Noriyuki Iwamoto

ApoA-I Facilitates ABCA1 Recycle/Accumulation to Cell Surface by Inhibiting Its Intracellular Degradation and Increases HDL Generation

Objective—Calpain-mediated proteolysis is one of the major regulatory factors for activity of ATP-binding cassette transporter (ABC) A1. Helical apolipoproteins protect ABCA1 against this degradation and increase generation of HDL. We investigated the mechanism for this reaction focusing on roles of endocytotic internalization of ABCA1. Methods and Results—Surface ABCA1 was labeled with biotin and traced for its internalization and degradation. ABCA1 in the cell surface was internalized within 10 minutes regardless of the presence of apoA-I. ABCA1 was intracellularly degraded and was protected against this only when exposed to extracellular apoA-I before its endocytosis. Consequently, recycle of ABCA1 to the surface was enhanced, and surface ABCA1 was increased by apoA-I. Direct inhibition of ABCA1 endocytosis led to decrease of its degradation and increase of surface ABCA1. Generation of HDL increased in parallel with surface ABCA1. Conclusion—Surface ABCA1 is internalized and degraded, and apoA-I interferes with only the latter step to recycle ABCA1 to the surface. Increase of surface ABCA1 results in the increase of generation of HDL.

ABCA7 expression is regulated by cellular cholesterol through the SREBP2 pathway and associated with phagocytosis

ABCA7 is highly homologous to ABCA1 and mediates cellular cholesterol and phospholipid release by apolipoproteins when transfected in vitro. However, expression of ABCA7 was downregulated by increased cellular cholesterol while ABCA1 was upregulated, and the results were consistent by forced expression or downregulation of sterol-responsive/regulatory element (SRE) binding proteins (SREBPs). We analyzed the promoter of the ABCA7 gene and identified the new exon encoding 96 bp (mouse) and 95 bp (human) of the 5′ untranslated region and the transcription start site at 1,122 bp (mouse) and 1,260 bp (human) upstream of the initiation methionine codon. At 5′ upstream of this exon is the ABCA7 proximal promoter containing multiple binding sites of transcription factors for hematopoiesis and SRE of 9 bp at 212 bp (mouse) and 179 bp (human) upstream of the new exon. The apolipoprotein A-I-mediated lipid release was not influenced by suppression of the endogenous ABCA7 with small interfering RNA in mouse fibroblasts or by its increase in ABCA1-deficient mouse cells. In contrast, phagocytic activity was altered in parallel to the ABCA7 expression in these cells. When phagocytosis was induced, the messages increased for SREBP2, ABCA7, and other SREBP2-regulated proteins. The ABCA1 message decreased in this condition. We conclude that the ABCA7 gene is regulated by sterol in the opposite direction to ABCA1 through SRE/SREBP2 and that expression of ABCA7 by this regulation is associated with phagocytic activity.

Glucocorticoid Receptor Regulates ATP-Binding Cassette Transporter-A1 Expression and Apolipoprotein-Mediated Cholesterol Efflux from Macrophages

Objective—The ATP-binding cassette transporter-A1 (ABCA1) regulates cholesterol efflux from cells and is involved in high-density lipoprotein metabolism and atherogenesis. The objective of this study was to investigate the effect of dexamethasone (Dex) and other glucocorticoid receptor (GR) ligands on apolipoprotein AI–mediated cholesterol efflux from macrophages and ABCA1 expression in them. Methods and Results—Dex, a GR agonist, decreased ABCA1 mRNA levels in a dose- and time-dependent fashion, and RU486, a GR antagonist, reversed the inhibitory effect of Dex. The effects of Dex and RU486 on ABCA1 protein levels and apolipoprotein AI–mediated cholesterol efflux from the macrophages were consistent with these changes in mRNA levels. Transfected RAW264.7, together with a human ABCA1 promoter–luciferase construct, inhibited transcriptional activity by Dex and overexpression of human GR. Transrepression by GR was not mediated by liver X receptor (LXR), because there were no differences in the effects of the GR ligands on promoter activity between a reporter construct with mutations at the LXR binding site and one without the mutations, and no changes were brought about in ABCG1 and ABCG4 expression by GR ligands. Conclusions—Our results showed that GR ligands affected ABCA1 expression and cholesterol efflux from macrophages, which are regulated by GR through a LXR-independent mechanism.

Serum amyloid A generates high density lipoprotein with cellular lipid in an ABCA1- or ABCA7-dependent manner

Serum amyloid A (SAA) is an amphiphilic helical protein that is found associated with plasma HDL in various pathological conditions, such as acute or chronic inflammation. Cellular lipid release and generation of HDL by this protein were investigated, in comparison with the reactions by apolipoprotein A-I (apoA-I) and several types of cells that appear with various specific profiles of cholesterol and phospholipid release. SAA mediated cellular lipid release from these cells with the same profile as apoA-I. Upregulation of cellular ABCA1 protein by liver X receptor/retinoid X receptor agonists resulted in an increase of cellular lipid release by apoA-I and SAA. SAA reacted with the HEK293-derived clones that stably express human ABCA1 (293/2c) or ABCA7 (293/6c) to generate cholesterol-containing HDL in a similar manner to apoA-I. Dibutyryl cyclic AMP and phorbol 12-myristate 13-acetate, which differentiate apoA-I-mediated cellular lipid release between 293/2c and 293/6c, also exhibited the same differential effects on the SAA-mediated reactions. No evidence was found for the ABCA1/ABCA7-independent lipid release by SAA. Characterization of physicochemical properties of the HDL revealed that SAA-generated HDL particles had higher density, larger diameter, and slower electrophoretic mobility than those generated by apoA-I. These results demonstrate that SAA generates cholesterol-containing HDL directly with cellular lipid and that the reaction is mediated by ABCA1 and ABCA7.

ATP-Binding Cassette Transporter A1 Gene Transcription Is Downregulated by Activator Protein 2α Doxazosin Inhibits Activator Protein 2α and Increases High-Density Lipoprotein Biogenesis Independent of α1-Adrenoceptor Blockade

ATP-binding cassette transporter A1 (ABCA1) is a rate-limiting factor for high-density lipoprotein (HDL) biogenesis. The ABCA1 gene expression is known to be upregulated by various transcriptional factors. However, negative regulation factors would be better targets for pharmacological modulation of HDL biogenesis. Doxazosin, an &agr;1-adrenoceptor blocker, increased ABCA1 mRNA, its protein, and apolipoprotein A-I–mediated HDL biogenesis in THP-1 macrophages and CHO-K1 cells, independent of &agr;1-adrenoceptor blockade. Analysis of the human ABCA1 promoter indicated that the region between the positions −368 and −147 that contains an activator protein (AP)2-binding site responsible for the effects of doxazosin. Overexpression of AP2&agr; inhibited ABCA1 transcription in a dose-dependent fashion. Mutation in the AP2-binding site caused increase of the basal promoter activity and canceling both the transactivation by doxazosin and the trans-repression by AP2&agr;. Doxazosin had no effect on ABCA1 mRNA level in HepG2 cells, which lack endogenous AP2&agr;, and it reversed the inhibitory effect of AP2&agr; expression in this type of cells. Chromatin immunoprecipitation and gel shift assays revealed that doxazosin reduced specific binding of AP2&agr; to the ABCA1 promoter, as it suppressed phosphorylation of AP2&agr;. Finally, doxazosin increased ABCA1 expression and plasma HDL in mice. We thus concluded that AP2&agr; negatively regulates the ABCA1 gene transcription. Doxazosin inhibits AP2&agr; activity independent of &agr;1-adrenoceptor blockade and increases the ABCA1 expression and HDL biogenesis. AP2&agr; is a potent pharmacological target for the increase of HDL.

Pharmacological inhibition of ABCA1 degradation increases HDL biogenesis and exhibits antiatherogenesis

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.

Biogenesis of HDL by SAA is dependent on ABCA1 in the liver in vivo

Serum amyloid A (SAA) was markedly increased in the plasma and in the liver upon acute inflammation induced by intraperitoneal injection of lipopolysaccharide (LPS) in mice, and SAA in the plasma was exclusively associated with HDL. In contrast, no HDL was present in the plasma and only a small amount of SAA was found in the VLDL/LDL fraction (d < 1.063 g/ml) after the induction of inflammation in ABCA1-knockout (KO) mice, although SAA increased in the liver. Primary hepatocytes isolated from LPS-treated wild-type (WT) and ABCA1-KO mice both secreted SAA into the medium. SAA secreted from WT hepatocytes was associated with HDL, whereas SAA from ABCA1-KO hepatocytes was recovered in the fraction that was >1.21 g/ml. The behavior of apolipoprotein A-I (apoA-I) was the same as that of SAA in HDL biogenesis by WT and ABCA1-KO mouse hepatocytes. Lipid-free SAA and apoA-I both stabilized ABCA1 and caused cellular lipid release in WT mouse-derived fibroblasts, but not in ABCA1-KO mouse-derived fibroblasts, in vitro when added exogenously. We conclude that both SAA and apoA-I generate HDL largely in hepatocytes only in the presence of ABCA1, likely being secreted in a lipid-free form to interact with cellular ABCA1. In the absence of ABCA1, nonlipidated SAA is seemingly removed rapidly from the extracellular space.

Helical apolipoproteins of high-density lipoprotein enhance phagocytosis by stabilizing ATP-binding cassette transporter A7

We previously reported that the endogenous ATP-binding cassette transporter (ABC)A7 strongly associates with phagocytic function rather than biogenesis of high-density lipoprotein (HDL), being regulated by sterol-regulatory element binding protein (SREBP)2. Phagocytic activity was found enhanced by apolipoprotein (apo)A-I and apoA-II more than twice the maximum in J774 and mouse peritoneal macrophages. Therefore we investigated the molecular basis of this reaction in association with the function of ABCA7. Similar to ABCA1, ABCA7 was degraded, likely by calpain, and apoA-I and apoA-II stabilize ABCA7 against degradation. Cell surface biotinylation experiments demonstrated that endogenous ABCA7 predominantly resides on the cell surface and that the apolipoproteins increase the surface ABCA7. The increase of phagocytosis by apolipoproteins was retained in the J774 cells treated with ABCA1 siRNA and in the peritoneal macrophages from ABCA1-knockout mice, but it was abolished in the J774 cells treated with ABCA7 siRNA and in the peritoneal macrophages from ABCA7-knockout mice. Phagocytosis was decreased in the cells in the peritoneal cavity of the ABCA7-knockout mouse compared with the wild-type control. We thus concluded that extracellular helical apolipoproteins augment ABCA7-associated phagocytosis by stabilizing ABCA7. The results demonstrated direct enhancement of the host defense system by HDL components.

Calmodulin Interacts With ATP Binding Cassette Transporter A1 to Protect From Calpain-Mediated Degradation and Upregulates High-Density Lipoprotein Generation

Objective—To investigate the interaction of ATP-binding cassette transporter A1 (ABCA1) with calmodulin in relation to its calpain-mediated degradation because many calpain substrates bind calmodulin to regulate cellular functions. Methods and Results—The activity of ABCA1 is regulated through proteolysis by calpain. An immunoprecipitation and glutathione S-transferase pull-down assay revealed that ABCA1 directly binds calmodulin in a Ca2+-dependent manner. The cytoplasmic loop of ABCA1 contains a typical calmodulin binding sequence of 1-5-8-14 motifs (1245 to 1257 amino acids). The peptide of this region showed binding to calmodulin, and deletion of the 1-5-8-14 motif abolished this interaction. This motif is located near the ABCA1 Pro-Glu-Ser-Thr sequence, and the presence of calmodulin/Ca2+ protected the peptides from proteolysis by calpain. The knockdown of calmodulin by a specific small and interfering RNA increased the degradation of ABCA1 and decreased ABCA1 protein and apolipoprotein A-I-mediated lipid release. Surprisingly, calmodulin inhibitor W7 increased calmodulin binding to ABCA1 and protected it from calpain-mediated degradation, consistent with our previous finding that this compound increased apolipoprotein A-I-mediated cell cholesterol release. Conclusion—Calmodulin directly binds and stabilizes ABCA1 in the presence of Ca2+ and increases the generation of high-density lipoprotein.

HMG-CoA reductase inhibitors enhance phagocytosis by upregulating ATP-binding cassette transporter A7.

We recently reported that the endogenous ATP-binding cassette transporter (ABC) A7 strongly associates with phagocytosis, being regulated by sterol regulatory element binding protein 2. We therefore examined the effect of statins on phagocytosis in vitro and in vivo through the SREBP-ABCA7. Phagocytosis was found to be enhanced by pravastatin, rosuvastatin and simvastatin and cyclodextrin in J774 macrophages, as cellular cholesterol was reduced and expressions of the cholesterol-related genes were modulated, including an increase of ABCA7 mRNA and decrease of ABCA1 mRNA. Conversely, knock-down of ABCA7 expression by siRNA ablated enhancement of phagocytosis by statins. In vivo, pravastatin enhanced phagocytosis in wild-type mice, but not in ABCA7-knockout mice. We thus concluded that statins enhance phagocytosis through the SREBP-ABCA7 pathway. These findings provide a molecular basis for enhancement of the host-defense system by statins showing that one of their pleiotropic effects is in fact achieved through their reaction to a primary target.