Adipocyte enhancer-binding protein 1 is a potential novel atherogenic factor involved in macrophage cholesterol homeostasis and inflammation

Abstract

Peroxisome proliferator-activated receptor gamma1 (PPARgamma1) and liver X receptor alpha (LXRalpha) play pivotal roles in macrophage cholesterol homeostasis and inflammation, key biological processes in atherogenesis. Herein we identify adipocyte enhancer-binding protein 1 (AEBP1) as a transcriptional repressor that impedes macrophage cholesterol efflux, promoting foam cell formation, via PPARgamma1 and LXRalpha down-regulation. Contrary to AEBP1 deficiency, AEBP1 overexpression in macrophages is accompanied by decreased expression of PPARgamma1, LXRalpha, and their target genes ATP-binding cassette A1, ATP-binding cassette G1, apolipoprotein E, and CD36, with concomitant elevation in IL-6, TNF-alpha, monocyte chemoattractant protein 1, and inducible NO synthase levels. AEBP1, but not the C-terminally truncated DNA-binding domain mutant (AEBP1DeltaSty), represses PPARgamma1 and LXRalpha in vitro. Expectedly, AEBP1-overexpressing transgenic (AEBP1TG) macrophages accumulate considerable amounts of lipids compared with AEBP1 nontransgenic macrophages, making them precursors for foam cells. Indeed, AEBP1-overexpressing transgenic macrophages exhibit diminished cholesterol efflux compared with AEBP1 nontransgenic macrophages, whereas AEBP1-knockout (AEBP1-/-) macrophages exhibit enhanced cholesterol efflux compared with wild-type (AEBP1+/+) macrophages. Our in vitro and ex vivo experimental data strongly suggest that AEBP1 plays critical regulatory roles in macrophage cholesterol homeostasis, foam cell formation, and proinflammation. Thereby, we speculate that AEBP1 may be critically implicated in the development of atherosclerosis, and it may serve as a molecular target toward developing antiinflammatory, antiatherogenic therapeutic approaches.

Fig. 1.

PPARγ1 and LXRα repression by AEBP1. (A) Sequence homology between AE-1 sequence and putative AEBP1-binding sequences within the promoter regions of mLXRα and hPPARγ1 genes. A vertical line represents an exact nucleotide match, and an asterisk represents a purine:purine or a pyrimidine:pyrimidine match. The underlined sequences are deleted in the pGL3–mLXRα–luciferase and pGL3–hPPARγ1–luciferase constructs, respectively. (B) The effect of AEBP1 on PPARγ1 and LXRα expression in CHO cells was assessed by luciferase assays. An empty vector was used to equalize the total amount of DNA transfected. (C) Densitometric analysis and immunoblotting of protein extracts obtained from transiently transfected CHO cells are shown. (D and E) Transcriptional activity of PPARγ1 (D) and LXRα (E) was assessed by luciferase assays by using PPRE–luciferase and LXRE–luciferase constructs, respectively. Statistical significance was determined relative to 0-ng AEBP1 transfection sample (B), empty vector (C), or DMSO treatment (D and E).

Fig. 2.

Repression of PPARγ1 and LXRα by AEBP1 requires DNA binding. (A) The ability of full-length and the C-terminally truncated form of AEBP1 (AEBP1^ΔSty) to repress PPARγ1 and LXRα in CHO cells is assessed by luciferase assays. (B and C) Deletion of putative AEBP1-binding sequences within the promoter regions of PPARγ1 (PPARγ1–M1) and LXRα (LXRα–M3) eliminates PPARγ1 (B) and LXRα (C) repression by AEBP1. Statistical significance was determined relative to empty vector transfection in PPARγ1–M1 and LXRα–M3. (D) EMSA shows that AEBP1 specifically binds to AE-1 homologous sequences in the promoter regions of hPPARγ1 and mLXRα but not to the mutated sequences (hPPARγ1-M and mLXRα-M). For each probe, lane 1 represents ³²P-labeled probe alone, lane 2 represents probe plus purified AEBP1 protein, and lanes 3 and 4 represent probe plus purified AEBP1 protein in presence of specific and nonspecific competitors, respectively.

Fig. 3.

AEBP1 down-regulates major macrophage cholesterol homeostasis mediators. Protein extracts from AEBP1^TG and AEBP1^NT macrophages (A), as well as AEBP1^+/+ and AEBP1^−/− macrophages (C), were subjected to immunoblotting. Densitometric analysis based on actin expression in AEBP1^+/+ and AEBP1^−/− macrophages (B), as well as in AEBP1^+/+ and AEBP1^−/− macrophages (D), is shown. (E–H) Semiquantitative RT-PCR was performed on RNA samples obtained from AEBP1^TG and AEBP1^NT macrophages (E), as well as AEBP1^+/+ and AEBP1^−/− macrophages (G). Densitometric analysis based on β-actin level in AEBP1^TG and AEBP1^NT macrophages (F), as well as AEBP1^+/+ and AEBP1^−/− macrophages (H), is shown. Statistical significance was determined relative to protein expression level or mRNA level in AEBP1^NT or AEBP1^+/+ macrophages.

Fig. 4.

Enhanced macrophage inflammatory responsiveness by AEBP1. (A–D) ELISA was performed to evaluate the secretion of IL-6 (A and C) and TNF-α (B and D) by macrophages. (E–H) Semiquantitative RT-PCR was performed to assess MCP-1 and iNOS expression in AEBP1^TG and AEBP1^NT (E) and AEBP1^+/+ and AEBP1^−/− (G) macrophages. Histograms illustrating MCP-1 and iNOS mRNA levels in AEBP1^TG and AEBP1^NT (F) and AEBP1^+/+ and AEBP1^−/− (H) macrophages are shown. Statistical significance was determined relative to IL-6 and TNF-α secretion and MCP-1 and iNOS mRNA levels in AEBP1^NT (A, B, and F) and AEBP1^+/+ (C, D, and H) macrophages.

Fig. 5.

Regulation of macrophage cholesterol efflux and foam cell formation by AEBP1. Macrophages isolated from 32-wk-old, HFD-fed AEBP1^TG (A) and AEBP1^NT (B) mice were cultured for 72 h in complete medium and subsequently stained with oil red O (red/pink, lipid; blue, nuclei). (C) Macrophage cholesterol efflux efficiency was determined by in vitro cholesterol efflux assays. Data are normalized based on macrophage cholesterol efflux in absence of apolipoprotein A-I.

Fig. 6.

A model implicating AEBP1 as a potentially critical player in macrophage cholesterol homeostasis and atherogenesis. In macrophages, PPARγ1 and LXRα cooperate to induce the expression of major cholesterol efflux mediators that are critically involved in transferring excess cholesterol to its acceptor (i.e., high-density lipoprotein) in plasma. PPARγ1 and LXRα also play imperative antiinflammatory functions by antagonizing the expression of key inflammatory mediators in macrophages. AEBP1 is proposed to impede macrophage cholesterol efflux, induce foam cell formation, and provoke proinflammation. Hence, AEBP1 is anticipated to function as a likely proatherogenic factor, promoting both metabolic and inflammatory processes involved in atherogenesis.