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Lipopolysaccharide Induces GFAT2 Expression to Promote O-Linked Β Lipopolysaccharide Induces GFAT2 Expression to Promote O-Linked β-N -Acetylglucosaminylation and Attenuate Inflammation in Macrophages This information is current as of September 28, 2021. Hasanain Al-Mukh, Léa Baudoin, Abdelouhab Bouaboud, José-Luis Sanchez-Salgado, Nabih Maraqa, Mostafa Khair, Patrick Pagesy, Georges Bismuth, Florence Niedergang and Tarik Issad J Immunol published online 25 September 2020 Downloaded from http://www.jimmunol.org/content/early/2020/09/22/jimmun ol.2000345 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2020/09/22/jimmunol.200034 Material 5.DCSupplemental Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on September 28, 2021 • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2020 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published September 25, 2020, doi:10.4049/jimmunol.2000345 The Journal of Immunology Lipopolysaccharide Induces GFAT2 Expression to Promote O-Linked b-N-Acetylglucosaminylation and Attenuate Inflammation in Macrophages Hasanain Al-Mukh, Le´a Baudoin, Abdelouhab Bouaboud, Jose´-Luis Sanchez-Salgado, Nabih Maraqa, Mostafa Khair, Patrick Pagesy, Georges Bismuth, Florence Niedergang, and Tarik Issad Glycosylation with O-linked b-N-acetylglucosamine (O-GlcNAcylation) is a reversible posttranslational modification that regulates the activity of intracellular proteins according to glucose availability and its metabolism through the hexosamine biosynthesis pathway. This modification has been involved in the regulation of various immune cell types, including macrophages. However, little is known concerning the mechanisms that regulate the protein O-GlcNAcylation level in these cells. In the present work, we Downloaded from demonstrate that LPS treatment induces a marked increase in protein O-GlcNAcylation in RAW264.7 cells, bone marrow–derived and peritoneal mouse macrophages, as well as human monocyte-derived macrophages. Targeted deletion of OGT in macrophages resulted in an increased effect of LPS on NOS2 expression and cytokine production, suggesting that O-GlcNAcylation may restrain inflammatory processes induced by LPS. The effect of LPS on protein O-GlcNAcylation in macrophages was associated with an increased expression and activity of glutamine fructose 6-phosphate amidotransferase (GFAT), the enzyme that catalyzes the rate- limiting step of the hexosamine biosynthesis pathway. More specifically, we observed that LPS potently stimulated GFAT2 isoform http://www.jimmunol.org/ mRNA and protein expression. Genetic or pharmacological inhibition of FoxO1 impaired the LPS effect on GFAT2 expression, suggesting a FoxO1-dependent mechanism. We conclude that GFAT2 should be considered a new LPS-inducible gene involved in regulation of protein O-GlcNAcylation, which permits limited exacerbation of inflammation upon macrophage activation. The Journal of Immunology, 2020, 205: 000–000. lycosylation with O-linked b-N-acetylglucosamine perturbations of O-GlcNAcylation participate in several important (O-GlcNAcylation) is a posttranslational modification human pathologies (2–4). Moreover, a number of studies indicated G that regulates the activity of cytosolic, nuclear, and mi- a link between O-GlcNAcylation and inflammatory conditions, in- by guest on September 28, 2021 tochondrial proteins. This modification is controlled by only two cluding diabetes, autoimmune diseases, and cancer (5–11). Moreover, enzymes: OGT, which adds N-acetylglucosamine (GlcNAc) on serine several lines of evidence have suggested a role for O-GlcNAcylation or threonine residues, and OGA, which removes it. O-GlcNAcylation, in immune cell signaling. Indeed, O-GlcNAcylation was first which regulates a wide array of biological processes (1), depends on discovered at the surface of lymphocytes (12), and dynamic the availability of glucose and its metabolism through the hexosamine changes upon lymphocyte activation were described at the be- biosynthesis pathway (HBP) (Fig. 1A). This modification has been ginning of the 1990s (13). More recently, several studies indicated involved in the modulation of various signaling pathways, and that protein O-GlcNAcylation plays major roles in the immune system. In T cells, TCR activation results in global elevation of Universite´ de Paris, Institut Cochin, CNRS, INSERM, F-75014 Paris, France protein O-GlcNAcylation (14, 15), increased O-GlcNAcylation of several transcription factors (16–19), and accumulation of OGT at ORCIDs: 0000-0002-2530-7995 (H.A.-M.); 0000-0002-4524-3933 (J.-L.S.-S.); 0000-0003-2248-8054 (N.M.); 0000-0002-7064-830X (F.N.); 0000-0002-2638- the immunological synapse (14). Moreover, in B lymphocytes, 6330 (T.I.). activation of the BCR induces O-GlcNAcylation of several signaling Received for publication March 31, 2020. Accepted for publication August 29, 2020. molecules involved in BCR signaling (20, 21). Overall, these studies This work was supported by the Socie´te´ Francophone du Diabe´te (SFD-2013), the strongly support a major role in O-GlcNAcylation in adaptive im- Fondation pour la Recherche Me´dicale (FRM-DEQ20130326518 and FRM- munity. However, the molecular mechanisms by which immune cell DEQ20150331744) and by the Programme Inter-Cochin (Institut Cochin, CNRS, Institut National de la Sante´ et de la Recherche Me´dicale, Universite´ de Paris). L.B. and activation modulates protein O-GlcNAcylation remain elusive. H.A.-M. held Ph.D. grants from the CORDDIM-Re´gion Ile-de-France. H.A.-M. was In macrophages, contradictory results have been obtained con- also funded by the Fondation pour la Recherche Me´dicale. J.-L.S.S. was a recipient of a postdoctoral grant from the Consejo Nacional de Ciencia y Tecnologia, Mexico cerning the effect of LPS on protein O-GlcNAcylation and its role (CONACYT-CVU 376926). in inflammatory processes. Whereas some studies indicated that Address correspondence and reprint requests to Tarik Issad, Department of Endocri- LPS induced a general increase in protein O-GlcNAcylation (22, nology, Metabolism and Diabetes, Institut Cochin, 24 Rue du Faubourg Saint- 23), others found that LPS decreases protein O-GlcNAcylation in Jacques, 75014 Paris, France. E-mail address: [email protected] macrophages (24, 25). Moreover, whereas several lines of evi- The online version of this article contains supplemental material. dence indicate that increased O-GlcNAcylation of signaling proteins Abbreviations used in this article: BMDM, bone marrow–derived macrophage; BRET, bioluminescence resonance energy transfer; ChIP, chromatin immunoprecip- in the NF-kB pathway potentiates inflammatory processes (26–29), itation; HBP, hexosamine biosynthesis pathway; GFAT, glutamine fructose-6-phosphate other studies indicate that increased O-GlcNAcylation may inhibit amidotransferase; GlcNAc, N-acetylglucosamine; KO, knockout; O-GlcNAcylation, proinflammatory signals (24, 30). More recently, O-GlcNAcyla- glycosylation with O-linked b-N-acetylglucosamine. tion of the signaling adaptor MAVS in macrophages has been Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 involved in antiviral signaling response (31), whereas increased www.jimmunol.org/cgi/doi/10.4049/jimmunol.2000345 2 GFAT2 IS A FoxO1-DEPENDENT LPS-INDUCIBLE GENE RIPK3 O-GlcNAcylation was shown to regulate necroptosis sig- previously coated with polylysine (Perkin Elmer), then cultured for 18 h at naling upon LPS stimulation (25). Thus, whereas much evidence 37˚C and 5% CO2 before treatments. suggests that modulation of O-GlcNAcylation signaling consti- HEK293T cells were cultured in DMEM and transfected with FuGENE as described previously (34). tutes an important facet of innate immune response, the mecha- nisms by which macrophage activation regulates O-GlcNAc and Chromatin immunoprecipitation its consequences on inflammatory processes remain to be firmly RAW264.7 cells (1 3 107 cells) were prepared for chromatin immuno- established. precipitation (ChIP) assay using HighCell# ChIP kit protein A (Dia- In the present work, using different macrophage cell models, genode). After cross-linking with formaldehyde, DNA was sonicated into we clearly demonstrated that LPS markedly stimulates protein 200–300-bp fragments, and protein–DNA complexes were immunopreci- pitated using either FoxO1-ChIP grade Ab (ab39670; Abcam) or control O-GlcNAcylation in macrophages through increased expression rabbit IgG. Protein–DNA cross-links were reversed by heating, and and activity of the rate-limiting enzyme of the HBP, glutamine precipitated DNA was quantified by real-time PCR using primers fructose-6-phosphate amidotransferase (GFAT). More specifically, (Supplemental Table I) that amplify either Foxo1 binding site 1 or 2 we showed that, whereas resting macrophages express mainly the present in the 500 bp upstream of the transcription start site of the GFAT1
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