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• W2081594562 abstract "To unravel the roles of LXRs in inflammation and immunity, we examined the function of LXRs in development of IFN-γ-mediated inflammation using cultured rat brain astrocytes. LXR ligands inhibit neither STAT1 phosphorylation nor STAT1 translocation to the nucleus but, rather, inhibit STAT1 binding to promoters and the expression of IRF1, TNFα, and IL-6, downstream effectors of STAT1 action. Immunoprecipitation data revealed that LXRβ formed a trimer with PIAS1-pSTAT1, whereas LXRα formed a trimer with HDAC4-pSTAT1, mediated by direct ligand binding to the LXR proteins. In line with the fact that both PIAS1 and HDAC4 belong to the SUMO E3 ligase family, LXRβ and LXRα were SUMO-conjugated by PIAS1 or HDAC4, respectively, and SUMOylation was blocked by transient transfection of appropriate individual siRNAs, reversing LXR-induced suppression of IRF1 and TNFα expression. Together, our data show that SUMOylation is required for the suppression of STAT1-dependent inflammatory responses by LXRs in IFN-γ-stimulated brain astrocytes. To unravel the roles of LXRs in inflammation and immunity, we examined the function of LXRs in development of IFN-γ-mediated inflammation using cultured rat brain astrocytes. LXR ligands inhibit neither STAT1 phosphorylation nor STAT1 translocation to the nucleus but, rather, inhibit STAT1 binding to promoters and the expression of IRF1, TNFα, and IL-6, downstream effectors of STAT1 action. Immunoprecipitation data revealed that LXRβ formed a trimer with PIAS1-pSTAT1, whereas LXRα formed a trimer with HDAC4-pSTAT1, mediated by direct ligand binding to the LXR proteins. In line with the fact that both PIAS1 and HDAC4 belong to the SUMO E3 ligase family, LXRβ and LXRα were SUMO-conjugated by PIAS1 or HDAC4, respectively, and SUMOylation was blocked by transient transfection of appropriate individual siRNAs, reversing LXR-induced suppression of IRF1 and TNFα expression. Together, our data show that SUMOylation is required for the suppression of STAT1-dependent inflammatory responses by LXRs in IFN-γ-stimulated brain astrocytes. Astrocytes and microglia are representative immunocompetent cells in the brain that play important roles in brain pathology, where inflammatory responses lead to the progression and aggravation of disease. Thus, it is therapeutically useful to regulate inflammatory responses for control of degenerative brain disease. In our previous studies, cultured astrocytes and microglia were activated, as measured by signal transduction activity and enhancement of transcription (STAT) signaling pathways, either directly by interferon (IFN)-γ or gangliosides or indirectly by lipopolysaccharide (LPS) (Kim et al., 2002Kim O.S. Park E.J. Joe E.H. Jou I. JAK-STAT signaling mediates gangliosides-induced inflammatory responses in brain microglial cells.J. Biol. Chem. 2002; 277: 40594-40601Crossref PubMed Scopus (139) Google Scholar, Lee et al., 2005Lee J.H. Park E.J. Kim O.S. Kim H.Y. Joe E.H. Jou I. Double-stranded RNA-activated protein kinase is required for the LPS-induced activation of STAT1 inflammatory signaling in rat brain glial cells.Glia. 2005; 50: 66-79Crossref PubMed Scopus (40) Google Scholar, Park et al., 2004Park E.J. Ji K.A. Jeon S.B. Choi W.H. Han I.O. You H.J. Kim J.H. Jou I. Joe E.H. Rac1 contributes to maximal activation of STAT1 and STAT3 in IFN-gamma-stimulated rat astrocytes.J. Immunol. 2004; 173: 5697-5703PubMed Google Scholar). Also, STAT activation in LPS-stimulated microglia and astrocytes was efficiently suppressed by oxysterol metabolites and synthetic liver X receptor (LXR) ligands, including TO901317 and GW3965 (Kim et al., 2006Kim O.S. Lee C.S. Joe E.H. Jou I. Oxidized low density lipoprotein suppresses lipopolysaccharide-induced inflammatory responses in microglia: oxidative stress acts through control of inflammation.Biochem. Biophys. Res. Commun. 2006; 342: 9-18Crossref PubMed Scopus (30) Google Scholar, Lee et al., 2006Lee C.S. Joe E.H. Jou I. Oxysterols suppress inducible nitric oxide synthase expression in lipopolysaccharide-stimulated astrocytes through liver X receptor.Neuroreport. 2006; 17: 183-187Crossref PubMed Scopus (9) Google Scholar). LXRα and LXRβ (NR1H3 and NR1H2, respectively) are ligand-activated nuclear receptors that play important roles in the control of cholesterol homeostasis (Castrillo and Tontonoz, 2004Castrillo A. Tontonoz P. Nuclear receptors in macrophage biology: at the crossroads of lipid metabolism and inflammation.Annu. Rev. Cell Dev. Biol. 2004; 20: 455-480Crossref PubMed Scopus (238) Google Scholar, Repa and Mangelsdorf, 2000Repa J.J. Mangelsdorf D.J. The role of orphan nuclear receptors in the regulation of cholesterol homeostasis.Annu. Rev. Cell Dev. Biol. 2000; 16: 459-481Crossref PubMed Scopus (576) Google Scholar). Once activated, LXRs upregulate transcription of genes involved in cholesterol transport such as those encoding ATP-binding cassette (ABC) transporters, including ATP-binding cassette protein A (ABCA)1 and apolipoprotein E (Apo E), in various brain diseases. ABCA1 is abundantly expressed in both glia and neurons, forms high-density lipoprotein (HDL)-like particles, and is believed to act as the major lipoprotein, carrying and delivering cholesterol to neurons through receptor-mediated endocytosis (Fagan et al., 1999Fagan A.M. Holtzman D.M. Munson G. Mathur T. Schneider D. Chang L.K. Getz G.S. Reardon C.A. Lukens J. Shah J.A. LaDu M.J. Unique lipoproteins secreted by primary astrocytes from wild type, apoE (−/−), and human apoE transgenic mice.J. Biol. Chem. 1999; 274: 30001-30007Crossref PubMed Scopus (157) Google Scholar). In Alzheimer's brain, both Apo E and the ABCA1 transporter are thought to be intimately involved in amyloid-β (Aβ) transport and clearance (Koistinaho et al., 2004Koistinaho M. Lin S. Wu X. Esterman M. Koger D. Hanson J. Higgs R. Liu F. Malkani S. Bales K.R. Paul S.M. Apolipoprotein E promotes astrocyte colocalization and degradation of deposited amyloid-beta peptides.Nat. Med. 2004; 10: 719-726Crossref PubMed Scopus (429) Google Scholar, Wahrle et al., 2005Wahrle S.E. Jiang H. Parsadanian M. Hartman R.E. Bales K.R. Paul S.M. Holtzman D.M. Deletion of Abca1 increases Abeta deposition in the PDAPP transgenic mouse model of Alzheimer disease.J. Biol. Chem. 2005; 280: 43236-43242Crossref PubMed Scopus (246) Google Scholar) because Apo E expressed in astrocytes resulted in a dose-dependent reduction of brain Aβ peptide burden (Holtzman et al., 1999Holtzman D.M. Bales K.R. Wu S. Bhat P. Parsadanian M. Fagan A.M. Chang L.K. Sun Y. Paul S.M. Expression of human apolipoprotein E reduces amyloid-beta deposition in a mouse model of Alzheimer's disease.J. Clin. Invest. 1999; 103: R15-R21Crossref PubMed Scopus (279) Google Scholar, Holtzman et al., 2000Holtzman D.M. Bales K.R. Tenkova T. Fagan A.M. Parsadanian M. Sartorius L.J. Mackey B. Olney J. McKeel D. Wozniak D. Paul S.M. Apolipoprotein E isoform-dependent amyloid deposition and neuritic degeneration in a mouse model of Alzheimer's disease.Proc. Natl. Acad. Sci. USA. 2000; 97: 2892-2897Crossref PubMed Scopus (687) Google Scholar). Thus, ABCA1 and Apo E genes activated by LXR ligands could play important roles in Aβ deposition and amyloid plaque formation. We also observed that both endogenous (7-ketocholesterol [7-KC] and 22R-hydroxycholesterol [22{R}]) and synthetic (GW3965 [GW] and TO901317 [TO]) ligands of LXR induced the ABCA1 gene in brain astrocytes (Kim et al., 2006Kim O.S. Lee C.S. Joe E.H. Jou I. Oxidized low density lipoprotein suppresses lipopolysaccharide-induced inflammatory responses in microglia: oxidative stress acts through control of inflammation.Biochem. Biophys. Res. Commun. 2006; 342: 9-18Crossref PubMed Scopus (30) Google Scholar, Lee et al., 2006Lee C.S. Joe E.H. Jou I. Oxysterols suppress inducible nitric oxide synthase expression in lipopolysaccharide-stimulated astrocytes through liver X receptor.Neuroreport. 2006; 17: 183-187Crossref PubMed Scopus (9) Google Scholar). In addition to functions in lipid metabolism, LXRs have also been found to modulate immune and inflammatory responses in many cell types (Castrillo and Tontonoz, 2004Castrillo A. Tontonoz P. Nuclear receptors in macrophage biology: at the crossroads of lipid metabolism and inflammation.Annu. Rev. Cell Dev. Biol. 2004; 20: 455-480Crossref PubMed Scopus (238) Google Scholar, Zelcer and Tontonoz, 2006Zelcer N. Tontonoz P. Liver X receptors as integrators of metabolic and inflammatory signaling.J. Clin. Invest. 2006; 116: 607-614Crossref PubMed Scopus (716) Google Scholar). Administration of LXR activators to mice inhibited expression of various proinflammatory genes in macrophages after challenge with LPS, tumor necrosis factor (TNF)α, and interleukin (IL)-1β (Joseph et al., 2003Joseph S.B. Castrillo A. Laffitte B.A. Mangelsdorf D.J. Tontonoz P. Reciprocal regulation of inflammation and lipid metabolism by liver X receptors.Nat. Med. 2003; 9: 213-219Crossref PubMed Scopus (952) Google Scholar). Furthermore, genetic loss of Lxrα or Lxrβ in Alzheimer's disease-transgenic mice exacerbated senile plaque pathology partly because no LXRs were available to inhibit the inflammatory response of glial cells to β-amyloid fibrils (fAβ) (Repa et al., 2007Repa J.J. Li H. Frank-Cannon T.C. Valasek M.A. Turley S.D. Tansey M.G. Dietschy J.M. Liver X receptor activation enhances cholesterol loss from the brain, decreases neuroinflammation, and increases survival of the NPC1 mouse.J. Neurosci. 2007; 27: 14470-14480Crossref PubMed Scopus (121) Google Scholar, Zelcer et al., 2007Zelcer N. Khanlou N. Clare R. Jiang Q. Reed-Geaghan E.G. Landreth G.E. Vinters H.V. Tontonoz P. Attenuation of neuroinflammation and Alzheimer's disease pathology by liver x receptors.Proc. Natl. Acad. Sci. USA. 2007; 104: 10601-10606Crossref PubMed Scopus (259) Google Scholar). As LXR-responsive elements (LXREs) have not been identified in the promoters of repressed genes (Zelcer and Tontonoz, 2006Zelcer N. Tontonoz P. Liver X receptors as integrators of metabolic and inflammatory signaling.J. Clin. Invest. 2006; 116: 607-614Crossref PubMed Scopus (716) Google Scholar), LXRs are suggested to indirectly inhibit inflammatory responses by transrepressing coactivators or by recruiting corepressors. However, the detailed mechanism underlying the ligand-dependent transrepression process is poorly understood. In this study, we demonstrate that both synthetic and oxysterol derivatives of LXR ligands efficiently reduce IFN-γ-induced STAT inflammatory signals of brain astrocytes in a receptor-dependent manner. The anti-inflammatory ligand actions were derived from inhibitory actions on STAT1 binding to the promoter regions of target genes; such binding is mediated by small ubiquitin-like modifier (SUMO)1 or SUMO2 conjugation to LXRβ and LXRα catalyzed by the protein inhibitor of activated STAT (PIAS)1 or histone diacetylase (HDAC)4, respectively. Given the role of inflammation in degenerative brain diseases, including Alzheimer's disease, the dual roles of the LXRs, as regulators of both lipid metabolism and inflammatory responses, could be synergistically or specifically exploited for combating degenerative brain disease. To examine anti-inflammatory actions mediated by LXRs, we stimulated cultured astrocytes with IFN-γ for 2 hr (for subsequent RT-PCR and western blot assays) or 12 hr (for subsequent enzyme-linked immunosorbent assays [ELISA]) in the absence or presence of individual LXR ligands. Next, transcript levels (Figure 1A) and protein secretion levels (Figure 1B) of TNFα and IL-6 were measured using RT-PCR or ELISA, respectively. All LXR ligands dose dependently suppressed transcript levels and protein secretion of TNFα and IL-6 in IFN-γ-activated astrocytes (Figures 1A and 1B). IFN-γ-induced protein expression of IRF1, a representative signal molecule and a target of IFN-γ, was also dose dependently suppressed by all LXR ligands upon western blot analysis (Figure 1C). To demonstrate that LXR ligands acted through IFN-γ-STAT1 signaling, we examined their effects on the expression of IFN-β and suppressors of cytokine signaling (SOCS)3, which are known as IFN-γ-induced STAT1 signaling-independent genes (Ramana et al., 2001Ramana C.V. Gil M.P. Han Y. Ransohoff R.M. Schreiber R.D. Stark G.R. Stat1-independent regulation of gene expression in response to IFN-gamma.Proc. Natl. Acad. Sci. USA. 2001; 98: 6674-6679Crossref PubMed Scopus (204) Google Scholar). None of the LXR ligands that were used suppressed IFN-β or SOCS3 expressions, suggesting that LXR ligands specifically suppressed STAT1-dependent gene expressions (Figure S1 available online). As LXR ligands suppressed IFN-γ-induced cytokine release and IRF1 expression, we next examined which upstream step might be affected by these ligands. Primary astrocytes were stimulated with IFN-γ in the absence or presence of individual LXR ligands for 90 min (for subsequent electrophoretic mobility shift assays [EMSA]) or 30 min (for subsequent western blotting to detect phosphorylation). STAT1 binding to the IRF1 promoter was suppressed by all LXR ligands in EMSA-based experiments, which employed competition with a 20× molar excess of unlabeled (“cold”) probe and anti-STAT1 antibody (Figure 2A). These findings were further confirmed in chromatin immunoprecipitation (ChIP) assays. The amount of promoter DNA present in immunoprecipitated chromatin fractions was analyzed by PCR using primer pairs derived from the pSTAT1-binding sites (−158 to −8) or p-cJun-binding site (−1452 to −1315) of rat IRF1. In line with the results of EMSA, LXR ligands reduced pSTAT1 binding to the IRF1 promoter. However, western blots using antibodies against tyrosine 701- or serine 727-phosphorylated STAT1 (pY-STAT1 and pS-STAT1, respectively) revealed that none of the LXR ligands suppressed tyrosine or serine phosphorylation of STAT1 (Figure 2C). Their inability to suppress the phosphorylation of STAT1 was maintained up to 4 hr in IFN-γ-stimulated astrocytes (Figure S2A). As LXR ligands did suppress STAT1 nuclear binding, but not STAT1 phosphorylation, we next examined, using immunohistochemistry, whether the ligands could suppress STAT1 nuclear translocation. Nuclear translocation of pSTAT1 in IFN-γ-stimulated astrocytes was not inhibited by any of the LXR ligands that were tested (Figure 2D). This was also confirmed in nucleus/cytoplasm fractionation experiments (Figure S2B). As LXR ligands did not suppress the phosphorylation or nuclear translocation of STAT1, ligand inhibitory actions on STAT1 inflammatory responses were thought to occur in the nucleus. Although both receptor-dependent and -independent actions of LXR ligands have been reported, LXRs are nuclear receptors, and our finding that the receptors were in the nucleus in either the absence or presence of their ligands (Figure S3) suggested that ligand inhibitory actions were LXR dependent. We explored this possibility using siRNA technology. Astrocytes transfected with specific siRNAs for LXRα, LXRβ, or a control siRNA for more than 72 hr were stimulated with IFN-γ in the presence or absence of 7KC or GW (LXR ligands) for 90 min, and proteins were next analyzed by western blotting. Gene expression was measured by RT-PCR. In both LXRα and LXRβ knockdown cells (achieved by transfection of individual siRNAs), LXR ligand-induced IRF1 suppression was reversed, suggesting that such suppression is LXRα or LXRβ receptor mediated (Figure 3A). In line with the IRF1 results, inhibition of TNFα transcription by LXR ligands was also reversed by transfection of individual siRNAs for LXRα or LXRβ (Figure 3B). siRNA experiments were confirmed using two more independent siRNAs for individual LXR, and LXR-induced suppression of TNFα transcripts was restored in either of the siRNA-transfected cells (Figure S4A). LXR ligands could not inhibit STAT1 phosphorylation or STAT1 nuclear translocation in astrocytes. Thus, the question remained as to how they suppressed STAT1 binding to the IRF1 promoter region in the nucleus, resulting in inhibition of IFN-γ-induced downstream gene expression including IRF1 and TNFα. A previous study demonstrated a role for PIAS1 as a physiologically important negative regulator of STAT1 in the nucleus (Chen et al., 2004Chen W. Daines M.O. Khurana Hershey G.K. Turning off signal transducer and activator of transcription (STAT): the negative regulation of STAT signaling.J. Allergy Clin. Immunol. 2004; 114: 476-489Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Based on this work, we asked whether PIAS1 might be involved in the inhibitory actions of the LXRs. To examine this possibility, we performed immunoprecipitation tests to validate interactions among PIAS1, pSTAT1, and LXRs. Although interactions between PIAS1, pSTAT1, and LXRβ were induced by LXR ligands, those between PIAS1, pSTAT1, and LXRα did not appear to be affected (Figures 4A and 4B), suggesting that a molecule other than PIAS1 is involved in interaction with LXRα. HDAC4 was a candidate, as members of the class II deacetylases, including HDAC4, have been reported to interact with nuclear receptors (Franco et al., 2001Franco P.J. Farooqui M. Seto E. Wei L.N. The orphan nuclear receptor TR2 interacts directly with both class I and class II histone deacetylases.Mol. Endocrinol. 2001; 15: 1318-1328Crossref PubMed Scopus (26) Google Scholar). As expected, although the binding of LXRα to HDAC4 (and not to PIAS1) was increased in the presence of LXR ligands, that of LXRβ with HDAC4 showed no change when LXR ligands were present (Figures 4A and 4B). The next question regarded how this trimer failed to bind to the promoter region of STAT1-dependent inflammatory genes. In other words, how were the inhibitory actions of the LXRs expressed at the molecular level? PIAS1 and HDAC4 are reported to act as SUMO E3 ligases (Chen et al., 2004Chen W. Daines M.O. Khurana Hershey G.K. Turning off signal transducer and activator of transcription (STAT): the negative regulation of STAT signaling.J. Allergy Clin. Immunol. 2004; 114: 476-489Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, Gregoire and Yang, 2005Gregoire S. Yang X.J. Association with class IIa histone deacetylases upregulates the sumoylation of MEF2 transcription factors.Mol. Cell. Biol. 2005; 25: 2273-2287Crossref PubMed Scopus (161) Google Scholar, Sharrocks, 2006Sharrocks A.D. PIAS proteins and transcriptional regulation—more than just SUMO E3 ligases?.Genes Dev. 2006; 20: 754-758Crossref PubMed Scopus (117) Google Scholar, Shuai and Liu, 2005Shuai K. Liu B. Regulation of gene-activation pathways by PIAS proteins in the immune system.Nat. Rev. Immunol. 2005; 5: 593-605Crossref PubMed Scopus (328) Google Scholar, Zhao et al., 2005Zhao X. Sternsdorf T. Bolger T.A. Evans R.M. Yao T.P. Regulation of MEF2 by histone deacetylase 4- and SIRT1 deacetylase-mediated lysine modifications.Mol. Cell. Biol. 2005; 25: 8456-8464Crossref PubMed Scopus (206) Google Scholar), and are thus involved in cellular regulatory mechanisms. Moreover, a recent report on the critical role of PIAS1-dependent SUMOylation of the nuclear receptor PPARγ in anti-inflammatory actions (Pascual et al., 2005Pascual G. Fong A.L. Ogawa S. Gamliel A. Li A.C. Perissi V. Rose D.W. Willson T.M. Rosenfeld M.G. Glass C.K. A SUMOylation-dependent pathway mediates transrepression of inflammatory response genes by PPAR-gamma.Nature. 2005; 437: 759-763Crossref PubMed Scopus (963) Google Scholar) suggested that SUMOylation might be involved in the interactions studied in this paper. We thus tested whether LXRα and LXRβ might be SUMOylated by HDAC4 and PIAS1, respectively. Astrocyte lysates were immunoprecipitated with anti-SUMO1 and anti-SUMO2/3 antibodies, followed by immunoblot analysis with anti-LXRα or anti-LXRβ antibodies. Whereas LXRβ was SUMO1 conjugated by PIAS1, LXRα was SUMO2 conjugated by HDAC4 in brain astrocytes (Figure 4C). These results were further confirmed by in vitro SUMOylation assays. To demonstrate the crucial role of LXRs ligands, we performed in vitro SUMOylation assays using the immunoprecipitated LXRα (or LXRβ) obtained from the lysates of control or IFN-γ-treated cells with or without LXR ligands in place of LXR proteins. LXR ligands promoted SUMO1 conjugation to LXRβ and SUMO2 conjugation to LXRα (Figure 4D). These results indicate that LXRα and LXRβ are SUMOylated by different SUMO E3 ligases in brain astrocytes. To explore the system further, we knocked down PIAS1 or HDAC4 by transfecting cells with PIAS1- or HDAC4-specific siRNA in the presence or absence of LXR ligands. PIAS1 knockdown completely abolishes the formation of the LXR ligand-induced PIAS1-LXRβ-pSTAT1 trimer and, at the same time, inhibits SUMO1 SUMOylation of LXRβ, whereas PIAS1 knockdown does not affect LXRα SUMOylation or pSTAT1 binding with LXRα (compare top and bottom panels in Figure 5A). In line with these findings, STAT1-mediated inflammatory gene expression, suppressed by LXR ligands, was also reversed by PIAS1 knockdown (Figure 5B). As with PIAS1, knockdown of HDAC4 expression using specific siRNA also suppressed both the HDAC4-LXRα-pSTAT1 trimer formation (Figure 5C) and subsequent STAT1-mediated inflammatory gene expression (Figure 5D). We also showed that HDAC4 siRNA did not alter LXRβ SUMOylation or pSTAT1 binding with LXRβ (bottom panels in Figure 5C). Also, STAT1-binding activity shown with ChIP (Figure 5E) and EMSA analysis (Figure 5F) was suppressed by LXR ligands but recovered after PIAS1 or HDAC4 siRNA transfection, revealing that restoration of STAT1-dependent inflammatory gene expression could be ascribed to the recovery of STAT1-binding activities to target genes. siRNA experiments with PIAS1 or HDAC4 were also confirmed by using two more independent siRNA for each, and LXR-induced suppression of TNFα was restored in either of the siRNA-transfected cells (Figure S4B). Overall, these data indicated that LXR-dependent suppression of STAT1-mediated inflammatory gene expression in astrocytes was achieved by PIAS1- or HDAC4-dependent SUMOylation of LXRs. As the data described above revealed that LXRα and LXRβ SUMOylation was essential for LXR-mediated anti-inflammatory actions, we further explored this point using SUMO1- and SUMO2/3-specific siRNA. Astrocytes transfected with specific siRNA for SUMO1, SUMO2/3, or control siRNA for 72 hr were stimulated with IFN-γ in the presence or absence of LXR ligands for 90 min and then examined by western blotting or RT-PCR analysis. In both SUMO1 and SUMO2/3 knockdown cells, LXR ligand-induced IRF1 suppression was reversed (Figure 6A, top), and in line with the IRF1 data, inhibition of TNFα transcription (Figures 6A, bottom, and S4C) and pSTAT1-binding activity (Figure 6B) by LXR ligands was also reversed by transfection of either of the siRNAs, leading to the conclusion that the anti-inflammatory effects of LXR ligands were attributable to LXR SUMOylation-dependent events. To further investigate the functional significance of LXR SUMOylation, we made a SUMOylation site mutant of LXRβ (LXRβ K30R), as described in the Experimental Procedures. The SUMO1 conjugation to LXRβ after ligand binding occurred in astrocytes transfected with wild-type LXRβ (LXRβ WT), but not in cells transfected with LXRβ K30R (Figure 6C). Immunocytochemistry images further demonstrated that TNFα expression suppressed by LXR ligands was restored in LXRβ K30R-transfected cells, but not in LXRβ WT-transfected cells (Figure 6D). These results clearly indicated that SUMOylation was indispensable when LXR ligands suppressed inflammatory gene expression and cytokine release in IFN-γ-stimulated rat astrocytes. Although recent reports have addressed the anti-inflammatory roles of LXR ligands, the exact mechanisms of action are largely unknown. In this study, we demonstrated that synthetic, as well as endogenous, LXR ligands inhibited STAT1-mediated inflammatory responses in IFN-γ-stimulated brain astrocytes, which resulted from LXR action on STAT1 after LXR translocation to the nucleus. Especially, ligand-dependent SUMOylations of LXRs are required for inhibitory action on STAT1, as STAT1 complexed with SUMOylated LXR become unable to bind to promoter regions of target genes, resulting in transcriptional failure. This is summarized in Figure 7. Different types of posttranslational modifications, such as phosphorylation, acetylation, and ubiquitination, regulate the transcriptional activation and/or stability of nuclear receptors (Jenuwein and Allis, 2001Jenuwein T. Allis C.D. Translating the histone code.Science. 2001; 293: 1074-1080Crossref PubMed Scopus (7211) Google Scholar). SUMO modification is a further covalent protein modification (Bossis and Melchior, 2006Bossis G. Melchior F. SUMO: regulating the regulator.Cell Div. 2006; 1: 13Crossref PubMed Scopus (121) Google Scholar, Gill, 2004Gill G. SUMO and ubiquitin in the nucleus: different functions, similar mechanisms?.Genes Dev. 2004; 18: 2046-2059Crossref PubMed Scopus (587) Google Scholar, Melchior et al., 2003Melchior F. Schergaut M. Pichler A. SUMO: ligases, isopeptidases and nuclear pores.Trends Biochem. Sci. 2003; 28: 612-618Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar) mediated by a set of SUMO-conjugating enzymes. Although similar mechanisms are involved, SUMOylation affects degradation, stability, localization, and transcriptional activity of target proteins, whereas ubiquitination promotes target protein degradation (Gill, 2004Gill G. SUMO and ubiquitin in the nucleus: different functions, similar mechanisms?.Genes Dev. 2004; 18: 2046-2059Crossref PubMed Scopus (587) Google Scholar). Interestingly, several recent studies have reported the mechanisms involved in SUMO-modification of nuclear receptors (Treuter and Gustafsson, 2007Treuter E. Gustafsson J.A. Wrestling rules in transrepression: as easy as SUMO-1, -2, -3?.Mol. Cell. 2007; 25: 178-180Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). The transcriptional activity of estrogen receptor alpha (ERα), which is a member of the steroid/thyroid hormone nuclear receptor superfamily, may be regulated via phosphorylation-dependent SUMOylation within the DNA-binding domain (DBD), affecting ERα response to coactivators (Sentis et al., 2005Sentis S. Le Romancer M. Bianchin C. Rostan M.C. Corbo L. Sumoylation of the estrogen receptor alpha hinge region regulates its transcriptional activity.Mol. Endocrin. 2005; 19: 2671-2684Crossref PubMed Scopus (164) Google Scholar, Vu et al., 2007Vu E.H. Kraus R.J. Mertz J.E. Phosphorylation-dependent sumoylation of estrogen-related receptor alpha1.Biochemistry. 2007; 46: 9795-9804Crossref PubMed Scopus (45) Google Scholar). PPARγ, another nuclear receptor with broad anti-inflammatory effects in various cell types, is SUMO1-SUMOylated by PIAS1 with ligand binding. SUMO1-SUMOylated PPARγ prevents ubiquitinylation of NCoR corepressor complexes, resulting in repression of iNOS gene expression (Pascual et al., 2005Pascual G. Fong A.L. Ogawa S. Gamliel A. Li A.C. Perissi V. Rose D.W. Willson T.M. Rosenfeld M.G. Glass C.K. A SUMOylation-dependent pathway mediates transrepression of inflammatory response genes by PPAR-gamma.Nature. 2005; 437: 759-763Crossref PubMed Scopus (963) Google Scholar). These findings led us to hypothesize that SUMOylation might underlie the inhibitory actions of nuclear receptors on the transcription of target genes. Among SUMO E3 ligases, PIAS1 was originally described as a potent negative regulator of STAT1. By interacting with STAT1 dimers through the C-terminal region of PIAS1 and the N-terminal region of STAT1, PIAS1 blocks STAT1 DNA binding and, therefore, also STAT1-dependent gene induction (Chen et al., 2004Chen W. Daines M.O. Khurana Hershey G.K. Turning off signal transducer and activator of transcription (STAT): the negative regulation of STAT signaling.J. Allergy Clin. Immunol. 2004; 114: 476-489Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). In addition to its role as a STAT1 negative regulator, PIAS1 acts as a SUMO E3 ligase on target proteins (Chen et al., 2004Chen W. Daines M.O. Khurana Hershey G.K. Turning off signal transducer and activator of transcription (STAT): the negative regulation of STAT signaling.J. Allergy Clin. Immunol. 2004; 114: 476-489Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, Sharrocks, 2006Sharrocks A.D. PIAS proteins and transcriptional regulation—more than just SUMO E3 ligases?.Genes Dev. 2006; 20: 754-758Crossref PubMed Scopus (117) Google Scholar, Shuai and Liu, 2005Shuai K. Liu B. Regulation of gene-activation pathways by PIAS proteins in the immune system.Nat. Rev. Immunol. 2005; 5: 593-605Crossref PubMed Scopus (328) Google Scholar). Our data show that PIAS1-mediated SUMOylation of LXRβ is indispensable when LXRβ suppresses STAT1-mediated inflammatory responses in IFN-γ-stimulated astrocytes. In contrast to LXRβ, LXRα was SUMO2-SUMOylated by HDAC4, which is both a SUMO E3 ligase and a protein deacetylase, as has been reported (Zhao et al., 2005Zhao X. Sternsdorf T. Bolger T.A. Evans R.M. Yao T.P. Regulation of MEF2 by histone deacetylase 4- and SIRT1 deacetylase-mediated lysine modifications.Mol. Cell. Biol. 2005; 25: 8456-8464Crossref PubMed Scopus (206) Google Scholar). Moreover, the high expression of HDAC4 in brain astrocytes (data not shown) led us to hypothesize that HDAC4 might mediate SUMO2-SUMOylation of LXRα in rat astrocytes. Ghisletti and colleagues have reported that LXRα and LXRβ were SUMO2-SUMOylated by HDAC4 and that these SUMOylated LXRs bound to NCoR complexes associated with target gene promoters, thus preventing the proteins from being ubiquitinated and degraded in mouse macrophages (Ghisletti et al., 2007Ghisletti S. Huang W. Ogawa S. Pascual G. Lin M.E. Willson T.M. Rosenfeld M.G. Glass C.K. P" @default.
• W2081594562 created "2016-06-24" @default.
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• W2081594562 date "2009-09-01" @default.
• W2081594562 modified "2023-10-18" @default.
• W2081594562 title "Differential SUMOylation of LXRα and LXRβ Mediates Transrepression of STAT1 Inflammatory Signaling in IFN-γ-Stimulated Brain Astrocytes" @default.
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