GPS2-dependent corepressor/SUMO pathways govern anti-inflammatory actions of LRH-1 and LXRbeta in the hepatic acute phase response - PubMed (original) (raw)
. 2010 Feb 15;24(4):381-95.
doi: 10.1101/gad.545110.
Tomas Jakobsson, Anna Ehrlund, Anastasios Damdimopoulos, Laura Mikkonen, Ewa Ellis, Lisa-Mari Nilsson, Paolo Parini, Olli A Jänne, Jan-Ake Gustafsson, Knut R Steffensen, Eckardt Treuter
Affiliations
- PMID: 20159957
- PMCID: PMC2816737
- DOI: 10.1101/gad.545110
GPS2-dependent corepressor/SUMO pathways govern anti-inflammatory actions of LRH-1 and LXRbeta in the hepatic acute phase response
Nicolas Venteclef et al. Genes Dev. 2010.
Abstract
The orphan receptor LRH-1 and the oxysterol receptors LXRalpha and LXRbeta are established transcriptional regulators of lipid metabolism that appear to control inflammatory processes. Here, we investigate the anti-inflammatory actions of these nuclear receptors in the hepatic acute phase response (APR). We report that selective synthetic agonists induce SUMOylation-dependent recruitment of either LRH-1 or LXR to hepatic APR promoters and prevent the clearance of the N-CoR corepressor complex upon cytokine stimulation. Investigations of the APR in vivo, using LXR knockout mice, indicate that the anti-inflammatory actions of LXR agonists are triggered selectively by the LXRbeta subtype. We further find that hepatic APR responses in small ubiquitin-like modifier-1 (SUMO-1) knockout mice are increased, which is due in part to diminished LRH-1 action at APR promoters. Finally, we provide evidence that the metabolically important coregulator GPS2 functions as a hitherto unrecognized transrepression mediator of interactions between SUMOylated nuclear receptors and the N-CoR corepressor complex. Our study extends the knowledge of anti-inflammatory mechanisms and pathways directed by metabolic nuclear receptor-corepressor networks to the control of the hepatic APR, and implies alternative pharmacological strategies for the treatment of human metabolic diseases associated with inflammation.
Figures
Figure 1.
LRH-1 and LXRs inhibit hepatic APR gene expression. (A–C) The potential of LRH-1 (GR8470) and LXR (GW3965) agonists to repress cytokine-induced APR gene expression was analyzed in human primary hepatocytes. Hepatocytes were pretreated with vehicle (DMSO), GR8470 (10 μM), or GW3965 (2 μM) for 24 h and stimulated with 10 nM IL1β + IL6 for 16 h. Haptoglobin, SAA, and PAI-1 mRNA levels were quantified by qPCR. Data are presented as mean ± SD of three independent experiments. (D) IL1β + IL6 induce the dissociation of N-CoR or SMRT corepressor complexes from APR promoters. Huh7 cells were stimulated with 10 nM IL1β + IL6 for 1 h. Protein recruitment to APR promoters was analyzed by ChIP. (E,F) Activation of LRH-1 (E) or LXR (F) prevents the dissociation of N-CoR complexes from the haptoglobin promoter. Huh7 cells were pretreated by GR8470 (10 μM) or GW3965 (2 μM) and treated with 10 nM IL1β + IL6 for 1 h. Protein recruitment was analyzed by ChIP and re-ChIP.
Figure 2.
APR transrepression by LRH-1 and LXR is dependent on SUMOylation. (A,B) siRNA depletion of SUMO-1 or SUMO-2/3 reverse transrepression of haptoglobin expression (A) and haptoglobin reporter activity (B) in Huh7 cells. Huh7 cells were pretreated with DMSO, GR8470 (10 μM), or GW3965 (2 μM) for 24 h and 10 nM IL1β + IL6 for 3 h. (C,D) siRNA depletion of SUMO-1 or SUMO-2/3 prevents recruitment of LRH-1 or LXR to the haptoglobin promoter. Huh7 cells were transfected with siRNA according to the figure, and were treated with LRH-1 or LXR agonists for 24 h and 10 nM IL1β + IL6 for 1 h. Protein recruitment was analyzed by ChIP. (E,F) SUMOylation of LRH-1 and LXRβ is required for transrepression. Huh7 cells were transfected by Myc-LRH-1 wild type or K224R mutant (E) or Flag-LXRβ wild type or K410R/K448R double mutant (F). After transfection, cells were treated with DMSO or agonists for 24 h and 10 nM IL1β + IL6 for 1 h. Receptor recruitment at the haptoglobin promoter was measured by ChIP using Myc or Flag antibodies.
Figure 3.
SUMO-1 KO mice have increased APR. (A–C) Wild-type and SUMO-1 KO mice were treated with 10 mg/kg LPS for 6 h. APR gene expression was analyzed by qPCR. (D,E) Protein recruitment onto the mouse haptoglobin promoter was analyzed by ChIP from liver extracts. (F,G) Gene expression of LRH-1 target genes SHP and CYP7A1 (left panel) and LRH-1 recruitment to the promoters (right panel) were analyzed in wild-type and SUMO-1 KO mice. Data are presented as mean ± SD from five individual mice per group.
Figure 4.
LXRβ mediates APR transrepression upon activation of the LXRs. (A,B) Wild-type, LXRαβ double-KO, or LXRα and LXRβ single-KO mice were pretreated with 30 mg/kg per day GW3965 for 4 d and then treated with 2 mg/kg LPS for 2.5 h. Gene expression was analyzed by qPCR. (C–F) Protein recruitment to the mouse haptoglobin promoter was analyzed by ChIP from liver extracts. Data are presented as mean ± SD from five individual mice per group. (G) LXRβ but not LXRα is recruited to the haptoglobin promoter. Huh7 cells were transfected with Flag-LXRα or Flag-LXRβ, and were treated with GW3965 (2 μM) for 24 h and 10 nM IL1β + IL6 for 1 h. Recruitment of LXRs to haptoglobin or ABCA1 promoters was analyzed by ChIP using Flag or LXR antibody. (H) LXRβ but not LXRα is modified by SUMO-2. Huh7 cells were cotransfected with Flag-LXRα or Flag-LXRβ and Myc-SUMO-2, and were treated with GW3965 (2 μM) for 6 h. Whole-cell lysates were immunoblotted using Flag antibody to detect modified LXR, or immunoprecipitated using Flag antibody followed by immunoblotting using Myc antibody to detect SUMO-2-conjugated LXRs.
Figure 5.
APR transrepression by LRH-1 and LXR requires GPS2. (A,B) siRNA depletion of N-CoR or GPS2 abolishes transrepression of haptoglobin expression (A) and haptoglobin reporter gene activity (B) in Huh7 cells. Cells were transfected with siRNA according to figure, and were treated with DMSO or agonists for 24 h and 10 nM IL1β + IL6 for 3 h. Data are represented as mean ± SD of triplicate experiments. (C) GPS2 is required for transrepression of the haptoglobin promoter by LRH-1 (top panel) and LXR (bottom panel). (D) Mapping of the GPS2 “transrepression” domain. Huh7 cells expressing siRNA targeting GPS2 were transfected with different HA-GPS2 derivatives (wild type, 1–105, and 100–327). Recruitment of LRH-1 and LXR to the haptoglobin promoter was analyzed by ChIP.
Figure 6.
GPS2 interacts with SUMO and SUMOylated LRH-1 or LXRβ. (A) Schematic structure of human GPS2 and mutations within the N-terminal transrepression domain. (B) Purified recombinant HIS-GPS2 (amino acids 1–105) was incubated with purified GST, GST-SUMO-1, or GST-SUMO-2 protein. Precipitated GPS2 was visualized using HIS antibody. (C) Agarose-SUMO-2 was incubated with 35S-labeled GPS2 or SUMO protease 1 (SuPr1) and GPS2 or RCOR peptides as indicated. Proteins were separated by SDS-PAGE and detected using autoradiography. (D) Lysates from HeLa cells transfected with Myc-SUMO-1 or Myc-SUMO-2 and HA-GPS2 were immunoprecipitated using HA or Myc antibodies. Precipitates were subjected to Western blot as indicated. (E) Lysates from Cos-7 cells transfected with Myc-SUMO-1 or Myc-SUMO-2 and HA-GPS2 derivatives (wild type and Δ61–94) were immunoprecipitated using Myc antibody. Precipitates were subjected to Western blot using HA antibody. (F) Lysates from Cos-7 cells transfected with Myc-SUMO-2, Myc-HDAC4, Flag-LXRβ, and HA-GPS2 were immunoprecipitated using Flag antibody. Precipitates were subjected to Western blot using HA and Myc antibodies. (G) Lysates from Cos-7 cells transfected with Myc-SUMO-2, Flag-LXRβ, and HA-GPS2 derivatives (wild type and Δ61–94) were immunoprecipitated using HA antibody. Precipitates were subjected to Western blot using Flag and Myc antibodies. (H) GST or GST-GPS2-N (amino acids 1–105) were bound to gluthathione sepharose and incubated with 35S-labeled LRH-1 and SUMO-1–LRH-1 for 2 h. Proteins were separated by SDS-PAGE and detected using autoradiography. (I) Lysates from Cos-7 cells transfected with Myc-SUMO-1, HA-GPS2, and Myc-LRH-1 (wild type or K224R) were immunoprecipitated using HA antibody. Precipitates were subjected to Western blot using LRH-1 antibody.
Figure 7.
In vivo characterization of transrepression domains in GPS2 and LRH-1. (A,B) Mapping of the GPS2 transrepression domain. Huh7 cells, depleted for GPS2 by siRNA, were transfected with different HA-GPS2 derivatives and treated with DMSO, GR8470 (10 μM), or GW3965 (2 μM) for 24 h and 10 nM IL1β + IL6 for 1 h. Recruitment of LXR, N-CoR, and GPS2 (HA) to the haptoglobin (A) and ABCA1 (B) promoters were analyzed by ChIP. (C) Mapping of the LRH-1 transrepression domain. The LRH-1 LBD is required to prevent the degradation of the N-CoR complex from the APR promoter. Huh7 cells were transfected with Myc-LRH-1 wild type, Myc-LRH-1 ΔLBD, or Myc-SUMO–1-LRH-1 and treated with DMSO or GR8470 (10 μM) (top panel), or cotransfected with GFP-SUMO-1 (bottom panel) for 24 h. Cells were treated with 10 nM IL1β + IL6 for 1 h. Recruitment of Myc-tagged LRH-1 derivatives, N-CoR, SUMO-1, and GPS2 to the haptoglobin promoter was analyzed by ChIP. (D) LRH-1 transrepression at APR promoters can be dissected into two separate steps. (Step 1) Ligand- and SUMO-dependent “docking” to the corepressor complex via GPS2. (Step 2) LRH-1-dependent inhibition of corepressor complex degradation.
Figure 8.
Suggested roles of GPS2, LRH-1, and LXRβ in the transrepression of hepatic APR. Treatment with agonists either induces specific SUMOylation of LXRβ or increases the levels of SUMOylated LRH-1. SUMOylated LXRβ or LRH-1 interacts with GPS2, thereby associating with the N-CoR complex and preventing its dissociation in an inflammatory state. Considered is the involvement of additional modulators (MOD) of GPS2 interactions. Furthermore, as a result of the anti-inflammatory transrepression, LXR and LRH-1 inhibit the production of APPs during inflammation and infection, thereby potentially reducing the biosynthesis of proatherogenic acute-phase HDL. (APO) Apolipoproteins (e.g., APOAI).
References
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