Lipoxins Inhibit Akt/PKB Activation and Cell Cycle Progression in Human Mesangial Cells
2004; Elsevier BV; Volume: 164; Issue: 3 Linguagem: Inglês
10.1016/s0002-9440(10)63181-1
ISSN1525-2191
AutoresDerick Mitchell, Karen Rodgers, Jennifer Hanly, Blaithin A. McMahon, Hugh R. Brady, Finian Martin, Catherine Godson,
Tópico(s)Kruppel-like factors research
ResumoLipoxins (LX) are endogenously produced eicosanoids with a spectrum of bioactions that suggest anti-inflammatory, pro-resolution roles for these agents. Mesangial cell (MC) proliferation plays a pivotal role in the pathophysiology of glomerular inflammation and is coupled to sclerosis and tubulointerstitial fibrosis. We have previously reported that LXA4 acts through a specific G-protein-coupled-receptor (GPCR) to modulate MC proliferation in response to the proinflammatory mediators LTD4 and platelet-derived growth factor (PDGF). Further investigations revealed that these effects were mediated by modulation of receptor tyrosine kinase activity. Here we have explored the underlying mechanisms and report inhibition of growth factor (PDGF; epithelial growth factor) activation of Akt/PKB by LXA4. LXA4 (10 nmol/L) modulates PDGF-induced (10 ng/ml, 24 hours) decrements in the levels of cyclin kinase inhibitors p21Cip1 and p27Kip1. PDGF-induced increases in CDK2-cyclin E complex formation are also inhibited by LXA4. The potential of LXA4 as an anti-inflammatory therapeutic is compromised by its degradation; this has been circumvented by synthesis of stable analogs. We report that 15-(R/S)-methyl-LXA4 and 16-phenoxy-LXA4 mimic the native compound with respect to modulation of cell proliferation and PDGF-induced changes in cell cycle proteins. In vivo, MC proliferation in response to PDGF is associated with TGFβ1 production and the subsequent development of renal fibrosis. Here we demonstrate that prolonged (24 to 48 hours) exposure to PDGF is associated with autocrine TGFβ1 production, which is significantly reduced by LXA4. In aggregate these data demonstrate that LX inhibit PDGF stimulated proliferation via modulation of the PI-3-kinase pathway preventing mitogen-elicited G1-S phase progression and suggest the therapeutic potential of LX as anti-fibrotic agents. Lipoxins (LX) are endogenously produced eicosanoids with a spectrum of bioactions that suggest anti-inflammatory, pro-resolution roles for these agents. Mesangial cell (MC) proliferation plays a pivotal role in the pathophysiology of glomerular inflammation and is coupled to sclerosis and tubulointerstitial fibrosis. We have previously reported that LXA4 acts through a specific G-protein-coupled-receptor (GPCR) to modulate MC proliferation in response to the proinflammatory mediators LTD4 and platelet-derived growth factor (PDGF). Further investigations revealed that these effects were mediated by modulation of receptor tyrosine kinase activity. Here we have explored the underlying mechanisms and report inhibition of growth factor (PDGF; epithelial growth factor) activation of Akt/PKB by LXA4. LXA4 (10 nmol/L) modulates PDGF-induced (10 ng/ml, 24 hours) decrements in the levels of cyclin kinase inhibitors p21Cip1 and p27Kip1. PDGF-induced increases in CDK2-cyclin E complex formation are also inhibited by LXA4. The potential of LXA4 as an anti-inflammatory therapeutic is compromised by its degradation; this has been circumvented by synthesis of stable analogs. We report that 15-(R/S)-methyl-LXA4 and 16-phenoxy-LXA4 mimic the native compound with respect to modulation of cell proliferation and PDGF-induced changes in cell cycle proteins. In vivo, MC proliferation in response to PDGF is associated with TGFβ1 production and the subsequent development of renal fibrosis. Here we demonstrate that prolonged (24 to 48 hours) exposure to PDGF is associated with autocrine TGFβ1 production, which is significantly reduced by LXA4. In aggregate these data demonstrate that LX inhibit PDGF stimulated proliferation via modulation of the PI-3-kinase pathway preventing mitogen-elicited G1-S phase progression and suggest the therapeutic potential of LX as anti-fibrotic agents. Lipoxins (LX) are endogenously produced eicosanoids with potent anti-inflammatory bioactions.1McMahon BM Mitchell S Brady HR Godson C Lipoxins: revelations on resolution.Trends Pharmacol Sci. 2001; 22: 391-395Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar LX are typically generated by transcellular metabolism initiated by the sequential actions of 15- and 5-, or 5- and 12-lipoxygenases on arachidonic acid, depending on the cellular context.2Serhan CN Lipoxins and novel aspirin-triggered 15-epi-lipoxins[ATL]: a jungle of cell-cell interactions or a therapeutic opportunity?.Prostaglandins. 1997; 53: 107-137Crossref PubMed Scopus (222) Google Scholar In a cytokine-primed milieu, aspirin-induced acetylation of COX-2 shifts its activity from an endoperoxide to a lipoxygenase, thereby promoting synthesis of 15-epi-LX [aspirin-triggered lipoxins (ATL)].1McMahon BM Mitchell S Brady HR Godson C Lipoxins: revelations on resolution.Trends Pharmacol Sci. 2001; 22: 391-395Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar It has been proposed that some of the desirable effects of aspirin independent of its anti-thrombotic actions may be attributed to the biosynthesis of anti-inflammatory lipid mediators such as ATLs.2Serhan CN Lipoxins and novel aspirin-triggered 15-epi-lipoxins[ATL]: a jungle of cell-cell interactions or a therapeutic opportunity?.Prostaglandins. 1997; 53: 107-137Crossref PubMed Scopus (222) Google Scholar It is increasingly appreciated that there are two distinct phases of lipid mediator production in inflammation, an initial phase characterized by the production of proinflammatory mediators essential to effective host defense, which is superseded by the production of anti-inflammatory mediators such as LX during the resolution phase.3Levy BD Clish CB Schmidt B Gronert K Serhan CN Lipid mediator class switching during acute inflammation: signals in resolution.Nat Med. 2001; 2: 612-619Google Scholar LXA4 and LXB4 are the principal LX formed in mammals. In particular, LXA4 has been shown to exert potent anti-inflammatory actions, modulating leukocyte trafficking and phagocytic clearance of apoptotic cells.1McMahon BM Mitchell S Brady HR Godson C Lipoxins: revelations on resolution.Trends Pharmacol Sci. 2001; 22: 391-395Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 3Levy BD Clish CB Schmidt B Gronert K Serhan CN Lipid mediator class switching during acute inflammation: signals in resolution.Nat Med. 2001; 2: 612-619Google Scholar LX biosynthesis has been demonstrated in many inflammatory conditions such as the glomerulonephritides (GN),4Papayianni A Serhan CN Phillips ML Rennke HG Brady HR Transcellular biosynthesis of lipoxin A4 during adhesion of platelets and neutrophils in experimental immune complex glomerulonephritis.Kidney Int. 1995; : 1295-1302Crossref PubMed Scopus (119) Google Scholar human pleural disease,3Levy BD Clish CB Schmidt B Gronert K Serhan CN Lipid mediator class switching during acute inflammation: signals in resolution.Nat Med. 2001; 2: 612-619Google Scholar and in experimental models where the potential for cell-cell interactions is significant.5Mayadas TN Mendrick DL Brady HR Tang T Papayianni A Assmann KJ Wagner DD Hynes RO Cotran RS Acute passive anti-glomerular basement membrane nephritis in P-selectin-deficient mice.Kidney Int. 1996; 49: 1342-1349Crossref PubMed Scopus (81) Google Scholar LXA4 may exert its anti-inflammatory effects through signals generated by binding to a high-affinity, G-protein-coupled LXA4 receptor (ALXR).2Serhan CN Lipoxins and novel aspirin-triggered 15-epi-lipoxins[ATL]: a jungle of cell-cell interactions or a therapeutic opportunity?.Prostaglandins. 1997; 53: 107-137Crossref PubMed Scopus (222) Google Scholar LX are rapidly metabolized, the major routes of degradation being dehydrogenation at C-15 and possibly ω-oxidation at C-20.2Serhan CN Lipoxins and novel aspirin-triggered 15-epi-lipoxins[ATL]: a jungle of cell-cell interactions or a therapeutic opportunity?.Prostaglandins. 1997; 53: 107-137Crossref PubMed Scopus (222) Google Scholar To circumvent such metabolic inactivation, stable synthetic analogs have been developed that are modified at C-15, C-16, and/or C-20.6Clish CB O'Brien JA Gronert K Stahl GL Petasis NA Serhan CN Local and systemic delivery of an aspirin-triggered lipoxin prevents neutrophil recruitment in vivo.Proc Natl Acad Sci USA. 1999; 96: 8247-8252Crossref PubMed Scopus (199) Google Scholar These compounds retain the biological activity of native LXs and have been shown to bind with higher affinity to the ALXR, resulting in greater potency.2Serhan CN Lipoxins and novel aspirin-triggered 15-epi-lipoxins[ATL]: a jungle of cell-cell interactions or a therapeutic opportunity?.Prostaglandins. 1997; 53: 107-137Crossref PubMed Scopus (222) Google Scholar, 6Clish CB O'Brien JA Gronert K Stahl GL Petasis NA Serhan CN Local and systemic delivery of an aspirin-triggered lipoxin prevents neutrophil recruitment in vivo.Proc Natl Acad Sci USA. 1999; 96: 8247-8252Crossref PubMed Scopus (199) Google Scholar The enhanced stability and improved efficacy of these analogs following local and systemic administration in models of inflammation and ischemia-reperfusion suggests significant therapeutic potential.4Papayianni A Serhan CN Phillips ML Rennke HG Brady HR Transcellular biosynthesis of lipoxin A4 during adhesion of platelets and neutrophils in experimental immune complex glomerulonephritis.Kidney Int. 1995; : 1295-1302Crossref PubMed Scopus (119) Google Scholar Mesangial cells (MC) are modified smooth muscle cells that play a pivotal role in renal physiology by regulating circulation and glomerular structural integrity. Mesangial cell proliferation and or matrix accumulation characterizes many forms of GN and other progressive renal diseases including diabetic nephropathy. MC proliferation can be induced by several mitogens, including platelet-derived growth factor (PDGF) isoforms,7Abboud HE Growth factors in glomerulonephritis.Kidney Int. 1993; 43: 252-267Crossref PubMed Scopus (183) Google Scholar epidermal growth factor (EGF)8Silver BJ Jaffer FE Abboud HE Platelet-derived growth factor synthesis in mesangial cells: induction by multiple growth factors.Proc Natl Acad Sci. 1989; 86: 1056-1060Crossref PubMed Scopus (226) Google Scholar and eicosanoids such as the cysteinyl leukotriene D4.9McMahon B Stenson C McPhillips F Fanning A Brady HR Godson C Lipoxin A4 antagonises the mitogenic effect of LTD4 on human renal mesangial cells: differential activation of MAP kinases through distinct receptors.J Biol Chem. 2000; 275: 27566-27575Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar PDGF has been widely implicated in the etiology of GN, triggering MC proliferation, migration, contraction, and synthesis of other cytokines (eg, TGFβ1 and IL-1). In this context, blockade of PDGF bioactions with anti-PDGF antibody10Johnson RJ Raines EW Floege J Yoshimura A Pritzl P Alpers C Ross R Inhibition of mesangial cell proliferation and matrix expansion in glomerulonephritis in the rat by antibody to platelet-derived growth factor.J Exp Med. 1992; 175: 1413-1416Crossref PubMed Scopus (353) Google Scholar aptamers,11Floege J Ostendorf T Janssen U Burg M Radeke HH Vargeese C Gill SC Green LS Janjic N Novel approach to specific growth factor inhibition in vivo: antagonism of platelet-derived growth factor in glomerulonephritis byaptamers.Am J Pathol. 1999; 154: 169-179Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar soluble receptors,12Ostendorf T van Roeyen CR Peterson JD Kunter U Eitner F Hamad AJ Chan G Jia XC Macaluso J Gazit-Bornstein G Keyt BA Lichenstein HS LaRochelle WJ Floege J A fully human monoclonal antibody (CR002) identifies PDGF-D as a novel mediator of mesangioproliferative glomerulonephritis.J Am Soc Nephrol. 2003; 14: 2237-2247Crossref PubMed Scopus (83) Google Scholar or inhibitors of receptor activation13Gilbert RE Kelly DJ McKay T Chadban S Hill PA Cooper ME Atkins RC Nikolic-Paterson DJ PDGF signal transduction inhibition ameliorates experimental mesangial proliferative glomerulonephritis.Kidney Int. 2001; 59: 1324-1332Crossref PubMed Scopus (120) Google Scholar have been proposed as therapeutic strategies in proliferative GN. Such blockade of PDGF activity inhibits mesangioproliferative changes, scarring and interstitial fibrosis.14Floege J Johnson RJ Multiple roles for platelet-derived growth factor in renal disease.Minor Electrolyte Metab. 1995; 21: 271-282PubMed Google Scholar The mitogenic actions of PDGF in MC are mediated via the PDGFR, a member of the receptor tyrosine kinase (RTK) family. Predominant among the PDGF isoforms that are mitogenic for MC is PDGF B which acts via the PDGFRβ.7Abboud HE Growth factors in glomerulonephritis.Kidney Int. 1993; 43: 252-267Crossref PubMed Scopus (183) Google Scholar Activation of the intrinsic tyrosine kinase activity of the receptor facilitates recruitment of several SH2 domain-containing molecules and associated proteins including the p85 subunit of PI-3-kinase, RasGAP and PLCγ1.15Heldin H Ostmann A Ronnstrand L Signal transduction via platelet-derived growth factor receptors.Biochim Biophysica Acta. 1998; 1378: F79-F113PubMed Google Scholar Our previous investigations have indicated that LXA4 inhibits MC proliferation in response to mitogens such as LTD49McMahon B Stenson C McPhillips F Fanning A Brady HR Godson C Lipoxin A4 antagonises the mitogenic effect of LTD4 on human renal mesangial cells: differential activation of MAP kinases through distinct receptors.J Biol Chem. 2000; 275: 27566-27575Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar and PDGF.16McMahon B Mitchell D Shattock R Martin F Brady HR Godson C Lipoxin, leukotriene, and PDGF receptors cross-talk to regulate mesangial cell proliferation.EMBO J. 2002; 16: 1817-1819Google Scholar These potential anti-inflammatory, pro-resolution bioactions of LX involve complex cross-talk between distinct GPCR and receptor tyrosine kinases.16McMahon B Mitchell D Shattock R Martin F Brady HR Godson C Lipoxin, leukotriene, and PDGF receptors cross-talk to regulate mesangial cell proliferation.EMBO J. 2002; 16: 1817-1819Google Scholar We have shown that LX modulate PI-3-kinase activation9McMahon B Stenson C McPhillips F Fanning A Brady HR Godson C Lipoxin A4 antagonises the mitogenic effect of LTD4 on human renal mesangial cells: differential activation of MAP kinases through distinct receptors.J Biol Chem. 2000; 275: 27566-27575Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar and recruitment of the p85 subunit of PI-3-kinase to the activated PDGFR.16McMahon B Mitchell D Shattock R Martin F Brady HR Godson C Lipoxin, leukotriene, and PDGF receptors cross-talk to regulate mesangial cell proliferation.EMBO J. 2002; 16: 1817-1819Google Scholar Here we have investigated the mechanisms underlying LX inhibition of MC proliferation and whether stable synthetic LX analogs can mimic the effects of the native compound in this regard. We report that 15-(R/S)-methyl-LXA4 and 16- phenoxy-LXA4 significantly inhibit PDGF and EGF-stimulated MC proliferation. We demonstrate that LXA4 modulates PDGF-induced decrements in the levels of p21Cip1 and p27Kip1 and promotes nuclear retention of these CKI. Importantly, LXA4 significantly modulated autocrine production of TGFβ1 by PDGF-stimulated MC. MC proliferation in response to PDGF and EGF was coupled to activation of the Ser/Thr kinase, Akt/PKB (Akt), a downstream target of PI-3-kinase. Interestingly, inhibition of PI-3-kinase with LY294002 mimicked the effects of LXA4 with respect to nuclear retention of p27Kip1 in PDGF-stimulated MC. Our data suggest that LXs modulate PDGF-induced proliferation by attenuation of Akt activation and prevention of G1-S progression. LXA4 and LTD4 were obtained from Biomol (Plymouth Meeting, PA). Human recombinant PDGF-BB and EGF were purchased from Upstate Biotechnology (Milton Keynes, UK). TGFβ1 and anti-TGFβ1 polyclonal antibody were from BD Biosciences (Oxford, UK). AG1296 and AG1478 were acquired from Calbiochem (Nottingham, UK). Transfer membranes were from Millipore (Bedford, MA). All other reagents were purchased from Sigma (Poole, Dorset, UK) unless otherwise stated. Stable synthetic LX analogs were a generous gift from Dr. Nicos Petasis, University of Southern California, Los Angeles, CA. Human kidneys were obtained from excess nephrectomy specimens according to the Mater Misericordiae University Hospital ethical guidelines. Renal glomeruli were isolated by differential sieving and mesangial cells were obtained and cultured in RPMI 1640 supplemented with 10% fetal calf serum (FCS), penicillin (100 U/ml), and streptomycin (100 μg/ml) (all purchased from Gibco BRL, Paisley, Scotland). Isolated cells retained the phenotypic characteristics of mesangial cells, including stellate morphology, stained positive for vimentin and α-smooth muscle actin expression, and were negative for factor VIII and cytokeratin excluding endothelial and epithelial cell contamination, respectively.17Mene P Mesangial cell cultures.J Nephrol. 2001; 14: 198-203PubMed Google Scholar The CHOK1 cell line stably expressing ALXR and a control cell line were established and cultured as previously described.16McMahon B Mitchell D Shattock R Martin F Brady HR Godson C Lipoxin, leukotriene, and PDGF receptors cross-talk to regulate mesangial cell proliferation.EMBO J. 2002; 16: 1817-1819Google Scholar For analysis of proliferation, primary cultures of mesangial cells and CHOK1 cells were grown to approximately 70% confluence on 24-well plates before serum restriction in 0.2% FCS RPMI-1640 for 48 hours (MC) or serum deprivation in 0% FCS Ham's F12 medium for 24 hours (CHOK1 cells). After this period, cells were stimulated with various agents in triplicate wells for indicated times (44 hours for MC or 20 hours for CHOK1 cells) as detailed in Figure legends. Proliferation of cells was measured by determining [3H]-thymidine incorporation as follows: 1 μCi [3H]-thymidine (90 to 120 Ci/mmol; NEN, Cambridge, UK) was added to each well and incubated for 4 hours. Cells were washed twice in DPBS, solubilized in 0.2% sodium dodecyl sulfate (SDS) and counts per minute (cpm) were measured in 10 ml of scintillant (σ-Fluor). Data provided are from 4 to 5 independent experiments, as indicated. Mesangial cells were serum restricted in 0.2% FCS RPMI 1640 for 48 hours and exposed to various agents for indicated times. Lysates were harvested in RIPA lysis buffer (20 mmol/L Tris-HCl, pH 7.4, 50 mmol/L NaCl, 5 mmol/L ethylene diaminetetraacetic acid, 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS, 5 mmol/L NaF, 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L Na3VO4, 1 μmol/L leupeptin, 0.3 μmol/L aprotinin). The lysates were clarified by centrifugation at 12,000 × g for 10 minutes and protein concentration in the supernatant was measured by Bradford protein assay. For Western blot analysis, 30 μg of MC protein extract was loaded onto each lane, separated under reducing conditions on an SDS-polyacrylamide gel electrophoresis (PAGE) gel, and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore) by electroblotting. To reduce non-specific antibody binding, the membranes were first blocked with 5% nonfat dried milk for 1 hour at room temperature. This was followed by an overnight incubation at 4°C with antibodies to either cyclin A (Upstate Biotech, Lake Placid, NY), cyclin E (BD Biosciences), CDK2, p21Cip1 or p27Kip1 (all Santa Cruz Biotechnology, Heidelberg, Germany). Controls included omitting the primary antibody and/or replacing the primary antibody with rabbit or mouse serum. Membranes were incubated with a horseradish-peroxidase-conjugated secondary antibody for 1 hour at room temperature and were visualized by chemiluminescence. To check for equal loading, membranes were either stained with Ponceau-S staining solution or were stripped and reprobed for β-actin (Sigma). For analysis of Akt phosphorylation, membranes were blocked with 5% bovine serum albumin (BSA) and probed with antibodies to either phospho-Akt (Ser473) or Akt protein (both New England Biosciences, Hertfordshire, UK). MC lysate (300 μg) from each condition was precleared with Protein-G agarose beads (Santa Cruz) for 1 hour at 4°C before incubation with antibody to CDK2 (Santa Cruz, 1:500 dilution) overnight at 4°C with constant rocking. Protein-G agarose beads (10 μl) were then added to each immunoprecipitation and incubated for 2 hours at 4°C with constant rocking. Immunocomplexes were washed three times in fresh RIPA lysis buffer, denatured in 5X reducing sample buffer and boiled for 5 minutes to elute protein off beads. Samples were electrophoresed on SDS-PAGE gels, transferred to PVDF membranes, and probed for either cyclin E or CDK2 (loading control). Quiescent MC were treated as indicated. At 24-, 48-, and 72-hour time points, medium was removed and assayed for TGFβ1 release by ELISA (R&D systems, Abingdon, UK) as per manufacturer's protocol. TGFβ1 produced was expressed as picogram TGFβ1 per microgram of cellular protein. Using differential centrifugation, nuclear and cytosolic fractions from stimulated cells were harvested using a Nuclear Extract kit (Activemotif, Rixensart, Belgium). Briefly, cell lysates were fractionated at 14,000 × g for 30 seconds. Protein concentration in the pellet and soluble fractions was measured by Bradford protein assay. Cytosolic fractions were concentrated using 10,000 MW cut-off filters (Millipore) to maximize protein yield. Nuclear-cytoplasmic translocation of p27Kip1 was assayed by immunoblotting of cytosolic lysates and densitometric analysis. MC were cultured in 4-well chamber slides (Nalge Nunc, Naperville, IL), rendered quiescent and stimulated as indicated. After stimulation, cells were washed with PBS, fixed with 2% paraformaldehyde (10 minutes) and permeabilized with 0.1% Triton X-100 (15 minutes). Fixed cells were then treated with blocking solution (5% BSA) for 1 hour. Localization of p27Kip1 was determined using anti-p27Kip1 polyclonal antibody (1:500). After washes, samples were treated with Oregon Green-conjugated anti-rabbit IgG (1:200). For DNA staining, samples were incubated with 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI) dye (1 μg/ml, 10 minutes). Cells were viewed and recorded in phase contrast (10× and 40× magnification) and corresponding fields using an Axiovert 200 fluorescent microscope (Carl Zeiss, Jena, Germany). Serum, PDGF, and EGF induced maximal DNA synthesis in primary human MC by 48 hours poststimulation. The mitogenic response of MCs to PDGF-BB and EGF was dose-dependent, with an apparent maximal concentration of 100 ng/ml and 250 ng/ml and an EC50 of 8 ng/ml and 44 ng/ml, respectively. In subsequent experiments, PDGF-BB and EGF were applied at 10 ng/ml and 50 ng/ml concentrations, respectively, which consistently induced a minimum of a 2.5-fold increase in MC DNA synthesis. Incubation of MCs with increasing concentrations of LXA4 did not induce MC DNA synthesis at any concentration tested (10−12 M to 10−8 M). Preincubation of quiescent MC with LXA4 (1 nmol/L) significantly inhibited mitogenesis induced by PDGF (10 ng/ml), as assessed by [3H]-thymidine uptake (Figure 1A). (The effect of LXA4 was found to be dose-dependent [10 pM to 1 μmol/L, data not shown] with an EC50 of approximately 10 nmol/L LXA4, in contrast to other LX responses,9McMahon B Stenson C McPhillips F Fanning A Brady HR Godson C Lipoxin A4 antagonises the mitogenic effect of LTD4 on human renal mesangial cells: differential activation of MAP kinases through distinct receptors.J Biol Chem. 2000; 275: 27566-27575Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar the dose:response was not bell-shaped). This effect was replicated by 16-phenoxy-LXA4 (10 pM), a stable synthetic analog of LXA4, and 15-(R/S)-LXA4 (10 pM), a stable synthetic analog of ATL (Figure 1A). These data indicate that stable LX analog can act as mimetics of endogenous and aspirin-triggered LXA4 in the context of counter regulation of mitogenic signals. AG1296, the tyrophostin inhibitor of PDGF receptor tyrosine kinase activity was included as a negative control in these experiments.18Goppelt-Struebe M Fickel S Reiser CO The platelet-derived-growth-factor receptor, not the epidermal-growth-factor receptor, is used by lysophosphatidic acid to activate p42/44 mitogen-activated protein kinase and to induce prostaglandin G/H synthase-2 in mesangial cells.Biochem J. 2000; 345: 217-224Crossref PubMed Scopus (42) Google Scholar To investigate whether the effect was specific to PDGF-induced proliferation we stimulated cells with LX analogs before treatment with both EGF and serum. As Figure 1B depicts, this anti-proliferative effect was replicated on EGF (50 ng/ml)-stimulated MC proliferation (n = 2). The reduction in DNA synthesis by LX on EGF-treated MC was less than the reduction seen in PDGF-stimulated MC (25% reduction vs. 48% reduction, respectively). Analagous to data with AG1296, the inhibitor of EGF receptor tyrosine kinase activity, AG1478, inhibited EGF-stimulated MC proliferation. The antiproliferative effect of LXA4 analogs was also observed in serum-stimulated CHO K1 cells stably expressing the lipoxin A4 receptor (ALXR) but not in control cells (Figure 1C). Preincubation of quiescent CHOK1 ± ALXR with LXA4 (1 nmol/L, 60′) significantly (P < 0.05, unpaired Student's t-test) inhibited mitogenesis (approximately 20% reduction) induced by serum, an effect replicated by 15-(R/S)-methyl-LXA4 (10 pM) and 16-phenoxy-LXA4 (10 pM). Further investigation of the antiproliferative effects of LX using an assay based on reduction of a tetrazolium component (MTT) to an insoluble formazan product indicate that LXA4 inhibition of serum-stimulated proliferation does not involve promotion of apoptosis (data not shown). We have previously observed a reduction in phosphorylation of the PDGFR in MC by LXA4.16McMahon B Mitchell D Shattock R Martin F Brady HR Godson C Lipoxin, leukotriene, and PDGF receptors cross-talk to regulate mesangial cell proliferation.EMBO J. 2002; 16: 1817-1819Google Scholar This, in conjunction with data showing LXA4-induced inhibition of LTD4-stimulated PI-3-kinase activation9McMahon B Stenson C McPhillips F Fanning A Brady HR Godson C Lipoxin A4 antagonises the mitogenic effect of LTD4 on human renal mesangial cells: differential activation of MAP kinases through distinct receptors.J Biol Chem. 2000; 275: 27566-27575Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar and p85 subunit recruitment to the PDGFR,16McMahon B Mitchell D Shattock R Martin F Brady HR Godson C Lipoxin, leukotriene, and PDGF receptors cross-talk to regulate mesangial cell proliferation.EMBO J. 2002; 16: 1817-1819Google Scholar suggested that the PI3-kinase pathway may be a locus of the inhibitory effect of LXA4 in MC. As indicated in Figure 2, both PDGF (10 ng/ml) (A) and EGF (50 ng/ml) (B) stimulated activation of Akt via phosphorylation at Ser473. Both PDGF- and EGF-stimulated activation was inhibited by preincubation of MC with LXA4 (10 nmol/L, 30 minutes and 60 minutes) and by a specific inhibitor of PI-3 K activity, LY294002 (3 μmol/L). The LXA4-dependent inhibition was not mimicked by two other GPCR-coupled agonists, LTD4 (10 nmol/L) (A) and 5-HT (10 μmol/L) (B) which have been previously shown to be involved in cross-talk with PDGF and EGF RTK, respectively, in mesangial cells.9McMahon B Stenson C McPhillips F Fanning A Brady HR Godson C Lipoxin A4 antagonises the mitogenic effect of LTD4 on human renal mesangial cells: differential activation of MAP kinases through distinct receptors.J Biol Chem. 2000; 275: 27566-27575Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 19Grewal JS Luttrell LM Raymond JR G protein-coupled receptors desensitize and downregulate EGF receptors in renal mesangial cells.J Biol Chem. 2001; 276: 27335-27344Crossref PubMed Scopus (45) Google Scholar Expression of the receptors for both of these GPCR agonists has been previously demonstrated in MC.16McMahon B Mitchell D Shattock R Martin F Brady HR Godson C Lipoxin, leukotriene, and PDGF receptors cross-talk to regulate mesangial cell proliferation.EMBO J. 2002; 16: 1817-1819Google Scholar, 20Nebigil CG Garnovskaya MN Spurney RF Raymond JR Identification of a rat glomerular mesangial cell mitogenic 5-HT2A receptor.Am J Physiol. 1995; 268: F122-F127PubMed Google Scholar The observation that LXA4 inhibition of PDGF and EGF-stimulated proliferation is coupled to modulation of Akt phosphorylation is noteworthy given that expression of dominant-negative Akt in rat mesangial cells inhibits mitogenic responses to PDGF.21Ghosh-Choudhury G Zhang JH Ghosh-Choudhury N Abboud HE Ceramide blocks PDGF-induced DNA synthesis in mesangial cells via inhibition of Akt kinase in the absence of apoptosis.Biochim Biophys Res Commun. 2001; 286: 1183-1190Crossref PubMed Scopus (7) Google Scholar The levels of G1 phase CKI p21Cip1 and p27Kip1 in quiescent and proliferating MC were determined by western blot analysis (Figure 3A). Quiescent MCs were stimulated with LXA4 (10 nmol/L, 60 minutes) or vehicle before addition of PDGF-BB (10 ng/ml, 24 hours). Detectable levels of p21Cip1 and p27 Kip1 were expressed in quiescent MCs and no change in their levels was observed over a 24-hour period in vehicle or LXA4-treated MC (data not shown). However, incubation with PDGF caused a decrease in detectable levels of both p21Cip1 and p27Kip1 at 24 hours poststimulation (Figure 3A). These decreases were significantly abrogated by preincubation with LXA4 (10 nmol/L, 60 minutes) or by co-stimulation with TGFβ1. TGFβ1 has previously been shown to act in an anti-mitotic manner in PDGF-stimulated MC, acting on G1-phase cell cycle proteins.22Schoecklmann HO Rupprecht HD Zauner I Sterzel RB TGF 1-induced cell cycle arrest in renal mesangial cells involves inhibition of cyclin E-cdk 2 activation and retinoblastoma protein phosphorylation.Kidney
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