Characterization of adjacent E-box and nuclear factor 1-like DNA binding sequence in the human CYP1A2 promoter

To better understand the molecular mechanisms of cytochrome P450 1A2 (CYP1A2) regulation, we have characterized a region of the promoter (+3 to -176) that contains a single E-box and an adjacent nuclear factor 1 (NF1)-like DNA binding site. The E-box was shown to specifically bind nuclear proteins that were recognized by antibodies against upstream stimulatory factor (USF) 1 and 2. Comparison of NF1 binding proteins in HepG2 cells and primary cultures of rat hepatocytes revealed different patterns of DNA-protein complexes, all of which were recognized by a general NF1 antibody. Mutations of the E-box resulted in substantial reduction of promoter activity in either primary hepatocytes or HepG2 cells regardless of the presence in the reporter constructs of other CYP1A2 regulatory elements, such as the hepatic nuclear factor 1 (HNF-1) binding site. In contrast, reporter gene activity of the promoter construct harboring the mutated NF1-like binding site was affected by upstream sequences when transfected into HepG2 cells, but not in primary hepatocytes. We conclude that both USF proteins and different isoforms of NF1 contribute to the constitutive expression of CYP1A2.     

Apolipoprotein E4 Is Deficient in Inducing Macrophage ABCA1 Expression and Stimulating the Sp1 Signaling Pathway

ATP binding cassette A1 (ABCA1) is a membrane protein that promotes cellular cholesterol efflux. Using RAW 264.7 macrophages, we studied the relative effects of apolipoprotein (apo) E3 and apoE4 on ABCA1 and on the signaling pathway that regulates its expression. Both lipid-associated and lipid-free apoE4 forms induced ∼30% lower levels of ABCA1 protein and mRNA than apoE3 forms. Phosphorylated levels of phosphoinositol 3-kinase (PI3K), protein kinase Cζ (PKCζ) and specificity protein 1 (Sp1) were also lower when treated with apoE4 compared to apoE3. The reduced ability of apoE4 to induce ABCA1 expression, PKCζ and Sp1 phosphorylation were confirmed in human THP-1 monocytes/macrophages. Sequential phosphorylation of PI3K, PKCζ and Sp1 has been suggested as a mechanism for upregulation of ABCA1 expression. Both apoE3 and apoE4 reduced total cholesterol and cholesterol esters in lipid-laden RAW 264.7 cells, and induced apoAI-mediated cholesterol efflux. However, the cholesterol esters and cholesterol efflux in apoE4-treated cells were ∼50% and ∼24% lower, respectively, compared to apoE3-treated cells. Accumulation of cholesterol esters in macrophages is a mechanism for foam cell formation. Thus the reduced ability of apoE4 to activate the PI3K-PKCζ-Sp1 signaling pathway and induce ABCA1 expression likely impairs cholesterol ester removal, and increases foam cell formation.     

Genomic organization and characterization of the promoter of the human ATP-binding cassette transporter-G1 (ABCG1) gene

The ATP-binding cassette transporter G1 (ABCG1) was recently identified as a regulator of macrophage cholesterol and phospholipid transport. This transporter together with ABCA1 belongs to a group of sterol-sensitive ABC proteins which are induced by lipid loading or specific oxysterols. We report here the genomic structure of ABCG1 along with the 5' flanking sequence using library screening and BLAST search analysis. The ABCG1 gene spans more than 70 kb and contains 15 exons. The exon size is between 30 and 1081 bp and the introns range in size from 137 bp to more than 45 kb. All exon-intron boundaries display the canonical GT/AG sequences. Using promoter-luciferase reporter assays in the myeloid cell lines THP-1 and RAW246.7 and the hepatoma cell line HepG2 we could demonstrate the functionality of the ABCG1 promoter and the minimal sequence requirements for gene expression. The TATA-less proximal promoter contains multiple Sp1 binding sites and a consensus sequence for sterol regulatory element binding protein.     

Transcriptional regulation of ATP-binding cassette transporter A1 expression by a novel signaling pathway

ATP-binding cassette transporter A1 (ABCA1) is a membrane-bound protein that regulates the efflux of cholesterol derived from internalized lipoproteins. Using a mouse macrophage cell line, this report studied the impact of low-density lipoproteins (LDL) on ABCA1 expression and the signaling pathway responsible for lipoprotein-induced ABCA1 expression. Our data demonstrated that treatment of macrophages with LDL increased ABCA1 mRNA and protein levels 4.3- and 3.5-fold, respectively. LDL also induced an ～2-fold increase in macrophage surface expression of ABCA1 and a 14-fold-increase in apolipoprotein AI-mediated cholesterol efflux. In addition, LDL significantly increased the level of phosphorylated specificity protein 1 (Sp1) and the amount of Sp1 bound to the ABCA1 promoter without alteration in total Sp1 protein level. Mutation of the Sp1 binding site in the ABCA1 promoter and inhibition of Sp1 DNA binding with mithramycin A suppressed the ABCA1 promoter activity and reduced the ABCA1 expression level induced by LDL. LDL treatment also elevated protein kinase C-ζ (PKC-ζ) phosphorylation and induced PKC-ζ binding with Sp1. Inhibition of PKC-ζ with kinase inhibitors or overexpression of kinase-dead PKC-ζ attenuated Sp1 phosphorylation and ABCA1 expression induced by LDL. These results demonstrate for the first time that activation of the PKCζ-Sp1 signaling cascade is a mechanism for regulation of LDL-induced ABCA1 expression.