1. Volume 35, Issue 6, Pages 806-817 (September 2009)
Differential SUMOylation of LXRα and LXRβ Mediates Transrepression of STAT1 Inflammatory Signaling in IFN-γ-Stimulated Brain Astrocytes 
Jee Hoon Lee, Sang Myun Park, Ohn Soon Kim, Chang Seok Lee, Joo Hong Woo, Soo Jung Park, Eun-hye Joe, Ilo Jou 
Molecular Cell 
Volume 35, Issue 6, Pages (September 2009)
DOI: /j.molcel
Copyright © 2009 Elsevier Inc. Terms and Conditions

2. Figure 1
Endogenous and Synthetic Ligands of Liver X Receptor Suppress IFN-γ-Induced Inflammatory Responses in Brain Astrocytes
(A and B) Primary astrocytes were stimulated with IFN-γ in the presence of the indicated levels of individual LXR ligands, and next, TNFα and IL-6 transcript levels (A) or TNFα and IL-6 protein secretion into media (B) were determined using RT-PCR or ELISA, respectively. ELISA data were presented as means ± SEMs of two independent experiments. ∗p < 0.05 and ∗∗p < 0.01 versus IFN-γ group.
(C) IRF1 protein expression under the same experimental conditions was measured by western blot analysis. The arrow indicates the IRF1 band.
7-KC, 7-ketocholesterol. 22(R), 22(R)-hydroxycholesterol. GW, GW3695. T0, T
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions

3. Figure 2
LXR Ligands Suppress the Promoter Binding, but Not the Phosphorylation or Nuclear Translocation, of STAT1 in IFN-γ-Stimulated Astrocytes
(A and B) Astrocytes were stimulated with IFN-γ in the presence of the indicated LXR ligands, and pSTAT1 binding to the IRF1 promoter was measured using EMSA (A) and ChIP assays (B) (see Experimental Procedures for details). ChIP assay was performed with antibody against STAT1 or control IgG. Immunoprecipitated DNA was amplified by using primer for pY-STAT1-binding site (−159 to −8) or p-cJun-binding site (−1452 to −1315) of IRF1. The input indicates the control PCR data and shows the amount of IRF1 promoter DNA present in each sample before ChIP. IgG- and p-cJun-binding site of IRF1 are used as negative controls. The asterisk denotes positive control for cJun binding.
(C) Western blot analysis showing the changes in tyrosine and serine phosphorylation of STAT1 in the presence of the indicated levels of LXR ligands in IFN-γ-stimulated astrocytes.
(D) Confocal microscopic images of astrocytes immunostained with pY-STAT1, GFAP (astrocyte marker), and Hoechst (nuclear marker) under the indicated conditions.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions

4. Figure 3
siRNA Experiments Reveal that the Anti-Inflammatory Actions of LXR Ligands Are LXRα and LXRβ Dependent
(A and B) Astrocytes were transfected with an LXRα- or LXRβ-specific siRNA duplex or a nonsilencing control siRNA using Oligofectamine. After 72 hr, cells were stimulated with IFN-γ for 2 hr in the absence or presence of LXR ligands. The levels of pSTAT1 and IRF1 proteins were detected by western blot analysis (A), and the transcript levels of TNFα, LXRα, and LXRβ were examined by RT-PCR (B).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions

5. Figure 4
STAT1-Interacting LXRα and LXRβ Form Trimers with HDAC4 and PIAS1, Respectively, and Become SUMOylated
(A–C) Astrocytes were stimulated with IFN-γ for 2 hr with individual LXR ligands, and cell lysates were immunoprecipitated with indicated antibodies; proteins were detected using the indicated antibodies.
(D) LXR SUMOylation was detected using in vitro SUMOylation Kit (see Experimental Procedures for details). Western blots against LXRα or LXRβ were loading controls for immunoprecipitated proteins. (1–6) The addition of immunoprecipitated LXRα (or LXRβ) obtained from cells treated with: (1) control, (2) IFN-γ, (3) IFN-γ+7-ketocholesterol, (4) IFN-γ+22(R)-hydroxycholesterol, (5) IFN-γ+GW3965, and (6) IFN-γ+T The asterisk denotes nonspecific bands that were not correlated with SUMOylation. MW, molecular weight marker. WT, wild-type. MT, mutated.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions

6. Figure 5
Knockdown of PIAS1 or HDAC4 Abrogates LXR-Induced STAT1 Binding with LXRs and LXR SUMOylation, Resulting in Restoration of Inflammatory Gene Expression
(A–D) Primary astrocytes were transfected with a PIAS1-specific (A and B) or HDAC4-specific siRNA duplex (C and D) or a nonsilencing control siRNA, using Oligofectamine. At 72 hr after transfection, cells were stimulated with IFN-γ for 2 hr in the absence or presence of LXR ligands, and next, interactions of PIAS1 (or HDAC4), pSTAT1, and LXRβ (or LXRα) were measured by immunoprecipitation using the indicated antibodies (A and C). Protein levels of IRF1 and pSTAT1 and transcript levels of TNFα and PIAS1 (or HDAC4) were determined by western blot analysis and RT-PCR, respectively (B and D).
(E and F) Cells were transfected with a PIAS1- or HDAC4-specific siRNA duplex, and next, DNA-binding activities of pSTAT1 were measured by ChIP assay (E) and EMSA (F). ChIP assay was performed with antibody against STAT1 or control IgG. Immunoprecipitated DNA was amplified by using primer for pY-STAT1-binding site (−159 to −8) or p-cJun-binding site (−1452 to −1315) of IRF1. The input indicates the control PCR data and shows the amount of IRF1 promoter DNA present in each sample before ChIP. IgG- and p-cJun-binding site of IRF1 are used as negative controls. The asterisk denotes positive control for cJun binding.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions

7. Figure 6
SUMO siRNA or an LXR Mutation Abrogates LXR-Dependent Suppression of IRF1 and TNFα Expression
(A and B) SUMO1- or SUMO2-specific siRNA-transfected astrocytes were stimulated with IFN-γ for 2 hr in the presence of individual LXR ligands. The levels of pY-STAT1 and IRF1 were assessed by western blot analysis (A, top), and the transcript levels of TNFα, SUMO1, and SUMO2 were examined by RT-PCR (A, bottom). DNA-binding activities of pSTAT1 were measured by ChIP assay (B). ChIP assay was performed with antibody against STAT1 or control IgG. Immunoprecipitated DNA was amplified by using primer for pY-STAT1-binding site (−159 to −8) or p-cJun-binding site (−1452 to −1315) as a negative control region. The input indicates the control PCR data and shows the amount of IRF1 promoter DNA present in each sample before ChIP. IgG- and p-cJun-binding site of IRF1 are used as negative controls. The asterisk denotes positive control for cJun binding.
(C and D) Genes encoding wild-type LXRβ (LXRβ WT) or mutant LXRβ (LXRβ K30R) were cloned into the pEGFP-C3 vector and transfected into rat astrocytes (see Experimental Procedures for details). After transfection, cells were treated with IFN-γ for 2 hr in the absence or presence of LXR ligands, and next, cell lysates from LXRβ WT- or LXRβ K30R-transfected astrocytes were immunoprecipitated with anti-GFP antibody and immunoblotted with the indicated antibodies (C). After transfection, cells were treated with IFN-γ for 8 hr in the absence or presence of LXR ligands, and TNFα levels were assessed by immunocytochemistry (D).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions

8. Figure 7
A Model Showing the Anti-Inflammatory Mechanisms of LXR Ligands in IFN-γ-Stimulated Astrocytes
An IFN-γ signal leads to an early response, in which STAT1 is phosphorylated and translocated to the nucleus, thus inducing inflammatory gene expression. Synthetic and oxysterol derivatives of LXR ligands trigger the formation of PIAS1 (or HDAC4)-pSTAT1-LXRβ (or LXRα) trimers, a process mediated by differential SUMO (Su) conjugation to individual LXRs. Finally, STAT1 binding to the promoters of target genes is prevented.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions