Abstract
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.
Highlights
Apolipoprotein E, known as arginine-rich apoprotein, was first characterized as a protein moiety of very low-density lipoproteins (VLDL) [1,2]
We previously reported that,70% of cholesterol was distributed in the VLDL fractions of apoE2/2 mice, when mouse plasma samples were directly analyzed by fast performance liquid chromatography (FPLC) [54]
The propensity of apoE4 to induce protein kinase Cf (PKCf) and specificity protein 1 (Sp1) phosphorylations was reduced relative to apoE3. These findings suggest that the PKCf-Sp1-ATP binding cassette A1 (ABCA1) signaling pathway is not limited to RAW 264.7 macrophages, and may represent a general cascade mediated by human Apolipoprotein E (apoE) isoforms
Summary
Apolipoprotein E (apoE), known as arginine-rich apoprotein, was first characterized as a protein moiety of very low-density lipoproteins (VLDL) [1,2]. Isoelectric focusing of the isolated proteins [8] demonstrated that human apoE contains more than one form; isoforms were subsequently named from the most acidic to the least acidic, i.e., E1 is more acidic than E2, while E2 is more acidic than E3, and so on [9,10,11]. ApoE3, with an allelic frequency of about 68–87%, depending on the population under study [12,13], is considered the normal isoform. The Arg158 in apoE3 is mutated to Cys158 in apoE2, while the Cys112 in apoE3 is mutated to Arg112 in apoE4 [14,15]
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