Inhibition Of Inositol Requiring Enzyme-1 Research Articles

Inhibition of IRE1 suppresses the catabolic effect of IL-1β on nucleus pulposus cell and prevents intervertebral disc degeneration in vivo

Neck pain and low back pain are two of the major diseases, which causes patients a low quantify of life and a heavy economic burden, intervertebral disc degeneration (IDD) contributes to them, and the mechanism is not totally clear. The increased inflammatory cytokines including interleukin (IL)-1β and tumor necrosis factor (TNF)α and downstream signaling pathways are involved. Inositol requiring enzyme 1 (IRE1) is a crucial enzyme that regulates endoplasmic reticulum (ER) stress. It is reported that IRE1 plays an important role in the activation of NF-κB, PI3K/Akt and MAPK signaling pathways. Considering this, we performed a series of experiments in vitro and in vivo to evaluate the role of IRE1 in the progress of IDD. We demonstrated that IRE1 pathway was induced by IL-1β, inhibition of IRE1 suppressed the matrix degeneration of NP cells and ameliorated IDD grade in the punctured rat model. Further results indicated that inhibition of IRE1 suppressed H2O2 induced cell senescence, IL-1β-induced cellular reactive oxygen species (ROS) level and the activation of NF-κB, PI3K/Akt and MAPK signaling pathways. It also played a crucial role in the apoptosis of NP cells and the progress of macrophage polarization. Our findings demonstrated that inhibition of IRE1 could suppress the degeneration of NP cells and prevent IDD in vivo. IRE1 may be a potential target for IDD treatment.

Qishen Granule alleviates endoplasmic reticulum stress-induced myocardial apoptosis through IRE-1-CRYAB pathway in myocardial ischemia

Ethnopharmacological relevanceQishen Granule (QSG) is a prevailing traditional Chinese medicine formula that displays impressive cardiovascular protection in clinical. However, underlying mechanisms by which QSG alleviates endoplasmic reticulum (ER) stress-induced apoptosis in myocardial ischemia still remain unknown. Aim of the studyThis study aims to elucidate whether QSG ameliorates ER stress-induced myocardial apoptosis to protect against myocardial ischemia via inositol requiring enzyme 1 (IRE-1)-αBcrystallin (CRYAB) signaling pathway. Materials and methodsLeft anterior descending (LAD) ligation induced-ischemic heart model and oxygen-glucose deprivation-reperfusion (OGD/R)-induced H9C2 cells injury model were established to clarify the effects and potential mechanism of QSG. Ethanol extracts of QSG (2.352 g/kg) were orally administered for four weeks and Ginaton Tablets (100 mg/kg) was selected as a positive group in vivo. In vitro, QSG (800 μg/ml) or STF080310 (an inhibitor of IRE-1, 10 μM) was co-cultured under OGD/R in H9C2 cells. Inhibition of IRE-1 was conducted in H9C2 cells to further confirm the exact mechanism. Finally, to define the active components of anti-cardiomyocyte apoptosis in QSG which absorbed into the blood, we furtherly used the OGD/R-induced cardiomyocyte apoptosis model to evaluate the effects. ResultsQSG treatment improved cardiac function, ameliorated inflammatory cell infiltration and myocardial apoptosis. Similar effects were revalidated in OGD/R-induced H9C2 injury model. Western blots demonstrated QSG exerted anti-apoptotic effects by regulating apoptosis-related proteins, including increasing Bcl-2 and caspase 3/12, reducing the expressions of Bax and cleaved-caspase 3/12. Mechanistically, the IRE-1-CRYAB signaling pathway was significantly activated by QSG. Co-treatment with STF080310, the IRE-1 specific inhibitor significantly compromised the protective effects of QSG in vitro. Especially, the active components of QSG including Formononetin, Tanshinone IIA, Tanshinone I, Cryptotanshinon and Harpagoside showed significantly anti-apoptosis effects. ConclusionQSG protected against ER stress-induced myocardial apoptosis via the IRE-1-CRYAB pathway, which is proposed as a promising therapeutic target for myocardial ischemia.

Abstract A119: Inhibition of IRE1-mediated pro-tumorigenic responses enhances the sensitivity of MDA-MB-231 cells to the novel arylurea-fatty acid CTU

Abstract There is an unmet need for new agents with low toxicity for treatment of triple-negative breast cancer (TNBC) because current approaches are limited to toxic drug combinations of low efficacy. We recently identified an aryl-substituted fatty acid analogue, termed CTU, that effectively killed TNBC cells in vitro and in mouse xenograft models in vivo without producing toxicity (Rawling et al., 2017). However, there was a residual cell population that survived CTU. The present study evaluated the mechanism underlying residual cell survival. From RNA-seq profiling, a major regulated process in CTU-treated cells was activation of the unfolded response and endoplasmic reticulum (ER)-stress. The ER transmembrane protein inositol-requiring enzyme 1 (IRE1) is a central mediator of the unfolded response and ER-stress that was activated by CTU. IRE1 is also implicated as a pro-tumorigenic factor that can activate the expansion of tumor-initiating cells. From RNA-seq several pro-tumorigenic factors, including cyclooxygenase-2 and the cytokines IL6, IL8 and GM-CSF, were strongly activated in CTU-treated cells; this was confirmed by real-time RT-PCR and Western blot analysis. Using the non-adherent, in vitro colony-forming mammosphere assay, that enables the quantification of cell self-renewal, it was found that CTU expanded tumor-initiating cells. Co-treatment with an inhibitor of IRE1 impaired IRE1 kinase and IRE1 RNase activities, as reflected by decreases in phospho-p65 and XBP1s expression. IRE1 inhibition also prevented the activation of pro-tumorigenic factors and self-renewal by CTU. Taken together, the present findings suggest that inhibition of IRE1 may be a novel approach to optimise the activity of CTU in TNBC. Citation Format: Md Khalilur Rahman, Tristan Rawling, Yassir Al-Zubaidi, Hassan Choucair, Bala Umashankar, Yongjuan Chen, Kirsi Bourget, Stanton Tam, Charlotte Smith, Michael Murray. Inhibition of IRE1-mediated pro-tumorigenic responses enhances the sensitivity of MDA-MB-231 cells to the novel arylurea-fatty acid CTU [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A119. doi:10.1158/1535-7163.TARG-19-A119

Carbon monoxide releasing molecule-3 alleviates neuron death after spinal cord injury via inflammasome regulation.

BackgroundGenetic overexpression or pharmacological activation of heme oxygenase (HO) are identified as potential therapeutic target for spinal cord injury (SCI); however, the role of carbon monoxide (CO), which is a major product of haem degenerated by HO, in SCI remains unknown. Applying hemin or chemicals which may regulate HO expression or activity to increase CO production are inadequate to elaborate the direct role of CO. Here, we assessed the effect of CO releasing molecule-3 (CORM-3), the classical donor of CO, in SCI and explained its possible protective mechanism.MethodsRat SCI model was performed with a vascular clip (30 g) compressing at T9 vertebral level for 1 min and CO was delivered immediately after SCI by CORM-3. The neurological deficits and neuron survival were assessed. Inflammasome and inositol-requiring enzyme 1 (IRE1) pathway were measured by western blot and immunofluorescence. For in vitro study, oxygen glucose deprivation (OGD) simulated the SCI-inflammasome change in cultured the primary neurons.FindingsCORM-3 suppressed inflammasome signaling and pyroptosis occurrence, which consequently alleviated neuron death and improved motor functional recovery following SCI. As a pivotal sensor involving in endoplasmic reticulum stress-medicated inflammasome signaling, IRE1 and its downstream X-box binding protein 1 (XBP1) were activated in SCI tissues as well as in OGD neurons; while inhibition of IRE1 by STF-083010 in SCI rats or by si-RNA in OGD neurons suppressed inflammasome signaling and pyroptosis. Interestingly, the SCI/OGD-stimulated IRE1 activation was attenuated by CORM-3 treatment.InterpretationsCO may alleviate neuron death and improve motor functional recovery in SCI through IRE1 regulation, and administration of CO could be a promising therapeutic strategy for SCI.

Inhibition of IRE1 modifies hypoxic regulation of G6PD, GPI, TKT, TALDO1, PGLS and RPIA genes expression in U87 glioma cells.

We have studied the effect of hypoxia on the expression level of mRNA of the basic enzymes of pentose-phosphate cycle (G6PD, TKT, TALDO1, PGLS and RPIA) and glucose-6-phosphate isomerase (GPI) in U87 glioma cells in relation to inhibition of IRE1 (inositol requiring enzyme 1). It was shown that hypoxia leads to up-regulation of the expression of GPI and PGLS genes and to down-regulation of TALDO1 and RPIA genes in control glioma cells. Changes for GPI gene were more significant than for other genes. At the same time, inhibition of IRE1 modified the effect of hypoxia on the expression of all studied genes. In particular, it increased sensitivity to hypoxia of G6PD and TKT genes expression and suppressed the effect of hypoxia on the expression of GPI and RPIA genes. Additionally, inhibition of IRE1 eliminated hypoxic regulation of PGLS gene and did not change significantly effect of hypoxia on the expression of TALDO1 gene in glioma cells. Present study demonstrated that hypoxia, which often contributes to tumor growth, affects the expression of most studied genes and inhibition of IRE1 modified the hypoxic regulation of pentose-phosphate cycle gene expressions in a gene specific manner and thus possibly contributes to slower glioma growth, but several aspects of this regulation warrant further investigation.

Abstract P2-04-06: Targeting of the unfolded protein response signaling arms differentially regulates macrophage proliferation, plasticity, and breast cancer cell clearance

Abstract The unfolded protein response (UPR) is an endoplasmic reticulum stress pathway controlled by the protein chaperone, glucose-regulated protein 78 (GRP78), to mediate inositol-requiring enzyme 1 (IRE1), PKR-like endoplasmic reticulum kinase (PERK), and activating transcription factor-6 (ATF6) signaling. UPR signaling has been shown to be upregulated in many different types of cancers, including breast cancer and melanoma, and is associated with the development of therapeutic resistance. These data suggest the importance of targeting UPR signaling as a possible cancer therapy. We have previously shown GRP78 to be upregulated in human breast tumor samples and leads to endocrine targeted therapy resistance. We recently showed inhibiting GRP78 in human orthotopic xenografts potentiates tamoxifen therapy effectiveness in sensitive tumors and restores endocrine therapy responsiveness in resistant tumors. In these GRP78-inhibited tumors there was a significant increase of CD68 positive macrophage population, suggesting that targeting UPR signaling has critical effects on the tumor microenvironment. Therefore, consideration of each UPR signaling component and how it effects the different cellular compartments of the tumor microenvironment need to be investigated to optimally induce both an antitumor immune effect and inhibit tumor epithelial cell growth. We now show deletion of GRP78, IRE1, and PERK through RNAi differentially regulates macrophage polarization. Specifically, PERK inhibition enhances macrophage proliferation and macrophage-mediated phagocytosis of 4T1 breast cancer cells, but not GRP78 or IRE1 inhibition. Targeting UPR signaling in the breast cancer cells also differentially affected macrophage cytolytic capacity; Specific breast cancer cell inhibition of IRE1 or GRP78 enhanced macrophage-mediated phagocytosis. Conditioned media from control or GRP78 silenced ZR-75-1 breast cancer cells indicated reciprocal regulation of CD206 and CD80; suggesting regulation of macrophage plasticity by GRP78-controlled secreted factors. GRP78 targeting in mice resulting in a cytokine shift and increased tumoral CD80+/CD68+ cells, suggesting that GRP78 inhibition favors a M1-like macrophage profile. Inhibition of UPR components in both macrophage and breast cancer cells, similar to what would be observed in systemic cancer therapies, indicated that either PERK or GRP78 inhibition enhances macrophage cytolytic clearance of breast cancer cells. Taken together, these data suggest that targeting GRP78 or PERK promotes an anti-tumor immune response by either directly promoting macrophage cytolytic activity (PERK targeting) or indirectly by shifting tumoral cytokine secretion (GRP78 targeting). Citation Format: Cook KL, Wilson A, Westwood B, Soto-Pantoja DR. Targeting of the unfolded protein response signaling arms differentially regulates macrophage proliferation, plasticity, and breast cancer cell clearance [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-04-06.

Hypoxic regulation of the expression of genes encoded estrogen related proteins in U87 glioma cells: eff ect of IRE1 inhibition

The aim of the present study was to examine the effect of inhibition of endoplasmic reticulum stress signaling, mediated by IRE1 (inositol requiring enzyme 1), which is a central mediator of the unfolded protein response on the expression of genes encoded estrogen related proteins (NRIP1/RIP140, TRIM16/EBBP, ESRRA/NR3B1, FAM162A/E2IG5, PGRMC2/PMBP, and SLC39A6/LIV-1) and their hypoxic regulation in U87 glioma cells for evaluation of their possible significance in the control of glioma cells proliferation. The expression of NRIP1, EBBP, ESRRA, E2IG5, PGRMC2, and SLC39A6 genes in U87 glioma cells, transfected by empty vector pcDNA3.1 (control) and cells without IRE1 signaling enzyme function (transfected by dnIRE1) upon hypoxia, was studied by a quantitative polymerase chain reaction. Inhibition of both enzymatic activities (kinase and endoribonuclease) of IRE1 signaling enzyme function up-regulates the expression of EBBP, E2IG5, PGRMC2, and SLC39A6 genes is in U87 glioma cells in comparison with the control glioma cells, with more significant changes for E2IG5 and PGRMC2 genes. At the same time, the expression of NRIP1 and ESRRA genes is strongly down-regulated in glioma cells upon inhibition of IRE1. We also showed that hypoxia increases the expression of E2IG5, PGRMC2, and EBBP genes and decreases NRIP1 and ESRRA genes expression in control glioma cells. Furthermore, the inhibition of IRE1 in U87 glioma cells decreases the eff ect of hypoxia on the expression of E2IG5 and PGRMC2 genes, eliminates hypoxic regulation of NRIP1 gene, and enhances the sensitivity of ESRRA gene to hypoxic condition. Furthermore, the expression of SLC39A6 gene is resistant to hypoxia in both the glioma cells with and without IRE1 signaling enzyme function. Results of this investigation demonstrate that inhibition of IRE1 signaling enzyme function affects the expression of NRIP1, EBBP, ESRRA, E2IG5, PGRMC2, and SLC39A6 genes in U87 glioma cells in gene specific manner and these changes possibly contribute to the suppression of the cell proliferation. Most of these genes are regulated by hypoxia and preferentially through IRE1 signaling pathway of endoplasmic reticulum stress.

Effect of Hypoxia on the Expression of a Subset of Proliferation Related Genes in IRE1 Knockdown U87 Glioma Cells

We have studied the expression of a subset of genes encoding important tumor growth related factors in U87 glioma cells with IRE1 (inositol requiring enzyme-1) knockdown as well as their hypoxic regulation. It was shown that the expression levels of activating transcription factor 6 (ATF6), clusterin (CLU), adhesion G protein-coupled receptor E5 (ADGRE5), transglutaminase 2, C polypeptide (TGM2), leukemia inhibitory factor (LIF), phosphoserine aminotransferase 1 (PSAT1), glyoxalase I (GLO1) and tetraspanin 13 (TSPAN13) are significantly down-regulated in glioma cells with the knockdown of IRE1 signaling enzyme. It was also shown that in glioma cells subjected to hypoxia, the expression levels of PSAT1, TSPAN13, EIF2AK3, and TGM2 genes were up-regulated, whereas the expression of ATF6 gene was down-regulated. At the same time, the expression levels of LIF, CLU, and ADGRE5 genes did not change in response to hypoxic treatment. Furthermore, inhibition of IRE1, a key effector of an unfolded protein response pathway, modified the effect of hypoxia on the expression of most studied genes. Present study demonstrates that IRE1 knockdown down-regulated the expression of most studied genes and modified their hypoxic regulation and that these changes possibly contributed to the suppression of glioma growth in cells without IRE1 signaling enzyme function.

The role of the TNF receptors and apoptosis inducing ligands in tumor growth.

Tumor necrosis factor (TNF) superfamily receptors and TNF apoptosis inducing ligands play an important role in the realization of TNF function and control tumor growth. The TNF-related pathways are controlled by endoplasmic reticulum stress signaling, which has a crucial role in the control of cell proliferation and tumor growth. Furthermore, the inhibition of IRE1 (inositol requiring enzyme-1), which is a central mediator of endoplasmic reticulum stress sand mainly responsible for cell proliferation and apoptosis, leads to suppression of tumor growth through specific changes in the expression of genes encoding transcription factors, tumor suppressors, angiogenesis and apoptosis related proteins, including TNF superfamily receptors and TNF apoptosis inducing ligands. Therefore, changes in the expression level of TNF-related genes encoding TNF superfamily receptors and apoptosis inducing ligands possibly reflect metabolic reprogramming of cancer cells upon inhibition of IRE1-mediated endoplasmic reticulum stress signaling and correlate with suppression of glioma cell proliferation.

Inhibition of IRE1 signaling affects the expression of genes encoded glucocorticoid receptor and some related factors and their hypoxic regulation in U87 glioma cells.

The aim of the present investigation was to examine the effect of inhibition of endoplasmic reticulum stress signaling, mediated by IRE1 (inositol requiring enzyme 1), which is a central mediator of the unfolded protein response on the expression of genes encoding glucocorticoid receptor (NR3C1) and some related proteins (SGK1, SGK3, NCOA1, NCOA2, ARHGAP35, NNT) and their hypoxic regulation in U87 glioma cells for evaluation of their possible significance in the control of the glioma growth. The expression of NR3C1,SGK1,SGK3, NCOA1, NCOA2, ARHGAP35, and NNT genes in U87 glioma cells, transfected by empty vector pcDNA3.1 (control) and cells without IRE1 signaling enzyme function (transfected by dnIRE1) upon hypoxia, was studied by quantitative polymerase chain reaction. Inhibition of IRE1 signaling enzyme function up-regulates the expression of NR3C1, SGK1, NCOA1, NCOA2, ARHGAP35, and NNT genes in U87 glioma cells in comparison with the control glioma cells, with more significant changes for NR3C1, SGK1, and NNT genes. At the same time, the expression of SGK3 gene is strongly down-regulated in glioma cells upon inhibition of IRE1. We have also shown that hypoxia increases the expression of NR3C1, SGK1, NCOA2, ARHGAP35, and NNT genes but decreases SGK3 and NCOA1 genes expression in control glioma cells. Moreover, the inhibition of both enzymatic activities (kinase and endoribonuclease) of IRE1 in U87 glioma cells enhances the eff ect of hypoxia on the expression of SGK1, SGK3, and NNT genes, but decreases the sensitivity of NR3C1 gene to hypoxic condition. Furthermore, the expression of NCOA1 gene is resistant to hypoxia in control glioma cells, but NCOA2 and ARHGAP35 genes are resistant to this condition in glioma cells without functional activity of IRE1 signaling enzyme. Results of this investigation demonstrate that inhibition of IRE1 signaling enzyme function affects the expression of NR3C1, SGK1, SGK3, NCOA1, NCOA2, ARHGAP35, and NNT genes in U87 glioma cells in gene specific manner and that all these genes are regulated by hypoxia preferentially through IRE1 signaling pathway of the endoplasmic reticulum stress.

Expression of IGFBP6, IGFBP7, NOV, CYR61, WISP1 and WISP2 genes in U87 glioma cells in glutamine deprivation condition.

We have studied gene expression of insulin-like growth factor binding proteins in U87 glioma cells upon glutamine deprivation depending on the inhibition of IRE1 (inositol requiring enzyme-1), a central mediator of endoplasmic reticulum stress. We have shown that exposure of control glioma cells upon glutamine deprivation leads to down-regulation of NOV/IGFBP9, WISP1 and WISP2 gene expressions and up-regulation of CYR61/IGFBP10 gene expression at the mRNA level. At the same time, the expression of IGFBP6 and IGFBP7 genes in control glioma cells was resistant to glutamine deprivation. It was also shown that the inhibition of IRE1 modifies the effect of glutamine deprivation on the expression of all studied genes. Thus, the inhibition of IRE1 signaling enzyme enhances the effect of glutamine deprivation on the expression of CYR61 and WISP1 genes and suppresses effect of the deprivation on WISP2 gene expression in glioma cells. Moreover, the inhibition of IRE1 introduces sensitivity of the expression of IGFBP6 and IGFBP7 genes to glutamine deprivation and removes this sensitivity to NOV gene. We have also demonstrated that the expression of all studied genes in glioma cells growing with glutamine is regulated by IRE1 signaling enzyme, because the inhibition of IRE1 significantly down-regulates IGFBP6 and NOV genes and up-regulates IGFBP7, CYR61, WISP1, and WISP2 genes as compared to control glioma cells. The present study demonstrates that glutamine deprivation condition affects most studied IGFBP and WISP gene expressions in relation to IRE1 signaling enzyme function and possibly contributes to slower glioma cell proliferation upon inhibition of IRE1.

Effect of hypoxia on the expression of nuclear genes encoding mitochondrial proteins in U87 glioma cells.

We have studied the effect of hypoxia on the expression of nuclear genes encoding mitochondrial proteins in U87 glioma cells under the inhibition of IRE1 (inositol requiring enzyme-1), which controls cell proliferation and tumor growth as a central mediator of endoplasmic reticulum stress. It was shown that hypoxia down-regulated gene expression of malate dehydrogenase 2 (MDH2), malic enzyme 2 (ME2), mitochondrial aspartate aminotransferase (GOT2), and subunit B of succinate dehydrogenase (SDHB) in control (transfected by empty vector) glioma cells in a gene specific manner. At the same time, the expression level of mitochondrial NADP+-dependent isocitrate dehydrogenase 2 (IDH2) and subunit D of succinate dehydrogenase (SDHD) genes in these cells does not significantly change in hypoxic conditions. It was also shown that the inhibition of ІRE1 signaling enzyme function in U87 glioma cells decreases the effect of hypoxia on the expression of ME2, GOT2, and SDHB genes and introduces the sensitivity of IDH2 gene to hypoxia. Furthermore, the expression of all studied genes depends on IRE1-mediated endoplasmic reticulum stress signaling in gene specific manner, because ІRE1 knockdown significantly decreases their expression in normoxic conditions, except for IDH2 gene, which expression level is strongly up-regulated. Therefore, changes in the expression level of nuclear genes encoding ME2, MDH2, IDH2, SDHB, SDHD, and GOT2 proteins possibly reflect metabolic reprogramming of mitochondria by hypoxia and IRE1-mediated endoplasmic reticulum stress signaling and correlate with suppression of glioma cell proliferation under inhibition of the IRE1 enzyme function.

Inhibition of IRE1 modifies the hypoxic regulation of GADD family gene expressions in U87 glioma cells.

We have studied hypoxic regulation of the expression of genes encoded GADD (growth arrest and DNA damage) family proteins in U87 glioma cells in relation to inhibition of IRE1 (inositol requiring enzyme-1), which controls cell proliferation and tumor growth as a central mediator of endoplasmic reticulum stress. We have shown that hypoxia up-regulates the expression of GADD34, GADD45A, GADD45B, and GADD153 genes, which are related to cell proliferation and apoptosis, in control (transfected by empty vector) glioma cells in gene specific manner. At the same time, the expression level of EIF2AK 1 (eukaryotic translation initiation factor 2-alpha kinase 1) and AI FM1 (apoptosis inducing factor, mitochondria associated 1) genes in these cells is down-regulated upon hypoxic condition. It was also shown that inhibition of ІRE1 signaling enzyme function in U87 glioma cells enhances the effect of hypoxia on these genes expression, except EIF2AK 1 and AI FM1 genes. Furthermore, the expression of all studied genes in ІRE1 knockdown cells is significantly decreased upon normoxic condition, except GADD45B gene, which expression level is strongly up-regulated. Therefore, the expression level of genes encoding GADD34, GADD45A, GADD45B, GADD153, EIF2AK 1, and AI FM1 is affected by hypoxia and by inhibition of IRE1-mediated endoplasmic reticulum stress signaling in gene specific manner and correlates with suppression of glioma cell proliferation upon inhibition of the IRE1 enzyme function.

Effect of hypoxia on the expression of genes encoding insulin-like growth factors and some related proteins in U87 glioma cells without IRE1 function.

The aim of the present study was to investigate the effect of hypoxia on the expression of genes encoding insulin-like growth factors (IGF1 and IGF2), their receptor (IGF1R), binding protein-4 (IGFBP4), and stanniocalcin 2 (STC2) in U87 glioma cells in relation to inhibition of endoplasmic reticulum stress signaling mediated by IRE1 (inositol requiring enzyme 1) for evaluation of their possible significance in the control of tumor growth. The expression of IGF1, IGF2, IGF1R, IGFBP4, and STC2 genes in U87 glioma cells transfected by empty vector pcDNA3.1 (control) and cells without IRE1 signaling enzyme function (transfected by dnIRE1) upon hypoxia was studied by qPCR. The expression of IGF1 and IGF2 genes is down-regulated in glioma cells without IRE1 signaling enzyme function in comparison with the control cells. At the same time, the expression of IGF1R, IGFBP4, and STC2 genes was up-regulated in glioma cells upon inhibition of IRE1, with more significant changes for IGFBP4 and STC2 genes. We also showed that hypoxia does not change significantly the expression of IGF1, IGF2, and IGF1R genes but up-regulated IGFBP4 and STC2 genes expression in control glioma cells. Moreover, the inhibition of both enzymatic activities (kinase and endoribonuclease) of IRE1 in glioma cells does not change significantly the effect of hypoxia on the expression of IGF1, IGF1R, and IGFBP4 genes but introduces sensitivity of IGF2 gene to hypoxic condition. Thus, the expression of IGF2 gene is resistant to hypoxia only in control glioma cells and significantly down-regulated in cells without functional activity of IRE1 signaling enzyme, which is central mediator of the unfolded protein response and an important component of the tumor growth as well as metabolic diseases. Results of this study demonstrate that the expression of IGF1 and IGF1R genes is resistant to hypoxic condition both in control U87 glioma cells and cells without IRE1 signaling enzyme function. However, hypoxia significantly up-regulates the expression of IGFBP4 gene independently on the inhibition of IRE1 enzyme. These data show that proteins encoded by these genes are resistant to hypoxia except IGFBP4 and participate in the regulation of metabolic and proliferative processes through IRE1 signaling.

Hypoxic regulation of the expression of cell proliferation related genes in U87 glioma cells upon inhibition of IRE1 signaling enzyme

We have studied the effect of inhibition of IRE1 (inositol requiring enzyme 1), which is a central mediator of endoplasmic reticulum stress and a controller of cell proliferation and tumor growth, on hypoxic regulation of the expression of different proliferation related genes in U87 glioma cells. It was shown that hypoxia leads to up-regulation of the expression of IL13RA2, CD24, ING1, ING2, ENDOG, and POLG genes and to down-regulation – of KRT18, TRAPPC3, TSFM, and MTIF2 genes at the mRNA level in control glioma cells. Changes for ING1 and CD24 genes were more significant. At the same time, inhibition of IRE1 modifies the effect of hypoxia on the expression of all studied genes. In particular, it increases sensitivity to hypoxia of the expression of IL13RA2, TRAPPC3, ENDOG, and PLOG genes and suppresses the effect of hypoxia on the expression of ING1 gene. Additionally, it eliminates hypoxic regulation of KRT18, CD24, ING2, TSFM, and MTIF2 genes expressions and introduces sensitivity to hypoxia of the expression of BET1 gene in glioma cells. The present study demonstrates that hypoxia, which often contributes to tumor growth, affects the expression of almost all studied genes. Additionally, inhibition of IRE1 can both enhance and suppress the hypoxic regulation of these gene expressions in a gene specific manner and thus possibly contributes to slower glioma growth, but several aspects of this regulation must be further clarified.

Inhibition of IRE1 signaling affects expression of a subset genes encoding for TNF-related factors and receptors and modifies their hypoxic regulation in U87 glioma cells

AbstractInhibition of IRE1 (inositol requiring enzyme-1), the major signaling pathway of endoplasmic reticulum stress, significantly decreases tumor growth and proliferation of glioma cells. To elucidate the role of IRE1- mediated glioma growth, we studied the expression of a subset genes encoding for TNF (tumor necrosis factor)- related factors and receptors and their hypoxic regulation in U87 glioma cells overexpressing dominant-negative IRE1 (dnIRE1). We demonstrated that the expression of TNFAIP1, TNFRSF10D, TNFRSF21, TNFRSF11B, TNFSF7, and LITAF genes is increased in glioma cells with modified IRE1; however, TNFRSF10B, TRADD, and TNFAIP3 is down-regulated in these cells as compared to their control counterparts. We did not find TNFRSF1A gene expression to change significantly under this experimental condition. In control glioma cells, hypoxia leads to the up-regulated expression of TNFAIP1, TNFAIP3, TRADD, and TNFRSF10D genes and the concomitant down-regulation of TNFRSF21, TNFRSF11B, and LITAF genes; while, TNFRSF10B and TNFRSF1A genes are resistant to hypoxic treatment. However, inhibition of IRE1 modifies the hypoxic regulation of LITAF, TNFRSF21, TNFRSF11B, and TRADD genes and introduces hypoxia-induced sensitivity to TNFRSF10B, TNFRSF1A, and TNFSF7 gene expressions. Furthermore, knockdown by siRNA of TNFRSF21 mRNA modifies the hypoxic effect on the IRE1-dependent rate of proliferation and cell death in U87 glioma cells. The present study demonstrates that fine-tuned manipulation of the expression of TNF-related factors and receptors directly relating to cell death and proliferation, is mediated by an effector of endoplasmic reticulum stress, IRE1, as well as by hypoxia in a gene-specific manner. Thus, inhibition of the kinase and endoribonuclease activities of IRE1 correlates with deregulation of TNF-related factors and receptors in a manner that is gene specific and thus slows tumor growth.

EFFECT OF HYPOXIA ON THE EXPRESSION OF GENES THAT ENCODE SOME IGFBP AND CCN PROTEINS IN U87 GLIOMA CELLS DEPENDS ON IRE1 SIGNALING.

We have studied hypoxic regulation of the expression of different insulin-like growth factor binding protein genes in U87 glioma cells in relation to inhibition of IRE1 (inositol requiring enzyme-1), a central mediator of endoplasmic reticulum stress, which controls cell proliferation and tumor growth. We have demonstrated that hypoxia leads to up-regulation of the expression of IGFBP6, IGFBP7, IGFBP10/CYR61, WISP1, and WISP2 genes and down-regulation--of IGFBP9/NOV gene at the mRNA level in control glioma cells, being more signifcant changes for IGFBP10/CYR61 and WISP2 genes. At the same time, inhibition of IRE1 modifies the effect of hypoxia on the expression of all studied genes: eliminates sensitivity to hypoxia the expression of IGFBP7 and IGFBP9/NOV genes, suppresses effect of hypoxia on IGFBP6, IGFBP10/CYR61, and WISP2 genes, and slightly enhances hypoxic regulation of WISP1 gene expression in glioma cells. We have also demonstrated that the expression of all studied genes in glioma cells is regulated by IRE1 signaling enzyme upon normoxic condition, because inhibition of IRE1 significantly up-regulates IGFBP7, IGFBP10/CYR61, WISP1, and WISP2 genes and down-regulates IGFBP6 and IGFBP9/NOV genes as compared to control glioma cells. The present study demonstrates that hypoxia, which contributes to tumor growth, affects all studied IGFBP and WISP gene expressions and that inhibition of IRE1 preferentially abolishes or suppresses the hypoxic regulation of these gene expressions and thus possibly contributes to slower glioma growth. Moreover, inhibition of IRE1, which correlates with suppression of cell proliferation and glioma growth, is down-regulated expression of pro-proliferative IGFBP genes, attesting to the fact that endoplasmic reticulum stress is a necessary component of malignant tumor growth.

INHIBITION OF IRE1 MODIFIES EFFECT OF GLUCOSE DEPRIVATION ON THE EXPRESSION OF TNFα-RELATED GENES IN U87 GLIOMA CELLS.

Inhibition of IRE1 (inositol requiring enzyme-1), the major signaling pathway of endoplasmic reticulm stress, significantly decreases glioma cell proliferation and tumor growth. We have studied the expression of TNFα-related genes and effect of glucose deprivation on these gene expressions in U87 glioma cells over-expressing dominant-negative IRE1 defective in both kinase and endonuclease (dn-IRE1) activity of IRE1 with hopes of elucidating its contribution to IRE1 mediated glioma growth. We have demonstrated that glucose deprivation condition leads to down-regulation of the expression of TNFRSF11B, TNFRSF1A, TNFRSF10D/TRAILR4, and LITAF genes and up-regulation of TNFRSF10B/TRAILR2/DR5 gene at the mRNA level in control glioma cells. At the same time, the expression of TNFRSF21/DR6, TNFAIP1, TNFAIP3, TRADD, and CD70/TNFSF7 genes in control glioma cells is resistant to glucose deprivation condition. The inhibition of IRE1 modifies the effect of glucose deprivation on LITAF, TNFRSF21, TNFRSF11B, and TRADD gene expressions and induces sensitivity to glucose deprivation condition the expression of TNFRSF10B, TNFRSF1A, and CD70 genes. We have also demonstrated that the expression of all studied genes is affected in glioma cells by inhibition of IRE1, except TNFRSF1A gene, as compared to control glioma cells. Moreover, the changes in the expression of TNFRSF1A, TNFRSF10D/TRAILR4, and LITAF genes induced by glucose deprivation condition have opposite orientation to that induced by inhibition of IRE1. The present study demonstrates that fine-tuning of the expression of TNFα-induced proteins and TNF receptor superfamily genes, which related to cell death and proliferation, is regulated by IRE1, an effector of endoplasmic reticulum stress, as well as depends on glucose deprivation in gene specific manner. Thus, the inhibition of kinase and endoribonuclease activity of IRE1 correlates with deregulation of TNFα-induced protein genes and TNF receptor superfamily genes in gene specific manner and thus slower the tumor growth.