Activating signal cointegrator 2 required for liver lipid metabolism mediated by liver X receptors in mice

Abstract

Activating signal cointegrator 2 (ASC-2), a cancer-amplified transcriptional coactivator of nuclear receptors and many other transcription factors, contains two LXXLL-type nuclear receptor interaction domains. Interestingly, the second LXXLL motif is highly specific to the liver X receptors (LXRs). In cotransfection, DN2, an ASC-2 fragment encompassing this motif, exerts a potent dominant-negative effect on transactivation by LXRs, which is rescued by ectopic coexpression of the full-length ASC-2 but not by other LXXLL-type coactivators, such as SRC-1 and TRAP220. In contrast, DN2/m, in which the LXXLL motif is mutated to LXXAA to abolish the interactions with LXRs, is without any effect. Accordingly, expression of DN2, but not DN2/m, in transgenic mice results in phenotypes that are highly homologous to those previously observed with LXRalpha(-/-) mice, including a rapid accumulation of large amounts of cholesterol and down-regulation of the known lipid-metabolizing target genes of LXRalpha in the liver upon being fed a high-cholesterol diet. These results identify ASC-2 as a physiologically important transcriptional coactivator of LXRs and demonstrate its pivotal role in the liver lipid metabolism.

FIG. 1.

The second LXXLL motif specifically binds to LXRα and LXRβ. (A) A series of chimeric proteins containing amino acids of the second LXXLL motif region within the context of ASC2-2c (i.e., the ASC-2 residues 849 to 1057) were constructed using two-step PCR procedures and synthesized as labeled proteins expressed by in vitro translation using the TNT-coupled transcription-translation system, with conditions as described by the manufacturer. These proteins were incubated with GST fusion to TRβ and LXRβ in the absence and presence of either 0.1 μM T3 or 10 μM T0901317, as previously reported (20). Twenty percent of the total reaction mixture was loaded as input. The ASC-2 residues 875 to 907 (shaded) and 1479 to 1511 (open), containing the first and second LXXLL motif, respectively, are shown. (B) The indicated B42 and LexA plasmids were transformed into a yeast strain containing an appropriate lacZ reporter gene, as described previously (24). The shaded and solid bars indicate the absence and presence of 1 μM of 22(R)-hydroxycholesterol, respectively. Better ligand responsiveness was observed with higher doses of 22(R)-hydroxycholesterol (data not shown). −, B42 alone.

FIG. 2.

DN2 as a specific dominant-negative repressor of LXR transactivation. (A and B) An oxysterol-responsive LXRE-LUC reporter construct was cotransfected into HeLa cells, along with lacZ expression vector (100 ng) and expression vectors for ASC-2, DN2 (i.e., the ASC-2 residues 1431 to 1511), DN2/m, TRAP220, and SRC-1, as indicated. The solid and shaded bars indicate the absence and presence of 10 μM 22(R)-hydroxycholesterol, respectively. Normalized luciferase expressions from triplicate samples were calculated relative to the lacZ expression. Similar results were obtained with CV-1 cells (data not shown). The error bars indicate standard deviations. (C) ASC-2 recruitment to the LXRE region of SREBP-1c. 293T cells were cotransfected with expression vectors for DN2 (50 and 100 ng) and DN2/m (50 and 100 ng) in the presence of 5 μM T0901317, as indicated. Chromatin from these cells was isolated and immunoprecipitated (IP) by the indicated antibodies. The endogenous SREBP-1c-LXRE region present in the immunoprecipitated samples was amplified by PCR, and the input PCR (10%) is shown for loading controls. Similar results were also obtained in the absence of ligand (data not shown).

FIG. 3.

Morphology and histology of livers from DN2-TG (TG) and wild-type (WT) mice on high-cholesterol diets. (A) Expression of DN2 was assessed with indirect immunofluorescence with αHA antibody. TG #4 indicates the DN2-TG line no. 4 (Table 1). (B) Gross morphology of livers from male DN2-TG and wild-type mice fed chow supplemented with 2% cholesterol (HCD, high-cholesterol diet) for 90 days. ND, normal chow diet. The development of fatty livers in the DN2-TG mice is evident after 7 days on the high-cholesterol diet. (C) Sections from livers shown in panel B were prepared for histology and stained with oil red O, as indicated. The unstained vacuoles visible in the hematoxylin and eosin (H & E)-stained sections of the livers from DN2-TG mice on the high-cholesterol diet stain positive (red) for lipids with oil red O. (D and E) Measurements of plasma and hepatic cholesterol quantitated enzymatically from extracts of the livers shown in panel B. All values are expressed as means plus standard errors of the mean; n = 5.

FIG. 4.

Impaired lipogenesis in DN2-TG mice. (A) Liver sections from wild-type (WT) and DN2-TG (TG) mice treated orally with vehicle or 50 mg of T0901317/kg for 6 days were prepared for histology and stained with oil red O, as indicated. #18, DN2-TG line no. 18. (B) Measurements of hepatic triglyceride quantitated enzymatically from extracts of the livers shown in panel A. All values are expressed as means plus standard errors of the mean; n = 5.

FIG. 5.

Confirmation of cDNA microarray results. Total RNA was prepared using Trizol reagent (Life Technologies), according to the instructions given by the manufacturer, from wild-type and DN2-TG mice treated with vehicle alone or 50 mg of T0901317/kg for 6 days (A), as well as Hepa-1c1c7 (B) and RAW264.7 (C) cells treated with either vehicle alone or 10 μM T0901317 for 16 h, as indicated. Semiquantitative RT-PCR was used to determine the relative levels of mRNA for insig-2, PPARγ, IKKɛ, IKKα, IKKβ, and GAPDH. #4, DN2-TG mouse line no. 4.

FIG. 6.

RT-PCR analyses of LXR target genes in DN2-TG mice. Total RNA was prepared from wild-type (WT) and DN2-TG (TG) mice untreated or treated with either a high-cholesterol diet for 60 days (A) or 50 mg of T0901317/kg for 6 days (B). Semiquantitative RT-PCR was used to determine the relative levels of mRNA for lipoprotein lipase (LPL), ABCA1, ABCG1, ABCG5, ABCG8, SREBP-1c, fatty acid synthase (FAS), Cyp7A1, LXRα, SCD-1, acetyl-CoA carboxylase (ACC), DN2, GAPDH, and β-actin.