Endoplasmic Reticulum Stress Markers CHOP Research Articles

Autophagy Limited Role In Mitigating Ubiquitin Proteasome Pathway Inhibition Induced ER Stress and mTORC1 Signaling.

The regulation of muscle mass is dictated by a balance between the rates of muscle protein synthesis (MPS) and muscle protein breakdown (MPB). The absolute rate of both measures fluctuates during catabolic and anabolic conditions. Decreasing the rate of MPB can but has not always resulted in increased muscle mass. This disparity may be the result of interactions between the ubiquitin proteasome pathway (UPP), autophagy, and endoplasmic reticulum (ER) stress pathways along with crosstalk with the mTORC1 signaling pathway, a primary mediator of MPS. PURPOSE: To determine how the inhibition of the ubiquitin proteasome alters autophagy, ER stress, mTORC1 signaling, and MPS within cultured myotubes. METHODS: MG132 was selected as a primary inhibitor of the UPP. Differentiated C2C12’s, were treated with 50μM of MG132 for 6 or 12hours(h), with DMSO volume and time match controls (n=3 per experiment). Cells were incubated with 1μM puromycin for 5min prior to collection, for determination of MPS via SuNSET. Equal aliquots of protein were separated by SDS-PAGE and protein levels determined by western blot. The effect of treatment was compared using a one way ANOVA with tukeys post hoc test. RESULTS: Inhibition of the ubiquitin proteasome pathway resulted in a 0.75- and 4.24-fold increase in total ubiquitinated protein following 6 and 12h of MG132 treatment respectively (P<0.05). Puromycin incorporation decreased ∼0.5-fold at both 6 and 12h (P<0.05). Phospho (Ser473)/total Akt increased 1.9- and 3-fold over corresponding controls at 6 and 12h (P<0.05). Phospho-S6K (Thr 389) increased 1.8 and 3.4 fold with MG132 treatment. Phospho-S6 (Ser 244/240) showed no increase in phosphorylation with treatment. Autophagy markers including Atg7, Beclin or LC3B exhibited no significant effect of treatment. However, Phospho (Ser575)/Total ULK decreased 0.33-fold at 12h, (P<0.05). The ER stress markers BIP and CHOP, exhibited marked increases in expression with a 2.4-fold increase at 12h (P<0.05) and a 2.2- and 4.9-fold increase in expression at 6 and 12h of treatment, respectively (P<0.05). CONCLUSION: Inhibition of the UPP decreased total protein synthesis in C2C12 myotubes even though Akt/mTORC1 signaling were partially activated. The changes in proteosomal activity did not immediately result in a compensatory increase in autophagy. More work will need to be done to determine the interconnection between these pathways.

Elevated Endoplasmic Reticulum Stress Response Contributes to Adipose Tissue Inflammation in Aging.

Adipose tissue inflammation has been linked to age-related metabolic diseases. However, the underlying mechanisms are poorly understood. Adipose tissue inflammation and insulin resistance in diet associated obesity has been correlated with aberrant endoplasmic reticulum (ER) stress. This study was undertaken to test our hypothesis that increased ER stress response contributes to age-associated adipose tissue inflammation. We found elevated ER stress response in adipose tissue of old (18-20 months) compared to young (4-6 months) mice. Elevated ER stress markers BIP (GRP78), CHOP, cleaved-ATF-6, phospho-IRE1α, and XBP-1 were observed in old compared to young adipose tissue stromal cells. Additionally, old adipose tissue stromal cells were more sensitive to an ER stress inducer, thapsigargin. Similar experiments with adipose tissue macrophages showed elevated Chop and Bip expression in old adipose tissue macrophages when induced with thapsigargin. Treatment of chemical chaperone 4-phenyle-butyric acid alleviated ER stress in adipose tissue stromal cells and adipose tissue macrophages and attenuated the production of IL-6 and MCP-1 by adipose tissue stromal cells, and TNF-α by adipose tissue macrophages from both young and old mice. Finally, old mice fed with 4-phenyle-butyric acid have reduced expression of ER stress and inflammatory cytokine genes. Our data suggests that an exaggerated ER stress response in aging adipose tissue contributes to age-associated inflammation that can be mitigated by treatment with chemical chaperones.

Inhibition of neutral sphingomyelinase decreases elevated levels of inducible nitric oxide synthase and apoptotic cell death in ocular hypertensive rats

Endoplasmic reticulum (ER) stress and excessive nitric oxide production via induction of inducible nitric oxide synthase (NOS2) have been implicated in the pathogenesis of neuronal retinal cell death in ocular hypertension. Neutral sphingomyelinase (N-SMase)/ceramide pathway can regulate NOS2 expression, hence this study determined the role of selective neutral sphingomyelinase (N-SMase) inhibition on retinal NOS2 levels, ER stress, apoptosis and visual evoked potentials (VEPs) in a rat model of elevated intraocular pressure (EIOP). NOS2 expression and retinal protein nitration were significantly greater in EIOP and significantly decreased with N-SMase inhibition. A significant increase was observed in retinal ER stress markers pPERK, CHOP and GRP78 in EIOP, which were not significantly altered by N-SMase inhibition. Retinal TUNEL staining showed increased apoptosis in all EIOP groups; however N-SMase inhibition significantly decreased the percent of apoptotic cells in EIOP. Caspase-3, -8 and -9 activities were significantly increased in EIOP and returned to baseline levels following N-SMase inhibition. Latencies of all VEP components were significantly prolonged in EIOP and shortened following N-SMase inhibition. Data confirm the role of nitrative injury in EIOP and highlight the protective effect of N-SMase inhibition in EIOP via down-regulation of NOS2 levels and nitrative stress.

Improved immunoblotting methods provide critical insights into phenotypic differences between two murine dysferlinopathy models

We adopted a proteomics-based approach to gain insights into phenotypic differences between A/J and B10.SJL murine dysferlinopathy models. We optimized immunoblotting of dysferlin by preparing homogenates of the tibialis anterior (TA) muscle under several different conditions. We compared TA muscles of control, A/J, and B10.SJL mice for levels of dysferlin; dysferlin's partners MG53, annexin-A2, and caveolin-3; and the endoplasmic reticulum (ER) stress marker CHOP. We performed immunoelectron microscopy on control rat TA muscle to determine the precise location of dysferlin. RIPA (radioimmunoprecipitation assay) buffer and sonication improves immunoblotting of dysferlin. The ER stress marker CHOP is elevated in A/J muscle. Dysferlin is localized mostly to membranes close to the Z-disk that have been reported to be part of the Golgi, ER, and sarcoplasmic reticulum (SR) networks. ER stress might underlie phenotypic differences between A/J and B10.SJL mice and play a role in human dysferlinopathies.

High MUC2 production in goblet cells causes increased susceptibility to ER stress and apoptosis (151.4)

MUC2, a large glycoprotein produced by goblet cells in the colon, forms a protective mucus blanket over the epithelium as the first line of host defense. During pathological conditions such as inflammatory bowel diseases, there is accelerated biosynthesis and secretion of MUC2. Surprisingly, little information is known on how MUC2 production is regulated and whether goblet cells undergo increased stress in response to high MUC2 biosynthesis and secretion. In this study we investigated the role of MUC2 in goblet cell stress using a high mucin producing cell line, HT29‐H, and a clone of HT29‐H (HT29‐L) in which MUC2 has been silenced using lentivirus shRNA. Cells were treated with tunicamycin and MG132, and markers for endoplasmic reticulum (ER) stress and apoptosis were quantified. Compared to HT29‐L cells, HT29‐H cells exhibited a greater dose‐and time‐dependent increase in ER stress markers GRP78, ATF4, CHOP, sXBP1 and AGR2 in response to both tunicamycin and MG132. ER stress also caused a decrease in phosphorylation of Akt and increased apoptosis in HT29‐H cells 6h after treatment. Phospho‐Akt in HT29‐L cells was however sustained, decreasing slightly only after 24h with less apoptosis compared to HT29‐H cells. Our findings indicate that MUC2 induces goblet cell stress and apoptosis which could subsequently lead to diminished mucus barrier function.Grant Funding Source: Supported by CIHR

FBXO6 attenuates cadmium toxicity in HEK293 cells by inhibiting ER stress and JNK activation.

Cadmium-induced cell death is associated with endoplasmic reticulum (ER) stress. We previously found that inhibition of FBXO6 expression, which is a ubiquitin ligase involved in ER-associated protein degradation (ERAD), induces high sensitivity to cadmium in HEK293 cells. However, the precise role of FBXO6 in ER stress remains unexplored. In this study, we investigated the role of FBXO6 in cadmium-induced ER stress in HEK293 cells. Our results showed that the cadmium-induced increase in expression of the ER stress marker proteins, BiP and CHOP, was further enhanced by inhibiting FBXO6 expression. Cadmium-induced c-Jun phosphorylation was also markedly increased by inhibition of FBXO6 expression. However, this c-Jun phosphorylation was almost entirely abolished by inhibition of c-Jun N-terminal kinase 1 (JNK1) expression. The level of high cadmium sensitivity induced by inhibition of FBXO6 expression was markedly lower in the JNK1-ablated cells than in the control cells. In addition, cadmium elevated the cellular level of ERAD substrate proteins, and this elevation was further enhanced by inhibiting FBXO6 expression. These results suggest that FBXO6 might inhibit cadmium-induced ER stress by functioning as a ubiquitin ligase in the ERAD system, thereby attenuating the cell death induced by subsequent JNK1 activation.

Selective, potent blockade of the IRE1 and ATF6 pathways by 4‐phenylbutyric acid analogues

4-Phenylbutyric acid (4-PBA) is a chemical chaperone that eliminates the accumulation of unfolded proteins in the endoplasmic reticulum (ER). However, its chaperoning ability is often weak and unable to attenuate the unfolded protein response (UPR) in vitro or in vivo. To develop more potent chemical chaperones, we synthesized six analogues of 4-PBA and evaluated their pharmacological actions on the UPR. NRK-52E cells were treated with ER stress inducers (tunicamycin or thapsigargin) in the presence of each of the 4-PBA analogues; the suppressive effects of these analogues on the UPR were assessed using selective indicators for individual UPR pathways. 2-POAA-OMe, 2-POAA-NO2 and 2-NOAA, but not others, suppressed the induction of ER stress markers GRP78 and CHOP. This suppressive effect was more potent than that of 4-PBA. Of the three major UPR branches, the IRE1 and ATF6 pathways were markedly blocked by these compounds, as indicated by suppression of XBP1 splicing, inhibition of UPRE and ERSE activation, and inhibition of JNK phosphorylation. Unexpectedly, however, these agents did not inhibit phosphorylation of PERK and eIF2α triggered by ER stress. These compounds dose-dependently inhibited the early activation of NF-κB in ER stress-exposed cells. 2-POAA-OMe and 2-POAA-NO2 also inhibited ER stress-induced phosphorylation of Akt. The 4-PBA analogues 2-POAA-OMe, 2-POAA-NO2 and 2-NOAA strongly inhibited activation of the IRE1 and ATF6 pathways and downstream pathogenic targets, including NF-κB and Akt, in ER stress-exposed cells. These compounds may be useful for therapeutic intervention in ER stress-related pathological conditions.

Synergistic effect of combined treatment with gamma-tocotrienol and statin on human malignant mesothelioma cells

The present study is the first to demonstrate the synergetic effect of statins (atorvastatin and simvastatin) and gamma-tocotrienol (γ-T3) on human malignant mesothelioma (MM). Statin+γ-T3 combinations induced greater cell growth inhibition more than each single treatment via inhibition of mevalonate pathway, a well-known target of both γ-T3 and statins. γ-T3 was necessary for endoplasmic reticulum stress markers CHOP and GRP78, whereas an intrinsic apoptotic marker, caspase 3 activation was induced only in the presence of statins. Overall, the combination of γ-T3 and statins could be useful for MM therapy and functions in a complementary style.

Nelfinavir and bortezomib inhibit mTOR activity via ATF4-mediated sestrin-2 regulation

Endoplasmic reticulum (ER) stress and autophagy are two basic cell survival mechanisms often occurring in concert. Extensive ER stress in cancer cells deliberately induced by chemotherapeutic drugs may lead to growth arrest and cell death. However, the link between ER stress and autophagy is not well understood. In this study, the treatment of cancer cells with ER stress-inducing drug nelfinavir resulted in the expression of endogenous mTOR inhibitor sestrin-2 (SESN2). Upregulation of SESN2 expression was associated with expression of ER stress markers ATF4, ATF3, and CHOP. SESN2 upregulation also occurred in cells treated with the proteasome inhibitor bortezomib. Ectopic expression of ATF4, but not of ATF3 or CHOP, caused transcriptional upregulation of SESN2 expression, indicating expressional regulation of SESN2 by ATF4. Transient overexpression of ectopic SESN2 resulted in mTOR inhibition and autophagy, confirming a link between ER stress, SESN2 upregulation, and mTOR inhibition. Accordingly, cancer cells treated with the ER stress-inducing agent nelfinavir showed reduced mTOR activity and associated increases in the expression levels of ATF4 and SESN2. These results show that ATF4-regulated SESN2 expression presents a new link between ER stress and mTOR inhibition and autophagy. mTOR inhibition by nelfinavir, which is currently in clinical trials for cancer patients, may also explain its observed ability to induce autophagy, growth arrest, and radiosensitization in cancer cells.

Calreticulin and other components of endoplasmic reticulum stress in rat and human inflammatory demyelination

BackgroundCalreticulin (CRT) is a chaperone protein, which aids correct folding of glycosylated proteins in the endoplasmic reticulum (ER). Under conditions of ER stress, CRT is upregulated and may be displayed on the surface of cells or be secreted. This ‘ecto-CRT’ may activate the innate immune response or it may aid clearance of apoptotic cells. Our and other studies have demonstrated upregulation of ER stress markers CHOP, BiP, ATF4, XBP1 and phosphorylated e-IF2 alpha (p-eIF2 alpha) in biopsy and post-mortem human multiple sclerosis (MS) samples. We extend this work by analysing changes in expression of CRT, BiP, CHOP, XBP1 and p-eIF2 alpha in a rat model of inflammatory demyelination. Demyelination was induced in the spinal cord by intradermal injection of recombinant mouse MOG mixed with incomplete Freund’s adjuvant (IFA) at the base of the tail. Tissue samples were analysed by semi-quantitative scoring of immunohistochemically stained frozen tissue sections. Data generated following sampling of tissue from animals with spinal cord lesions, was compared to that obtained using tissue derived from IFA- or saline-injected controls. CRT present in rat serum and in a cohort of human serum derived from 14 multiple sclerosis patients and 11 healthy controls was measured by ELISA.ResultsStained tissue scores revealed significantly (p<0.05) increased amounts of CRT, CHOP and p-eIF2 alpha in the lesion, lesion edge and normal-appearing white matter when compared to controls. CHOP and p-eIF2 alpha were also significantly raised in regions of grey matter and the central canal (p<0.05). Immunofluorescent dual-label staining confirmed expression of these markers in astrocytes, microglia or neurons. Dual staining of rat and human spinal cord lesions with Oil Red O and CRT antibody showed co-localisation of CRT with the rim of myelin fragments. ELISA testing of sera from control and EAE rats demonstrated significant down-regulation (p<0.05) of CRT in the serum of EAE animals, compared to saline and IFA controls. This contrasted with significantly increased amounts of CRT detected in the sera of MS patients (p<0.05), compared to controls.ConclusionThis data highlights the potential importance of CRT and other ER stress proteins in inflammatory demyelination.

Abstract 518: Inhibition of Endoplasmic Reticulum Stress Reduces Aneurysm Induction in Different Aneuryms Models

Abdominal aorta aneurysm (AAA) is a major cause of mortality in men. The role of endoplasmic reticulum (ER) stress in the pathogenesis of AAA is unknown. Human AAA specimens from diabetic patients were found to have increased expression of ER stress markers. The inhibition of ER stress signaling with (taurine-conjugated ursodeoxycholic acid, Tudca) administration reduces the development of aneurysm in Ang-II infused and calcium chloride (CaCl2)-induced mouse models. Type 2 diabetic (db-/db-) and control (db-/db+) mice were infused with angiotensin II (Ang-II, 1000ng/kg/min) and treated with or without the ER stress inhibitor Tudca for 4 weeks. Mice infused with Ang-II displayed an increase in blood pressure, plasma cholesterol and CRP levels, which were reduced after ER stress inhibition. Human AAA and Ang-II-induced AAA are characterized by an increase in lumen, cross sectional area and were associated with increased macrophage infiltration, expression of the ER stress marker CHOP and elastic fiber disruption, and these indicators of aneurysm disease were reduced by Tudca treatment. To determine the effect of ER stress inhibition on aneurysm formation in a model that did not exhibit blood pressure and metabolic changes, we exposed the adventitial surface of carotid arteries of db-/db- and db-/db+ mice to CaCl2 and then treated the mice with or without Tudca. Our results indicate that in the CaCl2 induced carotid artery aneurysm model that treatment with Tudca significantly reduced carotid artery aneurysm formation. These results provide evidence in support of a role for that ER stress activation in the induction of aneurysm in type 2 diabetes.

Oxidation of SERCA2 at cysteine 674 increases ER stress during hind limb ischemia in mice

Reversible glutathione adducts on the cysteine 674 (C674) thiol of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) stimulate Ca2+ uptake activity, endothelial cell (EC) angiogenic signaling, and migration. This study was to determine if oxidation of SERCA C674 and endoplasmic reticulum (ER) stress affects angiogenesis during hind limb ischemia. SERCA2 C674 serine mutant heterozygote knock in (SKI) mice, lacking one allele for the redox‐regulated C674 thiol, had impaired blood flow recovery after femoral artery ligation. Immunostaining demonstrated increased SERCA C674 thiol oxidation in microvascular endothelium of ischemic limbs, and double staining revealed increased BIP expression co‐localized with the EC marker isolectin B4. Staining was greater in SKI compared with WT, indicating thiol oxidation or mutation in ischemia is associated with ER stress activation. Western blotting showed ER stress markers p‐PERK and CHOP were greater in both non‐ischemic and ischemic SKI limbs compared to WT. Cultured SKI microvascular ECs showed smaller Ca2+ stores, and higher basal and hypoxia‐induced levels of BIP and CHOP. Collectively, these data suggest that lack of the SERCA C674 thiol caused by oxidation or mutation increases EC ER stress. Thus, oxidation of SERCA2 C674 and associated Ca2+ store depletion and ER stress during hind limb ischemia may contribute to impaired angiogenesis.Funding NIH RO1 HL31607

THE ENDOPLASMIC RETICULUM STRESS-JNK PATHWAY-MEDICATED APOPTOSIS IN MACROPHAGES CONTRIBUTES TO THE INSTABILITY OF ATHEROSCLEROTIC PLAQUES INDUCED BY COLD STRESS

ObjectivesTo elucidate whether and how the endoplasmic reticulum(ER) stress-JNK pathway in macrophages is involved in the instability of atherosclerotic plaques induced by cold stress.MethodsForty male New Zealand white rabbits fed...

Prodigiosin activates endoplasmic reticulum stress cell death pathway in human breast carcinoma cell lines

Prodigiosin is a bacterial tripyrrole pigment with potent cytotoxicity against diverse human cancer cell lines. Endoplasmic reticulum (ER) stress is initiated by accumulation of unfolded or misfolded proteins in the ER lumen and may induce cell death when irremediable. In this study, the role of ER stress in prodigiosin-induced cytotoxicity was elucidated for the first time. Comparable to the ER stress inducer thapsigargin, prodigiosin up-regulated signature ER stress markers GRP78 and CHOP in addition to activating the IRE1, PERK and ATF6 branches of the unfolded protein response (UPR) in multiple human breast carcinoma cell lines, confirming prodigiosin as an ER stress inducer. Prodigiosin transcriptionally up-regulated CHOP, as evidenced by its promoting effect on the CHOP promoter activity. Of note, knockdown of CHOP effectively lowered prodigiosin's capacity to evoke PARP cleavage, reduce cell viability and suppress colony formation, highlighting an essential role of CHOP in prodigiosin-induced cytotoxic ER stress response. In addition, prodigiosin down-regulated BCL2 in a CHOP-dependent manner. Importantly, restoration of BCL2 expression blocked prodigiosin-induced PARP cleavage and greatly enhanced the survival of prodigiosin-treated cells, suggesting that CHOP-dependent BCL2 suppression mediates prodigiosin-elicited cell death. Moreover, pharmacological inhibition of JNK by SP600125 or dominant-negative blockade of PERK-mediated eIF2α phosphorylation impaired prodigiosin-induced CHOP up-regulation and PARP cleavage. Collectively, these results identified ER stress-mediated cell death as a mode-of-action of prodigiosin's tumoricidal effect. Mechanistically, prodigiosin engages the IRE1–JNK and PERK–eIF2α branches of the UPR signaling to up-regulate CHOP, which in turn mediates BCL2 suppression to induce cell death.

Endoplasmic reticulum stress is activated in endometrial adenocarcinoma

ObjectivesEndometrial cancer is the most common malignancy of the female genital tract. However, in spite of a huge advance in our understanding of endometrial cancer biology, therapeutic modalities haven't significantly changed over the past 40years. The activation of the Unfolded Protein Response (UPR) and GRP78 increase following Endoplasmic Reticulum (ER) stress have been recently identified as mechanisms favoring growth, invasion and resistance to therapy of different types of cancer. However, a possible role of ER stress and GRP78 in endometrial cancer has never been investigated. MethodsTissue specimens from normal and neoplastic endometrium were analyzed for the expression of the ER stress markers GRP78, ATF6 and CHOP by Real-Time RT-PCR. In addition, GRP78 protein expression and localization were evaluated by Western blot and immunohistochemistry, respectively. The effect of GRP78 knock down on cell growth of Ishikawa cells was analyzed by proliferation curve analysis. ResultsIn this analysis, the expression levels of GRP78, ATF6 and CHOP mRNAs were significantly increased in specimens of endometrioid endometrial carcinomas. GRP78 and ATF6 protein expression levels were also increased in specimens of endometrial adenocarcinomas. GRP78 knock down caused a decrease of Ishikawa cells' growth. ConclusionsThe increased expression of ER stress markers in endometrioid endometrial carcinomas suggests a role for ER stress, the UPR and, possibly, GRP78 in endometrial cancer. Whether these mechanisms have a substantial function in the pathogenesis of malignant transformation of human endometrium is still under investigation in our laboratory.

151. LIPOTOXICITY MEDIATED ENDOPLASMIC RETICULUM STRESS, MITOCHONDRIAL DYSFUNCTION AND APOPTOSIS CONTRIBUTE IMPAIRED OOCYTE QUALITY IN RESPONSE TO OBESITY

In obesity, accumulation of lipid in non-adipose tissues, a process termed lipotoxicity, is associated with endoplasmic reticulum (ER) stress, mitochondrial dysfunction and ultimately apoptosis . We have previously shown that diet-induced obesity in mice causes impaired oocyte developmental competence, but whether this is due to activation of lipotoxicity pathways in the ovary is not known. The present study examined the hypothesis that diet-induced lipid accumulation in the cumulus oocyte complex (COC) disrupts ER homeostasis and mitochondrial membrane potential which leads to apoptosis. COCs and mural granulosa cells were collected from ovaries of adult mice fed a high fat (HFD) or control diet for 4 weeks. ER homeostasis was assessed by measuring expression of known ER stress marker genes, GRP78, ATF4 and CHOP. COCs from mice fed HFD showed significantly increased expression of GRP78 and ATF4. There was a similar trend towards increased expression in granulosa cells. Mitochondrial function was assessed by measuring membrane potential using the dual emission probe JC-1. In COCs from mice fed HFD there were reduced numbers of active mitochondria but instead large aggregated clusters of inactive mitochondria. Apoptosis in granulosa cells was determined by DNA laddering assay which showed significantly increased DNA fragmentation in cells from mice fed HFD. Apoptosis was also assessed by TUNEL staining of paraffin embedded ovaries from identical treatment groups. Ovaries from HFD mice appeared to have increased TUNEL positivity in both granulosa and cumulus cells. Our results demonstrate that the ER stress, mitochondrial dysfunction and apoptosis are markedly increased in granulosa cells and COCs from mice fed HFD, suggesting that lipotoxicity contributes to the impaired oocyte quality and reduced fertility observed in response to obesity.

HIV-1 Protease Inhibitors Nelfinavir and Atazanavir Induce Malignant Glioma Death by Triggering Endoplasmic Reticulum Stress

HIV type 1 (HIV-1) protease inhibitors (PI) have been shown to have anticancer activity in non-HIV-associated human cancer cells. The underlying mechanism of this effect is unclear. Here, we show that the PIs nelfinavir and atazanavir cause cell death in various malignant glioma cell lines in vitro. The underlying mechanism of this antitumor effect involves the potent stimulation of the endoplasmic reticulum (ER) stress response (ESR), as indicated by increased expression of two ESR markers, GRP78 and CHOP, and activation of ESR-associated caspase-4. Induction of ESR seems to play a central role in PI-induced cell death because small interfering RNA-mediated knockdown of the protective ER chaperone GRP78 sensitizes cells; whereas knockdown of proapoptotic caspase-4 protects cells from PI-induced cell death. Furthermore, the treatment of cells with PIs leads to aggresome formation and accumulation of polyubiquitinated proteins, implying proteasome inhibition. Thus, our results support a model whereby PIs cause tumor cell death via triggering of the ESR, inhibition of proteasome activity, and subsequent accumulation of misfolded proteins. Inhibition of glioma growth via ESR takes place in the in vivo setting as well, as nelfinavir inhibits the growth of xenografted human malignant glioma, with concomitant induction of the proapoptotic ER stress marker CHOP. Because ER stress has also been reported as the mechanism for insulin resistance and diabetes, our ER stress model of PI function may also explain why these drugs may induce insulin resistance as one of their most common side effects.

Role of TNF-? in ethanol-induced hyperhomocysteinemia and murine alcoholic liver injury

We previously reported a link between ethanol-induced elevation of homocysteine, endoplasmic reticulum (ER) stress, and alcoholic liver injury in the murine model of intragastric ethanol feeding. We studied the role of TNFalpha in this setting by using TNFR1 knockout mice (C57 BL/6). There was a 7.4-fold increase of homocysteine in wild-type and a 6-fold increase in TNFR1 knockout mice with intragastric alcohol exposure for 4 weeks. Plasma TNFalpha increased in the wild-type (18.4 +/- 3.3 pg/mL vs. 8.4 +/- 1.3 pg/mL (control)) and in the knockouts (12.9 +/- 1.4 pg/mL vs. 7.2 +/- 1.6 pg/mL (control)). Similar extent of fatty liver was observed in both types. Increased ALT was observed in both groups. Necroinflammatory foci were increased significantly in ethanol-fed knockouts but not to the same extent as in the ethanol-fed wild type. Increase of hepatic apoptosis and reduction of S-adenosyl-L-methionine was detected in both types of animals fed ethanol. ER stress demonstrated by RT-PCR of mRNA of selective ER stress markers GRP78, CHOP, and SREBP1 was increased equivalently in both types of mice. Betaine administration decreased ER stress in conjunction with attenuation of the elevated plasma homocysteine in both types of animals. Betaine increased hepatic S-adenosyl-L-methionine by 28 fold in the knockouts and by 24-fold in wild type. In conclusion, TNFalpha makes a moderate contribution to the ALT elevation, necroinflammation, apoptosis, a small contribution to the fatty liver and no contribution to hyperhomocysteinemia and ER stress in intragastric alcohol fed mice.