CHOP Knockout Research Articles

Loss-of-rescue of Ryr1I4895T-related pathology by the genetic inhibition of the ER stress response mediator CHOP

RYR1 is the gene encoding the ryanodine receptor 1, a calcium release channel of the endo/sarcoplasmic reticulum. I4898T in RYR1 is one of the most common mutations that give rise to central core disease (CCD), with a variable phenotype ranging from mild to severe myopathy to lethal early-onset core-rod myopathy. Mice with the corresponding I4895T mutation in Ryr1 present mild myopathy when the mutation is heterozygous while I4895T homozygous is perinatal-lethal. Here we show that skeletal muscles of I4895T homozygous mice at birth present signs of stress of the endoplasmic reticulum (ER stress) and of the related unfolded protein response (UPR) with increased levels of the maladaptive mediators CHOP and ERO1. To gain information on the role of CHOP in the pathogenesis of RYR1I4895T-related myopathy, we generated compound Ryr1I4895T, Chop knock-out (-/-) mice. However, the genetic deletion of Chop, although it attenuates ER stress in the skeletal muscle of the newborns, does not rescue any phenotypic or functional features of Ryr1I4895T in mice: neither the perinatal-lethal phenotype nor the inability of Ryr1I4895T to respond to its agonist caffeine, but protects from ER stress-induced apoptosis. These findings suggest that genetic deletion of the ER stress response mediator CHOP is not sufficient to counteract the pathological Ryr1I4895T phenotype.

The role of the ER stress response during C. rodentium infection

Abstract C. rodentium is a mucosal pathogen of mice that shares several pathogenic mechanisms with enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC), which are two clinically important human gastrointestinal pathogens. The C. rodentium model of infection is used to study host pathogen interactions in the gastrointestinal tract and to study ulcerative colitis (UC). Mutations in unfolded protein response (UPR) pathways have been linked to a higher risk of UC development. The ER is an important organelle for protein synthesis and protein folding. Dysregulation of ER stress results in the activation of the UPR to restore homeostasis. Chop is activated downstream of the PERK arm of the UPR and is a transcription factor for multiple targets including cell death and inflammatory pathways. We hypothesized that Chop activation increases cell death and inflammation during C. rodentium infection which then promotes infection and tissue damage. We found that when a mouse epithelial cell line was treated with an ER stress inducing drug or C. rodentium there was an upregulation of Il6 and Il23 but when we inhibited Chop expression the upregulation was lost. We next infected Chop knockout mice with C. rodentium and these mice showed less colon pathology and colon colonization at Day 10. These data suggest that Chop has a role in the ability of C. rodentium to trigger inflammation and in tissue pathology.

Abstract 3275: Inhibition of ER-stress factor C/EBP homologous protein (Chop) with LNAplus™ antisense-oligonucleotides to improve immunotherapy of cancer

Abstract The microenvironment generated by tumor cells and suppressive stromal cells creates unfavorable conditions for effector immune cells in order to escape anti-tumor immunity. Besides suppressive pathways like for example the CD39/CD73 axis, mechanisms including nutrient starvation, hypoxia, exposure to high levels of reactive oxygen species and acidosis contribute to the suppression of tumor-reactive immune cells. We have recently discovered that the ER-stress response, in particular the C/EBP homologous protein (Chop) plays an important role in the suppression of tumor-exposed T cells. Furthermore, it is upregulated in activated T cells in vitro and may hamper the efficacy of adoptive T cell therapies. As Chop is a transcription factor it falls into the category of “difficult to drug” and therefore represents an optimal target for antisense-oligonucleotides. In order to revert Chop-induced suppression of T cell activity, we designed locked nucleic acid (LNA) ASOs with specificity for mouse or human Chop using our Oligofyer™ bioinformatics system. Knockdown on mRNA level was investigated in cancer cell lines and in T cells in vitro and the most potent ASOs were selected for further experiments. Downstream effects of Chop knockdown were investigated by mRNA expression analysis and flow cytometry. We furthermore investigated the effect of ex vivo Chop knockdown on the efficacy of adoptively transferred Pmel-specific T cells (recognized target: gp10025-33 peptide) in a B16 melanoma model. Treatment of cells with selected Chop-specific ASO leads to potent knockdown of Chop in vitro. In accordance with observations made in T cells derived from Chop-knockout mice, we observed an increase in T cell-associated transcription factor (Tbet), Interferon-gamma (IFN-γ) and Granzyme B (GZMB) expression in T cells that have been treated with a Chop ASO compared to control oligo treated cells. Strikingly, this translated into improved tumor control by Pmel-specific T cells. We furthermore observed an increase in IFN-γ producing tumor-infiltrating T cells and increased frequency of IFN-γ producing T cells in gp100 restimulated splenocytes from animals that received Chop ASO-treated Pmel-specific T cells. Of note, the impact of Chop knockdown was comparable to the effects that we observed in T cells derived from Chop knockout animals. Taken together, we herein show that Chop is a highly promising novel target in immunotherapy and can effectively be targeted by LNAplus™ ASOs. As shown in a model of adoptive T cell therapy, Chop ASOs have a high potential to optimize the efficacy of T cell therapies, such as chimeric antigen receptor (CAR)- or T cell receptor (TCR)-transgenic T cells. We are currently investigating the effect of systemic Chop ASO treatment in murine tumor models to expand the spectrum of applications of this innovative therapeutic tool. Citation Format: Richard Klar, Yu Cao, Eslam Mohamed, Sven Michel, Monika Schell, Lisa Hinterwimmer, Stefanie Raith, Paulo Rodriguez, Frank Jaschinski. Inhibition of ER-stress factor C/EBP homologous protein (Chop) with LNAplus™ antisense-oligonucleotides to improve immunotherapy of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3275.

The transcription factor CHOP, an effector of the integrated stress response, is required for host sensitivity to the fungal intracellular pathogen Histoplasma capsulatum.

The ability of intracellular pathogens to manipulate host-cell viability is critical to successful infection. Some pathogens promote host-cell survival to protect their replicative niche, whereas others trigger host-cell death to facilitate release and dissemination of the pathogen after intracellular replication has occurred. We previously showed that the intracellular fungal pathogen Histoplasma capsulatum (Hc) uses the secreted protein Cbp1 to actively induce apoptosis in macrophages; interestingly, cbp1 mutant strains are unable to kill macrophages and display severely reduced virulence in the mouse model of Hc infection. To elucidate the mechanism of Cbp1-induced host-cell death, we performed a comprehensive alanine scanning mutagenesis and identified all amino acid residues that are required for Cbp1 to trigger macrophage lysis. Here we demonstrate that Hc strains expressing lytic CBP1 alleles activate the integrated stress response (ISR) in infected macrophages, as indicated by an increase in eIF2α phosphorylation as well as induction of the transcription factor CHOP and the pseudokinase Tribbles 3 (TRIB3). In contrast, strains bearing a non-lytic allele of CBP1 fail to activate the ISR, whereas a partially lytic CBP1 allele triggers intermediate levels of activation. We further show that macrophages deficient for CHOP or TRIB3 are partially resistant to lysis during Hc infection, indicating that the ISR is critical for susceptibility to Hc-mediated cell death. Moreover, we show that CHOP-dependent macrophage lysis is critical for efficient spread of Hc infection to other macrophages. Notably, CHOP knockout mice display reduced macrophage apoptosis and diminished fungal burden and are markedly resistant to Hc infection. Together, these data indicate that Cbp1 is required for Hc to induce the ISR and mediate a CHOP-dependent virulence pathway in the host.

746 Enhanced skin tumor development in CHOP knockout mice after UVB exposure involves modulation of multiple survival and apoptotic signaling pathways

746 Enhanced skin tumor development in CHOP knockout mice after UVB exposure involves modulation of multiple survival and apoptotic signaling pathways

PRESSURE-OVERLOAD INDUCED LV HYPERTROPHY AND DYSFUNCTION: CRITICAL ROLES OF CAMKII AND P38 MAP KINASE IN ER STRESS SIGNALING PATHWAY

The role of endoplasmic reticulum (ER) stress in pressure overload-induced LV hypertrophy and dysfunction is poorly understood. The specific roles of CaMKII and p38 signaling remain unclear. Transverse aortic constriction (TAC) was performed in age-matched CHOP knockout (KO) mice and controls.

Ethanol promotes saturated fatty acid-induced hepatoxicity through endoplasmic reticulum (ER) stress response

Serum palmitic acid (PA), a type of saturated fatty acid, causes lipid accumulation and induces toxicity in hepatocytes. Ethanol (EtOH) is metabolized by the liver and induces hepatic injury and inflammation. Herein, we analyzed the effects of EtOH on PA-induced lipotoxicity in the liver. Our results indicated that EtOH aggravated PA-induced apoptosis and lipid accumulation in primary rat hepatocytes in dose-dependent manner. EtOH intensified PA-caused endoplasmic reticulum (ER) stress response in vitro and in vivo, and the expressions of CHOP, ATF4, and XBP-1 in nucleus were significantly increased. EtOH also increased PA-caused cleaved caspase-3 in cytoplasm. In wild type and CHOP−/− mice treated with EtOH and high fat diet (HFD), EtOH worsened the HFD-induced liver injury and dyslipidemia, while CHOP knockout blocked toxic effects of EtOH and PA. Our study suggested that targeting UPR-signaling pathways is a promising, novel approach to reducing EtOH and saturated fatty acid-induced metabolic complications.

The CHOP conundrum: controversial discussion about the role of endoplasmic reticulum stress in hepatotoxicity.

Therefore, the recently published observation of Godoy and his team that CHOP does not play a pro-damage role in the liver is surprising (Campos et al. 2014). The authors used the well-characterized model of acute hepatotoxicity by carbon tetrachloride (CCl4), which is known to induce pericentral liver damage (Hohme et al. 2007; Hoehme et al. 2010; Hammad et al. 2014). In their study, CCl4 caused nuclear translocation of CHOP in the liver (Campos et al. 2014). Importantly, CHOP translocation to the nucleus occurred only in the pericentral compartment of the lobule, the region where CCl4 induces necrosis. Moreover, pro-apoptotic CHOP-regulated genes were observed, such as upregulated GADD34, TrB3, and erOIl (Campos et al. 2014). This scenario strongly suggests a situation of CCl4-induced er stress, where CHOP plays a pro-damage function. However, when Campos and colleagues repeated the experiment in CHOP knockout mice, they observed no major difference compared with wild-type animals. rather, a trend to slightly more liver damage was seen in the CHOP knockout mice. since the result was surprising, the experiment was repeated with various doses and exposure periods. However, the provocative negative result was reproduced (Campos et al. 2014). This leads to the question, whether the role of CHOP in hepatotoxicity is more complex than previously expected. It also remains difficult to understand, why CHOP plays a pro-damage role in acetaminophen, but not in CCl4-induced hepatotoxicity. The current study of Campos et al. (2014) shows that CHOP cannot be accepted as a well-understood pro-damage biomarker of hepatotoxicity.

The transcription factor CHOP, a central component of the transcriptional regulatory network induced upon CCl4 intoxication in mouse liver, is not a critical mediator of hepatotoxicity.

Since xenobiotics enter the organism via the liver, hepatocytes must cope with numerous perturbations, including modifications of proteins leading to endoplasmic reticulum stress (ER-stress). This triggers a signaling pathway termed unfolded protein response (UPR) that aims to restore homeostasis or to eliminate disturbed hepatocytes by apoptosis. In the present study, we used the well-established CCl4 hepatotoxicity model in mice to address the questions whether CCl4 induces ER-stress and, if so, whether the well-known ER-stress effector CHOP is responsible for CCl4-induced apoptosis. For this purpose, we treated mice with a high dose of CCl4 injected i.p. and followed gene expression profile over time using Affymetrix gene array analysis. This time resolved gene expression analysis allowed the identification of gene clusters with overrepresented binding sites for the three most important ER-stress induced transcription factors, CHOP, XBP1 and ATF4. Such result was confirmed by the demonstration of CCl4-induced XBP1 splicing, upregulation of CHOP at mRNA and protein levels, and translocation of CHOP to the nucleus. Two observations indicated that CHOP may be responsible for CCl4-induced cell death: (1) Nuclear translocation of CHOP was exclusively observed in the pericentral fraction of hepatocytes that deteriorate in response to CCl4 and (2) CHOP-regulated genes with previously reported pro-apoptotic function such as GADD34, TRB3 and ERO1L were induced in the pericentral zone as well. Therefore, we compared CCl4 induced hepatotoxicity in CHOP knockout versus wild-type mice. Surprisingly, genetic depletion of CHOP did not afford protection against CCl4-induced damage as evidenced by serum GOT and GPT as well as quantification of dead tissue areas. The negative result was obtained at several time points (8, 24 and 72 h) and different CCl4 doses (1.6 and 0.132 g/kg). Overall, our results demonstrate that all branches of the UPR are activated in mouse liver upon CCl4 treatment. However, CHOP does not play a critical role in CCl4-induced cell death and cannot be considered as a biomarker strictly linked to hepatotoxicity. The role of alternative UPR effectors such as XBP1 remains to be investigated.

Stromal interaction molecule 1 deletion in smooth muscle cells protects hypertension‐induced vascular dysfunction by endoplasmic reticulum stress‐dependent mechanism (1065.13)

Background: stromal interacting molecule‐1 (STIM1) and endoplasmic reticulum (ER) regulate intracellular calcium for vascular function. Hypertension is associated with vascular dysfunction raising the question of whether STIM1 has any role in vascular dysfunction through ER stress and oxidative stress in hypertension.Methods & results: We used male C57/BL6, homozygous/heterozygous STIM1 knockout specifically in smooth muscle cells (STIM1SMC‐/‐, STIM1SMC‐/+), and homozygous CHOP knockout (CHOP‐/‐) mice infused with and without angiotensin II (Ang II, 400 ng/Kg/min) for 4 weeks. Hypertension development was significantly delayed in STIM1SMC‐/‐ and CHOP‐/‐ mice infused with Ang II compared to control and STIM1SMC‐/+ with Ang II. The vasoconstriction of mesenteric resistance artery and thoracic aorta in response to phenylephrine was significantly reduced only in STIM1SMC‐/‐ mice infused with and without Ang II. However, vasoconstriction to thromboxane was identical in all groups and was augmented in hypertension with low effect in STIM1SMC‐/‐ and CHOP‐/‐. The endothelium‐dependent relaxation was similar in all groups but it was impaired in the control and STIMSMC‐/+ infused with Ang II, protected in STIM1SMC‐/‐ + Ang II and rescued in CHOP‐/‐ + Ang II. The NADPH oxidase activity was elevated, while eNOS phosphorylation, cGMP and nitrite levels were reduced in control with Ang II, compared to STIM1SMC‐/‐ and CHOP‐/‐ with and without Ang II. ER stress markers were only enhanced in control infused with Ang II. The expression of STIM1 was augmented in all groups of mice infused with Ang II.Conclusions: We conclude that in hypertension, the aberrant increase in STIM1 expression causes vascular dysfunction through ER stress and increases oxidative stress‐dependent mechanisms.

Combined treatment with SAHA, bortezomib, and clarithromycin for concomitant targeting of aggresome formation and intracellular proteolytic pathways enhances ER stress-mediated cell death in breast cancer cells

The ubiquitin–proteasome pathway and the autophagy–lysosome pathway are two major intracellular protein degradation systems. We previously reported that clarithromycin (CAM) blocks autophagy flux, and that combined treatment with CAM and proteasome inhibitor bortezomib (BZ) enhances ER-stress-mediated apoptosis in breast cancer cells, whereas treatment with CAM alone results in almost no cytotoxicity. Since HDAC6 is involved in aggresome formation, which is recognized as a cytoprotective response serving to sequester misfolded proteins and facilitate their clearance by autophagy, we further investigated the combined effect of vorinostat (suberoylanilide hydroxamic acid (SAHA)), which has a potent inhibitory effect for HDAC6, with CAM and BZ in breast cancer cell lines. SAHA exhibited some cytotoxicity along with an increased acetylation level of α-tubulin, a substrate of HDAC6. Combined treatment of SAHA, CAM, and BZ potently enhanced the apoptosis-inducing effect compared with treatment using each reagent alone or a combination of two of the three. Expression levels of ER-stress-related genes, including the pro-apoptotic transcription factor CHOP (GADD153), were maximally induced by the simultaneous combination of three reagents. Like breast cancer cell lines, a wild-type murine embryonic fibroblast (MEF) cell line exhibited enhanced cytotoxicity and maximally up-regulated Chop after combined treatment with SAHA, CAM, and BZ; however, a Chop knockout MEF cell line almost completely canceled this enhanced effect. The specific HDAC6 inhibitor tubacin also exhibited a pronounced cytocidal effect with a combination of CAM plus BZ. These data suggest that simultaneous targeting of intracellular proteolytic pathways and HDAC6 enhances ER-stress-mediated apoptosis in breast cancer cells.

CHOP is a critical regulator of acetaminophen-induced hepatotoxicity

The liver is a major site of drug metabolism and elimination and as such is susceptible to drug toxicity. Drug induced liver injury is a leading cause of acute liver injury, of which acetaminophen (APAP) is the most frequent causative agent. APAP toxicity is initiated by its toxic metabolite NAPQI. However, downstream mechanisms underlying APAP induced cell death are still unclear. Endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) have recently emerged as major regulators of metabolic homeostasis. UPR regulation of the transcription repressor CHOP promotes cell death. We analyzed the role of UPR and CHOP in mediating APAP hepatotoxicity. A toxic dose of APAP was orally administered to wild type (wt) and CHOP knockout (KO) mice and damage mechanisms were assessed. CHOP KO mice were protected from APAP induced damage and exhibited decreased liver necrosis and increased survival. APAP metabolism in CHOP KO mice was undisturbed and glutathione was depleted at similar kinetics to wt. ER stress and UPR activation were overtly seen 12h following APAP administration, a time that coincided with strong upregulation of CHOP. Remarkably, CHOP KO but not wt mice exhibited hepatocyte proliferation at sites of necrosis. In vitro, large T immortalized CHOP KO hepatocytes were protected from APAP toxicity in comparison to wt control cells. CHOP upregulation during APAP induced liver injury compromises hepatocyte survival in various mechanisms, in part by curtailing the regeneration phase following liver damage. Thus, CHOP plays a pro-damage role in response to APAP intoxication.

Abstract LB-157: Combined treatment with clarithromycin and bortezomib enhances cytotoxicity via endplasmic reticulum stress-meidated CHOP induction and autophagy in breast cancer cells

Abstract Bortezomib (BZ), a selective and potent inhibitor of the 26S proteasome, has been approved for treating multiple myeloma. It was reported that BZ also inhibited breast cancer cell growth. Autophagy is a highly conserved cellular process in eukaryotes. Intracellular proteins and organelles including endoplasmic reticulum (ER) are engulfed in a double-membrane vesicle called an autophagosome and delivered to lysosomes for degradation by lysosomal hydrolases. However, recent reports focused on that the selective degradation pathway of ubiquitinated protein through autophagy via p62 and the related protein NBR1, which are docking proteins having both a LC3-interacting region and a ubiquitin-associated domain. LC3 is essential for autophagy and is associated with autophagosome membranes after processing. Thus, after binding ubiquitin via their C-terminal ubiquitin-associated domains, p62-mediated degradation of ubiquitinated cargo occurs by selective autophagy. Clarithromycin (CAM) has been reported to inhibit autophagy flux and to exhibit some cell growth inhibition in myeloma cells. Based on previous data, we attempted to investigate whether combined treatment with CAM and BZ in terms of simultaneous inhibition of two major intracellular protein degradation systems enhances ER stress-mediated cell death in breast cancer cells. BZ potently induces apoptosis as well as autophagy in breast cancer cell lines such as MDA-MB-231 and MDA-MB-468. The combined treatment of CAM and BZ significantly enhances cytotoxicity in these cell lines. Although treatment with up to 100 μg/ml CAM alone had little effect on cell growth inhibition, the accumulation of autophagosomes and p62 was observed after treatment with 25 μg/ml CAM. This result indicated that CAM blocked autophagy flux. However, the combined treatment of BZ and CAM resulted in more pronounced autophagy induction, as assessed by increased expression ratios of LC3B-II to LC3B-I and clearance of intracellular p62, than treatment with BZ alone. This combination further enhanced induction of the pro-apoptotic transcription factor CHOP and the chaperone protein GRP78. Knockdown of CHOP by siRNA attenuated the death-promoting effect of BZ in MDA-MB-231 cells. A wildtype MEF cell line also exhibited enhanced BZ-induced cytotoxicity with the addition of CAM, whereas a Chop knockout MEF cell line completely abolished this enhancement and exhibited resistance to BZ treatment. These data suggest that ER-stress mediated CHOP induction is involved in pronounced cytotoxicity by combining these reagents. Simultaneously targeting two major intracellular protein degradation pathways such as the ubiquitin proteasome system by BZ and the autophagy-lysosome pathway by CAM may improve the therapeutic outcome in breast cancer patients via ER-stress mediated apoptosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-157. doi:1538-7445.AM2012-LB-157

Clarithromycin enhances bortezomib-induced cytotoxicity via endoplasmic reticulum stress-mediated CHOP (GADD153) induction and autophagy in breast cancer cells

The specific 26S proteasome inhibitor, bortezomib (BZ) potently induces apoptosis as well as autophagy in metastatic breast cancer cell lines such as MDA-MB-231 and MDA-MB-468. The combined treatment of clarithromycin (CAM) and BZ significantly enhances cytotoxicity in these cell lines. Although treatment with up to 100 μg/ml CAM alone had little effect on cell growth inhibition, the accumulation of autophagosomes and p62 was observed after treatment with 25 μg/ml CAM. This result indicated that CAM blocked autophagy flux. However, the combined treatment of BZ and CAM resulted in more pronounced autophagy induction, as assessed by increased expression ratios of LC3B-II to LC3B-I and clearance of intracellular p62, than treatment with BZ alone. This combination further enhanced induction of the pro-apoptotic transcription factor CHOP (CADD153) and the chaperone protein GRP78. Knockdown of CHOP by siRNA attenuated the death-promoting effect of BZ in MDA-MB-231 cells. A wild-type murine embryonic fibroblast (MEF) cell line also exhibited enhanced BZ-induced cytotoxicity with the addition of CAM, whereas a Chop knockout MEF cell line completely abolished this enhancement and exhibited resistance to BZ treatment. These data suggest that endoplasmic reticulum (ER)-stress mediated CHOP induction is involved in pronounced cytotoxicity by combining these reagents. Simultaneously targeting two major intracellular protein degradation pathways such as the ubiquitin-proteasome system by BZ and the autophagy-lysosome pathway by CAM may improve the therapeutic outcome in breast cancer patients via ER-stress mediated apoptosis.

Involvement of endoplasmic reticulum stress-mediated CHOP (GADD153) induction in the cytotoxicity of 2-aminophenoxazine-3-one in cancer cells

In this study, 2-aminophenoxazine-3-one (Phx-3) exhibited a potent cell growth inhibitory effect with apoptotic features in a dose-dependent manner in various cancer cell lines tested. Comparison of the expression profiles of endoplasmic reticulum (ER) stress-related genes in U266 multiple myeloma cells after treatment with Phx-3 and the ER stress inducers tunicamycin (TNM) and thapsigargin (TPG) indicated that although TNM and TPG potently induced pro-apoptotic transcription factor CHOP (GADD153) within 8 h of treatment, Phx-3 induced almost no CHOP within 48 h of treatment in U266 cells. However, murine embryonic fibroblast (MEF) cells and other cancer cell lines (e.g. A549 lung cancer cells and HL-60 acute leukemia cells) exhibited up-regulation of CHOP after treatment with Phx-3. The potency of CHOP induction in response to Phx-3 appeared to be partially correlated with the cytotoxic sensitivity of Phx-3 among various cell lines tested. MEF cells derived from CHOP knockout mice were more resistant to Phx-3 than wild-type MEF cells. Since Phx-3 has been shown to induce activation of NF-κB, a transcription factor functioning as a repressor of CHOP, we further treated U266 cells with a combination of Phx-3 and NF-κB inhibitors (e.g. BAY11-7082 or parthenolide). This enhanced cytotoxicity along with up-modulation of CHOP in U266 cells. These data suggest that ER stress-mediated CHOP induction by Phx-3 is involved in the cytotoxic effect. Regulation of CHOP expression appears to be a potent therapeutic target for cancer treatment.

18beta‐glycyrrhetinic acid prevents free fatty acid‐induced lipotoxicity by inhibiting ER stress and oxidative stress

The endoplasmic reticulum (ER) stress signal pathway has been linked to free fatty acid (FFA)‐induced inflammatory response and disease progression of nonalcoholic fatty liver disease in clinic. We have previously shown that 18?β‐glycyrrhetinic acid (GA) prevents FFA‐induced hepatic lipotoxicity by inhibiting cathepsin B expression and activity. But the underlying cellular mechanism remains unclear. This study determined if GA prevents FFA‐induced lipotoxicity by regulating ER stress signaling pathways.MethodsMouse macrophages, primary rat hepatocytes and Kupffer cells were used in this study. Cells were treated with palmitic acid (PA) and oleic acid (OA) in the presence or absence of GA. Activation of the UPR was determined by real time RT‐PCR and Western Blot analysis. The apoptosis was detected by fluorescence microscopy using Annexin V‐FITC/propidium iodide. Reactive oxygen species (ROS) was measured using dichlorofluorescein diacetate (DCFH‐DA).ResultsPA dose‐dependently induced UPR activation, ROS formation, and apoptosis both in macrophages and hepatocytes, while OA had no effect. GA not only inhibited PA‐induced UPR activation, but also inhibited PA‐induced ROS formation. PA‐induced ROS formation and cathepsin B expression were inhibited in CHOP knockout macrophages and hepatocytes.ConclusionGA prevents FFA‐induced lipotoxicity by inhibiting ER stress and oxidative stress.

W1269 HIV Protease Inhibitors Induce ER Stress and Disrupt Barrier Function in Intestinal Epithelial Cells

HIV protease inhibitor (PI)-induced diarrhea is one of the most frequent adverse side effects of these drugs and occurs in 16 to 62% of patients. Recently it has been reported that HIV PIs induce apoptosis and decrease barrier function in intestinal epithelial cells. However, the underlying cellular/molecular mechanisms remain unclear.We have previously demonstrated that HIV PIs induce endoplasmic reticulum (ER) stress, activate the unfolded protein response (UPR), and promote apoptosis in macrophages and hepatocytes. In the present study, we examined whether HIV PI-induced ER stress also plays a role in HIV PI-induced apoptosis and disruption of barrier function in intestinal epithelial cells. Methods: In Vitro studies were conducted in IEC6 cells (normal rat intestinal epithelial cells), differentiated IEC6 (stable Cdx2-transfected, IEC6-Cdx2L1), and stable SEAP-transfected IEC6 cells. HIV PIinduced ER stress was measured by decrease of SEAP activity. The mRNA and protein expression levels of ER stress-related genes were determined by real-time RT-PCR and western blot analysis, respectively. The paracellular permeability was measured by paracellular tracer flux assay and membrane-impermeable molecule, [14C]-mannitol, served as tracer. Lentiviral CHOP siRNA was used to knock down CHOP expression in IEC6 cells. Both C57/B6 wild type and CHOP knock out mice were used in In Vivo studies. Mice were gavaged with individual HIV PI (50 mg/kg) for three weeks. The pathological changes in intestine and colon were determined by histological evaluation. Results: Both ritonavir and lopinavir significantly induced ER stress, increased the expression of GRP78, CHOP, XBP-1 and ATF4, and promoted apoptosis in IEC6 and IEC6-Cdx2L1 cells. Paracellular permeability was increased by ~ 2.5 to 5 fold after cells were treated with 15 μM of ritonavir or lopinavir for 24 h. In contrast, amprenavir neither induced ER stress, nor increased paracellular permeability. Down-regulation of CHOP expression in IEC6 cells reduced HIV PI-induced apoptosis and disruption of barrier function. In Vivo studies demonstrated that ritonavir and lopinavir also significantly induced CHOP, XBP-1 and ATF4 expression in the intestine and colon of wild type mice. Furthermore, histological assessment indicated that ritonavir and lopinavir significantly induced intestinal epithelial damage in wild type mice, but had less effect in CHOP knock out mice. Conclusions: These findings indicate that HIV PI-induced ER stress and subsequent activation of the UPR represents an important cellular/molecular mechanism underlying HIV PI-induced disruption of intestinal barrier function.

HIV protease inhibitor‐induced inflammatory response is coupled to ER Stress and MAPKs signaling pathways in macrophages

Macrophages are the key players in the inflammatory response. Our previous studies have shown that HIV protease inhibitors (PIs) induce ER stress and increase TNF‐¦Á and IL‐6 expression in macrophages. However, the underlying signaling mechanisms remain unclear. The aim of present study was to determine if MAPKs are involved in HIV‐PI‐induced inflammatory response in macrophages. J774A.1 cells and primary mouse peritoneal macrophages isolated from wild type and CHOP knock out mice were used in this study. Cells were treated with individual HIV PI. The mRNA and protein levels of TNF‐¦Á and IL‐6 were measured by real‐time RT‐PCR and ELISA, respectively. Activation of MAPKs was detected by Western blot using phospho‐specific antibodies. Results: HIV PIs, atazanavir and lopinavir, activated ERK1/2 and JNK, but not p38. HIV PI‐induced TNF‐¦Áand IL‐6 expression was attenuated by PD98059 and JNKII, but not SB203580. Furthermore, HIV PI‐induced ERK1/2 activation was completely inhibited by CHOP knock down using siRNA or in CHOP knock out macrophages. However, HIV PI‐induced CHOP expression was only inhibited by JNKII but not PD98059 and SB203580. Conclusions: Both ERK1/2 and JNK signaling pathways are involved in HIV PIs‐induced increase of TNF‐¦Á and IL‐6 expression in macrophages. CHOP plays an essential role in HIV PI‐induced inflammatory cytokine release by activating ERK1/2 and JNK signaling pathways in macrophages.

C/EBP Homologous Protein (CHOP) Is Crucial for the Induction of Caspase-11 and the Pathogenesis of Lipopolysaccharide-Induced Inflammatio

C/EBP homologous protein (CHOP)/growth arrest and DNA damage-inducible gene 153 is a C/EBP family transcription factor which is involved in endoplasmic reticulum (ER) stress-mediated apoptosis. To determine whether the ER stress-CHOP pathway is involved in the pathogenesis of the lung inflammation, mice were given LPS intratracheally. Treatment with LPS induced mRNAs for CHOP and BiP. The LPS-induced inflammation in lung, including the IL-1beta activity in bronchoalveolar lavage fluid, was attenuated in the Chop knockout mice. Caspase-11, which is needed for the activation of procaspase-1 and pro-IL-1beta, was induced by LPS treatment in the lung and primary cultured macrophages. The induction of caspase-11 by LPS was suppressed in Chop knockout mice. Caspase-11 was also induced by such ER stress inducers as thapsigargin or tunicamycin. These results show that CHOP plays a crucial role in the pathogenesis of inflammation through the induction of caspase-11.

Hsp70-DnaJ chaperone pair prevents nitric oxide- and CHOP-induced apoptosis by inhibiting translocation of Bax to mitochondria.

We reported that the endoplasmic reticulum (ER) stress pathway involving CHOP, a member of the C/EBP transcription factor family, plays a key role in nitric oxide (NO)-mediated apoptosis of macrophages and pancreatic beta cells. We also showed that the cytosolic chaperone pair of hsp70 and dj1 (hsp40/hdj-1) or dj2 (HSDJ/hdj-2) prevents NO-mediated apoptosis upstream of cytochrome c release from mitochondria. To analyze roles of the chaperone pair in preventing apoptosis, RAW 264.7 macrophages stably expressing hsp70 and dj1 or dj2 were established. The chaperone pair prevented LPS/IFN-gamma-induced and NO-mediated apoptosis downstream of CHOP induction. hsp70 mutant protein lacking the ATPase domain or the C-terminal EEVD sequence were not effective in preventing CHOP-induced apoptosis. A mutant dj2 lacking the C-terminal prenylation CaaX motif, was also not effective. When wild-type RAW 264.7 cells were treated with LPS/IFN-gamma, NO-mediated apoptosis was induced, and proapoptotic Bcl-2 family protein Bax was translocated from cytosol to mitochondria. This translocation was prevented in cells stably expressing hsp70/dj2, and in CHOP knockout cells. Overexpression of CHOP in wild-type cells also induced translocation of Bax and this translocation was prevented in cells expressing hsp70/dj2. CHOP-induced apoptosis was prevented by Bax knock-down. Coimmunoprecipitation experiments showed that Bax interacts with both hsp70 and dj1/dj2. ATPase domain of hsp70 was necessary for the binding with Bax. These findings indicate that CHOP-induced apoptosis is mediated by translocation of Bax from the cytosol to the mitochondria, and hsp70/dj1 or dj2 chaperone pair prevents apoptosis by interacting with Bax and preventing translocation to the mitochondria.