Abstract ATP-binding Cassette Transporters Research Articles

Backbone NMR assignment of the nucleotide binding domain of the Bacillus subtilis ABC multidrug transporter BmrA in the post-hydrolysis state

ATP binding cassette (ABC) proteins are present in all phyla of life and form one of the largest protein families. The Bacillus subtilis ABC transporter BmrA is a functional homodimer that can extrude many different harmful compounds out of the cell. Each BmrA monomer is composed of a transmembrane domain (TMD) and a nucleotide binding domain (NBD). While the TMDs of ABC transporters are sequentially diverse, the highly conserved NBDs harbor distinctive conserved motifs that enable nucleotide binding and hydrolysis, interdomain communication and that mark a protein as a member of the ABC superfamily. In the catalytic cycle of an ABC transporter, the NBDs function as the molecular motor that fuels substrate translocation across the membrane via the TMDs and are thus pivotal for the entire transport process. For a better understanding of the structural and dynamic consequences of nucleotide interactions within the NBD at atomic resolution, we determined the 1H, 13C and 15N backbone chemical shift assignments of the 259 amino acid wildtype BmrA-NBD in its post-hydrolytic, ADP-bound state.

ATP-binding cassette transporters and neurodegenerative diseases.

ATP-binding cassette (ABC) transporters are one of the largest groups of transporter families in humans. ABC transporters mediate the translocation of a diverse range of substrates across cellular membranes, including amino acids, nucleosides, lipids, sugars and xenobiotics. Neurodegenerative diseases are a group of brain diseases that detrimentally affect neurons and other brain cells and are usually associated with deposits of pathogenic proteins in the brain. Major neurodegenerative diseases include Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis. ABC transporters are highly expressed in the brain and have been implicated in a number of pathological processes underlying neurodegenerative diseases. This review outlines the current understanding of the role of ABC transporters in neurodegenerative diseases, focusing on some of the most important pathways, and also suggests future directions for research in this field.

ATP hydrolysis and nucleotide exit enhance maltose translocation in the MalFGK2E importer

ATP binding cassette (ABC) transporters employ ATP hydrolysis to harness substrate translocation across membranes. The Escherichia coli MalFGK2E maltose importer is an example of a type I ABC importer and a model system for this class of ABC transporters. The MalFGK2E importer is responsible for the intake of malto-oligossacharides in E.coli. Despite being extensively studied, little is known about the effect of ATP hydrolysis and nucleotide exit on substrate transport. In this work, we studied this phenomenon using extensive molecular dynamics simulations (MD) along with potential of mean force calculations of maltose transport across the pore, in the pre-hydrolysis, post-hydrolysis and nucleotide-free states. We concluded that ATP hydrolysis and nucleotide exit trigger conformational changes that result in the decrease of energetic barriers to maltose translocation towards the cytoplasm, with a concomitant increase of the energy barrier in the periplasmic side of the pore, contributing for the irreversibility of the process. We also identified key residues that aid in positioning and orientation of maltose, as well as a novel binding pocket for maltose in MalG. Additionally, ATP hydrolysis leads to conformations similar to the nucleotide-free state. This study shows the contribution of ATP hydrolysis and nucleotide exit in the transport cycle, shedding light on ABC type I importer mechanisms.

ATP-BINDING CASSETTE TRANSPORTERS (ABC). NOMENCLATURE, STRUCTURE, MOLECULAR DIVERSITY, FUNCTION, CONTRIBUTION TO THE FUNCTIONING OF THE XENOBIOTICS TRANSFORMATION SYSTEM (А REWIEW)

ATP-binding cassette (ABC) transporters are membrane proteins responsible for removing various substrates from cells through cell membranes using the energy derived from ATP hydrolysis. The human genome codes for 48 ABC transporters, which are categorized into eight families: ABCA, ABCB, ABCC, ABCD, ABCE, ABCF, ABCG, ABCH. An ABC molecule consists of two nucleotide-binding domains (NBD) and two transmembrane domains (TMD) which transport substrates (NBD–TMD–NBD–TMD), or exists as a half-transporter consisting of one NBD and one TMD, which functions as a dimer. It is assumed that the ABC transport cycle is a four-stage one. The energy obtained in the hydrolysis of ATP is utilized to remove the substrate from the complex with the transporter molecule and restore it in its original state. In mammals, ABC were, apart from cancer cells, found in many tissues, such as the brain, adrenal glands, kidneys, lungs, testes, liver, placenta, intestines. ABC substrates encompass a wide range of compounds of both endo- and exogenous origin. Some ABC are involved in metabolizing xenobiotics, actively removing both lipophilic compounds that pass through the plasma membrane, and hydrophilic metabolites and conjugates formed in biotransformation phases I and II. A number of studies have demonstrated the interaction between the CYP3A4 enzyme from the P450 cytochrome group (biotransformation phase I) and the transporter АВСВ1. The ABCG2 transporter from the liver and intestines was found to actively export metabolites of biotransformation phase II.

GW27-e1151 ABCG1 protects vascular endothelial cells against high glucose-induced oxidative stress through the Nrf2-dependent pathway

ATP binding cassette transporter G1 (ABCG1), a regulator of cholesterol efflux to HDL, has been found its up-regulation reversed the high glucose-induced oxidative stress in endothelial cells in our previous study. The present study aimed to further implore whether the endogenous antioxidant gene

Abca7 deletion does not affect adult neurogenesis in the mouse.

ATP-binding cassette transporter A7 (ABCA7) is highly expressed in the brain. Recent genome-wide association studies (GWAS) have identified ABCA7 single nucleotide polymorphisms (SNPs) that increase Alzheimer's disease (AD) risk, however, the mechanisms by which ABCA7 may control AD risk remain to be fully elucidated. Based on previous research suggesting that certain ABC transporters may play a role in the regulation of neurogenesis, we conducted a study of cell proliferation and neurogenic potential using cellular bromodeoxyuridine (BrdU) incorporation and doublecortin (DCX) immunostaining in adult Abca7 deficient mice and wild-type-like (WT) littermates. In the present study counting of BrdU-positive and DCX-positive cells in an established adult neurogenesis site in the dentate gyrus (DG) indicated there were no significant differences when WT and Abca7 deficient mice were compared. We also measured the area occupied by immunohistochemical staining for BrdU and DCX in the DG and the subventricular zone (SVZ) of the same mice and this confirmed that ABCA7 does not play a significant role in the regulation of cell proliferation or neurogenesis in the adult mouse.

Abstract WMP38: ABCA1/HDL Pathway Mediates GW3965 Induced Neurorestoration and White Matter Remodeling After Stroke

ATP-binding cassette transporter A1 (ABCA1), a major cholesterol transporter in the central nervous system, plays an important role in the formation of brain HDL-cholesterol. ABCA1 is up-regulated by the transcription factors, liver X receptors (LXRs). Our previous study using brain-specific ABCA1 deficient (floxed, nestin-Cre positive, ABCA1-B/-B) and ABCA1 floxed-control (ABCA1fl/fl) mice showed that ABCA1 has anti-inflammatory effects and reduces white matter (WM) damage in ischemic stroke models. Here, we further test whether GW3965, a synthetic LXR agonist treatment of stroke increases ABCA1 and HDL, and thereby improves WM-remodeling and functional outcome. Adult male ABCA1fl/fl and ABCA1-B/-B mice were subjected to permanent extraluminal distal middle cerebral artery occlusion (dMCAo) by transcranial ligation method. Animals were gavaged with saline or GW3965 (10 mg/kg) starting 24h after dMCAo and daily till sacrificed 14 days after dMCAo. There were no differences in the lesion volume and blood levels of total cholesterol and triglyceride before and after dMCAo between ABCA1-B/-B and ABCA1fl/fl mice. However, GW3965 treatment significantly increased blood HDL levels in ABCA1fl/fl mice, but not in ABCA1-B/-B mice 14 days after dMCAo. GW3965 treatment of ABCA1fl/fl stroke mice, but not in ABCA1-B/-B stroke mice, also increased: 1) WM-density identified by Luxol Fast Blue (myelin marker), Bielshowsky silver (axon marker), and SMI31 (phosphorylated neurofilament marker) staining; 2) the number of platelet-derived growth factor receptor alpha positive oligodendrocyte progenitor cells; 3) ABCA1, synaptophysin and myelin basic protein expression in the ischemic brain; 4) functional outcome 7 and 14 days after dMCAo, compared with non-treatment ABCA1fl/fl stroke mice (p<0.05, n=9/group). GW3965 and HDL treatment significantly increase axonal/neurite outgrowth in cultured primary cortical neurons derived from ABCA1fl/fl embryos, but not in neurons derived from ABCA1-B/-B embryos. Treatment of stroke with GW3965 significantly increased blood level of HDL, increased ABCA1 expression and WM-remodeling in the ischemic brain. Increasing ABCA1 may contribute to GW3965 induced neurorestoration and WM remodeling after stroke.

The ABC exporter MsbA probed by solid state NMR – challenges and opportunities.

ATP binding cassette (ABC) transporters form a superfamily of integral membrane proteins involved in translocation of substrates across the membrane driven by ATP hydrolysis. Despite available crystal structures and extensive biochemical data, many open questions regarding their transport mechanisms remain. Therefore, there is a need to explore spectroscopic techniques such as solid state NMR in order to bridge the gap between structural and mechanistic data. In this study, we investigate the feasibility of using Escherichia coli MsbA as a model ABC transporter for solid state NMR studies. We show that optimised solubilisation and reconstitution procedures enable preparing stable and homogenous protein samples. Depending on the duration of solubilisation, MsbA can be obtained in either an apo- or in a native lipid A bound form. Building onto these optimisations, the first promising MAS-NMR spectra with narrow lines have been recorded. However, further sensitivity improvements are required so that complex NMR experiments can be recorded within a reasonable amount of time. We therefore demonstrate the usability of paramagnetic doping for rapid data acquisition and explore dynamic nuclear polarisation as a method for general signal enhancement. Our results demonstrate that solid state NMR provides an opportunity to address important biological questions related to complex mechanisms of ABC transporters.

Conformational changes in MetNI: steered molecular dynamic studies of the methionine ABC transporter with and without substrates

ATP-binding cassette (ABC) transporters are integral membrane proteins that utilised energy from ATP hydrolysis to translocate substrates across the membrane. In addition to the common nucleotide-binding domains (NBDs) and transmembrane domains (TMDs), the methionine ABC transporter has C-terminal regulatory domains (C2 domains) that belong to ACT protein family. When the amount of methionine in the cell is high, the transport stops. This phenomenon is called trans-inhibition. To understand how a trans-inhibited protein returns to an uninhibited, resting state, we performed steered molecular dynamic simulations with and without the substrates. From the simulations, we observed some important conformational changes in the whole ABC transporter, including the constriction in the translocation pathway in the TMDs and approach of the NBDs. However, the C2 domains behaved differently in two types of the simulations. These findings might help to explain the relationship of the conformational changes of the C2 domains with the rearrangements of the NBDs or TMDs, and provide a way to understand the trans-inhibition from an opposite direction.

Abstract 61: T Cell--Specific Deficiency of ABCG1 Protects Against Atherosclerosis

ATP-binding cassette transporter G1 (ABCG1) promotes cholesterol efflux from peripheral cells to HDL particles, which transport cholesterol to liver for processing for excretion. ABCG1, and not ABCA1, was found to be critical for the proliferation of T lymphocytes. Despite this, to date, very little is known about the role of ABCG1 in T cells during atherogenesis. In this study, we aim to understand how ABCG1 regulates T cell function and how absence of ABCG1 selectively in T cells impacts atherosclerosis. We found that, on a high cholesterol diet, mice with T cell-specific ABCG1 deficiency on the LDLR -/- background (LCK-Cre + /ABCG1 fl/fl /LDLR -/- ) developed 40% less atherosclerotic lesions than their littermate controls (LCK-Cre - /ABCG1 fl/fl /LDLR -/- ) (P<0.0001). Furthermore, we found that the percentage of CD4 + CD25 + Foxp3 + regulatory T cells was increased in the LCK-Cre + /ABCG1 fl/fl /LDLR -/- mice compared to the littermate controls (P<0.01). Since Tregs are considered anti-atherogenic, we hypothesize that the deficiency of ABCG1 in T cells confers changes in Tregs, which exerts our observed atheroprotective effect. We found that Tregs are hyperproliferative in the LCK-Cre + /ABCG1 fl/fl /LDLR -/- mice. We have also found increased conversion of naïve CD4 + T cells into Tregs in the LCK-Cre + /ABCG1 fl/fl /LDLR -/- mice both in vivo and in vitro . Furthermore, the Treg proliferation and differentiation can both be enhanced by the addition of excess cholesterol in vitro . To determine if deficiency of ABCG1 in Treg cells alone is sufficient to drive their induction, we examined the T cell compartment in wild-type and Treg-specific ABCG1 deletion mice (Foxp3-Cre + /ABCG1 fl/fl ). The percentage of Tregs in Foxp3-Cre + /ABCG1 fl/fl mice was significantly higher than that of wild-type controls (P<0.01). In short, we have provided evidence that ABCG1 deficiency in T cells leads to decreased atherosclerosis and induction of Tregs. By tying these two findings together, our study provides novel insights in sterol-mediated regulation of Tregs and its impact on atherosclerosis.

Nucleotide-free Crystal Structure of Nucleotide-binding Domain 1 from Human ABCC1 Supports a ‘General-Base Catalysis’ Mechanism for ATP Hydrolysis

ATP-binding cassette (ABC) transporters are molecular motors that transport substrates through biological membranes, using the energy of ATP binding and hydrolysis to foster a series of conformational changes that follow the alternating access mechanism principles. While this general scheme of substrate transport is well accepted, details about the ATP hydrolysis mechanism itself are still a matter of debate. Decades of biochemical and structural studies have identified well-conserved key residues involved in the enzymatic reaction, but their actual role, or order in the hydrolytic process, differ according to the mechanism involved. Here, we investigate the fundamental mechanisms of ATP hydrolysis, by reporting the nucleotide-free structure of the N-terminal nucleotide-Binding Domain (NBD1) of human Multidrug Resistance Protein 1 (MRP1/ABCC1), a transporter of chemotherapeutic drugs. Comparison with the nucleotide-bound structure clearly identified movements of H827 associated with nucleotide binding and release of the active site. These results, put in the context of other structural information and mutational studies of ABC transporters, support the general-base catalysis mechanism for ATP hydrolysis for ABCC1.

Abstract 49: Myeloid Cell Specific ABCA1 Deletion Does Not Significantly Accelerate Atherogenesis in LDL Receptor Knockout Mice

ATP binding cassette transporter A1 (ABCA1) functions as a cellular cholesterol exporter. Macrophage ABCA1 expression is important in reducing cellular cholesterol content and inflammatory response. Bone marrow (BM) transplantation studies suggest that leukocyte ABCA1 protects against atherosclerosis development. However, the in vivo effect of macrophage ABCA1 in atherogenesis is not fully understood due to the presence in BM of other leukocyte populations. Myeloid[[Unable to Display Character: ‐]]Specific ABCA1 Knockout (MSKO) mice in the LDL receptor knockout (LDLrKO) C57BL/6 background were developed to address this question. MSKO/LDLrKO (DKO) and LDLrKO (SKO) mice were fed chow or an atherogenic diet for 10-24wk to examine early to advanced stages of atherosclerosis. Basal (i.e. chow) plasma lipid levels were similar between genotypes, but relative to SKO mice, DKO mice had reduced plasma apoB[[Unable to Display Character: ‐]]containing lipoprotein (apoB Lp) levels throughout the diet-induced atherosclerosis progression phase resulting from decreased hepatic VLDL secretion. Despite a significant reduction in plasma apoB Lp levels, chow and atherogenic diet fed DKO mice had significantly higher cholesterol content in resident peritoneal macrophages and higher plasma proinflammatory cytokine and chemokine levels during atherogenic diet[[Unable to Display Character: ‐]]feeding compared to SKO mice. Aortic cholesterol content was similar between genotypes at early (i.e. 24wk chow-fed) or intermediate (i.e. atherogenic diet for 10wk or 16wk) stages and slightly increased at late stage atherosclerosis (i.e. 24wk atherogenic diet). Aortic root lesion area was also similar for both genotypes after 16wk of atherosclerosis induction. Transplantation of DKO or SKO BM into SKO mice followed by 16wk atherogenic diet feeding also showed similar extent of atherosclerosis. Collectively, these results suggest a novel role for myeloid cell ABCA1 in increasing hepatic VLDL secretion and plasma apoB Lp concentrations in atherogenic diet fed LDLrKO mice that offsets its atheroprotective role in decreasing macrophage cholesterol content and inflammatory response, resulting in no increase in atherosclerosis in its absence.

Quantitative evaluation of the combination between cytotoxic drug and efflux transporter inhibitors based on a tumour growth inhibition model

ATP-Binding Cassette transporters such as ABCG2 confer resistance to various anticancer drugs including irinotecan and its active metabolite, SN38. Early quantitative evaluation of efflux transporter inhibitors-cytotoxic combination requires quantitative drug-disease models. A proof-of-concept study has been carried out for studying the effect of a new ABCG2 transporter inhibitor, MBLI87 combined to irinotecan in mice xenografted with cells overexpressing ABCG2. Mice were treated with irinotecan alone or combined to MBLI87, and tumour size was periodically measured. To model those data, a tumour growth inhibition model was developed. Unperturbed tumour growth was modelled using Simeoni's model. Drug effect kinetics was accounted for by a Kinetic-Pharmacodynamic approach. Effect of inhibitor was described with a pharmacodynamic interaction model where inhibitor enhances activity of cytotoxic. This model correctly predicted tumour growth dynamics from our study. MBLI87 increased irinotecan potency by 20% per μmol of MBLI87. This model retains enough complexity to simultaneously describe tumour growth and effect of this type of drug combination. It can thus be used as a template to early evaluate efflux transporter inhibitors in-vivo.

Genome-wide RNA interference screening for the clock-related gene of ATP-binding cassette transporters in Drosophila melanogaster (Diptera: Drosophilidae)

ATP-binding cassette (ABC) transporters are widespread among organisms, and 56 genes encode ABC transporters in Drosophilamelanogaster (Meigen). Their functions are thought to be divergent. In this study, we examined whether there is a clock-related ABC transporter by performing genome-wide screening using tissue-specific RNA interference. We obtained five candidates when we used tim(UAS)-gal4, which expresses in virtually all clock-related cells. Because their phenotypes were principally reproducible even when we used pdf-gal4, which expresses in a subset of pacemaker neurons only, those transporters were presumed to function in pacemaker neurons. Those five candidates can be categorized into two groups according to the phenotype of the knockdown flies. In one group, CG9281 and CG15410 (E23), the circadian period of knockdown flies was altered. In the other group, CG5944, CG6052, and CG18633, some of the knockdown flies became arrhythmic whereas for others rhythmicity remained intact. Our results suggest that some ABC transporters that have a significant function in the Drosophila circadian system.

ATP binding cassette systems: structures, mechanisms, and functions

Abstract ATP-binding cassette (ABC) systems are found in all three domains of life and in some giant viruses and form one of the largest protein superfamilies. Most family members are transport proteins that couple the free energy of ATP hydrolysis to the translocation of solutes across a biological membrane. The energizing module is also used to drive non-transport processes associated, e.g., with DNA repair and protein translation. Many ABC proteins are of considerable medical importance. In humans, dysfunction of at least eighteen out of 49 ABC transporters is associated with disease, such as cystic fibrosis, Tangier disease, adrenoleukodystrophy or Stargardt’s macular degeneration. In prokaryotes, ABC proteins confer resistance to antibiotics, secrete virulence factors and envelope components, or mediate the uptake of a large variety of nutrients. Canonical ABC transporters share a common structural organization comprising two transmembrane domains (TMDs) that form the translocation pore and two nucleotide-binding domains (NBDs) that bind and hydrolyze ATP. In this Mini-Review, we summarize recent structural and biochemical data obtained from both prokaryotic and eukaryotic model systems.

Functional Rescue of a Misfolded Eukaryotic ATP-binding Cassette Transporter by Domain Replacement

ATP-binding cassette (ABC) transporters are integral membrane proteins that couple ATP binding/hydrolysis with the transport of hydrophilic substrates across lipid barriers. Deletion of Phe-670 in the first nucleotide-binding domain (NBD1) of the yeast ABC transporter, Yor1p, perturbs interdomain associations, reduces functionality, and hinders proper transport to the plasma membrane. Functionality of Yor1p-ΔF was restored upon co-expression of a peptide containing wild-type NBD1. To gain insight into the biogenesis of this important class of proteins, we defined the requirements for this rescue. We show that a misfolding lesion in NBD1 of the full-length protein is a prerequisite for functional rescue by exogenous NBD1, which is mediated by physical replacement of the dysfunctional domain by the soluble NBD1. This association does not restore trafficking of Yor1p-ΔF but instead confers catalytic activity to the small population of Yor1p-ΔF that escapes to the plasma membrane. An important coupling between the exogenous NBD1 and ICL4 within full-length aberrant Yor1p-ΔF is required for functional rescue but not for the physical interaction between the two polypeptides. Together, our genetic and biochemical data reveal that it is possible to modulate activity of ABC transporters by physically replacing dysfunctional domains.

Arabidopsis ABCG Transporters, Which Are Required for Export of Diverse Cuticular Lipids, Dimerize in Different Combinations

ATP binding cassette (ABC) transporters play diverse roles, including lipid transport, in all kingdoms. ABCG subfamily transporters that are encoded as half-transporters require dimerization to form a functional ABC transporter. Different dimer combinations that may transport diverse substrates have been predicted from mutant phenotypes. In Arabidopsis thaliana, mutant analyses have shown that ABCG11/WBC11 and ABCG12/CER5 are required for lipid export from the epidermis to the protective cuticle. The objective of this study was to determine whether ABCG11 and ABCG12 interact with themselves or each other using bimolecular fluorescence complementation (BiFC) and protein traffic assays in vivo. With BiFC, ABCG11/ABCG12 heterodimers and ABCG11 homodimers were detected, while ABCG12 homodimers were not. Fluorescently tagged ABCG11 or ABCG12 was localized in the stem epidermal cells of abcg11 abcg12 double mutants. ABCG11 could traffic to the plasma membrane in the absence of ABCG12, suggesting that ABCG11 is capable of forming flexible dimer partnerships. By contrast, ABCG12 was retained in the endoplasmic reticulum in the absence of ABCG11, indicating that ABCG12 is only capable of forming a dimer with ABCG11 in epidermal cells. Emerging themes in ABCG transporter biology are that some ABCG proteins are promiscuous, having multiple partnerships, while other ABCG transporters form obligate heterodimers for specialized functions.

Cholesterol efflux to apoA-I in ABCA1-expressing cells is regulated by Ca2+-dependent calcineurin signaling

ATP-binding cassette transporter A1 (ABCA1) is required for the lipidation of apolipoprotein A-I (apoA-I), although molecular mechanisms supporting this process remain poorly defined. In this study, we focused on the role of cytosolic Ca(2+) and its signaling and found that cytosolic Ca(2+) was required for cholesterol efflux to apoA-I. Removing extracellular Ca(2+) or chelating cytosolic Ca(2+) were equally inhibitory for apoA-I lipidation. We provide evidence that apoA-I induced Ca(2+) influx from the medium. We further demonstrate that calcineurin activity, the downstream target of Ca(2+) influx, was essential; inhibition of calcineurin activity by cyclosporine A or FK506 completely abolished apoA-I lipidation. Furthermore, calcineurin inhibition abolished apoA-I binding and diminished JAK2 phosphorylation, an established signaling event for cholesterol efflux to apoA-I. Finally, we demonstrate that neither Ca(2+) manipulation nor calcineurin inhibition influenced ABCA1's capacity to release microparticles or to remodel the plasma membrane. We conclude that this Ca(2+)-dependent calcineurin/JAK2 pathway is specifically responsible for apoA-I lipidation without directly modifying ABCA1 activity.

Paraoxon Down Regulates ATP-binding Cassette Transporter A1 Expression and Decreases Cholesterol Efflux Through Cyclic AMP Signaling Pathway in RAW 264.7 Macrophage-derived Foam Cells*

ATP-binding Cassette Transporter A1(ABCA1) plays a critical role in the reverse cholesterol transport(RCT).Previous studies showed that paraoxon,the active metabolite of organophosphorus insecticide,increased cholesterol retention in macrophages.However,its underlying mechanisms remain to be elucidated.The effect of paraoxon on ABCA1 expression and ABCA1-dependent cholesterol efflux was investigated,and then the role of cyclic adenosine monophospate(cAMP) signaling pathway in the regulation of ABCA1 expression and ABCA1-mediated cholesterol efflux was examined by paraoxon in RAW 264.7 macrophage-derived foam cells.Results showed that paraoxon significantly down regulated ABCA1 expression and reduced ABCA1-dependent cholesterol efflux and increased the levels of the total,free and esterified cholesterols in a time-and dose-dependent manner.Paraoxon also markedly reduced cAMP level and decreased adenylate cyclase(AC) activity and increased cAMP-specific phosphodiesterase(PDE) activity.Furthermore,cAMP analogs dibutyryl cyclic adenosine monophosphate(dBcAMP) markedly compensated the down-regulation of ABCA1 expression and partly compensated the reduction of ABCA1-mediated cholesterol efflux induced by paraoxon.Also,both adenylate cyclase agonist forskolin and phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine(IBMX) markedly compensated the suppression effect on cAMP level induced by paraoxon.In conclusion,the results mentioned above suggest that paraoxon down regulates ABCA1 expression and decreases ABCA1-mediated cholesterol efflux through cyclic AMP signaling pathway in RAW 264.7 macrophage-derived foam cells.

Abstract 1729: PPARγ Regulates ABCG1 Expression in Macrophages via LXR-Driven Dual Promoters

ATP binding cassette transporter G1 (ABCG1), which is expressed in macrophages, has been implicated in the efflux of cholesterol to high density lipoprotein. Peroxisome proliferator-activated receptor γ (PPARγ) has been reported to be involved in cholesterol efflux from macrophages, and increased expression of ABCG1 via liver receptor X (LXR)-dependent and independent pathways. However, the mechanisms by which ABCG1 expression is increased by PPARγ have not been fully characterized. We observed that pioglitazone, a PPARγ ligand, increases cholesterol efflux from THP-1 macrophages, as well as ABCG1 mRNA and protein levels. Treatment with actinomycin D abolished the inducible effect of pioglitazone on ABCG1, indicating that pioglitazone transcriptionally activated ABCG1 expression. To clarify how pioglitazone regulates ABCG1 expression, we investigated promoter activity using reporter constructs containing human ABCG1 promoter A and B (located upstream of exon 1 and 5, respectively), with or without mutated LXR-binding sites. The results indicated that pioglitazone activated both promoters in an LXR-dependent manner. We also observed that pioglitazone increased two major transcripts driven by promoter A and B using specific primers for each transcript. To determine whether PPARγ and LXRα were involved in these effects of pioglitazone, we performed siRNA-knockdown of PPARγ and LXRα in macrophages, which resulted in 75% and 91% decreases in PPARγ and LXRα mRNA levels, respectively. PPARγ and LXRα-knockdown, respectively, completely or partially abolished pioglitazone-induced ABCG1 expression. In conclusion, these results suggest that pioglitazone transcriptionally increased ABCG1 expression in macrophages by activating dual promoters in an LXR-dependent manner. Further studies are needed to assess LXR-independent mechanisms for the stimulatory effect of pioglitazone on ABCG1.