Endoplasmic Reticulum Stress In HepG2 Cells Research Articles

Copper exposure induces mitochondrial dysfunction and hepatotoxicity via the induction of oxidative stress and PERK/ATF4 -mediated endoplasmic reticulum stress

Copper is an essential trace element, and excessive exposure could result in hepatoxicity, however, the underlying molecular mechanisms remain incompletely understood. The present study is aimed to investigate the molecular mechanisms of copper sulfate (CuSO4) exposure-induced hepatoxicity both in vivo and in vitro. In vitro, HepG2 and L02 cells were exposed to various doses of CuSO4 for 24 h. Cell viability, ROS production, oxidative stress biomarkers, mitochondrial functions, ultrastructure, intracellular calcium (Ca2+) concentration, and the expression of proteins related to mitochondrial apoptosis and endoplasmic reticulum (ER) stress were assessed. In vivo, C57BL/6 mice were treated with CuSO4 at doses of 10 and 30 mg/kg BW/day and co-treated with 4-PBA at 100 mg/kg BW/day for 35 days. Subsequently, liver function, histopathological features, and protein expression were evaluated. Results found that exposure to CuSO4 at concentrations of 100–400 μM for 24 h significantly decreased the viabilities of HepG2 and L02 cells and it was in a dose-dependent manner. Additionally, CuSO4 exposure induced significant oxidative stress and mitochondrial dysfunction in HepG2 cells, which were partially ameliorated by the antioxidant N-acetylcysteine (NAC). Furthermore, CuSO4 exposure prominently triggered ER stress, as evidenced by the upregulation of GRP94, GRP78, phosphorylated forms of PERK and eIF2α, and CHOP proteins in livers of mice and HepG2 cells. NAC treatment significantly inhibited CuSO4 exposure -induced ER stress in HepG2 cells. Pharmacological inhibition of ER stress through co-treatment with 4-PBA and the PERK inhibitor GSK2606414, as well as genetic knockdown of ATF4, partially mitigated CuSO4-induced cytotoxicity in HepG2 cells by reducing mitochondrial dysfunction and inhibiting the mitochondrial apoptotic pathway. Moreover, 4-PBA treatment significantly attenuated CuSO4-induced caspase activation and hepatoxicity in mice. In conclusion, these results reveal that CuSO4-induced hepatotoxicity involves mitochondrial dysfunction and ER stress by activating oxidative stress induction and PERK/ATF4 pathway.

Kimchi attenuates endoplasmic reticulum stress-induced hepatic steatosis in HepG2 cells and C57BL/6N mice

Kimchi is a traditional fermented food that contains abundant nutrients and functional ingredients with various health benefits. We previously reported that kimchi active components suppress hepatic steatosis caused by endoplasmic reticulum (ER) stress in vitro and in vivo. Therefore, we assessed the effect of kimchi on the inhibition of hepatic steatosis caused by ER stress in HepG2 cells and C57BL/6N mice to verify the hypothesis that kimchi may potentially inhibit nonalcoholic fatty liver disease. We investigated the effect of kimchi on cell viability and triglyceride concentrations in cells and on lipid profile, lipid accumulation, and expression of related genes in cells and mice with hepatic steatosis. A mechanistic study was also performed using the liver X receptor α agonist T0901317 and the AMP-activated protein kinase agonist AICAR. Kimchi was noncytotoxic and effectively reduced triglyceride concentrations and suppressed hepatic steatosis-related gene expression in cells and mice. Additionally, kimchi recovered weight loss, lowered the serum and liver tissue lipid profiles, suppressed lipid accumulation, and reduced the effects of T0901317 and AICAR on lipogenic gene expression in tunicamycin-treated mice. Our results highlight that kimchi could prevent hepatic steatosis caused by ER stress in cells and mice.

Inhibition of ubiquitin specific peptidase 14 (USP14) promotes ER-phagy by inducing ER stress in human hepatoma HepG2 cells

ABSTRACT Autophagy plays a critical role in regulating the quality and quantity of cellular compartments; however, the mechanisms governing endoplasmic reticulum (ER) autophagy (ER-phagy) are still largely undefined. In this study, we identified several inhibitors of USP14, a ubiquitin-specific protease, including IU1 and b-AP15, as novel ER-phagy inducers through chemical library screening. While USP14 is known to act as an ER stress inducer, its precise role in ER-phagy remains unclear. Our findings demonstrate that treatment with either IU1 or b-AP15 induces ER-phagy by increasing ER stress in HepG2 cells. Similarly, depletion of USP14 augments ER-phagy and the ER stress response. The blockage of autophagy using an ULK1 inhibitor, SBI0206965, impedes ER-phagy. Moreover, inhibition of ER stress with tauroursodeoxycholic acid reverses ER-phagy by alleviating ER stress in HepG2 cells. We also found that suppression of the stress kinase JNK inhibited ER-phagy in IU1-treated cells. In conclusion, the results of this study suggest that the inhibition of USP14 accelerates ER-phagy by enhancing ER stress and JNK activation in HepG2 cells.

Emodin, an Emerging Mycotoxin, Induces Endoplasmic Reticulum Stress-Related Hepatotoxicity through IRE1α-XBP1 Axis in HepG2 Cells.

Emodin, an emerging mycotoxin, is known to be hepatotoxic, but its mechanism remains unclear. We hypothesized that emodin could induce endoplasmic reticulum (ER) stress through the inositol-requiring enzyme 1 alpha (IRE1α)-X-box-binding protein 1 (XBP1) pathway and apoptosis, which are closely correlated and contribute to hepatotoxicity. To test this hypothesis, a novel IRE1α inhibitor, STF-083010, was used. An MTT assay was used to evaluate metabolic activity, and quantitative PCR and western blotting were used to investigate the gene and protein expression of ER stress or apoptosis-related markers. Apoptosis was evaluated with flow cytometry. Results showed that emodin induced cytotoxicity in a dose-dependent manner in HepG2 cells and upregulated the expression of binding immunoglobulin protein (BiP), C/EBP homologous protein (CHOP), IRE1α, spliced XBP1, the B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax)/Bcl-2 ratio, and cleaved caspase-3. Cotreatment with emodin and STF-083010 led to the downregulation of BiP and upregulation of CHOP, the Bax/Bcl-2 ratio, and cleaved caspase-3 compared with single treatment with emodin. Furthermore, the apoptosis rate was increased in a dose-dependent manner with emodin treatment. Thus, emodin induced ER stress in HepG2 cells by activating the IRE1α-XBP1 axis and induced apoptosis, indicating that emodin can cause hepatotoxicity.

Effect of Flammulina velutipes polysaccharides on endoplasmic reticulum stress-mediated apoptosis by activating PLC-IP3 pathway in HepG2 cells.

Flammulina velutipes polysaccharides (FVP) have been proven to induce apoptosis in HepG2 cells. It is well known that endoplasmic reticulum stress (ERS) is involved in apoptosis. However, ERS mediates FVP-induced apoptosis in HepG2 cells remains unclear. In our study, the results indicated that FVP caused ERS in HepG2 cells. They showed that FVP were water-soluble polysaccharides with the weight average molecular weight of 1972kDa, which were mainly composed of mannose, gluconic acid, glucose, galactose, xylose and fructose in a molar ratio of 6.6 : 1.3 : 79.9 : 7.4 : 3.4 : 1.5. After FVP treatment, the expression levels of genes and proteins related to ERS were upregulated. The inhibition of ERS by 4-phenylbutyric acid (4-PBA) pretreatment could significantly reduce the role of FVP in inducing apoptosis. We further found the results of immunofluorescence and flow cytometry showing that Ca2+ in the ERS leaked out, and the intracellular Ca2+ concentration increased after FVP treatment. The pretreatment with the phospholipase C (PLC) inhibitor U73122 proved that FVP caused excessive intracellular Ca2+ concentration by activating the phospholipase C-inositol-1,4,5-triphosphate (PLC-IP3) pathway, resulting in ERS, and ultimately leading to apoptosis. In summary, our results indicated that FVP induced ERS-mediated apoptosis by activating PLC-IP3 pathway in HepG2 cells. PRACTICAL APPLICATION: This work may suggest that FVP could be used as an adjuvant therapy to anticancer drugs, providing new application prospects and possibilities.

Procyanidin B2 Alleviates Palmitic Acid-Induced Injury in HepG2 Cells via Endoplasmic Reticulum Stress Pathway.

Nonalcoholic fatty liver disease (NAFLD) is the hepatic manifestation of the metabolic syndrome featuring ectopic lipid accumulation in hepatocytes. NAFLD has been a severe threat to humans with a global prevalence of over 25% yet no approved drugs for the treatment to date. Previous studies showed that procyanidin B2 (PCB2), an active ingredient from herbal cinnamon, has an excellent hepatoprotective effect; however, the mechanism remains inconclusive. The present study aimed to investigate the protective effect and underlying mechanism of PCB2 on PA-induced cellular injury in human hepatoma HepG2 cells. Our results showed that PA-induced oxidative stress, calcium disequilibrium, and subsequent endoplasmic reticulum stress (ERS) mediated cellular injury, with elevated protein levels of GRP78, GRP94, CHOP, and hyperphosphorylation of PERK and IRE1α as well as the increased ratio of Bax/Bcl-2, which was restored by PCB2 in a concentration-dependent manner, proving the excellent antiapoptosis effect. In addition, 4-phenylbutyric acid (4-PBA), the ER stress inhibitor, increased cell viability and decreased protein levels of GRP78 and CHOP, which is similar to PCB2, and thapsigargin (TG), the ER stress agonist, exhibited conversely meanwhile partly counteracted the hepatic protection of PCB2. What is more, upregulated protein expression of p-IKKα/β, p-NF-κB p65, NLRP3, cleaved caspase 1, and mature IL-1β occurred in HepG2 cells in response to PA stress while rescued with the PCB2 intervention. In conclusion, our study demonstrated that PA induces ERS in HepG2 cells and subsequently activates downstream NLRP3 inflammasome-mediated cellular injury, while PCB2 inhibits NLRP3/caspase 1/IL-1β pathway, inflammation, and apoptosis with the presence of ERS, thereby promoting cell survival, which may provide pharmacological evidence for clinical approaches on NAFLD.

Natural Thiols, but Not Thioethers, Attenuate Patulin-Induced Endoplasmic Reticulum Stress in HepG2 Cells.

Patulin, a mycotoxin, is known to have cytotoxic effects, but few studies have focused on the involvement of the endoplasmic reticulum (ER) stress response in patulin toxicity and the natural compounds that attenuate it in HepG2 cells. This study tested the ability of patulin to induce ER stress, and that of four thiols and three thioethers to attenuate patulin-induced ER stress in HepG2 cells. Patulin dose-dependently inhibited cell proliferation (IC50, 8.43 μM). Additionally, patulin was found to increase the expression levels of ER stress-related genes and/or protein markers, including BiP, CHOP, and spliced XBP1, in HepG2 cells compared to the vehicle control, indicating its potential in ER stress induction. Patulin-induced cytotoxicity in HepG2 cells was reduced by naturally occurring thiol compounds (glutathione, L-acetyl-L-cysteine, cysteine, and captopril), but not by thioether compounds (sulforaphane, sulforaphene, and S-allyl-L-cysteine). Patulin-thiol co-treatment decreased CHOP expression and BiP and CHOP levels in HepG2 cells but did not alter BiP expression. Spliced XBP1 expression was decreased by patulin-thiol co-treatment. Thus, patulin induced ER stress in HepG2 cells and thiols, but not in thioethers, attenuated patulin-induced ER stress.

Sestrin2 protects against bavachin induced ER stress through AMPK/mTORC1 signaling pathway in HepG2 cells

Bavachin (BV), a natural flavonoid compound derived from Psoralea corylifolia L, has been reported to be a potential hepatotoxin. Our previous studies have found that BV can induce endoplasmic reticulum (ER) stress-related cell apoptosis, but the molecular mechanism underlying BV-induced ER stress remains obscure. Sestrin2, a highly conserved stress-inducible protein, is involved in the cellular responses of various stress conditions and homeostatic regulation. However, whether Sestrin2 participated in the ER stress related hepatotoxicity against BV is still elusive. In the present study, we aim to investigate the role of BV on liver injury of mice and the impact of Sestrin2 on BV-induced ER stress in HepG2 cells. The results in mice showed that BV induced ER stress related liver injury with increased Sestrin2 expression involvement. Knockdown of Sestrin2 with siRNA aggravated BV-induced ER stress significantly in HepG2 cells. Further mechanistic study uncovered that inhibition of mTORC1 with rapamycin blocked BV-induced ER stress, and treatment with Sestrin2 siRNA blocked the inhibition effect of AMPK to mTORC1. Therefore, constant mTORC1 would lead to accumulation of misfolded or unfolded proteins and aggravated ER stress. Collectively, our study indicates that Sestrin2 confers protection against BV-induced ER stress via activating of the AMPK/mTORC1 pathway.

Endoplasmic reticulum stress induces hepatic plasminogen activator inhibitor 1 in murine nonalcoholic steatohepatitis.

Plasminogen activator inhibitor 1 (PAI‐1) is a stress‐responsive gene that is highly induced in nonalcoholic steatohepatitis (NASH). Endoplasmic reticulum (ER) stress is a salient feature of NASH, yet it is unknown whether ER stress contributes to hepatic PAI‐1 induction in this disorder. Therefore, we aimed to (a) establish the role of ER stress in the regulation of hepatic Pai‐1 expression, and (b) determine whether induction of Pai‐1 in murine NASH is driven by ER stress. Hepatic Pai‐1 expression was measured in C57BL/6 J mice and human HepG2 cells subjected to acute or prolonged pharmacologic ER stress. We found that hepatic Pai‐1 expression was acutely suppressed in murine liver in response to severe ER stress followed by marked induction during the recovery phase of the ER stress response. Hepatic Pai‐1 expression was induced in response to prolonged low‐grade ER stress in mice. Induction of PAI‐1 by ER stress in HepG2 cells was prevented by pharmacologic inhibition of MEK1/ERK signaling or by siRNA‐mediated knockdown of XBP1, mediators of the recovery response to ER stress. Inhibiting ER stress with 4‐phenylbutyric acid prevented hepatic Pai‐1 induction in mice with diet‐induced steatohepatitis. We conclude that hepatic Pai‐1 is induced by ER stress via a pathway involving XBP1 and MEK1/ERK signaling, and induction of hepatic Pai‐1 in murine NASH is mediated by ER stress. These data implicate ER stress as a novel mechanistic link between Pai‐1 induction and NASH.

Catecholamines Induce Endoplasmic Reticulum Stress Via Both Alpha and Beta Receptors.

Severely burned patients suffer from a hypermetabolic syndrome that can last for years after the injury has resolved. The underlying cause of these metabolic alterations most likely involves the persistent elevated catecholamine levels that follow the surge induced by thermal injury. At the cellular level, endoplasmic reticulum (ER) stress in metabolic tissues is a hallmark observed in patients following burn injury and is associated with several detrimental effects. Therefore, ER stress could be the underlying cellular mechanism of persistent hypermetabolism in burned patients. Here, we show that catecholamines induce ER stress and that adreno-receptor blockers reduce stress responses in the HepG2 hepatocyte cell line. Our results also indicate that norepinephrine (NE) significantly induces ER stress in HepG2 cells and 3T3L1 mouse adipocytes. Furthermore, we demonstrate that the alpha-1 blocker, prazosin, and beta blocker, propranolol, block ER stress induced by NE. We also show that the effects of catecholamines in inducing ER stress are cell type-specific, as NE treatment failed to evoke ER stress in human fibroblasts. Thus, these findings reveal the mechanisms used by catecholamines to alter metabolism and suggest inhibition of the receptors utilized by these agents should be further explored as a potential target for the treatment of ER stress-mediated disease.

Anti-diabetic effect of 3-hydroxy-2-naphthoic acid, an endoplasmic reticulum stress-reducing chemical chaperone

Lots of experimental and clinical evidences indicate that chronic exposure to saturated fatty acids and high level of glucose is implicated in insulin resistance, beta cell failure and ultimately type 2 diabetes. In this study, we set up cell-based experimental conditions to induce endoplasmic reticulum (ER) stress and insulin resistance using high concentration of palmitate (PA). Hydroxynaphthoic acids (HNAs) were formerly identified as novel chemical chaperones to resolve ER stress induced by tunicamycin. In this study, we found the compounds have the same suppressive effect on PA-induced ER stress in HepG2 cells. The representing compound, 3-HNA reduced PA-induced phosphorylation of JNK, IKKβ and IRS1 (S307) and restored insulin signaling cascade which involves insulin receptor β, IRS1 and Akt. The insulin sensitizing effect of 3-HNA was confirmed in 3T3-L1 adipocytes, where the compound augmented insulin signaling and glucose transporter 4 (GLUT4) membrane translocation. 3-HNA also protected the pancreatic beta cells from PA-induced apoptosis by reducing ER stress. Upon 3-HNA treatment to ob/ob mice at 150mg/kg/day dosage, the diabetic parameters including glucose tolerance and systemic insulin sensitivity were significantly improved. Postmortem examination showed that 3-HNA markedly reduced ER stress and insulin resistance in the liver tissues and it sensitized insulin signaling in the liver and the skeletal muscle. Our results demonstrated that 3-HNA can sensitize insulin signaling by coping with lipotoxicity-induced ER stress as a chemical chaperone and suggested it holds therapeutic potential for insulin resistance and type 2 diabetes.

Perfluorooctanoic acid exposure induces endoplasmic reticulum stress in the liver and its effects are ameliorated by 4-phenylbutyrate

Perfluoroalkyl acids (PFAAs) are a group of widely used anthropogenic compounds. As one of the most dominant PFAAs, perfluorooctanoic acid (PFOA) has been suggested to induce hepatotoxicity and several other toxicological effects. However, details on the mechanisms for PFOA-induced hepatotoxicity still need to be elucidated. In this study, we observed the occurrence of endoplasmic reticulum (ER) stress in mouse livers and HepG2 cells after PFOA exposure using several familiar markers for the unfolded protein response (UPR). ER stress in HepG2 cells after PFOA exposure was not significantly influenced by autophagy inhibition or stimulation. The antioxidant defense system was significantly disturbed in mouse livers after PFOA exposure, and reactive oxygen species (ROS) were increased in cells exposed to PFOA for 24 h. However, N-acetyl-L-cysteine (NAC) pretreatment did not satisfactorily alleviate the UPR in cells exposed to PFOA even though the increase of ROS was less evident. Furthermore, exposure of HepG2 cells to PFOA in the presence of sodium 4-phenylbutyrate (4-PBA), a chemical chaperone and ER stress inhibitor, suggested that 4-PBA alleviated the UPR and autophagosome accumulation induced by PFOA in cells. In addition, several toxicological effects attributed to PFOA exposure, including cell cycle arrest, proteolytic activity impairment, and neutral lipid accumulation, were also improved by 4-PBA cotreatment in cells. In vivo study demonstrated that PFOA-induced lipid metabolism perturbation and liver injury were partially ameliorated by 4-PBA in mice after 28 days of exposure. These findings demonstrated that PFOA-induced ER stress leading to UPR might play an important role in PFOA-induced hepatotoxic effects, and chemical chaperone 4-PBA could ameliorate the effects.

CXC195 induces apoptosis and endoplastic reticulum stress in human hepatocellular carcinoma cells by inhibiting the PI3K/Akt/mTOR signaling pathway.

CXC195 exhibits strong protective effects against neuronal apoptosis by exerting antioxidant activity. However, the pharmacological function of CXC195 in cancer remains to be elucidated. The present study demonstrated that CXC195 exhibited significant cytotoxic effects, and induced cell cycle arrest and apoptosis in HepG2 human hepatocellular carcinoma (HCC) cell lines. Following treatment of HepG2 cells with 150µΜ CXC195 for 24, cell viability and the apoptotic rate were assessed using an MTT assay and AnnexinV/propidium iodide staining followed by flow cytometric analysis. Molecular markers of the cell cycle, apoptosis, mitochondrial function and endoplasmic reticulum (ER) stress were analyzed by western blot or polymerase chain reaction analysis. Caspase activation, cytochromec and apoptosis‑inducing factor release, and analysis of the Bcell lymphoma2 (Bcl‑2)‑associated X protein/Bcl‑2 ratio demonstrated that the anticancer effects of CXC195 in HepG2 cells were mediated by caspase and mitochondria‑dependent apoptosis. CXC195 also induced the expression of ER stress‑associated proteins, including CCAAT‑enhancer‑binding protein homologous protein, and glucose‑regulated proteins94 and78, and led to the activation of multiple branches of ER stress transducers, including inositol‑requiring enzyme1α‑apoptosis signal‑regulating kinase‑p38/c‑Jun N‑terminal kinase, and protein kinaseR‑like endoplasmic reticulum kinase‑eukaryotic translation initiation factor2α‑activating transcription factor (ATF)4 andATF6, in the HepG2 cells. In addition, CXC195 inhibited the phosphorylation of phosphoinositide 3‑kinase (PI3K), Akt and mammalian target of rapamycin (mTOR) in the HepG2 cells. These effects were enhanced following treatment with selected inhibitors of PI3K (LY294002), Akt (SH‑6) and mTOR (rapamycin). Furthermore, these inhibitors enhanced the pro‑apoptotic effects of CXC195 in the HepG2 cells. In conclusion, the results of the present study indicated that CXC195 induced apoptosis and ER stress in HepG2 cells through the inhibition of the PI3K/Akt/mTOR signaling pathway.

SIRT1 attenuates palmitate-induced endoplasmic reticulum stress and insulin resistance in HepG2 cells via induction of oxygen-regulated protein 150

Endoplasmic reticulum (ER) stress has been implicated in the pathology of type 2 diabetes mellitus (T2DM). Although SIRT1 has a therapeutic effect on T2DM, the mechanisms by which SIRT1 ameliorates insulin resistance (IR) remain unclear. In this study, we investigated the impact of SIRT1 on palmitate-induced ER stress in HepG2 cells and its underlying signal pathway. Treatment with resveratrol, a SIRT1 activator significantly inhibited palmitate-induced ER stress, leading to the protection against palmitate-induced ER stress and insulin resistance. Resveratrol and SIRT1 overexpression induced the expression of oxygen-regulated protein (ORP) 150 in HepG2 cells. Forkhead box O1 (FOXO1) was involved in the regulation of ORP150 expression because suppression of FOXO1 inhibited the induction of ORP150 by SIRT1. Our results indicate a novel mechanism by which SIRT1 regulates ER stress by overexpression of ORP150, and suggest that SIRT1 ameliorates palmitate-induced insulin resistance in HepG2 cells via regulation of ER stress.

Distinct gene expression profiles characterize cellular responses to palmitate and oleate

Obese individuals are both insulin resistant and have high levels of circulating free fatty acids (FFAs). In cell culture, saturated but not unsaturated fatty acids induce endoplasmic reticulum (ER) stress. We hypothesized that chronic exposure to low dose fatty acids would significantly attenuate the acute stress response to a saturated fatty acid challenge and that unsaturated fatty acids (oleate) would be more protective than saturated fatty acids (palmitate). The ER stress response to palmitate was reduced after low dose fatty acid exposure in human hepatoma cells. Palmitate and oleate gave distinctive transcript responses, both acutely and after chronic low dose exposure. Differentially regulated pathways included lipid, cholesterol, fatty acid, and triglyceride metabolism, and IkappaB kinase and nuclear factor kappaB kinase inflammatory cascades. Oleate reduced palmitate-induced changes significantly more than low dose palmitate and completely blocked palmitate-induced phosphoinositide 3 kinase inhibitor (PIK3IP1) as well as induction of GADD45A and B. These changes are predicted to alter the PI3 kinase pathway and the pro-apoptotic p38 MAPK pathway. We recapitulated the oleate response by small interfering RNA-mediated block of PIK3IP1 stimulation with palmitate and significantly protected cells from palmitate-mediated ER stress. We show that transcriptional responses to oleate and palmitate are distinct, broad, and often discordant. We identified several potential candidates that may direct the transcriptional networks and demonstrate that PIK3IP1 partially accounts for the protective effects of oleate.

XBP1U inhibits the XBP1S-mediated upregulation of the iNOS gene expression in mammalian ER stress response

Upregulation of the inducible nitric oxide synthase (iNOS) gene is associated with many pathological conditions such as endoplasmic reticulum (ER) stress, and X-box binding protein 1 (XBP1) is critical in mediating ER-stress responsive genes, including iNOS. Nonetheless, the mechanism by which XBP1 regulates iNOS during ER stress remains unexplored. Here we show that the active/spliced form of XBP1 protein, XBP1S, directly binds to the AABS (A-activator-binding site) in the iNOS promoter in vitro and in living cells. XBP1S exhibits dose-dependent activation of iNOS-specific reporter gene activity and endogenous iNOS expression. XBP1S is elevated whereas the unspliced form of XBP1, XBP1U, reduced in ER stress in HepG2 cells. In addition, XBP1U binds to XBP1S and this complex is associated with the iNOS promoter in response to ER stress. Furthermore, XBP1U acts as a negative mediator and suppresses XBP1S-mediated induction of iNOS. Collectively, we present the first evidence demonstrating the regulation of iNOS gene induction by the interaction between the spliced and unspliced forms of XBP1 in response to ER stress.

Influence of SelS gene silence on β-Mercaptoethanol-mediated endoplasmic reticulum stress and cell apoptosis in HepG2 cells

Selenoprotein S (SelS), a transmembrane selenoprotein, may be related to the response of endoplasmic reticulum (ER) stress. In this report, the influence of selenite supplementation and SelS gene silence on β-mercaptoethanol (β-ME)-mediated ER stress and cell apoptosis in HepG2 cells were examined. The results showed that SelS protein expression was markedly increased by 10 mM β-ME and 100 nM sodium selenite in HepG2 cells. GRP78 protein level was significantly increased after treatment with 10 mM β-ME in HepG2 cells, which suggested that β-ME was also an ER stress inducer. Meanwhile, β-ME (10 mM) was found to induce cell apoptosis, which was alleviated obviously when cells were pretreated with 100 nM selenite before exposure to β-ME. Moreover, the suppression of SelS gene by siRNA could aggravate HepG2 cell apoptosis induced by β-ME significantly. In conclusion, these results suggested that β-ME, also an ER stress agent, could induce cell apoptosis, and SelS may play an important role in protecting cells from apoptosis induced by ER stress in HepG2 cells.