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.

Extracellular apolipoprotein A-I removed cholesterol and phospholipid from cholesterol-loaded mouse peritoneal macrophage and thereby generated a prebeta high-density lipoprotein (HDL) particle having a weight ratio of cholesterol to phosphatidylcholine of approximately 1:1. Treatment of the cells with phorbol myristate slightly increased cholesterol efflux by this mechanism without influencing the nonspecific cholesterol efflux to the lipid microemulsion. When the cells were treated by protein kinase C (PKC) inhibitors, H7 and staurosporine, apolipoprotein-mediated cellular cholesterol efflux was substantially reduced without a significant change in phosphatidylcholine efflux, resulting in generation of cholesterol-poor prebeta-HDL particles having a weight ratio of cholesterol to phosphatidylcholine as low as 1:10. In spite of this change, specific binding of apoA-I to the cellular surface was unaffected. Cellular cholesterol available for acylCoA:cholesterol acyltransferase (ACAT) was rapidly depleted by adding apoA-I to the medium, and the PKC inhibitor treatment reversed this effect. In contrast, nonspecific cellular cholesterol efflux to the lipid microemulsion did not influence the ACAT-available cellular cholesterol pool, and it was not influenced by the PKC inhibitors. Thus, we concluded that apolipoprotein-mediated cellular cholesterol efflux is linked to mobilization of cholesterol from an intracellular pool used by ACAT to a specific pool for apolipoprotein-mediated prebeta-HDL generation, in response to apolipoprotein-cell interaction and subsequent intracellular signaling. Binding of apolipoprotein to the cell surface is required for assembly of the prebeta-HDL particle with cellular phospholipid, and the intracellular cholesterol mobilization is needed for enrichment with cholesterol of the prebeta-HDL. These reactions are largely independent of diffusion-mediated nonspecific cell cholesterol efflux.

We attempted to demonstrate selective modulation of lipid-free apolipoprotein-mediated cellular lipid efflux in order to test the hypothesis that it is an event independent of nonspecific physicochemical cholesterol exchange. Probucol, a unique cholesterol-lowering drug, was found to selectively suppress this pathway in vitro in mouse peritoneal macrophage. Probucol was given to the cells via the uptake of acetylated low-density lipoprotein (LDL) into which it had been incorporated. The uptake of lipoprotein-cholesteryl ester by the macrophage was the same whether the acetylated LDL was probucol-carrying or probucol-free, and probucol accumulated in the cell in parallel to cholesterol when carried by the lipoprotein. Incorporation of [35S]methionine into cell protein was unaffected by probucol accumulated in the cells. The efflux of cellular cholesterol and phospholipid mediated by lipid-free human apolipoproteins (apo) A-I, A-II, and E was all completely inhibited by probucol. Reversible binding of free apoA-I to the cellular surface was also completely blocked by probucol in this condition. On the other hand, nonspecific cholesterol exchange between LDL and macrophage was unaffected by probucol. Thus, probucol selectively inhibited apolipoprotein-mediated cellular lipid efflux by blocking specific binding of free apolipoprotein to the cell without influencing nonspecific lipid exchange. In the absence of lecithin: cholesterol acyltransferase (LCAT) reaction, apparent cellular cholesterol efflux to high-density lipoprotein (HDL) was reduced by probucol by 40-70% while the rate of cholesterol influx from HDL to the cells was unaffected, resulting in cancellation of the net cellular cholesterol efflux to HDL. However, the increase of the net cholesterol efflux to HDL by LCAT was unaffected by probucol. Net cellular cholesterol efflux to HDL in the absence of LCAT, therefore, seems to depend on an apolipoprotein-mediated mechanism.

generation of new HDL from cellular lipids is one of the major events in the initial step of this pathway, cellular cholesterol release ( 1, 2 ). This reaction was found to be defective in genetic HDL defi ciency, Tangier disease ( 3, 4 ), and mutations were identifi ed in the gene of ABCA1 as the cause of this disorder ( 5-7 ).Expression of ABCA1 is regulated at the transcriptional level and posttranslationally by calpain-mediated proteolysys. ABCA1 is stabilized against this degradation by helical apolipoproteins ( 8-10 ), and destabilized by unsaturated fatty acid ( 11 ) or excess unesterifi ed cholesterol ( 12 ). Inhibition of calpain increases HDL formation by cultured cells ( 8 ), suggesting inhibition of proteolytic degradation of ABCA1 is a potential drug target for increasing HDL. ABCA1 degradation takes place intracellularly and its inhibition results in increased ABCA1 recycling to the cell surface ( 13 ). Direct inhibition of internalization of ABCA1 also causes its accumulation in the cell surface ( 13 ). HDL formation increases in both cases, indicating that it takes place at the cell surface ( 13 ). Inhibition of ABCA1 degradation or internalization would therefore be a potential strategy to increase HDL biogenesis for prevention and/ or regression of atherosclerosis.The hypolipidemic drug, probucol, is known to reduce plasma HDL ( 14 ) by inhibiting ABCA1-mediated HDL biogenesis ( 15 ) and producing a Tangier disease-like state ( 16 ). Interestingly, probucol causes not only inactivation of ABCA1 but also inhibits its degradation ( 17 ). We found in preliminary experiments that the crude oxidized products of probucol enhanced HDL formation by cultured cells rather than inhibiting it. Based on these fi ndings, we Abstract Expression of ABCA1 is regulated by transcription of the gene and calpain-mediated proteolytic degradation, and inhibition ABCA1 degradation results in increased ABCA1 and HDL biogenesis in vitro. We examined whether this approach could be a potential antiatherogenic treatment. Although probucol inhibits both the activity and degradation of ABCA1, its oxidized products, spiroquinone and diphenoquinone, reduce degradation of ABCA1 without inhibiting its activity or altering transcription of the ABCA1 gene. Accordingly, both compounds enhanced apolipoprotein A-I/ABCA1-dependent generation of HDL in vitro, and increased hepatic ABCA1 and plasma HDL without increasing antioxidant activity in plasma when given to rabbits. Both compounds also decreased vascular lipid deposition in cholesterol-fed rabbits. We therefore conclude that stabilization of ABCA1 against calpain-mediated degradation is a novel and potentially important strategy to increase HDL formation and prevent atherosclerosis. Spiroquinone and diphenoquinone are potential seeds for development of such drugs.

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Fibric acid-shaped drugs raise high-density lipoprotein (HDL) cholesterol by upregulating the HDL-related genes through activating peroxisome proliferater activated receptor (PPAR)-alpha. We investigated the effects of fibrates to induce expression of adenosine triphosphate-binding cassette transporter A1 (ABCA1) and increase HDL biogenesis in hepatocytes. Fenofibrate, bezafibrate, gemfibrozil, and LY518674 were tested for HepG2 cells and primary-cultured mouse hepatocytes. All the compounds examined increased ABCA1 expression and HDL biogenesis dependent on PPARalpha in association with the liver X receptor alpha upregulation. While fenofibrate and LY518674 showed exclusive dependency on PPARalpha for these activities, bezafibrate and gemfibrozil exhibited dependency on PPARbeta/delta and PPARgamma as well. On the other hand, cholesterol-enrichment of HDL may involve PPARgamma for fenofibrate and bezafibrate, and PPARbeta/delta for the fibrates examined except for bezafibrate. We concluded that fibrates enhance expression of ABCA1 in hepatocytes to contribute to increase of the HDL biogenesis in a PPAR-dependent manner, whether exclusively or nonexclusively on PPARalpha.