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Originally published In Press as doi:10.1074/jbc.M611004200 on April 2, 2007

J. Biol. Chem., Vol. 282, Issue 21, 15606-15618, May 25, 2007
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The Lipoxin A4 Receptor Is Coupled to SHP-2 Activation

IMPLICATIONS FOR REGULATION OF RECEPTOR TYROSINE KINASES*Formula

Derick Mitchell{ddagger}1, Sarah J. O'Meara{ddagger}, Andrew Gaffney{ddagger}, John K. G. Crean§, B. Therese Kinsella§, and Catherine Godson{ddagger}2

From the {ddagger}School of Medicine and Medical Science and the §School of Biomolecular and Biomedical Science, Diabetes Research Centre, UCD Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland

Received for publication, November 29, 2006 , and in revised form, March 29, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Mesangial cell proliferation is pivotal to the pathology of glomerular injury in inflammation. We have previously reported that lipoxins, endogenously produced eicosanoids with anti-inflammatory and pro-resolution bioactions, can inhibit mesangial cell proliferation in response to several agents. This process is associated with elaborate receptor cross-talk involving modification receptor tyrosine kinase phosphorylation (McMahon, B., Mitchell, D., Shattock, R., Martin, F., Brady, H. R., and Godson, C. (2002) FASEB J. 16, 1817–1819). Here we demonstrate that the lipoxin A4 (LXA4) receptor is coupled to activation and recruitment of the SHP-2 (SH2 domain-containing tyrosine phosphatase-2) within a lipid raft microdomain. Using site-directed mutagenesis of the cytosolic domain of the platelet-derived growth factor receptor beta (PDGFRbeta), we report that mutation of the sites for phosphatidylinositol 3-kinase (Tyr740 and Tyr751) and SHP-2 (Tyr763 and Tyr1009) recruitment specifically inhibit the effect of LXA4 on the PDGFRbeta signaling; furthermore inhibition of SHP-2 expression with short interfering RNA constructs blocked the effect of LXA4 on PDGFRbeta phosphorylation. We demonstrate that association of the PDGFRbeta with lipid raft microdomains renders it susceptible to LXA4-mediated dephosphorylation by possible reactivation of oxidatively inactivated SHP-2. These data further elaborate on the potential mechanisms underlying the anti-inflammatory, proresolution, and anti-fibrotic bioactions of lipoxins.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
There is a growing appreciation that novel, endogenously produced lipid mediators can regulate inflammatory processes and their resolution (1). Lipoxins (LXs)3 are endogenously generated eicosanoids with potent anti-inflammatory properties (2). There is increasing evidence that LXs promote the resolution of inflammation and can elicit distinct anti-fibrotic responses within an inflammatory milieu (3–6). LXA4 exerts its actions via interaction with its cognate G protein-coupled receptor (GPCR), the LXA4 receptor (ALXR) (recently reviewed in Ref. 7). Aside from LXA4, a number of pleiotropic lipid and peptide agonists have been shown to activate the ALXR (8). The intracellular signaling pathways activated upon ALXR engagement in multiple cell types remain to be fully elucidated (2).

We have previously demonstrated that complex cross-talk exists between the ALXR and other GPCRs and receptor tyrosine kinases (RTKs) in primary human renal mesangial cells (2, 4, 9). In this context it is noteworthy that LX can inhibit MC proliferation in response to both growth factors and proinflammatory mediators such as the cysteinyl leukotriene D4 (9). MC proliferation is a prototypic inflammatory response coupled to matrix accumulation and further production of cytokines that may culminate in the onset of many subacute and chronic forms of glomerulonephritis and eventual fibrosis (10).

Activation of the PDGFRbeta and subsequent signaling events play a pivotal role in renal inflammation and fibrosis (11). In this regard modulation of PDGFRbeta activity is considered an appropriate therapeutic target (10, 12). PDGFRbeta activation leads to increased tyrosine phosphorylation and recruitment of Src homology 2 (SH2) domain-containing molecules to specific phosphotyrosine residues within the cytosolic domain of the receptor, initiating processes that promote cell proliferation and gene expression (13). We have previously reported that LXA4 treatment of MC modulates PDGF-BB-stimulated tyrosine phosphorylation of the PDGFRbeta and subsequent mitogenic events (9). Here we report that the ALXR may be coupled to reactivation of a protein-tyrosine phosphatase (PTP), SHP-2, that specifically dephosphorylates the recruitment sites of the p85 subunit of PI 3-kinase on the PDGFRbeta.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents, antibodies, plasmids/primers information, and cell culture techniques are described in the supplemental text.

Cell Transfection and Preparation of Lysates—Human embryonic kidney 293 (HEK293) cells and HEK293 cells stably overexpressing the HA epitope-tagged ALXR (designated ALXR cells) were transiently transfected with various plasmids using Genejuice® transfection reagent as per the manufacturer's protocol. Briefly, the cells were plated to 70% confluency, allowed to adhere for 24 h, and subsequently transfected with 1 µg of plasmid. 24 h post-transfection, the cells were serum-restricted in 0.01% FCS medium for 24 h prior to stimulation. For short interfering RNA (siRNA)-mediated knockdown of SHP-2 levels, ALXR cells were co-transfected with targeted SHP-2 PTP siRNA (sequence 5'-CCTCTGAAAGGTGGTTTCATGGACATCTCTCTGGGAAAGAAGCAGAGAAAT-3', residues 703–753 of the SHP-2 sequence) (Qiagen) using RNAfect® transfection reagent as per the manufacturer's protocol. As a control, a nonspecific siRNA duplex with a random nucleotide sequence) (Qiagen) was used. Two additional siRNA sequences complementary to the SHP-2 gene were designed using custom SMARTPools from Dharmacon (Lafayette, CO) (Dharmacon catalogue numbers J-003947-09 and J-003947-10) and were transfected using Lipofectamine (Invitrogen) as per the manufacturer's protocol.

Immunoprecipitation and Immunoblotting—MCs were serum-restricted in 0.2% FCS MCDB131 for 48 h, whereas HEK293 and ALXR cells were serum-restricted in 0.01% FCS modified Eagle's medium for 24 h prior to exposure to various agents. The lysates were harvested in RIPA lysis buffer (20 mM Tris-HCl, pH 7.4, 50 mM NaCl, 5 mM ethylene diaminetetraacetic acid, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 5 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4,1 µM leupeptin, 0.3 µM aprotinin). The lysates were clarified by centrifugation at 14,000 rpm for 20 min, and the samples were normalized for total protein. Thereafter, the lysates were precleared with Protein G-Plus Agarose beads for 1 h at 4 °C. For MC lysates, PDGFRbeta or epidermal growth factor receptor (EGFR) was immunopurified from 250 µg of precleared lysate with anti-PDGFRbeta or anti-EGFR (both 1:100) overnight with constant rocking at 4 °C. For HEK293 or ALXR cell lysates, PDGFRbeta (1:100), HA-tag (1:300), SHP-1, or SHP-2 (1:100) were immunopurified from 50–70 µg of precleared lysate overnight at 4 °C with constant rocking. Mouse IgG (1:100) was used as an antibody control. 10 µl of protein G-agarose beads were added to the protein antibody mixture, and the samples were rocked for a further 2 h at 4°C. Precipitated immunocomplexes were washed three times in fresh lysis buffer and boiled in sample buffer. The samples were subsequently resolved by SDS-PAGE, transferred onto polyvinylidene difluoride membrane, and probed for phosphotyrosine antigenicity (1:1000), total PDGFRbeta/EGFR (1:1000), SHP-2 (1:2500), or HA tag ALXR (1:2000).

RTK Activity Assay—MCs were serum-restricted and exposed to various agents for 0–60 min. The cells were lysed, and PDGFRbeta was immunoprecipitated as previously described. Thereafter, immunocomplexes were washed thrice in fresh RIPA buffer and once in kinase assay buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM dithiothreitol, 1 mM benzamidine, and 0.5 mM phenylmethylsulfonyl fluoride) before resuspending in 25 µl of kinase assay buffer. 10 µCi of [{gamma}-32P]ATP was subsequently added, and the reactions were incubated for 30 min at 37 °C. Immunocomplexes were then denatured in sample buffer and were resolved by SDS/PAGE. After electrophoresis, the gels were dried and subsequently subjected to autoradiography. Autoradiograms were scanned using Chemidoc Quantity One software (Bio-Rad). Normalization of data were achieved by an anti-PDGFRbeta immunoblot using a small amount of each sample collected after the immunoprecipitation stage.

Analysis of SHP-2 Activity—SHP-2 activity was measured in SHP-2 immunoprecipitates using a Malachite Green PTP activity assay (Upstate%20Biotechnology">Upstate Biotechnology Inc.) according to the manufacturer's instructions. Briefly, SHP-2 was immunoprecipitated from 200–300 µg of MC lysate using a specific monoclonal SHP-2 antibody (BD Transduction Laboratories) for 3 h at 4 °C. Protein G-agarose beads were subsequently added to each immunoprecipitate and incubated for further 2 h at 4°C with constant rocking. Immunocomplexes were washed three times in fresh lysis buffer and then twice in the phosphatase assay buffer (Tris-HCl, 50 mM, pH 7.0, 0.1% bovine serum albumin) before finally resuspending in 100 µl of assay buffer. To each sample, phosphopeptide substrate was added, and the samples were incubated for 30 min at 37 °C. The reactions were terminated by the addition of 100 µl of malachite green solution, allowing 15 min for color development followed by spectrophotometric analysis at 650 nm. The results were expressed as pmol of phosphate released per microgram of protein.

Sucrose Flotation Gradients—ALXR cells seeded in 150-mm plates were washed twice with ice-cold phosphate-buffered saline and scraped into 1 ml of phosphate-buffered saline. Thereafter, the cells were harvested and lysed with 1 ml of TNE buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1%Triton X-100, 1x CompleteTM protease inhibitor mixture) for 30 min at 4 °C. The lysates were subsequently homogenized with 15 strokes of a Dounce homogenizer, transferred to a microcentrifuge tube, and centrifuged at 4 °C for 10 min at 830 x g. 1 ml of the resultant cell supernatant was mixed with an equal volume of 80% (w/v) sucrose in TNE buffer, transferred to a 5-ml ultracentrifuge tube, and then carefully overlaid with 2 ml of 30% (w/v) sucrose in TNE buffer and finally with 1 ml of 5% (w/v) sucrose in TNE buffer. The tube was centrifuged at 4 °C for 17 h at 134,400 x g. Eight fractions (600 µl) were collected from the top to the bottom of the gradient. Protein from each fraction was precipitated using a standard methanolchloroform protocol. The resulting precipitates were resuspended in reducing sample buffer, heated to 90 °C for 5 min, and resolved on an SDS/PAGE gel or stored at -80 °C until required.

Detection of Reactive Oxygen Species—Reactive oxygen species (ROS) were detected in MCs stimulated with various agents using an Image-iTTM LIVE Green ROS detection kit. Briefly, subconfluent cultures of MC were plated on 8-well chamber slides and serum-restricted in 0.2% FCS for 48 h. Thereafter, MCs were treated with vehicle (30 min), PDGF-BB (10 ng/ml, 30 min), LXA4 (10 nM, 30 min), or with LXA4 (10 nM, 30 min) followed by PDGF-BB (10 ng/ml, 30 min). The cells were washed once with warm Dulbecco's phosphate buffered saline (DPBS) solution. Carboxy-H2DCFDA (10 µmol/liter) was added to each well, and the cells were further incubated for 30 min at 37 °C. In addition, the nuclei were countered stained with a Hoechst 33342 nuclei stain (1 µM) for 5 min at 37 °C. Subsequently, the slides were gently immersed and washed three times in a warm DPBS solution, after which one drop of Slowfade® Light Antifade solution was added before placing a coverslip over the wells. The fluorescence of the oxidized form of carboxy-H2DCFDA was visualized using a M.1 Zeiss microscope, and the images were captured using an Axiocam system and Axiovision Rel 4.4 software (Carl Zeiss).


Figure 1
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  		FIGURE 1.
RTK phosphorylation in MC is inhibited by LXA4. A and B, serum-restricted MC were stimulated with vehicle, LXA4 (10 nM, 30 min), PDGF-BB (10 ng/ml, 5 min), or LXA4 (100 pM to 100 nM, 30 min) followed by PDGF-BB (10 ng/ml, 5 min). C, MC were treated with vehicle, LXA4 (10 nM, 30 min), Ac2-26 (10 µg/ml, 30 min), PDGF-BB (10 ng/ml, 5 min), LXA4 (10 nM, 30 min) followed by Ac2-26 (10, 50, 100 µg/ml, 30 min) or AG1296 (10 µM, 30 min) followed by PDGF-BB (10 ng/ml, 5 min). D, MC were treated with vehicle, LXA4 (10 nM, 30 min), AG1478 (10 µM, 30 min), or EGF (10 ng/ml, 5 min), LXA4 (10 nM, 60 min) followed by EGF (10 ng/ml, 5 min) and AG1478 (10 µM, 30 min) followed by EGF (10 ng/ml, 5 min). The cells were lysed with RIPA buffer and immunoprecipitated with PDGFRbeta (A–C) and EGFR (D) antibodies, respectively. A, C, and D, immunocomplexes were denatured in sample buffer, resolved by SDS/PAGE, electroblotted onto polyvinylidene difluoride membrane, and probed for phosphotyrosine using a mouse monoclonal antibody against PY-7E1 and PY20 (upper panels). Total cellular PDGFRbeta and EGFR was determined by immunoblotting for PDGFRbeta and EGFR (lower panels), respectively. Quantitative data are representative of the means ± S.D. of three independent experiments. The extent of receptor phosphorylation is depicted as the ratio of phosphorylated receptor to total receptor, with PDGF-BB-stimulated PDGFRbeta taken as maximum value. #, p < 0.01 relative to PDGF stimulation. B, immunoprecipitates were subjected to an autophosphorylation assay as described under "Experimental Procedures." 32P-Labeled proteins were visualized by autoradiography. WB, Western blot; P-Y, phosphotyrosine; IP, immunoprecipitation.

 
Data Analysis—Statistical analysis was carried out using the unpaired Student's t test. All of the values are expressed as either the means ± S.D. or the means ± S.E. p values of less than or equal to 0.05 were considered to indicate a statistically significant difference.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Treatment of MCs with LXA4 resulted in a concentration-dependent (100 pM to 100 nM) decrease in activated PDGFRbeta upon stimulation with PDGF-BB. (The EC50 was ~10 nM LXA4; data not shown). Similarly, the amount of immunodetected PDGFRbeta in phosphotyrosine immunoprecipitates was decreased by 52 ± 8% following preincubation with LXA4 (10 nM) prior to stimulation with PDGF (Fig. 1A, inset). To investigate whether altered receptor phosphorylation reflected decreased autokinase activity or possible phosphatase activation, the effect of LXA4 on RTK activation was investigated. As shown in Fig. 1B, LXA4 did not impact receptor tyrosine kinase activity.

The ALXR binds both lipid and peptide ligands (8). Recent data have shown that the annexin 1-derived peptide Ac2-26 can bind the ALXR (14), suggesting that some of the anti-inflammatory pro-resolution effects of dexamethasone (15) may be mediated via the ALXR (16). Interestingly, we have found that Ac2-26 mimics the effect of LXA4 on PDGFR activation (Fig. 1C). In addition, we investigated whether LXA4 could impact EGFR activation, another important pathologic event in renal dysfunction (17). As shown in Fig. 1D, preincubation of MC with LXA4 (10 nM) caused a decrease in the EGF-activated tyrosine phosphorylation of the EGFR. Similarly, specific PDGFR and EGFR protein-tyrosine kinase inhibitors AG1296 and AG1478 inhibited PDGF- and EGF-induced activation of the PDGFRbeta and EGFR, respectively (Fig. 1, C and D).

To establish the molecular basis of ALXR-PDGFRbeta cross-talk, we generated a HEK293 cell line stably overexpressing the ALXR (ALXR cells) in a PDGFRbeta null background. Thereafter, we examined the effect of LXA4 on PDGFRbeta phosphorylation in ALXR and control cells that were transiently transfected with PDGFRbeta. Consistent with data generated in MCs, LXA4 significantly reduced PDGF-BB stimulated PDGFRbeta phosphorylation (Fig. 2A). In contrast, whereas stimulation of HEK293 cells transiently transfected with PDGFRbeta resulted in PDGFRbeta phosphorylation, preincubation of those cells with LXA4 had no significant effect on receptor phosphorylation (Fig. 2B). These data indicate that the observed effects of LXA4 are mediated via the ALXR.

To define the locus/loci at which LXA4 modulates growth factor receptor tyrosine phosphorylation and subsequent mitogenic events, a series of PDGFRbeta mutants were generated whereby specific tyrosines were substituted for phenylalanine at positions 579, 771, 740/751, and 763/1009. Although stimulation of ALXR cells transiently expressing Y579F and Y771F mutants displayed a clear increase in receptor phosphorylation when stimulated with PDGF-BB, preincubation with LXA4 reduced the extent of phosphorylation similar to that of vehicle (Fig. 2, C and D). These residues are specific for recruitment of Src and Ras GTPase-activating protein, respectively (13), and therefore we concluded that LXA4 was not acting at these loci. In contrast, mutation of the PDGFRbeta binding sites for p85 recruitment (Y740F/Y751F) blocked the effect of LXA4 on receptor phosphorylation (Fig. 2E). Interestingly, the effect of LXA4 in cells expressing the Y740F alone was maintained, whereas in cells expressing Y751F, the effect of LXA4 was partially reduced (supplemental data). These data suggest that the high affinity binding site for the recruitment of p85 (Tyr751) is the site most likely to be modulated by LXA4, although both sites must be mutated before a complete block in the effect of LXA4 is observed. These data are particularly noteworthy given that p85 is a component of the PI 3-kinase complex, and our previous data have demonstrated that LXA4 inhibits MC PI 3-kinase activity in response to leukotriene D4 and PDGF, respectively (4, 18). Consistent with our observations in ALXR cells expressing the Y740F/Y751F mutant, PDGF-induced phosphorylation of Tyr751 was significantly inhibited by preincubation with LXA4 in primary MC as assessed by immunoblot analysis using an antibody specific against PDGFRbeta-Y751 phosphorylation (supplemental data).

The phosphorylation status and signaling activity of RTKs is determined not only by the kinase activity of the RTK but also by the activities of PTPs (19). Several PTPs have been shown to be recruited to the PDGFR and EGFR and are also stimulated in response to ligand-activation of many GPCRs (20, 21). Herein, the attenuation of RTK tyrosine phosphorylation levels, without alteration in RTK activity (Fig. 1B), suggests the involvement of PTPs in LXA4 ability to modulate PDGFRbeta phosphorylation. Consistent with this hypothesis, the observed reduction in phosphorylation of the PDGFRbeta in MC in response to preincubation with LXA4 was abolished by the broad spectrum, nonspecific PTP inhibitors vanadate and phenylarsine oxide (Fig. 3A). Interestingly, incubation of MC with the inhibitor mixture alone resulted in a 4-fold maximal increase in basal activation of the PDGFRbeta (data not shown), suggesting a tonic inhibition of RTK activation by PTPs in quiescent MC. To test whether SHP-2 contributes to LXA4 modulation of PDGF-induced PDGFRbeta phosphorylation, ALXR cells were transfected with a double mutant for the recruitment sites for SHP-2 PTP (Y763F/Y1009F). Consistent with the non-specific PTP inhibitor data, LXA4 (10 nM, 30 min) had no effect on PDGF-induced receptor phosphorylation (Fig. 3B), suggesting that LXA4 may require the recruitment of SHP-2 PTP to the PDGFRbeta to exert its effect.


Figure 2
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  		FIGURE 2.
PDGFRbeta phosphorylation in ALXR cells is inhibited by LXA4. HEK293 cells (-ALXR) and ALXR cells (+ALXR) were transiently transfected with wild type pcDNA3-PDGFRbeta (A and B) or with PDGFRbeta mutant constructs Y579F (C), Y771F (D), or Y740F/Y751F (E), prior to serum restriction in 0.01% FCS for 24 h. Thereafter, the cells were treated with vehicle (5 min), PDGF-BB (10 ng/ml, 5 min), or LXA4 (10 nM, 30 min) followed by PDGF-BB (10 ng/ml, 5 min) and AG1296 (10 µM, 30 min) followed by PDGF-BB (10 ng/ml, 5 min). The cells were lysed with RIPA buffer and immunoprecipitated with a PDGFRbeta antibody. Thereafter, immunocomplexes were denatured in sample buffer, resolved by SDS/PAGE, electroblotted onto polyvinylidene difluoride membrane, and probed for phosphotyrosine using a mouse monoclonal antibody against PY-7E1 and PY20. Total cellular PDGFRbeta was determined by immunoblotting for PDGFRbeta. The blots are representative of three independent experiments, and the quantitative data are representative of the means ± S.D. of three independent experiments. The extent of receptor phosphorylation is depicted as fold/basal (vehicle-treated) phosphorylation. *, p < 0.01 relative to vehicle-treated cells; #, p < 0.05 relative to PDGF stimulation. WB, Western blot; P-Y, phosphotyrosine; Wt, wild type; Veh, vehicle.

 
To further investigate the role of SHP-2 on LXA4 modulation of PDGF-induced PDGFRbeta phosphorylation, specific SHP-2 activity was measured in SHP-2 immunoprecipitates from differentially treated MC. As seen in Fig. 3C, LXA4 induced significant increases in SHP-2 activity. Consistent with previous reports (22, 23), PDGF significantly increased SHP-2 activity; however, the kinetics at which it did so were considerably different to LXA4. Maximal activation in response to PDGF was observed at 5 min, after which no significant activation was detected. In contrast, LXA4 stimulated a prolonged activation of SHP-2 (Fig. 3C). To evaluate the role of the ALXR in the activation of SHP-2 in response to LXA4, MCs were treated with an ALXR antagonist, Boc-2 (24) prior to treatment with LXA4. Pretreatment with Boc-2 inhibited the SHP-2 activation in response to LXA4 (Fig. 3D).

Collectively, our data suggest that LXA4 regulation of PDGFRbeta activation is mediated via ALXR activation of SHP-2. To explore this hypothesis directly, we investigated whether inhibition of SHP-2 expression could block the effect of LXA4 on PDGFRbeta activation in ALXR cells. To do so, we used SHP-2-specific siRNA to silence the expression of SHP-2 by RNA interference. Treatment of ALXR cells with siRNA for 0–96 h resulted in strong reduction of SHP-2 levels relative to non-treated cells or cell treated with scrambled siRNA (Fig. 4A), with maximal inhibition at 50 nM occurring after 48 h. Thereafter, we compared the ability of LXA4 to modulate PDGFRbeta activation in cells transfected with SHP-2 siRNA and in cells transfected with scrambled siRNA (Fig. 4C). Importantly these effects on inhibition of SHP-2 expression were also seen with other SHP-2 siRNA constructs (supplemental Fig. S3). Consistent with Y740F/Y751F mutant data, LXA4 had no effect on PDGF-induced receptor phosphorylation in SHP-2 knock-out ALXR cells, whereas the effect of LXA4 was maintained in ALXR cells transfected with scrambled siRNA (Fig. 4C and supplemental data).

Several RTKs and GPCRs as well as their downstream effector proteins have been reported to be present in or recruit to lipid rafts following activation. Indeed, there is a growing body of evidence that caveolae-containing lipid rafts may provide platforms for specific signaling molecules (26). To investigate whether such microdomains are implicated in ALXR-PDGFRbeta cross-talk, we used a cholesterol-binding agent, methyl-beta-cyclodextrin, to sequester cholesterol and cause disassembly of cholesterol-rich rafts and caveolae. Incubation of MC with 10 mM methyl-beta-cyclodextrin for 30 min did not alter cell morphology or integrity as measured by trypan blue exclusion (data not shown). Fig. 5A shows that LXA4-dependent dephosphorylation of the PDGFRbeta was significantly reduced and approached nontreated, PDGF-stimulated levels after depletion of plasma membrane cholesterol. In contrast, methyl-beta-cyclodextrin did not significantly decrease PDGF-BB-stimulated PDGFRbeta phosphorylation (Fig. 5A), implying that it is the transinactivation process that requires cholesterol-rich microdomains. To examine whether the apparent importance of cholesterol for LXA4 activity reflects the association of the ALXR with lipid microdomains, we used sucrose gradient centrifugation to separate detergent-resistant rafts, which include caveolae, from ALXR cell lysates. Immunoblotting of the eight different fractions obtained revealed that fraction 3 was highly enriched in both caveolin-1 and flotillin-1 (Fig. 5B), whereas proteins such as clathrin and beta-actin, which are thought not to reside within caveolae in situ, localized to fractions 4–8 (bulk plasma membrane). Importantly, fraction 3 contained the highest levels of the ALXR, whereas significant levels of transfected PDGFRbeta and SHP-2 are also shown to be present (Fig. 5B).

To investigate whether the LXA4-driven dephosphorylation of the PDGFRbeta is localized to the lipid raft fraction, we isolated the soluble (fraction 7) and lipid raft fractions (fraction 3) from the sucrose gradient and immunoblotted for the aforementioned proteins. The PDGFRbeta in both fractions was phosphorylated in response to PDGF-BB treatment, but only the PDGFRbeta in the lipid raft fraction was dephosphorylated in response to preincubation with LXA4 (Fig. 5C). This reduction in PDGFRbeta phosphorylation in response to LXA4 was accompanied by concomitant increases in levels of SHP-2 within the lipid raft fraction. Interestingly, SHP-2 levels in the soluble fraction were increased in response to PDGF-BB (Fig. 5C), suggesting that the different ligands activate different subpopulations of the PTP.

Previous reports have suggested that the interaction between PTPs and GPCRs/RTKs might be controlled by regulated distribution of PTPs to lipid rafts (27). To investigate possible recruitment of SHP-2 to the lipid raft-bound ALXR, we immunoprecipitated the HA-tagged ALXR from the lipid raft (Fraction 3) and soluble (Fraction 7) fractions of ALXR cells and immunoblotted for SHP-2. As Fig. 5D demonstrates, SHP-2 was constitutively associated with the ALXR in the lipid raft fraction (fraction 3) of quiescent ALXR cells, whereas no association was seen within the soluble (fraction 7) fraction or with the mock-transfected HEK293 cell lysate control. Moreover, the SHP-2 association seen in the lipid raft fraction was increased upon activation of the receptor by LXA4 and returned to basal levels 30 min post-stimulation. In contrast, no SHP-1 recruitment to the ALXR was observed in either the lipid raft fraction (fraction 3) or in the soluble fraction (fraction 7) (data not shown).

To understand the molecular mechanism by which PDGF signaling is down-regulated by ALXR activation of SHP-2 within lipid microdomains, we analyzed endogenous H2O2 production, which has been shown to mediate site-selective amplification of PDGFR phosphorylation (25). Recent studies have implicated the involvement of a cellular protease, 2-Cys peroxiredoxin type II (Prx II), in the down-regulation of PDGF signaling via a decrease in localized endogenous H2O2 production (25). To assess the role of LXA4 within this signaling system, we examined the induction of ROS in MC. Upon PDGF stimulation, LXA4-pretreated cells exhibited a significant reduction in the amount of ROS released compared with MC treated with PDGF alone, as measured by reduced 5-(and-6)-carboxyl 2',7'dichlorodihydrofluorescein diacetate oxidation (Fig. 6A). Whereas MC exhibited significant PDGF-induced Prx II protein expression, pretreatment of cells with LXA4 prior to PDGF stimulation reduced Prx II expression to levels that were not significantly different from those generated by nontreated cells (Fig. 6B).


Figure 3
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  		FIGURE 3.
LXA4 inhibition of PDGFRbeta is PTP-dependent. A, serum-restricted MC were treated with vehicle or PDGF-BB (10 ng/ml, 5 min) or were preincubated with a mixture of phenylarsine oxide (30 µM) and activated Na3VO4 (100 µM) (30 min) and/or LXA4 (10 nM, 30 min) prior to stimulation with PDGF-BB (10 ng/ml, 5 min). The quantitative data are representative of the means ± S.D. of three independent experiments. The extent of receptor phosphorylation is depicted as the ratio of phosphorylated receptor to total receptor, with PDGF-BB-stimulated PDGFRbeta taken as maximum value. *, p < 0.01 relative to vehicle stimulation; #, p < 0.05 relative to PDGF stimulation; ##, p < 0.05 relative to PDGF+LXA4 stimulation. B, HEK293+ALXR cells were transiently transfected with mutant pcDNA3-PDGFRbeta construct Y763F/Y1009F prior to serum restriction in 0.01% FCS for 24 h and stimulated as indicated. The cells were lysed with RIPA buffer and immunoprecipitated with a PDGFRbeta antibody followed by immunoblotting with a phosphotyrosine antibody. The blots are representative of three independent experiments. C and D, serum-restricted MC were treated with vehicle, LXA4 (10 nM), PDGF (10 ng/ml), or Boc-2 (100 µM, 30 min) followed by LXA4 (10 nM) for 0–60 min. Thereafter, the cells were lysed and immunoprecipitated with a SHP-2 mAb. SHP-2 PTP activity in the immunoprecipitates was measured using a Malachite-Green PTP activity assay (Upstate%20Biotechnology">Upstate Biotechnology Inc.) as described under "Experimental Procedures." The results represent the means ± S.D. of six (C) and two (D) independent experiments and are expressed as pmol of phosphate released per microgram of protein (pmol P/mg protein). *, p < 0.05 relative to vehicle. WB, Western blot; P-Y, phosphotyrosine; Veh, vehicle; IP, immunoprecipitation.

 


Figure 4
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  		FIGURE 4.
siRNA knockdown of SHP-2 results in inhibition of LXA4 effect. A, ALXR cells were transiently transfected with siRNA duplexes for SHP-2 (100 nM to 10 nM) and/or scrambled siRNA (50 nM) for 48 h. B, ALXR cells were transfected with siRNA for SHP-2 (50 nM) for 0–96 h and/or scrambled siRNA (50 nM, 48 h). The cells were lysed, and both SHP-2 and beta-actin protein levels were measured by immunoblotting. C, ALXR cells were transfected with wild type pcDNA3-PDGFRbeta (lanes 1–3) or were co-transfected with scrambled siRNA (all lanes 2) or siRNA for SHP-2 (all lanes 3) for 48 h prior to serum restriction in 0.01% FCS for 24 h. Thereafter, the cells were treated with vehicle (5 min), PDGF-BB (10 ng/ml, 5 min), or LXA4 (10 nM, 30 min) followed by PDGF-BB (10 ng/ml, 5 min). The cells were lysed and immunoblotted for SHP-2 (upper panel). Alternatively, PDGFRbeta was immunoprecipitated and was subsequently immunoblotted for phosphotyrosine (middle panel). Total cellular PDGFRbeta was determined by immunoblotting for PDGFRbeta (lower panel). The blots are representative of three independent experiments. WB, Western blot; P-Y, phosphotyrosine; Veh, vehicle; IP, immunoprecipitation.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The mechanisms underlying the resolution of glomerular inflammation remain enigmatic. However, it has been proposed that regulation of MC proliferation by LX may play a key role (2, 3, 28).

We have previously demonstrated that the anti-proliferative effects of LXA4 are mediated through activation of the ALXR and result in modulation of growth factor (PDGF and EGF) receptor phosphorylation leading to down-regulation of the PI 3-kinase signaling pathway (4, 9, 18). Here, we report that the ability of LXA4 to reduce the tyrosine phosphorylation level of the PDGFRbeta is independent of inhibition of RTK activity but dependent on SHP-2 PTP activation. Moreover, we demonstrate that the effect of LXA4 is replicated by a low affinity agonist of the ALXR, Ac2-26, (Kd for Ac2-26 is 0.9 µM versus 1.7 nM for LXA4), a peptide derivative of annexin 1, and that these effects are not restricted to the PDGFR signaling pathway but encompass other pleiotropic RTK such as the EGFR.

The PDGFRbeta is activated by binding of PDGF and undergoes autophosphorylation at multiple tyrosine residues. The tyrosine-phosphorylated receptor associates with numerous SH2 domain-containing signal transduction molecules that include phospholipase C{gamma}1, the GTPase-activating protein of Ras, the p85 subunit of PI 3-kinase, the phosphotyrosine phosphatase SHP-2, and several other proteins (29, 30). Indeed, individual mutation of these specific tyrosine residues prevents association of its cognate effectors without altering the binding of other enzymes to the receptor. Thus, this phenomenon has afforded us a convenient method to assess the role of LXA4 transinhibition on the receptor-associated effector-binding sites of the PDGFRbeta in HEK293 cells overexpressing both the ALXR and wild type/mutant PDGFRbeta receptors. Our results suggest that, among the sites for binding of effector molecules (i.e. PI 3-kinase, Ras GTPase-activating protein, Src kinase, and SHP-2), only mutation of the sites for PI-3-kinase and SHP-2 affected the dephosphorylation of the PDGFRbeta by LXA4 pretreatment. Our data indicate that the observed LXA4-mediated transinhibition of PDGFRbeta does not reflect a diminution in receptor autokinase activity but a specific effect on particular phosphotyrosines residues.


Figure 5
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  		FIGURE 5.
The ALXR is localized to lipid rafts in HEK293 cells. A, subconfluent cultures of MC were serum-restricted in 0. 2% FCS for 48 h. MCs were then treated with vehicle (5 min), MbetaC (10 mg/ml, 30 min), or PDGF-BB (10 ng/ml, 5 min). MCs were also preincubated with MbetaC (10 mg/ml, 30 min) and/or LXA4 (10 nM, 30 min) prior to PDGF-BB (10 ng/ml, 5 min). The lysates were harvested, and PDGFRbeta was immunoprecipitated from all conditions, followed by immunoblotting as indicated. B, ALXR cells were transiently transfected with pcDNA3.1-PDGFRbeta prior to serum restriction in 0.01% FCS for 24 h. The cells were subsequently treated with vehicle (30 min) and lysed with TNE buffer, and a sucrose flotation experiment was carried out as described under "Experimental Procedures." Thereafter, immunoblotting for caveolin-1, flotillin-1, beta-actin, clathrin, HA-tagged ALXR, SHP-2, and PDGFRbeta was carried out on all gradient fractions. The blots shown are representatives of three independent experiments. C, ALXR cells transiently transfected with pcDNA3.1-PDGFRbeta were serum-restricted in 0.01% FCS for 24 h prior to treatment with vehicle (5 min), PDGF-BB (10 ng/ml, 5 min), or LXA4 (10 nM, 30 min) followed by PDGF-BB (10 ng/ml, 5 min). The cells were lysed with TNE buffer, and a sucrose flotation experiment was carried out as described under "Experimental Procedures." Lipid raft (Fraction 3) and soluble fractions (Fraction 7) were analyzed by immunoblotting for HA-tagged ALXR, SHP-2, PDGFRbeta, phosphotyrosine, caveolin-1, and beta-actin. D, ALXR cells were serum-restricted in 0.01% FCS for 24 h prior to treatment with vehicle (5 min) or LXA4 (10 nM, 5–30 min). Thereafter, the cells were lysed with TNE buffer, and a sucrose flotation experiment was carried out as previously described under "Experimental Procedures." To analyze the association of the ALXR with SHP-2, lipid raft fraction (Fraction 3) lysates were subject to immunoprecipitation with anti-HA serum to immunoprecipitate the ALXR. Soluble fractions (Fraction 7), lysates from mock-transfected HEK-293 cells and mouse IgG served as negative controls. Immunoprecipitates were resolved by SDS-PAGE/immunoblotting and screened with anti-SHP-2 as described under "Experimental Procedures." All of the blots are representatives of three independent experiments. WB, Western blot; P-Y, phosphotyrosine; Veh, vehicle; IP, immunoprecipitation; MbetaCD, methyl-beta-cyclodextrin; Cav-1, caveolin-1.

 


Figure 6
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  		FIGURE 6.
LXA4 inhibition of PDGFRbeta mediated ROS production. A, subconfluent cultures of MC were serum-restricted in 0. 2% FCS for 48 h. Thereafter, MCs were treated with vehicle (30 min), PDGF-BB (10 ng/ml, 30 min), LXA4 (10 nM, 30 min), or LXA4 (10 nM, 30 min) followed by PDGF-BB (10 ng/ml, 30 min). The cells were washed and labeled with carboxy-H2DCFDA. The nuclei were counterstained with 4',6'-diamino-2-phenylindole. The images are taken from a random field of view and are representative of three independent experiments. B, alternatively, MC cells were lysed with RIPA buffer and immunoblotted with a mouse monoclonal peroxiredoxin II antibody (12B1). Expression of beta-actin was used as a loading control. The blots are representative of three independent experiments. WB, Western blot.

 
SHP-2, a ubiquitously expressed PTP, is a critical intracellular regulator of cytokine and growth factor-induced cell survival, proliferation, and differentiation (31). Several PTPs have been shown to bind PDGFRbeta, with SHP-2 being the most efficiently binding PTP (32). SHP-2 binds to Tyr(P)763 and Tyr(P)1009 on activated PDGFRbeta and in turn may dephosphorylate selective docking sites that partake in the down-regulation of receptor signaling, including Tyr(P)751 and Tyr(P)740 (p85 recruitment sites) (33). However, the effect of SHP-2 on the PDGFRbeta is contradictory. Depending on the cell type and the nature of the agonist, SHP-2 either augments or antagonizes the signaling pathway initiated by activation of the PDGFRbeta. For example, SHP-2 can act as a positive regulator for PDGF signaling by acting as an adapter between the PDGF receptor and the Grb2-Sos complex, leading to activation of the Ras signaling pathway (34). In contrast, Zhao and Zhao (35) demonstrated that catalytically active forms of SHP-2 decreased the tyrosine phosphorylation of the PDGFRbeta but enhanced PDGF-induced activation of the mitogen-activated protein kinase pathway (35). From our results, activation of SHP-2 PTP by LXA4 via the ALXR leading to reduced PDGFRbeta phosphorylation is supported by multiple lines of evidence. First, the effect of LXA4 on PDGFRbeta phosphorylation in MC was diminished in the presence of PTP inhibitors vanadate and phenylarsine oxide. Second, we detected sustained elevation of SHP-2 PTP activity in immunoprecipitates from LXA4-treated MC. This effect was mediated via the ALXR given that inclusion of the nonspecific ALXR antagonist, Boc-2, blocked LXA4-induced SHP-2 activation. Third, we demonstrate that mutation of the SHP-2 recruitment sites on the PDGFRbeta results in an inhibition of the LXA4 effect and therefore suggests that SHP-2 is recruited to the PDGFRbeta in response to activation by LXA4. Furthermore, siRNA-directed modulation of SHP-2 expression resulted in inhibition of the LXA4 effect on the PDGFRbeta. Importantly, the EGFR, like the PDGFRbeta, also contains tyrosine residues for the recruitment of SHP-2 (37), suggesting that multiple phosphorylated growth factor receptors may act as substrates for SHP-2 and in turn may be modulated by LXA4.


Figure 7
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  		FIGURE 7.
Proposed mechanism for SHP-2 regulation of PDGFRbeta in response to LXA4. Ligand activation of the PDFGRbeta induces a transient increase in the intracellular concentration of H2O2 leading to enhanced phosphorylation of the PDGFRbeta, PI 3-kinase (PI 3-K) activation and mitogenic responses. Furthermore, H2O2 can oxidatively inactivate PTPases, including SHP-2, within the microenvironment of the PDGFRbeta. In contrast, activation of the lipid raft-bound ALXR induces recruitment to and activation/reactivation of SHP-2 through direct activation of PTPase and/or indirectly by attenuating H2O2 production. Thereafter, activated SHP-2 is released from the ALXR and thus diminishes subsequent PDGF-mediated mitogenic activities.

 
The temporal requirements of exposure to LXA4 for significant inhibition of phosphorylation of RTK (30–60 min) suggested that the effect may reflect RTK trafficking from the cell membrane (combined with PTP activation). Receptor down-regulation appears to be an important element in the control of cellular proliferation, because mutations that impair the ability of some receptors to internalize and degrade cause cell transformation (41). We have investigated the involvement of cholesterol-rich lipid microdomains in the LXA4 effect on the PDGFRbeta. These microdomains participate in endocytosis and are thought to facilitate recruitment and organization of downstream effector molecules into specialized signaling complexes (26). We have employed a cholesterol-depleting agent to disrupt lipid rafts and demonstrate that intact lipid rafts are necessary for LXA4 transinhibition of the PDGFRbeta. From our data, we identify that caveolae-enriched fractions of HEK293 cells contain both the ALXR and PDGFRbeta, and we conclude that the ALXR requires association with lipid rafts to initiate both the recruitment of SHP-2 and the dephosphorylation of the PDGFRbeta. The targeting of GPCRs and RTKs to lipid rafts following activation can occur through different mechanisms. The PDGFRbeta is known to be recruited to lipid raft fractions upon ligand activation (42), whereas other receptors exist in lipid rafts apparently as a result of their random distribution in the plasma membrane (43). Both the ALXR and PDGFRbeta were found to reside in the lipid raft fraction in ALXR cells. However, we found no enrichment of either receptor in the lipid raft fraction in response to ligand activation. A previous study has identified distinct mechanisms by which SHP-2 is recruited to the cell membrane in a multiprotein complex that is distributed differently inside and outside lipid rafts (44). In our study we found increased levels of SHP-2 in the lipid raft fraction of ALXR cells in response to LXA4 treatment, whereas PDGF stimulated an increase of SHP-2 levels in the soluble fraction, suggesting that both ligands target different cellular pools of the PTP. Interestingly, only the lipid raft-associated PDGFRbeta was found to be dephosphorylated in response to LXA4 pretreatment, indicating a specific deactivation process involving the lipid raft population of SHP-2.

In further studies, we uncovered a constitutive association of SHP-2 with the lipid raft-bound ALXR in HEK293 cells stably expressing the ALXR. This association was increased in response to ligand activation and returned to basal levels in a time-dependent manner. In this context, it is noteworthy that SHP-2 itself has been shown to be directly recruited and subsequently activated by the immunoreceptor tyrosine-based inhibitor motif (21) sequence in the bradykinin B2 receptor in MC, resulting in global dephosphorylation of tyrosine residues and inhibition of proliferation (20). We have identified a consensus immunoreceptor tyrosine-based inhibitor motif (45) in the ALXR (supplemental data), located at the interface of the N-terminal part of the second transmembrane domain and the first intracellular domain. Previous studies have demonstrated that occupation of the SH2 domain of SHP-2 by a ligand may stimulate both subcellular relocalization of the enzyme to the vicinity of substrates or may also stimulate the phosphatase activity (46). Thus, we envision that SHP-2 is recruited to the ALXR in lipid microdomains via Tyr(P)-SH2 interaction, and subsequent ALXR dephosphorylation eventually leads to dephosphorylation of the SH2-binding domain-binding sites and disruption of the complex.

Numerous studies suggest that ROS such as O2. and H2O2 function as mitogenic mediators of activated growth factor receptor signaling such as the PDGFR (38, 39). PDGF-induced tyrosine phosphorylation and DNA synthesis was inhibited when the growth factor-stimulated rise in H2O2 concentration was blocked. Moreover, Choi et al. (25) report that the mammalian Prx II allows oxidatively inactivated protein-tyrosine phosphatases to be reactivated by removing endogenous H2O2, resulting in decreased phosphorylation of key tyrosine residues in both PDGFRbeta and the downstream signaling molecule phospholipase C{gamma}1 (25). We have observed robust PrxII up-regulation in response to PDGF after 30 min of stimulation, a response similar to that reported for serum-stimulated epithelial cells (47). In an effort to elucidate the molecular mechanism by which PDGF signaling is down-regulated by ALXR activation of SHP-2, we examined the influence of LXA4 on ROS and Prx II levels produced in response to PDGF in MC. LXA4 attenuated PDGF-stimulated ROS production, consistent with previous data from experiments carried out in human polymorphonuclear leukocytes (40). Interestingly, although we have shown that LXA4 results in the prolonged activation of SHP-2, LXA4 abolished PDGF-induced Prx II protein expression. This leads us to suggest that LXA4 attenuation of ROS/H2O2 production and hence activation of SHP-2 may be independent of Prx II. Conversely, Prx II down-regulation is a direct result of LXA4 attenuation of ROS production (21, 38). Therefore, we propose that LXA4 possibly mimics Prx II in that it potentially reactivates oxidatively inactivated SHP-2 PTP by removing endogenous H2O2.

In summary, based on our general understanding of PDGFR signaling, we now propose a model for SHP-2 regulation of the PDGFRbeta in response to LXA4 (Fig. 7). LXA4 binding to the lipid raft-bound ALXR induces a recruitment to and reactivation of oxidatively inactivated SHP-2 in the lipid raft region. In a time-dependent manner, recruited SHP-2 is released from the ALXR whereby it is free to bind to and dephosphorylate the lipid raft population of the PDGFRbeta and hence diminish its subsequent mitogenic activities. Interestingly, SHP-2 has been shown to exert site-selective dephosphorylating activity, showing preference for the PI 3-kinase-binding sites (33). Our results support this hypothesis as mutation of the PDGFRbeta-binding sites for p85 recruitment (Y740F/Y751F) blocked the effect of LXA4 on receptor phosphorylation. Taken together, these data further elaborate on the potential mechanisms underlying the anti-inflammatory, pro-resolution, and anti-fibrotic bioactions of LX.


   FOOTNOTES
 
* This work was supported by the Health Research Board, Ireland, Enterprise Ireland, The Wellcome Trust, European Commission FP6 Grant LSHM-CT-2004-0050333, Science Foundation Ireland, and The Government of Ireland Programme for Research in Third Level Institutions. 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

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S3 and supplemental text. Back

1 Present address: Dept. of Pharmacology, New York University School of Medicine, New York, NY 10016. Back

2 To whom correspondence should be addressed. Tel.: 353-1-716-6731; E-mail: catherine.godson{at}ucd.ie.

3 The abbreviations used are: LX, lipoxin; MC, human renal mesangial cell; ALXR, LXA4 receptor; PDGF, platelet-derived growth factor; PDGFR, PDGF receptor; PI, phosphatidylinositol; siRNA, short interfering RNA; GPCR, G protein-coupled receptor; RTK, receptor tyrosine kinase; SH2, Src homology 2; PTP, protein-tyrosine phosphatase; HEK, human embryonic kidney; HA, hemagglutinin; FCS, fetal calf serum; RIPA, radioimmune precipitation assay; EGF, epidermal growth factor; EGFR, EGF receptor; ROS, reactive oxygen species; carboxy-H2DCFDA, 5-(and-6)-carboxyl 2',7'dichlorodihydrofluorescein diacetate; Prx II, peroxiredoxin type II. Back


   ACKNOWLEDGMENTS
 
We thank Prof. Finian Martin for critical review of the manuscript; Dr. Hugh Brady for continued support and advice, Prof. Mauro Perretti, William Harvey Research Institute, London, for provision of Ac2-26; and Dr. Susan K. Logan, New York University School of Medicine, for support.



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TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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