Expression of ATP binding cassette transporter A1 (ABCA1), a major regulator of high density lipoprotein (HDL) biogenesis, is known to be up-regulated by the transcription factor liver X receptor (LXR) α, and expression is further enhanced by activation of the peroxisome proliferator activated receptors (PPARs). We investigated this complex regulatory network using specific PPAR agonists: four fibrates (fenofibrate, bezafibrate, gemfibrozil and LY518674), a PPAR δ agonist (GW501516) and a PPAR γ agonist (pioglitazone). All of these compounds increased the expression of LXRs, PPARs and ABCA1 mRNAs, and associated apoA-I-mediated lipid release in THP-1 macrophage, WI38 fibroblast and mouse fibroblast. When mouse fibroblasts lacking expression of PPAR α were examined, the effects of fenofibrate and LY518674 were markedly diminished while induction by other ligands were retained. The PPAR α promoter was activated by all of these compounds in an LXR α-dependent manner, and partially in a PPAR α-dependent manner, in mouse fibroblast. The LXR responsive element (LXRE)-luciferase activity was enhanced by all the compounds in an LXR α-dependent manner in mouse fibroblast. This activation was exclusively PPAR α-dependent by fenofibrate and LY518674, but nonexclusively by the others. We conclude that PPARs and LXRs are involved in the regulation of ABCA1 expression and HDL biogenesis in a cooperative signal transduction pathway.

Probucol has been shown to inhibit the release of cellular lipid by helical apolipoprotein and thereby to reduce plasma high density lipoprotein. We attempted to explore the underlying mechanism for this effect in human fibroblast WI-38. Probucol inhibited the apoA-Imediated cellular lipid release and binding of apoA-I to the cells in a dose-dependent manner. It did not influence cellular uptake of low density lipoprotein, transport of cholesterol to the cell surface whether de novo synthesized or delivered as low density lipoprotein, and overall cellular content of cholesterol, although biosynthesis of lipids from acetate was somewhat increased. Probucol did not affect the mRNA level of ABCA1, and ABCA1 was recovered along with marker proteins for plasma membrane regardless of the presence of probucol. However, the protein level of ABCA1 increased, and the rate of its decay in the presence of cycloheximide was slower in the probucol-treated cells. ABCA1 in the probucol-treated cells was resistant to digestion by calpain but not by trypsin. We concluded that probucol inactivates ABCA1 in the plasma membrane with respect to its function in mediating binding of and lipid release by apolipoprotein and with respect to proteolytic degradation by calpain.Cholesterol is an essential molecule for animal cells to maintain and regulate function and structure of the biomembrane. It is synthesized in most somatic cells, whereas its catabolic site is limited to the liver and to the steroidogenic cells except for partial hydroxylation in some somatic cells. Accordingly, cholesterol is removed from the cells and transported to the liver for its conversion to bile acids, and this is one of the essential events in cholesterol homeostasis for the body and for the cells (1). High density lipoprotein (HDL) 1 is believed to play a central role in this system, and this is thought to be one of the antiatherogenic characteristics of HDL. This reaction takes place through at least two distinct mechanisms: 1) physicochemical release of cholesterol from the cell surface, which is driven by cholesterol esterification on HDL, and 2) the apolipoprotein-mediated pathway to remove cellular cholesterol and phospholipid to generate new HDL particles (2). HDL thus plays a central role in both mechanisms.Apolipoprotein-dependent cellular cholesterol release is absent in fibroblasts from patients with Tangier disease (3, 4), and mutations in the gene encoding the ATP-binding cassette transporter A1 (ABCA1) are the underlying cause of this disease (5-9). On the other hand, in vitro overexpression of functional ABCA1 in the cells (10, 11) and induction of ABCA1 expression by cyclic AMP analogues (12, 13) or by the ligands for the liver X receptor or retinoid X receptor (14, 15) enhanced the release of cellular cholesterol and phospholipid by apolipoprotein. The transgenic mice for ABCA1 had a significant increase in plasma HDL (16,17). These results indicate that this protein is a regulating factor for the plasma HDL level through generation of HDL b...

The mechanism for the assembly of HDL with cellular lipid by ABCA1 and helical apolipoprotein was investigated in hepatocytes. Both HepG2 cells and mouse primary culture hepatocytes produced HDL with apolipoprotein A-I (apoA-I) whether endogenously synthesized or exogenously provided. Probucol, an ABCA1 inactivator, inhibited these reactions, as well as the reversible binding of apoA-I to HepG2. Primary cultured hepatocytes of ABCA1-deficient mice also lacked HDL production regardless of the presence of exogenous apoA-I. HepG2 cells secreted apoA-I into the medium even when ABCA1 was inactivated by probucol, but it was all in a free form as HDL production was inhibited. When a lipid-free apoA-I-specific monoclonal antibody, 725-1E2, was present in the culture medium, production of HDL was suppressed, whether with endogenous or exogenously added apoA-I, and the antibody did not influence HDL already produced by HepG2 cells. We conclude that the main mechanism for HDL assembly by endogenous apoA-I in HepG2 cells is an autocrine-like reaction in which apoA-I is secreted and then interacts with cellular ABCA1 to generate HDL. -Tsujita, M., C-A. Wu, S. AbeDohmae, S. Usui, M. Okazaki, and S. Yokoyama. On the hepatic mechanism of HDL assembly by the ABCA1/apoA-I pathway. J. Lipid Res. 2005. 46: 154-162.