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Lipoxin, leukotriene, and PDGF receptors cross-talk to regulate mesangial cell proliferation ¹

BLAITHIN McMAHON^*,, DERICK MITCHELL^*,, RICHARD SHATTOCK^,, FINIAN MARTIN^,, HUGH R. BRADY^*, and D. CATHERINE GODSON^*,2

^* Centre for Molecular Inflammation and Vascular Research, Department of Medicine and Therapeutics, Mater Misericordiae Hospital and
The Conway Institute of Biomolecular and Biomedical Research, and
Department of Pharmacology, University College Dublin, and The Dublin Molecular Medicine Centre, Dublin, Ireland

²Correspondence: E-mail: cgodson{at}mater.ie

SPECIFIC AIMS

The aims of the current study were to investigate the mechanisms whereby LTD₄ stimulates proliferation of hMC and the mechanisms underlying LXA₄ modulation of MC proliferation.

Mesangial cell (MC) proliferation plays a pivotal role in the pathogenesis of glomerular inflammation and fibrosis. Dysregulated mesangial cell proliferation is an attractive target for therapeutic intervention in many subacute and chronic forms of glomerulonephritis (GN). Proinflammatory cytokines, eicosanoids, and growth factors generated locally by resident or infiltrating cells are important regulators of mesangial cell proliferation in this context.

The cysteinyl leukotrienes, particularly leukotriene D₄ (LTD₄), are proinflammatory mediators that modulate glomerular filtration, increase endothelial and mesangial cell adhesiveness for leukocytes, and stimulate MC proliferation. Agents that inhibit LT biosynthesis or bioactions confer protection in several experimental models of GN. Lipoxins (LXs) are endogenously produced eicosanoids typically formed by transcellular metabolism at sites of inflammation. LX act as putative "braking" and pro-resolution signals in host defense and inflammation, inhibiting PMN adhesion and transmigration, activating monocytes, and promoting macrophage phagocytosis of apoptotic neutrophils. We recently demonstrated that LXA₄, a major LX generated in GN, inhibits LTD₄-induced MC proliferation in vitro. In the present study, we propose a novel mechanism of action by LX, namely, inhibition of growth factor receptor tyrosine kinase activity. We have reported that mesangial cells express distinct G-protein-coupled receptors (GPCR) for LXA₄ and LTD_4. Both agents activate MAP kinases, but LTD_4.-induced MC proliferation can be modulated by LXA₄ in vitro. The mechanisms underlying GPCR activation of MAP kinases and proliferative responses remain enigmatic. However, trans-activation of receptor tyrosine kinases has been proposed.

PRINCIPAL FINDINGS

1-LTD₄ trans-activates the PDGFRß in hMC
We investigated whether LTD₄ stimulation of MC was associated with activation of the PDGF receptor ß (PDGFRß), the prototypic receptor tyrosine kinase implicated in MC proliferation. Using primary cultured human MC, we found that LTD₄ stimulates trans-activation of the PDGF receptor as observed by phosphotyrosine immunoblotting of immunoprecipitated PDGFRß (Fig. 1 A); this was blocked by the nonspecific CysLT receptor antagonist pobilukast (i.e., cysLT₁/cysLT₂ antagonist) (Fig. 1B ), the PKC inhibitor GF 102903X (Fig. 1C ), and the PDGF receptor kinase inhibitor (AG 1296). These data are consistent with our report that AG 1296 inhibits LTD₄-stimulated MC proliferation. To evaluate whether LTD₄ activation of the PDGFRß generated a signaling competent receptor, we investigated c-src association with immunoprecipitated PDGFRß from LTD₄-stimulated cells. We found that treatment of the cells with LTD₄ mimicked the effect of PDGF to increase tyrosine phosphorylated c-src association with the PDGFR. We have also found that LTD₄-stimulated ras activation was blocked by AG 1296, suggesting that ras activation in response to LTD₄ is downstream of PDGFRß trans-activation.

View larger version (32K): [in this window] [in a new window]   Figure 1. LTD4 stimulates PDGFRß trans-activation: inhibition by pobilukast and GF 109203X. A) Subconfluent cultures of human mesangial cells were serum starved for 48 h in 0.2% FCS before stimulation with LTD4 (1x10-8 M, 10 min), PDGF-BB (5 ng/ml, 5 min), or vehicle. PDGFRß phosphotyrosine content was assessed by anti-phosphotyrosine immunoblotting of PDGFRß immunoprecipitates (upper panel). PDGFRß immunoprecipitates were reblotted with PDGFRß antisera to ensure equal loading of PDGFRß (bottom panel). Serum-starved cells were preincubated in the presence of B) pobilukast (1x10-7 M, 5 min) or C) GF 109203X (1x10-5 M, 15 min) before a 10 min stimulation with LTD4 (1x10-8 M). Results depicted graphically represent the mean ± SE for 3 independent experiments; *P < 0.05, **P < 0.005 relative to vehicle control.

2-hMC express cysLT₁ and cysLT₂ receptors: PDGFR trans-activation is mediated through the cysLT₂ receptor
Recent data in the literature have indicated the existence of two distinct cysLT receptors in several tissues. We investigated the expression of these receptor subtypes in hMC and whether they might be differentially coupled to PDGFRß trans-activation. LTD₄-stimulated c-src activation was sensitive to pobilukast (a nonspecific cysLT receptor antagonist) but insensitive to Zafirlukast (a cysLT₁ antagonist) and pertussis toxin. These data coupled with PDGF receptor trans-activation in response to LTC₄ suggested the existence of two LTD₄ receptor subtypes. Using RT-PCR, we have detected transcripts for both cysLT₁ and cysLT₂ receptors in human mesangial cells.

3-LXA₄ modulates PDGFRß activation
Given the previous report that LX modulated LTD₄-stimulated proliferation of mesangial cells mediated through PDGFRß trans-activation, we investigated whether LXA₄ affected PDGFRß activation. As shown in Fig. 2 A, LXA₄ inhibited LTD₄ trans-activation of the PDGFRß. We then investigated whether LXA₄ directly modulated PDGFRß activation and found that pretreatment of the hMC with LXA₄ significantly inhibited PDGFRß activation (Fig. 2B ) and mitogenic responses to PDGF (Fig. 2C ). Consistent with this, we found a modest but significant inhibition of serum-stimulated proliferation in CHO K1 cells stably expressing the ALXR relative to untransfected cells.

View larger version (35K): [in this window] [in a new window]   Figure 2. LXA4 inhibits LTD4-induced trans-activation and PDGF BB-induced tyrosine phosphorylation of the PDGFRß and cell proliferation. A) Subconfluent cultures were serum starved for 48 h before stimulation with vehicle, LXA4 (1x10-8 M), LTD4 (1x10-8 M), or both. PDGFRß phosphotyrosine content was assessed by anti-phosphotyrosine immunoblotting of PDGFRß immunoprecipitates (upper panel). PDGFRß immunoprecipitates were blotted for PDGFRß to ensure that equal amounts of PDGFRß were loaded (bottom panel). Results are depicted graphically and represent the mean ± SE for 3 independent experiments; *P < 0.05 relative to vehicle. B) Subconfluent cultures of human mesangial cells were serum starved for 48 h in 0.2% FCS before stimulation with LXA4 (1x10-8 M, 60 min), AG 1296 (1x10-5 M, 30 min), and PDGF BB (10 ng/ml, 5 min), or vehicle. In double stimulations, cells were pretreated with LXA4 (1x10-8 M, 30 and 60 min), AG 1296 (1x10-5 M, 30 min) before stimulation with PDGF-BB (10 ng/ml, 5 min). PDGFRß phosphotyrosine content was assessed by anti-phosphotyrosine immunoblotting of PDGFRß immunoprecipitates (upper panel). PDGFRß immunoprecipitates were also blotted for PDGFRß to ensure that equal amounts of PDGFRß were loaded (bottom panel). Results depicted graphically represent the mean ± SE for 3 independent experiments. **P < 0.005 relative to vehicle, *P < 0.05 relative to PDGF-stimulated cells. Values are % phosphorylation (i.e., tyrosine phosphorylation of PDGFRß in lysates from PDGF-BB-treated cells). C) Subconfluent cultures were serum starved for 48 h before stimulation with LXA4 (1x10-8 M, 30 or 60 min) or vehicle before addition of PDGF-BB (10 ng/ml, 48 h). The number of proliferating cells was determined by [3H] thymidine incorporation. Data are mean ± SE of five independent experiments performed in quadruplicate; *P < 0.005 relative to vehicle-treated cells, **P < 0.05 relative to PDGF-BB-treated cells. D) CHOK1 cells stably expressing the ALXR were plated onto 24-well plates at 5 x 104cells/well. Subconfluent cultures were serum deprived for 24 h before stimulation with 10% FCS Ham’s F-12 media. Effects of LXA4 were investigated by pretreating the cells for 60 min with LXA4 (1x10-8 M) before stimulation with serum for 20 h. Results are expressed as % proliferation of mesangial cells relative to serum-stimulated cells (100%). Data are mean ± SE of 4 independent experiments performed in quadruplicate; *P < 0.005 relative to serum-treated cells.

CONCLUSIONS

We have shown that LTD₄ trans-activates the PDGFRß, apparently via the cysLT₂ receptor. LXA₄ modulated PDGFRß tyrosine kinase activity in response to PDGF BB and LTD₄. Furthermore, growth factor and serum-stimulated proliferation were both modulated by LXA₄. These data expand on the potential of LX to act in an anti-inflammatory capacity. Given the importance of MC proliferation in the development of glomerulosclerosis and renal fibrosis, the data suggest that LX possess novel anti-fibrotic actions that may be amenable to pharmacologic manipulation for therapeutic gain.

View larger version (16K): [in this window] [in a new window]   Figure 3. Lipoxin, leukotriene, and PDGF receptor cross-talk regulates mesangial cell proliferation. Schematic depicts LTD4-stimulated mesangial cell proliferation coupled to PDGFRß trans-activation, c-src recruitment, Ras, and PI 3-kinase activation. Mesangial cell proliferation is inhibited by the cysLT1/LT2 antagonist pobilukast, the PKC inhibitor GF 102903 X, and tyrophostin AG 1296. LXA4 inhibits activation of the PDGFRß in response to LTD4 and PDGF and thereby modulates mesangial cell proliferation.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0416fje; to cite this article, use FASEB J. (September 5, 2002) 10.1096/fj.02-0416fje

This Article Full Text (PDF) All Versions of this Article: 16/13/1817 02-0416fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by McMAHON, B. Articles by GODSON, D. C. Search for Related Content PubMed PubMed Citation Articles by McMAHON, B. Articles by GODSON, D. C.