Progesterone inhibits apolipoprotein-mediated cellular lipid release: a putative mechanism for the decrease of high-density lipoprotein

The liver is the major site of both apolipoprotein A-I (apoA-I) synthesis and ATP-binding cassette transporter A1 (ABCA1) expression. Here, we compare the lipidation with cholesterol and phospholipid of newly synthesized human apoA-I (hapoA-I) using adenoviral vector-mediated endogenous expression or exogenously added hapoA-I in wild type and ABCA1-null hepatocytes. 3 H]choline. ABCA1 deficiency decreased apoA-I phospholipidation by 80%, but acquisition of de novo synthesized and exogenous cholesterol only decreased by 40 -60%. The transfer of de novo synthesized cholesterol to apoA-I was decreased at all time points, but that of exogenously delivered cholesterol was independent of ABCA1 activity at the early time points. Progesterone does not affect apoA-I synthesis or its lipidation but inhibited the early phase of apoA-I cholesterol lipidation in both wild type and ABCA1-null hepatocytes. Fast protein liquid chromatography analysis of medium lipoproteins confirmed that with ABCA1 deficiency, the proportion of secreted high density lipoprotein-associated apoA-I and cholesterol decreased by about 50%. The very low density lipoprotein (VLDL)/ LDL size fraction also contained a significant level of cholesterol in ABCA1 deficiency, consistent with the result of immunoprecipitations showing the presence of lipoproteins with both apoA-I and murine apoB. ApoA-I lipidation with newly synthesized cholesterol in ABCA1-null hepatocytes was significantly decreased by brefeldin A and monensin. In conclusion, we demonstrate that: (i) whereas most hepatic phospholipidation of apoA-I is mediated by ABCA1, acquisition of cholesterol depends on active transfer from intracellular compartments by ABCA1-dependent and -independent pathways, both sensitive to progesterone and (ii) there is separate regulation of phospholipid and cholesterol lipidation of apoA-I in hepatocytes.The ATP-binding cassette transporter protein, ABCA1, 1 is the major determinant of the phospholipid and cholesterol lipidation of apolipoprotein A-I that enables formation of high density lipoprotein (HDL) (1). ABCA1 has been shown to regulate lipid efflux, and a deficiency in ABCA1, as in Tangier disease, results in nearly undetectable levels of HDL (2-6). Macrophages in various tissues are profoundly affected by this disease, first suggesting that ABCA1 function in macrophages was critical for the maintenance of HDL levels in the plasma, as part of the reverse cholesterol transport. However, Haghpassand et al. (7) demonstrated that macrophages account for only 20% of the generation of circulating HDL. ABCA1 (4, 8 -12) and apoA-I (13, 14) are both highly expressed in the liver and intestine, suggesting that ABCA1 contributes to the lipidation of newly secreted lipoproteins in those tissues. Basso et al. (15) used an ABCA1 adenovirus expression system to promote liver expression of ABCA1, thereby raising plasma HDLcholesterol and promoting cholesterol efflux from primary hepatocytes, supporting the role of the liver as a major source of HDL.As noted by Tall e...

Section: Effect Of Abca1 Deficiency On Distribution Of Hapoa-i and Chmentioning

confidence: 80%

Section: Effect Of Abca1 Deficiency On Distribution Of Hapoa-i and Chmentioning

confidence: 98%

ApoA-I Lipidation in Primary Mouse Hepatocytes

Kiss¹,

Franklin

Wang

et al. 2005

“…Immunobloting, Pulse Labeleing, and Phosphorylation of ABCA1-The cells were incubated with apolipoproteins or peptides for 24 h, and ABCA1 in the cells was analyzed by immunoblotting by using an antibody against the C-terminal peptide of human ABCA1 according to a previous method (20). For estimation of the rate of decay of ABCA1, the cells were pulse labeled with 35 S-labeled amino acids for 30 min and incubated with apolipoproteins or peptides prior to the analysis by electrophoresis-autoradiography of the immunoprecipitated fraction with the anti-ABCA1 antibody as described previously (8).…”

Section: Methodsmentioning

confidence: 99%

Phosphorylation and Stabilization of ATP Binding Cassette Transporter A1 by Synthetic Amphiphilic Helical Peptides

Arakawa

Hayashi

Remaley

et al. 2004

To investigate structural requirement of helical apolipoprotein to phosphorylate and stabilize ATP-binding cassette transporter A1 (ABCA1), synthetic peptides (Remaley, A. T., Thomas, F., Stonik, J. A., Demosky, S. J., Bark, S. E., Neufeld, E. B., Bocharov, A. V., Vishnyakova, T. G., Patterson, A. P., Eggerman, T. L., SantamarinaFojo, S., and Brewer, H. B. (2003) J. Lipid Res. 44, 828 -836) were examined for these activities. L37pA, an L amino acid peptide that contains two class-A amphiphilic helices, and D37pA, the same peptide with all D amino acids, both removed cholesterol and phospholipid from differentiated THP-1 cells more than apolipoproteins (apos) A-I, A-II, and E. Both peptides also mediated lipid release from human fibroblasts WI-38 similar to apoA-I. L2D37pA, an L-peptide whose valine and tyrosine were replaced with D amino acids also promoted lipid release from WI-38 but less so with THP-1, whereas L3D37pA, in which alanine, lysine, and asparatic acid were replaced with D amino acids was ineffective in lipid release for both cell lines. ABCA1 protein in THP-1 and WT-38 was stabilized against proteolytic degradation by apoA-I, apoA-II, and apoE and by all the peptides tested except for L3D37pA, and ABCA1 phosphorylation closely correlated with its stabilization. The analysis of the relationship among these parameters indicated that removal of phospholipid triggers signals for phosphorylation and stabilization of ABCA1. We thus concluded that an amphiphilic helical motif is the minimum structural requirement for a protein to stabilize ABCA1 against proteolytic degradation.Release of cellular cholesterol is one of the essential events of its homeostasis for the cells and also for the whole body, since cholesterol is catabolized only in the a few certain organs, mainly in the liver for the conversion to bile acids and in very limited amount in steroidogenic organs. This reaction is mediated by two distinct mechanisms: 1) nonspecific physicochemical exchange with cell surface and extracellular cholesterol acceptors and 2) by a specific mechanism for helical apolipoproteins such as apolipoprotein (apo) 1 A-I, which remove cellular phospholipid and cholesterol to generate new high density lipoprotein (HDL) (1). The latter reaction is known to be mediated by the ATP-binding cassette transporter A1 (ABCA1) (2-4) and is considered a rate-limiting step in regulating of the HDL concentration in blood plasma (5-7). It is therefore of clinical importance as it may influence this strong negative risk factor for atherosclerotic vascular disease.ABCA1 is degraded by calpain, and it appears to be one of the major mechanisms for regulation of its cellular level, and helical apolipoproteins protect ABCA1 against this degradation (8, 9). Helical apolipoproteins interact with ABCA1 and generate new HDL by removing cellular lipid, ABCA1 is phosphorylated and stabilized by a mechanism involving protein kinase C that is presumably activated by diacylglycerol generated by replenishment reaction for the removal of sphingomy...

“…Western Blotting-The membrane fraction and cellular subfractions were resuspended in 50 mM Tris-HCl (pH 7.5) containing 5 mM EDTA, 10 mM EGTA, 1 mM PMSF, 1 mM benzamidine, 1% Triton X-100, and 1% protease inhibitor cocktails (Sigma) and were sonicated for 5 s. After determination of the protein content by a BCA method (Pierce), the fractions were dissolved in 9 M urea, 2% Triton X-100, 1% dithiothreitol and were developed in 6 or 15% (w/v) polyacrylamide gel electrophoresis in the presence of 10% SDS, respectively, and the proteins were transferred to a polyvinylidene difluoride membrane (Bio-Rad) by semidry blotter in blotting buffer (25 mM Tris-HCl, 0.2 M glycine, and 10% methanol (v/v)) for 3.5 h. The membrane was blocked with 5% skim milk in 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 0.05% Tween 20 and was probed with the rabbit antiserum against the C-terminal peptide of human ABCA1 (26,39), rabbit anticaveolin-1 (N-20) (Santa Cruz Biotechnology), anti-GLUT-1, mouse anti-integrin ␤ 1 (CHEMICON International, Inc.), anti-GM130, and anti-Bip/GRP78 (BD Transduction Laboratories, BD Biosciences), respectively. The immunoreactive proteins were visualized by ECL or the ECL Plus system (Amersham Biosciences).…”

Section: Chemicals and Reagents-probucolmentioning

confidence: 99%

Probucol Inactivates ABCA1 in the Plasma Membrane with Respect to Its Mediation of Apolipoprotein Binding and High Density Lipoprotein Assembly and to Its Proteolytic Degradation

Chengai

Tsujita

Hayashi

et al. 2004

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...