Genome-wide CRISPR screen identifies suppressors of endoplasmic reticulum stress-induced apoptosis.

Sensing misfolded proteins in the endoplasmic reticulum (ER), cells initiate the ER stress response and, when overwhelmed, undergo apoptosis. However, little is known about how cells prevent excessive ER stress response and cell death to restore homeostasis. Here, we report the identification and characterization of cellular suppressors of ER stress-induced apoptosis. Using a genome-wide CRISPR library, we screen for genes whose inactivation further increases ER stress-induced up-regulation of C/EBP homologous protein 10 (CHOP)-the transcription factor central to ER stress-associated apoptosis. Among the top validated hits are two interacting components of the polycomb repressive complex (L3MBTL2 [L(3)Mbt-Like 2] and MGA [MAX gene associated]), and microRNA-124-3 (miR-124-3). CRISPR knockout of these genes increases CHOP expression and sensitizes cells to apoptosis induced by multiple ER stressors, while overexpression confers the opposite effects. L3MBTL2 associates with the CHOP promoter in unstressed cells to repress CHOP induction but dissociates from the promoter in the presence of ER stress, whereas miR-124-3 directly targets the IRE1 branch of the ER stress pathway. Our study reveals distinct mechanisms that suppress ER stress-induced apoptosis and may lead to a better understanding of diseases whose pathogenesis is linked to overactive ER stress response.

Apelin-13 Inhibits Methylglyoxal-Induced Unfolded Protein Responses and Endothelial Dysfunction via Regulating AMPK Pathway.

It has been suggested that methylglyoxal (MGO), a glycolytic metabolite, has more detrimental effects on endothelial dysfunction than glucose itself. Recent reports showed that high glucose and MGO induced endoplasmic reticulum (ER) stress and myocyte apoptosis in ischemic heart disease was inhibited by apelin. The goal of the study is to investigate the molecular mechanism by which MGO induces endothelial dysfunction via the regulation of ER stress in endothelial cells, and to examine whether apelin-13, a cytoprotective polypeptide ligand, protects MGO-induced aortic endothelial dysfunction. MGO-induced ER stress and apoptosis were determined by immunoblotting and MTT assay in HUVECs. Aortic endothelial dysfunction was addressed by en face immunostaining and acetylcholine-induced vasodilation analysis with aortic rings from mice treated with MGO in the presence or absence of apelin ex vivo. TUDCA, an inhibitor of ER stress, inhibited MGO-induced apoptosis and reduction of cell viability, suggesting that MGO signaling to endothelial apoptosis is mediated via ER stress, which leads to activation of unfolded protein responses (UPR). In addition, MGO-induced UPR and aortic endothelial dysfunction were significantly diminished by apelin-13. Finally, this study showed that apelin-13 protects MGO-induced UPR and endothelial apoptosis through the AMPK pathway. Apelin-13 reduces MGO-induced UPR and endothelial dysfunction via regulating the AMPK activating pathway, suggesting the therapeutic potential of apelin-13 in diabetic cardiovascular complications.

Hypoxia and ER stress promote Staufen1 expression through an alternative translation mechanism.

Under physiological stress conditions the cell protects itself through a global blockade on cap-dependent translation of mRNA. This allows cap-independent mechanisms such as internal ribosome entry site (IRES)-mediated translation to take over and initiate the translation of a specific pool of mRNAs that encode proteins involved in protecting the cell from stress. Staufen 1 (Stau1) is an RNA-binding protein that has been previously implicated in the regulation of stress granule formation and therefore could play a key role in protecting the cell against stress stimuli such as oxidative and endoplasmic reticulum (ER) stress. We hypothesized that Stau1 mRNA could, like many stress response genes, contain an IRES in its 5'UTR. Here we describe that a bona fide IRES element is present in the 5'UTR of Stau1 mRNA, which is activated under hypoxic and ER stress conditions. Further, we show that the activity of PERK kinase, a major effector of the ER stress response, is required for Stau1 IRES-mediated translation during ER stress. These results suggest that Stau1 is a stress response gene that remains efficiently translated during hypoxia and ER stress despite the substantial global inhibition of cap-dependent protein translation, promoting cell recovery following stress.

Calnexin, an ER stress-induced protein, is a prognostic marker and potential therapeutic target in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is a leading cause of cancer mortality in the Western world and commonly treated with genotoxic chemotherapy. Stress in the endoplasmic reticulum (ER) was implicated to contribute to chemotherapeutic resistance. Hence, ER stress related protein may be of prognostic or therapeutic significance. METHODS: The expression levels of ER stress proteins calnexin, calreticulin, GRP78 and GRP94 were determined in n = 23 Stage II and III colon cancer fresh frozen tumour and matched normal tissue samples. Data were validated in a cohort of n = 11 rectal cancer patients treated with radiochemotherapy in the neoadjuvant setting. The calnexin gene was silenced using siRNA in HCT116 cells. RESULTS: There were no increased levels of ER stress proteins in tumour compared to matched normal tissue samples in Stage II or III CRC. However, increased calnexin protein levels were predictive of poor clinical outcome in the patient cohort. Data were validated in the rectal cancer cohort treated in the neoadjuvant setting. Calnexin gene-silencing significantly reduced cell survival and increased cancer cell susceptibility to 5FU chemotherapy. CONCLUSION: Increased tumour protein levels of calnexin may be of prognostic significance in CRC, and calnexin may represent a potential target for future therapies.

Fingerprint of the oxido-reductase ERO1: A protein disulfide bond producer and supporter of cancer.

Endoplasmic reticulum oxidoreductin 1 (ERO1) alpha (ERO1A) is an endoplasmic reticulum (ER)-localized protein disulfide oxidoreductase, involved in the disulfide bond formation of proteins. ERO1's activity in oxidative protein folding is redundant in higher eukaryotes and its loss is well compensated. Although it is dispensable in non-cancer cells, high ERO1 levels are seen with different cancers and predict their malignant phenotype. ERO1 fosters tumor aggressiveness and the response to drug therapy in hypoxic and highly metastatic tumors. It regulates vascular endothelial growth factor (VEGF) levels, oxidative folding and N-glycosylation in hypoxic conditions, boosting tumor fitness and angiogenesis on multiple levels. In addition, ERO1 regulates protein death ligand-1 (PD-L1) on tumors, interfering with the related immune surveillance mechanism, hence acting on the tumors' response to immune check-point inhibitors (ICI). This all points to inhibition of ERO1 as an effective pharmacological tool, selectively targeting tumors while sparing non-cancer cells from cytotoxicity. The critical discussion here closely examines the molecular basis for ERO1's involvement in tumors and ERO1 inhibition strategies for their treatment.

Endoplasmic reticulum oxidoreductin 1-alpha deficiency and activation of protein translation synergistically impair breast tumour resilience.

BACKGROUND AND PURPOSE: Endoplasmic reticulum (ER) stress triggers an adaptive response in tumours which fosters cell survival and resilience to stress. Activation of the ER stress response, through its PERK branch, promotes phosphorylation of the α-subunit of the translation initiation factor eIF2, thereby repressing general protein translation and augmenting the translation of ATF4 with the downstream CHOP transcription factor and the protein disulfide oxidase, ERO1-alpha EXPERIMENTAL APPROACH: Here, we show that ISRIB, a small molecule that inhibits the action of phosphorylated eIF2alpha, activating protein translation, synergistically interacts with the genetic deficiency of protein disulfide oxidase ERO1-alpha, enfeebling breast tumour growth and spread. KEY RESULTS: ISRIB represses the CHOP signal, but does not inhibit ERO1. Mechanistically, ISRIB increases the ER protein load with a marked perturbing effect on ERO1-deficient triple-negative breast cancer cells, which display impaired proteostasis and have adapted to a low client protein load in hypoxia, and ERO1 deficiency impairs VEGF-dependent angiogenesis. ERO1-deficient triple-negative breast cancer xenografts have an augmented ER stress response and its PERK branch. ISRIB acts synergistically with ERO1 deficiency, inhibiting the growth of triple-negative breast cancer xenografts by impairing proliferation and angiogenesis. CONCLUSION AND IMPLICATIONS: These results demonstrate that ISRIB together with ERO1 deficiency synergistically shatter the PERK-dependent adaptive ER stress response, by restarting protein synthesis in the setting of impaired proteostasis, finally promoting tumour cytotoxicity. Our findings suggest two surprising features in breast tumours: ERO1 is not regulated via CHOP under hypoxic conditions, and ISRIB offers a therapeutic option to efficiently inhibit tumour progression in conditions of impaired proteostasis.

Assessment of the effects of organic vs. inorganic arsenic and mercury in Caenorhabditis elegans.

Exposures to mercury and arsenic are known to pose significant threats to human health. Effects specific to organic vs. inorganic forms of these toxic elements are less understood however, especially for organic dimethylarsinic acid (DMA), which has recently been detected in pups of rodent dams orally exposed to inorganic sodium (meta)arsenite (NaAsO2). Caenorhabditis elegans is a small animal alternative toxicity model. To fill data gaps on the effects of DMA relative to NaAsO2, C. elegans were exposed to these two compounds alongside more thoroughly researched inorganic mercury chloride (HgCl2) and organic methylmercury chloride (meHgCl). For timing of developmental milestone acquisition in C. elegans, meHgCl was 2 to 4-fold more toxic than HgCl2, and NaAsO2 was 20-fold more toxic than DMA, ranking the four compounds meHgCl > HgCl2 > NaAsO2 ≫ DMA for developmental toxicity. Methylmercury induced significant decreases in population locomotor activity levels in developing C. elegans. DMA was also associated with developmental hypoactivity, but at >100-fold higher concentrations than meHgCl. Transcriptional alterations in native genes were observed in wild type C. elegans adults exposed to concentrations equitoxic for developmental delay in juveniles. Both forms of arsenic induced genes involved in immune defense and oxidative stress response, while the two mercury species induced proportionally more genes involved in transcriptional regulation. A transgenic bioreporter for activation of conserved proteosome specific unfolded protein response was strongly activated by NaAsO2, but not DMA at tested concentrations. HgCl2 and meHgCl had opposite effects on a bioreporter for unfolded protein response in the endoplasmic reticulum. Presented experiments indicating low toxicity for DMA in C. elegans are consistent with human epidemiologic data correlating higher arsenic methylation capacity with resistance to arsenic toxicity. This work contributes to the understanding of the accuracy and fit-for-use categories for C. elegans toxicity screening and its usefulness to prioritize compounds of concern for further testing.

Ischemic preconditioning upregulates Mitofusin2 and preserves muscle strength in tourniquet-induced ischemia/reperfusion.

Background: Tourniquet-induced ischemia and reperfusion (I/R) has been related to postoperative muscle atrophy through mechanisms involving protein synthesis/breakdown, cellular metabolism, mitochondrial dysfunction, and apoptosis. Ischemic preconditioning (IPC) could protect skeletal muscle against I/R injury. This study aims to determine the underlying mechanisms of IPC and its effect on muscle strength after total knee arthroplasty (TKA). Methods: Twenty-four TKA patients were randomized to receive either sham IPC or IPC (3 cycles of 5-min ischemia followed by 5-min reperfusion). Vastus medialis muscle biopsies were collected at 30 â€‹min after tourniquet (TQ) inflation and the onset of reperfusion. Western blot analysis was performed in muscle protein for 4-HNE, SOD2, TNF-ɑ, IL-6, p-Drp1ser616, Drp1, Mfn1, Mfn2, Opa1, PGC-1ɑ, ETC complex I-V, cytochrome c, cleaved caspase-3, and caspase-3. Clinical outcomes including isokinetic muscle strength and quality of life were evaluated pre- and postoperatively. Results: IPC significantly increased Mfn2 (2.0 â€‹± â€‹0.2 vs 1.2 â€‹± â€‹0.1, p â€‹= â€‹0.001) and Opa1 (2.9 â€‹± â€‹0.3 vs 1.9 â€‹± â€‹0.2, p â€‹= â€‹0.005) proteins expression at the onset of reperfusion, compared to the ischemic phase. There were no differences in 4-HNE, SOD2, TNF-ɑ, IL-6, p-Drp1ser616/Drp1, Mfn1, PGC-1ɑ, ETC complex I-V, cytochrome c, and cleaved caspase-3/caspase-3 expression between the ischemic and reperfusion periods, or between the groups. Clinically, postoperative peak torque for knee extension significantly reduced in the sham IPC group (-16.6 [-29.5, -3.6] N.m, p â€‹= â€‹0.020), while that in the IPC group was preserved (-4.7 [-25.3, 16.0] N.m, p â€‹= â€‹0.617). Conclusion: In TKA with TQ application, IPC preserved postoperative quadriceps strength and prevented TQ-induced I/R injury partly by enhancing mitochondrial fusion proteins in the skeletal muscle. The translational potential of this article: Mitochondrial fusion is a potential underlying mechanism of IPC in preventing skeletal muscle I/R injury. IPC applied before TQ-induced I/R preserved postoperative quadriceps muscle strength after TKA.

A cyclodextrin-based nanoformulation achieves co-delivery of ginsenoside Rg3 and quercetin for chemo-immunotherapy in colorectal cancer.

The immune checkpoint blockade therapy has profoundly revolutionized the field of cancer immunotherapy. However, despite great promise for a variety of cancers, the efficacy of immune checkpoint inhibitors is still low in colorectal cancer (CRC). This is mainly due to the immunosuppressive feature of the tumor microenvironment (TME). Emerging evidence reveals that certain chemotherapeutic drugs induce immunogenic cell death (ICD), demonstrating great potential for remodeling the immunosuppressive TME. In this study, the potential of ginsenoside Rg3 (Rg3) as an ICD inducer against CRC cells was confirmed using in vitro and in vivo experimental approaches. The ICD efficacy of Rg3 could be significantly enhanced by quercetin (QTN) that elicited reactive oxygen species (ROS). To ameliorate in vivo delivery barriers associated with chemotherapeutic drugs, a folate (FA)-targeted polyethylene glycol (PEG)-modified amphiphilic cyclodextrin nanoparticle (NP) was developed for co-encapsulation of Rg3 and QTN. The resultant nanoformulation (CD-PEG-FA.Rg3.QTN) significantly prolonged blood circulation and enhanced tumor targeting in an orthotopic CRC mouse model, resulting in the conversion of immunosuppressive TME. Furthermore, the CD-PEG-FA.Rg3.QTN achieved significantly longer survival of animals in combination with Anti-PD-L1. The study provides a promising strategy for the treatment of CRC.

The regulatory and modulatory roles of TRP family channels in malignant tumors and relevant therapeutic strategies.

Transient receptor potential (TRP) channels are one primary type of calcium (Ca2+) permeable channels, and those relevant transmembrane and intracellular TRP channels were previously thought to be mainly associated with the regulation of cardiovascular and neuronal systems. Nowadays, however, accumulating evidence shows that those TRP channels are also responsible for tumorigenesis and progression, inducing tumor invasion and metastasis. However, the overall underlying mechanisms and possible signaling transduction pathways that TRP channels in malignant tumors might still remain elusive. Therefore, in this review, we focus on the linkage between TRP channels and the significant characteristics of tumors such as multi-drug resistance (MDR), metastasis, apoptosis, proliferation, immune surveillance evasion, and the alterations of relevant tumor micro-environment. Moreover, we also have discussed the expression of relevant TRP channels in various forms of cancer and the relevant inhibitors' efficacy. The chemo-sensitivity of the anti-cancer drugs of various acting mechanisms and the potential clinical applications are also presented. Furthermore, it would be enlightening to provide possible novel therapeutic approaches to counteract malignant tumors regarding the intervention of calcium channels of this type.

Therapeutic regulation of autophagy in hepatic metabolism.

Metabolic homeostasis requires dynamic catabolic and anabolic processes. Autophagy, an intracellular lysosomal degradative pathway, can rewire cellular metabolism linking catabolic to anabolic processes and thus sustain homeostasis. This is especially relevant in the liver, a key metabolic organ that governs body energy metabolism. Autophagy's role in hepatic energy regulation has just begun to emerge and autophagy seems to have a much broader impact than what has been appreciated in the field. Though classically known for selective or bulk degradation of cellular components or energy-dense macromolecules, emerging evidence indicates autophagy selectively regulates various signaling proteins to directly impact the expression levels of metabolic enzymes or their upstream regulators. Hence, we review three specific mechanisms by which autophagy can regulate metabolism: A) nutrient regeneration, B) quality control of organelles, and C) signaling protein regulation. The plasticity of the autophagic function is unraveling a new therapeutic approach. Thus, we will also discuss the potential translation of promising preclinical data on autophagy modulation into therapeutic strategies that can be used in the clinic to treat common metabolic disorders.

Mid-pregnancy maternal immune activation increases Pax6-positive and Tbr2-positive neural progenitor cells and causes integrated stress response in the fetal brain in a mouse model of maternal viral infection.

Maternal immune activation (MIA) in midpregnancy is a risk factor for neurodevelopmental disorders. Improper brain development may cause malformations of the brain; maldevelopment induced by MIA may lead to a pathology-related phenotype. In this study, a single intraperitoneal injection of 20 mg/kg polyriboinosinic-polyribocytidylic acid [poly(I:C)] was administered to C57BL/6J mice on embryonic day (E) 12.5 to mimic maternal viral infection. Histopathological analysis of neurogenesis was performed using markers for Pax6, Tbr2, and Tbr1. In these fetuses, significant increases were observed in the proportion of Pax6-positive neural progenitor cells and Pax6/Tbr2 double-positive cells 24 h after poly(I:C) injection. There were no differences in the proportion of Tbr1-positive postmitotic neurons 48 h after poly(I:C) injection. At E18.5, there were more Pax6-positive and Tbr2-positive neural progenitor cells in the poly(I:C)-injected group than in the saline-injected group. Gene ontology enrichment analysis of poly(I:C)-induced differentially expressed genes in the fetal brain at E12.5 demonstrated that these genes were enriched in terms including response to cytokine, response to decreased oxygen levels in the category of biological process. At E13.5, activating transcription factor 4 (Atf4), which is an effector of integrated stress response, was significantly upregulated in the fetal brain. Our results show that poly(I:C)-induced MIA at E12.5 leads to dysregulated neurogenesis and upregulates Atf4 in the fetal brain. These findings provide a new insight in the mechanism of MIA causing improper brain development and subsequent neurodevelopmental disorders.

A glycine substitution in the collagenous domain of Col4a3 in mice recapitulates late onset Alport syndrome.

Alport syndrome (AS) is a severe inherited glomerulopathy caused by mutations in the genes encoding the α-chains of type-IV collagen, the most abundant component of the extracellular glomerular basement membrane (GBM). Currently most AS mouse models are knockout models for one of the collagen-IV genes. In contrast, about half of AS patients have missense mutations, with single aminoacid substitutions of glycine being the most common. The only mouse model for AS with a homozygous knockin missense mutation, Col4a3-p.Gly1332Glu, was partly described before by our group. Here, a detailed in-depth description of the same mouse is presented, along with another compound heterozygous mouse that carries the glycine substitution in trans with a knockout allele. Both mice recapitulate essential features of AS, including shorten lifespan by 30-35%, increased proteinuria, increased serum urea and creatinine, pathognomonic alternate GBM thinning and thickening, and podocyte foot process effacement. Notably, glomeruli and tubuli respond differently to mutant collagen-IV protomers, with reduced expression in tubules but apparently normal in glomeruli. However, equally important is the fact that in the glomeruli the mutant α3-chain as well as the normal α4/α5 chains seem to undergo a cleavage at, or near the point of the mutation, possibly by the metalloproteinase MMP-9, producing a 35 kDa C-terminal fragment. These mouse models represent a good tool for better understanding the spectrum of molecular mechanisms governing collagen-IV nephropathies and could be used for pre-clinical studies aimed at better treatments for AS.

Calcium and Redox Liaison: A Key Role of Selenoprotein N in Skeletal Muscle.

Selenoprotein N (SEPN1) is a type II glycoprotein of the endoplasmic reticulum (ER) that senses calcium levels to tune the activity of the sarcoplasmic reticulum calcium pump (SERCA pump) through a redox-mediated mechanism, modulating ER calcium homeostasis. In SEPN1-depleted muscles, altered ER calcium homeostasis triggers ER stress, which induces CHOP-mediated malfunction, altering excitation-contraction coupling. SEPN1 is localized in a region of the ER where the latter is in close contact with mitochondria, i.e., the mitochondria-associated membranes (MAM), which are important for calcium mobilization from the ER to mitochondria. Accordingly, SEPN1-depleted models have impairment of both ER and mitochondria calcium regulation and ATP production. SEPN1-related myopathy (SEPN1-RM) is an inherited congenital muscle disease due to SEPN1 loss of function, whose main histopathological features are minicores, i.e., areas of mitochondria depletion and sarcomere disorganization in muscle fibers. SEPN1-RM presents with weakness involving predominantly axial and diaphragmatic muscles. Since there is currently no disease-modifying drug to treat this myopathy, analysis of SEPN1 function in parallel with that of the muscle phenotype in SEPN1 loss of function models should help in understanding the pathogenic basis of the disease and possibly point to novel drugs for therapy. The present essay recapitulates the novel biological findings on SEPN1 and how these reconcile with the muscle and bioenergetics phenotype of SEPN1-related myopathy.

Global transcriptome profiling reveals genes responding to overproduction of a small secretory, a high cysteine- and a high glycosylation-bearing protein in Yarrowia lipolytica.

Investigation of the yeast cell's response to recombinant secretory protein (rs-Prot) overproduction is relevant for both basic and applied research. Imbalance, overloading or stress within this process impacts the whole cell. In the present study, by using steady-state cultures and transcriptomics, we investigated the cellular response of Yarrowia lipolytica challenged with high-level expression of genes encoding proteins with significantly different biochemical characteristics: a small protein retained within the cell i) or secreted ii), a medium size secretory protein with a high number of disulfide bonds iii), or glycosylation sites iv). Extensive analysis of omics data, supported by careful manual curation, led to some anticipated observations on oxidative and unfolded protein stress (CTT1, PXMP2/4, HAC1), glycosylation (ALGs, KTRs, MNTs, MNNs), folding and translocation (SSAs, SSEs) but also generated new exciting knowledge on non-conventional protein secretion (NCE102), transcriptional regulators (FLO11, MHY1, D01353 g, RSFA, E23925g or MAF1), vacuolar proteolysis targets in Y. lipolytica (ATGs, VPSs, HSE1, PRB1, PRC1, PEP4) or growth arrest (CLN1) upon rs-Prots overproduction.

SMYD3-PARP16 axis accelerates unfolded protein response and mediates neointima formation.

Neointimal hyperplasia after vascular injury is a representative complication of restenosis. Endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR) is involved in the pathogenesis of vascular intimal hyperplasia. PARP16, a member of the poly(ADP-ribose) polymerases family, is correlated with the nuclear envelope and the ER. Here, we found that PERK and IRE1α are ADP-ribosylated by PARP16, and this might promote proliferation and migration of smooth muscle cells (SMCs) during the platelet-derived growth factor (PDGF)-BB stimulating. Using chromatin immunoprecipitation coupled with deep sequencing (ChIP-seq) analysis, PARP16 was identified as a novel target gene for histone H3 lysine 4 (H3K4) methyltransferase SMYD3, and SMYD3 could bind to the promoter of Parp16 and increased H3K4me3 level to activate its host gene's transcription, which causes UPR activation and SMC proliferation. Moreover, knockdown either of PARP16 or SMYD3 impeded the ER stress and SMC proliferation. On the contrary, overexpression of PARP16 induced ER stress and SMC proliferation and migration. In vivo depletion of PARP16 attenuated injury-induced neointimal hyperplasia by mediating UPR activation and neointimal SMC proliferation. This study identified SMYD3-PARP16 is a novel signal axis in regulating UPR and neointimal hyperplasia, and targeting this axis has implications in preventing neointimal hyperplasia related diseases.

A small molecule UPR modulator for diabetes identified by high throughput screening.

Unfolded protein response (UPR) is a stress response that is specific to the endoplasmic reticulum (ER). UPR is activated upon accumulation of unfolded (or misfolded) proteins in the ER's lumen to restore protein folding capacity by increasing the synthesis of chaperones. In addition, UPR also enhances degradation of unfolded proteins and reduces global protein synthesis to alleviate additional accumulation of unfolded proteins in the ER. Herein, we describe a cell-based ultra-high throughput screening (uHTS) campaign that identifies a small molecule that can modulate UPR and ER stress in cellular and in vivo disease models. Using asialoglycoprotein receptor 1 (ASGR) fused with Cypridina luciferase (CLuc) as reporter assay for folding capacity, we have screened a million small molecule library and identified APC655 as a potent activator of protein folding, that appears to act by promoting chaperone expression. Furthermore, APC655 improved pancreatic ß cell viability and insulin secretion under ER stress conditions induced by thapsigargin or cytokines. APC655 was also effective in preserving ß cell function and decreasing lipid accumulation in the liver of the leptin-deficient (ob/ob) mouse model. These results demonstrate a successful uHTS campaign that identified a modulator of UPR, which can provide a novel candidate for potential therapeutic development for a host of metabolic diseases.

Cardiomyocyte Specific Deletion of ADAR1 Causes Severe Cardiac Dysfunction and Increased Lethality.

Background: Adenosine deaminase acting on RNA 1 (ADAR1) is a double-stranded RNA-editing enzyme that is involved in several functions including the deamination of adenosine to inosine, RNA interference (RNAi) mechanisms and microRNA (miRNA) processing, rendering ADAR1 essential for life. Methods and Results: To investigate whether maintenance of ADAR1 expression is required for normal myocardial homeostasis, we bypassed the early embryonic lethality of ADAR1-null mice through the use of a tamoxifen-inducible Cre recombinase under the control of the cardiac-specific α-myosin heavy chain promoter (αMHC). Targeted ADAR1 deletion in adult mice caused a significant increase in lethality accompanied by severe ventricular remodeling and quick and spontaneous cardiac dysfunction, induction of stress markers and overall reduced expression of miRNAs. Administration of a selective inhibitor of the unfolded protein response (UPR) stress significantly blunted the deleterious effects and improved cardiac function thereby prolonging animal survival. In vitro restoring miR-199a-5p levels in cardiomyocytes lacking ADAR1 diminished UPR activation and concomitant apoptosis. Conclusions: Our findings demonstrate an essential role for ADAR1 in cardiomyocyte survival and maintenance of cardiac function through a mechanism that integrates ADAR1 dependent miRNA processing and the suppression of UPR stress.

Calreticulin is important for the development of renal fibrosis and dysfunction in diabetic nephropathy.

Previously, our lab showed that the endoplasmic reticulum (ER) and calcium regulatory protein, calreticulin (CRT), is important for collagen transcription, secretion, and assembly into the extracellular matrix (ECM) and that ER CRT is critical for TGF-ß stimulation of type I collagen transcription through stimulation of ER calcium release and NFAT activation. Diabetes is the leading cause of end stage renal disease. TGF-ß is a key factor in the pathogenesis of diabetic nephropathy. However, the role of calreticulin (Calr) in fibrosis of diabetic nephropathy has not been investigated. In current work, we used both in vitro and in vivo approaches to assess the role of ER CRT in TGF-ß and glucose stimulated ECM production by renal tubule cells and in diabetic mice. Knockdown of CALR by siRNA in a human proximal tubular cell line (HK-2) showed reduced induction of soluble collagen when stimulated by TGF-ß or high glucose as compared to control cells, as well as a reduction in fibronectin and collagen IV transcript levels. CRT protein is increased in kidneys of mice made diabetic with streptozotocin and subjected to uninephrectomy to accelerate renal tubular injury as compared to controls. We used renal-targeted ultrasound delivery of Cre-recombinase plasmid to knockdown specifically CRT expression in the remaining kidney of uninephrectomized Calr fl/fl mice with streptozotocin-induced diabetes. This approach reduced CRT expression in the kidney, primarily in the tubular epithelium, by 30-55%, which persisted over the course of the studies. Renal function as measured by the urinary albumin/creatinine ratio was improved in the mice with knockdown of CRT as compared to diabetic mice injected with saline or subjected to ultrasound and injected with control GFP plasmid. PAS staining of kidneys and immunohistochemical analyses of collagen types I and IV show reduced glomerular and tubulointerstitial fibrosis. Renal sections from diabetic mice with CRT knockdown showed reduced nuclear NFAT in renal tubules and treatment of diabetic mice with 11R-VIVIT, an NFAT inhibitor, reduced proteinuria and renal fibrosis. These studies identify ER CRT as an important regulator of TGF-ß stimulated ECM production in the diabetic kidney, potentially through regulation of NFAT-dependent ECM transcription.

Characterizing Cell Stress and GRP78 in Glioma to Enhance Tumor Treatment.

Glioblastoma (GBM) is the most common primary brain tumor, carrying a very poor prognosis, with median overall survival at about 12 to 15 months despite surgical resection, chemotherapy with temozolomide (TMZ), and radiation therapy. GBM recurs in the vast majority of patients, with recurrent tumors commonly displaying increase in resistance to standard of care chemotherapy, TMZ, as well as radiotherapy. One of the most commonly cited mechanisms of chemotherapeutic and radio-resistance occurs via the glucose-regulated protein 78 (GRP78), a well-studied mediator of the unfolded protein response (UPR), that has also demonstrated potential as a biomarker in GBM. Overexpression of GRP78 has been directly correlated with malignant tumor characteristics, including higher tumor grade, cellular proliferation, migration, invasion, poorer responses to TMZ and radiation therapy, and poorer patient outcomes. GRP78 expression is also higher in GBM tumor cells upon recurrence. Meanwhile, knockdown or suppression of GRP78 has been shown to sensitize cells to TMZ and radiation therapy. In light of these findings, various novel developing therapies are targeting GRP78 as monotherapies, combination therapies that enhance the effects of TMZ and radiation therapy, and as treatment delivery modalities. In this review, we delineate the mechanisms by which GRP78 has been noted to specifically modulate glioblastoma behavior and discuss current developing therapies involving GRP78 in GBM. While further research is necessary to translate these developing therapies into clinical settings, GRP78-based therapies hold promise in improving current standard-of-care GBM therapy and may ultimately lead to improved patient outcomes.