ABSTRACT
Atherosclerosis (AS) is a risky cardiovascular disease with limited treatment options. Various pan or type-selective histone deacetylase (HDAC) inhibitors are reportedly atheroprotective against atherosclerosis (AS); however, the key effectors and the main cellular processes that mediate the protective effects remain poorly defined. Here, we report that PPARγ (Peroxisome proliferator-activated receptor gamma), a transcription factor actively involved in lipid metabolism with strong tissue protective and anti-inflammation properties, is a critical mediator of the anti-AS effects by HDAC inhibition. We showed that a well-known pan-HDAC inhibitor TSA (Trichostatin A) reduced foam cell formation of macrophages that is accompanied by a marked elevation of PPARγ and its downstream cholesterol efflux transporter ABCA1 (ATP-binding membrane cassette transport protein A1) and ABCG1. In an AS model of ApoE-/- mice fed on high-fat diet, TSA treatment alleviated AS lesions, similarly increased PPARγ and the downstream cholesterol transporters and mitigated the induction of inflammatory cytokine TNFα and IL-1ß. Exploring the potential cause of PPARγ elevation revealed that TSA induced the acetylation of C/EBPα (CCAAT enhancer binding protein alpha), the upstream regulator of PPARγ, through which it increased PPARγ transactivation. More importantly, we generated a strain of PPARγ/ApoE double knockout mice and demonstrated that lack of PPARγ abrogated the protective effects of TSA on foam cell formation of peritoneal macrophages and the AS pathogenesis. Taken together, these results unravel that C/EBPα and PPARγ are the HDAC-sensitive components of an epigenetic signaling pathway mediating foam cell formation and AS development, and suggest that targeting C/EBPα/PPARγ axis by HDAC inhibitors possesses therapeutic potentials in retarding the progression of AS and the related cardiovascular diseases.
Subject(s)
Apolipoproteins E/deficiency , Apolipoproteins E/genetics , Atherosclerosis/prevention & control , Foam Cells/drug effects , Histone Deacetylase Inhibitors/pharmacology , Hydroxamic Acids/pharmacology , PPAR gamma/drug effects , ATP Binding Cassette Transporter 1/antagonists & inhibitors , ATP Binding Cassette Transporter, Subfamily G, Member 1/antagonists & inhibitors , Animals , CCAAT-Enhancer-Binding Protein-alpha/antagonists & inhibitors , Diet, High-Fat , Epigenesis, Genetic/drug effects , Macrophages, Peritoneal/drug effects , Mice , Mice, Knockout , RAW 264.7 CellsABSTRACT
BACKGROUND: The ATP binding cassette (ABC) transporters participate in the cholesterol and phospholipid transport within and through cell membranes of many cells including spermatozoa. Cholesterol efflux is important for capacitation of spermatozoa. ABCA1 expression has been assessed in canine spermatozoa previously but its role in capacitation still has to be determined. The aim of the study was to test whether inhibition of ABCA1 (1) decreases capacitation in ejaculated and epididymal canine sperm samples and (2) decreases cholesterol efflux in the same samples. Twenty-one ejaculates and sperm from 22 epididymal tails were collected from healthy dogs. Motility was measured by CASA and viability assessed after staining with SYBR-14/PI. Samples from ejaculated sperm and sperm from epididymal tails were aliquoted. One part was incubated with the ABCA1 inhibitor probucol, the other served as a negative control. In all samples, capacitation was evaluated by chlortetracyclin (CTC) assay and cholesterol was measured by cholesterol efflux assay and colorimetric enzymatic assay. RESULTS: In ejaculated sperm, blockade of ABCA1 with 100 µM of probucol/mL of sample resulted in a significantly higher percentage of uncapacitated and acrosome reacted spermatozoa (P < 0.001 and P = 0.031), capacitation was significantly decreased (35% in probucol samples vs 54.2% in controls, P < 0.001). In probucol inhibited sperm samples from epididymal tails, the percentage of capacitated spermatozoa did not differ between groups but the percentage of acrosome reacted spermatozoa increased significantly (P = 0.014). The cholesterol measurement revealed significantly lower cholesterol concentration in the probucol group when compared to the controls (P = 0.035), however only in ejaculated sperm samples. CONCLUSIONS: CTC assay and cholesterol measurement revealed significant differences between groups; we conclude that inhibition of ABCA1 significantly decreased capacitation and cholesterol efflux in ejaculated canine spermatozoa. The inhibition was not complete but ABCA1 is supposed to contribute to capacitation in canine ejaculated spermatozoa. ABCA1 is probably not important for capacitation of epididymal spermatozoa but might exert other functions during spermatozoa ripening.
Subject(s)
ATP Binding Cassette Transporter 1/antagonists & inhibitors , Cholesterol/metabolism , Probucol/pharmacology , Sperm Capacitation/drug effects , Spermatozoa/drug effects , Animals , Anticholesteremic Agents/pharmacology , Dogs , MaleABSTRACT
Calpains, intracellular proteases specifically inhibited by calpastatin, play a major role in neoangiogenesis involved in tumor invasiveness and metastasis. They are partly exteriorized via the ATP-binding cassette transporter A1(ABCA1) transporter, but the importance of this process in tumor growth is still unknown. The aim of our study was to investigate the role of extracellular calpains in a model of melanoma by blocking their extracellular activity or exteriorization. In the first approach, a B16-F10 model of melanoma was developed in transgenic mice expressing high extracellular levels of calpastatin. In these mice, tumor growth was inhibited by â¼ 3-fold compared with wild-type animals. In vitro cytotoxicity assays and in vivo tumor studies have demonstrated that this protection was associated with a defect in tumor neoangiogenesis. Similarly, in wild-type animals given probenecid to blunt ABCA1 activity, melanoma tumor growth was inhibited by â¼ 3-fold. Again, this response was associated with a defect in neoangiogenesis. In vitro studies confirmed that probenecid limited endothelial cell migration and capillary formation from vascular explants. The observed reduction in fibronectin cleavage under these conditions is potentially involved in the response. Collectively, these studies demonstrate that probenecid, by blunting ABCA1 activity and thereby calpain exteriorization, limits melanoma tumor neoangiogenesis and invasiveness.
Subject(s)
ATP Binding Cassette Transporter 1/antagonists & inhibitors , Calpain/metabolism , Melanoma, Experimental/drug therapy , Neovascularization, Pathologic/drug therapy , Probenecid/pharmacology , Skin Neoplasms/drug therapy , ATP Binding Cassette Transporter 1/genetics , ATP Binding Cassette Transporter 1/metabolism , Animals , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/metabolism , Cell Line, Tumor/transplantation , Cell Proliferation/drug effects , Humans , Male , Melanoma, Experimental/blood supply , Melanoma, Experimental/pathology , Mice , Mice, Transgenic , Neovascularization, Pathologic/pathology , Probenecid/therapeutic use , Skin Neoplasms/blood supply , Skin Neoplasms/pathologyABSTRACT
The mosquito-borne chikungunya virus (CHIKV) causes acute pain and joint inflammation, and in recent years the virus has caused large epidemics in previously CHIKV-free geographic areas. To advance the understanding of host factors that antagonize CHIKV, we show that synthetic agonist of liver X receptor (LXR-623) inhibits CHIKV replication by upregulating the cholesterol exporter ABCA1 and that endogenous and pharmacological activation of interferon signaling pathway partners with LXR-623 to generate a superior antiviral state.
Subject(s)
ATP Binding Cassette Transporter 1/genetics , Antiviral Agents/pharmacology , Chikungunya virus/drug effects , Fibroblasts/drug effects , Host-Pathogen Interactions/drug effects , Indazoles/pharmacology , Liver X Receptors/genetics , ATP Binding Cassette Transporter 1/antagonists & inhibitors , ATP Binding Cassette Transporter 1/metabolism , ATP Binding Cassette Transporter, Subfamily G, Member 1/genetics , ATP Binding Cassette Transporter, Subfamily G, Member 1/metabolism , Apolipoproteins E/genetics , Apolipoproteins E/metabolism , Chikungunya virus/growth & development , Chikungunya virus/metabolism , Cholesterol/metabolism , Cholesterol/pharmacology , Fibroblasts/metabolism , Fibroblasts/virology , Gene Expression Regulation , Humans , Interferons/genetics , Interferons/metabolism , Liver X Receptors/antagonists & inhibitors , Liver X Receptors/metabolism , Primary Cell Culture , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Signal Transduction , Virus Replication/drug effectsABSTRACT
We have previously shown that blockade of ATP-binding cassette transporter A1 (ABCA1) with cyclosporine A (CsA) stimulates the epithelial sodium channel (ENaC) in cultured distal nephron cells. Here we show that CsA elevated systolic blood pressure in both wild-type and apolipoprotein E (ApoE) knockout (KO) mice to a similar level. The elevated systolic blood pressure was completely reversed by inhibition of cholesterol (Cho) synthesis with lovastatin. Inside-out patch-clamp data show that intracellular Cho stimulated ENaC in cultured distal nephron cells by interacting with phosphatidylinositol4,5bisphosphate (PIP2), an ENaC activator. Confocal microscopy data show that both αENaC and PIP2 were localized in microvilli via a Cho-dependent mechanism. Deletion of membrane Cho reduced the levels of γENaC in the apical membrane. Reduced ABCA1 expression and elevated intracellular Cho were observed in old mice, compared to young mice. In parallel, cell-attached patch-clamp data from the split-open cortical collecting ducts (CCD) show that ENaC activity was significantly increased in old mice. These data suggest that elevation of intracellular Cho due to blockade of ABCA1 stimulates ENaC, which may contribute to CsA-induced hypertension. This study also implies that reduced ABCA1 expression may mediate age-related hypertension by increasing ENaC activity via elevation of intracellular Cho.
Subject(s)
Cholesterol/metabolism , Cyclosporine/adverse effects , Enzyme Inhibitors/adverse effects , Epithelial Sodium Channels/metabolism , Hypertension/chemically induced , ATP Binding Cassette Transporter 1/antagonists & inhibitors , ATP Binding Cassette Transporter 1/metabolism , Animals , Blood Pressure/drug effects , Cell Line , Hypertension/metabolism , Mice , Mice, Inbred C57BL , Phosphatidylinositol Phosphates/metabolism , XenopusABSTRACT
The pathogenesis of Alzheimer's disease (AD) is characterized by overproduction, impaired clearance, and deposition of amyloid-ß peptides (Aß) and connected to cholesterol homeostasis. Since the blood-brain barrier (BBB) is involved in these processes, we investigated effects of the retinoid X receptor agonist, bexarotene (Bex), and the peroxisome proliferator-activated receptor α agonist and antioxidant, astaxanthin (Asx), on pathways of cellular cholesterol metabolism, amyloid precursor protein processing/Aß production and transfer at the BBB in vitro using primary porcine brain capillary endothelial cells (pBCEC), and in 3xTg AD mice. Asx/Bex downregulated transcription/activity of amyloidogenic BACE1 and reduced Aß oligomers and ~80â¯kDa intracellular 6E10-reactive APP/Aß species, while upregulating non-amyloidogenic ADAM10 and soluble (s)APPα production in pBCEC. Asx/Bex enhanced Aß clearance to the apical/plasma compartment of the in vitro BBB model. Asx/Bex increased expression levels of ABCA1, LRP1, and/or APOA-I. Asx/Bex promoted cholesterol efflux, partly via PPARα/RXR activation, while cholesterol biosynthesis/esterification was suppressed. Silencing of LRP-1 or inhibition of ABCA1 by probucol reversed Asx/Bex-mediated effects on levels of APP/Aß species in pBCEC. Murine (m)BCEC isolated from 3xTg AD mice treated with Bex revealed elevated expression of APOE and ABCA1. Asx/Bex reduced BACE1 and increased LRP-1 expression in mBCEC from 3xTg AD mice when compared to vehicle-treated or non-Tg treated mice. In parallel, Asx/Bex reduced levels of Aß oligomers in mBCEC and Aß species in brain soluble and insoluble fractions of 3xTg AD mice. Our results suggest that both agonists exert beneficial effects at the BBB by balancing cholesterol homeostasis and enhancing clearance of Aß from cerebrovascular endothelial cells.
Subject(s)
Amyloid beta-Peptides/metabolism , Bexarotene/pharmacology , Blood-Brain Barrier/drug effects , Cholesterol/metabolism , Protective Agents/pharmacology , ADAM10 Protein/metabolism , ATP Binding Cassette Transporter 1/antagonists & inhibitors , ATP Binding Cassette Transporter 1/metabolism , Alzheimer Disease/drug therapy , Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Amyloid Precursor Protein Secretases/metabolism , Animals , Apolipoproteins E/metabolism , Bexarotene/therapeutic use , Blood-Brain Barrier/metabolism , Down-Regulation/drug effects , Endothelial Cells/cytology , Endothelial Cells/metabolism , Female , Mice , Mice, Inbred C57BL , Mice, Transgenic , Probucol/pharmacology , Swine , Xanthophylls/pharmacologyABSTRACT
High-density lipoprotein (HDL), also known as antiatherogenic lipoprotein, consists of heterogeneous particles in terms of size, density and composition, suggesting differences among HDL subclasses in characteristics and functions. We investigated the role of apolipoprotein E (apoE)-containing HDL, a minor HDL subclass, in the cholesterol efflux capacity (CEC) of HDL, which is its predominant atheroprotective function. The CEC of apoE-containing HDL was similar to that of apoE-deficient HDL, but the former exhibited a greater rate increase (1.48-fold) compared to that of the latter (1.10-fold) by the stimulation of THP-1 macrophages with the Liver X Receptor (LXR) agonist. No difference in CEC was observed without the LXR agonist between apoA-I, the main apolipoprotein in HDL, and apoE, whereas the increase in CEC in response to treatment with the LXR agonist was greater for apoA-I (4.25-fold) than for apoE (2.22-fold). Furthermore, the increase in the CEC of apoE-containing HDL induced by the LXR agonist was significantly reduced by treatment with glyburide, an inhibitor of ATP-binding cassette transporter A1 (ABCA1). These results suggest that apoE-containing HDL, unlike apoE-deficient HDL, is involved in cholesterol efflux via ABCA1.
Subject(s)
Apolipoproteins E/metabolism , Cholesterol/metabolism , Lipoproteins, HDL/metabolism , ATP Binding Cassette Transporter 1/antagonists & inhibitors , Glyburide/pharmacology , Humans , Hypoglycemic Agents/pharmacology , Liver X Receptors/agonists , Macrophages/metabolism , THP-1 CellsABSTRACT
BACKGROUND: HIV-associated atherosclerosis is a major comorbidity due, in part, to systemic effects of the virus on cholesterol metabolism. HIV protein Nef plays an important role in this pathology by impairing maturation of the main cellular cholesterol transporter ATP-Binding Cassette (ABCA) 1. ABCA1 maturation critically depends on calnexin, an integral endoplasmic reticulum membrane chaperone, and Nef binds to the cytoplasmic domain of calnexin and impairs interaction of calnexin with ABCA1. Overarching goal of the present study was to model Nef-calnexin interaction interface, and identify small molecule compounds potentially inhibiting this interaction. METHODS: Molecular dynamics was utilized to build structure model of calnexin cytoplasmic domain, followed by global docking combined with application of QASDOM software developed by us for efficient analysis of receptor-ligand complexes. Structure-based virtual screening was performed for all sites identified by docking. A soluble analogue of a compound from the screening results list was tested for ability to down-regulate ABCA1. RESULTS: We identified major interaction sites in calnexin and reciprocal sites in Nef. Virtual screening yielded a number of small-molecule compounds potentially blocking a calnexin site. Interestingly, one of the compounds, NSC13987, was previously identified by us as an inhibitor targeting a Nef site. An analogue of NSC13987, AMS-55, potently reversed the negative effect of Nef on ABCA1 abundance. CONCLUSIONS: We have modelled Nef-calnexin interaction, predicted small molecule compounds that can potentially inhibit this interaction, and experimentally tested one of these compounds, confirming its effectiveness. These findings provide a platform for searching for new therapeutic agents to treat HIV-associated comorbidities.
Subject(s)
Calnexin/metabolism , HIV-1/pathogenicity , Host-Pathogen Interactions , nef Gene Products, Human Immunodeficiency Virus/metabolism , ATP Binding Cassette Transporter 1/antagonists & inhibitors , Enzyme Inhibitors/isolation & purification , Enzyme Inhibitors/pharmacology , Humans , Models, Molecular , Molecular Docking Simulation , Molecular Dynamics Simulation , Protein Binding/drug effects , Protein Interaction Mapping , nef Gene Products, Human Immunodeficiency Virus/antagonists & inhibitorsABSTRACT
Hepatic ATP-binding cassette A1 (ABCA1) transporter is the modulator of intrahepatic cholesterol levels via the efflux of cholesterol into plasma. This study aimed to determine the expression of hepatic ABCA1 levels in a cholestatic rat model and patients with primary biliary cholangitis (PBC). A cholesterol efflux study was conducted with Abca1 knock down using siRNA in WIF9 cells. Cholesterol levels in the ABCA1 siRNA cells in the medium were significantly decreased compared with those in controls (P < 0.05). Hepatic ABCA1 mRNA levels were significantly higher in BDL rats than in control rats (P < 0.05). Furthermore, the protein expression level of hepatic ABCA1 was also significantly increased by 200% in BDL rats (P < 0.05). In PBC patients, expression of hepatic ABCA1 mRNA was 2.2-fold higher than that in controls (P < 0.05). The level of hepatic liver X receptor (LXR)ß mRNA was correlated with ABCA1 mRNA levels in PBC patients. The expression of hepatic ABCA1 transporter was upregulated in both the cholestatic rat model and PBC patients. Upregulated hepatic ABCA1 may lead to efflux of cholesterol into plasma, thus explaining the mechanism of cholestasis leading to hypercholesterolemia.
Subject(s)
ATP Binding Cassette Transporter 1/genetics , Cholestasis, Intrahepatic/genetics , Cholesterol/metabolism , Hypercholesterolemia/genetics , Liver Cirrhosis, Biliary/genetics , Liver/metabolism , ATP Binding Cassette Transporter 1/antagonists & inhibitors , ATP Binding Cassette Transporter 1/metabolism , Animals , Biological Transport , Cell Line, Tumor , Cholestasis, Intrahepatic/metabolism , Cholestasis, Intrahepatic/pathology , Disease Models, Animal , Gene Expression Regulation , Hepatocytes/metabolism , Hepatocytes/pathology , Humans , Hypercholesterolemia/metabolism , Hypercholesterolemia/pathology , Liver/pathology , Liver Cirrhosis, Biliary/metabolism , Liver Cirrhosis, Biliary/pathology , Liver X Receptors/genetics , Liver X Receptors/metabolism , Male , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Rats , Rats, Wistar , Signal TransductionABSTRACT
BACKGROUND AND AIMS: Micro-particles (MP) are secreted by various cells. Their biological roles in health and in disease remain unknown. Here we describe formation of MP in the process of ABCA1-dependent cholesterol efflux in different cell types. METHODS: The ATP-binding cassette transporter, subfamily A, member 1 (ABCA1) is the rate-limiting step in the biogenesis of high-density lipoproteins (HDL). We have found that ABCA1 and apoA-I contribute to the formation of MP. Using cell-based systems with overexpression and selective inactivation of ABCA1, pharmacological blockade and modulation of membrane cholesterol content, we characterized MP release from various cell lines. We studied MP release in BHK cells stably expressing ABCA1 under mifepristone control, human THP-1 macrophages and HepG2 cells without, or with incubation with human apoA-I. RESULTS: ABCA1 mediates the production of MPs containing cholesterol. This was also confirmed in primary human monocyte-derived macrophages (MDMs). Adding apoA-I markedly increases MP release from cells. Inhibition of ABCA1 with probucol or decreasing plasma membrane cholesterol with methyl-ß cyclodextrin (CDX) markedly reduced MP release and nascent HDL formation. MPs do not contain apoA-I, but contain flotilin-2, a marker of plasma membrane, and CD63, an exosome marker. MPs exhibit considerable size heterogeneity (50-250 nm). CONCLUSIONS: We show that MPs are lipoprotein-sized structures created by the ABCA1 transporter, and contribute approximately 30% of ABCA1-and apoA-I mediated cholesterol efflux. In addition, we found that MPs release from cells consists, in part, of exosomes and depends on the same pathway used for HDL biogenesis.
Subject(s)
ATP Binding Cassette Transporter 1/metabolism , Cell-Derived Microparticles/metabolism , Lipoproteins, HDL/biosynthesis , Macrophages/metabolism , ATP Binding Cassette Transporter 1/antagonists & inhibitors , ATP Binding Cassette Transporter 1/genetics , Animals , Apolipoprotein A-I/metabolism , Cell-Derived Microparticles/drug effects , Cholesterol/metabolism , Cricetinae , Exosomes/metabolism , Hep G2 Cells , Humans , Macrophages/drug effects , Membrane Proteins/metabolism , Particle Size , Probucol/pharmacology , Tetraspanin 30/metabolism , Time Factors , Transfection , beta-Cyclodextrins/pharmacologyABSTRACT
Sphingosine-1-phosphate (S1P), which has emerged as a pivotal signaling mediator that participates in the regulation of multiple cellular processes, is derived from various cells, including vascular endothelial cells. S1P accumulates in lipoproteins, especially HDL, and the majority of free plasma S1P is bound to HDL. We hypothesized that HDL-associated S1P is released through mechanisms associated with the HDL maturation process. ApoA-I, a major HDL apolipoprotein, is a critical factor for nascent HDL formation and lipid trafficking via ABCA1. Moreover, apoA-I is capable of promoting bidirectional lipid movement through SR-BI. In the present study, we confirmed that apoA-I can facilitate the production and release of S1P by HUVECs. Furthermore, we demonstrated that ERK1/2 and SphK activation induced by apoA-I is involved in the release of S1P from HUVECs. Inhibitor and siRNA experiments showed that ABCA1 and SR-BI are required for S1P release and ERK1/2 phosphorylation induced by apoA-I. However, the effects triggered by apoA-I were not suppressed by inhibiting ABCA1/JAK2 or the SR-BI/Src pathway. S1P released due to apoA-I activation can stimulate the (ERK1/2)/SphK1 pathway through S1PR (S1P receptor) 1/3. These results indicated that apoA-I not only promotes S1P release through ABCA1 and SR-BI but also indirectly activates the (ERK1/2)/SphK1 pathway by releasing S1P to trigger their receptors. In conclusion, we suggest that release of S1P induced by apoA-I from endothelial cells through ABCA1 and SR-BI is a self-positive-feedback process: apoA-I-(ABCA1 and SR-BI)-(S1P release)-S1PR-ERK1/2-SphK1-(S1P production)-(more S1P release induced by apoA-I).
Subject(s)
ATP Binding Cassette Transporter 1/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Apolipoprotein A-I/pharmacology , Lysophospholipids/metabolism , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Scavenger Receptors, Class B/metabolism , Sphingosine/analogs & derivatives , ATP Binding Cassette Transporter 1/antagonists & inhibitors , ATP Binding Cassette Transporter 1/genetics , Adaptor Proteins, Signal Transducing/genetics , Apolipoprotein A-I/metabolism , Dose-Response Relationship, Drug , Feedback, Physiological , Gene Expression Regulation , Human Umbilical Vein Endothelial Cells , Humans , Mitogen-Activated Protein Kinase 1/genetics , Mitogen-Activated Protein Kinase 3/genetics , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Scavenger Receptors, Class B/antagonists & inhibitors , Scavenger Receptors, Class B/genetics , Signal Transduction , Sphingosine/metabolismABSTRACT
Accumulation of unesterified cholesterol-rich lipid vesicles in the subendothelial space contributes to atherogenesis. Transport of cholesterol from the subendothelial intima back to the circulating blood inhibits atherosclerosis development; however, the mechanism for this process has not been fully defined. Using cultured mouse aortic endothelial cells (MAECs), we observed that unesterified cholesterol can be transported across the endothelial cell monolayer from the basolateral to the apical compartment. Administration of high-density lipoprotein (HDL) or apolipoprotein AI (apoAI) to the apical compartment enhanced transendothelial cholesterol transport in a concentration-dependent manner. Knockdown of ATP-binding cassette transporter G1 (ABCG1) or scavenger receptor class B type I (SR-B1), or inhibition of SR-B1 diminished HDL-induced transendothelial cholesterol transport; while knockdown of ABCA1 reduced apoAI-mediated cholesterol transport. HDL enhanced phosphorylation of phosphatidylinositol 3-kinase (PI3K) and Akt in MAECs. However, inhibition of PI3K or Akt did not reduce HDL-induced transendothelial cholesterol transport. These results suggest that HDL enhances transendothelial cholesterol transport by activation of a mechanism involving ABCA1, ABCG1 and SR-B1 but not involving PI3K and Akt.
Subject(s)
ATP Binding Cassette Transporter 1/metabolism , ATP-Binding Cassette Transporters/metabolism , Aorta/drug effects , Endothelial Cells/drug effects , Lipoproteins, HDL/pharmacology , Lipoproteins/metabolism , Scavenger Receptors, Class B/metabolism , ATP Binding Cassette Transporter 1/antagonists & inhibitors , ATP Binding Cassette Transporter 1/genetics , ATP Binding Cassette Transporter, Subfamily G, Member 1 , ATP-Binding Cassette Transporters/antagonists & inhibitors , ATP-Binding Cassette Transporters/genetics , Animals , Aorta/cytology , Aorta/metabolism , Apolipoprotein A-I/metabolism , Apolipoprotein A-I/pharmacology , Biological Transport/drug effects , Cell Polarity , Dose-Response Relationship, Drug , Endothelial Cells/cytology , Endothelial Cells/metabolism , Gene Expression , Lipoproteins/antagonists & inhibitors , Lipoproteins/genetics , Lipoproteins, HDL/metabolism , Mice , Mice, Inbred C57BL , Phosphatidylinositol 3-Kinase/genetics , Phosphatidylinositol 3-Kinase/metabolism , Phosphorylation/drug effects , Primary Cell Culture , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Scavenger Receptors, Class B/antagonists & inhibitors , Scavenger Receptors, Class B/geneticsABSTRACT
We previously reported that cholesterol-enriched macrophages excrete cholesterol into the extracellular matrix. A monoclonal antibody that detects cholesterol microdomains labels the deposited extracellular particles. Macro-phage deposition of extracellular cholesterol depends, in part, on ABCG1, and this cholesterol can be mobilized by HDL components of the reverse cholesterol transport process. The objective of the current study was to determine whether ABCA1 also contributes to macrophage deposition of extracellular cholesterol. ABCA1 functioned in extracellular cholesterol deposition. The liver X receptor agonist, TO901317 (TO9), an ABCA1-inducing factor, restored cholesterol deposition that was absent in cholesterol-enriched ABCG1(-/-) mouse macrophages. In addition, the ABCA1 inhibitor, probucol, blocked the increment in cholesterol deposited by TO9-treated wild-type macrophages, and completely inhibited deposition from TO9-treated ABCG1(-/-) macrophages. Lastly, ABCA1(-/-) macrophages deposited much less extracellular cholesterol than wild-type macrophages. These findings demonstrate a novel function of ABCA1 in contributing to macrophage export of cholesterol into the extracellular matrix.
Subject(s)
ATP Binding Cassette Transporter 1/genetics , Atherosclerosis/genetics , Cholesterol/metabolism , ATP Binding Cassette Transporter 1/antagonists & inhibitors , ATP Binding Cassette Transporter 1/metabolism , ATP Binding Cassette Transporter, Subfamily G, Member 1 , ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/metabolism , Animals , Apolipoprotein A-I/genetics , Apolipoprotein A-I/metabolism , Atherosclerosis/metabolism , Atherosclerosis/pathology , Cholesterol/genetics , Extracellular Matrix/metabolism , Humans , Hydrocarbons, Fluorinated/administration & dosage , Lipoproteins/genetics , Lipoproteins/metabolism , Lipoproteins, HDL/genetics , Lipoproteins, HDL/metabolism , Liver X Receptors , Macrophages/metabolism , Mice , Orphan Nuclear Receptors/agonists , Orphan Nuclear Receptors/genetics , Probucol/administration & dosage , Sulfonamides/administration & dosageABSTRACT
The transcription of the ATP-binding cassette transporter A1 (ABCA1) gene, which plays a key anti-atherogenic role, is known to be induced by agonists of liver X receptors (LXRs). LXRs form obligate heterodimers with retinoid X receptors (RXRs) and interact with their recognition sequences in the regulatory regions of key genes implicated in the control of cholesterol, fatty acid and glucose homeostasis. We have previously shown a novel role for c-Jun N-terminal kinase (JNK) and phosphoinositide 3-kinase (PI3K) in the LXRs-mediated induction of macrophage gene expression. Protein kinase C (PKC) is often found to regulate the action of nuclear receptors and cross talk between this kinase family and JNK and/or PI3K has been shown in several settings. We have, therefore, investigated a potential role for PKC in the action of LXR/RXR agonist 22-(R)-hydroxycholesterol (22-(R)-HC)/9-cis-retinoic acid (9cRA) in THP-1 macrophages, including the induction of ABCA1 expression. The pan PKC inhibitor bisindoylmaleimide was found to attenuate the induction of ABCA1 protein expression, the activation of the JNK signaling pathway and the stimulation of activator protein-1 (AP-1) DNA binding activity in macrophages treated with 22-(R)-HC and 9cRA. The role of PKC in the action of these ligands was confirmed further by the use of more isotype-specific inhibitors. These studies, therefore, reveal a potentially important role for PKC in the action of 22-(R)-HC and 9cRA in human macrophages.
Subject(s)
ATP Binding Cassette Transporter 1/metabolism , Hydroxycholesterols/pharmacology , Macrophages/drug effects , Protein Kinase C/metabolism , Tretinoin/pharmacology , ATP Binding Cassette Transporter 1/antagonists & inhibitors , Alitretinoin , Cell Line , Gene Expression Regulation/drug effects , Humans , Indoles/pharmacology , Liver X Receptors , MAP Kinase Signaling System/drug effects , Macrophages/cytology , Maleimides/pharmacology , Orphan Nuclear Receptors/agonists , Orphan Nuclear Receptors/metabolism , Retinoid X Receptors/agonists , Retinoid X Receptors/metabolismABSTRACT
ABCB4, which is specifically expressed on the canalicular membrane of hepatocytes, exports phosphatidylcholine (PC) into bile. Because SM depletion increases cellular PC content and stimulates PC and cholesterol efflux by ABCA1, a key transporter involved in generation of HDL, we predicted that SM depletion also stimulates PC efflux through ABCB4. To test this prediction, we compared the lipid efflux activity of ABCB4 and ABCA1 under SM depletion induced by two different types of inhibitors for SM synthesis, myriocin and (1R,3S)-N-(3-hydroxy-1-hydroxymethyl-3-phenylpropyl)dodecanamide, in human embryonic kidney 293 and baby hamster kidney cells. Unexpectedly, SM depletion exerted opposite effects on ABCB4 and ABCA1, suppressing PC efflux through ABCB4 while stimulating efflux through ABCA1. Both ABCB4 and ABCA1 were recovered from Triton-X-100-soluble membranes, but ABCB4 was mainly recovered from CHAPS-insoluble SM-rich membranes, whereas ABCA1 was recovered from CHAPS-soluble membranes. These results suggest that a SM-rich membrane environment is required for ABCB4 to function. ABCB4 must have evolved to exert its maximum activity in the SM-rich membrane environment of the canalicular membrane, where it transports PC as the physiological substrate.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B/metabolism , Cell Membrane/metabolism , Phosphatidylcholines/metabolism , Sphingomyelins/metabolism , ATP Binding Cassette Transporter 1/antagonists & inhibitors , ATP Binding Cassette Transporter 1/genetics , ATP Binding Cassette Transporter 1/metabolism , ATP Binding Cassette Transporter, Subfamily B/antagonists & inhibitors , ATP Binding Cassette Transporter, Subfamily B/genetics , Animals , Biological Transport, Active/physiology , Cell Membrane/genetics , Cricetinae , HEK293 Cells , Humans , Phosphatidylcholines/genetics , Sphingomyelins/geneticsABSTRACT
RATIONALE: High-density lipoprotein (HDL) is a heterogeneous population of particles. Differences in the capacities of HDL subfractions to remove cellular cholesterol may explain variable correlations between HDL-cholesterol and cardiovascular risk and inform future targets for HDL-related therapies. The ATP binding cassette transporter A1 (ABCA1) facilitates cholesterol efflux to lipid-free apolipoprotein A-I, but the majority of apolipoprotein A-I in the circulation is transported in a lipidated state and ABCA1-dependent efflux to individual HDL subfractions has not been systematically studied. OBJECTIVE: Our aims were to determine which HDL particle subfractions are most efficient in mediating cellular cholesterol efflux from foam cell macrophages and to identify the cellular cholesterol transporters involved in this process. METHODS AND RESULTS: We used reconstituted HDL particles of defined size and composition, isolated subfractions of human plasma HDL, cell lines stably expressing ABCA1 or ABCG1, and both mouse and human macrophages in which ABCA1 or ABCG1 expression was deleted. We show that ABCA1 is the major mediator of macrophage cholesterol efflux to HDL, demonstrating most marked efficiency with small, dense HDL subfractions (HDL3b and HDL3c). ABCG1 has a lesser role in cholesterol efflux and a negligible role in efflux to HDL3b and HDL3c subfractions. CONCLUSIONS: Small, dense HDL subfractions are the most efficient mediators of cholesterol efflux, and ABCA1 mediates cholesterol efflux to small dense HDL and to lipid-free apolipoprotein A-I. HDL-directed therapies should target increasing the concentrations or the cholesterol efflux capacity of small, dense HDL species in vivo.
Subject(s)
ATP Binding Cassette Transporter 1/physiology , Cholesterol, HDL/metabolism , Cholesterol/metabolism , Lipoproteins, HDL/metabolism , Macrophages/metabolism , ATP Binding Cassette Transporter 1/antagonists & inhibitors , ATP Binding Cassette Transporter 1/deficiency , ATP Binding Cassette Transporter 1/genetics , ATP Binding Cassette Transporter, Subfamily G, Member 1 , ATP-Binding Cassette Transporters/deficiency , ATP-Binding Cassette Transporters/physiology , Animals , Apolipoprotein A-I/metabolism , Biological Transport , CHO Cells , Cell Line , Cricetinae , Cricetulus , Foam Cells/metabolism , Gene Silencing , Humans , Lipoproteins/deficiency , Lipoproteins/physiology , Lipoproteins, HDL2/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Particle Size , Recombinant Fusion Proteins/metabolism , Tangier Disease/enzymology , Tangier Disease/geneticsABSTRACT
RATIONALE: Macrophage accumulation of cholesterol leads to foam cell formation which is a major pathological event of atherosclerosis. Recent studies have shown that microRNA (miR)-19b might play an important role in cholesterol metabolism and atherosclerotic diseases. Here, we have identified miR-19b binding to the 3'UTR of ATP-binding cassette transporter A1 (ABCA1) transporters, and further determined the potential roles of this novel interaction in atherogenesis. OBJECTIVE: To investigate the molecular mechanisms involved in a miR-19b promotion of macrophage cholesterol accumulation and the development of aortic atherosclerosis. METHODS AND RESULTS: We performed bioinformatics analysis using online websites, and found that miR-19b was highly conserved during evolution and directly bound to ABCA1 mRNA with very low binding free energy. Luciferase reporter assay confirmed that miR-19b bound to 3110-3116 sites within ABCA1 3'UTR. MiR-19b directly regulated the expression levels of endogenous ABCA1 in foam cells derived from human THP-1 macrophages and mouse peritoneal macrophages (MPMs) as determined by qRT-PCR and western blot. Cholesterol transport assays revealed that miR-19b dramatically suppressed apolipoprotein AI-mediated ABCA1-dependent cholesterol efflux, resulting in the increased levels of total cholesterol (TC), free cholesterol (FC) and cholesterol ester (CE) as revealed by HPLC. The excretion of (3)H-cholesterol originating from cholesterol-laden MPMs into feces was decreased in mice overexpressing miR-19b. Finally, we evaluated the proatherosclerotic role of miR-19b in apolipoprotein E deficient (apoE(-/-)) mice. Treatment with miR-19b precursor reduced plasma high-density lipoprotein (HDL) levels, but increased plasma low-density lipoprotein (LDL) levels. Consistently, miR-19b precursor treatment increased aortic plaque size and lipid content, but reduced collagen content and ABCA1 expression. In contrast, treatment with the inhibitory miR-19b antisense oligonucleotides (ASO) prevented or reversed these effects. CONCLUSION: MiR-19b promotes macrophage cholesterol accumulation, foam cell formation and aortic atherosclerotic development by targeting ABCA1.
Subject(s)
ATP Binding Cassette Transporter 1/antagonists & inhibitors , Aortic Diseases/etiology , Atherosclerosis/etiology , Cholesterol/metabolism , Macrophages/metabolism , MicroRNAs/physiology , ATP Binding Cassette Transporter 1/genetics , ATP Binding Cassette Transporter 1/physiology , Animals , Aortic Diseases/genetics , Aortic Diseases/metabolism , Apolipoprotein A-I/metabolism , Apolipoproteins E/deficiency , Atherosclerosis/genetics , Atherosclerosis/metabolism , Base Sequence , Cell Line , Cholesterol Esters/metabolism , Collagen/analysis , Foam Cells/metabolism , Macrophages, Peritoneal/metabolism , Male , Mice , Mice, Knockout , Molecular Sequence Data , Plaque, Atherosclerotic/metabolism , RNA, Messenger/metabolism , Sequence Homology, Nucleic AcidABSTRACT
Cholesterol efflux from macrophages is a critical mechanism to prevent the development of atherosclerosis. Although PPARγ is known to be a potent sterol sensor that play a fundamental role in cholesterol metabolism, the potential effects of PPARγ responsive miRNA still need to be revealed. In this study, we found that miR-613 is inversely correlated with LXRα and ABCA1 in PPARγ activated THP-1 cells. PPARγ negatively regulates the expression of miR-613 at transcriptional level, and miR-613 suppressed LXRα and ABCA1 by targeting the 3'-UTR of their mRNAs. Furthermore, downregulation of LXRα and ABCA1 by miR-613 inhibited cholesterol efflux from PPARγ activated THP-1 macrophages. These results revealed an alternative mechanism for PPARγ regulation and provided a potential target for the treatment of cholesterol metabolic diseases.
Subject(s)
ATP Binding Cassette Transporter 1/antagonists & inhibitors , ATP Binding Cassette Transporter 1/genetics , Cholesterol/metabolism , MicroRNAs/genetics , Orphan Nuclear Receptors/antagonists & inhibitors , Orphan Nuclear Receptors/genetics , PPAR gamma/metabolism , 3' Untranslated Regions , Atherosclerosis/genetics , Atherosclerosis/metabolism , Base Sequence , Binding Sites/genetics , Biological Transport, Active , Cell Line , Down-Regulation , Humans , Liver X Receptors , Macrophage Activation , Macrophages/metabolism , MicroRNAs/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sequence Homology, Nucleic AcidABSTRACT
OBJECTIVE: This study questions whether high-density lipoproteins (HDLs) and apolipoprotein A-I inhibit joint inflammation in streptococcal cell wall peptidoglycan-polysaccharide (PG-PS)-induced arthritis in female Lewis rats. APPROACH AND RESULTS: Administration of PG-PS to female Lewis rats caused acute joint inflammation after 4 days, followed by remission by day 8. The animals subsequently developed chronic joint inflammation that persisted until euthanasia at day 21. Treatment with apolipoprotein A-I 24 hours before and 24 hours after PG-PS administration reduced the acute and chronic joint inflammation. Treatment with apolipoprotein A-I at days 7, 9, and 11 after PG-PS administration reduced the chronic joint inflammation. Treatment with apolipoprotein A-I or reconstituted HDLs consisting of apolipoprotein A-I complexed with phosphatidylcholine 24 hours before and at days 1, 7, 9, and 11 after PG-PS administration reduced acute and chronic joint inflammation. Treatment with apolipoprotein A-I also reduced the inflammatory white blood cell count, synovial fluid proinflammatory cytokine levels, synovial tissue macrophage accumulation, as well as toll-like receptor 2, and inflammatory cytokine expression. At the molecular level, preincubation of human monocyte-derived macrophages with apolipoprotein A-I or reconstituted HDLs before PG-PS stimulation inhibited the PG-PS-induced increase in toll-like receptor 2 and myeloid differentiation primary response gene (88) mRNA levels, nuclear factor-κB activation, and proinflammatory cytokine production. The effects of apolipoprotein A-I and reconstituted HDLs were abolished by transfecting the human monocyte-derived macrophages with ATP-binding cassette transporter A1 or G1 siRNA. CONCLUSIONS: Apolipoprotein A-I and reconstituted HDLs attenuate PG-PS-induced arthritis in the rat. Studies in human monocyte-derived macrophages indicate that this benefit may be because of the inhibition of toll-like receptor 2 expression and decreased nuclear factor-κB activation in macrophages.