Poly(ADP-ribose) Polymerase 1 Represses Liver X Receptor-mediated ABCA1 Expression and Cholesterol Efflux in Macrophages

Abstract

Liver X receptors (LXR) are oxysterol-activated nuclear receptors that play a central role in reverse cholesterol transport through up-regulation of ATP-binding cassette transporters (ABCA1 and ABCG1) that mediate cellular cholesterol efflux. Mouse models of atherosclerosis exhibit reduced atherosclerosis and enhanced regression of established plaques upon LXR activation. However, the coregulatory factors that affect LXR-dependent gene activation in macrophages remain to be elucidated. To identify novel regulators of LXR that modulate its activity, we used affinity purification and mass spectrometry to analyze nuclear LXRα complexes and identified poly(ADP-ribose) polymerase-1 (PARP-1) as an LXR-associated factor. In fact, PARP-1 interacted with both LXRα and LXRβ. Both depletion of PARP-1 and inhibition of PARP-1 activity augmented LXR ligand-induced ABCA1 expression in the RAW 264.7 macrophage line and primary bone marrow-derived macrophages but did not affect LXR-dependent expression of other target genes, ABCG1 and SREBP-1c. Chromatin immunoprecipitation experiments confirmed PARP-1 recruitment at the LXR response element in the promoter of the ABCA1 gene. Further, we demonstrated that LXR is poly(ADP-ribosyl)ated by PARP-1, a potential mechanism by which PARP-1 influences LXR function. Importantly, the PARP inhibitor 3-aminobenzamide enhanced macrophage ABCA1-mediated cholesterol efflux to the lipid-poor apolipoprotein AI. These findings shed light on the important role of PARP-1 on LXR-regulated lipid homeostasis. Understanding the interplay between PARP-1 and LXR may provide insights into developing novel therapeutics for treating atherosclerosis.

Keywords: ABC transporter; ABCA1; ADP-ribosylation; LXR; PARP-1; atherosclerosis; cholesterol regulation; gene expression; nuclear receptor; transcription corepressor.

FIGURE 1.

Interaction of PARP-1 with LXRs. A and B, vector alone, FLAG-tagged LXRα (A), or LXRβ (B) was transiently expressed in HEK293 cells. Whole cell extracts were prepared, and FLAG-tagged protein complexes were immunoprecipitated with FLAG antibody and eluted by FLAG peptides. The eluted proteins were analyzed by Western blotting for LXRα (A) or LXRβ (B) and PARP-1. The lysates used for immunoprecipitation were Western blotted with PARP-1 antibody, as well as LXRα or LXRβ antibody. The blots in the top two panels in B were spliced as marked by the black line to show only the lanes that are relevant.

FIGURE 2.

Effects of modulating PARP-1 level and activity on LXR-mediated ABCA1 expression in HEK293 cells. A, PARP-1 protein levels were measured by Western blot in the whole cell lysates of 293-WT and 293-PARP-1-KO cells generated by using CRISPR/Cas9. Clones 1 and 2 represent cell lines generated from two independent CRISPR clones. B and C, 293-WT and 293-PARP-1-KO cells were transiently transfected with LXRα (B) or LXRβ (C) and treated with DMSO or 5 μm T0901317 (T) for 4 h. ABCA1 steady state mRNA levels were measured by qPCR. The expression levels of ABCA1 were normalized to RPL19 levels. D, ABCA1 nascent mRNA levels were measured by qPCR in WT and KO cells that were transiently transfected with LXRα. E, protein extracts prepared from 293-WT and 293-PARP-1-KO cells in parallel with RNA samples in D were analyzed for global PARylation by immunoblotting with anti-PAR antibody. F, global PARylation levels were measured in whole cell extracts from 293T-LXRα cells pretreated with 0 mm or 5 mm of pan-PARP inhibitor, 3-AB for 16 h. G, 293T-LXRα cells were pretreated with 5 mm 3-AB and were stimulated with vehicle (DMSO) or 5 μm T for 4 h. ABCA1 mRNA expression levels were measured by qPCR similarly as above. Experiments were performed three times, and the values were averaged. The error bars represent S.E. Significance was determined using the two-tailed Student's t test. *, p < 0.05; **, p < 0.005; ***, p < 0.0005. For B, significance was determined by comparing values with WT clone 1.

FIGURE 3.

Effects of PARP-1 depletion, overexpression, and inhibition on expression of LXR target genes in macrophages. A, whole cell lysates from siRNA nontargeting control and siRNA PARP-1 transfected RAW-LXRα cells were analyzed by immunoblotting for the PARP-1 protein levels (top left). RAW-LXRα macrophages transfected with si control or si PARP-1 were treated with DMSO (D) or 5 μm T for 4 h, and ABCA1 (top right), SREBP1c (bottom left), and ABCG1 (bottom right) mRNA levels were measured by qPCR. The expression levels were normalized to cyclophilin A levels. B, RAW-LXRα cells were overexpressed with PARP-1 or vector control and treated with 0, 0.001, 0.01, 0.1, and 1 μm T0901317 (T), and ABCA1 mRNA levels were measured by qPCR. C–F, RAW-LXRα cells (C and D), WT primary BMDMs (E), and LXRα^−/− LXRβ^−/− BMDMs (F) were pretreated with 5 mm 3-AB and were stimulated with vehicle (DMSO), 5 μm T0901317 (T), 1 μm 9-cis-RA (9-cis), or a combination of T0901317 and 9-cis-retinoic acid (T+9-cis) as indicated for 4 h (C, E, and F) or 24 h (D). ABCA1, SREBP1c, and/or ABCG1 mRNA expression levels were measured by qPCR as indicated similarly as above. Experiments were performed three times, and the values were averaged. The error bars represent S.E. Significance was determined using the two-tailed Student's t test. *, p < 0.05; **, p < 0.005; ***, p < 0.0005.

FIGURE 3.

Effects of PARP-1 depletion, overexpression, and inhibition on expression of LXR target genes in macrophages. A, whole cell lysates from siRNA nontargeting control and siRNA PARP-1 transfected RAW-LXRα cells were analyzed by immunoblotting for the PARP-1 protein levels (top left). RAW-LXRα macrophages transfected with si control or si PARP-1 were treated with DMSO (D) or 5 μm T for 4 h, and ABCA1 (top right), SREBP1c (bottom left), and ABCG1 (bottom right) mRNA levels were measured by qPCR. The expression levels were normalized to cyclophilin A levels. B, RAW-LXRα cells were overexpressed with PARP-1 or vector control and treated with 0, 0.001, 0.01, 0.1, and 1 μm T0901317 (T), and ABCA1 mRNA levels were measured by qPCR. C–F, RAW-LXRα cells (C and D), WT primary BMDMs (E), and LXRα^−/− LXRβ^−/− BMDMs (F) were pretreated with 5 mm 3-AB and were stimulated with vehicle (DMSO), 5 μm T0901317 (T), 1 μm 9-cis-RA (9-cis), or a combination of T0901317 and 9-cis-retinoic acid (T+9-cis) as indicated for 4 h (C, E, and F) or 24 h (D). ABCA1, SREBP1c, and/or ABCG1 mRNA expression levels were measured by qPCR as indicated similarly as above. Experiments were performed three times, and the values were averaged. The error bars represent S.E. Significance was determined using the two-tailed Student's t test. *, p < 0.05; **, p < 0.005; ***, p < 0.0005.

FIGURE 4.

PARP-1 occupancy at the ABCA1 promoter. RAW-LXRα cells were incubated with DMSO or 5 μm T0901317 (T) for 1 h. Chromatin immunoprecipitation was performed using PARP-1 antibody or rabbit IgG. Precipitated DNA was quantified by qPCR using the primers spanning a non-LXRE site 4 kb upstream of the transcription start site of the ABCA1 gene, LXRE site at the ABCA1 promoter 85 bp upstream of the transcription start site, and ABCG1 promoter LXRE and SREBP-1c LXRE sites; normalized to total input chromatin levels; and measured as a percentage of input. The experiment was performed three times, and the values were averaged. The error bars represent S.E. Significance was determined using the two-tailed Student's t test. *, p < 0.05; **, p < 0.005; ***, p < 0.0005.

FIGURE 5.

Effects of depletion of PARP-1 on LXRα occupancy at ABCA1 promoter LXRE. RAW LXRα macrophages transfected with siRNA control (sicon) or siRNA PARP-1 (siPARP1) were treated with DMSO (D) or 5 μm T0901317 (T) for 1 h. Chromatin immunoprecipitation was performed using LXRα antibody or mouse IgG. Precipitated DNA was quantified by qPCR using primers spanning ABCA1 LXRE or a control non-LXRE site, normalized to total input chromatin levels, and measured as a percentage of input. The experiment was performed three times, and the values were averaged. The error bars represent S.E. The two-tailed Student's t test showed no significant (ns) differences in LXRα occupancy between si control and si PARP1 groups.

FIGURE 6.

Poly(ADP-ribosyl)ation of LXRα by PARP-1. A, recombinant LXRα (100 ng) and PARP-1 (40 ng) together or alone were incubated in PARylation buffer in the presence or absence of 300 μm NAD⁺ for 30 min. PARylation was stopped by the addition of SDS sample buffer. The samples were run on a 10% SDS-polyacrylamide gel. PARylation was detected by Western blot with anti-poly-ADP-ribose antibody. B, 293-WT and 293-PARP-1-KO cells were transiently transfected with FLAG-tagged LXRα or vector alone. LXRα was immunoprecipitated by FLAG antibody and immunoblotted with anti-PAR antibody. C, whole cell extracts from BMDMs treated with vehicle or 5 mm 3-AB were subjected to immunoprecipitation using control IgG or LXRα antibody. The samples were analyzed for total LXRα and PARylation by Western blot. D, LXRα was ectopically expressed in HEK293 cells, which were then treated with vehicle (DMSO) or 5 μm T0901317 (T) for 4 h. LXRα was immunoprecipitated and analyzed by Western blot for PARylation. Whole cell lysates were also analyzed for FLAG-tagged LXRα and PARP-1 protein levels by Western blot. Hsp90 was used as a loading control. The experiments were performed at least three times with similar results, and a representative experiment is shown.

FIGURE 7.

Inhibition of PARP-1 increases cholesterol efflux in macrophages. A, ABCA1 protein levels were measured in the whole cell lysates from RAW-LXRα cells pretreated with vehicle or 5 mm 3-AB and stimulated with vehicle (D), 5 μm T0901317 (T), or a combination of 5 μm T0901317 and 1 μm 9-cis-retinoic acid (T+9). Two exposures (short and long) are shown. Hsp90 levels were measured as a loading control. The Western blot was quantitated using ImageJ software, and the ratio of ABCA1 to HSP90 is shown with DMSO and non-3-AB-treated sample set to 1. B, RAW-LXRα cells were labeled with medium containing BODIPY cholesterol, methyl-β-cyclodextrin, and unlabeled cholesterol for 1 h. Then the cells were equilibrated with vehicle or 3-AB in combination with DMSO or 5 μm T0901317 for 18 h before incubating for 4 h with the efflux medium containing extracellular acceptor apoA-I (10 μg/ml). apoA-I-mediated efflux was calculated as a percentage of total cholesterol cleared from the cells after accounting for the cholesterol taken out from the cells in the absence of acceptors. C, WT, Lxrα^−/− Lxrβ^−/−, and Abca1^−/− primary BMDMs were loaded with [³H]cholesterol, and specific efflux to apoA-I was measured in the presence or absence of 5 mm 3-AB. The experiments in B and C were performed three times with similar results. Each efflux experiment was performed in triplicate, and a representative experiment is shown. The error bars represent S.D. Significance was determined using the two-tailed Student's t test. *, p < 0.05; **, p < 0.005.