Hyo-Sung Ro

PPARgamma1 and LXRalpha face a new regulator of macrophage cholesterol homeostasis and inflammatory responsiveness, AEBP1.

Peroxisome proliferator-activated receptor γ1 (PPARγ1) and liver X receptor α (LXRα) are nuclear receptors that play pivotal roles in macrophage cholesterol homeostasis and inflammation; key biological processes in atherogenesis. The activation of PPARγ1 and LXRα by natural or synthetic ligands results in the transactivation of ABCA1, ABCG1, and ApoE; integral players in cholesterol efflux and reverse cholesterol transport. In this review, we describe the structure, isoforms, expression pattern, and functional specificity of PPARs and LXRs. Control of PPARs and LXRs transcriptional activity by coactivators and corepressors is also highlighted. The specific roles that PPARγ1 and LXRα play in inducing macrophage cholesterol efflux mediators and antagonizing macrophage inflammatory responsiveness are summarized. Finally, this review focuses on the recently reported regulatory functions that adipocyte enhancer-binding protein 1 (AEBP1) exerts on PPARγ1 and LXRα transcriptional activity in the context of macrophage cholesterol homeostasis and inflammation.

Transcriptional regulation by the γ5 subunit of a heterotrimeric G protein during adipogenesis

The adipocyte enhancer‐binding protein (AEBP1) is a novel transcriptional repressor with carboxypeptidase activity. A two‐hybrid screen was conducted to identify components of AEBP1 that might be important in regulating its activity. The γ5 subunit of a heterotrimeric G protein was shown to bind specifically to AEBP1 and to attenuate its transcriptional repression activity. Adipogenic stimulation selectively decreased the Gγ5 level and enhanced the transcriptional repression activity of AEBP1 during mitotic clonal expansion at the onset of adipogenesis. Thus, the actions of Gγ5 and AEBP1 are directly linked, which could provide the basis for the regulation of transcription at the onset of differentiation. This report shows that a signal‐transducing molecule is involved, by direct protein–protein interaction, in the regulation of transcription during adipogenesis.

The trans-10, cis-12 isomer of conjugated linoleic acid decreases adiponectin assembly by PPARγ-dependent and PPARγ-independent mechanisms

The adipocyte-derived secretory protein adiponectin functions as an insulin-sensitizing agent. In plasma, adiponectin exists as low, medium, and high molecular weight oligomers. Treatment with trans-10, cis-12 conjugated linoleic acid (t-10, c-12 CLA) reduces levels of adiponectin as well as triglyceride (TG) in mice and adipocyte cell culture models. The aim of this study was to determine whether the effects of t-10, c-12 CLA on adiponectin and TG are mediated through modulation of the transcription factor peroxisome proliferator-activated receptor γ (PPARγ). 3T3-L1 cells were treated either during or after differentiation into adipocytes with 100 μM t-10, c-12 CLA with or without 10 μM troglitazone, a PPARγ agonist, or 1 μM GW9662, a PPARγ antagonist, and adiponectin and TG levels were analyzed. Treatment with t-10, c-12 CLA reduced TG as well as cellular and secreted adiponectin levels and impaired the assembly of adiponectin oligomers. These changes were accompanied by decreases in PPARγ mass. Troglitazone was able to reverse the t-10, c-12 CLA-mediated decrease in TG levels and restore the assembly of adiponectin oligomers but was unable to restore adiponectin synthesis. Conversely, treatment with GW9662 decreased TG mass and impaired adiponectin oligomer assembly but did not decrease total adiponectin mass. In a reporter assay, t-10, c-12 CLA appeared to be a partial PPARγ agonist and prevented the stimulation of reporter activity by troglitazone. Therefore, the t-10, c-12 CLA isomer appears to alter adipocyte adiponectin metabolism through PPARγ-dependent and PPARγ-independent mechanisms.

Cloning and Characterization of a Novel Zinc Finger Transcriptional Repressor A DIRECT ROLE OF THE ZINC FINGER MOTIF IN REPRESSION

We have identified a novel transcriptional repressor, AEBP2, that binds to a regulatory sequence (termed AE-1) located in the proximal promoter region of the aP2 gene that encodes the adipose fatty acid-binding protein. Sequence analysis of AEBP2 cDNA revealed that it encodes a protein containing three Gli-Krüppel (Cys2-His2)-type zinc fingers. Northern blot analysis revealed two transcripts (4.5 and 3.5 kilobases) which were ubiquitously expressed in every mouse tissue examined. In co-transfection assays, AEBP2 repressed transcription from the homologous aP2 promoter containing multiple copies of the AE-1 sequence. Moreover, a chimeric construct encoding a fusion AEBP2 protein with the Gal4 DNA-binding domain was able to repress the transcriptional activity of a heterologous promoter containing the Gal4-binding sequence. The transcriptional repression function of AEBP2 was completely abolished when one of the conserved histidine residues and a flanking serine residue in the middle zinc finger were replaced with an arginine residue. The defective transcriptional repression function of the mutant derivative was due neither to lack of expression nor to a failure to localize to the nucleus. Moreover, both the wild-type and mutant derivative of either the histidine-tagged recombinant AEBP2 proteins or the in vitro translated Gal4-AEBP2 fusion proteins were equally able to bind to the target DNA. These results suggest that a portion of the zinc finger structure may play a direct role in transcriptional repression function, but not in DNA binding.

Regulation of IkappaBalpha function and NF-kappaB signaling: AEBP1 is a novel proinflammatory mediator in macrophages.

NF-κB comprises a family of transcription factors that are critically involved in various inflammatory processes. In this paper, the role of NF-κB in inflammation and atherosclerosis and the regulation of the NF-κB signaling pathway are summarized. The structure, function, and regulation of the NF-κB inhibitors, IκBα and IκBβ, are reviewed. The regulation of NF-κB activity by glucocorticoid receptor (GR) signaling and IκBα sumoylation is also discussed. This paper focuses on the recently reported regulatory function that adipocyte enhancer-binding protein 1 (AEBP1) exerts on NF-κB transcriptional activity in macrophages, in which AEBP1 manifests itself as a potent modulator of NF-κB via physical interaction with IκBα and a critical mediator of inflammation. Finally, we summarize the regulatory roles that recently identified IκBα-interacting proteins play in NF-κB signaling. Based on its proinflammatory roles in macrophages, AEBP1 is anticipated to serve as a therapeutic target towards the treatment of various inflammatory conditions and disorders.

LPS-induced suppression of macrophage cholesterol efflux is mediated by adipocyte enhancer-binding protein 1.

Macrophages facilitate clearance of cholesterol from the body via reverse cholesterol transport (RCT). The first event in RCT is internalization of modified low density lipoprotein by macrophages, upon which PPARgamma1 and LXRalpha signaling pathways are turned on, leading to the transactivation of a cascade of genes (e.g. ABCA1 and ABCG1), whose products promote macrophage cholesterol efflux. Down-regulation of macrophage cholesterol efflux mediators leads to an imbalance in cholesterol homeostasis, promoting foam cell formation. Lipopolysaccharide (LPS) has been shown to suppress PPARgamma1 and its downstream target genes in macrophages, inducing foam cell formation; a key mechanism proposed to underlie bacterial infection-induced atherosclerosis. Herein, we show that adipocyte enhancer-binding protein 1 (AEBP1) is up-regulated during monocyte differentiation. Moreover, we provide experimental evidence suggesting that AEBP1 expression is induced by LPS, and that LPS-induced down-regulation of pivotal macrophage cholesterol efflux mediators, leading to foam cell formation, is largely mediated by AEBP1. Although AEBP1-independent pathways seem to contribute to these LPS effects, such pathways can only mediate lesser and delayed effects of LPS on macrophage cholesterol efflux and development of foam cells. We speculate that AEBP1 may serve as a potential therapeutic target for the prevention/treatment of bacterial infection-induced atherosclerosis.

Regulation of IκBα Function and NF-κB Signaling: AEBP1 Is a Novel Proinflammatory Mediator in Macrophages

NF-κB comprises a family of transcription factors that are critically involved in various inflammatory processes. In this paper, the role of NF-κB in inflammation and atherosclerosis and the regulation of the NF-κB signaling pathway are summarized. The structure, function, and regulation of the NF-κB inhibitors, IκBα and IκBβ, are reviewed. The regulation of NF-κB activity by glucocorticoid receptor (GR) signaling and IκBα sumoylation is also discussed. This paper focuses on the recently reported regulatory function that adipocyte enhancer-binding protein 1 (AEBP1) exerts on NF-κB transcriptional activity in macrophages, in which AEBP1 manifests itself as a potent modulator of NF-κB via physical interaction with IκBα and a critical mediator of inflammation. Finally, we summarize the regulatory roles that recently identified IκBα-interacting proteins play in NF-κB signaling. Based on its proinflammatory roles in macrophages, AEBP1 is anticipated to serve as a therapeutic target towards the treatment of various inflammatory conditions and disorders.

Adipocyte Enhancer‐Binding Protein 1 Modulates Adiposity and Energy Homeostasis

Objective: To determine whether adipocyte enhancer binding protein (AEBP) 1, a transcriptional repressor that is down‐regulated during adipogenesis, functions as a critical regulator of adipose tissue homeostasis through modulation of phosphatase and tensin homolog deleted on chromosome ten (PTEN) tumor suppressor activity and mitogen‐activated protein kinase (MAPK) activation.

Gene structure and expression of the mouse adipocyte enhancer-binding protein.

The adipocyte enhancer-binding protein (AEBP1) is a transcriptional repressor with carboxypeptidase activity. AEBP1 expression is down-regulated during adipogenesis. Aortic carboxypeptidase-like protein (ACLP) is a non-nuclear isoform of AEBP1 that has an N-terminal extension of 380 amino acids. ACLP expression is up-regulated during vascular smooth muscle cell differentiation. To gain insight into the regulation of AEBP1 isoform expression, we have determined the structural organization of the mouse AEBP1 gene. This gene extends over 10 kb, has 21 exons, and gives rise to two mRNAs (AEBP1 and ACLP). The 9th intron is retained in the mature AEBP1 transcript. Thus, ACLP encodes an additional 380 amino acids N-terminal to the first ATG codon of AEBP1 which is located in exon 10. RT-PCR experiments showed that both transcripts are expressed ubiquitously in all mouse tissues examined, while Western blot analysis suggested that expression is translationally regulated. Our results provide evidence that two isoforms of AEBP1 with very different functions are produced by an alternative splicing mechanism. This represents a new example of regulation of subcellular localization by protein truncation.

Adipocyte enhancer-binding protein 1 (AEBP1) (a novel macrophage proinflammatory mediator) overexpression promotes and ablation attenuates atherosclerosis in ApoE (-/-) and LDLR (-/-) mice.

Atherogenesis is a long-term process that involves inflammatory response coupled with metabolic dysfunction. Foam cell formation and macrophage inflammatory response are two key events in atherogenesis. Adipocyte enhancer-binding protein 1 (AEBP1) has been shown to impede macrophage cholesterol efflux, promoting foam cell formation, via peroxisome proliferator-activated receptor (PPAR)-γl and liver X receptor α (LXRα) downregulation. Moreover, AEBP1 has been shown to promote macrophage inflammatory responsiveness by inducing nuclear factor (NF)-κB activity via IκBα downregulation. Lipopolysaccharide (LPS)-induced suppression of pivotal macrophage cholesterol efflux mediators, leading to foam cell formation, has been shown to be mediated by AEBP1. Herein, we showed that AEBP1-transgenic mice (AEBP1TG) with macrophage-specific AEBP1 overexpression exhibit hyperlipidemia and develop atherosclerotic lesions in their proximal aortas. Consistently, ablation of AEBP1 results in significant attenuation of atherosclerosis (males: 3.2-fold, P = 0.001 (en face)), 2.7-fold, P = 0.0004 (aortic roots); females: 2.1-fold, P = 0.0026 (en face), 1.7-fold, P = 0.0126 (aortic roots)) in the AEBP1−/−/low-density lipoprotein receptor (LDLR)−/− double-knockout (KO) mice. Bone marrow (BM) transplantation experiments further revealed that LDLR−/− mice reconstituted with AEBP1−/−/LDLR−/− BM cells (LDLR−/−/KO-BM chimera) display significant reduction of atherosclerosis lesions (en face: 2.0-fold, P = 0.0268; aortic roots: 1.7-fold, P = 0.05) compared with control mice reconstituted with AEBP1+/+/LDLR−/− BM cells (LDLR−/−/WT-BM chimera). Furthermore, transplantation of AEBP1TG BM cells with the normal apolipoprotein E (ApoE) gene into ApoE−/− mice (ApoE−/−/TG-BM chimera) leads to significant development of atherosclerosis (males: 2.5-fold, P = 0.0001 (en face), 4.7-fold, P = 0.0001 [aortic roots]; females: 1.8-fold, P = 0.0001 (en face), 3.0-fold, P = 0.0001 [aortic roots]) despite the restoration of ApoE expression. Macrophages from ApoE−/−/TG-BM chimeric mice express reduced levels of PPARγ1, LXRα, ATP-binding cassette A1 (ABCA1) and ATP-binding cassette G1 (ABCG1) and increased levels of the inflammatory mediators interleukin (IL)-6 and tumor necrosis factor (TNF)-α compared with macrophages of control chimeric mice (ApoE−/−/NT-BM) that received AEBP1 nontransgenic (AEBP1NT) BM cells. Our in vivo experimental data strongly suggest that macrophage AEBP1 plays critical regulatory roles in atherogenesis, and it may serve as a potential therapeutic target for the prevention or treatment of atherosclerosis.