PAF enhances MMP-2 production in rat aortic VSMCs via a β-arrestin2-dependent ERK signaling pathway

Abstract

Platelet-activating factor (PAF), 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, is a potent phospholipid mediator and has been reported to be localized in atherosclerotic plaque. However, its role in the progression of atherosclerosis remains unclear. In the present study, we investigated the role of PAF in the production of matrix metalloproteinase (MMP) in primary vascular smooth muscle cells (VSMCs). When rat aortic primary VSMCs were stimulated with PAF (1 nmol/l), the expressions of MMP-2 mRNA and protein, but not of MMP-9, were significantly increased, and these upregulations were markedly attenuated by inhibiting extracellular signal-regulated kinases (ERKs) using molecular and pharmacological inhibitors, but not by using inhibitors of p38 mitogen-activated protein kinase or c-Jun N-terminal kinase. Likewise, ERK phosphorylation was markedly enhanced in PAF-stimulated VSMCs, and this was attenuated by WEB2086, but not by EGF receptor inhibitor, demonstrating the specificity of PAF receptor (PAFR) in PAF-induced ERK phosphorylation. In immunofluorescence studies, β-arrestin2 in PAF-stimulated VSMCs colocalized with PAFR and phosphorylated ERK (P-ERK). Coimmunoprecipitation results suggest that β-arrestin2-bound PAFRs existed as a complex with P-ERK. In addition, PAF-induced ERK phosphorylation and MMP-2 production were significantly attenuated by β-arrestin2 depletion. Taken together, the study shows that PAF enhances MMP-2 production in VSMCs via a β-arrestin2-dependent ERK signaling pathway. Platelet-activating factor (PAF), 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, is a potent phospholipid mediator and has been reported to be localized in atherosclerotic plaque. However, its role in the progression of atherosclerosis remains unclear. In the present study, we investigated the role of PAF in the production of matrix metalloproteinase (MMP) in primary vascular smooth muscle cells (VSMCs). When rat aortic primary VSMCs were stimulated with PAF (1 nmol/l), the expressions of MMP-2 mRNA and protein, but not of MMP-9, were significantly increased, and these upregulations were markedly attenuated by inhibiting extracellular signal-regulated kinases (ERKs) using molecular and pharmacological inhibitors, but not by using inhibitors of p38 mitogen-activated protein kinase or c-Jun N-terminal kinase. Likewise, ERK phosphorylation was markedly enhanced in PAF-stimulated VSMCs, and this was attenuated by WEB2086, but not by EGF receptor inhibitor, demonstrating the specificity of PAF receptor (PAFR) in PAF-induced ERK phosphorylation. In immunofluorescence studies, β-arrestin2 in PAF-stimulated VSMCs colocalized with PAFR and phosphorylated ERK (P-ERK). Coimmunoprecipitation results suggest that β-arrestin2-bound PAFRs existed as a complex with P-ERK. In addition, PAF-induced ERK phosphorylation and MMP-2 production were significantly attenuated by β-arrestin2 depletion. Taken together, the study shows that PAF enhances MMP-2 production in VSMCs via a β-arrestin2-dependent ERK signaling pathway. Platelet-activating factor (PAF) has been reported to be present in human atherosclerotic plaque, and to induce the production of reactive oxygen species and elastase in macrophages that degrade the extracellular matrix of intima (1Stengel D. Antonucci M. Arborati M. Hourton D. Griglio S. Chapman M.J. Ninio E. Expression of the PAF receptor in human monocyte-derived macrophages is downregulated by oxidized LDL: relevance to the inflammatory phase of atherogenesis.Arterioscler. Thromb. Vasc. Biol. 1997; 17: 954-962Crossref PubMed Scopus (24) Google Scholar). Furthermore, PAF is known to act as an initial trigger in atherosclerosis, and to play a critical role during disease progression (2Nasopoulou C. Tsoupras A.B. Karantonis H.C. Demopoulos C.A. Zabetakis I. Fish polar lipids retard atherosclerosis in rabbits by down-regulating PAF biosynthesis and up-regulating PAF catabolism.Lipids Health Dis. 2011; 10: 213Crossref PubMed Scopus (42) Google Scholar, 3Demopoulos C.A. Karantonis H.C. Antonopoulou S. Platelet activating factor - a molecular link between atherosclerosis theories.Eur. J. Lipid Sci. Technol. 2003; 105: 705-716Crossref Scopus (127) Google Scholar). The biological actions of PAF are regulated by a specific surface 7-transmembrane-domain receptor (7TMR), which couples to G proteins (4Honda Z. Ishii S. Shimizu T. Platelet-activating factor receptor.J. Biochem. 2002; 131: 773-779Crossref PubMed Scopus (175) Google Scholar). However, although the PAF receptor (PAFR) is known to be present in smooth muscle cells, the functional significance of intracellular PAFR has not been clarified (5Montrucchio G. Alloatti G. Camussi G. Role of platelet-activating factor in cardiovascular pathophysiology.Physiol. Rev. 2000; 80: 1669-1699Crossref PubMed Scopus (325) Google Scholar). Numerous G protein-coupled receptors (GPCRs) activate mitogen-activated protein kinase (MAPK) after specific agonist stimulation (6Kovacs J.J. Hara M.R. Davenport C.L. Kim J. Lefkowitz R.J. Arrestin development: Emerging roles for β-arrestins in developmental signaling pathways.Dev. Cell. 2009; 17: 443-458Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 7Pierce K.L. Maudsley S. Daaka Y. Luttrell L.M. Lefkowitz R.J. Role of endocytosis in the activation of the extracellular signal-regulated kinase cascade by sequestering and nonsequestering G protein-coupled receptor.Proc. Natl. Acad. Sci. 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Platelet-activating factor induces ovine fetal pulmonary venous smooth muscle cell proliferation: role of epidermal growth factor receptor transactivation.Am. J. Physiol. Heart Circ. Physiol. 2007; 292: H2773-H2781Crossref PubMed Scopus (28) Google Scholar). Extracellular signal-regulated kinase (ERK) activated by 7TMR may translocate from the cytosol to the nucleus where it activates transcription factors that modulate transcription programs such as that of cAMP response element binding protein (11Manson M.E. Corey D.A. Rymut S.M. Kelley T.J. β-arrestin-2 regulation of the cAMP response element binding protein.Biochemistry. 2011; 50: 6022-6029Crossref PubMed Scopus (17) Google Scholar). On the other hand, phosphorylated ERK (P-ERK) retained in cytosol activates various cytosolic substrates to modulate cell shape and motility (12Ge L. Ly Y. Hollenberg M. DeFea K. A β-arrestin-dependent scaffold is associated with prolonged MAPK activation in Pseudopodia during protease-activated receptor-2-induced chemotaxis.J. Biol. Chem. 2003; 278: 34418-34426Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). Moreover, it has recently been found that 7TMR can use multifunctional adaptor proteins such as β-arrestins to activate many substrates in cellular pathways (13Lefkowitz R.J. Shenoy S.K. Transduction of receptor signals by beta-arrestins.Science. 2005; 308: 512-517Crossref PubMed Scopus (1427) Google Scholar). Initially, β-arrestin was found to bind activated GRK-phosphorylated 7TMRs and thus block or “desensitize” G protein activation induced by agonist stimulation (14Vroon A. Heijnen C.J. Kavelaars A. GRKs and arrestins: regulators of migration and inflammation.J. Leukoc. Biol. 2006; 80: 1214-1221Crossref PubMed Scopus (136) Google Scholar). β-Arrestin acts as a bifunctional cellular mediator, that is, it not only terminates G protein signaling, but can also function as a scaffold for the signal transduction of MAPKs including ERK (15DeFea K.A. Zalevsky J. Thoma M.S. Déry O. Mullins R.D. Bunnett N.W. β-Arrestin-dependent endocytosis of proteinase-activated receptor 2 is required for intracellular targeting of activated ERK1/2.J. Cell Biol. 2000; 148: 1267-1281Crossref PubMed Scopus (687) Google Scholar, 16Luttrell L.M. Roudabush F.L. Choy E.W. Miller W.E. Field M.E. Pierce K.L. Lefkowitz R.J. Activation and targeting of extracellular signal regulated kinases by β-arrestin scaffolds.Proc. Natl. Acad. Sci. USA. 2001; 98: 2449-2454Crossref PubMed Scopus (703) Google Scholar). Matrix metalloproteinases (MMPs), especially MMP-2 and MMP-9, destabilize lesions and erode the fibrous cap of atherosclerotic plaque by digesting extracellular matrix proteins (17Johnson J.L. Matrix metalloproteinases: influence on smooth muscle cells and atherosclerotic plaque stability.Expert Rev. Cardiovasc. Ther. 2007; 5: 265-282Crossref PubMed Scopus (134) Google Scholar). In addition, MMP-2 can function as a mediator of vascular structural changes associated with physiologic and pathologic processes (18Risinger Jr, G.M. Hunt T.S. Updike D.L. Bullen E.C. Howard E.W. Matrix metalloproteinase-2 expression by vascular smooth muscle cells is mediated by both stimulatory and inhibitory signals in response to growth factors.J. Biol. Chem. 2006; 281: 25915-25925Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). In vivo studies have shown that MMP-2 expression in vascular smooth muscle cell (VSMCs) is associated with various pathologic conditions, especially with atherosclerotic plaque, which exhibit significantly enhanced MMP-2 expression and activation in vulnerable regions (19Shah P.K. Galis Z.S. Matrix metalloproteinase hypothesis of plaque rupture-players keep piling up but questions remain.Circulation. 2001; 104: 1878-1880Crossref PubMed Scopus (120) Google Scholar, 20Caird J. Napoli C. Taggart C. Farrell M. Bouchier-Hayes D. Matrix metalloproteinases 2 and 9 in human atherosclerotic and non-atherosclerotic cerebral aneurysms.Eur. J. Neurol. 2006; 13: 1098-1105Crossref PubMed Scopus (57) Google Scholar), thus suggesting MMP-2 plays a critical role in the fate of atherosclerotic plaque. To determine the importance of PAF in the progression of atherosclerosis, we investigated the roles of PAF and PAFR on the production of MMPs in rat aortic primary VSMCs. In addition, we sought to identify the signal pathways involved in PAF-mediated MMP production in VSMCs. PAF, norepinephrine (NE), Bay11-7082, and gelatin were purchased from Sigma (St. Louis, MO). The various signal pathway inhibitors were purchased from Calbiochem (La Jolla, CA). MMP-2 antibody, MAPK antibodies, and phosphospecific antibodies against MAPKs were from Cell Signaling Technology (Beverly, MA). β-Arrestin2 and PAFR antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Calponin and α-smooth muscle actin antibodies were purchased from Sigma. Horseradish peroxidase (HRP)-conjugated IgG (Santa Cruz Biotechnology) was used as the secondary antibody. All animal procedures were performed in accordance with our institutional guidelines for animal research and approved by our institutional animal care and use committee. VSMCs were isolated by enzymatic dissociation from the aortas of 7- to 8-week-old male Sprague-Dawley rats (Charles River Breeding Laboratories, Kingston, NY). Briefly, aortas were dissected, cut into ~1 mm2 segments, and then placed as explants in cell culture dishes containing DMEM (Gibco BRL, Grand Island, NY) with 10% fetal bovine serum (FBS). VSMC purity was determined by staining with smooth-muscle-specific-actin monoclonal antibodies (Sigma). Cells were maintained in DMEM containing 10% FBS at 37°C in a 5% CO2 incubator. Human aortic VSMCs, obtained from American Type Culture Collection, were maintained using a growth medium kit (American Type Culture Collection) at 37°C in a 5% CO2 incubator. Protein levels of MMP-2, β-arrestin2, and phosphorylated MAPKs were determined by Western blotting. Briefly, cell lysates of VSMCs were separated on 8% sodium dodecyl sulfate (SDS)-polyacrylamide gels, transferred electrophoretically onto nitrocellulose membranes, which were then blocked with 5% skim milk and incubated with primary antibodies in blocking buffer. Blots were incubated with HRP-conjugated secondary antibody and chemiluminescence intensities were measured using a LAS-3000 SYSTEM (Fuji Photo Film, Japan). Membranes were reblotted with anti-β-actin antibody (MP Biomedicals, Aurora, OH) for control purposes. To assess MMP-2 activities, extracellular medium from cultured VSMCs was collected, concentrated 30-fold using a Vivaspin2 Centricon (Sartorius Biolab products, Sartorius AG) and electrophoretically separated in 8% SDS-polyacrylamide gels containing 1 mg/ml gelatin. After washing with wash buffer (2.5% Triton X-100 in 50 mM Tris-HCl, pH 7.4), gels were stained with 0.2% Coomassie Brilliant Blue R-250 (Sigma) for 2 and then in the which were obtained using are as MMP The expressions of MMP-2 and mRNA in VSMCs were by using mRNA as an in cultured cells was isolated using and into using the transcription of by was performed using specific for MMP-2 and The from was by using the and the the and are in were using from and The obtained was into and the of the were by and factor binding the were using a from was using a kit CA). After cells been with MMP-2 using in cell lysates were determined using a and a for ERK and control were purchased from β-Arrestin2 was purchased from Santa Cruz cells were in and for Cells were then with ERK β-arrestin2 or using to the VSMCs on were with and binding were blocked with The cells were then incubated with specific antibodies, in and incubated with IgG and IgG was stained with in for at Cells were then in and a cells were with the by Kim S.M. with to a complex that signals to the cell growth 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). Briefly, cell were with 2 of and incubated for 2 were with buffer and with buffer. were then by Western as are as were by by for of of were on the that the VSMC present in atherosclerotic lesions from that in we of VSMCs to different shown in and were in 1 VSMCs, but not in cells, that VSMC from the to the during When VSMCs were stimulated with PAF (1 nmol/l), MMP-2 mRNA and MMP-2 protein in and VSMCs were significantly increased, On the other hand, PAF-induced MMP-2 production in VSMCs was to that in VSMCs. we used cells which levels of cells in the To MMP-2 in PAF-stimulated VSMCs, were into VSMCs and was MMP-2 in PAF-stimulated VSMCs to at 1 and was significantly after stimulation for 2 shown in MMP-2 mRNA expression and protein production and after and of PAF The of MMP-2 were also by 1 of PAF and at In addition, VSMCs were with of PAF nmol/l), MMP-2 mRNA and protein levels in a up to a PAF of 1 a in MMP-2 which at a PAF of 1 However, the mRNA expression and protein production were by PAF PAF-stimulated production and were significantly by 2 of PAFR that PAFR plays a role in PAF-induced production in VSMCs. To assess the of MAPK in PAF-induced MMP-2 production in VSMCs, cells were with inhibitors of that is, ERK p38 MAPK or c-Jun N-terminal kinase for and then stimulated with PAF (1 for shown in PAF-induced MMP-2 production was significantly attenuated by but not by or Moreover, PAF-induced MMP-2 production was by other signaling inhibitors, or factor which of the ERK signaling pathway in MMP-2 by to that PAF-induced MMP-2 production is mediated via ERK signaling we VSMCs with ERK targeting It was found that VSMCs with ERK in ERK expression PAF-induced MMP-2 production and were significantly by ERK the of ERK pathways in PAF-induced MMP-2 also investigated the of the of PAF on the of and c-Jun N-terminal kinase with PAF (1 nmol/l), levels from 1 at and However, and levels up to To determine is involved in PAF-induced ERK VSMCs were with or WEB2086, and then stimulated with PAF (1 shown in ERK phosphorylation in cells was significantly attenuated by WEB2086, but not by thus demonstrating PAFR in PAF-induced ERK phosphorylation in VSMCs. The immunofluorescence study that β-arrestin2 colocalized with PAFR after of PAF in VSMCs, and that colocalized with β-arrestin2 at suggest that β-arrestin2 and PAFRs be with after to of to PAF In addition, we these in human aortic VSMCs. shown in β-arrestin2 colocalized with PAFR after of PAF Furthermore, in VSMCs was activated after of PAF and colocalized with β-arrestin2 after In addition, PAF-induced MMP-2 production was attenuated by in human VSMCs used to study the of PAF on the between and β-arrestin2 in VSMCs using PAFR antibody as and or β-arrestin2 as we significantly between these proteins in VSMCs in suggest that β-arrestin2-bound PAFRs are with P-ERK. To the of β-arrestin2 on PAF-induced ERK VSMCs with β-arrestin2 were stimulated with PAF or agonist of ERK activation to G protein and ERK were shown in PAF not ERK phosphorylation in VSMCs with β-arrestin2 levels in cells were significantly by In addition, PAF to MMP-2 or production in cells The present study the signal pathways by which PAF enhances MMP-2 production in rat aortic primary VSMCs. MMP-2 production in PAF-stimulated VSMCs was attenuated by inhibiting the ERK pathway and by In addition, PAF enhanced ERK and this was significantly by β-arrestin2 in VSMCs. results the hypothesis that PAF enhances MMP-2 production in VSMCs via the β-arrestin2-dependent activation of ERK signaling The primary function of VSMCs is Furthermore, VSMCs have and a of T. of vascular smooth muscle cell 2005; PubMed Scopus Google Scholar). However, the of VSMCs to a of that cells to a of and induce changes that to the of cardiovascular T. of vascular smooth muscle cell 2005; PubMed Scopus Google Scholar). VSMC in atherosclerotic lesions from in of we of VSMCs that such as and at different In with a which significantly and levels in cells L. J. Y. Y. J. C.J. Y. Y. matrix protein the of vascular smooth muscle cells by with PubMed Scopus Google Scholar), the expression of and in VSMCs was markedly cells, that the VSMC from to during However, PAF-induced MMP-2 production in VSMCs was to that in VSMCs, we used cells in the MMP-2 is in VSMCs in and in atherosclerotic (18Risinger Jr, G.M. Hunt T.S. Updike D.L. Bullen E.C. Howard E.W. Matrix metalloproteinase-2 expression by vascular smooth muscle cells is mediated by both stimulatory and inhibitory signals in response to growth factors.J. Biol. Chem. 2006; 281: 25915-25925Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). In in vivo studies, MMP-2 production by VSMCs was found to be to a of conditions, including vulnerable regions in atherosclerotic plaque M.R. Kim enhances MMP-2 production in vascular smooth muscle cells via activation of the signaling Biol. PubMed Scopus Google Scholar, Matrix metalloproteinases and of vascular smooth muscle cells by matrix and 2006; PubMed Scopus Google Scholar). PAF and its are factors during atherosclerotic (2Nasopoulou C. Tsoupras A.B. Karantonis H.C. Demopoulos C.A. Zabetakis I. Fish polar lipids retard atherosclerosis in rabbits by down-regulating PAF biosynthesis and up-regulating PAF catabolism.Lipids Health Dis. 2011; 10: 213Crossref PubMed Scopus (42) Google Scholar), and are by cells and cells localized in human atherosclerotic (1Stengel D. Antonucci M. Arborati M. Hourton D. Griglio S. Chapman M.J. Ninio E. Expression of the PAF receptor in human monocyte-derived macrophages is downregulated by oxidized LDL: relevance to the inflammatory phase of atherogenesis.Arterioscler. Thromb. Vasc. Biol. 1997; 17: 954-962Crossref PubMed Scopus (24) Google Scholar). However, the role of PAF in the progression of atherosclerosis is we to the role by PAF in the production of MMPs in VSMCs. It was found PAF 1 significantly enhanced MMP-2 production in VSMCs. the of PAF at a of 1 a in ERK but in of p38 MAPK or are with a by and Raj (10Zhou W. Ibe B.O. Raj J.U. Platelet-activating factor induces ovine fetal pulmonary venous smooth muscle cell proliferation: role of epidermal growth factor receptor transactivation.Am. J. Physiol. Heart Circ. Physiol. 2007; 292: H2773-H2781Crossref PubMed Scopus (28) Google Scholar), in which PAF at 1 not the phosphorylation of p38 in fetal ovine venous VSMCs. studies have reported the importance of ERK pathways in MMP-2 production in VSMCs W. J.S. E. J.L. P.K. Jr, phosphorylation in the ERK signaling pathway is associated with in rat aortic smooth muscle Full Text Full Text PDF PubMed Scopus Google Scholar), and in VSMCs Kim M.R. Kim of in MMP-2 production in vascular smooth muscle Full Text Full Text PDF PubMed Scopus Google Scholar). Likewise, we found that PAF-induced MMP-2 production was significantly attenuated by ERK using molecular and pharmacological inhibitors, but not of p38 and which the ERK signaling pathway is for PAF-induced MMP-2 production in VSMCs. in human vascular cells and cells, ERK was found to play a role in the regulation of PAF-induced expression and via a activation S. Platelet-activating factor induces matrix expression or signaling pathway in a human vascular cell 2005; PubMed Scopus Google Scholar, Platelet-activating factor the of in the expression of in human Sci. 2007; PubMed Scopus Google Scholar). However, our results that a specific not MMP-2 production induced by PAF in VSMCs, which that MMP-2 production by PAF be independent on the pathway. are required to identify the transcription factors involved in MMP-2 production in PAF-stimulated VSMCs. The signaling activation are complex and may from the of classical G from between 7TMRs and receptor tyrosine kinases the EGF (10Zhou W. Ibe B.O. Raj J.U. Platelet-activating factor induces ovine fetal pulmonary venous smooth muscle cell proliferation: role of epidermal growth factor receptor transactivation.Am. J. Physiol. Heart Circ. Physiol. 2007; 292: H2773-H2781Crossref PubMed Scopus (28) Google or from β-arrestin on D. M. E. S. S. C.L. Luttrell L.M. and G pathways for Biol. Chem. 2006; 281: Full Text Full Text PDF PubMed Scopus Google reported that in ERK activation are receptor and cell In phosphorylation was found to be mediated by a β-arrestin-dependent but in cells β-arrestin-dependent and G were found to to the activation of In the present study, PAF-induced ERK phosphorylation was significantly attenuated by a PAFR but not by an EGF receptor inhibitor, thus the importance of PAFR for PAF-induced ERK phosphorylation in VSMCs. The intracellular and of GPCRs may with Furthermore, are required for PAFR after specific agonist stimulation Z. C. M. J. of the platelet-activating factor receptor is on but independent of Role of the and the Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). In is to the nucleus via the G not translocate to the nucleus β-arrestin as a scaffold for and (13Lefkowitz R.J. Shenoy S.K. Transduction of receptor signals by beta-arrestins.Science. 2005; 308: 512-517Crossref PubMed Scopus (1427) Google Scholar, extracellular signal-regulated kinases (ERKs) translocate to nucleus in to G ERK PubMed Scopus Google Scholar). In addition, a in the the of the which activated to translocate to the nucleus extracellular signal-regulated kinases (ERKs) translocate to nucleus in to G ERK PubMed Scopus Google Scholar, A. Pierce K.L. Choy E.W. Lefkowitz R.J. Luttrell L.M. of the ERK cascade enhances cytosolic ERK but transcription receptor Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). In the present study, we found that in VSMCs with PAF, β-arrestin2-bound PAFRs colocalized with P-ERK. Furthermore, as was these not exhibit from cytosol to the In addition, in cells, the activation of ERK by was but ERK phosphorylation induced in PAF-stimulated VSMCs was markedly results that β-arrestin2 is for PAF-induced ERK phosphorylation in VSMCs. results are by a by A. Pierce K.L. Choy E.W. Lefkowitz R.J. Luttrell L.M. of the ERK cascade enhances cytosolic ERK but transcription receptor Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar), in which mediated by β-arrestin was found to be retained in the Taken together, we that MMP-2 production in rat aortic primary VSMCs via the activation of a β-arrestin-dependent ERK signaling pathway. these a for the of an to atherosclerotic plaque with epidermal growth factor receptor extracellular signal-regulated kinase G protein-coupled receptor c-Jun N-terminal kinase mitogen-activated protein kinase matrix metalloproteinase norepinephrine factor 1 platelet-activating factor platelet-activating factor receptor phosphorylated extracellular signal-regulated kinase 7-transmembrane-domain receptor vascular smooth muscle cell