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• W2157738629 abstract "It has been suggested that the signal transduction initiated by apolipoprotein A-I (apoA-I) activates key proteins involved in cholesterol efflux. ABCA1 serves as a binding partner for apoA-I, but its participation in apoA-I-induced signaling remains uncertain. We show that the exposure of human fibroblasts to ABCA1 ligands (apolipoproteins and amphipathic helical peptides) results in the generation of intracellular signals, including activation of the small G-protein Cdc42, protein kinases (PAK-1 and p54JNK), and actin polymerization. ApoA-I-induced signaling was abrogated by glyburide, an inhibitor of the ABC transporter family, and in fibroblasts from patients with Tangier disease, which do not express ABCA1. Conversely, induction of ABCA1 expression with the liver X receptor agonist, T0901317, and the retinoid X receptor agonist, R0264456, potentiated apoA-I-induced signaling. Similar effects were observed in HEK293 cells overexpressing ABCA1-green fluorescent protein (GFP) fusion protein, but not ABCA1-GFP (K939M), which fails to hydrolyze ATP, or a nonfunctional ABCA1-GFP with a truncated C terminus. We further found that Cdc42 coimmunoprecipitates with ABCA1 in ABCA1-GFP-expressing HEK293 cells exposed to apoA-I but not in cells expressing ABCA1 mutants. We conclude that ABCA1 transduces signals from apoA-I by complexing and activating Cdc42 and downstream kinases and, therefore, acts as a full apoA-I receptor. It has been suggested that the signal transduction initiated by apolipoprotein A-I (apoA-I) activates key proteins involved in cholesterol efflux. ABCA1 serves as a binding partner for apoA-I, but its participation in apoA-I-induced signaling remains uncertain. We show that the exposure of human fibroblasts to ABCA1 ligands (apolipoproteins and amphipathic helical peptides) results in the generation of intracellular signals, including activation of the small G-protein Cdc42, protein kinases (PAK-1 and p54JNK), and actin polymerization. ApoA-I-induced signaling was abrogated by glyburide, an inhibitor of the ABC transporter family, and in fibroblasts from patients with Tangier disease, which do not express ABCA1. Conversely, induction of ABCA1 expression with the liver X receptor agonist, T0901317, and the retinoid X receptor agonist, R0264456, potentiated apoA-I-induced signaling. Similar effects were observed in HEK293 cells overexpressing ABCA1-green fluorescent protein (GFP) fusion protein, but not ABCA1-GFP (K939M), which fails to hydrolyze ATP, or a nonfunctional ABCA1-GFP with a truncated C terminus. We further found that Cdc42 coimmunoprecipitates with ABCA1 in ABCA1-GFP-expressing HEK293 cells exposed to apoA-I but not in cells expressing ABCA1 mutants. We conclude that ABCA1 transduces signals from apoA-I by complexing and activating Cdc42 and downstream kinases and, therefore, acts as a full apoA-I receptor. ABCA1 mediates the active transfer of excess cellular cholesterol and phospholipids from cells to exchangeable apolipoproteins, such as apolipoprotein A-I (apoA-I), apoC-III, and apoE (1Hersberger M. von Eckardstein A. Low high-density lipoprotein cholesterol: physiological background, clinical importance and drug treatment.Drugs. 2003; 63: 1907-1945Crossref PubMed Scopus (59) Google Scholar, 2Assmann G. Nofer J-R. Atheroprotective effects of high-density lipoproteins.Annu. Rev. Med. 2003; 54: 321-341Crossref PubMed Scopus (301) Google Scholar, 3Nofer J-R. Remaley A.T. Tangier disease: still more questions than answers.Cell. Mol. Life Sci. 2005; 62: 2150-2160Crossref PubMed Scopus (68) Google Scholar). The nascent HDL particles formed in this process initiate reverse cholesterol transport, one of the major mechanisms by which HDL protects against the development of atherosclerosis. The importance of ABCA1 for reverse cholesterol transport has been underscored by the identification of ABCA1 defects in Tangier disease, a severe HDL deficiency syndrome characterized by cholesterol deposition in tissue macrophages and premature atherosclerosis (1Hersberger M. von Eckardstein A. Low high-density lipoprotein cholesterol: physiological background, clinical importance and drug treatment.Drugs. 2003; 63: 1907-1945Crossref PubMed Scopus (59) Google Scholar, 2Assmann G. Nofer J-R. Atheroprotective effects of high-density lipoproteins.Annu. Rev. Med. 2003; 54: 321-341Crossref PubMed Scopus (301) Google Scholar, 3Nofer J-R. Remaley A.T. Tangier disease: still more questions than answers.Cell. Mol. Life Sci. 2005; 62: 2150-2160Crossref PubMed Scopus (68) Google Scholar). The mechanism by which ABCA1 facilitates cholesterol egress from cells is still a matter of debate. There is emerging evidence, however, that both binding of apolipoproteins to ABCA1 and apolipoprotein-induced cellular signaling play important roles in initiating lipid efflux. Cross-linking studies documented that apoA-I and other exchangeable apolipoproteins interact directly with ABCA1 on the cell surface (4Wang N. Silver D.L. Costet P. Tall A.R. Specific binding of apoA-I, enhanced cholesterol efflux, and altered plasma membrane morphology in cells expressing ABC1.J. Biol. Chem. 2000; 275: 33053-33058Abstract Full Text Full Text PDF PubMed Scopus (511) Google Scholar, 5Oram J.F. Lawn R.M. Garvin M.R. Wade D.P. ABCA1 is the cAMP-inducible apolipoprotein receptor that mediates cholesterol secretion from macrophages.J. Biol. Chem. 2000; 275: 34508-34511Abstract Full Text Full Text PDF PubMed Scopus (476) Google Scholar, 6Fitzgerald M.L. Morris A.L. Rhee J.S. Andersson L.P. Mendez A.J. Freeman M.W. Naturally occurring mutations in the largest extracellular loops of ABCA1 can disrupt its direct interaction with apolipoprotein A-I.J. Biol. Chem. 2002; 277: 33178-33187Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 7Fitzgerald M.L. Morris A.L. Chroni A. Mendez A.J. Zannis V.I. Freeman M.W. ABCA1 is the cAMP-inducible apolipoprotein receptor that mediates cholesterol secretion from macrophages.J. Lipid Res. 2004; 45: 287-294Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). In addition, apoA-I was shown to activate several serine/threonine protein kinases, including protein kinase A (PKA), protein kinase C (PKC), and Janus kinase 2 (JAK2) (8Haidar B. Denis M. Marcil M. Krimbou L. Genest Jr., J. Apolipoprotein A-I activates cellular cAMP signaling through the ABCA1 transporter.J. Biol. Chem. 2004; 279: 9963-9969Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 9Mendez A.J. Oram J.F. Bierman E.L. Protein kinase C as a mediator of high density lipoprotein receptor-dependent efflux of intracellular cholesterol.J. Biol. Chem. 1991; 266: 10104-10111Abstract Full Text PDF PubMed Google Scholar, 10Yamauchi Y. Hayashi M. Abe-Dohmae S. Yokoyama S. Apolipoprotein A-I activates protein kinase C alpha signaling to phosphorylate and stabilize ATP binding cassette transporter A1 for the high density lipoprotein assembly.J. Biol. Chem. 2003; 278: 47890-47897Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 11Tang C. Vaughan A.M. Oram J.F. Janus kinase 2 modulates the apolipoprotein interactions with ABCA1 required for removing cellular cholesterol.J. Biol. Chem. 2004; 279: 7622-7628Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Both PKA and PKC directly phosphorylate ABCA1 and thereby control its expression level and activity (10Yamauchi Y. Hayashi M. Abe-Dohmae S. Yokoyama S. Apolipoprotein A-I activates protein kinase C alpha signaling to phosphorylate and stabilize ATP binding cassette transporter A1 for the high density lipoprotein assembly.J. Biol. Chem. 2003; 278: 47890-47897Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 12See R.H. Caday-Malcolm R.A. Singaraja R.R. Zhou S. Silverston A. Huber M.T. Moran J. James E.R. Janoo R. Savill J.M. et al.Protein kinase A site-specific phosphorylation regulates ATP-binding cassette A1 (ABCA1)-mediated phospholipid efflux.J. Biol. Chem. 2002; 277: 41835-41842Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 13Haidar B. Denis M. Krimbou L. Marcil M. Genest Jr., J. cAMP induces ABCA1 phosphorylation activity and promotes cholesterol efflux from fibroblasts.J. Lipid Res. 2002; 43: 2087-2094Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 14Martinez L.O. Agerholm-Larsen B. Wang N. Chen W. Tall A.R. Phosphorylation of a PEST sequence in ABCA1 promotes calpain degradation and is reversed by apoA-I.J. Biol. Chem. 2003; 278: 37368-37374Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). It has not been unequivocally established, however, whether the binding of apolipoproteins to ABCA1 is directly involved in the activation of intracellular signaling pathways by apolipoproteins. We previously demonstrated that exposure of cells to apoA-I induces the activation of Rho family small G-proteins, including Cdc42 and Rac1, as well as actin polymerization, which is known to be controlled by Cdc42 (15Nofer J.R. Feuerborn R. Levkau B. Sokoll A. Seedorf U. Assmann G. Involvement of Cdc42 signaling in apoA-I-induced cholesterol efflux.J. Biol. Chem. 2003; 278: 53055-53062Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). In addition, protein kinases localized downstream of Cdc42 in the signaling cascade, such as PAK-1 and p54JNK, were activated in the presence of apoA-I. Inhibition of Cdc42 or p54JNK partially inhibited apoA-I-induced cholesterol efflux, indicating that these signal transduction pathways are obligatory for the optimal ABCA1-dependent transport of lipids from cells to apolipoproteins. However, it is not known whether ABCA1 is directly required for Cdc42 activation or is localized distally to the Cdc42 signaling cascade, where it functions solely as a cholesterol and/or phospholipid transporter. In this study, we provide evidence that ABCA1 directly links apoA-I binding to activation of the Cdc42 signaling cascade and thereby to cholesterol efflux in fibroblasts. Phosphospecific antibodies against p54JNK were purchased by Cell Signaling Technologies (Beverly, MA). Polyclonal antibodies against nonphosphorylated p54JNK and ABCA1 were from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal antibodies against green fluorescent protein (GFP) were from Roche (Mannheim, Germany). Anti-Cdc42 antibodies were from BD Biosciences (Erembodegem, Belgium). Glutathione S-transferase-p21 binding domain (GST-PBD) beads were purchased by Upstate Biotechnology (Chicago, IL). ApoA-I, apoC-III, and apoE were obtained from Merck Biosciences (Schwalbach, Germany). 4,4-difluror-4-bora-3a,4a-diaza-s-indacene (BODIPY)-phalloidin and Alexa Fluor 594 succinimidyl ester were from Molecular Probes (Eugene, OR). [3H]cholesterol was obtained from Amersham (Braunschweig, Germany). DMEM, FBS, and BSA were from PAA Laboratories (Pasching, Germany). The liver X receptor (LXR) agonist, T0901317, and the retinoid X receptor (RXR) agonist, R0264456, were generous gifts of Dr. Michael Pech (F. Hoffman-La Roche, Basel, Switzerland). All others chemicals were from Sigma (Deisenhofen, Germany). Dermal fibroblasts were obtained from a 65 year old male patient with Tangier disease, who presented with dyslipidemia (total cholesterol < 100 mg/ml, HDL-cholesterol < 2 mg/ml) and coronary heart disease. Characterization of the ABCA1 defect at the molecular level revealed a homozygous 1 bp deletion in exon 14, leading to a stop codon at amino acid position 635 and the deletion of most of the protein sequence, including both ATP cassettes. Human skin fibroblasts cultured from biopsies of adult human hip skin were grown and maintained in DMEM containing 10% (v/v) FBS, 2 mmol/l l-glutamine, and 1% (v/v) antibiotic/antimycotic solution. Once separated, the dermis was cut into small pieces (0.5 mm on each side) and placed in a flask in DMEM. When these primary cultures were confluent, they were expanded by passage. For experiments, cells between passage levels three and six were used. HEK293 cells were purchased from the American Tissue Cell Culture Collection and grown to confluence in DMEM supplemented with FBS (10%, v/v). For experiments, cells were plated on 6- or 24-well plates coated with collagen. Both human fibroblasts and HEK293 cells were enriched with cholesterol by incubation in serum-free DMEM with 2 mg/ml BSA and 30 μg/ml nonlipoprotein cholesterol for 24 h. The coding sequence of human ABCA1 (NM_005502.2, bp 311–7,096) without stop codon was amplified from human macrophage cDNA in three ∼2 kb fragments, which were reassembled in pBluescriptII KS+, using endogenous restriction sites BclI and Bsu36I, at positions 1,961 and 4,187, respectively, within the coding sequence of the ABCA1-cDNA. The whole coding sequence of ABCA1 was cut with ApaI and NotI restriction sites, which had been introduced during amplification of the cDNA fragments at the 5′ and 3′ ends and ligated into pcDNA3.1(−) vector. hGFP was amplified from phrGFP (Stratagene, La Jolla, CA) with flanking NotI and PmeI restriction sites and introduced in-frame with the ABCA1 open reading frame into the pcDNA3.1(−)-ABCA1 plasmid. ABCA1-W-GFP variant with the disrupted first Walker A motif of ABCA1 was constructed by PCR-based mutagenesis, creating a missense mutation of K939M. ABCA1-ΔC-GFP variant with a deleted C terminus was generated by PCR using a reverse primer annealing at nucleotide position 5,559 and introducing a stop codon adjacent to amino acid 1,873, which led to a deletion of the terminal 388 amino acids. Plasmid DNA was checked by DNA sequencing, using the ABI PRISM® BigDye™ Terminator 3.0 cycle sequencing kit and the ABI-Prism 3700 DNA analyzer (Applied Biosystems, Weiterstadt, Germany). HEK293 cells were transfected by electroporation, using commercially available reagent (Amaxa, Koäln, Germany), and selected with 0.5 mg/ml G418. Antibiotic-resistant cells were screened for the expression of the fusion protein by fluorescence microscopy, and positive clones were purified by limiting dilution. ApoA-I was labeled with Alexa Fluor 594 according to the manufacturer’s protocol. Briefly, apoA-I (2 mg) was mixed with 1 mg of the dye, followed by 1 h of incubation at room temperature with continuous stirring. The unbound dye was separated from the conjugate using a Sephadex G-25 column (Amersham) equilibrated with PBS buffer. Human fibroblasts or HEK293 cells stably expressing ABCA1 (5 × 105 cells/ml) were incubated with labeled apoA-I (0.01 mg/ml) for 15 min, washed twice with PBS, and collected for fluorescence measurement using a Hitachi F-2000 fluorescence spectrometer (excitation wavelength, 590 nm; emission wavelength, 620 nm). Actin polymerization was quantified as described by Ha and Exton (16Ha K.S. Exton J.H. Activation of actin polymerization by phosphatidic acid derived from phosphatidylcholine in IIC9 fibroblasts.J. Cell Biol. 1993; 123: 1789-1796Crossref PubMed Scopus (154) Google Scholar). Briefly, cells were treated with agonists and fixed with 4% (v/v) formaldehyde containing 2.5% (v/v) octyl glucopyranoside for 15 min on ice. Cells were then stained with 0.6 ml/well BODIPY-phalloidin (15 nmol/l) for 30 min, and bound BODIPY-phalloidin was extracted with 0.1 ml of methanol for 1 h on ice. Fluorescence intensity measurements were performed using a Hitachi F-2000 fluorescence spectrometer with excitation and emission wavelengths of 505 and 515 nm, respectively. SDS-PAGE and Western blotting were performed exactly as described previously (17Nofer J.R. Levkau B. Wolinska I. Junker R. Fobker M. von Eckardstein A. Seedorf U. Assmann G. Suppression of endothelial cell apoptosis by high density lipoproteins (HDL) and HDL-associated lysosphingolipids.J. Biol. Chem. 2001; 276: 34480-34485Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar). For each blot with anti-phosphospecific antibodies, loading controls were used, with an antibody against a nonphosphorylated isoform of p54JNK. Stimulated fibroblasts were lysed in a buffer containing 20 mmol/l HEPES (pH 7.4), 150 mmol/l NaCl, 2% (v/v) Nonidet P-40, 20% (v/v) glycerol, 8.0 mmol/l EGTA, 8.0 mmol/l EDTA, 10 mmol/l MgCl2, 1 mmol/l orthovanadate, and the Complete® protease inhibitor mixture. Cell lysates were homogenized by three freeze-thaw cycles, cleared by centrifugation (14,000 rpm, 4°C), and incubated for 1 h at 4°C with 10 μg/sample GST-PBD beads for Cdc42 precipitation. The beads were collected by centrifugation (14,000 rpm, 4°C) and washed, and captured proteins were removed by boiling for 5 min in Laemmli sample buffer. Samples were then subjected to Western blotting as described above. Human dermal fibroblasts treated with LXR/RXR agonists or HEK293 cells stably expressing ABCA1-GFP were stimulated with apoA-I (0.01 mg/ml) for 10 min, washed, and scraped into 0.4 ml of assay buffer (20 mmol/l Tris-HCl, 250 mmol/l NaCl, 3 mmol/l EDTA, and 3 mmol/l EGTA, pH 7.6) containing 0.5% (v/v) Nonidet P-10 and protease inhibitors. After lysis on ice (10 min) and three freeze-thaw cycles, insoluble material was cleared by centrifugation. The soluble fraction was incubated for 1 h on ice with polyclonal anti-ABCA1 antibody (5.0 μg) or anti-GFP antibody (2.0 μg). Thereafter, 0.01 ml of protein agarose G was added, and samples were incubated overnight. Agarose beads were washed four times with assay buffer and once with high-salt assay buffer containing 350 mmol/l NaCl. Captured proteins were resuspended in Laemmli buffer, boiled, and separated using 12% SDS-PAGE. Proteins were analyzed by Western blotting using polyclonal antibodies against ABCA1 and Cdc42, as described above. Cholesterol efflux was measured according to established methods (15Nofer J.R. Feuerborn R. Levkau B. Sokoll A. Seedorf U. Assmann G. Involvement of Cdc42 signaling in apoA-I-induced cholesterol efflux.J. Biol. Chem. 2003; 278: 53055-53062Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Briefly, [3H]cholesterol (1 μCi/well) was added to cells grown on 24-well plates for 24 h. Cells were washed and incubated for 4 h at 37°C with DMEM containing BSA (0.2%, v/v) and apoA-I at desired concentrations. The efflux medium was collected and centrifuged to remove cell debris. Cells were solubilized in 0.1 mol/l NaOH. Radioactivity in efflux medium and cell lysates was determined by scintillation counting. The results are reported as percentages of efflux relative to the radioactivity fraction present in efflux medium in the absence of apoA-I. Data are presented as means ± SD from three separate experiments or as representative immunoblots for at least three repetitions, unless indicated otherwise. Recent investigations have demonstrated that in addition to apoA-I, several other exchangeable apolipoproteins, such as apoC-I, apoC-II, and apoC-III, as well as apoE are capable of effluxing cholesterol from cells via the ABCA1-dependent pathway (18Remaley A.T. Stonik J.A. Demosky S.J. Neufeld E.B. Bocharov A.V. Vishnyakova T.G. Eggerman T.L. Patterson A.P. Duverger N.J. Santamarina-Fojo S. et al.Apolipoprotein specificity for lipid efflux by the human ABCAI transporter.Biochem. Biophys. Res. Commun. 2001; 280: 818-823Crossref PubMed Scopus (278) Google Scholar, 19Krimbou L. Denis M. Haidar B. Carrier M. Marcil M. Genest Jr., J. Molecular interactions between apoE and ABCA1: impact on apoE lipidation.J. Lipid Res. 2004; 45: 839-848Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Moreover, in cross-linking and competition experiments, these apolipoproteins were shown to physically bind to the extracellular domains of ABCA1 (7Fitzgerald M.L. Morris A.L. Chroni A. Mendez A.J. Zannis V.I. Freeman M.W. ABCA1 is the cAMP-inducible apolipoprotein receptor that mediates cholesterol secretion from macrophages.J. Lipid Res. 2004; 45: 287-294Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 19Krimbou L. Denis M. Haidar B. Carrier M. Marcil M. Genest Jr., J. Molecular interactions between apoE and ABCA1: impact on apoE lipidation.J. Lipid Res. 2004; 45: 839-848Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). To assess the role of ABCA1 in the generation of intracellular signals, we first examined the effect of ABCA1-interacting apolipoproteins on the activation of Cdc42 and its downstream effector kinases PAK-1 and p54JNK, as well as on the process of actin polymerization, which is directly regulated by Cdc42. As expected, in addition to apoA-I, two other exchangeable apolipoproteins, apoC-III and apoE, also stimulated cholesterol efflux to a similar extent. Under similar conditions, exposure of fibroblasts to all apolipoproteins tested increased the amount of extractable BODIPY-phalloidin, indicating that the intracellular content of polymerized actin increased after stimulation (Fig. 1B). To assess directly whether exchangeable apolipoproteins affect the activity of small G-proteins, we examined the amount of activated Cdc42 in fibroblasts treated with apoA-I, apoC-III, and apoE. The GST-PBD beads precipitated only marginal amounts of Cdc42 from unstimulated fibroblasts. By contrast, exposure of cells to apolipoproteins markedly stimulated the amount of active Cdc42. In addition, autophosphorylation of PAK-1 and phosphorylation of p54JNK were observed in fibroblasts exposed to apoA-I, apoC-III, and apoE (Fig. 1C). The ability of apolipoproteins to act as a lipid acceptor is most likely related to the shared secondary structure of these proteins, characterized by the presence of amphipathic helices. Small synthetic peptides, which do not have significant sequence homology with apolipoproteins but contain at least two amphipathic helices, are capable of interacting directly with ABCA1 and thereby inducing cholesterol egress from cells (7Fitzgerald M.L. Morris A.L. Chroni A. Mendez A.J. Zannis V.I. Freeman M.W. ABCA1 is the cAMP-inducible apolipoprotein receptor that mediates cholesterol secretion from macrophages.J. Lipid Res. 2004; 45: 287-294Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 20Remaley 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. et al.Synthetic amphipathic helical peptides promote lipid efflux from cells by an ABCA1-dependent and an ABCA1-independent pathway.J. Lipid Res. 2003; 44: 828-836Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 21Arakawa R. Hayashi M. Remaley A.T. Brewer B.H. Yamauchi Y. Yokoyama S. Phosphorylation and stabilization of ATP binding cassette transporter A1 by synthetic amphiphilic helical peptides.J. Biol. Chem. 2004; 279: 6217-6220Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). To establish whether ligand amphipathicity is required for the effective induction of intracellular signaling in fibroblasts, we made use of two synthetic peptides: D-37pA, which was synthesized solely from d amino acids and contains two octadecameric A class amphipathic helices linked by a proline, and L3D-37pA, which contains both d and l amino acids (20Remaley 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. et al.Synthetic amphipathic helical peptides promote lipid efflux from cells by an ABCA1-dependent and an ABCA1-independent pathway.J. Lipid Res. 2003; 44: 828-836Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). The introduction of d stereoisomers into a peptide that otherwise contains l stereoisomers is known to interfere with its ability to form an α-helical structure. Accordingly, only D-37pA, and not L3D-37pA, has been reported previously to induce cholesterol efflux and to compete with apoA-I for ABCA1 binding (20Remaley 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. et al.Synthetic amphipathic helical peptides promote lipid efflux from cells by an ABCA1-dependent and an ABCA1-independent pathway.J. Lipid Res. 2003; 44: 828-836Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). In our hands, D-37pA fully mimicked apoA-I in its capacity to induce cholesterol efflux and actin polymerization in fibroblasts (Fig. 1A, B). Furthermore, Cdc42 activation, autophosphorylation of PAK-1, and phosphorylation of p54JNK were observed in fibroblasts exposed to D-37pA (Fig. 1C). By contrast, L3D-37pA remained totally inactive with respect to all responses tested. The heterogeneous distribution of cholesterol in the cell membrane contributes to the formation of transverse domains, which are potentially involved in transmembrane signaling. For instance, cholesterol-rich membrane rafts host several signal transduction intermediates, such as receptors, protein kinases, and adaptor molecules. To investigate the possibility that apolipoprotein- or amphipathic peptide-induced signaling arises solely as a result of changes in the domain structure of the plasma membrane brought about by the reduction of its cholesterol content, we examined the intracellular signal generation in cells exposed to cyclodextrin or phosphatidylcholine (PC)-containing liposomes, which deplete membrane cholesterol by passive diffusion that does not involve ABCA1 (22Yancey P.G. Bortnick A.E. Kellner-Weibel G. de la Llera-Moya M. Phillips M.C. Rothblat G.H. Importance of different pathways of cellular cholesterol efflux.Arterioscler. Thromb. Vasc. Biol. 2003; 23: 712-719Crossref PubMed Scopus (440) Google Scholar). Both cyclodextrin and PC liposomes were applied in sufficient concentrations to induce cholesterol efflux at a magnitude similar to that of apoA-I (Fig. 1A). Under these experimental conditions, no effect of cyclodextrin or PC liposomes on the polymerization of actin was observed (Fig. 1B). In addition, both compounds failed to induce Cdc42 activation and PAK-1 and p54JNK phosphorylation in human fibroblasts. To further evaluate the relationship between ABCA1 and apoA-I-induced signal transduction, we sought to examine Cdc42 signaling under conditions in which ABCA1 activity is eliminated. To this aim, we used glyburide, a sulfonylthiourea derivative, which binds to and effectively blocks several ABC transporters, including cystic fibrosis transmembrane conductance regulator and multidrug resistance proteins. Previous studies demonstrated inhibitory effects of glyburide on ABCA1-dependent cholesterol and phospholipid efflux, as well as on apoA-I binding to ABCA1 in HEK293 cells (4Wang N. Silver D.L. Costet P. Tall A.R. Specific binding of apoA-I, enhanced cholesterol efflux, and altered plasma membrane morphology in cells expressing ABC1.J. Biol. Chem. 2000; 275: 33053-33058Abstract Full Text Full Text PDF PubMed Scopus (511) Google Scholar, 23Nieland T.J. Chroni A. Fitzgerald M.L. Maliga Z. Zannis V.I. Kirchhausen T. Krieger M. Cross-inhibition of SR-BI- and ABCA1-mediated cholesterol transport by the small molecules BLT-4 and glyburide.J. Lipid Res. 2004; 45: 1256-1265Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). In agreement with this report, glyburide treatment markedly reduced apoA-I-induced cholesterol efflux in human fibroblasts (Fig. 2A). The inhibitory effects of glyburide were dose-dependent, with a maximum seen at a concentration of 1.0 mmol/l. As shown in Fig. 2B, at the same concentration range, glyburide inhibited apoA-I-induced actin polymerization, as inferred from the reduced amount of BODIPY-phalloidin extracted from pretreated cells. Moreover, a reduced amount of activated Cdc42 could be precipitated from fibroblasts exposed to 1.0 mmol/l glyburide before stimulation with apoA-I (Fig. 2C). In addition, glyburide (1.0 mmol/l) markedly suppressed the apoA-I-induced autophosphorylation of PAK-1 and phosphorylation of p54JNK in human fibroblasts (Fig. 2C). To confirm the importance of ABCA1 for apoA-I-induced intracellular signaling, we conducted experiments in fibroblasts obtained from a Tangier disease patient, in which a stop codon at amino acid position 635 prevents ABCA1 expression (Fig. 3A, inset). As a consequence, cell binding of apoA-I conjugated to Alexa 596 was completely abolished in Tangier fibroblasts, whereas the fluorescence-labeled apolipoprotein bound to normal fibroblasts in a concentration-dependent manner (Fig. 3A). As shown in Fig. 3B, for all concentrations tested, apoA-I failed to induce cholesterol efflux from Tangier fibroblasts, unlike normal fibroblasts, which efficiently effluxed cholesterol in the presence of apoA-I. Next, the time course of actin polymerization in control and Tangier fibroblasts exposed to apoA-I was examined. Figure 3B demonstrates that the amounts of polymerized actin were increased significantly in response to apolipoprotein stimulation in normal but not in Tangier cells. Finally, the effect of apoA-I on the activation of Cdc42 and its downstream kinases was investigated (Fig. 3D). In marked contrast to normal fibroblasts, Tangier fibroblasts failed to respond to apoA-I stimulation with Cdc42 activation, autophosphorylation of PAK-1, or phosphorylation of p54JNK. The prediction from the postulated role of ABCA1 in facilitating signal transduction in response to apoA-I is that apoA-I-induced Cdc42 signaling should be enhanced in cells expressing this transporter. To test this proposition, we applied T0901317 and R0264456, which are synthetic agonists of LXR and RXR, respectively, two ubiquitously expressed nuclear transcription factors known to control ABCA1 gene expression (24Millatt L.J. Bocher V. Fruchart J.C. Staels B. Liver X receptors and the control of cholesterol homeostasis: potential therapeutic targets for the treatment of atherosclerosis.Biochim. Biophys. Acta. 2003; 1631: 107-118Crossref PubMed Scopus (80) Google Scholar). As shown in Fig. 4A(inset), combined treatment for 24 h with T0901317 (1.0 μmol" @default.
• W2157738629 created "2016-06-24" @default.
• W2157738629 creator A5021746678 @default.
• W2157738629 creator A5027046285 @default.
• W2157738629 creator A5035295431 @default.
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• W2157738629 date "2006-04-01" @default.
• W2157738629 modified "2023-10-15" @default.
• W2157738629 title "Apolipoprotein A-I activates Cdc42 signaling through the ABCA1 transporter" @default.
• W2157738629 cites W1581642890 @default.
• W2157738629 cites W1964094832 @default.
• W2157738629 cites W1974864595 @default.
• W2157738629 cites W1978488816 @default.
• W2157738629 cites W1978726944 @default.
• W2157738629 cites W1979717214 @default.
• W2157738629 cites W1986439388 @default.
• W2157738629 cites W1991073529 @default.
• W2157738629 cites W1993028907 @default.
• W2157738629 cites W2013348589 @default.
• W2157738629 cites W2015613856 @default.
• W2157738629 cites W2016399150 @default.
• W2157738629 cites W2017002005 @default.
• W2157738629 cites W2025573887 @default.
• W2157738629 cites W2025716299 @default.
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• W2157738629 cites W2057847302 @default.
• W2157738629 cites W2059472147 @default.
• W2157738629 cites W2059612359 @default.
• W2157738629 cites W2061826009 @default.
• W2157738629 cites W2066557706 @default.
• W2157738629 cites W2072568946 @default.
• W2157738629 cites W2084074011 @default.
• W2157738629 cites W2094418570 @default.
• W2157738629 cites W2096861375 @default.
• W2157738629 cites W2099227158 @default.
• W2157738629 cites W2103714932 @default.
• W2157738629 cites W2111482014 @default.
• W2157738629 cites W2112977127 @default.
• W2157738629 cites W2122062568 @default.
• W2157738629 cites W2123151772 @default.
• W2157738629 cites W2127510556 @default.
• W2157738629 cites W2139192607 @default.
• W2157738629 cites W2139795441 @default.
• W2157738629 cites W2147429205 @default.
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