Hepatic TRAP80 selectively regulates lipogenic activity of liver X receptor

Inflammation in response to excess low-density lipoproteins in the blood is an important driver of atherosclerosis development. Due to its ability to enhance ATP–binding cassette A1–dependent (ABCA1-dependent) reverse cholesterol transport (RCT), liver X receptor (LXR) is an attractive target for the treatment of atherosclerosis. However, LXR also upregulates the expression of sterol regulatory element–binding protein 1c (SREBP-1c), leading to increased hepatic triglyceride synthesis, an independent risk factor for atherosclerosis. Here, we developed a strategy to separate the favorable and unfavorable effects of LXR by exploiting the specificity of the coactivator thyroid hormone receptor–associated protein 80 (TRAP80). Using human hepatic cell lines, we determined that TRAP80 selectively promotes the transcription of SREBP-1c but not ABCA1. Adenovirus-mediated expression of shTRAP80 inhibited LXR-dependent SREBP-1c expression and RNA polymerase II recruitment to the LXR responsive element (LXRE) of SREBP-1c, but not to the LXRE of ABCA1. In murine models, liver-specific knockdown of TRAP80 ameliorated liver steatosis and hypertriglyceridemia induced by LXR activation and maintained RCT stimulation by the LXR ligand. Together, these data indicate that TRAP80 is a selective regulator of hepatic lipogenesis and is required for LXR-dependent SREBP-1c activation. Moreover, targeting the interaction between TRAP80 and LXR should facilitate the development of potential LXR agonists that effectively prevent atherosclerosis.

Liver X receptor (LXR) is a member of the nuclear receptor superfamily and acts as a cholesterol sensor to regulate the expression of several key proteins involved in lipid metabolism and inflammation (1, 2). Ligand-induced transactivation by nuclear receptors is a finely organized process that requires a variety of coactivators (3–5). Preferential functional association between particular nuclear receptors and coactivators may promote the specific regulation of individual genes under various conditions (6, 7). At present, a limited amount of information is available regarding coactivators of LXR and their mechanistic and physiological roles in LXR-mediated transactivation (8, 9).

Atherosclerosis is the leading cause of mortality in developed countries and is poised to become a worldwide health problem (10). LXR is a promising target for the treatment of atherosclerosis due to its ability to inhibit macrophage-driven inflammation and enhance ABCA1-dependent reverse cholesterol transport (RCT) (11–14). RCT is a pathway by which excess cellular cholesterol is transported from peripheral tissues to the liver for excretion in the bile and ultimately the feces, thereby helping to prevent atherosclerosis (15). Increased RCT mediated by ABCA1 upon treatment with the LXR ligands T0901317 or GW3965 contributes to an increase in serum HDL and a decrease in atherosclerosis (16, 17). Moreover, LXR exerts a further atheroprotective effect through inhibition of the macrophage inflammatory gene response (11, 18). These favorable effects of LXR agonists may be therapeutic in patients with atherosclerotic disease. However, LXR activation is also associated with increased mRNA levels of SREBP-1c, the master regulator of de novo fatty acid biosynthesis, leading to elevated serum and liver triglyceride levels (19–21). To optimize the therapeutic benefits of LXR agonists, it will be necessary to inhibit the unfavorable effects of LXR on fatty acid metabolism.

This study was initiated to clarify the mechanism of LXR-mediated transcription of metabolic genes, with particular focus on the details of the coactivator complex and a view toward modulating specific target gene expression. Here, we present 4 LXRα-interacting coactivators and demonstrate that 1 of these LXRα-interacting coactivators, TRAP80, is selectively recruited to the LXR responsive element (LXRE) of SREBP-1c, but not of ABCA1. TRAP80 knockdown by adenoviral shTRAP80 infection inhibited the transcription of lipogenic genes, including SREBP-1c, fatty acid synthase (FAS), and stearoyl CoA desaturase 1 (SCD1), but not the transcription of RCT-related genes such as ABCA1, ABCG5, and ABCG8. Furthermore, hepatic delivery of adenoviral shTRAP80 (Ad-shTRAP80) ameliorated hepatic steatosis induced by LXR-activated lipogenesis. Collectively, our data provide insight into the coactivator selectivity for transcriptional activation and suggest a strategy that could be exploited to amplify the beneficial effects of LXR agonists.

Identification of TRAP80 and TRAP220 as direct LXRα-interacting proteins. To isolate nuclear proteins as possible coactivators that interact independently or cooperatively with LXRα, we incubated purified GST-fused LXRα ligand–binding domain (LBD) with HepG2 nuclear extracts in the presence or absence of the LXR agonist T0901317 (Figure 1A and Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI73615DS1). Compared with GST alone, 4 proteins bound specifically to GST-LXRα LBD in a ligand-dependent manner (Figure 1A). Using matrix-assisted laser desorption ionization–time-of-flight (MALDI-TOF) analysis, we identified these LXRα LBD–interacting proteins as TRAP230 (ARC240 and MED12), TRAP220 (DRIP205, CRSP200, PBP, and MED1), CRSP130 (DRIP130, ARC130, SUR2, and MED23), and TRAP80 (DRIP80, ARC77, CRSP77, and MED17) (Supplemental Table 1 and refs. 22–26). We confirmed the identities of the GST-LXRα LBD–bound proteins by immunoblot analyses with specific antibodies (Figure 1B). To verify that these interactions took place inside the cells, total lysates from FLAG-LXRα–transfected cells were immunoprecipitated with anti-FLAG antibodies and subsequently probed with antibodies against each interacting protein (Figure 1C). All 4 proteins interacted with LXRα in a ligand-dependent manner in the cells. These results demonstrate that LXRα interacts with TRAP230, TRAP220, CRSP130, and TRAP80 via its LBD in a ligand-dependent manner.

Figure 1

Purification and identification of proteins interacting with LXRα LBD. (A) Ligand-dependent interactions between LXRα LBD and several nuclear proteins from HepG2 cells. Immobilized GST-LXRα LBD was incubated with HepG2 nuclear extracts in the presence or absence of T0901317. Immobilized GST was used as a control protein in the presence of ligand. Arrowheads indicate LXRα-interacting proteins analyzed by MALDI-TOF. (B) Identification of LXRα LBD interactors by immunoblot analysis. The thin black lines indicate that the lanes were run on the same gel but were noncontiguous. The TRAP80 blot was derived from samples run on a parallel gel. (C) Immunoblot analyses of proteins immunoprecipitated from FLAG-LXRα–transfected HEK293T cells using anti-FLAG antibodies. (D) GST pull-down assays were used to evaluate the binding between GST-LXRα LBD and [³⁵S]-methionine–labeled interactors. GST-LBD, GST-LXRα LBD; T1317, T0901317.

Next, we examined whether any of these proteins bind direct–ly to the LXRα LBD using in vitro–translated and [³⁵S]-labeled interactors. Only 2 of the proteins, TRAP220 and TRAP80, interacted directly with the LXRα LBD (Figure 1D). Interestingly, [³⁵S]-TRAP220 bound to GST-LXRα LBD in a ligand-dependent manner, whereas the [³⁵S]-TRAP80 interaction with GST-LXRα LBD was ligand independent in a 2-component protein-protein interaction assay.

TRAP80 stimulates the transcriptional activity of the LXRE of SREBP-1c but not of the LXRE of ABCA1. To determine the functional relevance of the interaction of TRAP220 and TRAP80 with LXRα, we performed cotransfection and luciferase reporter assays. Both TRAP220 and TRAP80 stimulated the transcriptional activity of LXRα on a synthetic LXRE promoter in a ligand-dependent manner (Figure 2A). We also examined their coactivator functions on 2 physiologically important natural promoters containing LXRE, specifically those of SREBP-1c and ABCA1. We observed that TRAP220 enhanced ligand-dependent transactivation of both the SREBP-1c and ABCA1 promoters (Figure 2, B and D). By contrast, TRAP80 increased the transcriptional activity of the SREBP-1c promoter, but not that of the ABCA1 promoter (Figure 2, B and D). Importantly, neither TRAP220 nor TRAP80 stimulated the transcription of SREBP-1c and ABCA1 promoters containing mutations in the LXRE, indicating that their coactivator function, at least in these cases, is specifically dependent on LXR recruitment to the LXRE (Figure 2, C and E). We also examined this target gene–specific activity of TRAP80 using GW3965, a specific LXR ligand structurally unrelated to T0901317. Again, TRAP80 showed LXR-mediated selective activation of the SREBP-1c promoter by GW3965 in a dose-dependent manner (Figure 2F). Dose-dependent activation of the ABCA1 promoter by GW3965 was not affected by TRAP80 (Figure 2G). Meanwhile, we found that TRAP230 and CRSP130, the indirect interactors of LXRα, stimulated both SREBP-1c and ABCA1 LXRE promoters (Supplemental Figure 2).

Figure 2

TRAP80 selectively stimulates SREBP-1c promoter activity in a ligand-dependent manner via LXRE. HepG2 cells were transfected with a luciferase reporter plasmid under the control of synthetic LXRE (A, n = 7), SREBP-1c WT promoter (B, n = 9; F, n = 6), ABCA1 WT promoter (D, n = 10; G, n = 6), SREBP-1c mutant promoter (C, n = 6), or ABCA1 mutant promoter (E, n = 5) with mutated DR-4 LXRE. T0901317 (A–E) or GW3965 (F and G) was used to activate the LXRs. Data are presented as the mean ± SEM. T1317, T0901317; Luc, luciferase; mt, mutant. **P < 0.01 and ***P < 0.001, by 1-way ANOVA.

In vivo transcription occurs on a nucleosomal template, i.e., chromatin. To investigate the recruitment of TRAP220 and TRAP80 in endogenous chromatin, we performed ChIP analyses of the Srebp-1c and Abca1 promoter regions (Figure 3A). Both TRAP220 and TRAP80 were recruited to the LXRE of the endogenous Srebp-1c promoter in response to treatment with T0901317 or GW3965 (Figure 3, B and D). By contrast, TRAP220 was recruited to the LXRE of the Abca1 promoter, but TRAP80 was not (Figure 3, C and E). We also investigated the recruitment of TRAP220 and TRAP80 under the condition of LXRα knockdown to confirm whether the recruitment of those coactivators is LXRα dependent. LXRα-specific knockdown in mouse primary hepatocytes by siRNA was validated by quantitative reverse transcriptase PCR (qRT-PCR) (Supplemental Figure 3). As a result, the recruitment of both TRAP220 and TRAP80 to Srebp-1c LXRE was not stimulated by LXR ligands under LXRα-knockdown conditions (Figure 3, B and D). We found that TRAP80 was not recruited to Abca1 LXRE, regardless of the expression of LXRα (Figure 3E). Interestingly, we observed that the recruitment of TRAP220 to the Abca1 LXRE was increased by LXR ligands, even under LXRα-knockdown conditions (Figure 3C). In conclusion, TRAP80 was selectively recruited in a ligand- and LXRα-dependent manner to the Srebp-1c promoter, but not to the Abca1 promoter.

Figure 3

TRAP80 is selectively recruited to the LXRE of the Srebp-1c promoter. (A) Schematic diagram of primer locations used for the ChIP assay across the Srebp-1c or Abca1 genomic structure. (B–E) ChIP assays were performed with anti-TRAP220 (B and C) or anti-TRAP80 (D and E) antibodies in mouse primary hepatocytes following LXRα knockdown by siRNA (sc-38829). Recruitment of TRAP220 or TRAP80 to the LXREs of the Srebp-1c and Abca1 genes was determined by qPCR (n = 3). The level of recruitment is expressed as the fold enrichment relative to the recruitment of TRAP220 to the LXREs in the presence of DMSO alone. Data are presented as the mean ± SEM. T1317, T0901317. *P < 0.05, **P < 0.01, and ***P < 0.001, by 1-way ANOVA.

TRAP80 regulates the recruitment of RNA polymerase II and histone modification of the LXRE region of Srebp-1c, but not that of Abca1. To address the consequences of the differential effects of TRAP80 on the Srebp-1c and Abca1 promoters, we introduced TRAP80 cDNA or Ad-shTRAP80 into the cells and determined the levels of Srebp-1c and Abca1 transcripts by qRT-PCR. TRAP80 overexpression significantly increased Srebp-1c mRNA expression, but did not affect Abca1 mRNA levels (Figure 4A). Consistent with this, Ad-shTRAP80 significantly suppressed the T0901317-dependent increase of Srebp-1c transcription, but did not affect Abca1 transcription (Figure 4B). Knockdown of TRAP80 using siRNA yielded results similar to those obtained with Ad-shTRAP80 (Supplemental Figure 4). To further investigate the role of TRAP80 in Srebp-1c and Abca1 promoter activation, we performed ChIP assays using antibodies against RNA polymerase II (Pol II) or acetyl-histone H4 (H4Kac). While Ad-shTRAP80 abolished the T0901317-dependent recruitment of Pol II to the LXRE of the Srebp-1c promoter, we found that recruitment of Pol II to the LXRE of the Abca1 promoter was not significantly altered (Figure 4C). The recruitment of transcriptional regulators to the promoter chromatin is coordinated by epigenetic information encoded in covalent modifications of histone proteins (27). Histone acetylation is a central switch that allows interconversion between transcriptionally permissive or repressive chromatin states (28). We found that infection of mouse primary hepatocytes with Ad-shTRAP80 consistently decreased T0901317-dependent acetylation of histone H4 lysine (H4K) in the vicinity of the LXRE of the Srebp-1c promoter (Figure 4D). However, ligand-dependent acetylation of H4K near the LXRE of the Abca1 promoter was still significantly increased in the presence of shTRAP80, although this acetylation was slightly attenuated (Figure 4D). Meanwhile, we observed that LXR was recruited to the LXRE regions of the Srebp-1c and Abca1 genes in a ligand-dependent manner, regardless of TRAP80 expression (Figure 4E). Taken together, these results suggest that TRAP80 selectively stimulates SREBP-1c expression by Pol II recruitment and chromatin remodeling.

Figure 4

TRAP80 regulates Srebp-1c transcript levels by recruiting RNA Pol II. (A and B) Changes in Srebp-1c and Abca1 mRNA levels following TRAP80 overexpression (A, n = 5) or infection with Ad-shTRAP80 (B, n = 6) in mouse primary hepatocytes. (C–E) ChIP assays were performed with anti–Pol II (C, n = 5), anti-H4Kac (D, n = 5), or anti-LXRα antibodies (E, n = 4) in mouse primary hepatocytes infected with Ad-shTRAP80. Abundance of each protein bound to Srebp-1c or Abca1 promoters was determined by qPCR. Enrichment levels are expressed as the fold enrichment relative to the enrichment observed in controls (shCon/DMSO). Data are presented as the mean ± SEM. T1317, T0901317. *P < 0.05, **P < 0.01, and ***P < 0.001 by 1-way ANOVA.

Hepatic knockdown of TRAP80 represses LXR-dependent lipogenesis in the liver. To investigate the physiological effects of TRAP80 modulation, we administered Ad-shTRAP80 to the liver via tail vein injection (Supplemental Figure 5). Five days after Ad-shTRAP80 injection, GW3965 was administered for 3 days. Surprisingly, we found that Ad-shTRAP80 prevented GW3965-induced hepatic steatosis (Figure 5A) and significantly reduced hepatic triglyceride accumulation (Figure 5B). In addition, Ad-shTRAP80 reduced the GW3965-induced increase in serum triglyceride levels (Figure 5C). Separation of serum lipoprotein classes by fast protein liquid chromatography (FPLC) indicated that the decrease of serum triglycerides in Ad-shTRAP80–administered mice treated with GW3965 was attributed to a decrease of triglycerides in the VLDL fraction (Figure 5D). We then determined the effects of hepatic TRAP80 knockdown on the RCT process. The increases in both biliary cholesterol output and fecal neutral sterol loss by GW3965 were not affected by TRAP80 knockdown (Figure 5, E and F). In addition, we also observed an increase in HDL cholesterol by GW3965 in the mice injected with Ad-shTRAP80 (Figure 5G). The T0901317 treatment of Ad-shTRAP80–injected mice did not increase hepatic lipogenesis or hypertriglyceridemia, either (Supplemental Figure 6), whereas the level of anti-atherogenic HDL cholesterol was still increased by T0901317 under hepatic TRAP80–knockdown conditions (Supplemental Figure 6F). We also examined the effects of TRAP80 knockdown on the lipoprotein cholesterol and triglyceride profile in Apoe^–/– mice, an experimental animal model of atherosclerosis, and found that the increase in VLDL cholesterol and triglycerides by GW3965 was diminished in Ad-shTRAP80–injected Apoe^–/– mice (Supplemental Figure 7). These results suggest that the detrimental effects of LXR activation could be selectively controlled by TRAP80 modulation.

Figure 5

Knockdown of hepatic TRAP80 ameliorates GW3965-induced fatty liver and an aberrant serum lipid profile. (A) Representative images of H&E and oil red O–stained liver of Ad-shTRAP80–injected mice after administration of 50 mg/kg GW3965 for 3 days. Original magnification, ×100. (B–D) Changes in hepatic triglyceride (B) and serum triglyceride (C and D) levels in mice injected with Ad-shTRAP80 via the tail vein (n = 5 for each group). (E–G) Effects of Ad-shTRAP80 on GW3965-mediated RCT. Biliary cholesterol output (E), fecal neutral sterol (F), and HDL cholesterol (G) were measured (n = 5 for each group). The lipoprotein profile of mouse serum was analyzed by FPLC (D and G). Data are presented as the mean ± SEM. TG, triglyceride. *P < 0.05, **P < 0.01, and ***P < 0.001, by 2-tailed Student’s t test.

Ad-shTRAP80 selectively inhibits lipogenic gene expression in the liver, but does not affect the expression of RCT-related genes or antiinflammatory effects in macrophages. In line with the serum lipid profile, the GW3965- and T0901317-induced transcription of lipogenic genes (Srebp-1c, Fas, and Scd1) was repressed by Ad-shTRAP80 expression in the liver (Figure 6A and Supplemental Figure 8A). On the other hand, Ad-shTRAP80 did not decrease GW3965- or T0901317-induced transcription of RCT-related genes (Abca1, Abcg5, and Abcg8) (Figure 6B and Supplemental Figure 8B). Recently, inducible degrader of low-density lipoprotein receptor (Idol) was reported as a new LXR target gene that regulates hepatic cholesterol uptake by ubiquitination of the LDL receptor (LDLR) (29). The IDOL-mediated inhibition of cholesterol uptake is independent of and complementary to the SREBP pathway (30). Interestingly, we found that the increases in Idol expression by GW3965 or T0901317 were differently affected by TRAP80 knockdown (Figure 6B and Supplemental Figure 8B). Meanwhile, macrophages are the primary cell type that becomes overloaded with cholesterol in atherosclerotic lesions and thus mediates the activation of the inflammatory pathway (15, 31). We found that the T0901317-dependent transcription of RCT-related genes in mouse peritoneal macrophages was not affected by Ad-shTRAP80 (Figure 6C). In contrast, the LXR-dependent increases of lipogenic genes were repressed in Ad-shTRAP80–infected macrophages (Figure 6D). Importantly, the inhibitory effects of T0901317 on the inflammatory gene response to LPS were reproduced in mouse peritoneal macrophages, even under TRAP80-knockdown conditions (Figure 6E). Taken together, these results suggest that TRAP80 selectively regulates the lipogenic activity of LXR, but not RCT or the antiinflammatory effects induced by LXR activation. On the basis of the results described herein, we propose a model for selective activation of the RCT process by simultaneous TRAP80 knockdown and treatment with LXR ligand (Figure 7).

Figure 6

Effects of TRAP80 on the expression of metabolic and inflammatory genes in the liver and peritoneal macrophages. (A and B) Effects of TRAP80 knockdown on the transcript levels of lipogenesis- (A) or RCT-related genes (B) in the livers of mice treated with GW3965 (n = 5 for each group). mRNA levels were measured by qRT-PCR. (C and D) Effects of Ad-shTRAP80 on the transcript levels of RCT- (C) or lipogenesis-related genes (D) in mouse peritoneal macrophages treated with DMSO or 1 μM T0901317 (n = 4). (E) Effects of Ad-shTRAP80 on the transcript levels of inflammation-related genes in mouse peritoneal macrophages (n = 4). Macrophages were pretreated with 1 μM T0901317 for 18 hours and then challenged with 100 ng/ml LPS for another 24 hours. Data are presented as the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, by 1-way ANOVA.

Figure 7

Working model for selective activation of RCT-related genes by simultaneous TRAP80 knockdown and treatment with LXR ligand. TRAP80 is essential for the activation of lipogenic genes including SREBP-1c, but not for RCT-related genes, such as ABCA1. Therefore, under conditions of TRAP80 knockdown, LXR ligand treatment selectively activates RCT-related genes.

In the present study, we identified LXRα-interacting nuclear proteins in HepG2 cells in an effort to elucidate the lipogenic processes mediated by LXR in the liver. There are 2 subtypes of LXR: LXRα and LXRβ. No obvious phenotype has yet been attributed to Lxrb-knockout mice. However, Lxra-knockout mice exhibited decreased serum triglyceride levels as well as decreased hepatic mRNA levels of multiple enzymes that participate in fatty acid synthesis, including FAS (32). These findings suggest that LXRα is primarily responsible for hepatic triglyceride accumulation and hypertriglyceridemia, effects that have hampered the development and use of LXR agonists as therapeutic agents (13). The adverse lipogenic effects of LXRα are in part mediated by LXR-induced transcription of SREBP-1c, a transcription factor that also induces the transcription of numerous lipogenic genes (33–35). To circumvent the adverse effect of LXR agonists, it is necessary to understand the mechanism by which SREBP-1c is activated by LXRα and identify the proteins involved in this activation process.

In this regard, we report for the first time to our knowledge the role of TRAP80 in the transcriptional regulation of SREBP-1c by LXRα. TRAP80 was first isolated as a component of the thyroid hormone receptor–associated protein (TRAP), which was initially identified as a large multimeric complex that copurifies with the thyroid hormone receptor (TR) from HeLa cells and distinctly increases TR-mediated transcription in vitro (23). Later, TRAP80 was also shown to interact directly with p53 and VP16 activation domains (22). TRAP80, also called DRIP77, ARC77, or CRSP77, is also identified as a subunit of vitamin D receptor–interacting protein (DRIP), activator-recruited cofactor (ARC), or cofactor required for Sp1 activation (CRSP), respectively (24, 26, 36). ARC can be purified by affinity chromatography using the SREBP-1α activation domain and has been shown to interact directly with VP16 and the NF-κB p65 subunit (26). Although more recent studies implicate TRAP as a coactivator of a wide range of nuclear hormone receptors as well as other classes of transcription factors, most studies focus on TRAP220/DRIP205, because it is thought to form an interface between the nuclear receptor and the TRAP complex and interact with nuclear receptors in a ligand-dependent manner (37). Consequently, very little is known about the other subunits of the TRAP complex, including TRAP80. TRAP80 is a 651–amino acid protein and is equivalent to the p78 component of the mouse Mediator (38). Our study shows that TRAP80 interacts with LXRα LBD in a ligand-dependent manner in nuclear extract–binding experiments and in a ligand-independent manner in 2-component GST pull-down assays. The same interaction pattern was seen between vitamin D receptor–LBD (VDR-LBD) and TRAP80/DRIP77 (36). This suggests that the ligand-dependent interactions between TRAP80 and LXR and VDR require nuclear component(s) other than TRAP80 and LXR or VDR alone. Interestingly, although full-length dTRAP80 of Drosophila interacts with retinoid X receptor (RXR) LBD only in the presence of the RXR ligand, 9-cis retinoic acid, a fragment of dTRAP80 possesses a sticky region that interacts with RXR LBD, regardless of the presence of ligand (39).

Here, we purified 4 coactivators with LXRα in the presence of ligand from hepatoma HepG2 cells and from HEK 293T cells transfected with FLAG-LXRα. Two of the coactivators, TRAP220 and TRAP80, interacted with LXRα directly and enhanced ligand-dependent transcription of the synthetic LXRE–driven reporter gene. However, while TRAP220 activated the transcription of both SREBP-1c and ABCA1, TRAP80 selectively activated SREBP-1c, but not ABCA1, although both are LXR target genes. This specificity of TRAP80 is attributed to selective recruitment to the SREBP-1c promoter, but not to the ABCA1 promoter. The ligand-stimulated recruitment of TRAP80 to the SREBP-1c LXRE was completely dependent on LXRα. On the other hand, it is noteworthy that LXRα-specific knockdown did not block TRAP220 recruitment to the ABCA1 promoter, while it completely blunted ligand-dependent recruitment of both TRAP220 and TRAP80 to the SREBP-1c promoter. Based on these observations, we hypothesized that LXRα plays a major role in T0901317- or GW3965-dependent activation of the SREBP-1c gene and recruitment of relevant coactivators to the LXRE region of SREBP-1c, while LXRβ has a main role in the LXR ligand–dependent activation of the ABCA1 gene via the recruitment of TRAP220.

The ChIP assay using anti–RNA Pol II and -H4KAc antibodies showed that TRAP80 could facilitate the recruitment of RNA Pol II via chromatin remodeling through acetylation of H4 near the LXRE region of the Srebp-1c gene. However, we observed that LXR was recruited to the LXRE regions of both Srebp-1c and Abca1 genes, irrespective of TRAP80 expression. This suggests that TRAP80 stimulates SREBP-1c transcription through the increase in RNA Pol II recruitment and histone acetylation, at least in part, but not through the enhancement of LXR binding. Consequently, Ad-shTRAP80 selectively decreased lipogenic gene expression, but did not affect the expression of RCT-related genes or the antiinflammatory effects of the LXR ligand. Meanwhile, IDOL is the E3 ubiquitin ligase that stimulates LDLR degradation and thus inhibits cholesterol uptake. In our study, Idol expression was differently regulated by Ad-shTRAP80, depending on the LXR ligands. The meaning of this differential regulation of IDOL by Ad-shTRAP80 in the context of different LXR ligands remains to be determined.

These differential effects of TRAP80 on LXR target gene expression resulted in alleviation of fatty liver and potentiation of RCT activity in the combinatorial treatment with LXR agonist and shTRAP80. In terms of RCT, we measured the expression of ABC transporters (ABCA1, ABCG1, ABCG5, and ABCG8) and the levels of biliary cholesterol and fecal sterol. RCT is a multistep process that occurs across several tissues including macrophages, liver, and intestine. Moreover, the ABC transporters are involved in the overall RCT process, from macrophages to feces, not just in cholesterol efflux from macrophages. Thus, we investigated the very last steps in RCT — biliary cholesterol secretion and fecal sterol loss — to evaluate the effect of TRAP80 on LXR-stimulated RCT.

This study suggests that the manipulation of TRAP80 represents a mechanism by which SREBP-1c–mediated lipogenesis can be selectively repressed while preserving RCT stimulation by LXRα. Therefore, TRAP80 could be a promising target not only for the treatment of fatty liver, but also for the development of LXR agonists that effectively prevent atherosclerosis. In addition, the mechanism of coactivator selectivity described here might be used to modulate the specific expression of other key regulatory proteins responsible for the pathogenesis of a diverse array of diseases.

View Supplemental data

We are grateful to Robert Roeder for providing TRAP230; Mononari Uesugi for providing DRIP130 (CRSP130); and Jae U. Jung for providing TRAP80 (CRSP77). We also thank Han Choe (Department of Physiology, University of Ulsan College of Medicine) and Hyun Ju Yoo (Metabolomics Core, Asan Institute for Life Sciences) for their help with FPLC lipoprotein analysis and GC analysis of fecal neutral sterol, respectively. This study was supported by grants from the Korea Healthcare Technology R&D Project (A084335); the Ministry for Health, Welfare & Family Affairs of the Republic of Korea; the National Research Foundation of Korea funded by the Korean Government (NRF-2006-2005402); and by intramural grants 2009-352 and 2010-352 (to S.W. Kim) from the Asan Institute for Life Sciences.

Address correspondence to: Seung-Whan Kim, 88, Olympic-Ro 43-gil, Songpa-Gu, Seoul 138-736, Republic of Korea. Phone: 822.3010.4297; E-mail: swkim7@amc.seoul.kr.

Conflict of interest: The authors have declared that no conflict of interest exists.

Reference information: J Clin Invest. 2015;125(1):183–193. doi:10.1172/JCI73615.

Geun Hyang Kim’s present address is: Sanford-Burnham Medical Research Institute at Lake Nona, Orlando, Florida, USA.

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