ER Stress Genes Research Articles

Is the lack of adiponectin associated with increased ER/SR stress and inflammation in the heart?

Objective To study whether there is an association between adiponectin and endoplasmic reticulum/sarcoplasmic reticulum (ERSR) stress. Research design Eleven-month-old male wild-type (WT) and adiponectin knockout (ADKO) mice were placed on chow or high fat diet for 12 weeks. The changes in ER stress and inflammatory genes were determined in the epididymal adipose, as well as heart tissue of adult WT and ADKO mice. To understand the role of ER/SR stress in the regulation of adiponectin, we studied the effect of tunicamycin or palmitate on H9C2 cardiomyoblasts in culture. To demonstrate the protective role of adiponectin, we studied the effect of purified adiponectin on the regulation of ERSR stress genes and inflammation in H9C2 cardiomyoblasts. Results (1) High fat diet increased TNFα in adipose tissue of ADKO mice. (2) ERSR stress genes, HSPa5, ERN1, and GADD34, and inflammation response genes, TNFα and CD68, were increased in heart of ADKO mice. High fat diet did not further increase the effect. (3) Induction of ERSR stress by tunicamycin in H9C2 resulted in the upregulation of ERSR stress response genes along with downregulation of adiponectin, adiponectin receptors 1 and 2, and Serca2A. ER stress was accompanied by down regulation of Iкβα and an increase in HSPa5 proteins. (4) Adiponectin decreased ERSR stress and inflammation response genes and increased Serca2A in to H9C2 cardiomyoblasts. Conclusion The lack of adiponectin is associated with increased ER/SR stress and inflammation in the heart. Adiponectin provides a protective effect by lowering inflammation and ER/SR stress along with increasing Serca2A in H9C2 cells.

Iron depletion increases manganese uptake and potentiates apoptosis through ER stress

Iron deficiency is a risk factor for manganese (Mn) accumulation. Excess Mn promotes neurotoxicity but the mechanisms involved and whether iron depletion might affect these pathways is unknown. To study Mn intoxication in vivo, iron deficient and control rats were intranasally instilled with 60mg MnCl2/kg over 3 weeks. TUNEL staining of olfactory tissue revealed that Mn exposure induced apoptosis and that iron deficiency potentiated this effect. In vitro studies using the dopaminergic SH-SY5Y cell line confirmed that Mn-induced apoptosis was enhanced by iron depletion using the iron chelator desferrioxamine. Mn has been reported to induce apoptosis through endoplasmic reticulum stress. In SH-SY5Y cells, Mn exposure induced the ER stress genes glucose regulated protein 94 (GRP94) and C/EBP homologous protein (CHOP). Increased phosphorylation of the eukaryotic translation initiation factor 2α (phospho-eIF2α) was also observed. These effects were accompanied by the activation of ER resident enzyme caspase-12, and the downstream apoptotic effector caspase-3 was also activated. All of the Mn-induced responses were enhanced by DFO treatment. Inhibitors of ER stress and caspases significantly blocked Mn-induced apoptosis and its potentiation by DFO, indicating that ER stress and subsequent caspase activation underlie cell death. Taken together, these data reveal that Mn induces neuronal cell death through ER stress and the UPR response pathway and that this apoptotic effect is potentiated by iron deficiency most likely through upregulation of DMT1.

Integrative genomic analysis of CREB defines a critical role for transcription factor networks in mediating the fed/fasted switch in liver.

BackgroundMetabolic homeostasis in mammals critically depends on the regulation of fasting-induced genes by CREB in the liver. Previous genome-wide analysis has shown that only a small percentage of CREB target genes are induced in response to fasting-associated signaling pathways. The precise molecular mechanisms by which CREB specifically targets these genes in response to alternating hormonal cues remain to be elucidated.ResultsWe performed chromatin immunoprecipitation coupled to high-throughput sequencing of CREB in livers from both fasted and re-fed mice. In order to quantitatively compare the extent of CREB-DNA interactions genome-wide between these two physiological conditions we developed a novel, robust analysis method, termed the ‘single sample independence’ (SSI) test that greatly reduced the number of false-positive peaks. We found that CREB remains constitutively bound to its target genes in the liver regardless of the metabolic state. Integration of the CREB cistrome with expression microarrays of fasted and re-fed mouse livers and ChIP-seq data for additional transcription factors revealed that the gene expression switches between the two metabolic states are associated with co-localization of additional transcription factors at CREB sites.ConclusionsOur results support a model in which CREB is constitutively bound to thousands of target genes, and combinatorial interactions between DNA-binding factors are necessary to achieve the specific transcriptional response of the liver to fasting. Furthermore, our genome-wide analysis identifies thousands of novel CREB target genes in liver, and suggests a previously unknown role for CREB in regulating ER stress genes in response to nutrient influx.

Gene expression analysis of human islets in a subject at onset of type 1 diabetes

Swollen islet cells have been repeatedly described at onset of type 1 diabetes, but the underlying mechanism of this observation, termed hydropic degeneration, awaits characterization. In this study, laser capture microdissection was applied to extract the islets from an organ donor that died at onset of type 1 diabetes and from an organ donor without pancreatic disease. Morphologic analysis revealed extensive hydropic degeneration in 73% of the islets from the donor with type 1 diabetes. Expression levels of genes involved in apoptosis, ER stress, beta cell function, and inflammation were analyzed in isolated and laser-captured islets by qPCR. The chemokine MCP-1 was expressed in islets from the donor with type 1 diabetes while undetectable in the control donor. No other signs of inflammation were detected. There were no signs of apoptosis on the gene expression level, which was also confirmed by negative immunostaining for cleaved caspase-8. There was an increased expression of the transcription factor ATF4, involved in transcription of ER stress genes, in the diabetic islets, but no further signs of ER stress were identified. In summary, on the transcription level, islets at onset of type 1 diabetes in which many beta cells display hydropic degeneration show no obvious signs of apoptosis, ER stress, or inflammation, supporting the notion that these cells are responding normally to high glucose and eventually succumbing to beta cell exhaustion. Also, this study validates the feasibility of performing qPCR analysis of RNA extracted from islets from subjects with recent onset of T1D and healthy controls by laser capture microdissection.

Increased expression of ER stress genes in patients with limited cutaneous Systemic Sclerosis and Pulmonary Arterial Hypertension

ObjectivePrevious studies have implicated ER stress and UPR in autoimmune and inflammatory diseases.The goal of this study was to assess whether markers of ER stress/UPR are present in PBMCs from limited cutaneous systemic sclerosis (lcSSc) patients with pulmonary arterial hypertension (PAH).MethodsPBMCs were purified from healthy controls (HC n=36) and lcSSc patients (n=38 with PAH and n=39 without PAH).Gene expression in HC PBMCs stimulated with ER stress inducer, thapsigargin (TG) was analyzed by DNA microarray.Expression levels of select ER stress/UPR genes and inflammation genes were analyzed by qPCR.ResultsER stress/UPR genes, including BiP, transcription factors ATF4 and ATF6, and a spliced form of XBP1 were upregulated in lcSSc PBMCs, with the highest levels in patients with PAH.TG induced expression of HSPs and IFN‐regulated genes in control PBMCs.Selected HSP genes, particularly DNAJB1, and IFN‐related genes were significantly elevated in lcSSc PBMCs.There was a positive correlation between DNAJB1 and severity of PAH disease (PAP) (r = 0.59, p<0.05) and between BiP and IL‐6 levels (r = 0.64, p< 0.0001) in lcSSc PBMCs.ConclusionThese studies demonstrate a possible association between ER stress/UPR gene activation and lcSSc‐PAH suggesting that ER stress/UPR contribute to the interferon signature and altered function of circulating immune cells in patients with lcSSc.

NRF2 Protection against Liver Injury Produced by Various Hepatotoxicants

To investigate the role of Nrf2 as a master defense against the hepatotoxicity produced by various chemicals, Nrf2-null, wild-type, Keap1-knock down (Keap1-Kd) and Keap1-hepatocyte knockout (Keap1-HKO) mice were used as a “graded Nrf2 activation” model. Mice were treated with 14 hepatotoxicants at appropriate doses, and blood and liver samples were collected thereafter (6 h to 7 days depending on the hepatotoxicant). Graded activation of Nrf2 offered a Nrf2-dependent protection against the hepatotoxicity produced by carbon tetrachloride, acetaminophen, microcystin, phalloidin, furosemide, cadmium, and lithocholic acid, as evidenced by serum alanine aminotransferase (ALT) activities and by histopathology. Nrf2 activation also offered moderate protection against liver injury produced by ethanol, arsenic, bromobenzene, and allyl alcohol but had no effects on the hepatotoxicity produced by D-galactosamine/endotoxin and the Fas ligand antibody Jo-2. Graded Nrf2 activation reduced the expression of inflammatory genes (MIP-2, mKC, IL-1β, IL-6, and TNFα), oxidative stress genes (Ho-1, Egr1), ER stress genes (Gadd45 and Gadd153), and genes encoding cell death (Noxa, Bax, Bad, and caspase3). Thus, this study demonstrates that Nrf2 prevents the liver from many, but not all, hepatotoxicants. The Nrf2-mediated protection is accompanied by induction of antioxidant genes, suppression of inflammatory responses, and attenuation of oxidative stress.

Pomegranate (Punica granatum L.) reduces endoplasmic reticulum stress induced by renal ischemia/reperfusion injury in rat

Ischemia/reperfusion (I/R) is one of the main causes of acute renal failure, leading to generation of reactive oxygen species (ROS) and ensuing oxidative stress, which results in unfolded/misfolded protein accumulation and its dependent response pathways, generally known as unfolded protein response (UPR), in the endoplasmic reticulum (ER). We studied the effects of renal I/R on the expression of ER-stress genes, as well as concomitant effects of oral pretreatment with pomegranate (Punica granatum L.) pith and carpellary membrane (PPCM). An in vivo model of rat kidney I/R followed by conventional and real-time RT-PCR was used to evaluate the modulation of GRP78 and XBP1 as central UPR and ER-stress markers. Ischemia followed by reperfusion (60 and 150 min, respectively) resulted in decreased antioxidant properties of plasma (lowered ferric reducing ability of plasma [FRAP] value) and GRP78 transcript levels. Oral administration of PPCM aqueous extract for 10 days with or without ischemia (PPCM and PPCM/Isc groups, respectively) was able to further decrease the GRP78 expression, while it increased the FRAP value. PPCM pretreatment also reduced the XBP1 splicing in the PPCM/Isc group compared to the Isc group. These results suggest that UPR is activated during oxidative insults induced by I/R, while PPCM can reduce I/R-induced ER stress in rat kidneys via antioxidant defense mechanisms.

Abstract 29: An Orally Active Epoxyeicosatrienoic Acid (eet) Analog Attenuates Kidney Injury in Hypertensive Dahl Salt Sensitive Rat by Reducing Oxidative Stress, Inflammation and Endoplasmic Reticulum Stress

Renal injury is associated with salt-sensitive hypertension. The Dahl salt-sensitive hypertensive rat (Dahl SS) is a model of salt-sensitive hypertension and the related kidney injury. The kidney injury in Dahl SS hypertension is associated with oxidative stress, inflammation and ER stress. We have recently developed orally active epoxyecosatrienoic acid (EET) analogs that provide organ protection in hypertension and other pathologies through multiple signaling pathways. We hypothesized that these EET analogs with their anti-inflammatory, anti-oxidant and anti-ER stress effects will protect the kidney in Dahl SS hypertension. Dahl SS rats received high salt diet and treated either with an EET analog (10 mg/kg/d) or vehicle (0.05% EtOH+0.1% PEG 400) in drinking water for 14 days. Blood pressure (SBP) was measured by tail-cuff plethysmography and urine, plasma and tissue samples were collected at the end of the treatment protocol. Urinary creatinine, albumin, nephrin, and renal tissue TBARS content were measured using ELISA or colorimetric assays. Renal expression of oxidative, inflammatory and ER stress marker genes were examined using RT-PCR. Histological analysis was done to assess renal injury. In Dahl SS rats, EET analog treatment did not affect the SBP compared to vehicle (183±6 vs. 185±12 mmHg). Interestingly, the EET analog treatment in Dahl SS reduced albumin-creatinine ratio (49±17 vs. 12±3) and nephrinuria (11±1 vs. 4±1 mg/d). EET analog treatment also reduced glomerular injury, intra-tubular cast formation and kidney fibrosis by 30-60% in Dahl SS rats compared to vehicle. In Dahl SS rats, EET analog treatment caused a 40% reduction in kidney TBARS content compared to vehicle. Moreover, the EET analog treatment in Dahl SS rats resulted 2-3 fold attenuation in the renal expression of oxidative ( p47PHOX ), inflammation ( IL-6, TGF-β ), and ER stress ( CHOP, GADD34 ) genes. These data, demonstrate that a novel orally active EET analog provides kidney protection in Dahl SS hypertension by reducing oxidative stress, inflammation and ER stress, and this kidney protection was independent of blood pressure reduction.

Required for Adaptive ER-Stress During Testosterone Production in Testis.

Male infertility has many causes from hormonal imbalances, to physical problems, to psychological and/or behavioral problems. Heat exposure initiates low serum testosterone levels that could be attributed to downregulated steroidogenic gene such as 3β-HSD and 17β-HSD1 in the testis.Testosterone are required for spermatogenesis; most males with impaired spermatogenesis have low serum testosterone levels. It has been reported that the Leydig cell, which producing testosterone by Luteinizing Hormone(LH), have a highly developed ER. Generally, Endoplasmic Reticulum (ER) stress has been observed in mainly secretory cell type. So we sought to clarify the roles of ER- stress response pathways whether of steroidogenesis and ER stress in male infertility induced repeatedly heat exposure. ER-stress induced by heat exposure are decreased testosterone level, steroidogenic gene but increase expression of ER-stress gene; Bip, p50ATF6.

Scavenger Receptor CD36 Expression Contributes to Adipose Tissue Inflammation and Cell Death in Diet-Induced Obesity

ObjectiveThe enlarged adipose tissue in obesity is characterized by inflammation, including the recruitment and infiltration of macrophages and lymphocytes. The objective of this study was to investigate the role of the scavenger receptor CD36 in high fat diet-induced obesity and adipose tissue inflammation and cell death.Experimental ApproachObesity and adipose tissue inflammation was compared in CD36 deficient (CD36 KO) mice and wild type (WT) mice fed a high fat diet (60% kcal fat) for 16 weeks and the inflammatory response was studied in primary adipocytes and macrophages isolated from CD36 KO and WT mice.ResultsCompared to WT mice, CD36 KO mice fed a high fat diet exhibited reduced adiposity and adipose tissue inflammation, with decreased adipocyte cell death, pro-inflammatory cytokine expression and macrophage and T-cell accumulation. In primary cell culture, the absence of CD36 expression in macrophages decreased pro-inflammatory cytokine, pro-apoptotic and ER stress gene expression in response to lipopolysaccharide (LPS). Likewise, CD36 deficiency in primary adipocytes reduced pro-inflammatory cytokine and chemokine secretion in response to LPS. Primary macrophage and adipocyte co-culture experiments showed that these cell types act synergistically in their inflammatory response to LPS and that CD36 modulates such synergistic effects.ConclusionsCD36 enhances adipose tissue inflammation and cell death in diet-induced obesity through its expression in both macrophages and adipocytes.

AVP infusion increases the expression of urea transporters and ER stress pathway genes in medullae of female mice with urinary concentrating defect

Abnormal proliferation in collecting duct cells plays a crucial role in polycystic kidney disease (PKD) and in lithium‐induced renal dysfunction. Females with PKD demonstrate lower proliferation rates than males, resulting in a less severe phenotype. We previously reported that vasopressin induced an increase in renal cell proliferation in male AQP1KO mice in addition to ER stress gene activation. In this study we infused AVP for 14 days into female AQP1KO mice. AVP induced a small but significant increase in urine osmolality (155 mosmol/kgH20 vs control). 14 days AVP infusion significantly increased the expression of UTA1 and UTA3 mRNA 2.97 ± 0.29 and 3.34 ± 0.57, respectively, vs control. AQP2 and AQP3 mRNA also increased 3.52 ± 0.28 and 2.59 ± 0.23, vs control. AVP infusion significantly increased the mRNA expression of early growth response gene Egr‐1 2.49 ± 0.49 and transcription factors ATF3 and ATF4 mRNA; 4.91 ± 0.70, and 3.36 ± 0.25, vs control. AVP infusion significantly increased the expression of ER stress factor GADD153 mRNA 2.53 ± 0.41 vs control. AVP did not increase the mRNA expression of PCNA in female kidneys; PCNA is a marker of proliferation. These data suggest that vasopressin regulation of urea transporters is intact in female AQP1KO mice. Further studies will determine if vasopressin induced proliferative pathways are blunted in female AQP1KO mice.Support: DK073611 (HLB)

Endoplasmic reticulum stress mediating downregulated StAR and 3-beta-HSD and low plasma testosterone caused by hypoxia is attenuated by CPU86017-RS and nifedipine

BackgroundHypoxia exposure initiates low serum testosterone levels that could be attributed to downregulated androgen biosynthesizing genes such as StAR (steroidogenic acute regulatory protein) and 3-beta-HSD (3-beta-hydroxysteroid dehydrogenase) in the testis. It was hypothesized that these abnormalities in the testis by hypoxia are associated with oxidative stress and an increase in chaperones of endoplasmic reticulum stress (ER stress) and ER stress could be modulated by a reduction in calcium influx. Therefore, we verify that if an application of CPU86017-RS (simplified as RS, a derivative to berberine) could alleviate the ER stress and depressed gene expressions of StAR and 3-beta-HSD, and low plasma testosterone in hypoxic rats, these were compared with those of nifedipine.MethodsAdult male Sprague-Dawley rats were randomly divided into control, hypoxia for 28 days, and hypoxia treated (mg/kg, p.o.) during the last 14 days with nifedipine (Nif, 10) and three doses of RS (20, 40, 80), and normal rats treated with RS isomer (80). Serum testosterone (T) and luteinizing hormone (LH) were measured. The testicular expressions of biomarkers including StAR, 3-beta-HSD, immunoglobulin heavy chain binding protein (Bip), double-strand RNA-activated protein kinase-like ER kinase (PERK) and pro-apoptotic transcription factor C/EBP homologous protein (CHOP) were measured.ResultsIn hypoxic rats, serum testosterone levels decreased and mRNA and protein expressions of the testosterone biosynthesis related genes, StAR and 3-beta-HSD were downregulated. These changes were linked to an increase in oxidants and upregulated ER stress chaperones: Bip, PERK, CHOP and distorted histological structure of the seminiferous tubules in the testis. These abnormalities were attenuated significantly by CPU86017-RS and nifedipine.ConclusionDownregulated StAR and 3-beta-HSD significantly contribute to low testosterone in hypoxic rats and is associated with ER stress which mediates testis damage caused by oxygen deprivation. CPU86017-RS is potential in ameliorating hypoxia-induced testicular injuries, possibly by its calcium antagonist effects on the testis.

Insulin resistance, ceramide accumulation, and endoplasmic reticulum stress in human chronic alcohol-related liver disease.

Background. Chronic alcohol-related liver disease (ALD) is mediated by insulin resistance, mitochondrial dysfunction, inflammation, oxidative stress, and DNA damage. Recent studies suggest that dysregulated lipid metabolism with accumulation of ceramides, together with ER stress potentiate hepatic insulin resistance and may cause steatohepatitis to progress. Objective. We examined the degree to which hepatic insulin resistance in advanced human ALD is correlated with ER stress, dysregulated lipid metabolism, and ceramide accumulation. Methods. We assessed the integrity of insulin signaling through the Akt pathway and measured proceramide and ER stress gene expression, ER stress signaling proteins, and ceramide profiles in liver tissue. Results. Chronic ALD was associated with increased expression of insulin, IGF-1, and IGF-2 receptors, impaired signaling through IGF-1R and IRS1, increased expression of multiple proceramide and ER stress genes and proteins, and higher levels of the C14, C16, C18, and C20 ceramide species relative to control. Conclusions. In human chronic ALD, persistent hepatic insulin resistance is associated with dysregulated lipid metabolism, ceramide accumulation, and striking upregulation of multiple ER stress signaling molecules. Given the role of ceramides as mediators of ER stress and insulin resistance, treatment with ceramide enzyme inhibitors may help reverse or halt progression of chronic ALD.

Involvement of Dopamine Receptors in Binge Methamphetamine-Induced Activation of Endoplasmic Reticulum and Mitochondrial Stress Pathways

Single large doses of methamphetamine (METH) cause endoplasmic reticulum (ER) stress and mitochondrial dysfunctions in rodent striata. The dopamine D1 receptor appears to be involved in these METH-mediated stresses. The purpose of this study was to investigate if dopamine D1 and D2 receptors are involved in ER and mitochondrial stresses caused by single-day METH binges in the rat striatum. Male Sprague-Dawley rats received 4 injections of 10 mg/kg of METH alone or in combination with a putative D1 or D2 receptor antagonist, SCH23390 or raclopride, respectively, given 30 min prior to each METH injection. Rats were euthanized at various timepoints afterwards. Striatal tissues were used in quantitative RT-PCR and western blot analyses. We found that binge METH injections caused increased expression of the pro-survival genes, BiP/GRP-78 and P58IPK, in a SCH23390-sensitive manner. METH also caused up-regulation of ER-stress genes, Atf2, Atf3, Atf4, CHOP/Gadd153 and Gadd34. The expression of heat shock proteins (HSPs) was increased after METH injections. SCH23390 completely blocked induction in all analyzed ER stress-related proteins that included ATF3, ATF4, CHOP/Gadd153, HSPs and caspase-12. The dopamine D2-like antagonist, raclopride, exerted small to moderate inhibitory influence on some METH-induced changes in ER stress proteins. Importantly, METH caused decreases in the mitochondrial anti-apoptotic protein, Bcl-2, but increases in the pro-apoptotic proteins, Bax, Bad and cytochrome c, in a SCH23390-sensitive fashion. In contrast, raclopride provided only small inhibition of METH-induced changes in mitochondrial proteins. These findings indicate that METH-induced activation of striatal ER and mitochondrial stress pathways might be more related to activation of SCH23390-sensitive receptors.

Neuroinflammation and Endoplasmic Reticulum Stress Are Coregulated by Crocin To Prevent Demyelination and Neurodegeneration

Endoplasmic reticulum (ER) stress is a homeostatic mechanism, which is used by cells to adapt to intercellular and intracellular changes. Moreover, ER stress is closely linked to inflammatory pathways. We hypothesized that ER stress is an integral component of neuroinflammation and contributes to the development of neurological diseases. In autopsied brain specimens from multiple sclerosis (MS) and non-MS patients, XBP-1 spliced variant (XBP-1/s) was increased in MS brains (p < 0.05) and was correlated with the expression of the human endogenous retrovirus-W envelope transcript, which encodes the glycoprotein, Syncytin-1 (p < 0.05). In primary human fetal astrocytes transfected with a Syncytin-1-expressing plasmid, XBP-1/s, BiP, and NOS2 were induced, which was suppressed by crocin treatment (p < 0.05). Crocin also protected oligodendrocytes exposed to cytotoxic supernatants derived from Syncytin-1-expressing astrocytes (p < 0.05) and NO-mediated oligodendrocytotoxicity (p < 0.05). During experimental autoimmune encephalomyelitis (EAE), the transcript levels of the ER stress genes XBP-1/s, BiP, PERK, and CHOP were increased in diseased spinal cords compared with healthy littermates (p < 0.05), although CHOP expression was not involved in the EAE disease phenotype. Daily treatment with crocin starting on day 7 post-EAE induction suppressed ER stress and inflammatory gene expression in spinal cords (p < 0.05), which was accompanied by preserved myelination and axonal density, together with reduced T cell infiltration and macrophage activation. EAE-associated neurobehavioral deficits were also ameliorated by crocin treatment (p < 0.05). These findings underscored the convergent roles of pathogenic ER stress and immune pathways in neuroinflammatory disease and point to potential therapeutic applications for crocin.

C-Rel Deficiency Increases Caspase-4 Expression and Leads to ER Stress and Necrosis in EBV-Transformed Cells

LMP1-mediated activation of nuclear factor of kappaB (NF-κB) is critical for the ligand independent proliferation and cell survival of in vitro EBV-transformed lymphoblastoid cell lines (LCLs). Previous experiments revealed that a majority of LMP1-dependent responses are regulated by NF-κB. However, the extent that individual NF-κB family members are required for these responses, in particular, c-Rel, whose expression is restricted to mature hematopoietic cells, remains unclear. Here we report that low c-Rel expression in LCLs derived from a patient with hyper-IgM syndrome (Pt1), resulted in defects in proliferation and cell survival. In contrast to studies that associated loss of NF-κB with increased apoptosis, Pt1 LCLs failed to initiate apoptosis and alternatively underwent autophagy and necrotic cell death. Whereas the proliferation defect appeared linked to a c-Rel-associated decrease in c-myc expression, identified pro-survival and pro-apoptotic targets were expressed at or near control levels consistent with the absence of apoptosis. Ultrastructural examination of Pt1 LCLs revealed a high level of cellular and ER stress that was further supported by gene expression profiling showing the upregulation of several genes involved in stress and inflammation. Apoptosis-independent cell death was accompanied by increased expression of the inflammatory marker, caspase-4. Using gene overexpression and siRNA knockdown we demonstrated that levels of c-Rel directly modulated expression of caspase-4 as well as other ER stress genes. Overall, these findings reveal the importance of c-Rel in maintaining LCL viability and that decreased expression results in ER stress and a default response leading to necrotic cell death.

Altered hepatic lipid and apoB homeostasis is related to ER-stress gene downregulation leading to caspase-12 inhibition during NAFLD in MAT1A-KO mice

Altered hepatic lipid and apoB homeostasis is related to ER-stress gene downregulation leading to caspase-12 inhibition during NAFLD in MAT1A-KO mice

Gene expression profiling of microglia infected by a highly neurovirulent murine leukemia virus: implications for neuropathogenesis

BackgroundCertain murine leukemia viruses (MLVs) are capable of inducing progressive spongiform motor neuron disease in susceptible mice upon infection of the central nervous system (CNS). The major CNS parenchymal target of these neurovirulent retroviruses (NVs) are the microglia, whose infection is largely coincident with neuropathological changes. Despite this close association, the role of microglial infection in disease induction is still unknown. In this paper, we investigate the interaction of the highly virulent MLV, FrCasE, with microglia ex vivo to evaluate whether infection induces specific changes that could account for neurodegeneration. Specifically, we compared microglia infected with FrCasE, a related non-neurovirulent virus (NN) F43/Fr57E, or mock-infected, both at a basic virological level, and at the level of cellular gene expression using quantitative real time RT-PCR (qRT-PCR) and Afffymetrix 430A mouse gene chips.ResultsBasic virological comparison of NN, NV, and mock-infected microglia in culture did not reveal differences in virus expression that provided insight into neuropathogenesis. Therefore, microglial analysis was extended to ER stress gene induction based on previous experiments demonstrating ER stress induction in NV-infected mouse brains and cultured fibroblasts. Analysis of message levels for the ER stress genes BiP (grp78), CHOP (Gadd153), calreticulin, and grp58 in cultured microglia, and BiP and CHOP in microglia enriched fractions from infected mouse brains, indicated that FrCasE infection did not induce these ER stress genes either in vitro or in vivo. To broadly identify physiological changes resulting from NV infection of microglia in vitro, we undertook a gene array screen of more than 14,000 well-characterized murine genes and expressed sequence tags (ESTs). This analysis revealed only a small set of gene expression changes between infected and uninfected cells (<18). Remarkably, gene array comparison of NN- and NV-infected microglia revealed only 3 apparent gene expression differences. Validation experiments for these genes by Taqman real-time RT-PCR indicated that only single Ig IL-1 receptor related protein (SIGIRR) transcript was consistently altered in culture; however, SIGIRR changes were not observed in enriched microglial fractions from infected brains.ConclusionThe results from this study indicate that infection of microglia by the highly neurovirulent virus, FrCasE, does not induce overt physiological changes in this cell type when assessed ex vivo. In particular, NV does not induce microglial ER stress and thus, FrCasE-associated CNS ER stress likely results from NV interactions with another cell type or from neurodegeneration directly. The lack of NV-induced microglial gene expression changes suggests that FrCasE either affects properties unique to microglia in situ, alters the expression of microglial genes not represented in this survey, or affects microglial cellular processes at a post-transcriptional level. Alternatively, NV-infected microglia may simply serve as an unaffected conduit for persistent dissemination of virus to other neural cells where they produce acute neuropathogenic effects.

Optimization of tetracycline‐responsive recombinant protein production and effect on cell growth and ER stress in mammalian cells

The inducible T-REx system and other inducible expression systems have been developed in order to control the expression levels of recombinant protein in mammalian cells. In order to study the effects of heterologous protein expression on mammalian host behavior, the gene for recombinant Human transferrin (hTf) was integrated into HEK-293 cells and expressed under the control of the T-REx inducible technology (293-TetR-Hyg-hTf) or using a constitutive promoter (293-CMV-hTf). A number of inducible clones with variable expression levels were identified for the T-REx system with levels of hTf for the high expressing clones nearly double those obtained using the constitutive cytomegalovirus (CMV) promoter. The level of transferrin produced was found to increase proportionately with tetracycline concentration between 0 and 1 mug/mL with no significant increases in transferrin production above 1 mug/mL. As a result, the optimal induction time and tetracycline concentrations were determined to be the day of plating and 1 mug/mL, respectively. Interestingly, the cells induced to express transferrin, 293-TetR-Hyg-hTf, exhibited lower viable cell densities and percent viabilities than the uninduced cultures for multiple clonal isolates. In addition, the induction of transferrin expression was found to cause an increase in the expression of the ER-stress gene, BiP, that was not observed in the uninduced cells. However, both uninduced and induced cell lines containing the hTf gene exhibited longer survival in culture than the control cells, possibly as a result of the positive effects of hTf on cell survival. Taken together, these results suggest that the high level expression of complex proteins in mammalian cells can limit the viable cell densities of cells in culture as a result of cellular stresses caused by generating proteins that may be difficult to fold or are otherwise toxic to cells. The application of inducible systems such as the T-REx technology will allow us to optimize protein production while limiting the negative effects that result from these cellular stresses.

293 Activation of the ER-stress gene GADD153 by HCV. Effects on cell cycle and oxidative stress

293 Activation of the ER-stress gene GADD153 by HCV. Effects on cell cycle and oxidative stress