Serum Amyloid A Binding to CLA-1 (CD36 and LIMPII Analogous-1) Mediates Serum Amyloid A Protein-induced Activation of ERK1/2 and p38 Mitogen-activated Protein Kinases
2004; Elsevier BV; Volume: 280; Issue: 9 Linguagem: Inglês
10.1074/jbc.m405009200
ISSN1083-351X
AutoresIrina N. Baranova, Tatyana G. Vishnyakova, Alexander V. Bocharov, Roger Kurlander, Zhigang Chen, Michael Kimelman, Alan T. Remaley, György Csákó, Thomas Fairwell, Thomas L. Eggerman, Amy P. Patterson,
Tópico(s)Pancreatitis Pathology and Treatment
ResumoSerum amyloid A protein (SAA) is an acute-phase reactant, known to mediate pro-inflammatory cellular responses. This study reports that CLA-1 (CD36 and LIMPII Analogous-1; human orthologue of the Scavenger Receptor Class B Type I (SR-BI)) mediates SAA uptake and downstream SAA signaling. Flow cytometry experiments revealed more than a 5-fold increase of Alexa-488 SAA uptake in HeLa cells stably transfected with CLA-1. Alexa 488-HDL uptake directly correlated with SAA uptake when determined in several CLA-1 stably transfected HeLa cell clones expressing various levels of CLA-1. SAA directly binds to CLA-1 as determined by cross-linking and colocalization of anti-CLA-1 antibody with SAA. SAA was co-internalized with transferrin to the endocytic recycling compartment pointing to a potential site of SAA metabolism. Alexa-488 SAA uptake in the CLA-1-overexpressing HeLa cells, as well as in THP-1 monocyte cell line, can be efficiently blocked by unlabeled SAA, high density lipoprotein, and other CLA-1 ligands. At the same time, markedly enhanced levels of phosphorylation of the mitogen-activated protein kinases (MAPKs), ERK1/2, and p38, were observed in cells stably transfected with CLA-1 cells following SAA stimulation when compared with mock transfected cells. The levels of the SAA-induced interleukin-8 (IL-8) secretion by CLA-1-overexpressing cells also significantly exceeded (5- to 10-fold) those detected for control cells. Synthetic amphipathic peptides possessing a structural α-helical motif inhibited SAA-induced activation of both MAPKs and IL-8 secretion in THP-1 cells. The results of this study demonstrate for the first time that CLA-1 functions as an endocytic SAA receptor and is involved in SAA-mediated cell signaling events associated with the immune-related and inflammatory effects of SAA. Serum amyloid A protein (SAA) is an acute-phase reactant, known to mediate pro-inflammatory cellular responses. This study reports that CLA-1 (CD36 and LIMPII Analogous-1; human orthologue of the Scavenger Receptor Class B Type I (SR-BI)) mediates SAA uptake and downstream SAA signaling. Flow cytometry experiments revealed more than a 5-fold increase of Alexa-488 SAA uptake in HeLa cells stably transfected with CLA-1. Alexa 488-HDL uptake directly correlated with SAA uptake when determined in several CLA-1 stably transfected HeLa cell clones expressing various levels of CLA-1. SAA directly binds to CLA-1 as determined by cross-linking and colocalization of anti-CLA-1 antibody with SAA. SAA was co-internalized with transferrin to the endocytic recycling compartment pointing to a potential site of SAA metabolism. Alexa-488 SAA uptake in the CLA-1-overexpressing HeLa cells, as well as in THP-1 monocyte cell line, can be efficiently blocked by unlabeled SAA, high density lipoprotein, and other CLA-1 ligands. At the same time, markedly enhanced levels of phosphorylation of the mitogen-activated protein kinases (MAPKs), ERK1/2, and p38, were observed in cells stably transfected with CLA-1 cells following SAA stimulation when compared with mock transfected cells. The levels of the SAA-induced interleukin-8 (IL-8) secretion by CLA-1-overexpressing cells also significantly exceeded (5- to 10-fold) those detected for control cells. Synthetic amphipathic peptides possessing a structural α-helical motif inhibited SAA-induced activation of both MAPKs and IL-8 secretion in THP-1 cells. The results of this study demonstrate for the first time that CLA-1 functions as an endocytic SAA receptor and is involved in SAA-mediated cell signaling events associated with the immune-related and inflammatory effects of SAA. The acute phase serum amyloid A protein (SAA) 1The abbreviations used are: SAA, serum amyloid A; CLA-1, CD36 and LIMPII analogous-1; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, MAPK/ERK kinase; HDL, high density lipoprotein; IL, interleukin; SR-BI, Scavenger Receptor Class B Type I; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)-ethyl]glycine; PBS, phosphate-buffered saline; ERC, endoplasmic recycling compartment; LPS, lipopolysaccharide. is a 12- to 14-kDa apolipoprotein encoded by SAA1 and SAA2 allelic variants (1Kumon Y. Hosokawa T. Suehiro T. Ikeda Y. Sipe J.D. Hashimoto K. Amyloid. 2002; 9: 237-241Crossref PubMed Scopus (26) Google Scholar) and is found predominantly in the plasma high density lipoprotein fraction (2Malle E. Steinmetz A. Raynes J.G. Atherosclerosis. 1993; 102: 131-146Abstract Full Text PDF PubMed Scopus (176) Google Scholar, 3Patel H. Fellowes R. Coade S. Woo P. Scand. J. Immunol. 1998; 48: 410-418Crossref PubMed Scopus (145) Google Scholar). SAA is normally present in the bloodstream at 0.1 μm. In response to various injuries, including trauma, infection, inflammation, and neoplasia (4Urieli-Shoval S. Linke R.P. Matzner Y. Curr. Opin. Hematol. 2000; 7: 64-69Crossref PubMed Scopus (371) Google Scholar), the levels of SAA increase up to 1000-fold. As with other acute-phase reactants, the liver is the major site of SAA expression (5Gabay C. Kushner I. N. Engl. J. Med. 1999; 340: 448-454Crossref PubMed Scopus (5110) Google Scholar). However, SAA is also expressed in cells within human atherosclerotic lesions (6Yamada T. Kakihara T. 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A. 1994; 91: 3186-3190Crossref PubMed Scopus (276) Google Scholar, 14Uhlar C.M. Whitehead A.S. Eur. J. Biochem. 1999; 265: 501-523Crossref PubMed Scopus (893) Google Scholar). Prolonged or repeated inflammatory conditions leading to elevated serum SAA levels can cause a reactive form of amyloidosis in peripheral tissues, resulting in progressive loss of organ function. SAA fragments resulting from the enzymatic degradation of SAA form amyloid A, a major constituent of amyloid fibrils (13Husebekk A. Skogen B. Husby G. Marhaug G. Scand. J. Immunol. 1985; 21: 283-287Crossref PubMed Scopus (205) Google Scholar, 14Uhlar C.M. Whitehead A.S. Eur. J. Biochem. 1999; 265: 501-523Crossref PubMed Scopus (893) Google Scholar, 15Tape C. Tan R. Nesheim M. Kisilevsky R. Scand. J. Immunol. 1988; 28: 317-324Crossref PubMed Scopus (94) Google Scholar). In atherosclerosis, SAA accumulates in macrophages, macrophage-derived “foam cells,” adipocytes, endothelial cells, and smooth muscle cells within the vascular plaque (9Meek R.L. Urieli-Shoval S. Benditt E.P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3186-3190Crossref PubMed Scopus (276) Google Scholar). The exact role of SAA in atherogenesis is complicated by its effect upon the metabolism of HDL and other lipoprotein fractions (16Banka C.L. Yuan T. de Beer M.C. Kindy M. Curtiss L.K. de Beer F.C. J. Lipid Res. 1995; 36: 1058-1065Abstract Full Text PDF PubMed Google Scholar, 17Cabana V.G. Siegel J.N. Sabesin S.M. J. Lipid Res. 1989; 30: 39-49Abstract Full Text PDF PubMed Google Scholar). At lower levels, SAA associates with HDL forming a heterogeneous HDL population containing both SAA and apoA-I. At elevated concentrations, SAA displaces apoA-I and produces lipoprotein fractions containing predominantly SAA, lipid-poor apoA-I, and lipoprotein-free SAA. Persistently high SAA levels are usually associated with significantly reduced apoA-I and HDL cholesterol levels due to enhanced HDL metabolism (18Salazar A. Mana J. Fiol C. Hurtado I. Argimon J.M. Pujol R. Pinto X. Atherosclerosis. 2000; 152: 497-502Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 19Coetzee G.A. Strachan A.F. van der Westhuyzen D.R. Hoppe H. Jeenah M.S. de Beer F.C. J. Biol. Chem. 1986; 261: 9644-9651Abstract Full Text PDF PubMed Google Scholar). SAA may also act as a signal for redirecting HDL to the sites of tissue destruction and cholesterol accumulation (20Kisilevsky R. Subrahmanyan L. Lab. Invest. 1992; 66: 778-785PubMed Google Scholar). Additionally SAA may contribute to HDL-mediated clearance of cellular cholesterol by modulating LCAT activity (21Steinmetz A. Hocke G. Saile R. Puchois P. Fruchart J.C. Biochim. Biophys. Acta. 1989; 1006: 173-178Crossref PubMed Scopus (112) Google Scholar). Moreover, SAA has been shown to modulate cholesterol transport by serving as a cholesterol-binding protein (22Liang J.S. Sipe J.D. J. Lipid Res. 1995; 36: 37-46Abstract Full Text PDF PubMed Google Scholar), as well as a direct activator of the ABCA1-dependent pathway of cholesterol translocation to an extracellular cholesterol acceptor (23Tam S.P. Flexman A. Hulme J. Kisilevsky R. J. Lipid Res. 2002; 43: 1410-1420Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). SAA in its lipid-poor form has been shown to modify immune responses. In vitro studies have provided compelling evidence that SAA can act as a chemoattractant for such immune cells as monocytes, polymorphonuclear leukocytes, mast cells, and T lymphocytes (24Badolato R. Wang J.M. Murphy W.J. Lloyd A.R. Michiel D.F. Bausserman L.L. Kelvin D.J. Oppenheim J.J. J. Exp. Med. 1994; 180: 203-209Crossref PubMed Scopus (430) Google Scholar, 25Xu L. Badolato R. Murphy W.J. Longo D.L. Anver M. Hale S. Oppenheim J.J. 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SAA has been recently demonstrated to be a chemoattractant ligand for the human N-formyl peptide receptor like-1, a transmembrane G-protein-coupled receptor expressed on phagocytes (28Su S.B. Gong W. Gao J.L. Shen W. Murphy P.M. Oppenheim J.J. Wang J.M. J. Exp. Med. 1999; 189: 395-402Crossref PubMed Scopus (374) Google Scholar). Additionally, the cytokine-like activity of SAA has been reported to be directly mediated by N-formyl peptide receptor like-1/LXA4R, the receptor recognized as a mediator of the anti-inflammatory effects of lipoxin (1Kumon Y. Hosokawa T. Suehiro T. Ikeda Y. Sipe J.D. Hashimoto K. Amyloid. 2002; 9: 237-241Crossref PubMed Scopus (26) Google Scholar, 29He R. Sang H. Ye R.D. Blood. 2003; 101: 1572-1581Crossref PubMed Scopus (275) Google Scholar). CLA-1 and rodent scavenger receptor BI (SR-BI), are HDL receptors, highly expressed in liver, adrenal gland, ovary (30Landschulz K.T. Pathak R.K. Rigotti A. Krieger M. Hobbs H.H. J. Clin. Invest. 1996; 98: 984-995Crossref PubMed Scopus (469) Google Scholar), and atherosclerotic lesions of apoE-deficient mice (31Ji Y. Jian B. Wang N. Sun Y. de la Llera Moya M. Philips M.C. Rothblat G.H. Swaney J.B. Tall A.R. J. Biol. Chem. 1997; 272: 20982-20985Abstract Full Text Full Text PDF PubMed Scopus (636) Google Scholar). In addition, CLA-1 and SR-BI are highly expressed on monocytes/macrophages (32Pearson A.M. Curr. Opin. Immunol. 1996; 8: 20-28Crossref PubMed Scopus (250) Google Scholar), cells known to be the primary sites of SAA uptake. Like CD36, SR-BI/CLA-1 has been shown to be a multifunctional receptor able to bind a broad variety of ligands, including native, oxidized, and acetylated low density lipoprotein (33Acton S.L. Scherer P.E. Lodish H.F. Krieger M. J. Biol. Chem. 1994; 269: 21003-21009Abstract Full Text PDF PubMed Google Scholar), native very low density lipoprotein (34Krieger M. Annu. Rev. Biochem. 1999; 68: 523-558Crossref PubMed Scopus (460) Google Scholar), anionic phospholipids, lipopolysaccharide, and apoptotic cells (35Murao K. Terpstra V. Green S.R. Kondratenko N. Steinberg D. Quehenberger O. J. Biol. Chem. 1997; 272: 17551-17557Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar, 36Svensson P.A. Johnson M.S. Ling C. Carlsson L.M. Billig H. Carlsson B. Endocrinology. 1999; 140: 2494-2500Crossref PubMed Scopus (34) Google Scholar, 37Vishnyakova T.G. Bocharov A.V. Baranova I.N. Chen Z. Remaley A.T. Csako G. Eggerman T.L. Patterson A.P. J. Biol. Chem. 2003; 278: 22771-22780Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). The presence of amphipathic helices is a common feature of exchangeable apolipoproteins, known to be the primary ligands for SR-BI. Utilizing this knowledge, our group and others (38Williams D.L. de La Llera-Moya M. Thuahnai S.T. Lund-Katz S. Connelly M.A. Azhar S. Anantharamaiah G.M. Phillips M.C. J. Biol. Chem. 2000; 275: 18897-18904Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar) have provided evidence that synthetic amphipathic peptides, possessing one or more class A amphipathic helices in their structure, are potent CLA-1 ligands and effective competitors for apoA-I and HDL binding to SR-BI/CLA-1 (39Bocharov A.V. Baranova I.N. Vishnyakova T.G. Remaley A.T. Csako G. Thomas F. Patterson A.P. Eggerman T.L. J. Biol. Chem. 2004; 279: 36072-36082Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Because SAA is known to be an amphipathic protein, having two amphipathic α-helical regions corresponding to the 1–18 N-terminal and 72–86 C-terminal sequences (14Uhlar C.M. Whitehead A.S. Eur. J. Biochem. 1999; 265: 501-523Crossref PubMed Scopus (893) Google Scholar), we evaluated whether CLA-1 is a potential SAA receptor involved in binding, internalization, and pro-inflammatory signaling. Only recently, a new function for SR-BI has emerged: the activation of cell signaling pathways upon ligand binding (40Yuhanna I.S. Zhu Y. Cox B.E. Hahner L.D. Osborne-Lawrence S. Lu P. Marcel Y.L. Anderson R.G. Mendelsohn M.E. Hobbs H.H. Shaul P.W. Nat. Med. 2001; 7: 853-857Crossref PubMed Scopus (645) Google Scholar, 41Li X.A. Titlow W.B. Jackson B.A. Giltiay N. Nikolova-Karakashian M. Uittenbogaard A. Smart E.J. J. Biol. Chem. 2002; 277: 11058-11063Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 42Grewal T. de Diego I. Kirchhoff M.F. Tebar F. Heeren J. Rinninger F. Enrich C. J. Biol. Chem. 2003; 278: 16478-16481Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). The concept of CLA-1 being involved in the signal transduction process results from a recently observed interaction between CLA-1 and the cytoplasmic PDZK1 adaptor protein (43Ikemoto M. Arai H. Feng D. Tanaka K. Aok I.J. Dohmae N. Takio K. Adachi H. Tsujimoto M. Inoue K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6538-6543Crossref PubMed Scopus (141) Google Scholar). This adaptor protein is known to play a crucial role in regulating various biological processes, including signal transduction, adhesion, membrane trafficking, and cellular transport (44Pawson T. Scott J.D. Science. 1997; 278: 2075-2080Crossref PubMed Scopus (1900) Google Scholar). The initial report indicated that one of the four domains of PDZK1 is associated with the SR-BI cytoplasmic C terminus (43Ikemoto M. Arai H. Feng D. Tanaka K. Aok I.J. Dohmae N. Takio K. Adachi H. Tsujimoto M. Inoue K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6538-6543Crossref PubMed Scopus (141) Google Scholar). Additionally, the ability of HDL to stimulate the endothelial nitric-oxide synthase activity in an SR-BI-dependent manner has been demonstrated by the studies of Yuhanna et al. (40Yuhanna I.S. Zhu Y. Cox B.E. Hahner L.D. Osborne-Lawrence S. Lu P. Marcel Y.L. Anderson R.G. Mendelsohn M.E. Hobbs H.H. Shaul P.W. Nat. Med. 2001; 7: 853-857Crossref PubMed Scopus (645) Google Scholar) and Li et al. (41Li X.A. Titlow W.B. Jackson B.A. Giltiay N. Nikolova-Karakashian M. Uittenbogaard A. Smart E.J. J. Biol. Chem. 2002; 277: 11058-11063Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). Furthermore, the report of Grewal et al. (42Grewal T. de Diego I. Kirchhoff M.F. Tebar F. Heeren J. Rinninger F. Enrich C. J. Biol. Chem. 2003; 278: 16478-16481Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar) provided additional evidence for SR-BI signaling by describing SR-BI as a primary candidate for HDL-initiated signaling through activating Ras in a protein kinase C-independent manner. These multiple observations have led us to investigate the role of CLA-1 as a potential SAA-signaling receptor, contributing to the SAA-induced pro-inflammatory response. In this study, using the CLA-1-overexpressing HeLa cell model and human monocyte cell line, we demonstrate that lipid-poor SAA directly binds to CLA-1 and that CLA-1 ligands efficiently compete with SAA for CLA-1 binding. Furthermore, SAA is found to activate p44/p42 and p38 kinases, two members of the mitogen-activated protein kinase (MAPK) superfamily in both HeLa and THP-1 cells in a CLA-1-dependent manner. Reagents—All media, sera, and antibiotics were from Invitrogen. The MAPK inhibitors PD98059 and SB203580 were purchased from BIOMOL Research Laboratories (Plymouth Meeting, PA), human HDL was obtained from Calbiochem, and recombinant synthetic human apoSAA was purchased from PeproTech (Rocky Hill, NJ). Lipid content of the recombinant apoSAA was analyzed by the Phospholipids B enzymatic method (Wako, Richmond, VA) and an enzymatic cholesterol method (Roche Applied Science) on a Cobas Fara II analyzer (Roche Applied Science). According to these assays, the SAA preparation contained small amounts of phospholipids (5 ng/μg) and cholesterol (<2 ng/μg) and was considered as a lipid-poor form of SAA throughout this study. The endotoxin level in both SAA and HDL preparations were <0.1 ng/μg (1 endotoxin unit/μg). The synthetic amphipathic peptides were synthesized by a solid-phase procedure (45Merrifield R.B. JAMA. 1969; 210: 1247-1254Crossref PubMed Scopus (11) Google Scholar, 46Fairwell T. Hospattankar A.V. Brewer Jr., H.B. Khan S.A. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 4796-4800Crossref PubMed Scopus (19) Google Scholar). Peptide sequences were described in a previous report (39Bocharov A.V. Baranova I.N. Vishnyakova T.G. Remaley A.T. Csako G. Thomas F. Patterson A.P. Eggerman T.L. J. Biol. Chem. 2004; 279: 36072-36082Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Cell Cultures—Human HeLa (Tet-off) cells (Clontech, Palo Alto, CA) and CLA-1-overexpressing HeLa cells were cultured in Dulbecco's modified Eagle's medium, supplemented with 10% fetal calf serum, 2 mm glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, and 100 μg/ml G418 at 37 °C in a 5% CO2 humidified atmosphere. The CLA-1-overexpressing HeLa cell (4C2 clone) was characterized in our previous report (37Vishnyakova T.G. Bocharov A.V. Baranova I.N. Chen Z. Remaley A.T. Csako G. Eggerman T.L. Patterson A.P. J. Biol. Chem. 2003; 278: 22771-22780Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). To further confirm the results obtained using the 4C2 clone, additional CLA-1-overexpressing HeLa cell clones were analyzed for Alexa 488-HDL binding and CLA-1 expression by Western blotting. As an additional model, HEK 293 cells with low endogenous CLA-1 levels were stably transfected with CLA-1 pIRES-hrGFP-2a plasmid (Stratagene) followed by selecting cells with highest green fluorescent protein expression. Human monocytic THP-1 cells (TIB-202 from ATTC) were maintained in complete RPMI 1640 medium containing 10% fetal calf serum, 2 mm l-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin. Differentiated human monocytes were prepared as previously reported (47Buechler C. Ritter M. Quoc C.D. Agildere A. Schmitz G. Biochem. Biophys. Res. Commun. 1999; 262: 251-254Crossref PubMed Scopus (60) Google Scholar). Alexa 488-labeled Ligand Uptake and Competition Experiments— HDL and SAA were conjugated with Alexa Fluor® 488, using a protein labeling kit (Molecular Probes, Eugene, OR) following the vendor's instructions. The Alexa-labeled preparations were analyzed by SDS-PAGE, using 10–20% Tricine pre-cast gels (Invitrogen). Gels were scanned using a Fluorocsan (model A, Hitachi). Alexa-labeled HDL apolipoproteins, and SAA were detected in the appropriate position with molecular masses of 28, 18, and 12 kDa for apoA-I, apoA-II, and SAA, respectively (data not shown). All incubations were performed in Dulbecco's modified Eagle's medium (HeLa cells) or RPMI 1640 (THP-1 cells), containing 0.1% bovine serum albumin at 37 °C. Uptake experiments with HeLa cells were performed by using Alexa 488-SAA at concentrations between 1.25 and 10 μg/ml. After 2 h of SAA incubation, the cells were rinsed with ice-cold PBS and detached by a 30-min incubation in EDTA-containing Cell stripper (Mediatech, Inc., Herndon, VA). Cells were resuspended and added to an equal volume of 4% paraformaldehyde in PBS. A competition assay was performed, using 5 μg/ml fluorescence-labeled SAA and unlabeled ligands ranging in concentration from 5 to 100 μg/ml. Following a 2-h incubation, HeLa cells were treated as described above, while the samples of THP-1 cell suspensions were transferred to centrifuge tubes and pelleted by a brief centrifugation (5000 rpm, 5 min at 4 °C). After washing with ice-cold PBS, the cells were centrifuged one more time, and the resulting pellets were again resuspended in PBS and mixed with an equal volume of 4% paraformaldehyde. Cell-associated fluorescence was analyzed by a flow cytometry using a FACScan (BD Biosciences) and analyzed using FlowJo software (Tree Star, San Carlos, CA). Detection of Activated ERK1/2 and p38 by Western Blot Analyses— Mock (Tet-off) and CLA-1-overexpressing HeLa cells were grown in 6-well culture plates to confluence. Before the MAPK activation assay, the cells were incubated overnight in Dulbecco's modified Eagle's medium. The cells were stimulated for the indicated periods of time with SAA (25 μg/ml), HDL (50 μg/ml), or phorbol 12-myristate 13-acetate (25 ng/ml) at 37 °C. After stimulation, the culture medium was immediately aspirated; the cells were placed on ice and washed three times with ice-cold PBS. Afterward, the cells were scrapped into 100 μl of lysis buffer (20 mm Tris-HCl, pH 7.5, 2 mm EDTA, 0.5% (v/v) Triton X-100, 1 mm NaF, 1 mm Na3VO4, 50 mm 2β-mercaptoethanol, 5 mm MgCl2, 1 mm phenylmethylsulfonyl fluoride, and 2% (v/v) protease inhibitor mixture set III (Calbiochem, San Diego, CA)). After 10-min incubation on ice, the samples were sonicated for 5–6 s and centrifuged at 12,000 rpm for 10 min at 4 °C. The cell extracts were collected and mixed with the 2× SDS sample buffer. The samples were separated on SDS-PAGE in 10% Tris-glycine pre-cast gels (Invitrogen) and then transferred to nitrocellulose membranes. The membranes were blocked in Tris-buffered saline with 0.1% Tween 20 and 5% (w/v) nonfat dry milk and further incubated either with the primary anti-phospho-p44/42 or anti-phospho-p38 MAPK antibodies or with the corresponding antibodies that recognize both active and inactive forms of each subfamily of kinases (Cell Signaling Technology, Beverly, MA) overnight at 4 °C. Immunoreactive bands were detected with a horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology) and the enhanced chemiluminescence (ECL) system (Amersham Biosciences). Alternatively, the immunoreactive bands were detected using an alkaline phosphatase-conjugated secondary antibody and Western Blue Stabilized Substrate for Alkaline Phosphatase (Promega, Madison, MI). For measurement of MAPK activation, THP-1 cells were incubated overnight in serum-free RPMI with 0.1% bovine serum albumin, and further incubated in the same fresh medium for the next 4 h. Before the experiment, the cells were placed into 12-well culture plates (2 × 106 cells per well) and treated with the corresponding stimuli for the indicated duration of time. The cell suspensions were then collected and centrifuged (12,000 rpm, 5 min at 4 °C). The cells were then washed with ice-cold PBS, and subsequently pelleted by a brief centrifugation. The resulting cell pellets were resuspended in the lysis buffer and analyzed according to the procedure described above for HeLa cells. Other Procedures—IL-8 secretion was analyzed in culture media after a 24-h incubation utilizing commercial enzyme-linked immunosorbent assay kits (BioSource International, Camarillo, CA). Cross-linking and colocalization experiments were conducted as reported previously (39Bocharov A.V. Baranova I.N. Vishnyakova T.G. Remaley A.T. Csako G. Thomas F. Patterson A.P. Eggerman T.L. J. Biol. Chem. 2004; 279: 36072-36082Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Briefly, fluorescently labeled ligands (Alexa 488-HDL or Alexa 488-SAA) were added in 1 ml of Dulbecco's modified Eagle's medium containing 2 mg/ml lipid-free bovine serum albumin, and the cells were incubated for 90 min at 37 °C in a CO2 incubator. Cells were washed three times with ice-cold PBS and incubated with 250 μm disuccinimidyl suberate in PBS for 15 min at room temperature. After washing and a 2% Triton X-100 extraction, the complexes were precipitated using rabbit anti-CLA-1 (raised against the C-terminal 15-AA peptide) or non-immune rabbit serum. Tris-glycine (12%)/SDS-PAGE, gels were utilized for protein separation, and Alexa 488 signal was detected using a Typhoon 9200 imager (Amersham Biosciences). For colocalization experiments, CLA-1-overexpressing and mock transfected HeLa cells were incubated with 2.5 μg/ml Alexa 488-SAA for 1 h at 37 °C followed by fixation using 4% paraformaldehyde. Cells were permeabilized by incubating with 0.1% Triton X-100 in PBS for 10 min at room temperature and further incubated with 10 mg/ml bovine serum albumin, 1% goat serum in PBS to prevent nonspecific antibody absorption. CLA-1 was detected utilizing rabbit anti-CLA-1 antiserum as a first antibody. Alexa 488/568-labeled goat anti-rabbit IgG were used as a second antibody (39Bocharov A.V. Baranova I.N. Vishnyakova T.G. Remaley A.T. Csako G. Thomas F. Patterson A.P. Eggerman T.L. J. Biol. Chem. 2004; 279: 36072-36082Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Fluorescence was viewed with a Zeiss 510 laser scanning confocal microscope, using a krypton-argon-Omnichrome laser with excitation wavelengths of 488 and 568 nm for Alexa-488 and Alexa-568 labels, respectively. Alexa 488-SAA Uptake Is Enhanced in CLA-1-overexpressing HeLa Cells—It is known that SR-BI/CLA-1 overexpression increases uptake of SR-BI ligands (33Acton S.L. Scherer P.E. Lodish H.F. Krieger M. J. Biol. Chem. 1994; 269: 21003-21009Abstract Full Text PDF PubMed Google Scholar, 36Svensson P.A. Johnson M.S. Ling C. Carlsson L.M. Billig H. Carlsson B. Endocrinology. 1999; 140: 2494-2500Crossref PubMed Scopus (34) Google Scholar, 48Calvo D. Gomez-Coronado D. Suarez Y. Lasuncion M.A. Vega M.A. J. Lipid Res. 1998; 39: 777-788Abstract Full Text Full Text PDF PubMed Google Scholar). The functional activity of CLA-1-overexpressing HeLa cells has been demonstrated previously by observing increased HDL binding and HDL cholesterol ester uptake (37Vishnyakova T.G. Bocharov A.V. Baranova I.N. Chen Z. Remaley A.T. Csako G. Eggerman T.L. Patterson A.P. J. Biol. Chem. 2003; 278: 22771-22780Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). To evaluate if SAA could be a potential ligand for CLA-1, we determined Alexa 488-SAA uptake by the CLA-1-overexpressing HeLa cells compared with mock transfected control cells. As seen in Fig. 1A, CLA-1-overexpressing HeLa cells demonstrated a 5-fold increase in SAA-uptake when compared with the mock transfected control. SAA-uptake calculated as an arbitrary unit of fluorescence at 488 nm per cell appears to be dose-dependent, approaching a plateau at 2.5 μg/ml SAA (Fig. 1A). Alexa 488-HDL uptake demo
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