Blocking CHOP-dependent TXNIP shuttling to mitochondria attenuates albuminuria and mitigates kidney injury in nephrotic syndrome.

Albuminuria is a hallmark of glomerular disease of various etiologies. It is not only a symptom of glomerular disease but also a cause leading to glomerulosclerosis, interstitial fibrosis, and eventually, a decline in kidney function. The molecular mechanism underlying albuminuria-induced kidney injury remains poorly defined. In our genetic model of nephrotic syndrome (NS), we have identified CHOP (C/EBP homologous protein)-TXNIP (thioredoxin-interacting protein) as critical molecular linkers between albuminuria-induced ER dysfunction and mitochondria dyshomeostasis. TXNIP is a ubiquitously expressed redox protein that binds to and inhibits antioxidant enzyme, cytosolic thioredoxin 1 (Trx1), and mitochondrial Trx2. However, very little is known about the regulation and function of TXNIP in NS. By utilizing Chop-/- and Txnip-/- mice as well as 68Ga-Galuminox, our molecular imaging probe for detection of mitochondrial reactive oxygen species (ROS) in vivo, we demonstrate that CHOP up-regulation induced by albuminuria drives TXNIP shuttling from nucleus to mitochondria, where it is required for the induction of mitochondrial ROS. The increased ROS accumulation in mitochondria oxidizes Trx2, thus liberating TXNIP to associate with mitochondrial nod-like receptor protein 3 (NLRP3) to activate inflammasome, as well as releasing mitochondrial apoptosis signal-regulating kinase 1 (ASK1) to induce mitochondria-dependent apoptosis. Importantly, inhibition of TXNIP translocation and mitochondrial ROS overproduction by CHOP deletion suppresses NLRP3 inflammasome activation and p-ASK1-dependent mitochondria apoptosis in NS. Thus, targeting TXNIP represents a promising therapeutic strategy for the treatment of NS.

Imeglimin Ameliorates ß-Cell Apoptosis by Modulating the Endoplasmic Reticulum Homeostasis Pathway.

The effects of imeglimin, a novel antidiabetes agent, on ß-cell function remain unclear. Here, we unveiled the impact of imeglimin on ß-cell survival. Treatment with imeglimin augmented mitochondrial function, enhanced insulin secretion, promoted ß-cell proliferation, and improved ß-cell survival in mouse islets. Imeglimin upregulated the expression of endoplasmic reticulum (ER)-related molecules, including Chop (Ddit3), Gadd34 (Ppp1r15a), Atf3, and Sdf2l1, and decreased eIF2α phosphorylation after treatment with thapsigargin and restored global protein synthesis in ß-cells under ER stress. Imeglimin failed to protect against ER stress-induced ß-cell apoptosis in CHOP-deficient islets or in the presence of GADD34 inhibitor. Treatment with imeglimin showed a significant decrease in the number of apoptotic ß-cells and increased ß-cell mass in Akita mice. Imeglimin also protected against ß-cell apoptosis in both human islets and human pluripotent stem cell-derived ß-like cells. Taken together, imeglimin modulates the ER homeostasis pathway, which results in the prevention of ß-cell apoptosis both in vitro and in vivo.

CHOP deletion and anti-neuroinflammation treatment with hesperidin synergistically attenuate NMDA retinal injury in mice.

Glaucoma is a leading cause of blindness worldwide and is characterized by degeneration associated with the death of retinal ganglion cells (RGCs). It is believed that glaucoma is a group of heterogeneous diseases with multifactorial pathomechanisms. Here, we investigate whether anti-inflammation treatment with an ER stress blockade can selectively promote neuroprotection against NMDA injury in the RGCs. Retinal excitotoxicity was induced with an intravitreal NMDA injection. Microglial activation and neuroinflammation were evaluated with Iba1 immunostaining and cytokine gene expression. A stable HT22 cell line transfected with an NF-kB reporter was used to assess NF-kB activity after hesperidin treatment. CHOP-deficient mice were used as a model of ER stress blockade. Retinal cell death was evaluated with a TUNEL assay. As results, in the NMDA injury group, Iba1-positive microglia increased 6 h after NMDA injection. Also at 6 h, pro-inflammatory cytokines and chemokine increased, including TNFα, IL-1b, IL-6 and MCP-1. In addition, the MCP-1 promoter-driven EGFP signal, which we previously identified as a stress signal in injured RGCs, also increased; hesperidin treatment suppressed this inflammatory response and reduced stressed RGCs. In CHOP-deficient mice that received an NMDA injection, the gene expression of pro-inflammatory cytokines, chemokines, markers of active microglia, and inflammatory regulators was greater than in WT mice. In WT mice, hesperidin treatment partially prevented retinal cell death after NMDA injury; this neuroprotective effect was enhanced in CHOP-deficient mice. These findings demonstrate that ER stress blockade is not enough by itself to prevent RGC loss due to neuroinflammation in the retina, but it has a synergistic neuroprotective effect after NMDA injury when combined with an anti-inflammatory treatment based on hesperidin.

DDIT3/CHOP promotes autophagy in chondrocytes via SIRT1-AKT pathway.

Endoplasmic reticulum (ER) stress can initiate autophagy via unfolded protein response (UPR). As a key downstream gene of UPR, DDIT3/CHOP is expressed in chondrocytes. However, the regulation mechanism of DDIT3/CHOP on autophagy in chondrocytes remains unclear. In this study, the expression levels of autophagic markers Beclin1 and LC3B were found to decrease while p62 increase in the tibial growth plate and costal primary chondrocytes from DDIT3/CHOP KO mice. In vitro, overexpressing DDIT3/CHOP induced autophagy in ATDC5 chondrocytes, displaying an elevated immunofluorescence signal of LC3B and elevated numbers of autophagosomes and autolysosomes. Analysis of the gain- and loss-of-function indicated that the protein level of Beclin1 and the ratio of LC3BII/I increased in DDIT3/CHOP overexpression cells, whereas decreased in DDIT3/CHOP knockdown cells. The decreased level of p62 and additional accumulation of LC3BII caused by chloroquine (CQ) further indicated that DDIT3/CHOP enhanced autophagic flux. Mechanistically, we found that DDIT3/CHOP binds directly to the promoter of SIRT1 to promote its expression by CHIP, qRT-PCR, and Western blot analysis. In addition, SIRT1 enhanced autophagic activity in ATDC5 cells, and inhibition or activation of SIRT1 partially reversed the effect of overexpressing or downregulating DDIT3/CHOP on autophagy. Furthermore, AKT signaling was found to be responsible for DDIT3/CHOP-regulated autophagy in ATDC5 cells. SIRT1 knockdown reversed the effect of DDIT3/CHOP overexpression on AKT signaling. In conclusion, our data clarifies that DDIT3/CHOP promotes autophagy in ATDC5 chondrocytes through the SIRT1-AKT pathway. These results were also confirmed in the primary chondrocytes.

DDIT3 Targets Innate Immunity via the DDIT3-OTUD1-MAVS Pathway To Promote Bovine Viral Diarrhea Virus Replication.

DNA damage-inducible transcript 3 (DDIT3) plays important roles in endoplasmic reticulum (ER) stress-induced apoptosis and autophagy, but its role in innate immunity is not clear. Here, we report that DDIT3 inhibits the antiviral immune response during bovine viral diarrhea virus (BVDV) infection by targeting mitochondrial antiviral signaling (MAVS) in Madin-Darby bovine kidney (MDBK) cells and in mice. BVDV infection induced high DDIT3 mRNA and protein expression. DDIT3 overexpression inhibited type I interferon (IFN-I) and IFN-stimulated gene production, thereby promoting BVDV replication, while DDIT3 knockdown promoted the antiviral innate immune response to suppress viral replication. DDIT3 promoted NF-κB-dependent ovarian tumor (OTU) deubiquitinase 1 (OTUD1) expression. Furthermore, OTUD1 induced upregulation of the E3 ubiquitin ligase Smurf1 by deubiquitinating Smurf1, and Smurf1 degraded MAVS in MDBK cells in a ubiquitination-dependent manner, ultimately inhibiting IFN-I production. Moreover, knocking out DDIT3 promoted the antiviral innate immune response to reduce BVDV replication and pathological changes in mice. These findings provide direct insights into the molecular mechanisms by which DDIT3 inhibits IFN-I production by regulating MAVS degradation.IMPORTANCE Extensive studies have demonstrated roles of DDIT3 in apoptosis and autophagy during viral infection. However, the role of DDIT3 in innate immunity remains largely unknown. Here, we show that DDIT3 is positively regulated in bovine viral diarrhea virus (BVDV)-infected Madin-Darby bovine kidney (MDBK) cells and could significantly enhance BVDV replication. Importantly, DDIT3 induced OTU deubiquitinase 1 (OTUD1) expression by activating the NF-κB signaling pathway, thus increasing intracellular Smurf1 protein levels to degrade MAVS and inhibit IFN-I production during BVDV infection. Together, these results indicate that DDIT3 plays critical roles in host innate immunity repression and viral infection facilitation.

Roles of Autophagy and Pancreatic Secretory Trypsin Inhibitor in Trypsinogen Activation in Acute Pancreatitis.

The focus of the review is on roles of autophagy and pancreatic secretory trypsin inhibitor (PSTI), an endogenous trypsin inhibitor, in trypsinogen activation in acute pancreatitis. Acute pancreatitis is a disease in which tissues in and around the pancreas are autodigested by pancreatic digestive enzymes. This reaction is triggered by the intrapancreatic activation of trypsinogen. Autophagy causes trypsinogen and cathepsin B, a trypsinogen activator, to colocalize within the autolysosomes. Consequently, if the resultant trypsin activity exceeds the inhibitory activity of PSTI, the pancreatic digestive enzymes are activated, and they cause autodigestion of the acinar cells. Thus, autophagy and PSTI play important roles in the development and suppression of acute pancreatitis, respectively.

CAAT/enhancer binding protein-homologous protein deficiency attenuates liver ischemia/reperfusion injury in mice.

Ischemia/reperfusion injury (IRI) is one of the main causes of liver dysfunction after liver surgery. Involvement of endoplasmic reticulum (ER) stress in various diseases has been demonstrated, and CAAT/enhancer binding protein-homologous protein (CHOP) is a transcriptional regulator that is induced by ER stress. It is also a key regulator of ER stress-mediated apoptosis. The aim of this study was to investigate the role of CHOP in liver IRI. Wild type (WT) and CAAT/enhancer binding protein-homologous protein knockout (CHOP-/-) mice were subjected to 70% liver warm ischemia/reperfusion for 60 minutes. At different times after reperfusion, liver tissues and blood samples were collected for evaluation. Induction of ER stress including CHOP expression was ascertained. Liver damage was evaluated based on serum liver enzymes, liver histology, and neutrophil infiltration. Hepatocyte death including apoptosis was assessed. Liver warm IRI induced ER stress in both WT and CHOP-/- mice. In addition, CHOP expression was up-regulated in WT mice. At 6 hours after reperfusion, liver damage was attenuated in CHOP-/- mice. On the basis of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining, apoptotic and necrotic cells were significantly reduced in CHOP-/- mice. CHOP deficiency also reduced the cleavage of caspase 3 and expression of the proapoptotic protein B cell lymphoma 2-associated X protein. Liver IRI induces CHOP expression, and CHOP deficiency attenuates liver IRI by inhibiting apoptosis. Elucidation of the function of CHOP in liver IRI may contribute to further investigation for a therapy against liver IRI associated with the ER stress pathway. Liver Transplantation 24 645-654 2018 AASLD.

Deficiency of CCAAT/enhancer-binding protein homologous protein (CHOP) prevents diet-induced aortic valve calcification in vivo.

Aortic valve (AoV) calcification is common in aged populations. Its subsequent aortic stenosis has been linked with increased morbidity, but still has no effective pharmacological intervention. Our previous data show endoplasmic reticulum (ER) stress is involved in AoV calcification. Here, we investigated whether deficiency of ER stress downstream effector CCAAT/enhancer-binding protein homology protein (CHOP) may prevent development of AoV calcification. AoV calcification was evaluated in Apoe-/- mice (n = 10) or in mice with dual deficiencies of ApoE and CHOP (Apoe-/- CHOP-/- , n = 10) fed with Western diet for 24 weeks. Histological and echocardiographic analysis showed that genetic ablation of CHOP attenuated AoV calcification, pro-calcification signaling activation, and apoptosis in the leaflets of Apoe-/- mice. In cultured human aortic valvular interstitial cells (VIC), we found oxidized low-density lipoprotein (oxLDL) promoted apoptosis and osteoblastic differentiation of VIC via CHOP activation. Using conditioned media (CM) from oxLDL-treated VIC, we further identified that oxLDL triggered osteoblastic differentiation of VIC via paracrine pathway, while depletion of apoptotic bodies (ABs) in CM suppressed the effect. CM from oxLDL-exposed CHOP-silenced cells prevented osteoblastic differentiation of VIC, while depletion of ABs did not further enhance this protective effect. Overall, our study indicates that CHOP deficiency protects against Western diet-induced AoV calcification in Apoe-/- mice. CHOP deficiency prevents oxLDL-induced VIC osteoblastic differentiation via preventing VIC-derived ABs releasing.

CHOP Deficiency Ameliorates ERK5 Inhibition-Mediated Exacerbation of Streptozotocin-Induced Hyperglycemia and Pancreatic ß-Cell Apoptosis.

Streptozotocin (STZ)-induced murine models of type 1 diabetes have been used to examine ER stress during pancreatic ß-cell apoptosis, as this ER stress plays important roles in the pathogenesis and development of the disease. However, the mechanisms linking type 1 diabetes to the ER stress-modulating anti-diabetic signaling pathway remain to be addressed, though it was recently established that ERK5 (Extracellular-signal-regulated kinase 5) contributes to the pathogeneses of diabetic complications. This study was undertaken to explore the mechanism whereby ERK5 inhibition instigates pancreatic ß-cell apoptosis via an ER stress-dependent signaling pathway. STZ-induced diabetic WT and CHOP deficient mice were i.p. injected every 2 days for 6 days under BIX02189 (a specific ERK5 inhibitor) treatment in order to evaluate the role of ERK5. Hyperglycemia was exacerbated by co-treating C57BL/6J mice with STZ and BIX02189 as compared with mice administered with STZ alone. In addition, immunoblotting data revealed that ERK5 inhibition activated the unfolded protein response pathway accompanying apoptotic events, such as, PARP-1 and caspase-3 cleavage. Interestingly, ERK5 inhibition-induced exacerbation of pancreatic ß-cell apoptosis was inhibited in CHOP deficient mice. Moreover, transduction of adenovirus encoding an active mutant form of MEK5α, an upstream kinase of ERK5, inhibited STZ-induced unfolded protein responses and ß-cell apoptosis. These results suggest that ERK5 protects against STZ-induced pancreatic ß-cell apoptosis and hyperglycemia by interrupting the ER stress-mediated apoptotic pathway.

Listeria monocytogenes induces an interferon-enhanced activation of the integrated stress response that is detrimental for resolution of infection in mice.

Type I interferons (IFNs) induce a detrimental response during Listeria monocytogenes (L. monocytogenes) infection. We were interested in identifying mechanisms linking IFN signaling to negative host responses against L. monocytogenes infection. Herein, we found that infection of myeloid cells with L. monocytogenes led to a coordinated induction of type I IFNs and activation of the integrated stress response (ISR). Infected cells did not induce Xbp1 splicing or BiP upregulation, indicating that the unfolded protein response was not triggered. CHOP (Ddit3) gene expression was upregulated during the ISR activation induced by L. monocytogenes. Myeloid cells deficient in either type I IFN signaling or PKR activation had less upregulation of CHOP following infection. CHOP-deficient mice showed lower expression of innate immune cytokines and were more resistant than wild-type counterparts following L. monocytogenes infection. These findings indicate that L. monocytogenes infection induces type I IFNs, which activate the ISR through PKR, which contributes to a detrimental outcome in the infected host.

Palmitate Increases ß-site AßPP-Cleavage Enzyme 1 Activity and Amyloid-ß Genesis by Evoking Endoplasmic Reticulum Stress and Subsequent C/EBP Homologous Protein Activation.

Epidemiological studies implicate diets rich in saturated free fatty acids (sFFA) as a potential risk factor for developing Alzheimer's disease (AD). In particular, high plasma levels of the sFFA palmitic acid (palmitate) were shown to inversely correlate with cognitive function. However, the cellular mechanisms by which sFFA may increase the risk for AD are not well known. Endoplasmic reticulum (ER) stress has emerged as one of the signaling pathways initiating and fostering the neurodegenerative changes in AD by increasing the aspartyl protease ß-site AßPP cleaving enzyme 1 (BACE1) and amyloid-ß (Aß) genesis. In this study, we determined the extent to which palmitate increases BACE1 and Aß levels in vitro and in vivo as well as the potential role of ER stress as cellular mechanism underlying palmitate effects. We demonstrate, in palmitate-treated SH-SY5Y neuroblastoma cells and in the hippocampi of palmitate-enriched diet-fed mice, that palmitate evokes the activation of the C/EBP Homologous Protein (CHOP), a transcription factor that is specifically responsive to ER stress. Induction of CHOP expression is associated with increased BACE1 mRNA, protein and activity levels, and subsequent enhanced amyloidogenic processing of amyloid-ß protein precursor (AßPP) that culminates in a substantial increase in Aß genesis. We further show that CHOP is an indispensable molecular mediator of palmitate-induced upregulation in BACE1 activity and Aß genesis. Indeed, we show that Chop-/- mice and CHOP knocked-down SH-SY5Y neuroblastoma cells do not exhibit the same commensurate degree of palmitate-induced increase in BACE1 expression levels and Aß genesis.

Endoplasmic reticulum stress inhibition attenuates hypertensive chronic kidney disease through reduction in proteinuria.

Endoplasmic reticulum (ER) stress is implicated in chronic kidney disease (CKD) development in patients and in animal models. Here we show that ER stress inhibition through 4-phenylbutyric acid (4-PBA) administration decreases blood pressure, albuminuria, and tubular casts in an angiotensin II/deoxycorticosterone acetate/salt murine model of CKD. Lower albuminuria in 4-PBA-treated mice was associated with higher levels of cubilin protein in renal tissue membrane fractions. 4-PBA decreased renal interstitial fibrosis, renal CD3+ T-cell and macrophage infiltration, mRNA expression of TGFß1, Wnt signaling molecules, and ER stress-induced pro-inflammatory genes. CHOP deficient mice that underwent this model of CKD developed hypertension comparable to wild type mice, but had less albuminuria and tubular casts. CHOP deficiency resulted in higher nephrin levels and decreased glomerulosclerosis compared to wild type mice; this effect was accompanied by lower macrophage infiltration and fibrosis. Our findings portray ER stress inhibition as a means to alleviate hypertensive CKD by preserving glomerular barrier integrity and tubular function. These results demonstrate ER stress modulation as a novel target for preserving renal function in hypertensive CKD.

Essential Role of Smooth Muscle STIM1 in Hypertension and Cardiovascular Dysfunction.

OBJECTIVES: Chronic hypertension is the most critical risk factor for cardiovascular disease, heart failure, and stroke. APPROACH AND RESULTS: Here we show that wild-type mice infused with angiotensin II develop hypertension, cardiac hypertrophy, perivascular fibrosis, and endothelial dysfunction with enhanced stromal interaction molecule 1 (STIM1) expression in heart and vessels. All these pathologies were significantly blunted in mice lacking STIM1 specifically in smooth muscle (Stim1(SMC-/-)). Mechanistically, STIM1 upregulation during angiotensin II-induced hypertension was associated with enhanced endoplasmic reticulum stress, and smooth muscle STIM1 was required for endoplasmic reticulum stress-induced vascular dysfunction through transforming growth factor-ß and nicotinamide adenine dinucleotide phosphate oxidase-dependent pathways. Accordingly, knockout mice for the endoplasmic reticulum stress proapoptotic transcriptional factor, CCAAT-enhancer-binding protein homologous protein (CHOP(-/-)), were resistant to hypertension-induced cardiovascular pathologies. Wild-type mice infused with angiotensin II, but not Stim1(SMC-/-) or CHOP(-/-) mice showed elevated vascular nicotinamide adenine dinucleotide phosphate oxidase activity and reduced phosphorylated endothelial nitric oxide synthase, cGMP, and nitrite levels. CONCLUSIONS: Thus, smooth muscle STIM1 plays a crucial role in the development of hypertension and associated cardiovascular pathologies and represents a promising target for cardiovascular therapy.

C/EBP homologous protein modulates liraglutide-mediated attenuation of non-alcoholic steatohepatitis.

The CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP), a major transcriptional regulator of endoplasmic reticulum (ER) stress-mediated apoptosis, is implicated in lipotoxicity-induced ER stress and hepatocyte apoptosis in non-alcoholic fatty liver disease (NAFLD). We have previously demonstrated that the glucagon-like peptide-1 (GLP-1) agonist, liraglutide, protects steatotic hepatocytes from lipotoxicity-induced apoptosis by improved handling of free fatty acid (FFA)-induced ER stress. In the present study, we investigated whether CHOP is critical for GLP-1-mediated restoration of ER homeostasis and mitigation of hepatocyte apoptosis in a murine model of NASH (non-alcoholic steatohepatitis). Our data show that despite similar caloric intake, CHOP KO (CHOP(-/-)) mice fed a diet high in fat, fructose, and cholesterol (HFCD) for 16 weeks developed more severe histological features of NASH compared with wild-type (WT) controls. Severity of NASH in HFCD-fed CHOP(-/-) mice correlated with significant decrease in peroxisomal ß-oxidation, and increased de novo lipogenesis and ER stress-mediated hepatocyte apoptosis. Four weeks of liraglutide treatment markedly attenuated steatohepatitis in HFCD-fed WT mice by improving insulin sensitivity, and suppressing de novo lipogenesis and ER stress-mediated hepatocyte apoptosis. However, in the absence of CHOP, liraglutide did not improve insulin sensitivity, nor suppress peroxisomal ß-oxidation or ER stress-mediated hepatocyte apoptosis. Taken together, these data indicate that CHOP protects hepatocytes from HFCD-induced ER stress, and has a significant role in the mechanism of liraglutide-mediated protection against NASH pathogenesis.

C/EBP homologous protein (CHOP) deficiency ameliorates renal fibrosis in unilateral ureteral obstructive kidney disease.

Renal tubulointerstitial fibrosis is an important pathogenic feature in chronic kidney disease and end-stage renal disease, regardless of the initiating insults. A recent study has shown that CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP) is involved in acute ischemia/reperfusion-related acute kidney injury through oxidative stress induction. However, the influence of CHOP on chronic kidney disease-correlated renal fibrosis remains unclear. Here, we investigated the role of CHOP in unilateral ureteral obstruction (UUO)-induced experimental chronic tubulointerstital fibrosis. The CHOP knockout and wild type mice with or without UUO were used. The results showed that the increased expressions of renal fibrosis markers collagen I, fibronectin, α-smooth muscle actin, and plasminogen activator inhibitor-1 in the kidneys of UUO-treated wild type mice were dramatically attenuated in the kidneys of UUO-treated CHOP knockout mice. CHOP deficiency could also ameliorate lipid peroxidation and endogenous antioxidant enzymes depletion, tubular apoptosis, and inflammatory cells infiltration in the UUO kidneys. These results suggest that CHOP deficiency not only attenuates apoptotic death and oxidative stress in experimental renal fibrosis, but also reduces local inflammation, leading to diminish UUO-induced renal fibrosis. Our findings support that CHOP may be an important signaling molecule in the progression of chronic kidney disease.

Stage-dependent effects of exogenous TRAIL on atherogenesis: role of ER stress-mediated sensitization of macrophage apoptosis.

Deletion of the gene of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in apolipoprotein E-deficient (ApoE-/-) mice increased atherosclerosis. However, the effect of TRAIL at a supra-physiological level on early atherogenesis is unknown. ApoE-/- mice were divided into Early (high-fat diet with concomitant TRAIL treatment for 4 weeks) and Late (high-fat diet for 16 weeks with TRAIL being given during the last 4 weeks) groups. It was found that TRAIL stimulated atherogenesis in the Early group but not in the Late group. TRAIL did not change the intra-plaque macrophage content in Early group, but decreased it in the Late group. In cultured macrophages, induction of endoplasmic reticulum (ER) stress increased death receptor 5 (DR5) expression and TRAIL-induced apoptosis, which were mediated by the transcription factor CCAAT/enhancer-binding protein homologous protein (CHOP). The expression levels of CHOP, 78 kDa glucose-regulated protein (GRP78) and DR5 were all elevated in the Late group. TRAIL treatment in vivo also increased intra-plaque apoptotic only in Late lesions. Moreover, the chemical chaperone 4-phenylbutyrate blocked the development of ER stress and upregulation of DR5 in Late lesions in vivo. In conclusion, TRAIL at a supra-physiological level has a stimulatory effect on early atherogenesis, but not in the advanced lesions. The differential effects of TRAIL may be related to differences in ER stress, DR5 expression, and the sensitivity of macrophage apoptosis in response to TRAIL in early versus advanced lesions. The results presented here raise the possibility that treatment with exogenous TRAIL as a therapeutic agent may be detrimental in patients with increased risk of atherosclerosis.

Chop Deficiency Protects Mice Against Bleomycin-induced Pulmonary Fibrosis by Attenuating M2 Macrophage Production.

C/EBP homologous protein (Chop) has been shown to have altered expression in patients with idiopathic pulmonary fibrosis (IPF), but its exact role in IPF pathoaetiology has not been fully addressed. Studies conducted in patients with IPF and Chop(-/-) mice have dissected the role of Chop and endoplasmic reticulum (ER) stress in pulmonary fibrosis pathogenesis. The effect of Chop deficiency on macrophage polarization and related signalling pathways were investigated to identify the underlying mechanisms. Patients with IPF and mice with bleomycin (BLM)-induced pulmonary fibrosis were affected by the altered Chop expression and ER stress. In particular, Chop deficiency protected mice against BLM-induced lung injury and fibrosis. Loss of Chop significantly attenuated transforming growth factor ß (TGF-ß) production and reduced M2 macrophage infiltration in the lung following BLM induction. Mechanistic studies showed that Chop deficiency repressed the M2 program in macrophages, which then attenuated TGF-ß secretion. Specifically, loss of Chop promoted the expression of suppressors of cytokine signaling 1 and suppressors of cytokine signaling 3, and through which Chop deficiency repressed signal transducer and activator of transcription 6/peroxisome proliferator-activated receptor gamma signaling, the essential pathway for the M2 program in macrophages. Together, our data support the idea that Chop and ER stress are implicated in IPF pathoaetiology, involving at least the induction and differentiation of M2 macrophages.

Ablation of Chop Transiently Enhances Photoreceptor Survival but Does Not Prevent Retinal Degeneration in Transgenic Mice Expressing Human P23H Rhodopsin.

RHO (Rod opsin) encodes a G-protein coupled receptor that is expressed exclusively by rod photoreceptors of the retina and forms the essential photopigment, rhodopsin, when coupled with 11-cis-retinal. Many rod opsin disease -mutations cause rod opsin protein misfolding and trigger endoplasmic reticulum (ER) stress, leading to activation of the Unfolded Protein Response (UPR) signal transduction network. Chop is a transcriptional activator that is induced by ER stress and promotes cell death in response to chronic ER stress. Here, we examined the role of Chop in transgenic mice expressing human P23H rhodopsin (hP23H Rho Tg) that undergo retinal degeneration. With the exception of one time point, we found no significant induction of Chop in these animals and no significant change in retinal degeneration by histology and electrophysiology when hP23H Rho Tg animals were bred into a Chop (-/-) background. Our results indicate that Chop does not play a significant causal role during retinal degeneration in these animals. We suggest that other modules of the ER stress-induced UPR signaling network may be involved photoreceptor disease induced by P23H rhodopsin.

Chop deficiency prevents UUO-induced renal fibrosis by attenuating fibrotic signals originated from Hmgb1/TLR4/NFκB/IL-1ß signaling.

Renal fibrosis, particularly tubulointerstitial fibrosis is considered to be the final manifestation of almost all chronic kidney diseases (CKDs). Herein we demonstrated evidence that CHOP-related ER stress is associated with the development of renal fibrosis in both CKD patients and unilateral ureteral obstruction (UUO)-induced animals, and specifically, mice deficient in Chop were protected from UUO-induced renal fibrosis. Mechanistic studies revealed that loss of Chop protected tubular cells from UUO-induced apoptosis and secondary necrosis along with attenuated Hmgb1 passive release and active secretion. As a result, Chop deficiency suppressed Hmgb1/TLR4/NFκB signaling, which then repressed UUO-induced IL-1ß production. Consequently, the IL-1ß downstream Erk1/2 activity and its related c-Jun transcriptional activity were reduced, leading to attenuated production of TGF-ß1 following UUO insult. It was further noted that reduced IL-1ß production also inhibited UUO-induced PI3K/AKT signaling, and both of which ultimately protected mice from UUO-induced renal fibrosis. Together, our data support that suppression of CHOP expression could be a viable therapeutic strategy to prevent renal fibrosis in patients with CKDs.

Ablation of C/EBP homologous protein increases the acute phase mortality and doesn't attenuate cardiac remodeling in mice with myocardial infarction.

Endoplasmic reticulum stress is a proapoptotic and profibrotic stimulus. Ablation of C/EBP homologous protein (CHOP) is reported to reverse cardiac dysfunction by attenuating cardiac endoplasmic reticulum stress in mice with pressure overload or ischemia/reperfusion, but it is unclear whether loss of CHOP also inhibits cardiac remodeling induced by permanent-infarction. In mice with permanent ligation of left coronary artery, we found that ablation of CHOP increased the acute phase mortality. For the mice survived to 4 weeks, left ventricular anterior (LV) wall thickness was larger in CHOP knockout mice than in the wildtype littermates, while no difference was noted on posterior wall thickness, LV dimensions, LV fractional shortening and ejection fraction. Similarly, invasive assessment of LV hemodynamics, morphological analysis of heart and lung weight indexes, myocardial fibrosis and TUNEL-assessed apoptosis showed no significant differences between CHOP knockout mice and their wildtype ones, while in mice with ischemia for 45 min and reperfusion for 1 week, myocardial fibrosis and apoptosis in the infarct area were significantly attenuated in CHOP knockout mice. These findings indicate that ablation of CHOP doesn't ameliorate cardiac remodeling induced by permanent-myocardial infarction, which implicates that early reperfusion is a prerequisite for ischemic myocardium to benefit from CHOP inhibition.