We have investigated whether a raft heterogeneity exists in human monocyte-derived macrophages and fibroblasts and whether these microdomains are modulated by lipid efflux. Triton X-100 (Triton) or Lubrol WX (Lubrol) detergent-resistant membranes from cholesterol-loaded monocytes were associated with the following findings: (i) Lubrol-DRM contained most of the cellular cholesterol and at least 75% of Tritondetergent-resistant membranes. (ii) 'Lubrol rafts', defined by their solubility in Triton but insolubility in Lubrol, were enriched in unsaturated phosphatidylcholine and showed a lower cholesterol to choline-phospholipid ratio compared to Triton rafts. (iii) CD14 and CD55 were recovered in Triton-and Lubrol-detergent-resistant membranes, whereas CD11b was found exclusively in Triton DRM. ABCA1 implicated in apo AI-mediated lipid efflux and CDC42 were partially localized in Lubrol-but not in Triton-detergent-resistant membranes. (iv) Apo AI preferentially depleted cholesterol and choline-phospholipids from Lubrol rafts, whereas HDL 3 additionally decreased the cholesterol content of Triton rafts. In fibroblasts, neither ABCA1 nor CDC42 was found in Lubrol rafts, and both apo AI and HDL 3 reduced the lipid content in Lubrol-as well as in Tritondetergent-resistant membranes. In summary, we provide evidence for the existence of compositionally distinct membrane microdomains in human cells and their modulation by apo AI/ABCA1-dependent and HDL 3 -mediated lipid efflux. Key words: ABCA1, CDC42, microvilli, monocytes, rafts Received 12 November 2001, revised and accepted for publication 21 January 2002More than 10 years ago the raft hypothesis was introduced, suggesting that horizontal interaction of certain membrane lipids forms the basis for plasma membrane microdomains 268(termed rafts) that are characterized by the enrichment of cholesterol, sphingolipids and saturated phospholipids (1-3). The specific lipid composition renders these microdomains resistant to solubilization in Triton X-100 (Triton) at 4 aeC, a property that has been widely used to study lipid rafts designated 'Triton rafts' (4). As recently reviewed by Simons and Toomre, the most important role of rafts may be their function in signal transduction and cell regulation by providing a platform for ligand-triggered clustering of receptors and signaling molecules (5). There are, however, still many open issues, including the question whether more than one kind of raft exists on the cell surface of different cell types (6). In support of the existence of different types of rafts, Röper et al. recently identified a novel type of cholesterol-based microdomain that is discriminated from the classical lipid rafts by its solubility in Triton but its resistance to another non-ionic detergent, Lubrol WX (Lubrol) (7). This microdomain has been designated 'Lubrol-raft', and current data suggest that it serves as a building unit for different types of plasma membrane protrusions. The formation of membrane protrusions, like lamellipodia, microvilli, or filopodia...
The zinc finger gene 202 (ZNF202) located within a hypoalphalipoproteinemia susceptibility locus on chromosome 11q23 is a transcriptional repressor of various genes involved in lipid metabolism. To provide further evidence for a functional linkage between ZNF202 and hypoalphalipoproteinemia, we investigated the effect of ZNF202 expression on ATP binding cassette transporter A1 (ABCA1) and ABCG1. ABCA1 is a key regulator of the plasma high density lipoprotein pool size, whereas ABCG1 is another mediator of cellular cholesterol and phospholipid efflux in human macrophage. We demonstrate here that the full-length ZNF202m1 isoform binds to GnT repeats within the promotors of ABCA1 (؊229/ ؊210) and ABCG1 (؊572/؊552). In past years, evidence has accumulated to suggest that a number of transcription factors play critical roles in the coordinate transcriptional regulation of genes involved in lipid metabolism (7,8). Linkage analysis in large Utah pedigrees led to the identification of a low HDL-cholesterol locus on chromosome 11q23 that is distinct from the apoAI/C-III/AIV gene cluster (9). This novel familial susceptibility locus for hypoalphalipoproteinemia contains the zinc finger protein 202 (ZNF202) originally described as a testis-specific transcription factor (9, 10). ZNF202 is expressed in two common splice variants. The m1 splice form encodes a full-length protein of 648 amino acids with an amino-terminal SCAN domain, a central KRAB repression domain, and 8 carboxyl-terminal Cys 2 -His 2 zinc finger motifs. The m3 splice form encodes a carboxylterminal-truncated protein of 133 amino acids that contains only the SCAN domain. The SCAN domain of ZNF202 has been shown to mediate selective protein oligomerization and the zinc finger motifs to bind to specific DNA elements (9, 11). Intriguingly, the ZNF202 DNA binding elements are present in promotors of various genes involved in lipid metabolism including apolipoproteins and lipid-processing enyzmes. ZNF202 has been therefore proposed to function as a transcriptional regulator of lipid metabolism (9).Based on this information, we tested the hypothesis of whether the ABC lipid transporters ABCA1 and ABCG1 are transcriptionally regulated by ZNF202. Our results provide evidence that ZNF202 acts as a transcriptional repressor of both ABCA1 and ABCG1 and, thus, establish a functional link between ZNF202 and hypoalphalipoproteinemia. EXPERIMENTAL PROCEDURESCell Culture-HepG2 and RAW264.7 cells (American Type Culture Collection) were cultured in Dulbecco's modified Eagle's medium (BioWhittaker) supplemented with 10% fetal calf serum (Sigma) in a 5% CO 2 atmosphere at 37°C. Cells (1 ϫ 10 6 cells/2-ml medium) were
The ATP-binding cassette transporter A1 (ABCA1) shows a differentiation-, cAMP-, and sterol-dependent up-regulation in human monocytes. As part of an ongoing study, we investigated the proximal promoter regions that are highly conserved between the human and murine ABCA1 genes. Using reporter gene assays, we show here that a TATA box 24 bp upstream of the transcription initiation site is essential for promoter activity in RAW 264.7 and HepG2 cells, whereas further enhancement of transcriptional activity is mediated by the ؊175 bp promoter region. Gel shift assays revealed in vitro binding of Sp1 to a ؊91 GnC motif as well as binding of Sp1 and Sp3 to a ؊157 GnC promoter region. In cotransfection experiments using Drosophila S2 cells, we demonstrate that Sp3 competes with Sp1 for binding to the ؊157 GnC motif and acts as a repressor. On the other hand, overexpression of Sp1 increased ABCA1 mRNA expression in HeLa cells and enhanced cellular cholesterol and phospholipid efflux in RAW 246.7 macrophages. We also show here that the conserved E-box at position ؊140 binds upstream stimulatory factors 1 and 2 and hepatic nuclear factor 1␣ and that mutagenesis of the E-box enhanced constitutive ABCA1 expression in RAW 264.7 cells, implying a role for this element in silencing ABCA1 expression. Besides the functional importance for basal gene expression, we have identified that the core promoter region (؊175 to ؉224) is also responsible for the induction of ABCA1 by the cytokine oncostatin M, resulting in a rapid increase in ABCA1 mRNA levels in HepG2 cells. Interestingly, this oncostatin M-induced expression is not dependent on the currently known sequence motifs in the ABCA1 promoter. In conclusion, a functional complex of cis-elements within the proximal human ABCA1 promoter associated with the transcription factors Sp1/3, upstream stimulatory factors 1 and 2, and hepatic nuclear factor 1␣ has been characterized, which allows a subtle tissue-specific regulation of ABCA1 gene expression.The ATP-binding cassette transporter A1 (ABCA1) 1 was recently identified as a key regulator of cellular cholesterol efflux (1-3). Mutations of the ABCA1 gene are the causative defect in genetic HDL deficiency syndromes, and affected subjects have a defect in cellular cholesterol removal, which results in the almost complete absence of plasma HDL cholesterol (4). Cells lacking functional ABCA1 are characterized by structural and functional abnormalities including impaired raft/caveolar processing and Golgi-dependent lipid export processes due to impaired vesicular budding and excess lipid storage in the trans-Golgi network (5).Expression of ABCA1 gene transcription is up-regulated in human monocytes during phagocytic differentiation, and its expression is further increased by loading with modified lipoproteins (6) or cAMP treatment (7,8). In addition, peroxisome-proliferator-activated receptor (PPAR) agonists and interferon-␥ modulate ABCA1 expression (9, 10). Following earlier papers describing the ABCA1 promoter sequence (11, 12) (for ...
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