathway.
    Full-text · Article · Nov 2013 · Antiviral research
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    • "In accord with published evidence indicating that CYP2E1 is a positive regulator of UPR [21], our results lent support to the notion that CYP2E1 induces UPR in MCF7 breast cancer cells transfected with CYP2E1. UPR is altered in many types of cancer [69], including breast cancer, and in some cases contributes to chemoresistance [24,35,70,71], highlighting the importance of our findings for cancer therapy. "
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    ABSTRACT: The cytochrome P450 (CYP) enzymes are a class of heme-containing enzymes involved in phase I metabolism of a large number of xenobiotics. The CYP family member CYP2E1 metabolises many xenobiotics and procarcinogens, it is not just expressed in the liver but also in many other tissues such as the kidney, the lung, the brain, the gastrointestinal tract and the breast tissue. It is induced in several pathological conditions including cancer, obesity, and type II diabetes implying that this enzyme is implicated in other biological processes beyond its role in phase I metabolism. Despite the detailed description of the role of CYP2E1 in the liver, its functions in other tissues have not been extensively studied. In this study we investigated the functional significance of CYP2E1 in breast carcinogenesis. Cellular levels of reactive oxygen species (ROS) were measured by H2DCFDA (2 2.9.2 2[prime],7[prime]-dichlorodihydrofluorescein diacetate ) staining and autophagy was assessed by tracing the cellular levels of autophagy markers using western blot assays. The endoplasmic reticulum stress and the unfolded protein response (UPR) were detected by luciferase assays reflecting the splicing of mRNA encoding the X-box binding protein 1 (XBP1) transcription factor and cell migration was evaluated using the scratch wound assay. Gene expression was recorded with standard transcription assays including luciferase reporter and chromatin immunoprecipitation. Ectopic expression of CYP2E1 induced ROS generation, affected autophagy, stimulated endoplasmic reticulum stress and inhibited migration in breast cancer cells with different metastatic potential and p53 status. Furthermore, evidence is presented indicating that CYP2E1 gene expression is under the transcriptional control of the p53 tumor suppressor. These results support the notion that CYP2E1 exerts an important role in mammary carcinogenesis, provide a potential link between ethanol metabolism and breast cancer and suggest that progression, and metastasis of advanced stages of breast cancer can be modulated by induction of CYP2E1 activity.
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    • "P450 2E1 induces oxidative stress-mediated cell death; the latter may be one mechanism for ER stress in the cell (Kim et al., 2009). It has been reported that P450 2E1-mediated oxidative stress downregulates expression of GRP proteins and is an important mechanism causing ER dysfunction (Dey et al., 2006). Our results are consistent with the theory that P450 2E1 increases ROS and the pathophysiological state of liver cells (Villeneuve et al., 2004). "
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    ABSTRACT: The role of ER stress on hepatic steatosis was investigated in a rat model. We injected CCl(4) into rats and found that CCl(4) could induce hepatic lipid accumulation, confirmed by Oil Red O staining and by measurement of triglyceride and cholesterol. The expression of ApoB, an apolipoprotein, was decreased in plasma and increased in the liver of CCl(4)-treated animals. The ER stress response was also significantly increased by CCl(4). P450 2E1 expression and activity were increased through interactions of P450 2E1 with NADPH-dependent P450 reductase (NPR) under CCl(4)-treated conditions. In HepG2 cells, intracellular lipid accumulation and its signaling were comparable to in vivo results. In order to elucidate the effect of the ER stress response itself, tunicamycin, an N-acetyl-glycosylation inhibitor, was injected into rats, followed by Oil Red O staining, lipid/triglyceride/cholesterol accumulation analysis, and examination of ApoB expression. Additionally, the ER stress response and upregulation of P450 2E1 were also confirmed in the tunicamycin-treated rats. All of the responses were similar to those seen with CCl(4). The P450 2E1 inhibitor diallyl sulphide (DAS), N-acetylcysteine (NAC), and reduced glutathione (GSH) antioxidants also regulated processes, including ApoB expression and lipid accumulation in CCl(4)-treated animals. In the presence of tunicamycin, DAS or NAC/GSH regulated all of the pathological phenomena with the exception of the ER stress response. In summary, CCl(4) induces liver steatosis, a process involving ER stress-induced P450 2E1 activation and ROS production.
    Full-text · Article · Jun 2011 · The international journal of biochemistry & cell biology
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