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RECEPTORS AND SIGNAL TRANSDUCTION

Class A scavenger receptor-mediated cell adhesion requires the sequential activation of Lyn and PI3-kinase

¹Department of Molecular and Biomedical Pharmacology, ²Graduate Center for Nutritional Sciences, and ³Department of Physiology, The University of Kentucky, Lexington, Kentucky

Submitted 24 July 2006 ; accepted in final form 22 December 2006

	ABSTRACT

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Class A scavenger receptors (SR-A) participate in multiple macrophage functions including macrophage adhesion to modified proteins. SR-A-mediated adhesion may therefore contribute to chronic inflammation by promoting macrophage accumulation at sites of protein modification. The mechanisms that couple SR-A binding to modified proteins with increased cell adhesion have not been defined. In this study, SR-A expressing HEK cells and SR-A+/+ or SR-A–/– macrophages were used to delineate the signaling pathways required for SR-A-mediated adhesion to modified protein. Inhibiting G_i/o activation, which decreases initial SR-A-mediated cell attachment, did not prevent the subsequent spreading of attached cells. In contrast, inhibition of Src kinases or PI3-kinase abolished SR-A-dependent cell spreading without affecting SR-A-mediated cell attachment. Consistent with these results, the Src kinase Lyn and PI3-kinase were sequentially activated during SR-A-mediated cell spreading. Furthermore, activation of both Lyn and PI3-kinase was required for enhancing paxillin phosphorylation. Activation of a Src kinase-PI3-kinase-Akt pathway was also observed in cells expressing a truncated SR-A protein that does not internalize indicating that SR-A-mediated activation of intracellular signaling cascades following adhesion to MDA-BSA is independent of receptor internalization. Thus SR-A binding to modified protein activates signaling cascades that have distinct roles in regulating initial cell attachment and subsequent cell spreading.

macrophage; inflammation; intracellular signaling

Receptor-mediated cell adhesion, which progresses from initial attachment of cells to subsequent cell spreading, requires coupling of cell surface adhesion molecules to the actin cytoskeleton. The best-studied adhesion molecules are the integrin proteins. Integrins are ubiquitously expressed multimeric proteins that mediate cell adhesion through interaction with the extracellular matrix. Integrin-mediated attachment and subsequent firm adhesion of macrophages are a dynamic process involving the activation of intracellular signaling cascades that regulate the formation of focal adhesion complexes, cytoskeletal rearrangements, and extension of membrane projections (reviewed in Refs. 2, 7, 27, 32). SR-A-dependent cell adhesion induces changes in cell morphology similar to those observed following integrin engagement (24). However, the signaling pathways required for SR-A-mediated adhesion have not been defined.

In addition to SR-A, macrophages express other receptors that bind modified proteins. Therefore, in the current study the signals involved in SR-A-mediated cell adhesion (attachment and spreading) were examined in peritoneal macrophages isolated from wild-type SR-A+/+ or SR-A–/– mice and human embryonic kidney cells (HEK-293) stably transfected that lack SR-A expression (HEK^SR-A–) or inducibly express SR-A (HEK^SR-A+). Our results support a model in which SR-A-mediated cell adhesion progresses from initial cell attachment to firm adhesion, characterized by increased cell surface area, via the differential activation of intracellular signaling pathways.

	EXPERIMENTAL PROCEDURES

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Chemicals. DMEM with L-glutamine, DMEM with 25 mM HEPES without phenol red, penicillin, streptomycin, heat-inactivated FBS, and human plasma fibronectin were purchased from GIBCO BRL (Grand Island, NY). Ultra-low attachment polystyrene six-well plates and six-well tissue culture plates were purchased from Costar (Corning, NY). Pertussis toxin (PTX; Bordetella pertussis), PP2 {4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3, 4-d] pyrimidine}, and PP3 {4-amino-7-phenylpyrazolo [3, 4-d] pyrimidine} were purchased from Calbiochem (La Jolla, CA). LY294002 and wortmannin were purchased from Biomol Research Laboratories (Plymouth, PA). Fluorescently labeled phalloidin and DAPI nuclear acid stain (4',6'-diamidino-2-phenylindole, dihydrochloride) were purchased from Molecular Probes (Eugene, OR). Methyl cellulose and malondialdehyde bis (dimethyl acetal) were purchased from Sigma (St. Louis, MO). Goat polyclonal anti-malondialdehyde antibody was purchased from Academy Bio-Medical (Houston, TX). Rabbit polyclonal anti-Lyn antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal antibodies against Akt, phospho-specific Ser⁴⁷³-Akt, phospho-GSK3, and the mouse monoclonal anti-phospho-Tyr antibody were purchased from Cell Signaling Technology (Beverly, MA). Mouse anti-paxillin antibody was from BD Transduction Laboratories.

Malondialdehyde modification of BSA. Malondialdehyde-modified proteins (e.g., MDA-BSA) have been used previously to study SR-A function (13, 14). Malondialdehyde removes positive charges from the -amino groups of proteins and thereby is able to convert a weakly anionic protein into a strongly anionic one. It is thought that this enhanced negative charge of MDA-modified proteins is responsible for their binding to SR-A. MDA-BSA was prepared using a previously described method (14). Briefly, hydrochloric acid (0.2 ml; 12 M) was added to malondialdehyde bis (dimethyl acetal; 0.165 ml) and incubated for 5 min while stirring on ice. Then, sodium phosphate buffer (4.8 ml; 0.1 M; pH 6.4) was added and the pH was adjusted to 6.4 with NaOH. An equal volume of BSA (10 mg/ml in PBS; pH 7.4) was added, and the reaction was incubated at 37°C for 3 h. The solution was then dialyzed (MW cut off: 12,000–14,000) against PBS and the protein concentration was determined using the Bio-Rad DC protein assay (Hercules, CA). Protein modification was confirmed by immunoblotting with anti-MDA-specific antibody (data not shown).

Cell isolation and culture. MPM were harvested from either SR-A–/– (kindly provided by Dr. M. Freeman, Massachusetts General Hospital, Harvard Medical School) or wild-type C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME) via peritoneal lavage with ice-cold sterile saline and cultured in DMEM containing FBS (10% vol/vol), penicillin, and streptomycin as previously described (30). Animal care and use for all procedures were done according to protocols reviewed and approved by the Institutional Animal Care and Use Committee at the University of Kentucky.

HEK-293 cells expressing the murine SR-A under inducible control of the ecdysone receptor were maintained in DMEM as previously described (24). We also used HEK-293 cells expressing either wild-type or truncated SR-A_1–49, which lacks the distal 49 amino acids of the 55-amino acid cytoplasmic tail, under the control of tetracycline inducible promoter (18). In the absence of inducing agent, these transfected HEK cells do not express SR-A (HEK^SR-A–). The addition of inducing agent (muristerone 4 µM; or tetracycline 0.5 µg/ml) for 16 h induces SR-A expression (HEK^SR-A+). For growth in suspension, HEK cells were detached and resuspended in DMEM supplemented with 10% FBS and cultured overnight in ultra-low adherent six-well plates. To minimize the aggregation of suspended cells, methylcellulose (0.8% wt/vol) was included in the culture medium and cells were filtered through cell strainers (40-µm nylon) before use in adhesion assays. Trypan blue exclusion was assessed to confirm that none of the treatments altered cell viability.

Cell adhesion assays. To assess cell attachment, MPM or transfected HEK cells (10⁶ cells/ml) were pretreated as indicated with inhibitors and then plated into tissue culture plates that were precoated with either fibronectin (65 µg/ml) or MDA-BSA (400 µg/ml) overnight at 4°C. Cells were allowed to attach for 10 min (HEK) or 30 min (macrophages) at 37°C; conditions that were optimized in preliminary experiments to assess SR-A-dependent attachment. Following incubation, nonattached cells were removed by washing, adherent cells were detached with trypsin, and cell number was determined using hemacytometer as described previously (24).

To assess cell spreading, MPM or transfected HEK cells were plated (30,000 cells/well) into four-chambered LAB-TEK slides (Nalge Nunc International, Naperville, IL) precoated with either MDA-BSA or fibronectin. Following treatments, cells were gently washed with warm PBS and fixed with 4% paraformaldehyde. Fixed cells were permeabilized with 0.1% Triton X-100 and then blocked with 1% BSA for 30 min. Polymerized actin (F actin) was stained with Alexa-Fluor⁵⁶⁸-conjugated phalloidin, and nuclei were stained with DAPI. Cells were mounted in the embedding medium Mowiol containing 1% n-propyl gallate and dried overnight at 4°C. Images were digitally captured using a Leica TCS SP confocal microscope. Cell surface area was quantified using Metamorph software. Images of at least 30 cells from at least three independent experiments were used for quantification.

Detection of protein phosphorylation. Following treatment, cells were washed with ice-cold PBS and lysed in RIPA buffer containing phosphatase and protease inhibitors for 30 min on ice. Cell lysates were centrifuged for 15 min (4°C; 13,000 g), and the pellet was discarded. Cell lysate protein was denatured by boiling for 5 min in reducing sample buffer, and proteins were resolved by 12% Bis-Tris gel SDS-PAGE. Proteins were electrophoretically transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA), blocked with 5% nonfat milk in TBST, and incubated overnight at 4°C with the indicated primary antibodies. Proteins were visualized by enhanced chemiluminescence using species-specific horseradish peroxidase-coupled secondary antibodies. Images were digitally captured and quantified using a Kodak Image Station 2000R.

To detect Lyn and paxillin phosphorylation, cell lysates were incubated overnight at 4°C with agarose-coupled Lyn antibody or paxillin antibody followed by protein A/G-coupled sepharose beads and washed with ice-cold RIPA buffer. Immunoprecipitated proteins were dissolved in SDS-loading buffer and processed as described above. The phosphorylated forms of Lyn and paxillin were detected by immunoblotting with an anti-phospho-Tyr antibody.

Phosphorylation of Akt and GSK3 was used to assess activation of the PI3-kinase pathway. To detect Akt phosphorylation, total cell lysates were immunoblotted with a rabbit polyclonal phospho-specific Ser⁴⁷³-Akt antibody. The same blots were reprobed with total Akt antibody and the amount of phosphorylated Akt was normalized to the total amount of Akt in each sample. Similarly, GSK3 phosphorylation in cell lysates was quantified by immunoblotting with a specific phospho-GSK3 antibody.

Statistical analysis. Data were analyzed using GraphPad Prism. Experiments were repeated at least three times, and significance between treatment groups was determined by one-way ANOVA. Values with P < 0.05 were considered to be statistically significant.

	RESULTS

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To study the regulation of SR-A-dependent adhesion, the ability of cells that express SR-A to adhere to tissue culture plates coated with the SR-A ligand MDA-BSA was compared with cells that do not express SR-A. For this, we first used stably transfected HEK cells that inducibly express SR-A. As shown in Fig. 1A, we found that only 5.4 ± 3.0% of HEK^SR-A– cells attached to MDA-BSA-coated plates following a 10-min incubation, compared with 64.0 ± 4.5% of HEK^SR-A+ cells (P < 0.05). Moreover, the enhanced attachment of HEK^SR-A+ cells to MDA-BSA was inhibited by the SR-A antagonist polyinosine but was not affected by EDTA, which chelates divalent cations required for integrin-mediated cell attachment. In contrast, integrin-mediated cell attachment to fibronectin was not affected by inducing SR-A expression or by adding SR-A antagonist but was abolished by EDTA (Fig. 1B). Next, the ability of SR-A to mediate cell attachment in macrophages that endogenously express the receptor was examined. As shown in Fig. 1C, the initial attachment of peritoneal macrophages isolated from SR-A+/+ mice to MDA-BSA-coated tissue culture plates for 30 min was significantly greater than macrophages isolated from SR-A–/– mice (50.2 ± 4.0 vs. 31.4 ± 4.2% of macrophages plated, P < 0.05). The addition of polyinosine reduced the attachment of SR-A+/+ macrophages to the level observed in SR-A–/– macrophages without significantly affecting the attachment of SR-A–/– macrophages. In contrast, integrin-mediated macrophage attachment to fibronectin was not affected by the absence of SR-A or the addition of SR-A antagonist but was abolished by EDTA (data not shown). Thus these results demonstrate that SR-A expression is required for the increased attachment of HEK^SR-A+ and SR-A+/+ macrophages to MDA-BSA-coated dishes.

View larger version (11K): [in this window] [in a new window]   Fig. 1. Class A scavenger receptors (SR-A) enhance the attachment of induced human embryonic kidney (HEK)SR-A cells and mouse peritoneal macrophages (MPM) to immobilized MDA-BSA. As indicated, HEKSR-A– cells, HEKSR-A+ cells, or MPM isolated from wild-type SR-A+/+ or SR-A–/– C57BL/6 mice were resuspended in either the absence or presence of EDTA (10 mM). As indicated, cells were pretreated with the SR-A antagonist polyinosine (10 µg/ml) for 5 min. Following pretreatment, macrophages were resuspended either in media without EDTA or in media containing EDTA (10 mM) and the number of cells that attached to MDA-BSA (A, C)- or fibronectin (B)-coated plates was determined. Results are expressed as the percentage of the total number of cells plated that remained attached and represent means ± SE of at least 3 separate experiments. *Significant difference (P < 0.05) from buffer control values.

View larger version (52K): [in this window] [in a new window]   Fig. 2. SR-A enhances the spreading of HEKSR-A cells and macrophages on MDA-BSA. As indicated, HEKSR-A– or HEKSR-A+ cells were cultured in suspension overnight and then plated into MDA-BSA-coated slides for either 10 min or 2 h at 37°C (A). B: MPM isolated from C57BL/6 wild-type (top) or SR-A–/– mice (bottom) were plated into MDA-BSA-coated slides and allowed to spread for the indicated times at 37°C. Following incubations, cells were fixed and cell morphology was visualized by staining with Alexa-Fluor568-conjugated phalloidin. Nuclei were stained with DAPI. Confocal images (x40) were digitally captured and are representative of at least 3 separate experiments. The scale bar represents 40 µm.

View larger version (36K): [in this window] [in a new window]   Fig. 3. SR-A-mediated cell spreading is independent of Gi/o protein activation. As indicated, HEKSR-A+ cells (A) or MPM isolated from wild-type C57BL/6 mice (B) were pretreated in suspension with pertussis toxin (PTX; 100 ng/ml) for 16 h. Cells were then plated for the indicated times on MDA-BSA-coated slides at 37°C. Following incubations, cell morphology was visualized by staining cells with Alexa-Fluor568-conjugated phalloidin and nuclei with DAPI. Confocal images (x40) were digitally captured and are representative of at least 3 separate experiments. The scale bar represents 40 µm for HEK cells and 50 µm for MPM.

View larger version (46K): [in this window] [in a new window]   Fig. 4. SR-A-mediated cell spreading requires activation of Src and PI-3-kinase. HEKSR-A– cells, HEKSR-A+ cells, or MPM isolated from SR-A+/+ or SR-A–/– mice were pretreated as indicated with wortmannin (200 nM) or PP2 (50 µM) for 30 min in suspension. To assess cell spreading (A), pretreated cells were plated on MDA-BSA-coated slides for 2 h at 37°C and cell morphology was visualized by staining cells with Alexa-Fluor568-conjugated phalloidin and nuclei with DAPI. Images (x100 for HEK cells; x40 for MPM) were digitally captured and are representative of at least 3 separate experiments. The scale bars represent 20 µm for HEK cells and 40 µm for MPM. To assess cell attachment (B), the number of pretreated cells that attached to MDA-BSA-coated plates was determined. Results are expressed as the percentage of the total number of cells plated that remained attached and represent means ± SE.

View larger version (19K): [in this window] [in a new window]   Fig. 5. SR-A-mediated adhesion activates the Src kinase Lyn. A: MPM isolated from wild-type C57BL/6 mice were pretreated as indicated with wortmannin (200 nM) or PP2 (50 µM) for 30 min in suspension. Following pretreatments, cells were either kept in suspension or plated into MDA-BSA-coated dishes for various times and cell lysates were prepared. Lyn activation was assessed by immunoprecipitating Lyn protein from cell lysates and detecting the active form of Lyn kinase by immunoblotting with an anti-phospho-Tyr-specific antibody. The amount of phosphorylated Lyn kinase was quantified and normalized to total amount of the Lyn kinase protein in each immunoprecipitate. A representative blot of Lyn phosphorylation and means ± SE of 3 separate experiments are shown. B: to determine whether Lyn kinase becomes specifically activated by SR-A-dependent adhesion, MPM isolated from wild-type or SR-A–/– mice were either kept in suspension or plated into MDA-BSA-coated dishes for 2 h. Cell lysates were prepared and activation of Lyn was determined as described above. A blot of Lyn phosphorylation is shown and is representative of 3 separate experiments.

View larger version (20K): [in this window] [in a new window]   Fig. 6. SR-A-mediated adhesion activates PI3-kinase. MPM isolated from wild-type C57BL/6 mice were either left untreated or pretreated as indicated with wortmannin (200 nM) or PP2 (50 µM) for 30 min in suspension. Following pretreatments, cells were either kept in suspension or plated into MDA-BSA-coated dishes for various times and cell lysates were prepared. A: to assess PI3-kinase activation, cell lysates were immunoblotted with an anti-phospho-Ser473-Akt antibody. The amount of phosphorylated Akt was quantified and normalized to total amount of the Akt in each sample. A representative blot of Akt phosphorylation and means ± range of 2 separate experiments are shown. B: similarly, GSK3 phosphorylation in cell lysates prepared from macrophages that were kept in suspension or adhered to MDA-BSA for 120 min was quantified by immunoblotting with a specific phospho-GSK3 antibody. A representative blot of GSK3 phosphorylation and means ± SE of 3 independent experiments are shown. *Significant difference (P < 0.05) from cells kept in suspension.

View larger version (13K): [in this window] [in a new window]   Fig. 7. SR-A-mediated adhesion enhances paxillin phosphorylation. MPM isolated from wild-type C57BL/6 mice were either left untreated or pretreated as indicated with wortmannin (200 nM) or PP2 (50 µM) for 30 min in suspension. Following pretreatments, cells were either kept in suspension or plated into MDA-BSA-coated dishes for 60 min and cell lysates were prepared. To detect paxillin phosphorylation, paxillin was immunoprecipitated from cell lysates and phosphorylation was assessed by immunoblotting with an anti-phospho-Tyr-specific antibody. A representative blot of paxillin phosphorylation and means ± SE of 3 separate experiments are shown. *Significant difference (P < 0.05) from cells kept in suspension.

1–49

1–49

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View larger version (19K): [in this window] [in a new window]   Fig. 8. Membrane proximal amino acids are sufficient for SR-A-mediated activation of adhesion signals. HEK cells were grown in suspension for 16 h and induced to express either wild-type or truncated SR-A1–49 by tetracycline. Cells were then pretreated with PP2 (50 µM) or wortmannin (200 nM) for 30 min and plated into MDA-BSA-coated dishes for 30 min. Cell lysates were prepared and Akt activation was determined by immunoblotting with an anti-phospho-Ser473-Akt antibody. The amount of phosphorylated Akt was quantified and normalized to total amount of the Akt in each sample. A representative blot of Akt phosphorylation and the means ± SE of 3 separate experiments are shown. *Significant difference (P < 0.05) from cells kept in suspension. #Significant difference (P < 0.05) from cells adhered in the absence of inhibitors.

	DISCUSSION

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To better understand the mechanism by which SR-A promotes macrophage adhesion, we examined the activation of signaling cascades required for SR-A-mediated macrophage adhesion. The regulation of SR-A function by intracellular signals is indicated by the previous findings that SR-A-mediated AcLDL uptake and initial cell attachment are reduced by inhibiting G_i/o activation (24, 30). In the current study, we found that the spreading of SR-A expressing cells is insensitive to G_i/o inhibition indicating that G_i/o activation enhances SR-A-dependent cell attachment but is not required for the subsequent cytoskeletal changes associated with cell spreading and firm adhesion. These results indicate that following initial SR-A-mediated cell attachment, activation of additional signals is required to mediate cell spreading.

In many cell types, membrane and cytoskeletal dynamics are regulated by the activation of PI3-kinase and Src kinases. We found that when macrophages adhered to SR-A ligand, both PI3-kinase and the Src kinase Lyn were activated and that inhibitors of Src kinases or PI3-kinase abolished SR-A-mediated macrophage spreading. Furthermore, the time course for Lyn and PI3-kinase activation coincided with that of macrophage spreading on MDA-BSA. However, SR-A-dependent Lyn activation occurred more rapidly than PI3-kinase activation suggesting that Lyn activation precedes PI3-kinase activation. The sequential activation of Lyn and PI3-kinase is supported by the findings that inhibiting Lyn abolished PI3-kinase activation, whereas inhibiting PI3-kinase did not affect Lyn activation.

Src family kinases play a predominant role in signaling triggered by integrin-mediated adhesion in macrophages (19, 21). Primary macrophages isolated from mice that do not express the Src kinases Hck and Fgr showed impaired macrophage spreading on fibronectin demonstrating an important role for these Src kinases in integrin-mediated cell adhesion (28). In contrast, the Src kinase Lyn has been previously associated with SR-A in THP-1 macrophages (20). Our results demonstrate that Lyn, but not other Src kinase isoforms, is activated during SR-A-mediated adhesion. Furthermore, paxillin phosphorylation induced by SR-A-mediated adhesion was inhibited by PP2 indicating that Lyn activation is required for SR-A-dependent PI3-kinase paxillin phosphorylation. Thus Lyn plays an essential role in coupling surface SR-A to the formation of focal adhesion complexes and the actin cytoskeleton.

In addition to Lyn, our data demonstrate an important role for PI3-kinase in SR-A-dependent paxillin phosphorylation and macrophage adhesion. The involvement of Src family kinases in regulating the PI3-kinase/Akt cascade has been previously demonstrated (4, 15, 19, 23). For example, when plated on fibronectin, macrophages isolated from Hck/Fgr/Lyn–/– mice displayed impaired PI3-kinase translocation to the cytoskeleton when compared with wild-type macrophages (19). This indicates that membrane localization of PI3-kinase may be critical in regulating cytoskeletal rearrangements and implies that the defective spreading of these macrophages on fibronectin might be due to impaired PI3-kinase function. Our results demonstrating that inhibiting Lyn blocks PI3-kinase activation in macrophages adhered to MDA-BSA indicate that Lyn may play a similar role in translocating PI3-kinase during SR-A-mediated cell adhesion. Our data further demonstrate that both Lyn and PI3-kinase activation are involved in paxillin phosphorylation. This is similar to previous results demonstrating that both Src and PI3-kinase were required for paxillin phosphorylation induced by LPS (31). The specific role of PI3-kinase in paxillin phosphorylation during SR-A-mediated adhesion is not clear but may involve the activation of additional kinases that are involved in the adhesion process.

Using a truncated receptor that does not internalize (SRA_1–49), we show that SR-A-mediated activation of intracellular signaling cascades following adhesion to MDA-BSA is independent of receptor internalization. Cell spreading mediated by both SR-A and SRA_1–49 induces the Src-kinase-dependent activation of PI3-kinase. Interestingly, Src inhibition does not affect SR-A-mediated AcLDL endocytosis (data not shown) providing additional evidence that the different SR-A functions, ligand uptake and cell adhesion, are regulated by distinct signaling pathways. Taken further, this would suggest that SR-A-mediated cell adhesion, but not ligand uptake, can be specifically inhibited to affect an inflammatory process. This is particularly intriguing given the recent association of SR-A-mediated ligand binding and internalization with a reduced inflammatory response (1, 5, 22).

Overall, our results support a model in which SR-A-mediated cell adhesion progresses from an initial cell attachment to subsequent cell spreading and that these processes are regulated by different signals. Following interaction with an immobilized ligand (e.g., MDA-BSA), G_i/o proteins are activated thereby enhancing SR-A-dependent cell attachment. Subsequently, additional signals are generated to acquire the spread morphology characteristic of macrophage firm adhesion. These signals include the sequential activation of the Src kinase Lyn and PI3-kinase. Activation of Lyn and PI3-kinase mediates paxillin phosphorylation, which in turn couples SR-A to the formation of focal adhesion complexes and the actin cytoskeleton.

SR-A-mediated macrophage adhesion may have important physiological and pathological roles, particularly at sites rich in modified extracellular matrix proteins. Modifications of the extracellular matrix that enhance interaction with SR-A can occur in chronic inflammation, atherosclerosis, smoking, and diabetes. Our results indicate that in addition to enhancing macrophage accumulation and retention at the sites of matrix modification, SR-A-mediated cell adhesion might contribute to the pathophysiology of inflammatory diseases by activating intracellular signaling pathways (e.g., Lyn-PI3-kinase-Akt-GSK3) that regulate macrophage function. Evaluation of the role of SR-A in vivo therefore requires consideration of not only its ability to mediate ligand internalization but also its ability to mediate macrophage adhesion (attachment and spreading) and the consequent activation of multiple intracellular signaling pathways.

	GRANTS

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This work was supported by National Institutes of Health Grants HL-65708 and HL-75241 and an Established Investigator Award from American Heart Association (AHA). D. Nikolic is a recipient of a predoctoral fellowship from AHA (Ohio Valley Affiliate).

	ACKNOWLEDGMENTS

 
The authors acknowledge members of the Cardiovascular Research Group at the University of Kentucky for helpful comments and suggestions.

	FOOTNOTES

 

Address for reprint requests and other correspondence: S. R. Post, Dept. of Molecular and Biomedical Pharmacology, UK Medical Center-MS305, Lexington, KY 40536-0298 (e-mail: spost{at}uky.edu)

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

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