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• W3095319430 abstract "Article2 November 2020free access Source DataTransparent process Inositol-requiring enzyme-1 regulates phosphoinositide signaling lipids and macrophage growth Syed Muhammad Hamid Syed Muhammad Hamid Smidt Heart Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA Search for more papers by this author Mevlut Citir Mevlut Citir The Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany Search for more papers by this author Erdem Murat Terzi Erdem Murat Terzi Department of Pathology, Laura & Isaac Perlmutter Cancer Center, New York University School of Medicine, New York, NY, USA Search for more papers by this author Ismail Cimen Ismail Cimen orcid.org/0000-0002-8182-6968 Institute for Cardiovascular Prevention, LMU Munich, German Cardiovascular Research Centre, partner site Munich Heart Alliance Munich, Munich, Germany Search for more papers by this author Zehra Yildirim Zehra Yildirim Smidt Heart Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA Department of Molecular Biology and Genetics, Bilkent University, Ankara, Turkey National Nanotechnology Center, Bilkent University, Ankara, Turkey Search for more papers by this author Asli Ekin Dogan Asli Ekin Dogan Smidt Heart Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA Department of Molecular Biology and Genetics, Bilkent University, Ankara, Turkey National Nanotechnology Center, Bilkent University, Ankara, Turkey Search for more papers by this author Begum Kocaturk Begum Kocaturk orcid.org/0000-0003-3657-6055 Department of Pediatrics and Medicine, Division of Infectious Diseases and Immunology, and Infectious and Immunologic Diseases Research Center, Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA, USA Search for more papers by this author Umut Inci Onat Umut Inci Onat Department of Molecular Biology and Genetics, Bilkent University, Ankara, Turkey National Nanotechnology Center, Bilkent University, Ankara, Turkey Search for more papers by this author Moshe Arditi Moshe Arditi Smidt Heart Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA Department of Pediatrics and Medicine, Division of Infectious Diseases and Immunology, and Infectious and Immunologic Diseases Research Center, Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA, USA Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA, USA Search for more papers by this author Christian Weber Christian Weber orcid.org/0000-0003-4610-8714 Institute for Cardiovascular Prevention, LMU Munich, German Cardiovascular Research Centre, partner site Munich Heart Alliance Munich, Munich, Germany Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, The Netherlands Munich Cluster for Systems Neurology (SyNergy), Munich, Germany Search for more papers by this author Alexis Traynor-Kaplan Alexis Traynor-Kaplan Department of Medicine, University of Washington School of Medicine, Seattle, WA, USA ATK Innovation, Analytics and Discovery, North Bend, WA, USA Search for more papers by this author Carsten Schultz Carsten Schultz orcid.org/0000-0002-5824-2171 The Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany Department of Chemical Physiology and Biochemistry, Oregon Health & Science University, Portland, OR, USA Search for more papers by this author Ebru Erbay Corresponding Author Ebru Erbay [email protected] orcid.org/0000-0001-9584-1803 Smidt Heart Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA, USA Search for more papers by this author Syed Muhammad Hamid Syed Muhammad Hamid Smidt Heart Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA Search for more papers by this author Mevlut Citir Mevlut Citir The Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany Search for more papers by this author Erdem Murat Terzi Erdem Murat Terzi Department of Pathology, Laura & Isaac Perlmutter Cancer Center, New York University School of Medicine, New York, NY, USA Search for more papers by this author Ismail Cimen Ismail Cimen orcid.org/0000-0002-8182-6968 Institute for Cardiovascular Prevention, LMU Munich, German Cardiovascular Research Centre, partner site Munich Heart Alliance Munich, Munich, Germany Search for more papers by this author Zehra Yildirim Zehra Yildirim Smidt Heart Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA Department of Molecular Biology and Genetics, Bilkent University, Ankara, Turkey National Nanotechnology Center, Bilkent University, Ankara, Turkey Search for more papers by this author Asli Ekin Dogan Asli Ekin Dogan Smidt Heart Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA Department of Molecular Biology and Genetics, Bilkent University, Ankara, Turkey National Nanotechnology Center, Bilkent University, Ankara, Turkey Search for more papers by this author Begum Kocaturk Begum Kocaturk orcid.org/0000-0003-3657-6055 Department of Pediatrics and Medicine, Division of Infectious Diseases and Immunology, and Infectious and Immunologic Diseases Research Center, Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA, USA Search for more papers by this author Umut Inci Onat Umut Inci Onat Department of Molecular Biology and Genetics, Bilkent University, Ankara, Turkey National Nanotechnology Center, Bilkent University, Ankara, Turkey Search for more papers by this author Moshe Arditi Moshe Arditi Smidt Heart Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA Department of Pediatrics and Medicine, Division of Infectious Diseases and Immunology, and Infectious and Immunologic Diseases Research Center, Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA, USA Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA, USA Search for more papers by this author Christian Weber Christian Weber orcid.org/0000-0003-4610-8714 Institute for Cardiovascular Prevention, LMU Munich, German Cardiovascular Research Centre, partner site Munich Heart Alliance Munich, Munich, Germany Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, The Netherlands Munich Cluster for Systems Neurology (SyNergy), Munich, Germany Search for more papers by this author Alexis Traynor-Kaplan Alexis Traynor-Kaplan Department of Medicine, University of Washington School of Medicine, Seattle, WA, USA ATK Innovation, Analytics and Discovery, North Bend, WA, USA Search for more papers by this author Carsten Schultz Carsten Schultz orcid.org/0000-0002-5824-2171 The Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany Department of Chemical Physiology and Biochemistry, Oregon Health & Science University, Portland, OR, USA Search for more papers by this author Ebru Erbay Corresponding Author Ebru Erbay [email protected] orcid.org/0000-0001-9584-1803 Smidt Heart Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA, USA Search for more papers by this author Author Information Syed Muhammad Hamid1, Mevlut Citir2, Erdem Murat Terzi3, Ismail Cimen4, Zehra Yildirim1,5,6, Asli Ekin Dogan1,5,6, Begum Kocaturk7, Umut Inci Onat5,6, Moshe Arditi1,7,8, Christian Weber4,9,10, Alexis Traynor-Kaplan11,12, Carsten Schultz2,13 and Ebru Erbay *,1,8 1Smidt Heart Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA 2The Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany 3Department of Pathology, Laura & Isaac Perlmutter Cancer Center, New York University School of Medicine, New York, NY, USA 4Institute for Cardiovascular Prevention, LMU Munich, German Cardiovascular Research Centre, partner site Munich Heart Alliance Munich, Munich, Germany 5Department of Molecular Biology and Genetics, Bilkent University, Ankara, Turkey 6National Nanotechnology Center, Bilkent University, Ankara, Turkey 7Department of Pediatrics and Medicine, Division of Infectious Diseases and Immunology, and Infectious and Immunologic Diseases Research Center, Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA, USA 8Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA, USA 9Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, The Netherlands 10Munich Cluster for Systems Neurology (SyNergy), Munich, Germany 11Department of Medicine, University of Washington School of Medicine, Seattle, WA, USA 12ATK Innovation, Analytics and Discovery, North Bend, WA, USA 13Department of Chemical Physiology and Biochemistry, Oregon Health & Science University, Portland, OR, USA *Corresponding author. Tel: +1 310 4237483; E-mail: [email protected] EMBO Reports (2020)21:e51462https://doi.org/10.15252/embr.202051462 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The ER-bound kinase/endoribonuclease (RNase), inositol-requiring enzyme-1 (IRE1), regulates the phylogenetically most conserved arm of the unfolded protein response (UPR). However, the complex biology and pathology regulated by mammalian IRE1 cannot be fully explained by IRE1’s one known, specific RNA target, X box-binding protein-1 (XBP1) or the RNA substrates of IRE1-dependent RNA degradation (RIDD) activity. Investigating other specific substrates of IRE1 kinase and RNase activities may illuminate how it performs these diverse functions in mammalian cells. We report that macrophage IRE1 plays an unprecedented role in regulating phosphatidylinositide-derived signaling lipid metabolites and has profound impact on the downstream signaling mediated by the mammalian target of rapamycin (mTOR). This cross-talk between UPR and mTOR pathways occurs through the unconventional maturation of microRNA (miR) 2137 by IRE1’s RNase activity. Furthermore, phosphatidylinositol (3,4,5) phosphate (PI(3,4,5)P3) 5-phosphatase-2 (INPPL1) is a direct target of miR-2137, which controls PI(3,4,5)P3 levels in macrophages. The modulation of cellular PI(3,4,5)P3/PIP2 ratio and anabolic mTOR signaling by the IRE1-induced miR-2137 demonstrates how the ER can provide a critical input into cell growth decisions. Synopsis miR-2137 is a new RNA substrate of IRE1, the master regulator of the Unfolded Protein Response, and connects IRE1 to phosphatidylinositide-derived signaling lipid metabolism and growth signaling. Endoplasmic reticulum-anchored IRE1’s endoribonuclease activity leads to maturation of a micro RNA, miR-2137. IRE1 plays an unprecedented role in regulating the metabolism of phosphatidylinositide-derived signaling lipids in macrophages. miR-2137 is induced by high fat diet-activated IRE1 in lipid-laden, foamy macrophages that are found in atherosclerotic plaques and is blocked by an IRE1 endoribonuclease-specific inhibitor, suggesting miR-2137 could be a mediator of macrophage IRE1’s known pro-atherogenic action. Introduction Endoplasmic reticulum (ER) plays an essential role in protein folding, lipid synthesis, and cellular calcium homeostasis. The disruption of ER’s functions can induce an adaptive stress response, known as the unfolded protein response (UPR). The mammalian UPR is an elaborate cellular signaling initiated at the ER membranes that involves transcriptional and translational layers geared to reinstate cellular homeostasis (Ron & Walter, 2007; Schuck et al, 2009; Tabas & Ron, 2011; Walter & Ron, 2011). Inositol-requiring enzyme-1 (IRE1), one of the three proximal ER stress sensors sitting on the ER membranes, senses misfolded proteins through its ER luminal domain, leading to IRE1 oligomerization and activation of its kinase and endoribonuclease (RNase) activities that reside in its cytoplasmic domain. On the other hand, the incorporation of saturated fatty acids (SFA) or free cholesterol leads to another type of ER stress known as the lipid bilayer stress and sensed by IRE1 transmembrane domain, leading to IRE1 oligomerization and activation (Volmer et al, 2013; Volmer & Ron, 2015). Although ER plays a central role in lipid metabolism (cholesterol and phospholipid synthesis, fatty acid desaturation), ER membranes are poor in cholesterol and SFA content (Hotamisligil & Erbay, 2008; Walter & Ron, 2011). As such, the ER membrane is not only uniquely poised to sense the changes in cellular cholesterol and SFA levels, but also sufficiently equipped with lipid-sensing transcription factors and key lipid metabolism enzymes to maintain membrane lipid equilibrium (Lee et al, 2003). As we have previously shown, ER membrane fatty acid saturation index provides a critical input for IRE1 oligomerization on the ER membranes and IRE1 activation state in vivo (Cimen et al, 2016). Moreover, IRE1, through its RNase activity’s target, the X box-binding protein-1 (XBP1), regulates the expression of key phospholipid metabolism enzymes. A well-known role of yeast Ire1 involves monitoring and promoting phosphatidylinositol (PI) levels, but no such regulation has been reported in mammalian cells (Chang et al, 2002). A zebrafish mutant that is defective in de novo PI synthesis exhibits overt ER stress and develops hepatosteatosis, but how UPR is connected to PI metabolism has not been investigated beyond yeast (Thakur et al, 2011). De novo PI synthesis is known to be crucial for intracellular availability of phosphatidylinositol 4,5-biphosphate (PI(4,5)P2) and phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) and proper functioning of downstream growth-promoting signaling pathways regulated by these lipids (Rameh & Mackey, 2016; Fruman et al, 2017). PI is synthesized in the ER and transported to other cellular membranes and phosphorylated and dephosphorylated by kinases and phosphatases to yield phosphatidylinositol phosphates (PIPn). Depending on the phosphorylation state, different protein effectors are recruited to regulate the signaling events. For example, PI(3,4,5)P3 recruits AKT serine/threonine kinase 1 (AKT) to the plasma membrane, where AKT is phosphorylated (on Serine 473) by the mammalian target of rapamycin complex-2 (mTORC2; Shimobayashi & Hall, 2014; Manning & Toker, 2017). Upon activation, AKT induces mTORC1-regulated anabolic signaling through 70-kDa ribosomal S6 protein kinase-1 (p70S6K) and eukaryotic translation initiation factor 4E-binding protein (4E-BP1), leading to increased protein synthesis and growth (Mehrpour et al, 2010; Zoncu et al, 2011; Knaevelsrud & Simonsen, 2012). Numerous studies have shown mTOR signaling shares extensive cross-talk with the UPR, but how this contributes to mammalian cell growth is not clear (Wullschleger et al, 2006; Rutkowski & Hegde, 2010; Zoncu et al, 2011; Tabas & Ron, 2011; Appenzeller-Herzog & Hall, 2012). A common stimulus that controls both mTOR and UPR is the availability of nutrients and growth signals (Hotamisligil, 2010). For example, SFA that are prevalent in obese persons activate both UPR and mTOR (Pineau & Ferreira, 2010). Also, UPR-mediated lipogenesis has been reported to be mTORC1-dependent in the obese mouse liver (Pfaffenbach et al, 2010). The collective evidence indicates that the UPR and mTOR can act synergistically to regulate cell growth, but no such direct link has been reported yet. A tight control of PIPn signaling lipid levels is critical for a spatiotemporally regulated signaling mechanism (Balla et al, 2009; Kim et al, 2011). In this study, we analyzed the impact of IRE1 on the production of PIPn signaling lipids from the ER-synthesized PI in macrophages. The results of our lipidomic analysis did not support a role for the mammalian IRE1 in the regulation of cellular PI production, however, we made the striking observation that IRE1’s RNase activity significantly impacts cellular PI(3,4,5)P3 levels. Our data further show that IRE1 RNAase activity generates microRNA (miR)-2137, that leads to post-transcriptional inhibition of a lipid phosphatase INPPL1 (PI(3,4,5)P3 5-phosphatase-2 (INPPL1) and accumulation of PI(3,4,5)P3 in bone marrow-derived macrophages (BMDM). The inhibition of IRE1 RNase activity or miR-2137 reduces macrophage cell size and proliferation in accord with the suppression of AKT-mTOR signaling. These results demonstrate IRE1-generated miR-2137 modulates an ER to plasma membrane, PI(3,4,5)P3-dependent, anabolic signaling that contributes to mammalian cell growth decision. Results Ablating macrophage IRE1 RNase activity can significantly reduce PI(3,4,5)P3 and downstream growth signaling We previously demonstrated that targeting IRE1’s RNase activity with a highly specific, small molecule inhibitor, 4µ8c, inhibits only its RNase activity and effectively prevents macrophage inflammation and atherosclerosis progression in mice (Tufanli et al, 2017). Similar to other cells, in macrophages, UPR arms display basal signaling activity (assessed by sXBP1—a read out for IRE1 activity and glucose regulated protein-78 (GRP78)—a read out for activating transcription factor-6 (ATF6)-regulated UPR arm) without stress induction, and this basal IRE1 RNase signaling can be inhibited by 4µ8c (Fig EV1A and B). Traditionally, UPR has been studied under acute ER stress conditions induced by chemical ER toxins (such as thapsigargin and tunicamycin), but very little is known about the basal IRE1 signaling in cells and tissues and its consequences (Iwawaki et al, 2004; Hayashi et al, 2007; Brunsing et al, 2008; Iwawaki et al, 2009; Cretenet et al, 2010; Onat et al, 2019a). Due to its atherosclerosis promoting functions, there is a great interest in understanding IRE1 signaling in macrophages and as it relates to macrophage biology. In this study, we set out to investigate the role of basal IRE1 RNase activity in the metabolism of PI and PI-derived signaling lipids by using a quantitative lipidomics approach in non-stressed BMDMs (Traynor-Kaplan et al, 2017; Tufanli et al, 2017). We observed that the inhibition of IRE1 RNase activity with a specific inhibitor, 4µ8c, did not alter cellular PI levels, however, we noted a striking reduction in the cellular PI(3,4,5)P3 to PIP2 ratio (Figs 1A, and EV1C and D). The decrease in PI(3,4,5)P3 to PI(3,4)P2 was also confirmed by ELISA (Fig EV1E). Moreover, the ratio of cellular PI(3,4,5)P3 to PI and to PIP (Figs 1B and C, and EV1F and G) was also decreased upon IRE1 RNase inhibition. As expected, the spliced form of X box-binding protein 1 (sXBP1) mRNA expression, a direct RNA substrate and a measure of IRE1 RNase activity, was also significantly reduced by 4µ8c treatment in the analyzed cells (Fig EV1H). This finding reveals an unprecedented role for IRE1 in the regulation of PI-derived signaling lipids in macrophages. Click here to expand this figure. Figure EV1. Expanded view figure related to Fig 1 A, B. BMDMs were treated with thapsigargin (100 nm), 4µ8c (100 µM), or DMSO for 12 h as indicated in figure. RNA lysates were analyzed by qRT–PCR for (A) sXBP1 and (B) GRP78 mRNA expression (n = 3 biological replicates). C, D. BMDMs were treated with DMSO or 4µ8c (100 µM) for 24 h, and cells were subjected to lipidomics analysis as described in Materials and Methods section. Fatty acyl species composition data are shown for (C) PIP3 and (D) PIP2 (n = 3 biological replicates). E. Ratio of PIP3 to PI(3,4)P2 as measured by PIP3 and PI(3,4)P2 mass ELISA kit (Echelon) as described in Materials and Methods section (n = 3 biological replicates). F, G. From the same samples in (C, D), fatty acyl species composition data are shown for (F) PIP and (G) PI (n = 3 biological replicates). H. BMDMs were treated with 4µ8c (100 µM) or DMSO for 24 h. RNA lysates were analyzed by qRT–PCR for sXBP1 mRNA expression (n = 3 biological replicates). I. Wild type C57BL/6 mice were injected with tunicamycin (1 mg/kg) and 4µ8c (10 µg/kg) for 8 h prior to collection of thiogylcolate-elicited peritoneal macrophages, and RNA lysates were analyzed by qRT–PCR for sXbp1 expression (n = 3 mice per group). J, K. BMDMs were treated with 4µ8c (100 µM) or DMSO for 24 h, and cells were analyzed for (J) protein synthesis by protein synthesis assay and (K) cell size by flow cytometry (n = 3 biological replicates). Data information: All data are mean ± SEM; (n = 3); unpaired t-test with Welch’s correction. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. Download figure Download PowerPoint Figure 1. Inhibition of IRE1 RNase activity leads to significant reduction in PI(3,4,5)P3 and downstream growth signaling A–C. Bone marrow-derived mouse macrophages (BMDMs) from wild type C57BL/6 mice were treated with 4µ8c (100 µM) or vehicle (dimethyl sulfoxide; DMSO) for 24 h prior to sample processing for lipidomics analysis: (A) PI(3,4,5)P3 to PIP2; (B) PI(3,4,5)P3 to PI; (C) PI(3,4,5)P3 to PIP ratio are shown. Data represent mean values ± standard error of mean (SEM) of peak areas normalized to internal standards (n = 3 biological replicates). D. BMDMs were treated with 4µ8c (100 µM) or DMSO for 24 h and protein lysates were analyzed by Western blotting using specific antibodies against, pAKTS473, AKT, p70S6KT389, p70S6K, pS6S235/236, S6, p4E-BP1S65, 4E-BP1, and β-actin. Western blot quantifications are shown next to the figure (n = 3 biological replicates; a representative blot is shown). E. Wild type C57BL/6 mice were injected with tunicamycin (1 mg/kg) and 4µ8c (10 µg/kg) for 8 h prior to sacrifice and collection of thiogylcolate-elicited peritoneal macrophages. Protein lysates were analyzed by Western blotting using specific antibodies against, pIRE1S742, IRE1, pAKTS473, AKT, p70S6KT389, p70S6K, pS6S235/236, S6, p4E-BP1S65, 4E-BP1, and β-actin (n = 3 biological replicates; a representative blot is shown). Western blot quantifications are shown next to the figure. F. IRE1+/+ and IRE1−/− BMDMs were treated with PA (500 µM) for 6 h, and protein lysates were analyzed by Western blotting using specific antibodies against pIRE1S742, IRE1, p70S6KT389, p70S6K, pS6S235/236, S6, p4E-BP1S65, 4E-BP1 and β-actin. Western blot quantifications are shown next to the figure (n = 3 biological replicates, a representative blot is shown). Data information: All data are mean ± SEM; (n = 3). Unpaired t-test with Welch’s correction. *P ≤ 0.05, **P ≤ 0.01. Download figure Download PowerPoint We next tried to gain insight into the biological consequences of reduced PI(3,4,5)P3/PIP2 ratio in IRE1-inhibited cells. Decreased level of PI(3,4,5)P3 can inhibit the phosphorylation of AKT and its downstream target mTORC1, which further phosphorylates p70S6K and 4E-BP1 (Ben-Sahra & Manning, 2017). The phosphorylation of AKT, p70S6K, S6 and 4E-BP1 were all inhibited in the IRE1 RNase inhibitor-treated BMDMs (Fig 1D). We further assessed this cross-talk in vivo using peritoneal macrophages isolated from mice that were injected with an ER toxin (tunicamycin—an inhibitor of protein N-linked glycosylation),and treated with or without 4µ8c (Gomez & Rutkowski, 2016; Harnoss et al, 2019). This acute ER stress resulted in IRE1 activation (evident by an increase in IRE1 phosphorylation and sXBP1 mRNA levels) and increased phosphorylation of p70S6K, S6 and 4E-BP1, which was significantly blocked by 4µ8c (Fig 1E). sXBP1 was also partially reduced by 4µ8c (Fig EV1I). This result shows IRE1-dependent regulation of the mTOR signaling pathway by ER stress also occurs in vivo. To confirm IRE1-dependent regulation of mTOR signaling, we next analyzed this pathway in BMDMs that were obtained from mice with myeloid-specific IRE1 gene deletion (IRE1−/−; IRE1Flox/Flox, LyzM cre+/+) and compared with BMDMs obtained from age-matched, control littermates (IRE+/+; IRE1Flox/Flox) (Iwawaki et al, 2009). In these cells, ER bilayer membrane stress and IRE1 activity was induced by treating with a SFA, palmitate (PA) (Wei et al, 2006; Diakogiannaki et al, 2008; Ishiyama et al, 2011; Tufanli et al, 2017). We observed that non-stressed BMDMs from IRE1−/− mice displayed significantly lower p70S6K phosphorylation, in comparison to BMDMs from IRE1+/+ mice (Fig 1F). PA activated IRE1 (as evident by IRE1 autophosphorylation) and Akt-mTOR signaling, but this was significantly reduced in the IRE1−/− BMDMs when compared to IRE1+/+ BMDMs, confirming our findings obtained with the IRE1 RNase inhibitor (Fig 1F). In alignment with the important function of p70S6K and S6 in cell growth and protein synthesis, inhibition of IRE1 RNase domain by 4µ8c resulted in a significant decrease in protein synthesis and cell size (in BMDMs) (Fig EV1J and K). Collectively, these findings reveal a novel role for IRE1’s RNase activity in regulating PI-derived signaling lipids in macrophages. IRE1-dependent miRNA expression changes in palmitate-stressed macrophages The role of IRE1-XBP1 in regulating phospholipid synthesis is conserved from yeast to mammals, but this is not the case in regulating PI production (Walter & Ron, 2011), Another striking difference between yeast Ire1 and mammalian IRE1 is that the latter was shown to alter the expression of a select group of ER stress-induced microRNA (miRNAs) (Upton et al, 2012). Many other studies have shown ER stress can either induce or reduce the expression of miRNAs, but a direct contribution of IRE1 to miRNA generation has not been eliminated (Behrman et al, 2011; Maurel & Chevet, 2013; Acosta-Alvear et al, 2018). We hypothesized that IRE1-regulated miRNAs may play a role in mediating IRE1’s novel regulatory function in PI-derived signaling lipid metabolism. For this, we utilized primary BMDMs from IRE−/− and IRE1+/+ mice that were either stressed with PA (to induce IRE1 activity) or not stressed (to assess basal IRE1 activity) and their respective impact on miRNA expression. RNA isolated from these BMDMs was analyzed for changes in miRNAs’ differential expression using a microarray platform and the PA upregulated miRNAs in IRE1+/+ are shown along with their corresponding levels in IRE−/− cells (Fig 2A). The PA downregulated miRNAs in IRE1+/+ and their corresponding expression in IRE−/− cells are also shown (Appendix Fig S1). Based on this analysis, miR-2137 was the highest, induced miRNA by PA in IRE1+/+ BMDMs. A smaller, but not significant, induction of miR-2137 was observed in the IRE1−/− BMDMs. We further confirmed IRE1-dependent upregulation of miR-2137 by PA in these cells by quantitative reverse transcriptase polymerase chain reaction (qRT–PCR) method (Fig 2B). In conjunction with findings from a previously published study that showed two different chemical ER toxins induce miR-2137, our results demonstrate miR-2137 is responsive to multiple modes of ER stress activation and is regulated by IRE1 (Behrman et al, 2011). In our experiment, PA-induced IRE1 kinase activity (as assessed by IRE1 phosphorylation) and only one of the IRE1 RNase outputs, XBP1 mRNA splicing, but not the other, RIDD activity (as evident from the unchanged mRNA levels of the Biogenesis of Lysosome-Related Organelles Complex-1 Subunit-1 (BLOC1S1) and Scavenger Receptor Class-A Receptor-3 (SCARA3)) (Fig 2C–F). Collectively, these results show that IRE1 RNase activation leads to a significant increase in miR2137 expression in BMDMs. Figure 2. IRE1-dependent changes in miRNA expression in ER-stressed macrophages IRE1−/− and IRE1+/+ BMDMs were treated with PA (500 µM) or vehicle for 6 h prior to RNA isolation for miRNA analysis using a microarray platform. A. Table showing fold change (log2) of statistically significant (P-value < 0.050) top PA-upregulated miRNAs in IRE1+/+ and IRE1−/− cells. NS indicates microRNAs that did not show statistically significant difference. Fold change was calculated by comparing mean intensity values of microarray signal for vehicle treatment with PA treatment (n = 3 biological replicates). B. RNA lysates from the same experiment were analyzed by qRT–PCR for miR-2137 expression. C. Protein lysates were analyzed by Western blotting using specific antibodies for pIRE1S742, IRE1, and β-actin (n = 3 biological replicates). D. RNA lysates were analyzed by qRT–PCR for sXBP1 expression (n = 3 biological replicates). E, F. RNA lysates from the same experiment were analyzed by qRT–PCR for (E) BLOC1S1 and (F) SCARA3 expression (n = 3 biological replicates). Data information: All data are mean ± SEM (n = 3); unpaired t-test with Welch’s correction; *P ≤ 0.05). Source data are available online for this figure. Source Data for Figure 2 [embr202051462-sup-0003-SDataFig2.xls] Download figure Download PowerPoint miR-2137 is also regulated by hyperlipidemia-induced IRE1 in vivo Saturated and monounsaturated fatty acids (MUFA) reprogram gene expression in different ways to alter macrophage functions (Duplus et al, 2000). Whereas SFA can induce ER stress response, MUFA do not (Wei et al, 2006; Diakogiannaki et al, 2008; Ben-Dror & Birk, 2019). While miR-2137 is induced by SFA like PA and stearic acid (SA) in BMDMs, it cannot be induced with MUFA such as palmitoleic acid (PAO) and oleic acid (OA) (Fig 3A). sXBP1 mRNA, a measure of IRE1 RNase activity, paralleled the changes in miR-2137 levels in these treatments (Fig 3B). These results suggest that miR-2137 expression (similar to IRE1 oligomerization and RNase activity) is uniquely responsive to t" @default.
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• W3095319430 title "Inositol‐requiring enzyme‐1 regulates phosphoinositide signaling lipids and macrophage growth" @default.
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