A, PPARγ driven reporter activity is increased by aP2 deficiency. Macrophage cell lines were co-transfected with pPPRE-Luc and phRL-null-Luc reporter constructs and stimulated for 24 h with 25 μm ciglitazone (Cig) or 25 μm 9-cis-retinoic acid (RA). Relative promoter activities are reported as a ratio of firefly to Renilla luciferase activities ± S.D. of triplicate determinations. B, the PPARγ-specific antagonist, GW9662 (GW), represses agonist driven reporter activity. Macrophage cell lines were transfected as in A and preincubated for 1 h with 100 nm GW9662 before treatment with Cig and 9-cis-RA. Relative promoter activities are reported as a ratio of firefly to Renilla luciferase activities ± S.D. of triplicate determinations. C, aP2 deficiency increases mRNA expression of PPARγ-regulated genes. The expression of CD36, LXRα, LXRβ, and ABCA1 was detected by Northern blot analysis using RNA isolated from macrophage cell lines, untreated aP2^+/+ and aP2^−/− (left), and aP2^−/− and aP2^−/− R (right) with the exception of LXRα on the right (treated for 24 h with 10 ng/ml IL-4). D, aP2 deficiency increases CD36 protein expression. Western blot was used to determine CD36 protein expression in splenic macrophages (unstim) treated with 20 ng/ml IL-4 for 48 h. E, expression of ABCA1 protein is elevated by aP2 deficiency. Western blot was used to determine ABCA1 protein expression in aP2^+/+ and aP2^−/− macrophage cell lines (unstim) incubated for 24 h with 25 μm Cig.

A, deficiency of aP2 diminishes production of proteins from NF-κB-regulated genes. aP2^+/+ and aP2^−/− macrophages were left unstimulated (un) or incubated for 12 h with 100 ng/ml LPS (left panel) or 100 ng/ml LPS + 10 units/ml murine IFNγ (right panel) before Western blot analysis. B, production of COX-2 and iNOS enzymatic products is reduced by aP2 deficiency. aP2^+/+ and aP2^−/− macrophage cell lines were stimulated with LPS at the concentrations shown for 24 h, and supernatants were assayed for PGE₂ content by ELISA (left panel). Data shown are mean ± S.D. of triplicate determinations. aP2^+/+ and aP2^−/− macrophage cell lines were stimulated with LPS (500 ng/ml) and/or IFNγ (1 units/ml) for 48 h before measuring nitrite content of supernatants. Data shown are mean ± S.D. of triplicate determinations (right panel).

A hypothetical model of the role of aP2 in macrophages is depicted. In the presence of aP2 (left panel), the availability of fatty acids (FA) or other bioactive lipid mediators is restricted, hence fatty acids are unable to interact with targets such as PPARγ and components of the IKK-NF-κB pathway. As such, aP2 may act to control signaling through these pathways. In the absence of aP2 (right panel), increased availability of FA inhibits the NF-κB pathway, resulting in the blockade of inflammatory responses, and enhances PPARγ activity, resulting in accelerated cholesterol trafficking. Although the identity of the key aP2 ligands and the specific mechanisms of action remain to be determined, this model suggests that aP2 may coordinately control macrophage signaling pathways that contribute to the pathogenesis of metabolic syndrome and atherosclerosis.

A, internalization of acLDL is enhanced by aP2 deficiency. aP2^+/+, aP2^−/−, and aP2^−/−R macrophages (Brightfield) were treated with 10 μg/ml DiI-acLDL (red) for 4 h. B, aP2 deficiency enhances flux of cholesterol into macrophages. oxLDL internalization by thioglycollate-elicited macrophages was assessed by treatment with ¹²⁵I-oxLDL in the presence or absence of excess unlabeled oxLDL for 24 h. Specific degradation is the percent uptake normalized to milligram of cellular protein ± S.E. C, stable expression of aP2 into aP2^−/− macrophages (aP2^−/−R) restores oxLDL uptake to aP2^+/+ levels. Macrophage cell lines were treated the same as in B. D, cholesterol efflux from macrophages is increased by aP2 deficiency at base line and with induction. Thiogly-collate-elicited aP2^+/+ and aP2^−/− macrophages were loaded for 24 h with [³H]AcLDL, and cholesterol efflux to 0 (black bar) or 10 μg/ml (gray bar) apoA1 proceeded for 3 h. Percent efflux was normalized to cellular protein content and expressed as a percentage relative to aP2^+/+ cultured with no apoA1 (100%) ± S.E. E, stable restoration of aP2 into aP2^−/− macrophages reduces induced cholesterol efflux. Macrophage cell lines were treated as in D with the exception that 0 (black bar) or 15 μg/ml (gray bar) apoA1 was used.

A, kinase activity of IKK is impaired by aP2 deficiency. The activity of IKK was determined by in vitro kinase phosphorylation of an IκBα-GST substrate after macrophage lines were stimulated by 1 μg/ml LPS for the indicated times. Western blot analysis of immunoprecipitated IKKα/β is shown on the right. B, reconstitution of aP2 restores kinase activity of IKK. aP2^+/+, aP2^−/−, and aP2^−/−R macrophage cell lines were stimulated by 1 μg/ml LPS for 10 min, and kinase activity of IKK was assayed as in A. Data shown are representative of three independent kinase assays. C, NF-κB binding to DNA is abrogated by aP2 deficiency. EMSA was used to determine NF-κB DNA binding activity of nuclear protein extracts from aP2^+/+ and aP2^−/− macrophages (−) stimulated with 1 μg/ml LPS (+) for 30 min using radiolabeled or non-labeled (cold competition, CC) NF-κB consensus-binding sequences (top panel). Nuclear extracts from the Burkitt's lymphoma cell line, Raji, served as a positive control. Western blot analysis of NF-κB in untreated macrophage lines (bottom panel). D, NF-κB driven reporter activity is impaired by aP2 deficiency. Macrophage lines were co-transfected with pNF-κB-Luc and phRL-null-Luc reporter constructs and activated with 1 μg/ml LPS (top panel) or with 0 (unstim), 25, 50, or 75 μg of membrane extracts from CHO-control cells or CHO-CD154 (bottom panel) for 16 h. Relative promoter activities are reported as the ratio of firefly to Renilla luciferase activity ± S.D. of triplicate samples (top) or fold induction by CHO-CD154 relative to CHO control (bottom).

A, production of inflammatory cytokine RNA is reduced by aP2 deficiency. aP2^+/+ and aP2^−/− macrophages were stimulated by 1 μg/ml LPS for 0, 2, 4, and 8 h, and cytokine mRNA expression was analyzed by RNase protection assay (top panel). IL-12p40 is shown in a darker exposure in lower panel. Band density of specific RNA is expressed as a percentage of L32 density (bottom panel). GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MIF, migration inhibitory factor. B, LPS induction of inflammatory cytokines and chemokine is reduced by aP2 deficiency. LPS stimulation of cytokines and chemokine production from aP2^+/+ and aP2^−/− macrophages stimulated for 18 h for IL-6 and MCP-1 and 6 h for tumor necrosis factor α were analyzed by ELISA. C, macrophages were stimulated with CD154 and assayed as in B. Data shown are mean ± S.D. of triplicate determinations. D, aP2 reconstitution into aP2^−/− macrophages restores MCP-1 production. Supernatants from aP2^+/+ and aP2^−/− macrophage cell lines were evaluated for MCP-1 secretion by ELISA after 0 (unstim), 2.5, or 25 nm PMA treatment for 6 h. Data are presented as mean ± S.E. of three determinations.