Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M405009200 on December 2, 2004

J. Biol. Chem., Vol. 280, Issue 9, 8031-8040, March 4, 2005
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/9/8031    most recent
M405009200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Baranova, I. N.
Right arrow Articles by Patterson, A. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Baranova, I. N.
Right arrow Articles by Patterson, A. P.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

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*

Irina N. Baranova{ddagger}, Tatyana G. Vishnyakova§, Alexander V. Bocharov{ddagger}, Roger Kurlander{ddagger}, Zhigang Chen§, Michael L. Kimelman{ddagger}, Alan T. Remaley§, Gyorgy Csako{ddagger}, Fairwell Thomas§, Thomas L. Eggerman{ddagger}, and Amy P. Patterson§||

From the {ddagger}Department of Laboratory Medicine, W. G. Magnuson Clinical Center, §NHLBI, and the Division of Diabetes, Endocrinology and Metabolic Diseases, NIDDK, National Institutes of Health, Bethesda, Maryland 20892

Received for publication, May 5, 2004 , and in revised form, November 19, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 {alpha}-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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The acute phase serum amyloid A protein (SAA)1 is a 12- to 14-kDa apolipoprotein encoded by SAA1 and SAA2 allelic variants (1) and is found predominantly in the plasma high density lipoprotein fraction (2, 3). SAA is normally present in the bloodstream at 0.1 µM. In response to various injuries, including trauma, infection, inflammation, and neoplasia (4), the levels of SAA increase up to 1000-fold. As with other acute-phase reactants, the liver is the major site of SAA expression (5). However, SAA is also expressed in cells within human atherosclerotic lesions (6, 7), histologically normal epithelial, endothelial (8), and monocytic cells, macrophage cell lines such as THP-1 cells (4, 9), and human tumor cells (4). SAA is also expressed in the brain of patients with Alzheimer disease (10) and in synovial tissue from patients with rheumatoid arthritis (11).

Multiple studies have demonstrated a significant association of altered SAA levels with several pathological states, particularly chronic inflammatory diseases such as secondary amyloidosis (12, 13) and atherosclerosis (6, 9, 14). 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 (13–15). In atherosclerosis, SAA accumulates in macrophages, macrophage-derived "foam cells," adipocytes, endothelial cells, and smooth muscle cells within the vascular plaque (9). The exact role of SAA in atherogenesis is complicated by its effect upon the metabolism of HDL and other lipoprotein fractions (16, 17). 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 (18, 19). SAA may also act as a signal for redirecting HDL to the sites of tissue destruction and cholesterol accumulation (20). Additionally SAA may contribute to HDL-mediated clearance of cellular cholesterol by modulating LCAT activity (21). Moreover, SAA has been shown to modulate cholesterol transport by serving as a cholesterol-binding protein (22), as well as a direct activator of the ABCA1-dependent pathway of cholesterol translocation to an extracellular cholesterol acceptor (23).

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 (24–26). Furthermore, it has been reported that SAA significantly stimulates the secretion of the pro-inflammatory cytokines tumor necrosis factor-{alpha}, IL-8 and IL-1{beta} by cultured human neutrophils, as well as the release of tumor necrosis factor-{alpha} from lymphocytes (27) and of IL-1{beta} from THP-1 monocytic cells (3). The importance of SAA in various physiological and pathological processes has raised a considerable interest in the identity of the one or more cell surface receptors that bind, internalize, and mediate SAA-induced pro-inflammatory effects. 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 (28). 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 (1, 29).

CLA-1 and rodent scavenger receptor BI (SR-BI), are HDL receptors, highly expressed in liver, adrenal gland, ovary (30), and atherosclerotic lesions of apoE-deficient mice (31). In addition, CLA-1 and SR-BI are highly expressed on monocytes/macrophages (32), 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 (33), native very low density lipoprotein (34), anionic phospholipids, lipopolysaccharide, and apoptotic cells (35, 36, 37). 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 (38) 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 (39). Because SAA is known to be an amphipathic protein, having two amphipathic {alpha}-helical regions corresponding to the 1–18 N-terminal and 72–86 C-terminal sequences (14), 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 (40–42). 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 (43). This adaptor protein is known to play a crucial role in regulating various biological processes, including signal transduction, adhesion, membrane trafficking, and cellular transport (44). The initial report indicated that one of the four domains of PDZK1 is associated with the SR-BI cytoplasmic C terminus (43). 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. (40) and Li et al. (41). Furthermore, the report of Grewal et al. (42) 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.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
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 (45, 46). Peptide sequences were described in a previous report (39).

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 (37). 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 (47).

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 2{beta}-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 2x 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 x 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 (39). 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 (39). 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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
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 (33, 36, 48). The functional activity of CLA-1-overexpressing HeLa cells has been demonstrated previously by observing increased HDL binding and HDL cholesterol ester uptake (37). 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 demonstrates a similar dose-dependent accumulation of fluorescent signal in CLA-1-overexpressing HeLa cells, approaching a plateau at 2.5 µg/ml HDL (Fig. 1B). To further confirm the increased SAA uptake in CLA-1-overexpressing cells, several clones expressing various CLA-1 levels were incubated either with 2.5 µg/ml Alexa 488-HDL or 1 µg/ml Alexa 488-SAA followed by FACScan analysis of bound ligand (see "Materials and Methods" section). As seen in Fig. 1C, HDL and SAA uptake demonstrated a linear correlation indicating a direct association between SAA uptake and a primary HDL-binding function of CLA-1.



View larger version (23K):
[in this window]
[in a new window]
  		FIG. 1.
Uptake of Alexa 488-HDL and SAA in CLA-1-overexpressing and mock transfected HeLa cells. Cells were incubated with the increasing (0–10 µg/ml) concentrations of fluorescently labeled SAA (A) or HDL (B)for2hat37 °C. Stably transfected CLA-1 HeLa clones (C) were incubated with 2.5 µg/ml Alexa 488-SAA or 5 µg/ml Alexa 488-HDL for at 37 °C. Cell-associated fluorescence was estimated by FACScan analyses. The data (mean ± S.D.) represent one of three separate experiments that yielded similar results.

 
SAA Cross-linking—To evaluate the direct interaction of CLA-1 with SAA, CLA-1-overexpressing, and mock transfected HeLa cells were incubated with Alexa 488-SAA- or Alexa 488-protein-labeled HDL as positive control using disuccinimidyl suberate as a cross-linker (39). As seen in Fig. 2 (lane 1) cross-linked complexes of CLA-1 and HDL were detected predominantly as one major band with molecular mass of ~200 kDa. This approximates the theoretical masses of HDL proteins (~100 kDa) cross-linked to CLA-1 monomer (~82 kDa). Incubations in the presence of 20-fold excess of HDL drastically reduced the signal intensity in that band, indicating a direct competition for CLA-1 binding (data not shown). The signal for Alexa 488-SAA (molecular mass ~12 kDa) occurred predominantly in a band with molecular mass of ~95–110 kDa (lane 2). An appearance of minor upper band may represent the CLA-1 cross-linking to a dimer SAA, whereas the lower one to monomer SAA. The signal intensity in both bands was significantly reduced when the fluorescent ligands were incubated with 20-fold excess of unlabeled HDL and SAA, respectively (lanes 3 and 4). L3D-37pA peptide, which contains three D-amino acid substitutions that disturb the helical structure, did not compete for CLA-1 with SAA (lane 5), whereas both helical L-37pA and D-37pA markedly reduced the SAA signal (lanes 6 and 7). Not surprisingly, SAA cross-linked to mouse SR-BI demonstrated bands with similar mobility (lane 8). These observations implicate both CLA-1 and SR-BI as potent SAA-binding proteins. Mock transfected cells demonstrated detectable complexes only when the cell extracts were prepared from 10-fold more cells (data not shown).



View larger version (67K):
[in this window]
[in a new window]
  		FIG. 2.
Electrophoretic analyses of cross-linked fluorescently labeled HDL and SAA. CLA-1/mouse SR-BI-overexpressing HeLa cells were incubated with either 5 µg/ml Alexa 488-HDL or 2.5 µg/ml Alexa 488-SAA for 90 min at 37 °C, washed, and cross-linked with disuccinimidyl suberate as described under "Experimental Procedures." Cell extracts were immunoprecipitated with an anti-CLA-1 antibody, and the immunoprecipitates were analyzed by a reducing 12% SDS-PAGE. Lane 1, Alexa 488-HDL/CLA-1 complexes; lane 2, Alexa 488-SAA/CLA-1 complexes; lane 3, Alexa 488-SAA/CLA-1 complexes formed in the presence of 20-fold excess HDL; lane 4, Alexa 488-SAA/CLA-1 complexes formed in the presence of 20-fold excess SAA; lane 5, Alexa 488-SAA/CLA-1 complexes formed in the presence of 20-fold excess L3D-37pA; lane 6, Alexa 488-SAA/CLA-1 complexes formed in the presence of 20-fold excess L-37pA; lane 7, Alexa 488-SAA/CLA-1 complexes formed in the presence of 20-fold excess D-37pA; and lane 8, Alexa 488-SAA/mouse SR-BI complex.

 
Colocalization of Alexa 488-SAA and Anti-CLA-1 Antibody in CLA-1-overexpressing HeLa Cells—To further characterize the interaction of CLA-1 with SAA, CLA-1-expressing and mock transfected HeLa cells were incubated with fluorescently labeled SAA or HDL as a positive control. After 1-h incubation, cells were fixed with 4% paraformaldehyde, permeabilized, and stained using anti-CLA-1 antibody. As seen at Fig. 3, a significant portion of SAA (panel A) was internalized (panel B). The merge images demonstrated that the majority of the SAA (yellow) is strongly colocalized with CLA-1, primary intracellularly and to lesser extent at the cell surface (panel C), whereas only a very weak signal was found in mock transfected cells (panel D).



View larger version (29K):
[in this window]
[in a new window]
  		FIG. 3.
CLA-1 colocalization with bound and internalized Alexa 488-SAA in CLA-1-overexpressing HeLa cells. Using CLA-1-overexpressing cells, the individual localizations are seen for Alexa 488-SAA (green, A) and CLA-1 (red, B). The merged image for SAA and CLA-1 is shown in C with colocalization seen as yellow. SAA uptake in mock transfected cells is shown in D. Scale bar: 10 µm.

 
SAA Colocalizes with the Endoplasmic Recycling Compartment Marker, Transferrin—In contrast to LPS and L-37pA, which bind to CLA-1 and are transported predominantly to the Golgi, SAA was found in the endoplasmic recycling compartment (ERC) and lysosomes (49, 50). Confocal microscopy demonstrated (Fig. 4) extensive overlap between fluorescent transferrin and SAA (panel C) in CLA-1-expressing HeLa cells, thus confirming an involvement of CLA-1 with SAA internalization and distribution to the ERC. Additionally, differentiated human monocytes also demonstrated a strong colocalization of Alexa 488-SAA with internalized transferrin, further confirming SAA internalization to the ERC (panels D–F).



View larger version (49K):
[in this window]
[in a new window]
  		FIG. 4.
Colocalization of Alexa 488-SAA and transferrin in CLA-1-overexpressing HeLa cells and differentiated human monocytes. The individual confocal microscopy images for Alexa 488-SAA (A and D), Bodipy-transferrin (B and E) are shown as green and red. Merge images are seen as yellow in C and F. A–C correspond to CLA-1-overexpressing HeLa cells, and D–F correspond to differentiated human monocytes. Scale bars: 10 µm.

 
Amphipathic {alpha}-Helical Peptides Compete for Alexa 488-SAA Uptake in HeLa and THP-1 Cells—As seen in Fig. 5 (A and B), two SR-BI ligands, apoA-I and the synthetic amphipathic helical peptide, L-37pA, competed with Alexa 488-SAA for CLA-1 in both mock transfected and CLA-1-overexpressing HeLa cells. Cold SAA was also an efficient competitor. No competition was found with the L3D-37pA peptide, which contains three D-amino acid substitutions and does not interact with CLA-1 (51). Two other peptides, L1D-37pA and L2D-37pA, containing, respectively, 1 and 2 D-amino acid substitutions, which partially disturb the amphipathic {alpha}-helical motif, were also without effect (data not shown). HDL also competed against SAA in a dose-dependent manner in both CLA-1-overexpressing HeLa cells and mock transfected HeLa cells (data not shown).



View larger version (32K):
[in this window]
[in a new window]
  		FIG. 5.
Competition of CLA-1 ligands with Alexa 488-SAA uptake in CLA-1-overexpressing (A) and mock transfected (B) HeLa cells. Cells were incubated with 5 µg/ml Alexa 488-SAA in the presence of the increasing concentrations (1–100 µg/ml) of unlabeled ligands for 2 h at 37 °C. Cell-associated fluorescence was estimated by FACScan analyses. The data (mean ± S.D.) represent one of three separate experiments that yielded similar results.

 
CLA-1 was next tested as a potential receptor for SAA, using the THP-1 monocyte cell line. This cell line represents a well recognized physiologically relevant model of phagocytes and expresses high levels of CLA-1 (52, 53). Unlabeled SAA, as well as other CLA-1 ligands, including HDL, apoA-I, and L-37pA peptide, potently inhibited Alexa Fluor 488-SAA uptake by THP-1 cells in a dose-dependent manner (Fig. 6). Importantly, the peptide with an impaired {alpha}-helical motif in its structure, L3D-37pA, was unable to compete for SAA uptake.



View larger version (18K):
[in this window]
[in a new window]
  		FIG. 6.
Competition of the CLA-1 ligands with Alexa 488-SAA uptake in THP-1 cells. Cells were incubated with 3.6 µg/ml of Alexa 488-SAA without any competitors or in the presence of the increasing concentrations (3.6–100 µg/ml) of unlabeled ligands for 2 h at 37 °C. Cell-associated fluorescence was estimated by FACS analyses. The data represent one of two separate experiments that yielded similar results.

 
SAA-induced IL-8 Secretion Is Increased in CLA-1-overexpressing HeLa and HEK 293 Cells—According to recently reported data, SAA in its lipid-poor form possesses "cytokine-like" activity, being able to induce the release of several pro-inflammatory cytokines by cultured human neutrophils (27, 29), lymphocytes (54), as well as by THP-1 monocytic cells (3). To evaluate if the CLA-1-mediated SAA binding can induce increased cytokine production, the levels of IL-8 secretion in CLA-1-overexpressing HeLa cells were determined in comparison with mock transfected cells. SAA stimulation resulted in a 3- to 5-fold increase of IL-8 release in CLA-1-overexpressing cells when compared with mock transfected controls (Fig. 7A). At the same time (Fig. 7B), we were able to demonstrate increased levels of IL-8 secretion by CLA-1-overexpressing cells as a result of their treatment with HDL (20–100 µg/ml), using HDL concentrations recently reported to mediate signaling (42). Importantly, HDL preparations contained negligible levels of LPS as determined by using the Limulus Amebocyte Lysate assay. These levels of LPS did not induce IL-8 secretion in either HeLa or THP-1 cells (data not shown). The results of the experiments using HEK 293 cells yielded similar differences in SAA-induced IL-8 secretion levels between the control mock transfected and CLA-1-overexpressing cells (Fig. 7C) and had a lower basal IL-8 expression.



View larger version (15K):
[in this window]
[in a new window]
  		FIG. 7.
Dose-dependent IL-8 secretion induced by SAA and HDL in CLA-1-overexpressing and mock transfected cells. HeLa cells were incubated with increasing SAA concentrations (A) or HDL (B) for 16 h. HEK 293 cells were treated by SAA in the same way as HeLa cells (C). IL-8 levels were determined in conditioned media. Data represent one of three separate experiments that yielded similar results.

 
Amphipathic {alpha}-Helical Peptides Block SAA-induced IL-8 Secretion in THP-1 Cells—To demonstrate that CLA-1 antagonists, such as the amphipathic {alpha}-helical peptides, block SAA-induced IL-8 secretion, THP-1 cells were used, which demonstrated higher reactivity to lower doses of SAA (0.25–2.5 µg/ml; data not shown) than was observed with HeLa cells. Because HeLa cell IL-8 secretion required a relatively high SAA concentration of 2.5 µg/ml, the same experiment would have required the use of amphipathic {alpha}-helical peptides of much higher (toxic at prolonged incubations (51)) concentrations. The results plotted in Fig. 8 demonstrate that amphipathic {alpha}-helical peptides inhibit SAA-induced IL-8 secretion in a dose-dependent manner. L-37pA peptide and its D-isomer, D-37pA, efficiently inhibited IL-8 release induced by SAA. On the other hand, L-37pA demonstrated a higher degree of inhibition (80%) compared with its D-analog (60%), respectively, and a lower dose of L-37pA than D-37pA was required (5 µg/ml versus 25 µg/ml) to reach a maximum blocking effect. At the same time, L3D-37pA, the synthetic peptide lacking an {alpha}-helical motif, failed to inhibit SAA-induced IL-8 secretion at any of the concentrations tested.



View larger version (24K):
[in this window]
[in a new window]
  		FIG. 8.
Inhibition of SAA-induced IL-8 secretion by synthetic peptides in THP-1 cells. Cells were incubated with 0.5 µg/ml SAA in the presence of increasing concentrations of helical L-37pA and L3D-37pA for 16 h. The data represent the mean ± S.D. of three separate experiments, each performed in duplicate.

 
SAA-initiated Activation of MAPKs (ERK1/2 and p38) Is Enhanced in CLA-1-overexpressing HeLa Cells—The MAPKs ERK1/2 and p38, members of the MAPK superfamily, are known to participate in the signal transduction cascades controlling cell growth and differentiation (55, 56), as well as cellular responses to cytokines and stress (57, 58). It has been previously reported that proximal signaling events induced by SAA include activation of MAPKs, ERK1/2 and p38, a requirement for induced IL-8 release (29).

To test whether increased IL-8 production induced by SAA is associated with activation of both MAPKs, we compared the degree of ERK1/2 and p38 phosphorylation in CLA-1-overexpressing and control HeLa cells after SAA stimulation. Fig. 9 demonstrates a higher level of ERK1/2 (panel A) and p38 (panel B) activation in CLA-1-overexpressing cells after SAA treatment for 5–30 min. The MAPK phosphorylation increase was also observed in the CLA-1-overexpressing cells after incubation with HDL for 10 min. This HDL effect is consistent with the recently reported data demonstrating HDL-mediated, SRBI-dependent MAPK activation (42). Treatment of HeLa cells with the specific MAPK inhibitors, PD-98059 and SB-203580, which selectively block MEK1, the upstream kinase of ERK1/2, and p38 kinase activity, respectively, resulted in the decrease of both SAA-induced as well as HDL-induced MAPK phosphorylation (Fig. 10, A and B). This finding suggests the involvement of the CLA-1-mediated MAPK signaling for both SAA and HDL. Despite SB-203580 being a direct inhibitor of p38 activity, our data demonstrate a marked decrease in the phosphorylated form of p38 kinase upon treatment with this compound. These results can be possibly explained by the nonspecific inhibitory effect of the large excess (50 µM) of SB-203580 upon the activity of some other upstream kinases. With lower doses of SB-203580 (10 µM and 20 µM), we did not observe any consistent changes in the phosphorylation levels of p38 kinase (data not shown). At the same time, lower doses of this inhibitor partially blocked (up to 20% from control levels) SAA-induced IL-8 secretion, the downstream event of the p38 kinase signaling cascade (Fig. 11B). As opposed to SB-203580, the effects of the MEK1 inhibitor PD-98059 on SAA-induced secretion of IL-8 in both mock and CLA-1-overexpressing HeLa cells were less effective, with the maximum inhibition being 50–60% with respect to control levels (Fig. 11A). This observation could possibly reflect the differential contribution of these MAPK pathways to the downstream IL-8 production.



View larger version (25K):
[in this window]
[in a new window]
  		FIG. 9.
Time course of SAA-induced phosphorylation of ERK1/2 (A) and p38 (B) in CLA-1-overexpressing and mock transfected HeLa cells. Following an overnight incubation in serum-free medium, the cells were stimulated with 25 µg/ml SAA for the indicated time intervals. HDL (50 µg/ml) was used as a positive control. ERK1/2 and p38 phosphorylation was detected by Western blotting using specific ERK1/2 and p38 antibodies. The non-phosphorylated forms of both kinases were also detected. Simultaneously, the {beta}-actin levels in the corresponding samples were detected to monitor protein loading. The resulting bands were quantified by scanning densitometry using GelPro Analyzer software. Data are presented as the ratio of integral optic density for activated MAPK bands to the corresponding integral optic density values for {beta}-actin bands. The data represent one of three separate experiments that yielded similar results.

 



View larger version (25K):
[in this window]
[in a new window]
  		FIG. 10.
Specific MAPK inhibitors block CLA-1-mediated phosphorylation of ERK1/2 (A) and p38 (B) induced by SAA in CLA-1-overexpressing HeLa cells. CLA-1-overexpressing and mock transfected cells were treated with the MEK1 inhibitor PD-98059 (25 µM) or p38 kinase inhibitor SB-203580 (50 µM) for 1 h prior to a 20-min stimulation with SAA (25 µg/ml) or a 10-min stimulation with HDL (50 µg/ml). Data are presented as the ratio of integral optic density for activated MAPK bands to the corresponding integral optic density values for {beta}-actin bands. The data represent one of three separate experiments that yielded similar results.

 



View larger version (29K):
[in this window]
[in a new window]
  		FIG. 11.
Effects of MAPK inhibitors on SAA-induced IL-8 secretion in mock and CLA-1-overexpressing HeLa cells. Cells were treated with the indicated doses of MEK1 inhibitor PD-98059 (A)orp38 kinase inhibitor SB-203580 (B) for 1 h prior to stimulation with SAA (10 or 25 µg/ml). Levels of the secreted IL-8 were measured after 20 h. Data are presented as means ± S.D. of one of three separate experiments performed in duplicates and yielded similar results.

 
Amphipathic {alpha}-Helical Peptide L-37pA Inhibits SAA-induced MAPKs Activation in THP-1 Cells—ERK1/2 and p38 can be activated in THP-1 monocyte cells, using SAA concentrations as low as 5 µg/ml, whereas a 4–5 times higher dose of SAA was required in epithelial HeLa cells (data not shown). Because monocytes/macrophages are more likely physiological targets than epithelial cells, we used THP-1 cells to demonstrate that L-37pA inhibits the CLA-1-dependent activation of ERK1/2 and p38 by SAA. Fig. 12 demonstrates that with the SAA stimulatory effect on both MAPKs (ERK1/2 (panel A) and p38 (panel B)), phosphorylation was reduced to basal levels in the presence of a 10-fold excess of the L-37pA peptide, whereas the non-helical peptide L3D-37pA did not demonstrate any inhibitory effect.



View larger version (27K):
[in this window]
[in a new window]
  		FIG. 12.
Inhibition of SAA-induced phosphorylation of ERK1/2 (A) and p38 (B) MAPKs by L-37pA and L3D-37pA synthetic peptides in THP-1 cells. Following an overnight incubation in serum-free medium, cells were stimulated with 5 µg/ml SAA in the presence of 5- and 10-fold peptide excess. Phosphorylated and non-phosphorylated forms of ERK1/2 and p38 were detected by the Western blot analyses. The data represent one of three separate experiments that yielded similar results.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
SAA is a major acute phase plasma protein involved in multiple physiological and pathological processes. Previous in vitro studies have demonstrated the cytokine-like properties of SAA, which induces the release of several cytokines in human THP-1 cells and neutrophils (3, 27). This along with experimental data demonstrating that SAA is a potent chemotactic agent for several types of inflammatory cells (24, 26, 59) provides compelling evidence for an important regulatory role of SAA in inflammatory and immune responses. Recently, the seven-transmembrane-spanning G-protein-coupled FPRL1/LXA4R protein has been reported as the candidate signaling receptor mediating some of cytokine-like and chemoattractant effects of SAA (1, 28, 29). However, no data has been reported on the possible involvement of CLA-1 in SAA-induced signaling. The possibility that SAA could be a potent ligand for CLA-1 was based on the current concept that CLA-1 like other scavenger receptors are pattern recognition receptors. CLA-1 binds a broad range of ligands, exhibiting various biological activities but sharing common structural features. SAA is an amphipathic polypeptide, containing two amphipathic {alpha}-helices in its structure (14). These features also are present in several other known CLA-1 ligands, including major HDL apolipoproteins, and apparently are determinants recognized by ligand-binding domain/domains of the CLA-1 receptor.

Our study provides evidence that the HDL receptor, CLA-1, may function as a receptor for lipid-poor SAA, being involved in both binding and downstream signaling, triggered by the ligand-receptor interaction. We have demonstrated that CLA-1-overexpressing cells show an enhanced SAA uptake when compared with mock transfected cells. Alexa 488-SAA specifically bound to CLA-1-expressing HeLa cells with high affinity (Kd ~ 1 µg/ml) and a 5-fold increased capacity when compared with vector-transfected control cells. Due to its amphipathic nature, SAA as well as L-37pA or LPS is oligomerized into micelles in physiological saline solution with an estimated molecular mass of 100–200 kDa. Using these estimated molecular masses, SAA has an approximate Kd value of 1 x 10-8 to 5 x 10-9 M, which is compatible with previously reported values for HDL, L-37pA, and LPS (37, 39). We also studied a correlation between the HDL-binding and SAA-binding function of CLA-1 in several HeLa cell lines stably transfected with CLA-1. As seen at Fig. 1, HDL and SAA binding correlated well with all of the clones used in the experiments. When transfecting another cell line, HEK 293, CLA-1 also dramatically increased SAA binding and internalization (data not shown). The direct interaction between CLA-1 and SAA was further supported by the results of the cross-linking experiments. SAA cross-linked predominantly to monomer CLA-1; this observation contrasts with our previously reported data regarding L-37pA and LPS, which demonstrated a significant signal associated with the dimer form of CLA-1 (39). Because LPS and L-37pA were distributed primarily to the Golgi, whereas SAA was distributed primarily to the ERC, this monomer/dimer binding difference may suggest that monomeric and dimeric CLA-1 internalize CLA-1 ligands toward distinct intracellular compartments. Both transferrin/SAA and anti-Cla-1/SAA colocalization experiments strongly suggest endocytic functions for CLA-1, which is now being further investigated in our laboratory. Importantly, differentiated human monocytes also demonstrated a rapid SAA internalization and distribution to the ERC.

CLA-1 ligands, including HDL, its major apolipoprotein apoA-I and {alpha}-helical amphipathic peptides, effectively competed for SAA binding in CLA-1-transfected HeLa and THP-1 cells. Our results demonstrate that the markedly increased SAA binding, observed in CLA-1-overexpressing HeLa cells, correlates well with their higher IL-8 secretion compared with control cells. IL-8 secretion is known to be a distal event of the ERK1/2 and p38 MAPK signaling cascades activated by numerous agents, including lipid-poor SAA (29, 60, 61). In our study, we were able to demonstrate that phosphorylation of both MAPKs was markedly increased in CLA-1-overexpressing cells as a result of their exposure to SAA. These results imply that CLA-1 may act as an SAA receptor, mediating SAA-induced activation of the MAPK signaling pathway. This suggestion is consistent with the recently reported results demonstrating SR-BI involvement in the HDL-induced downstream activation of Ras with the subsequent induction of the MAPK signaling cascade (42). In addition, CLA-1 has been reported to be associated with the PDZ-containing adaptor protein (43), whose function is associated with signal transduction (44). Furthermore, it has been demonstrated that in some cell types CLA-1 is located in the plasma membrane caveolae (62), specialized surface microdomains implicated in a number of transport and signaling processes (63). Abundant morphological and biochemical evidence indicates that a variety of proteins known to be involved directly or indirectly in signal transduction are enriched in caveolae (64). The implications of data linking CLA-1 and activation of the MAPK signaling cascades may go beyond CLA-1 participation in the SAA cytokine-like activity. In view of prior reports demonstrating that p38 MAPK activation can be involved in the actin cytoskeleton reorganization (65) as well as in filamentous actin polymerization (66), CLA-1 may be potentially involved in intracellular actin-based trafficking processes, initiated upon ligand binding by the CLA-1 receptor.

In our study, we used a lipid-poor recombinant synthetic human SAA. SAA is known to exist in normal plasma almost exclusively bound to HDL (67). This raises the question of the physiological relevance of the lipid-poor SAA form. Evidence for the relevance of the lipid-poor SAA comes from the analysis of serum SAA distribution during an acute phase reaction induced by LPS in mice (68). In this study, 15% of SAA was present in the lipid-poor (not associated with any class of lipoproteins) form. Considering the high levels of SAA occurring with an acute phase reaction, this portion of SAA could easily account for the "cytokine-like" activity of the lipid-poor SAA demonstrated in our study as well as by others (3, 27, 29). The association of SAA with HDL (3) or phosphatidylcholine vesicles (data not shown) greatly reduces its pro-inflammatory/cytokine-like activity. Under physiological conditions (serum level of HDL 1.4–1.6 mg/ml and SAA is <1.0 µg/ml) there is an over 1000-fold excess of HDL relative to SAA, which apparently acts as a natural inhibitor of the chemoattractant and cytokine-like activity of SAA (27). During the acute phase response, the serum concentration of SAA may reach levels up to 80–1000 µg/ml (25), exhausting the capacity of HDL to bind all of the SAA and allowing some of the SAA to be in a lipid-poor form. Interestingly, we have found that HDL can also induce IL-8 secretion. However, HDL itself was a significantly weaker stimulator when compared with SAA.

There are conditions when there is a greater propensity for SAA to be in a lipid-poor form. In genetically determined or acquired HDL deficiency conditions there is a diminished capacity for SAA binding by HDL. In patients during the acute phase of Kawasaki disease reductions (up to 70%) in plasma HDL-cholesterol and in apolipoprotein A-I are observed (69). Several studies (70, 71) indicate that HDL lipids and apolipoproteins measured in intravascular tissue fluids are 80–90% lower than those in plasma. This reduction creates an environment more prone for lipid-poor SAA formation. Additionally, SAA concentrations can be dramatically elevated locally due to local injury, inflammation, or infection (72). Considering that SAA synthesis occurs in several extra hepatic sites, this may also contribute to high local concentrations of lipid-poor SAA at the sites of inflammation (72).

The findings of this study provide new insights into the possible biological role of the HDL receptor, CLA-1. Our results demonstrate that SAA is a ligand for the CLA-1 receptor and that CLA-1 is able to mediate SAA binding, uptake, and pro-inflammatory activity. These findings warrant additional work evaluating the potential significance of CLA-1 as a mediator of inflammatory and immune-related responses as well as the development of anti-inflammatory therapeutic agents targeting CLA-1.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| To whom correspondence should be addressed: Office of Biotechnology Activities, Office of Science Policy, Office of the Director, National Institutes of Health, 6705 Rockledge Dr., St. 750, Rockville, MD 20892-7985. Tel.: 301-496-9838; Fax: 301-496-9839; E-mail: pattersa{at}od.nih.gov (to A. P. P.) and abocharov{at}mail.cc.nih.gov (to A. V. B.).

1 The 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. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Christian A. Combs, Director of the NHLBI, National Institutes of Health Light Microscopy facility, for the great expertise and generous help with confocal microscopy analyses.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Kumon, Y., Hosokawa, T., Suehiro, T., Ikeda, Y., and Sipe, J. D., and Hashimoto, K. (2002) Amyloid 9, 237-241[Medline] [Order article via Infotrieve]
  2. Malle, E., Steinmetz, A., and Raynes, J. G. (1993) Atherosclerosis 102, 131-146[CrossRef][Medline] [Order article via Infotrieve]
  3. Patel, H., Fellowes, R., Coade, S., and Woo, P. (1998) Scand. J. Immunol. 48, 410-418[CrossRef][Medline] [Order article via Infotrieve]
  4. Urieli-Shoval, S., Linke, R. P., and Matzner, Y. (2000) Curr. Opin. Hematol. 7, 64-69[CrossRef][Medline] [Order article via Infotrieve]
  5. Gabay, C., and Kushner, I. (1999) N. Engl. J. Med. 340, 448-454[Free Full Text]
  6. Yamada, T., Kakihara, T., Kamishima, T., Fukuda, T., and Kawai, T. (1996) Pathol. Int. 46, 797-800[Medline] [Order article via Infotrieve]
  7. Kumon, Y., Sipe, J. D., Brinckerhoff, C. E., and Schreiber, B. M. (1997) Scand. J. Immunol. 46, 284-291[CrossRef][Medline] [Order article via Infotrieve]
  8. Urieli-Shoval, S., Cohen, P., Eisenberg, S., and Matzner, Y. J. (1998) Histochem. Cytochem. 46, 1377-1384[Abstract/Free Full Text]
  9. Meek, R. L., Urieli-Shoval, S., and Benditt, E. P. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 3186-3190[Abstract/Free Full Text]
  10. Liang, J. S., Sloane, J. A., Wells, J. M., Abraham, C. R., Fine, R. E., and Sipe, J. D. (1997) Neurosci. Lett. 225, 73-76[CrossRef][Medline] [Order article via Infotrieve]
  11. Kumon, Y., Suehiro, T., Hashimoto, K., Nakatani, K., and Sipe, J. D. (1999) J. Rheumatol. 26, 785-790[Medline] [Order article via Infotrieve]
  12. Pepys, M. B., and Baltz, M. L. (1983) Adv. Immunol. 34, 141-212[Medline] [Order article via Infotrieve]
  13. Husebekk, A., Skogen, B., Husby, G., and Marhaug, G. (1985) Scand. J. Immunol. 21, 283-287[CrossRef][Medline] [Order article via Infotrieve]
  14. Uhlar, C. M., and Whitehead, A. S. (1999) Eur. J. Biochem. 265, 501-523[Medline] [Order article via Infotrieve]
  15. Tape, C., Tan, R., Nesheim, M., and Kisilevsky, R. (1988) Scand. J. Immunol. 28, 317-324[CrossRef][Medline] [Order article via Infotrieve]
  16. Banka, C. L., Yuan, T., de Beer, M. C., Kindy, M., Curtiss, L. K., and de Beer, F. C. (1995) J. Lipid Res. 36, 1058-1065[Abstract]
  17. Cabana, V. G., Siegel, J. N., and Sabesin, S. M. (1989) J. Lipid Res. 30, 39-49[Abstract]
  18. Salazar, A., Mana, J., Fiol, C., Hurtado, I., Argimon, J. M., Pujol, R., and Pinto, X. (2000) Atherosclerosis 152, 497-502[CrossRef][Medline] [Order article via Infotrieve]
  19. Coetzee, G. A., Strachan, A. F., van der Westhuyzen, D. R., Hoppe, H., Jeenah, M. S., and de Beer, F. C. (1986) J. Biol. Chem. 261, 9644-9651[Abstract/Free Full Text]
  20. Kisilevsky, R., and Subrahmanyan, L. (1992) Lab. Invest. 66, 778-785[Medline] [Order article via Infotrieve]
  21. Steinmetz, A., Hocke, G., Saile, R., Puchois, P., and Fruchart, J. C. (1989) Biochim. Biophys. Acta 1006, 173-178[Medline] [Order article via Infotrieve]
  22. Liang, J. S., and Sipe, J. D. (1995) J. Lipid Res. 36, 37-46[Abstract]
  23. Tam, S. P., Flexman, A., Hulme, J., and Kisilevsky, R. (2002) J. Lipid Res. 43, 1410-1420[Abstract/Free Full Text]
  24. Badolato, R., Wang, J. M., Murphy, W. J., Lloyd, A. R., Michiel, D. F., Bausserman, L. L., Kelvin, D. J., and Oppenheim, J. J. (1994) J. Exp. Med. 180, 203-209[Abstract/Free Full Text]
  25. Xu, L., Badolato, R., Murphy, W. J., Longo, D. L., Anver, M., Hale, S., Oppenheim, J. J., and Wang, J. M. (1995) J. Immunol. 155, 1184-1190[Abstract]
  26. Olsson, N., Siegbahn, A., and Nilsson, G. (1999) Biochem. Biophys. Res. Commun. 254, 143-146[CrossRef][Medline] [Order article via Infotrieve]
  27. Furlaneto, C. J., and Campa, A. (2000) Biochem. Biophys. Res. Commun. 268, 405-408[CrossRef][Medline] [Order article via Infotrieve]
  28. Su, S. B., Gong, W., Gao, J. L., Shen, W., Murphy, P. M., Oppenheim, J. J., and Wang, J. M. (1999) J. Exp. Med. 189, 395-402[Abstract/Free Full Text]
  29. He, R., Sang, H., and Ye, R. D. (2003)) Blood 101, 1572-1581[Abstract/Free Full Text]
  30. Landschulz, K. T., Pathak, R. K., Rigotti, A., Krieger, M., and Hobbs, H. H. (1996) J. Clin. Invest. 98, 984-995[Medline] [Order article via Infotrieve]
  31. Ji, Y., Jian, B., Wang, N., Sun, Y., de la Llera Moya, M., Philips, M. C., Rothblat, G. H., Swaney, J. B., and Tall, A. R. (1997) J. Biol. Chem. 272, 20982-20985[Abstract/Free Full Text]
  32. Pearson, A. M. (1996)) Curr. Opin. Immunol. 8, 20-28[CrossRef][Medline] [Order article via Infotrieve]
  33. Acton, S. L., Scherer, P. E., Lodish, H. F., and Krieger, M. (1994) J. Biol. Chem. 269, 21003-21009[Abstract/Free Full Text]
  34. Krieger, M. (1999) Annu. Rev. Biochem. 68, 523-558[CrossRef][Medline] [Order article via Infotrieve]
  35. Murao, K., Terpstra, V., Green, S. R., Kondratenko, N., Steinberg, D., and Quehenberger, O. (1997) J. Biol. Chem. 272, 17551-17557[Abstract/Free Full Text]
  36. Svensson, P. A., Johnson, M. S., Ling, C., Carlsson, L. M., Billig, H., and Carlsson, B. (1999) Endocrinology 140, 2494-2500[Abstract/Free Full Text]
  37. Vishnyakova, T. G., Bocharov, A. V., Baranova, I. N., Chen, Z., Remaley, A. T., Csako, G, Eggerman, T. L., and Patterson, A. P. (2003) J. Biol. Chem. 278, 22771-22780[Abstract/Free Full Text]
  38. Williams, D. L., de La Llera-Moya, M., Thuahnai, S. T., Lund-Katz, S., Connelly, M. A., Azhar, S., Anantharamaiah, G., M., and Phillips, M. C. (2000) J. Biol. Chem. 275, 18897-18904[Abstract/Free Full Text]
  39. Bocharov, A. V., Baranova, I. N., Vishnyakova, T. G., Remaley, A. T., Csako, G., Thomas, F., Patterson, A., P., and Eggerman, T. L. (2004) J. Biol. Chem. 279, 36072-36082[Abstract/Free Full Text]
  40. Yuhanna, 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., and Shaul, P. W. (2001) Nat. Med. 7, 853-857[CrossRef][Medline] [Order article via Infotrieve]
  41. Li, X. A., Titlow, W. B., Jackson, B. A., Giltiay, N., Nikolova-Karakashian, M., Uittenbogaard, A., and Smart, E. J. (2002) J. Biol. Chem. 277, 11058-11063[Abstract/Free Full Text]
  42. Grewal, T., de Diego, I., Kirchhoff, M. F., Tebar, F., Heeren, J., Rinninger, F., and Enrich, C. (2003) J. Biol. Chem. 278, 16478-16481[Abstract/Free Full Text]
  43. Ikemoto, M., Arai, H., Feng, D., Tanaka, K., Aok, I. J., Dohmae, N., Takio, K., Adachi, H., Tsujimoto, M., and Inoue, K. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 6538-6543[Abstract/Free Full Text]
  44. Pawson, T., and Scott, J. D. (1997) Science 278, 2075-2080[Abstract/Free Full Text]
  45. Merrifield, R. B. (1969) JAMA 210, 1247-1254[Abstract/Free Full Text]
  46. Fairwell, T., Hospattankar, A. V., Brewer, H. B., Jr., and Khan, S. A. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 4796-4800[Abstract/Free Full Text]
  47. Buechler, C., Ritter, M., Quoc, C. D., Agildere, A., and Schmitz, G. (1999) Biochem. Biophys. Res. Commun. 262, 251-254[CrossRef][Medline] [Order article via Infotrieve]
  48. Calvo, D., Gomez-Coronado, D., Suarez, Y., Lasuncion, M. A., and Vega, M. A. (1998) J. Lipid Res. 39, 777-788[Abstract/Free Full Text]
  49. Takahashi, M., Yokota, T., Kawano, H., Gondo, T., Ishihara, T., and Uchino, F. (1989) Virch. Arch. A Pathol. Anat. Histopathol. 415, 411-419
  50. Kluve-Beckerman, B., Manaloor, J. J., and Liepnieks, J. J. (2002) Arthritis Rheum. 46, 1905-1913[CrossRef][Medline] [Order article via Infotrieve]
  51. Remaley, 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., Santamarina-Fojo, S., and Brewer, H. B. (2003) J. Lipid Res. 44, 828-836[Abstract/Free Full Text]
  52. Baranova, I., Vishnyakova, T., Bocharov, A., Chen, Z., Remaley, A. T., Stonik, J., Eggerman, T. L., and Patterson, A. P. (2002) Infect. Immun. 70, 2995-3003[Abstract/Free Full Text]
  53. Schmitz, G., Kaminski, W. E., Porsch-Ozcurumez, M., Klucken, J., Orso, E., Bodzioch, M., Buchler, C., and Drobnik, W. (1999) Pathobiology 67, 236-240[CrossRef][Medline] [Order article via Infotrieve]
  54. Yavin, E. J., Preciado-Patt, L., Rosen, O., Yaron, M., Suessmuth, R. D., Levartowsky, D., Jung, G., Lider, O., and Fridkin, M. (2000) FEBS Lett. 472, 259-262[CrossRef][Medline] [Order article via Infotrieve]
  55. Hill, C. S., and Treisman, R. (1995) Cell 80, 199-211[CrossRef][Medline] [Order article via Infotrieve]
  56. Marshall, C. J. (1995) Cell 80, 179-185[CrossRef][Medline] [Order article via Infotrieve]
  57. Kyriakis, J. M., and Avruch, J. (2001) Physiol. Rev. 81, 807-869[Abstract/Free Full Text]
  58. Tibbles, L. A., and Woodgett, J. R. (1999) Cell. Mol. Life Sci. 55, 1230-1254[CrossRef][Medline] [Order article via Infotrieve]
  59. Liang, T. S., Wang, J. M., Murphy, P. M., and Gao, J. L. (2000) Biochem. Biophys. Res. Commun. 270, 331-335[CrossRef][Medline] [Order article via Infotrieve]
  60. Akhtar, M., Watson, J. L., Nazli, A., and McKay, D. M. (2003) FASEB J. 17, 1319-1321[Abstract/Free Full Text]
  61. Bhattacharyya, A., Pathak, S., Datta, S., Chattopadhyay, S., Basu, J., and Kundu, M. (2002) Biochem. J. 368, 121-129[CrossRef][Medline] [Order article via Infotrieve]
  62. Babitt, J., Trigatti, B., Rigotti, A., Smart, E. J., Anderson, R. G., Xu, S., and Krieger, M. (1997) J. Biol. Chem. 272, 13242-13249[Abstract/Free Full Text]
  63. Schnitzer, J. E., Oh, P., and McIntosh, D. P. (1996) Science 274, 239-242[Abstract/Free Full Text]
  64. Anderson, R. G. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 10909-10913[Abstract/Free Full Text]
  65. Schafer, C., Ross, S. E., Bragado, M. J., Groblewski, G. E., Ernst, S. A., and Williams, J. A. (1998) J. Biol. Chem. 273, 24173-24180[Abstract/Free Full Text]
  66. Wang, Q., and Doerschuk, C. M. (2001) J. Immunol. 166, 6877-6884[Abstract/Free Full Text]
  67. Hoffman, J. S., and Benditt, E. P. (1982) J. Biol. Chem. 257, 10510-10517[Abstract/Free Full Text]
  68. Cabana, V. G., Feng, N., Reardon, C. A., Lukens, J., Webb, N. R., de Beer, F. C., and Getz, G. S. (2004) J. Lipid Res. 45, 317-325[Abstract/Free Full Text]
  69. Cabana, V. G., Gidding, S. S., Getz, G. S., Chapman, J., and Shulman, S. T. (1997) Pediatr. Res. 42, 651-655[Medline] [Order article via Infotrieve]
  70. Sloop, C. H., Dory, L., Krause, B. R., Castle, C., and Roheim, P. S. (1983) Atherosclerosis 49, 9-21[CrossRef][Medline] [Order article via Infotrieve]
  71. Nanjee, M. N., Cooke, C. J., Olszewski, W. L., and Miller, N. E. (2000) J. Lipid Res. 41, 1317-1327[Abstract/Free Full Text]
  72. Meek, R. L., Eriksen, N., and Benditt, E. P. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 7949-7952[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Pharmacol. Rev.Home page
R. D. Ye, F. Boulay, J. M. Wang, C. Dahlgren, C. Gerard, M. Parmentier, C. N. Serhan, and P. M. Murphy
International Union of Basic and Clinical Pharmacology. LXXIII. Nomenclature for the Formyl Peptide Receptor (FPR) Family
Pharmacol. Rev., June 1, 2009; 61(2): 119 - 161.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
R. L. He, J. Zhou, C. Z. Hanson, J. Chen, N. Cheng, and R. D. Ye
Serum amyloid A induces G-CSF expression and neutrophilia via Toll-like receptor 2
Blood, January 8, 2009; 113(2): 429 - 437.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
X. Wang, H. Chai, Z. Wang, P. H. Lin, Q. Yao, and C. Chen
Serum amyloid A induces endothelial dysfunction in porcine coronary arteries and human coronary artery endothelial cells
Am J Physiol Heart Circ Physiol, December 1, 2008; 295(6): H2399 - H2408.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
N. Cheng, R. He, J. Tian, P. P. Ye, and R. D. Ye
Cutting Edge: TLR2 Is a Functional Receptor for Acute-Phase Serum Amyloid A
J. Immunol., July 1, 2008; 181(1): 22 - 26.
[Abstract] [Full Text] [PDF]

Home page
 IOVSHome page
W.-H. Wang, L. G. McNatt, I.-H. Pang, P. E. Hellberg, J. H. Fingert, M. D. McCartney, and A. F. Clark
Increased Expression of Serum Amyloid A in Glaucoma and Its Effect on Intraocular Pressure
Invest. Ophthalmol. Vis. Sci., May 1, 2008; 49(5): 1916 - 1923.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
S. Sandri, D. Rodriguez, E. Gomes, H. P. Monteiro, M. Russo, and A. Campa
Is serum amyloid A an endogenous TLR4 agonist?
J. Leukoc. Biol., May 1, 2008; 83(5): 1174 - 1180.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
L. Bjorkman, J. Karlsson, A. Karlsson, M.-J. Rabiet, F. Boulay, H. Fu, J. Bylund, and C. Dahlgren
Serum amyloid A mediates human neutrophil production of reactive oxygen species through a receptor independent of formyl peptide receptor like-1
J. Leukoc. Biol., February 1, 2008; 83(2): 245 - 253.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
K. Christenson, L. Bjorkman, C. Tangemo, and J. Bylund
Serum amyloid A inhibits apoptosis of human neutrophils via a P2X7-sensitive pathway independent of formyl peptide receptor-like 1
J. Leukoc. Biol., January 1, 2008; 83(1): 139 - 148.
[Abstract] [Full Text] [PDF]

Home page
 Arterioscler. Thromb. Vasc. Bio.Home page
Y. Zhao, S. Zhou, and C.-K. Heng
Impact of Serum Amyloid A on Tissue Factor and Tissue Factor Pathway Inhibitor Expression and Activity in Endothelial Cells
Arterioscler. Thromb. Vasc. Biol., July 1, 2007; 27(7): 1645 - 1650.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
Z. Cai, L. Cai, J. Jiang, K.-S. Chang, D. R. van der Westhuyzen, and G. Luo
Human Serum Amyloid A Protein Inhibits Hepatitis C Virus Entry into Cells
J. Virol., June 1, 2007; 81(11): 6128 - 6133.
[Abstract] [Full Text] [PDF]

Home page
 J. Lipid Res.Home page
Y. Zhang, A. M. Ahmed, N. McFarlane, C. Capone, D. R. Boreham, R. Truant, S. A. Igdoura, and B. L. Trigatti
Regulation of SR-BI-mediated selective lipid uptake in Chinese hamster ovary-derived cells by protein kinase signaling pathways
J. Lipid Res., February 1, 2007; 48(2): 405 - 416.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
H. Cai, C. Song, I. Endoh, J. Goyette, W. Jessup, S. B. Freedman, H. P. McNeil, and C. L. Geczy
Serum Amyloid A Induces Monocyte Tissue Factor
J. Immunol., February 1, 2007; 178(3): 1852 - 1860.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
T. G. Vishnyakova, R. Kurlander, A. V. Bocharov, I. N. Baranova, Z. Chen, M. S. Abu-Asab, M. Tsokos, D. Malide, F. Basso, A. Remaley, et al.
CLA-1 and its splicing variant CLA-2 mediate bacterial adhesion and cytosolic bacterial invasion in mammalian cells
PNAS, November 7, 2006; 103(45): 16888 - 16893.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
M.-S. Lee, S.-A. Yoo, C.-S. Cho, P.-G. Suh, W.-U. Kim, and S. H. Ryu
Serum Amyloid A Binding to Formyl Peptide Receptor-Like 1 Induces Synovial Hyperplasia and Angiogenesis
J. Immunol., October 15, 2006; 177(8): 5585 - 5594.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
R. He, L. W. Shepard, J. Chen, Z. K. Pan, and R. D. Ye
Serum Amyloid A Is an Endogenous Ligand That Differentially Induces IL-12 and IL-23
J. Immunol., September 15, 2006; 177(6): 4072 - 4079.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
C. Shah, R. Hari-Dass, and J. G. Raynes
Serum amyloid A is an innate immune opsonin for Gram-negative bacteria
Blood, September 1, 2006; 108(5): 1751 - 1757.
[Abstract] [Full Text] [PDF]

Home page
 J. Lipid Res.Home page
S. Abe-Dohmae, K. H. Kato, Y. Kumon, W. Hu, H. Ishigami, N. Iwamoto, M. Okazaki, C.-A. Wu, M. Tsujita, K. Ueda, et al.
Serum amyloid A generates high density lipoprotein with cellular lipid in an ABCA1- or ABCA7-dependent manner
J. Lipid Res., July 1, 2006; 47(7): 1542 - 1550.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
D. R. van der Westhuyzen, L. Cai, M. C. de Beer, and F. C. de Beer
Serum Amyloid A Promotes Cholesterol Efflux Mediated by Scavenger Receptor B-I
J. Biol. Chem., October 28, 2005; 280(43): 35890 - 35895.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
R. Hari-Dass, C. Shah, D. J. Meyer, and J. G. Raynes
Serum Amyloid A Protein Binds to Outer Membrane Protein A of Gram-negative Bacteria
J. Biol. Chem., May 13, 2005; 280(19): 18562 - 18567.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/9/8031    most recent
M405009200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Baranova, I. N.
Right arrow Articles by Patterson, A. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Baranova, I. N.
Right arrow Articles by Patterson, A. P.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2005 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement