1. bookVolume 54 (2020): Issue 3 (July 2020)
Journal Details
First Published
30 Mar 2016
Publication timeframe
4 times per year
Open Access

Expression of IDE and PITRM1 genes in ERN1 knockdown U87 glioma cells: effect of hypoxia and glucose deprivation

Published Online: 27 Aug 2020
Volume & Issue: Volume 54 (2020) - Issue 3 (July 2020)
Page range: 183 - 195
Journal Details
First Published
30 Mar 2016
Publication timeframe
4 times per year

Objective. The aim of the present investigation was to study the expression of genes encoding polyfunctional proteins insulinase (insulin degrading enzyme, IDE) and pitrilysin metallopeptidase 1 (PITRM1) in U87 glioma cells in response to inhibition of endoplasmic reticulum stress signaling mediated by ERN1/IRE1 (endoplasmic reticulum to nucleus signaling 1) for evaluation of their possible significance in the control of metabolism through ERN1 signaling as well as hypoxia, glucose and glutamine deprivations.

Methods. The expression level of IDE and PITRM1 genes was studied in control and ERN1 knockdown U87 glioma cells under glucose and glutamine deprivations as well as hypoxia by quantitative polymerase chain reaction.

Results. It was found that the expression level of IDE and PITRM1 genes was down-regulated in ERN1 knockdown (without ERN1 protein kinase and endoribonuclease activity) glioma cells in comparison with the control glioma cells, being more significant for PITRM1 gene. We also found up-regulation of microRNA MIR7-2 and MIRLET7A2, which have specific binding sites in 3’-untranslated region of IDE and PITRM1 mRNAs, correspondingly, and can participate in posttranscriptional regulation of these mRNA expressions. Only inhibition of ERN1 endoribonuclease did not change significantly the expression of IDE and PITRM1 genes in glioma cells. The expression of IDE and PITRM1 genes is preferentially regulated by ERN1 protein kinase. We also showed that hypoxia down-regulated the expression of IDE and PITRM1 genes and that knockdown of ERN1 signaling enzyme function modified the response of these gene expressions to hypoxia. Glucose deprivation increased the expression level of IDE and PITRM1 genes, but ERN1 knockdown enhanced only the effect of glucose deprivation on PITRM1 gene expression. Glutamine deprivation did not affect the expression of IDE gene in both types of glioma cells, but up-regulated PITRM1 gene and this up-regulation was stronger in ERN1 knockdown cells.

Conclusions. Results of this investigation demonstrate that ERN1 knockdown significantly decreases the expression of IDE and PITRM1 genes by ERN1 protein kinase mediated mechanism. The expression of both studied genes was sensitive to hypoxia as well as glucose deprivation and dependent on ERN1 signaling in gene-specific manner. It is possible that the level of these genes expression under hypoxia and glucose deprivation is a result of complex interaction of variable endoplasmic reticulum stress related and unrelated regulatory factors and contributed to the control of the cell metabolism.


Almanza A, Carlesso A, Chintha C, Creedican S, Doultsinos D, Leuzzi B, Luis A, McCarthy N, Montibeller L, More S, Papaioannou A, Puschel F, Sassano ML, Skoko J, Agostinis P, de Belleroche J, Eriksson LA, Fulda S, Gorman AM, Healy S, Kozlov A, Munoz-Pinedo C, Rehm M, Chevet E, Samali A. Endoplasmic reticulum stress signalling - from basic mechanisms to clinical applications. FEBS J 286, 241–278, 2019.10.1111/febs.14608Search in Google Scholar

Auf G, Jabouille A, Guerit S, Pineau R, Delugin M, Bouchecareilh M, Favereaux A, Maitre M, Gaiser T, von Deimling A, Czabanka M, Vajkoczy P, Chevet E, Bikfalvi A, Moenner M. A shift from an angiogenic to invasive phenotype induced in malignant glioma by inhibition of the unfolded protein response sensor IRE1. Proc Natl Acad Sci U S A 107, 15553–15558, 2010.10.1073/pnas.0914072107Search in Google Scholar

Auf G, Jabouille A, Delugin M, Guerit S, Pineau R, North S, Platonova N, Maitre M, Favereaux A, Vajkoczy P, Seno M, Bikfalvi A, Minchenko D, Minchenko O, Moenner M. High epiregulin expression in human U87 glioma cells relies on IRE1alpha and promotes autocrine growth through EGF receptor. BMC Cancer 13, 597, 2013.10.1186/1471-2407-13-597Search in Google Scholar

Bravo R, Parra V, Gatica D, Rodriguez AE, Torrealba N, Paredes F, Wang ZV, Zorzano A, Hill JA, Jaimovich E, Quest AF, Lavandero S. Endoplasmic reticulum and the unfolded protein response: dynamics and metabolic integration. Int Rev Cell Mol Biol 301, 215–290, 2013.10.1016/B978-0-12-407704-1.00005-1Search in Google Scholar

Brunetti D, Torsvik J, Dallabona C, Teixeira P, Sztromwasser P, Fernandez-Vizarra E, Cerutti R, Reyes A, Preziuso C, D’Amati G, Baruffini E, Goffrini P, Viscomi C, Ferrero I, Boman H, Telstad W, Johansson S, Glaser E, Knappskog PM, Zeviani M, Bindoff LA. Defective PITRM1 mitochondrial peptidase is associated with Aβ amyloidotic neurodegeneration. EMBO Mol Med 8, 176–190, 2016.10.15252/emmm.201505894Search in Google Scholar

Chevet E, Hetz C, Samali A. Endoplasmic reticulum stress-activated cell reprogramming in oncogenesis. Cancer Discov 5, 586–597, 2015.10.1158/2159-8290.CD-14-1490Search in Google Scholar

Chow KM, Gakh O, Payne IC, Juliano MA, Juliano L, Isaya G, Hersh LB. Mammalian pitrilysin: substrate specificity and mitochondrial Targeting. Biochemistry 48, 2868–2877, 2009.10.1021/bi8016125Search in Google Scholar

da Costa IB, de Labio RW, Rasmussen LT, Viani GA, Chen E, Villares J, Turecki G, Smith MAC, Payao SLM. Change in INSR, APBA2 and IDE gene expressions in brains of Alzheimer’s disease patients. Curr Alzheimer Res 14, 760–765, 2017.10.2174/1567205014666170203100734Search in Google Scholar

Doultsinos D, Avril T, Lhomond S, Dejeans N, Guedat P, Chevet E. Control of the unfolded protein response in health and disease. SLAS Discov 22, 787–800, 2017.10.1177/2472555217701685Search in Google Scholar

Falkevall A, Alikhani N, Bhushan S, Pavlov PF, Busch K, Johnson KA, Eneqvist T, Tjernberg L, Ankarcrona M, Glaser E. Degradation of the amyloid beta-protein by the novel mitochondrial peptidasome, PreP. J Biol Chem 281, 29096–29104, 2006.10.1074/jbc.M602532200Search in Google Scholar

Fernandez-de Frutos M, Galan-Chilet I, Goedeke L, Kim B, Pardo-Marques V, Perez-Garcia A, Herrero JI, Fernandez-Hernando C, Kim J, Ramirez CM. MicroRNA 7 impairs insulin signaling and regulates Aβ levels through posttranscriptional regulation of the insulin receptor substrate 2, insulin receptor, insulin-degrading enzyme, and liver X receptor pathway. Mol Cell Biol 39. pii: e00170–19, 2019.10.1128/MCB.00170-19Search in Google Scholar

Fernandez-Diaz CM, Merino B, Lopez-Acosta JF, Cidad P, de la Fuente MA, Lobaton CD, Moreno A, Leissring MA, Perdomo G, Cozar-Castellano I. Pancreatic β-cell-specific deletion of insulin-degrading enzyme leads to dysregulated insulin secretion and β-cell functional immaturity. Am J Physiol Endocrinol Metab 317, E805–E819, 2019.10.1152/ajpendo.00040.2019Search in Google Scholar

Garcia-Gonzalez P, Cabral-Miranda F, Hetz C, Osorio F. Interplay between the unfolded protein response and immune function in the development of neurodegenerative diseases. Front Immunol 9, 2541, 2018.10.3389/fimmu.2018.02541Search in Google Scholar

Gerakis Y, Hetz C. Emerging roles of ER stress in the etiology and pathogenesis of Alzheimer’s disease. FEBS J 285, 995–1011, 2018.10.1111/febs.14332Search in Google Scholar

Hassler JR, Scheuner DL, Wang S, Han J, Kodali VK, Li P, Nguyen J, George JS, Davis C, Wu SP, Bai Y, Sartor M, Cavalcoli J, Malhi H, Baudouin G, Zhang Y, Yates Iii JR, Itkin-Ansari P, Volkmann N, Kaufman RJ. The IRE1α/ XBP1s Pathway Is Essential for the Glucose Response and Protection of β Cells. PLoS Biol 13, e1002277, 2015.10.1371/journal.pbio.1002277Search in Google Scholar

Hetz C, Chevet E, Harding HP. Targeting the unfolded protein response in disease. Nat Rev Drug Discov 12, 703–719, 2013.10.1038/nrd3976Search in Google Scholar

Hetz C, Axten JM, Patterson JB. Pharmacological targeting of the unfolded protein response for disease intervention. Nat Chem Biol 15, 764–775, 2019.10.1038/s41589-019-0326-2Search in Google Scholar

Hughes D, Mallucci GR. The unfolded protein response in neurodegenerative disorders – therapeutic modulation of the PERK pathway. FEBS J 286, 342–355, 2019.10.1111/febs.14422Search in Google Scholar

Kazkayasi I, Burul-Bozkurt N, Ismail MA, Merino-Serrais P, Pekiner C, Cedazo-Minguez A, Uma S. Insulin deprivation decreases insulin degrading enzyme levels in primary cultured cortical neurons and in the cerebral cortex of rats with streptozotocin-induced diabetes. Pharmacol Rep 70, 677–683, 2018.10.1016/j.pharep.2018.01.008Search in Google Scholar

King JV, Liang WG, Scherpelz KP, Schilling AB, Meredith SC, Tang WJ. Molecular basis of substrate recognition and degradation by human presequence protease. Structure 22, 996–1007, 2014.10.1016/j.str.2014.05.003Search in Google Scholar

Kulas JA, Franklin WF, Smith NA, Manocha GD, Puig KL, Nagamoto-Combs K, Hendrix RD, Taglialatela G, Barger SW, Combs CK. Ablation of amyloid precursor protein increases insulin-degrading enzyme levels and activity in brain and peripheral tissues. Am J Physiol Endocrinol Metab 316, E106–E120, 2019.10.1152/ajpendo.00279.2018Search in Google Scholar

Kurochkin IV, Guarnera E, Wong JH, Eisenhaber F, Berezovsky IN. Toward allosterically increased catalytic activity of insulin-degrading enzyme against amyloid peptides. Biochemistry 56, 228–239, 2017.10.1021/acs.biochem.6b00783Search in Google Scholar

Lee J, Ozcan U. Unfolded protein response signaling and metabolic diseases. J Biol Chem 289, 1203–1211, 2014.10.1074/jbc.R113.534743Search in Google Scholar

Liu Z, Dai J, Shen H. Systematic analysis reveals long noncoding RNAs regulating neighboring transcription factors in human cancers. Biochim Biophys Acta Mol Basis Dis 1864, 2785–2792, 2018.10.1016/j.bbadis.2018.05.006Search in Google Scholar

Logue SE, McGrath EP, Cleary P, Greene S, Mnich K, Almanza A, Chevet E, Dwyer RM, Oommen A, Legembre P, Godey F, Madden EC, Leuzzi B, Obacz J, Zeng Q, Patterson JB, Jager R, Gorman AM, Samali A. Inhibition of IRE1 RNase activity modulates the tumor cell secretome and enhances response to chemotherapy. Nat Commun 9, 3267, 2018.10.1038/s41467-018-05763-8Search in Google Scholar

Manie SN, Lebeau J, Chevet E. Cellular mechanisms of endoplasmic reticulum stress signaling in health and disease. 3. Orchestrating the unfolded protein response in oncogenesis: an update. Am J Physiol Cell Physiol 307, C901–C907, 2014.10.1152/ajpcell.00292.2014Search in Google Scholar

Marciniak SJ. Endoplasmic reticulum stress: a key player in human disease. FEBS J 286, 228–231, 2019.10.1111/febs.14740Search in Google Scholar

Martinez A, Lopez N, Gonzalez C, Hetz C. Targeting of the unfolded protein response (UPR) as therapy for Parkinson’s disease. Biol Cell 111, 161–168, 2019.10.1111/boc.201800068Search in Google Scholar

McMahon M, Samali A, Chevet E. Regulation of the unfolded protein response by noncoding RNA. Am J Physiol Cell Physiol 313, C243–C254, 2017.10.1152/ajpcell.00293.2016Search in Google Scholar

Mercado G, Castillo V, Soto P, Lopez N, Axten JM, Sardi SP, Hoozemans JJM, Hetz C. Targeting PERK signaling with the small molecule GSK2606414 prevents neurodegeneration in a model of Parkinson’s disease. Neurobiol Dis 112, 136–148, 2018.10.1016/j.nbd.2018.01.004Search in Google Scholar

Minchenko DO, Danilovskyi SV, Kryvdiuk IV, Bakalets TV, Lypova NM, Karbovskyi LL, Minchenko OH. Inhibition of ERN1 modifies the hypoxic regulation of the expression of TP53-related genes in U87 glioma cells. Endoplasm Reticul Stress Dis 1, 18–26, 2014.10.2478/ersc-2014-0001Search in Google Scholar

Minchenko DO, Kharkova AP, Tsymbal DO, Karbovskyi LL, Minchenko OH. Expression of insulin-like growth factor binding protein genes and its hypoxic regulation in U87 glioma cells depends on ERN1 mediated signaling pathway of endoplasmic reticulum stress. Endocr Regul 49, 73–83, 2015a.10.4149/endo_2015_02_73Search in Google Scholar

Minchenko DO, Tsymbal DO, Riabovol OO, Viletska YM, Lahanovska YO, Sliusar MY, Bezrodnyi BH, Minchenko OH. Hypoxic regulation of EDN1, EDNRA, EDNRB, and ECE1 gene expressions in IRE1 knockdown U87 glioma cells. Endocr Reg 53, 250–262, 2019.10.2478/enr-2019-0025Search in Google Scholar

Minchenko OH, Tsymbal DO, Minchenko DO, Kovalevska OV, Karbovskyi LL, Bikfalvi A. Inhibition of ERN1 signaling enzyme affects hypoxic regulation of the expression of E2F8, EPAS1, HOXC6, ATF3, TBX3 and FOXF1 genes in U87 glioma cells. Ukr Biochem J 87, 76–87, 2015b.10.15407/ubj87.02.076Search in Google Scholar

Minchenko OH, Tsymbal DO, Minchenko DO, Moenner M, Kovalevska OV, Lypova NM. Inhibition of kinase and endoribonuclease activity of ERN1/IRE1 affects expression of proliferation-related genes in U87 glioma cells. Endoplasm Reticul Stress Dis 2, 18–29, 2015c.10.1515/ersc-2015-0002Search in Google Scholar

Minchenko OH, Kryvdiuk IV, Minchenko DO, Riabovol OO, Halkin OV. 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. Endoplasm Reticul Stress Dis 3, 1–15, 2016.10.1515/ersc-2016-0001Search in Google Scholar

Minchenko OH, Luzina OY, Hnatiuk OS, Minchenko DO, Garmash YA, Ratushna OO. Expression of tumor growth related genes in IRE1 knockdown U87 glioma cells: effect of hypoxia. Ukr Biochem J 89, 40–51, 2017.10.15407/ubj89.05.040Search in Google Scholar

Minchenko OH, Kharkova AP, Hnatiuk OS, Luzina OY, Kryvdiuk IV, Kuznetsova AY. ERN1 modifies effect of gluta-mine deprivation on tumor growth related factors expression in U87 glioma cells. Ukr Biochem J 90, 49–61, 2018a.10.15407/ubj90.03.049Search in Google Scholar

Minchenko OH, Tsymbal DO, Minchenko DO, Hnatiuk OS, Prylutskyy YI, Prylutska SV, Ritter U. Single-walled carbon nanotubes affect the expression of genes associated with immune response in normal human astrocytes. Toxicol In Vitro 52, 122–130, 2018b.10.1016/j.tiv.2018.06.011Search in Google Scholar

Moenner M, Pluquet O, Bouchecareilh M, Chevet E. Integrated endoplasmic reticulum stress responses in cancer. Cancer Res 67, 10631–10634, 2007.10.1158/0008-5472.CAN-07-1705Search in Google Scholar

Ohyagi Y, Miyoshi K, Nakamura N. Therapeutic strategies for Alzheimer’s disease in the view of diabetes mellitus. Adv Exp Med Biol 1128, 227–248, 2019.10.1007/978-981-13-3540-2_11Search in Google Scholar

Papaioannou A, Chevet E. Driving cancer tumorigenesis and metastasis through UPR signaling. Curr Top Microbiol Immunol 414, 159–192, 2018.10.1007/82_2017_36Search in Google Scholar

Pinho CM, Teixeira PF, Glaser E. Mitochondrial import and degradation of amyloid-β peptide. Biochim Biophys Acta 1837, 1069–1074, 2014.10.1016/j.bbabio.2014.02.007Search in Google Scholar

Pivovarova O, Gogebakan O, Pfeiffer AF, Rudovich N. Glucose inhibits the insulin-induced activation of the insulin-degrading enzyme in HepG2 cells. Diabetologia 52, 1656–1664, 2009.10.1007/s00125-009-1350-7Search in Google Scholar

Pivovarova O, von Loeffelholz C, Ilkavets I, Sticht C, Zhuk S, Murahovschi V, Lukowski S, Docke S, Kriebel J, de las Heras Gala T, Malashicheva A, Kostareva A, Lock JF, Stockmann M, Grallert H, Gretz N, Dooley S, Pfeiffer AF, Rudovich N. Modulation of insulin degrading enzyme activity and liver cell proliferation. Cell Cycle 14, 2293–2300, 2015.10.1080/15384101.2015.1046647Search in Google Scholar

Pivovarova O, Hohn A, Grune T, Pfeiffer AF, Rudovich N. Insulin-degrading enzyme: new therapeutic target for diabetes and Alzheimer’s disease? Ann Med 48, 614–624, 2016.Sandoval K, Umbaugh D, House A, Crider A, Witt K. Somatostatin receptor subtype-4 regulates mRNA expression of amyloid-beta degrading enzymes and microglia mediators of phagocytosis in brains of 3xTg-AD mice. Neurochem Res 44, 2670–268, 2019.10.1007/s11064-019-02890-6Search in Google Scholar

Semenza GL. A compendium of proteins that interact with HIF-1α. Exp Cell Res 356, 128–135, 2017.10.1016/j.yexcr.2017.03.041Search in Google Scholar

Smith-Carpenter JE, Alper BJ. Functional requirement for human pitrilysin metallopeptidase 1 arginine 183, mutated in amyloidogenic neuropathy. Protein Sci 27, 861–873, 2018.10.1002/pro.3380Search in Google Scholar

Sun RC, Denko NC. Hypoxic regulation of glutamine metabolism through HIF1 and SIAH2 supports lipid synthesis that is necessary for tumor growth. Cell Metab 19, 285–292, 2014.10.1016/j.cmet.2013.11.022Search in Google Scholar

Tang WJ. Targeting insulin-degrading enzyme to treat Type 2 Diabetes Mellitus. Trends Endocrinol Metab 27, 24–34, 2016.10.1016/j.tem.2015.11.003Search in Google Scholar

Tundo GR, Sbardella D, Ciaccio C, Grasso G, Gioia M, Coletta A, Polticelli F, Di Pierro D, Milardi D, Van Endert P, Marini S, Coletta M. Multiple functions of insulin-degrading enzyme: a metabolic crosslight? Crit Rev Biochem Mol Biol 52, 554–582, 2017.10.1080/10409238.2017.1337707Search in Google Scholar

Villa-Perez P, Merino B, Fernandez-Diaz CM, Cidad P, Lobaton CD, Moreno A, Muturi HT, Ghadieh HE, Najjar SM, Leissring MA, Cozar-Castellano I, Perdomo G. Liver-specific ablation of insulin-degrading enzyme causes hepatic insulin resistance and glucose intolerance, without affecting insulin clearance in mice. Metabolism 88, 1–11, 2018.10.1016/j.metabol.2018.08.001Search in Google Scholar

Wang M, Kaufman RJ. Protein misfolding in the endoplasmic reticulum as a conduit to human disease. Nature 529, 326–335, 2016.10.1038/nature17041Search in Google Scholar

Womeldorff M, Gillespie D, Jensen RL. Hypoxia-inducible factor–1 and associated upstream and downstream proteins in the pathophysiology and management of glioblastoma. Neurosurg Focus 37, E8, 2014.10.3171/2014.9.FOCUS14496Search in Google Scholar

Yamamoto N, Ishikuro R, Tanida M, Suzuki K, Ikeda-Matsuo Y, Sobue K. Insulin-signaling pathway regulates the degradation of amyloid β-protein via astrocytes. Neuroscience 385, 227–236, 2018.10.1016/j.neuroscience.2018.06.018Search in Google Scholar

Zhang Z, Liang WG, Bailey LJ, Tan YZ, Wei H, Wang A, Farcasanu M, Woods VA, McCord LA, Lee D, Shang W, Deprez-Poulain R, Deprez B, Liu DR, Koide A, Koide S, Kossiakoff AA, Li S, Carragher B, Potter CS, Tang WJ. Ensemble cryoEM elucidates the mechanism of insulin capture and degradation by human insulin degrading enzyme. Elife 7, e33572, 2018.10.7554/eLife.33572Search in Google Scholar

Zhang S, Xiao T, Yu Y, Qiao Y, Xu Z, Geng J, Liang Y, Mei Y, Dong Q, Wang B, Wei J, Suo G. The extracellular matrix enriched with membrane metalloendopeptidase and insulin-degrading enzyme suppresses the deposition of amyloid-beta peptide in Alzheimer’s disease cell models. J Tissue Eng Regen Med 13, 1759–1769, 2019.10.1002/term.2906Search in Google Scholar

Zhao S, Cai J, Li J, Bao G, Li D, Li Y, Zhai X, Jiang C, Fan L. Bioinformatic profiling identifies a glucose-related risk signature for the malignancy of glioma and the survival of patients. Mol Neurobiol 54, 8203–8210, 2017.10.1007/s12035-016-0314-4Search in Google Scholar

Zingale GA, Bellia F, Ahmed IMM, Mielczarek P, Silberring J, Grasso G. IDE degrades Nociceptin/Orphanin FQ through an insulin regulated mechanism. Int J Mol Sci 20, E4447, 2019.10.3390/ijms20184447Search in Google Scholar

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