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• W2604143793 abstract "•Celastrol binds Nur77 to inhibit inflammation by autophagy•Celastrol induces Nur77 translocation to mitochondria and interaction with TRAF2•An LxxLL motif in TRAF2 mediates its interaction with Nur77•Celastrol promotes Nur77 ubiquitination, p62/SQSTM1 interaction, and mitophagy Mitochondria play an integral role in cell death, autophagy, immunity, and inflammation. We previously showed that Nur77, an orphan nuclear receptor, induces apoptosis by targeting mitochondria. Here, we report that celastrol, a potent anti-inflammatory pentacyclic triterpene, binds Nur77 to inhibit inflammation and induce autophagy in a Nur77-dependent manner. Celastrol promotes Nur77 translocation from the nucleus to mitochondria, where it interacts with tumor necrosis factor receptor-associated factor 2 (TRAF2), a scaffold protein and E3 ubiquitin ligase important for inflammatory signaling. The interaction is mediated by an LxxLL motif in TRAF2 and results not only in the inhibition of TRAF2 ubiquitination but also in Lys63-linked Nur77 ubiquitination. Under inflammatory conditions, ubiquitinated Nur77 resides at mitochondria, rendering them sensitive to autophagy, an event involving Nur77 interaction with p62/SQSTM1. Together, our results identify Nur77 as a critical intracellular target for celastrol and unravel a mechanism of Nur77-dependent clearance of inflamed mitochondria to alleviate inflammation. Mitochondria play an integral role in cell death, autophagy, immunity, and inflammation. We previously showed that Nur77, an orphan nuclear receptor, induces apoptosis by targeting mitochondria. Here, we report that celastrol, a potent anti-inflammatory pentacyclic triterpene, binds Nur77 to inhibit inflammation and induce autophagy in a Nur77-dependent manner. Celastrol promotes Nur77 translocation from the nucleus to mitochondria, where it interacts with tumor necrosis factor receptor-associated factor 2 (TRAF2), a scaffold protein and E3 ubiquitin ligase important for inflammatory signaling. The interaction is mediated by an LxxLL motif in TRAF2 and results not only in the inhibition of TRAF2 ubiquitination but also in Lys63-linked Nur77 ubiquitination. Under inflammatory conditions, ubiquitinated Nur77 resides at mitochondria, rendering them sensitive to autophagy, an event involving Nur77 interaction with p62/SQSTM1. Together, our results identify Nur77 as a critical intracellular target for celastrol and unravel a mechanism of Nur77-dependent clearance of inflamed mitochondria to alleviate inflammation. Nur77 (also called TR3, NGFIB, and NR4A1), an orphan member of the nuclear receptor superfamily and an immediate early response gene, plays a critical role in a plethora of cellular processes in response to diverse stimuli such as mitogens, cytokines, and stress, metabolic, and apoptotic signals (Beard et al., 2015Beard J.A. Tenga A. Chen T. The interplay of NR4A receptors and the oncogene-tumor suppressor networks in cancer.Cell. Signal. 2015; 27: 257-266Crossref PubMed Scopus (58) Google Scholar, Evans, 2009Evans P.C. Nur77: orphaned at birth but adopted by the nuclear factor kappaB signaling pathway.Circ. Res. 2009; 104: 707-709Crossref PubMed Scopus (13) Google Scholar, Hamers et al., 2013Hamers A.A. Hanna R.N. Nowyhed H. Hedrick C.C. de Vries C.J. NR4A nuclear receptors in immunity and atherosclerosis.Curr. Opin. Lipidol. 2013; 24: 381-385Crossref PubMed Scopus (55) Google Scholar, Lee et al., 2011Lee S.O. Li X. Khan S. Safe S. Targeting NR4A1 (TR3) in cancer cells and tumors.Expert Opin. Ther. Targets. 2011; 15: 195-206Crossref PubMed Scopus (75) Google Scholar, McMorrow and Murphy, 2011McMorrow J.P. Murphy E.P. Inflammation: a role for NR4A orphan nuclear receptors?.Biochem. Soc. Trans. 2011; 39: 688-693Crossref PubMed Scopus (127) Google Scholar, Moll et al., 2006Moll U.M. Marchenko N. Zhang X.K. p53 and Nur77/TR3 - transcription factors that directly target mitochondria for cell death induction.Oncogene. 2006; 25: 4725-4743Crossref PubMed Scopus (210) Google Scholar, Zhang, 2007Zhang X.K. Targeting Nur77 translocation.Expert Opin. Ther. Targets. 2007; 11: 69-79Crossref PubMed Scopus (96) Google Scholar). Recent interest has focused on its potent anti-inflammatory effect in inflammatory diseases and cancer. Genetic studies have revealed a critical role of Nur77 in controlling the inflammatory responses, highlighted by its protective function in atherosclerosis (Hamers et al., 2012Hamers A.A. Vos M. Rassam F. Marinković G. Kurakula K. van Gorp P.J. de Winther M.P. Gijbels M.J. de Waard V. de Vries C.J. Bone marrow-specific deficiency of nuclear receptor Nur77 enhances atherosclerosis.Circ. Res. 2012; 110: 428-438Crossref PubMed Scopus (138) Google Scholar, Hanna et al., 2012Hanna R.N. Shaked I. Hubbeling H.G. Punt J.A. Wu R. Herrley E. Zaugg C. Pei H. Geissmann F. Ley K. Hedrick C.C. NR4A1 (Nur77) deletion polarizes macrophages toward an inflammatory phenotype and increases atherosclerosis.Circ. Res. 2012; 110: 416-427Crossref PubMed Scopus (290) Google Scholar), obesity (Perez-Sieira et al., 2013Perez-Sieira S. Martinez G. Porteiro B. Lopez M. Vidal A. Nogueiras R. Dieguez C. Female Nur77-deficient mice show increased susceptibility to diet-induced obesity.PLoS ONE. 2013; 8: e53836Crossref PubMed Scopus (30) Google Scholar), diabetes (Chao et al., 2009Chao L.C. Wroblewski K. Zhang Z. Pei L. Vergnes L. Ilkayeva O.R. Ding S.Y. Reue K. Watt M.J. Newgard C.B. et al.Insulin resistance and altered systemic glucose metabolism in mice lacking Nur77.Diabetes. 2009; 58: 2788-2796Crossref PubMed Scopus (110) Google Scholar), asthma (Kurakula et al., 2015Kurakula K. Vos M. Logiantara A. Roelofs J.J. Nieuwenhuis M.A. Koppelman G.H. Postma D.S. van Rijt L.S. de Vries C.J. Nuclear receptor Nur77 attenuates airway inflammation in mice by suppressing NF-κB activity in lung epithelial cells.J. Immunol. 2015; 195: 1388-1398Crossref PubMed Scopus (51) Google Scholar), arthritis (De Silva et al., 2005De Silva S. Han S. Zhang X. Huston D.P. Winoto A. Zheng B. Reduction of the incidence and severity of collagen-induced arthritis by constitutive Nur77 expression in the T cell lineage.Arthritis Rheum. 2005; 52: 333-338Crossref PubMed Scopus (28) Google Scholar), and inflammatory bowel disease (Hamers et al., 2015Hamers A.A. van Dam L. Teixeira Duarte J.M. Vos M. Marinković G. van Tiel C.M. Meijer S.L. van Stalborch A.M. Huveneers S. Te Velde A.A. et al.Deficiency of nuclear receptor Nur77 aggravates mouse experimental colitis by increased NFκB activity in macrophages.PLoS ONE. 2015; 10: e0133598Crossref Scopus (43) Google Scholar, Wu et al., 2016Wu H. Li X.M. Wang J.R. Gan W.J. Jiang F.Q. Liu Y. Zhang X.D. He X.S. Zhao Y.Y. Lu X.X. et al.NUR77 exerts a protective effect against inflammatory bowel disease by negatively regulating the TRAF6/TLR-IL-1R signalling axis.J. Pathol. 2016; 238: 457-469Crossref PubMed Scopus (52) Google Scholar). The NF-κB signaling pathway plays a pivotal role in inflammation (Chen, 2012Chen Z.J. Ubiquitination in signaling to and activation of IKK.Immunol. Rev. 2012; 246: 95-106Crossref PubMed Scopus (306) Google Scholar, Hayden and Ghosh, 2008Hayden M.S. Ghosh S. Shared principles in NF-kappaB signaling.Cell. 2008; 132: 344-362Abstract Full Text Full Text PDF PubMed Scopus (3526) Google Scholar, Karin and Gallagher, 2009Karin M. Gallagher E. TNFR signaling: ubiquitin-conjugated TRAFfic signals control stop-and-go for MAPK signaling complexes.Immunol. Rev. 2009; 228: 225-240Crossref PubMed Scopus (150) Google Scholar). Stimulation of the tumor necrosis factor receptor (TNFR) with the proinflammatory cytokine results in NF-κB activation, which is mediated by members of the TNFR-associated factor (TRAF) family characterized by a highly conserved TRAF domain and a RING domain. TRAF2, the most widely studied TRAF member, serves as a scaffold protein and E3 ubiquitin ligase by binding to numerous TNFR-family proteins to mediate the activation of the inhibitor of κB (IκB) kinase (IKK) and NF-κB (Borghi et al., 2016Borghi A. Verstrepen L. Beyaert R. TRAF2 multitasking in TNF receptor-induced signaling to NF-κB, MAP kinases and cell death.Biochem. Pharmacol. 2016; 116: 1-10Crossref PubMed Scopus (116) Google Scholar, Bradley and Pober, 2001Bradley J.R. Pober J.S. Tumor necrosis factor receptor-associated factors (TRAFs).Oncogene. 2001; 20: 6482-6491Crossref PubMed Scopus (516) Google Scholar). Previous studies have revealed extensive interaction between Nur77 and NF-κB signaling pathways. Ectopic expression of Nur77 in macrophages increases the expression of the inducible inhibitor of NF-κB kinase (Pei et al., 2006Pei L. Castrillo A. Tontonoz P. Regulation of macrophage inflammatory gene expression by the orphan nuclear receptor Nur77.Mol. Endocrinol. 2006; 20: 786-794Crossref PubMed Scopus (166) Google Scholar), while Nur77 could inhibit inflammatory gene expression (Harant and Lindley, 2004Harant H. Lindley I.J. Negative cross-talk between the human orphan nuclear receptor Nur77/NAK-1/TR3 and nuclear factor-kappaB.Nucleic Acids Res. 2004; 32: 5280-5290Crossref PubMed Scopus (52) Google Scholar). However, the precise mechanism underlying the potent anti-inflammatory effect of Nur77 remains obscure. Nur77 can function in the nucleus as a transcriptional factor to modulate target gene transcription (Beard et al., 2015Beard J.A. Tenga A. Chen T. The interplay of NR4A receptors and the oncogene-tumor suppressor networks in cancer.Cell. Signal. 2015; 27: 257-266Crossref PubMed Scopus (58) Google Scholar, Evans, 2009Evans P.C. Nur77: orphaned at birth but adopted by the nuclear factor kappaB signaling pathway.Circ. Res. 2009; 104: 707-709Crossref PubMed Scopus (13) Google Scholar, Hamers et al., 2013Hamers A.A. Hanna R.N. Nowyhed H. Hedrick C.C. de Vries C.J. NR4A nuclear receptors in immunity and atherosclerosis.Curr. Opin. Lipidol. 2013; 24: 381-385Crossref PubMed Scopus (55) Google Scholar, Lee et al., 2011Lee S.O. Li X. Khan S. Safe S. Targeting NR4A1 (TR3) in cancer cells and tumors.Expert Opin. Ther. Targets. 2011; 15: 195-206Crossref PubMed Scopus (75) Google Scholar, McMorrow and Murphy, 2011McMorrow J.P. Murphy E.P. Inflammation: a role for NR4A orphan nuclear receptors?.Biochem. Soc. Trans. 2011; 39: 688-693Crossref PubMed Scopus (127) Google Scholar, Moll et al., 2006Moll U.M. Marchenko N. Zhang X.K. p53 and Nur77/TR3 - transcription factors that directly target mitochondria for cell death induction.Oncogene. 2006; 25: 4725-4743Crossref PubMed Scopus (210) Google Scholar, Zhang, 2007Zhang X.K. Targeting Nur77 translocation.Expert Opin. Ther. Targets. 2007; 11: 69-79Crossref PubMed Scopus (96) Google Scholar). Regulation of gene transcription by nuclear receptors is mediated through their interaction with coactivators or corepressors via the LxxLL motif present in coactivators or the L/IxxI/VI motif present in corepressors, respectively (Dasgupta et al., 2014Dasgupta S. Lonard D.M. O’Malley B.W. Nuclear receptor coactivators: master regulators of human health and disease.Annu. Rev. Med. 2014; 65: 279-292Crossref PubMed Scopus (141) Google Scholar, Hermanson et al., 2002Hermanson O. Glass C.K. 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Dev. 1999; 9: 140-147Crossref PubMed Scopus (811) Google Scholar). Recent studies have revealed a critical role of mitochondria in regulating inflammatory processes. The organelle is susceptible to damage from inflammatory signals; it in turn releases danger signals, including reactive oxygen species (ROS), for the assembly and activation of inflammasome (Green et al., 2011Green D.R. Galluzzi L. Kroemer G. Mitochondria and the autophagy-inflammation-cell death axis in organismal aging.Science. 2011; 333: 1109-1112Crossref PubMed Scopus (840) Google Scholar, Gurung et al., 2015Gurung P. Lukens J.R. Kanneganti T.D. Mitochondria: diversity in the regulation of the NLRP3 inflammasome.Trends Mol. Med. 2015; 21: 193-201Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar, Levine et al., 2011Levine B. Mizushima N. Virgin H.W. Autophagy in immunity and inflammation.Nature. 2011; 469: 323-335Crossref PubMed Scopus (2415) Google Scholar, Nunnari and Suomalainen, 2012Nunnari J. Suomalainen A. Mitochondria: in sickness and in health.Cell. 2012; 148: 1145-1159Abstract Full Text Full Text PDF PubMed Scopus (1847) Google Scholar, Pinton and Kroemer, 2014Pinton P. Kroemer G. Cancer therapy: altering mitochondrial properties.Nat. Chem. Biol. 2014; 10: 89-90Crossref PubMed Scopus (15) Google Scholar). We previously showed that Nur77 could translocate from the nucleus to mitochondria to induce cytochrome c release and apoptosis (Kolluri et al., 2008Kolluri S.K. Zhu X. Zhou X. Lin B. Chen Y. Sun K. Tian X. Town J. Cao X. Lin F. et al.A short Nur77-derived peptide converts Bcl-2 from a protector to a killer.Cancer Cell. 2008; 14: 285-298Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, Li et al., 2000Li H. Kolluri S.K. Gu J. Dawson M.I. Cao X. Hobbs P.D. Lin B. Chen G. Lu J. Lin F. et al.Cytochrome c release and apoptosis induced by mitochondrial targeting of nuclear orphan receptor TR3.Science. 2000; 289: 1159-1164Crossref PubMed Scopus (588) Google Scholar, Lin et al., 2004Lin B. Kolluri S.K. Lin F. Liu W. Han Y.H. Cao X. Dawson M.I. Reed J.C. Zhang X.K. Conversion of Bcl-2 from protector to killer by interaction with nuclear orphan receptor Nur77/TR3.Cell. 2004; 116: 527-540Abstract Full Text Full Text PDF PubMed Scopus (561) Google Scholar, Zhang, 2007Zhang X.K. Targeting Nur77 translocation.Expert Opin. Ther. Targets. 2007; 11: 69-79Crossref PubMed Scopus (96) Google Scholar). Whether Nur77 participates in modulating mitochondrial quality control and mitochondria-mediated inflammatory signaling remains unknown. Identifying agents that bind Nur77 to induce its mitochondrial targeting will facilitate the investigation of the complex role of Nur77 at mitochondria. Here we report our discovery of celastrol, a triterpenoid quinine methide isolated from the root of Tripterygium wilfordii, which is commonly known as “Thunder God Vine” (Corson and Crews, 2007Corson T.W. Crews C.M. Molecular understanding and modern application of traditional medicines: triumphs and trials.Cell. 2007; 130: 769-774Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar, Kannaiyan et al., 2011Kannaiyan R. Shanmugam M.K. Sethi G. Molecular targets of celastrol derived from Thunder of God Vine: potential role in the treatment of inflammatory disorders and cancer.Cancer Lett. 2011; 303: 9-20Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, Liu et al., 2015Liu J. Lee J. Salazar Hernandez M.A. Mazitschek R. Ozcan U. Treatment of obesity with celastrol.Cell. 2015; 161: 999-1011Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar, Salminen et al., 2010Salminen A. Lehtonen M. Paimela T. Kaarniranta K. Celastrol: molecular targets of Thunder God Vine.Biochem. Biophys. Res. Commun. 2010; 394: 439-442Crossref PubMed Scopus (248) Google Scholar, Wong et al., 2012Wong K.F. Yuan Y. Luk J.M. Tripterygium wilfordii bioactive compounds as anticancer and anti-inflammatory agents.Clin. Exp. Pharmacol. Physiol. 2012; 39: 311-320Crossref PubMed Scopus (115) Google Scholar), as a Nur77 modulator. Our results demonstrate that celastrol confers its anti-inflammatory effect by inducing Nur77 mitochondrial translocation and, subsequently, Nur77-dependent elimination of damaged mitochondria through autophagy. We used surface plasmon resonance (SPR)-based assay to screen natural compounds from marine and terrestrial sources with known anti-inflammatory activity for binding to Nur77 (Figure S1A) and identified celastrol (Figure 1A ) as a potent binder with a Kd of 0.29 μM (Figure 1B). The binding was confirmed by circular dichroism (CD) spectroscopy, which showed an altered CD spectrum of the Nur77-LBD by celastrol (Figure S1B). High-performance liquid chromatography (HPLC) analysis also revealed a direct binding of celastrol to purified Nur77-LBD, but not to the LBD of retinoid X receptor (RXRα-LBD) (Figure S1C). In the reporter assay, celastrol inhibited transactivation of Nur77, but not glucocorticoid receptor (GR) (Figure S1D). Molecular docking studies suggested that celastrol binds to a previously identified, hydrophobic groove on the surface of Nur77 protein (Lee et al., 2014Lee S.O. Li X. Hedrick E. Jin U.H. Tjalkens R.B. Backos D.S. Li L. Zhang Y. Wu Q. Safe S. Diindolylmethane analogs bind NR4A1 and are NR4A1 antagonists in colon cancer cells.Mol. Endocrinol. 2014; 28: 1729-1739Crossref PubMed Scopus (69) Google Scholar, Zhan et al., 2012Zhan Y.Y. Chen Y. Zhang Q. Zhuang J.J. Tian M. Chen H.Z. Zhang L.R. Zhang H.K. He J.P. Wang W.J. et al.The orphan nuclear receptor Nur77 regulates LKB1 localization and activates AMPK.Nat. Chem. Biol. 2012; 8: 897-904Crossref PubMed Scopus (114) Google Scholar; Figure S1E). Celastrol antagonized the effects of inflammatory cytokine TNFα, including its induction of IκBα degradation, nuclear translocation of the p65 subunit of NF-κB, and NF-κB transactivation. Inhibition of TNFα-induced IκBα degradation by celastrol correlated with its suppression of IKKα/β phosphorylation (Figure 1C), demonstrating that celastrol acts at or upstream of IKK activation. The role of Nur77 was illustrated by data showing that transfection of Nur77 siRNA into HepG2 cells, which inhibited Nur77 expression, abrogated the inhibitory effect of celastrol on TNFα-induced IκBα degradation (Figure S1F), while transfection of RXRα siRNA had no effect (Figure S1G). In addition, celastrol inhibited TNFα-induced IκBα degradation (Figure 1D) in mouse embryonic fibroblasts (MEFs), but not in MEFs lacking Nur77 (Nur77−/− MEFs). To determine the role of Nur77 in vivo, we examined the effect of celastrol on lipopolysaccharide (LPS) and D-galactosamine (D-GalN)-induced hepatic inflammatory injury using Nur77-null mice. Administration of celastrol to wild-type mice reduced LPS- and D-GalN-induced serum levels of hepatic injury markers alanine aminotransferase (ALT) and aspartate aminotransferase (AST) by 35% and 47%, respectively (Figure S1H). Although Nur77−/− mice had higher serum ALT and AST than wild-type mice when injected with LPS and D-GalN, celastrol showed a much reduced inhibitory effect in these mice. LPS- and D-GalN-induced serum production of the proinflammatory cytokines IL-1β and IL-6 (Figure S1I) and their hepatic mRNA expression (Figure S1J) were significantly inhibited by celastrol in wild-type mice, while such effects were largely attenuated in Nur77−/− mice. Celastrol also alleviated LPS- and D-GalN-induced downregulation of IκBα expression (Figure 1E), destruction of hepatic architecture (Figure 1F), and p65 nuclear translocation (Figure S1K) in wild-type but not Nur77−/− mice. Histological analysis of lung tissue revealed a Nur77-dependent inhibition of LPS- and D-GalN-induced lung inflammation and neutrophil infiltration by celastrol (not shown). Thus, celastrol inhibition of LPS- and D-GalN-induced acute inflammation and NF-κB activation is Nur77 dependent. We next determined the effect of celastrol and Nur77 using the high-fat diet (HFD)-induced obesity animal model, which represents a state of chronic low-grade inflammation (Hotamisligil, 2006Hotamisligil G.S. Inflammation and metabolic disorders.Nature. 2006; 444: 860-867Crossref PubMed Scopus (6244) Google Scholar, Nathan and Ding, 2010Nathan C. Ding A. Nonresolving inflammation.Cell. 2010; 140: 871-882Abstract Full Text Full Text PDF PubMed Scopus (1418) Google Scholar). In agreement with recent results (Liu et al., 2015Liu J. Lee J. Salazar Hernandez M.A. Mazitschek R. Ozcan U. Treatment of obesity with celastrol.Cell. 2015; 161: 999-1011Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar, Ma et al., 2015Ma X. Xu L. Alberobello A.T. Gavrilova O. Bagattin A. Skarulis M. Liu J. Finkel T. Mueller E. Celastrol protects against obesity and metabolic dysfunction through activation of a HSF1-PGC1α transcriptional axis.Cell Metab. 2015; 22: 695-708Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar), administration of celastrol significantly reduced body weight, adipose tissue mass, the size of adipocytes, and ameliorated fatty liver caused by HFD in wild-type mice (Figures 1G and S1L). Nur77−/− mice exhibited increased susceptibility to HFD-induced obesity, gaining more body weight and fat mass than wild-type mice, consistent with the metabolic role of Nur77 (Chao et al., 2007Chao L.C. Zhang Z. Pei L. Saito T. Tontonoz P. Pilch P.F. Nur77 coordinately regulates expression of genes linked to glucose metabolism in skeletal muscle.Mol. Endocrinol. 2007; 21: 2152-2163Crossref PubMed Scopus (137) Google Scholar, Chao et al., 2009Chao L.C. Wroblewski K. Zhang Z. Pei L. Vergnes L. Ilkayeva O.R. Ding S.Y. Reue K. Watt M.J. Newgard C.B. et al.Insulin resistance and altered systemic glucose metabolism in mice lacking Nur77.Diabetes. 2009; 58: 2788-2796Crossref PubMed Scopus (110) Google Scholar, Chen et al., 2015Chen Y. Wu R. Chen H.Z. Xiao Q. Wang W.J. He J.P. Li X.X. Yu X.W. Li L. Wang P. et al.Enhancement of hypothalamic STAT3 acetylation by nuclear receptor Nur77 dictates leptin sensitivity.Diabetes. 2015; 64: 2069-2081Crossref PubMed Scopus (32) Google Scholar, Perez-Sieira et al., 2013Perez-Sieira S. Martinez G. Porteiro B. Lopez M. Vidal A. Nogueiras R. Dieguez C. Female Nur77-deficient mice show increased susceptibility to diet-induced obesity.PLoS ONE. 2013; 8: e53836Crossref PubMed Scopus (30) Google Scholar). Importantly, Nur77−/− mice were resistant to the anti-obesity effects of celastrol, as celastrol showed only about 4% inhibition on body weight of Nur77−/− mice, compared to 22% inhibition in wild-type mice (Figure 1G). The ability of celastrol to attenuate HFD-induced adiposity (not shown) and serum production of ALT and AST (Figure S1M) was also compromised in Nur77−/− mice. Furthermore, celastrol administration inhibited the effect of HFD on activating the inflammatory pathway—including the expression and production of IL-1β and IL-6 (Figures S1N and S1O), IκBα expression (Figure 1H), p65 nuclear translocation (Figure S1P), hepatic inflammation (Figure 1I), and accumulation of hepatic neutrophil (Figure S1Q) and macrophage (Figure S1R)—in wild-type, but not Nur77−/− mice. Thus, celastrol inhibition of chronic inflammation in obese animals is also Nur77 dependent. Recent developments reveal a crucial role of the autophagy pathway and proteins in regulating inflammation (Green et al., 2011Green D.R. Galluzzi L. Kroemer G. Mitochondria and the autophagy-inflammation-cell death axis in organismal aging.Science. 2011; 333: 1109-1112Crossref PubMed Scopus (840) Google Scholar, Levine et al., 2011Levine B. Mizushima N. Virgin H.W. Autophagy in immunity and inflammation.Nature. 2011; 469: 323-335Crossref PubMed Scopus (2415) Google Scholar). We next determined whether the anti-inflammatory effect of celastrol could be attributed to its induction of autophagy. Western blotting (WB) revealed a strong induction of LC3-II, a marker for autophagy, upon treatment of HepG2 cells with celastrol for 6 hr (Figure 2A ). Although treatment with TNFα alone had no effect, its combination with celastrol resulted in a stronger induction of LC3-II expression, suggesting a role of inflammation in promoting the autophagic effect of celastrol. The autophagic effect of celastrol was also illustrated by its induction of the formation of punctate green fluorescent protein LC3 (GFP-LC3) (Figure 2B) or red fluorescent protein LC3 (RFP-LC3) (Figure S2A), a hallmark of autophagy induction. Nur77 was essential for the autophagic effect of celastrol, as the induction of LC3-II expression by celastrol was observed in MEFs, but not Nur77−/− MEFs (Figure 2C). In vivo, celastrol administration induced LC3-II expression (Figures 2D and S2B) and aggregated distribution of LC3B (Figures 2E and S2C) in liver tissues from LPS- and D-GalN-injected wild-type mice, but not Nur77−/− mice. Similar induction of aggregated LC3B immunostaining (Figure 2F) and LC3-II expression (Figure 2G) by celastrol was found in HFD-induced wild-type, but not Nur77−/− obese mice. The concurrent effects of celastrol on inhibiting inflammation and inducing autophagy suggested that both events might converge. Indeed, treatment of HepG2 cells with the autophagic inhibitors chloroquine, a lysosomotropic agent that prevents fusion of endosomes and lysosomes, and wortmannin, which inhibits both class I phosphoinositide 3-kinase (PI3K) and class III PtdIns3K, impaired the ability of celastrol to inhibit TNFα-induced IκBα degradation (Figure 2H). Thus, Nur77-dependent induction of autophagy by celastrol contributes to its anti-inflammatory function. To determine whether mitochondrial targeting of Nur77 accounts for its anti-inflammatory and autophagic functions, we analyzed the effect of celastrol on the subcellular localization of Nur77. Nur77 predominantly resided in the nuclei of MEFs, which were not affected by TNFα treatment (Figure 3A ). However, celastrol treatment induced diffused distribution of Nur77 throughout cells. Addition of TNFα led to punctate Nur77 staining, which colocalized extensively with mitochondria (Figure 3A). The combination treatment also resulted in speckled Nur77 staining in the cytoplasm of several cancer cell lines (Figures S3A and S3B). GFP-Nur77, which was found exclusively in the nuclei of control cells, displayed a speckled pattern in the cytoplasm of cells treated with celastrol and TNFα (Figure S3C). In contrast, the combination treatment had no effect on the nuclear localization of transfected Myc-RXRα (Figure S3D), demonstrating a specific effect of the treatment. Furthermore, cellular fractionation assay revealed an accumulation of an upshifted Nur77 band in the mitochondrial fraction prepared from celastrol- and TNFα-treated cells (Figure 3B). Recent studies indicate a critical role of TRAF2 at mitochondria. TRAF2 was recruited to mitochondria by the mitochondrial adaptor protein MAVS or the mitochondrial E3 ligase MULAN (Hou et al., 2011Hou F. Sun L. Zheng H. Skaug B. Jiang Q.X. Chen Z.J. MAVS forms functional prion-like aggregates to activate and propagate antiviral innate immune response.Cell. 2011; 146: 448-461Abstract Full Text Full Text PDF PubMed Scopus (843) Google Scholar, Liu et al., 2013Liu S. Chen J. Cai X. Wu J. Chen X. Wu Y.T. Sun L. Chen Z.J. MAVS recruits multiple ubiquitin E3 ligases to activate antiviral signaling cascades.eLife. 2013; 2: e00785Crossref Scopus (17) Google Scholar, Zemirli et al., 2014Zemirli N. Pourcelot M. Ambroise G. Hatchi E. Vazquez A. Arnoult D. Mitochondrial hyperfusion promotes NF-κB activation via the mitochondrial E3 ligase MULAN.FEBS J. 2014; 281: 3095-3112Crossref PubMed Scopus (43) Google Scholar) to mediate mitochondrion-based inflammatory signaling, including IKK-dependent NF-κB activation. Previous studies (Kim et al., 2010Kim J.J. Lee S.B. Park J.K. Yoo Y.D. TNF-alpha-induced ROS production triggering apoptosis is directly linked to Romo1 and Bcl-X(L).Cell Death Differ. 2010; 17: 1420-1434Crossref PubMed Scopus (237) Google Scholar, Yang et al., 2015Yang K.C. Ma X. Liu H. Murphy J. Barger P.M. Mann D.L. Diwan A. Tumor necrosis factor receptor-associated factor 2 mediates mitochondrial autophagy.Circ Heart Fail. 2015; 8: 175-187Crossref PubMed Scopus (38) Google Scholar) and our data (Figure S3E) showed that TNFα could enhance TRAF2 mitochondrial accumulation in a time-dependent manner. Prolonged exposure of HepG2 cells to TNFα (6 hr or longer) resulted in mitochondrial dysfunction, revealed by a decrease in mitochondrial membrane potential (ΔΨm) (Figure S3F) and an altered mitochondrial network (Figure S3G). Transfection of TRAF2 siRNA impaired LC3-II induction by celastrol and TNFα treatment (Figure 3C) similarly to the effect of knocking down Nur77, implying that Nur77 might act in concert with TRAF2 to mediate the autophagic effect of celastrol. Our immunostaining revealed an extensive colocalization of endogenous TRAF2 with Nur77 at mitochondria in MEFs treated with celastrol and TNFα (Figure 3A). Induction of TRAF2 accumulation at mitochondria by celastrol and TNFα was suppressed in Nur77−/− MEFs, revealing a role of Nur77. Nur77 colocalization with TRAF2 was also observed in several other cell lines (Figures S3A and S3B). In addition, transfected Flag-TRAF2 formed punctate structures colocalizing with endogenous Nur77 (Figure 3D) and mitochondria (Figure 3E) in celastrol- and TNFα-treated cells. Cotranslocation of Nur77 and TRAF2 to mitochondria was further illustrated by immunostaining showing extensive colocalization of transfected GFP-Nur77 and Flag-TRAF2 with mitochondria (Figure 3F) and by cellular fractionation assay revealing coaccumulation of TRAF2 and the upshifted Nur77 band in the purified mitochondrial fraction (Figure" @default.
• W2604143793 created "2017-04-14" @default.
• W2604143793 creator A5007517619 @default.
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• W2604143793 title "Celastrol-Induced Nur77 Interaction with TRAF2 Alleviates Inflammation by Promoting Mitochondrial Ubiquitination and Autophagy" @default.
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