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Integrin-linked kinase functions as a downstream signal of platelet-derived growth factor to regulate… Esfandiarei, Mitra; Yazdi, Abdoli S; Gray, Virginia; Dedhar, Shoukat; van Breemen, Cornelis Feb 23, 2010

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RESEARCH ARTICLE Open AccessIntegrin-linked kinase functions as a downstreamsignal of platelet-derived growth factor toregulate actin polymerization and vascularsmooth muscle cell migrationMitra Esfandiarei1*, Sahar Abdoli Yazdi1, Virginia Gray2, Shoukat Dedhar2, Cornelis van Breemen1AbstractBackground: Vascular smooth muscle cell migration and accumulation in response to growth factors extensivelycontribute to the development of intimal thickening within the vessel wall. Cumulative evidence has shown thatactin cytoskeleton polymerization and rearrangement are critical steps during cellular spreading and migration.Integrin-linked kinase, an intracellular serine/threonine kinase, is a cytoplasmic interactor of integrin beta-1 andbeta-3 receptors regulating cell-cell and/or cell-extracellular matrix interaction, cell contraction, extracellular matrixmodification, and cell spreading and migration in response to various stimuli. However, the regulatory role of ILKduring vascular smooth muscle cell migration and the importance of integrin signaling in occlusive vasculardiseases are not yet fully elucidated.Results: In the present study, we report that integrin-linked kinase controls mouse aortic smooth muscle cellmigration in response to platelet-derived growth factor. We have also identified p38 mitogen activated proteinkinase as a downstream signaling pathway of the integrin-linked kinase that regulates platelet-derived growthfactor-induced actin polymerization and smooth muscle cell migration.Conclusion: This study will provide new insights into the potential therapeutic value of modulating integrinsignaling in an attempt to block or delay smooth muscle cell migration and the progression of vascular diseases.BackgroundThe initiation and progression of intimal thickening inarterial walls is largely due to migration and subsequentproliferation of smooth muscle cells (SMCs) in the sub-intimal space in response to various stimuli includingoxidized low density lipoprotein (oxLDL), circulatinggrowth factors, and inflammatory cytokines such as pla-telet-derived growth factor (PDGF), tumor necrosis fac-tor (TNF)-a, and interleukin-1 (IL-1) [1,2]. Of these,PDGF, a growth factor released by vascular SMC,endothelial cells, and platelets, has been reported as themost potent inducer of SMC migration within theinjured area of the vascular wall.PDGF is a heparin-binding growth factor composed ofpolypeptide chains that can be assembled into homodi-mers (PDGF-AA, -BB) or heterodimer (PDGF-AB)structures and bind to two related cell-surface receptorswith tyrosine kinase activity, PDGF receptor a and b[3-5]. In the last few years two additional homodimersPDGF-CC and PDGF-DD were also discovered [6-9].PDGF binding to its cognate receptors results in dimeri-zation, and subsequent auto-phosphorylation of specifictyrosine residues outside the kinase domain, creating adocking site for SH2 domain-containing signaling pro-teins. A large number of SH2 domain containing pro-teins including the phsophatidylinositol-3 kinase (PI3K),phospholipase C-g (PLC-g), the tyrosine phosphataseSHP-2, Ras GTPase activating protein (Ras-GAP), Grb2,Grb7, Nck, and the Src family of tyrosine kinases havebeen shown to bind to cytoplasmic tails of PDGF recep-tors activating various downstream signaling proteins* Correspondence: mesfandiarei@gmail.com1Child & Family Research Institute, Department of Anesthesiology,Pharmacology, and Therapeutics, University of British Columbia, 950 West28th Avenue, Vancouver, BC, V5Z 4H4, CanadaEsfandiarei et al. BMC Cell Biology 2010, 11:16http://www.biomedcentral.com/1471-2121/11/16© 2010 Esfandiarei et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.involved in cell growth, proliferation, survival, andmigration [10].Cellular migration is regulated via a complex interac-tion between growth factors that attach to their cognatereceptors, transmembrane integrins that bind to thecomponents of extracellular matrix, and mechanicalstress, all of which cooperatively induce polymerizationand reorganization of the actin cytoskeleton. Theseevents are coordinated by mechanisms involved inassembly or disassembly of local adhesion sites, transientchanges in actin filaments dynamics, and formation ofdiscrete structures such as stress actin fibers, membraneruffles, lamellipodia, and filopodia [11-13]. Integrin-linked kinase (ILK), a serine-threonine protein kinasecontaining a catalytic domain at C terminus, a centralpleckstrin homology (PH)-like domain, and fourankyrin-like repeats at the N terminus, is an importantcomponent of focal adhesion complex anchoring actinfilaments to integrin receptors and the cell membrane[14]. Previous studies have demonstrated that ILK regu-lates fibroblast migration through the phosphatidylinosi-tol-3 kinase (PI3K) [15], osteosarcoma cell spreadingand motility via Rho-associated kinase (ROCK) [16], andmammary epithelial cell migration through the guaninenucleotide exchange factor a-PIX [17].There is also growing evidence on the cooperationbetween PDGF receptor and integrins in regulating cel-lular survival and adhesion [18-20]. However, little isknown about the existence of cross-talk between ILKand PDGF signaling in vascular smooth muscle cellsand the probable regulatory role of ILK during PDGF-induced SMC migration. In the present study, we haveexamined the potential function for ILK as an upstreamprotein regulating the migratory response to PDGF in aprimary mouse aortic SMC culture. We have also char-acterized one downstream pathway that mediates theregulatory role of ILK in modulation actin polymeriza-tion and SMC migration.ResultsPDGF activates integrin-linked kinase in mouseaortic SMCsTo study the effect of PDGF treatment on ILK activa-tion, mouse aortic SMCs were treated with 25 ng/ml ofPDGF-BB for various timepoints and cell lysates werecollected and subjected to the kinase activity assay. Asshown, PDGF treatment increased ILK kinase activityaround 30 minutes post treatment in the SMC culturewithout having any significant effect on total ILK pro-tein expression. PDGF-induced ILK kinase activitydeclined around 60 minutes after treatment (figure 1A).To ensure the specificity of the test and as a negativecontrol, a group of SMCs culture was transfected withspecific ILK siRNA for 96 hours. Such treatmentcompletely abolished ILK protein expression in SMCs(figure 1B). As shown, very little kinase activity wasdetected in ILK-siRNA group confirming that the resultsof the kinase activity assay is specific. It is noteworthythat we carefully monitored the morphology of SMCstransfected with ILK siRNA for the incubation periodprior to PDGF treatment (96 hours) to assure thatsiRNA treatment had no cytotoxic effect on mouseSMC (data not shown).PDGF induces SMC migration through anILK-dependent pathwayTo investigate the potential function of ILK in regula-tion of PDGF-induced SMC migration, cells were trans-fected with specific ILK siRNA, and then treated with25 ng/ml of PDGF-BB. The migratory response wasmeasured using both modified Transwell Boyden cham-ber and wound healing assays. Inhibition of ILK proteinexpression markedly decreased SMC migration indicat-ing that the presence of ILK was required for transdu-cing the migratory signal stimulated by PDGF-BB(figures 2A &2B).PDGF is considered as a very potent mitogen. Theobserved effect of PDGF-BB on wound closure in figure2B could be due to the effect of this growth factor onSMC proliferation and/or migration. Thus, to determinewhether the proliferatory effect of PDGF-BB had con-tributed to the observed increase in cell migration., cellswere treated with increasing doses of PDGF-BB for 24hours (the maximum time required for wound closure),and cell proliferation and migration were measuredusing the modified Transwell Boyden chamber assayand MTS cell proliferation assay, respectively. Treat-ment of primary mouse SMCs with 25 ng/ml of PDGF-BB significantly increased cell migration without havingany noticeable effect on SMC proliferation. To assurethe accuracy of both assays and as a positive control,mouse SMC culture were also treated with 10% serum.These findings emphasized that the observed increase incell migration in both assays used in this study (24hours post injury or following loading onto TranswellBoyden Chambers) was due to the pro-migratory, notproliferatory, effect of PDGF-BB on mouse aortic SMC(additional file 1, figure S1).ILK inhibition abolishes PDGF-induced p38 MAPKactivation in mouse aortic SMC cultureStudies in various in vitro models, have shown thatmembers of the family of the mitogen activated proteinkinases (MAPKs) become activated and play a crucialrole in regulating cell migration in response to PDGFtreatment [21-24]. However, the actual mechanismsunderlying this activation and the identity of theupstream kinases involved are not fully elucidated. Here,Esfandiarei et al. BMC Cell Biology 2010, 11:16http://www.biomedcentral.com/1471-2121/11/16Page 2 of 12Figure 1 PDGF treatment increases integrin-linked kinase activity in mouse aortic smooth muscle cell culture. A) Primary mouse aorticSMCs were serum-starved overnight and then treated with 25 ng/ml of PDGF-BB. ILK kinase activity was measured at various timepointsfollowing treatment. PDGF-BB increased ILK kinase activity (two fold increase). SMC culture transfected with ILK siRNA was used as negativecontrol in the assay. As shown in first lane of the blot, ILK expression was completely abolished in negative control group that led to asignificant decrease in ILK kinase activity in this group. B) Transfection of mouse aortic SMCs with ILK siRNA for 96 hours completely blocked ILKprotein expression. Data presents three independent experiments and is presented as mean ± STDEV (n = 3), where P < 0.05 was consideredsignificant.Figure 2 PDGF induces SMC migration through anILK-dependent pathway. A) Inhibition of ILK protein expression with 20 nM ILK siRNAmarkedly decreased SMC migration in response to PDGF-BB. Data represents three independent experiments (n = 3 culture plates in eachreplicate) and the value is presented as mean ± STDEV where P < 0.05 was considered significant. B) ILK inhibition decreased the number ofmigrated SMCs into the site of injury in response to PDGF-BB treatment as compared to the control group (contorl siRNA). Originalmagnification, ×40. Data present one of three independent experiments.Esfandiarei et al. BMC Cell Biology 2010, 11:16http://www.biomedcentral.com/1471-2121/11/16Page 3 of 12we first characterized the dynamics of MAPKs familyactivation in response to PDGF-BB treatment in our pri-mary mouse aortic SMC model. In agreement with pre-vious reports in various cell models, PDGF-BBtreatment resulted in the phosphorylation of all threemembers of MAPKs family, extracellular signal-regu-lated protein kinase 1/2 (Erk1/2), Jun N-terminus kinase(JNK), and p38 MAPK starting around 5 minutes andwith a peak activation at 20-30 minutes post-treatment(additional file 2, figure S2).To investigate the role of each member of MAPKsfamily in regulating PDGF-induced cell migration, SMCswere treated with specific MEK/Erk1/2 inhibitor (20 μMU0126), JNK inhibitor (50 μM SP600125), and p38MAPK inhibitor (10 μM SB202190) for two hours priorto PDGF-BB treatment and cell migration was measuredusing the modified Transwell Boyden Chamber assay.Inhibition of all members of MAPKs family markedlyreduced mouse SMC migration in response to PDGF-BB treatment (figure 3A).Various components of focal adhesion complex havebeen shown to regulate or cross talk with members ofMAPKs family [24]. ILK, as a key constituent of thefocal adhesion complex, also plays an important func-tion in transducing inside-out and outside-in signals[25]. To investigate the role of ILK in regulatingMAPKs activation in response to PDGF, SMCs weretransfected with specific ILK siRNA prior to PDGF-BBtreatment. Inhibition of ILK expression with specificsiRNA resulted in significant decrease in PDGF-inducedp38 MAPK phosphorylation (figure 3B), while having noeffect on Erk1/2 and JNK phosphorylation (figure 3C).This observation suggests that ILK presence is requiredfor PDGF-induced phosphorylation of p38 MAPK inmouse SMC.ILK mediates PDGF-induced mouse aortic SMC migrationthrough a p38 MAPK-dependent pathwayTaking into account that PDGF-BB activates both ILKand p38 MAPK in mouse aortic SMCs (figures 1A &Additional file 1, figure S1), and the observation thatp38 MAPK has a crucial role in PDGF-induced cellmigration (Figure 3A); we sought to determine whetherILK can also modulate the migratory effect of p38MAPK activation in response to PDGF-BB. To examinethis hypothesis, we utilized a gain and loss of functionapproach using adenoviral constructs of ILK and/or p38MAPK.In order to revalidate the important role of p38MAPK activation during the migratory response, andalso to exploit a more target-specific approach, we firstinfected primary mouse SMCs with adenoviral con-structs expressing either a dominant negative form ofp38 MAPK (Ad-Dn-p38), a wild type form of p38MAPK (Ad-Wt-p38), or a GFP (Ad-GFP) protein. Wes-tern blot analysis (measuring protein expression) andfluorescent microscopy (visualizing GFP expression)were used to evaluate the transfection efficiency at 36hours post transfection (Figures 4A &4B).In concurrence with our previous observations, over-expression of a dominant negative mutant of p38MAPK significantly reduced PDGF-induced SMC migra-tion (Figure 4C). Furthermore, over-expression of a wildtype form of p38 rescued SMC migration in response toPDGF-BB, corroborating the specificity of p38 MAPKinhibition in mouse SMC culture transfected with theAd-Dn-p38 MAPK construct (Figure 4C).Moreover, a kinase-deficient form of ILK (E359K),which is also incapable of binding to paxillin and parvinand therefore unable to participate in formation of thefocal adhesion complex, resulted in a significant declinein PDGF-induced SMC migration, an event that can bereversed by a wild type form of ILK (Figure 5A).Finally, to establish a cause and effect associationbetween inhibition of ILK, a downstream decrease inp38 MAPK phosphorylation, and the subsequent declinein cell migration, we co-transfected cells with a kinasedeficient form of ILK (E359K) along with an active formof MKK3/6 (the specific upstream activator of p38MAPK) or a wild type form of p38 MAPK. As shown,expression of Ad-Ca-MKK3/6 significantly reversed theinhibitory effect of ILK inhibition (Ad-Kd-ILK) inPDGF-treated SMCs (Figure 5B). In contrast, over-expression of the wild type p38 MAPK in SMCs alreadytransfected with Ad-Kd-ILK, did not rescue cell migra-tion, confirming our previous observation that the exis-tence of an active form of ILK was required to facilitatethe p38 MAPK-mediated migratory response to PDGF-BB (Figure 5B).ILK and p38 MAPK regulate PDGF-induced actincytoskeleton polymerization in vascular SMCActin polymerization and re-organization is a criticalstep in cell motility and migration. To characterize therole of ILK and p38 MAPK in regulating actin polymeri-zation in response to PDGF, cells were transfected withspecific ILK siRNA (20 nM) or treated with specific p38MAPK inhibitor (10 μM SB202190) prior to PDGFtreatment. Inhibition of ILK expression and p38 MAPKactivity significantly blocked PDGF-induced actin poly-merization and re-organization in mouse SMC, indicat-ing a crucial role for ILK and p38 MAPK in regulationactin-mediated cell migration (Figures 6A, B).DiscussionVascular SMC migration contributes to the intimalthickening in occlusive vascular diseases. Following vas-cular injury, SMC migratory response to various stimuliEsfandiarei et al. BMC Cell Biology 2010, 11:16http://www.biomedcentral.com/1471-2121/11/16Page 4 of 12Figure 3 ILK inhibition abolishes PDGF-induced p38 MAPK activation in mouse aortic SMCs. A) Mouse aortic SMCs were treated withspecific MAPKs inhibitors at specified concentrations for 2 hours prior to seeding onto Transwell Boyden Chambers and PDGF-BB treatment (25ng/ml) and during the incubation period (12 hours), and cell migration was measured at 12 hours post culture. As shown, inhibition of all threemembers of MAPKs family significantly reduced SMC migration in response to PDGF-BB. Data is the representative of three independentexperiments (n = 3 cell culture replicates for each experiments). B) Mouse aortic SMCs were transfected with 20 nM of specific ILK siRNA andthen treated with 25 ng/ml of PDGF-BB for specified timepoints. Inhibition of ILK protein expression markedly reduced PDGF-induced p38 MAPKphosphorylation in mouse aortic SMCs. The blot presents one of three independent experiments. The value in the graph is presented as themean ± STDEV of three independent experiments. C) ILK inhibition by specific siRNA had no detectable effect on Erk1/2 and JNKphosphorylation in response to PDGF-BB treatment in mouse aortic SMC culture. Data presents one of three independent experiments (n = 3cell cultures for each independent experiments).Esfandiarei et al. BMC Cell Biology 2010, 11:16http://www.biomedcentral.com/1471-2121/11/16Page 5 of 12begins with activation of cell surface receptors followedby remodeling events that involve focal adhesion com-plex and filament actin remodeling. Various cytokines,peptide growth factors, and components of the extracel-lular matrix (ECM) have been characterized as pro-migratory molecules.PDGF is a very potent stimulant for SMC migrationthrough activation of several signaling pathways [26-28].In vascular SMC, PDGF activates multiple signalingcascades including the PI3K/Akt, protein kinase A(PKA), Src, Ras, and MAPKs signaling. The family ofMAPKs comprises three distinct protein kinases, p38MAPK, JNK, and Erk1/2, all of which have been shownto be involved in vascular diseases and remodeling [23].On another note, integrin clustering and its down-stream signals are necessary for SMC adhesion to ECM,an event that also initiates pro-migratory responseswithin the vascular wall. Mawatari and colleagues haveFigure 4 Dominant negative form of p38 MAPK significantly decreases SMC migration in response to PDGF. A) Photomicrograph ofmouse aortic SMCs showing the morphology and GFP expression at 36 hours following transfection. Sub-confluent SMCs were infected withadenoviral vector at the multiplicity of infection of 100. Note the high level of GFP expression as well as the normal phenotype of Ad-GFP-transfected SMCs. Original magnification, ×200. B) Over-expression of a dominant negative form of p38 MAPK markedly increased total p38MAPK expression while blocking p38 MAPK phosphorylation in response to serum treatment in SMCs (serum is used as a strong activator forp38 MAPK). Also, note the considerable increase in ILK expression level in SMCs transfected with adenoviral ILK constructs. C) For migrationassay, transfected SMCs were cultured in Transwell Boyden Chambers in the presence or absence of 25 ng/ml of PDGF-BB and cell migrationwas measured 12 hours post culture. Dominant negative p38 MAPK significantly decreases SMC migration in response to PDGF-BB treatmentwhile expression of a wild type form of p38 MAPK increases SMC migration in response to PDGF-BB. In addition, over-expression of a wild typemutant of p38 MAPK subverted the inhibitory effect of p38 inhibition on SMC migration. Data represents three independent experiments (n = 3cell cultures for each independent experiments).Esfandiarei et al. BMC Cell Biology 2010, 11:16http://www.biomedcentral.com/1471-2121/11/16Page 6 of 12shown that in human saphenous vein SMCs beta-3integrin is required for growth factor and cytokine-induced proliferation [29]. It is believed that a coordi-nated cross-talk between integrins and growth factortyrosine kinase receptors is necessary for the effectivemodulation of cell survival, growth, proliferation, andmigration [30].ILK is a key protein of focal adhesion complex con-necting b1 and b3 integrins in plasma membrane to theactin cytoskeletal machinery. ILK also interacts withgrowth factor receptors through the NH2-terminalankyrin repeat domain and small GTPase signalingmolecules such as Nck-2 [14,31]. Previous reports onthe effect of PDGF on ILK expression and activity innon-muscle cell cultures were controversial. WhileCampana et al reported on a PDGF-induced increase inILK activity in primary rat Schwann cells [32], Janji andcolleagues proposed that PDGF had no effect on ILKexpression in melanoma HT-144 cells [33], indicatingthat the outcome of PDGF treatment on ILK signalingis cell specific.In the present study, we have shown that PDGF-BBtreatment results in an increase in ILK kinase activity inmouse aortic SMC culture. It is of importance that theincrease in ILK kinase activity in SMCs is associatedwith a considerable increase in expression of both b1and b3 integrins (Esfandiarei & van Breemen, unpub-lished observation). It is well known that the adhesivefunction of integrin receptors affects cytoskeleton rear-rangement and cell motility. Therefore, it is presumablethat increase in b1 and b3 integrins expression contri-butes to the pro-migratory effect of PDGF-BB in SMCculture. However, further investigation is required toevaluate the potential cross-talk between b1 and b3integrins and PDGF receptors and the significance ofsuch events in vascular SMC migration.Numerous studies have provided a wealth of informa-tion on the crucial regulatory role for ILK as a proteinFigure 5 ILK regulates PDGF-induced SMC migration through a p38 MAPK-dependent pathway. A) Kinase deficient mutant of ILKsignificantly reduced PDGF-induced SMC migration in a Traswell Boyden Chamber migration assay. As shown, expression of a wild type form ofILK rescued SMC migratory response indicating the specificity of ILK inhibition by the adenoviral construct. B) Over-expression of an active formof MKK3/MKK6 (specific upstream activators of p38 MAPK), but not a wild type form of p38 MAPK, rescued SMC migration in response to PDGF-BB indicating that an active form of ILK is required for the initiation of a p38 MAPK-dependent migratory response to PDGF-BB in mouse aorticSMCs. (n = 3 cell cultures for each independent experiments).Esfandiarei et al. BMC Cell Biology 2010, 11:16http://www.biomedcentral.com/1471-2121/11/16Page 7 of 12kinase and also as a molecular scaffold and adaptor pro-tein during cell spreading and migration in a variety ofcancer cells [13,34]. Moreover, A recent study hasshown that an increase in ILK expression coincides withhigher rate of SMC migration within the atheroscleroticplaque in ApoE -/- mice, a reliable model that mimicsthe progression of atherosclerosis in humans [35]. Onthe contrary, Ho et al has shown that following ballooncatheter injury of the rat carotid artery, a significantdecrease in ILK expression is associated with an eleva-tion in vascular SMC proliferation and migration [36].Nevertheless, there is no clear understanding as to howILK contributes to the intimal thickening in response togrowth factors and cytokines released during the devel-opment of the atherosclerotic plaque, and the mechan-isms by which ILK may modulate vascular SMCmigration to the sub-intimal layer of the vascular wall.In this study, we examined the role of ILK in cellmigration, by inhibiting (using siRNA) or increasing(using an adenoviral vector expressing wild type ILK)ILK expression. Inhibition of ILK by different meanssignificantly decreased cell migration in response toFigure 6 ILK and p38 MAPK regulate PDGF-induced actin cytoskeleton polymerization in vascular SMCs. A) Mouse SMCs were treatedwith either vehicle (DMSO) or with 10 μM of p38 MAPK specific inhibitor SB202190 for 1 hour prior to PDGF treatment. Following treatment,cells were washed and fixed and then stained for actin filaments. Inhibition of p38 MAPK markedly blocks PDGF-induced actin polymerizationand reorganization (original magnification ×400). B) Mouse aortic SMCs were transfected with 20 nM of specific ILK siRNA and then treated with25 ng/ml of PDGF-BB for 20 minutes. Cells were then fixed and stained for actin filaments. Inhibition of ILK expression in SMCs significantlyreduced PDGF-induced actin polymerization and reorganization in mouse aortic SMCs. Images are representative image of three independentexperiments (original magnification ×400).Esfandiarei et al. BMC Cell Biology 2010, 11:16http://www.biomedcentral.com/1471-2121/11/16Page 8 of 12PDGF-BB, demonstrating that ILK plays an importantrole in regulating PDGF-induced vascular SMC migra-tion. Expression of a wild type form of ILK increasedSMC migration even in the absence of growth factor. Itis noteworthy that the effect of wild type ILK in increas-ing SMC migration is more evident when SMCs weretreated with PDGF-BB (figure 6A). These observationsare in agreement with previous report by Dwivedi et al[37] that over-expression of a dominant negative formof ILK markedly reduced intimal thickening in humansaphenous vein organ culture.ILK mutation within the catalytic domain (E359K) dis-rupts the interaction and binding of ILK with twoimportant components of focal adhesion complex, b-parvin and paxillin. This interaction is required for theassembly of a functional focal adhesion complex andsubsequent actin polymerization and reorganization[38,39]. Over-expression of the E395K mutant of ILKresulted in significant reduction in PDGF-induced SMCmigration. The effect was fully reversed by a wild typeform of ILK. These findings further underscore the cru-cial function of ILK as molecular scaffold during SMCmigration.In an effort to understand how ILK mediates the pro-migratory effect of PDGF-BB in primary mouse SMCusing RNA silencing technique, we were able to showthat ILK inhibition could lead to a considerable decreasein p38 MAPK phosphorylation, while having no visibleeffect on Erk1/2 and JNK phosphorylation in responseto PDGF-BB treatment. We also demonstrated thatover-expression of an active form of MKK3/6 (specificupstream activator of p38 MAPK) dramatically reversedthe inhibitory effect of ILK inhibition on SMC migra-tion. Expression of a wild type form of p38 MAPKmarkedly increased SMC migration in the presence ofPDGF-BB; however, it could not restore migratoryresponse in cells over-expressing a kinase deficient formof ILK. It is of importance that the latter observationaccentuates the view that the presence of a functionalILK is required for the initiation of a p38 MAPK-depen-dent migratory response to PDGF-BB in mouse aorticSMCs. Further studies are required to characterize theprecise mechanisms underlying the ILK-mediated regu-lation of p38 MAPK in SMCs.Actin polymerization and reorganization is a crucialstep in cell migration. We have shown that PDGFincreases actin polymerization and filament reorganiza-tion in SMCs. This event was significantly blocked byboth ILK siRNA and p38 MAPK inhibitor, confirmingthe importance of this pathway in regulating SMCmigration. The actual mechanism by which p38 MAPKmay regulate cell migration is not well understood.Actin polymerization and reorganization is under rigor-ous regulation by a group of protein kinases and/orphosphatases that coordinate the constant turnover ofactin filaments in a stimulated cell. Among these, cofilinis a conserved actin-binding protein that enhances actinfilament reorganization through de-polymerization andsevering of preexisting actin filaments [40]. It is wellknown that phosphorylation of cofilin (serine 3) by LIMkinase (LIMK) and testicular protein kinase 2 (TESK-2)results in deactivation of cofilin and subsequent inhibi-tion of actin reorganization [41,42]. In mouse aorticSMCs, PDGF treatment resulted in rapid de-phosphory-lation and activation of cofilin around 5-15 minutespost-treatment (additional file 3, figure S3). PDGF alsoincreased LIMK phosphorylation leading to phosphory-lation and deactivation of cofilin (additional file 3, figureS3). The feedback mechanism provides a strict controlsystem for cell responses to migratory stimuli such asPDGF. Our preliminary observation suggests that p38MAPK may control actin reorganization partly via regu-lating the de-phosphorylation and consequent activationof cofilin (Esfandiarei & van Breemen, unpublishedobservation). Further detailed studies are ongoing in ourlaboratory to characterize the signaling network anddownstream pathways mediating such regulatory events.ConclusionIn summary, we have provided evidence that ILK is animportant regulator of mouse vascular smooth musclecell migration in response to PDGF-BB by modulatingp38 MAPK phosphorylation. To our knowledge, this isthe first report of the regulation of p38 MAPK by integ-rin signaling in mediating platelet-derived growth fac-tor-induced migration in vascular smooth muscles.Future in vivo studies using specific small molecule inhi-bitors for ILK and/or p38 MAPK as well as developingtransgenic animal models will provide additional infor-mation on the potential therapeutic value of integrinsignaling, in general, and the integrin-linked kinase, inparticular, in controlling vascular smooth muscle cellmigration during the progression of occlusive vasculardiseases.MethodsPreparation of Primary SMC CultureAll surgical procedures and animal care were conductedaccording to the Guidelines for Animal Experiments ofFaculty of Medicine, University of British Columbiaapproved by the University Committee on Ethics of Ani-mal Experiments. Primary mouse aortic SMCs were iso-lated as described previously [43]. Breifly, aorticsegments were isolated, cleaned of excess advential tis-sue, and digested using collagenase II (0.5 mg/ml). Iso-lated cells were pelleted, resuspended, and grown inDulbecco’s modified Eagle’s medium. Sub-confluent (60-80%) cutlures were used for all experiments. ILKEsfandiarei et al. BMC Cell Biology 2010, 11:16http://www.biomedcentral.com/1471-2121/11/16Page 9 of 12recombinant adenoviral constructs were kind gifts fromDr. Hyo-Soo Kim (Seoul National University Hospital,Korea). Adenoviral constructs for recombinant p38MAPK and constitutively active forms of MKK3/6 werekindly provided by Dr. Donald R. Menick (Medical Uni-versity of South Carolina, USA).Transient TransfectionFor transient transfection experiments, cells wereinfected with adenoviruses encoding dominant negativep38 MAPK (Ad-Dn-p38), wild type of p38 MAPK (Ad-Wt-p38), constitutively active MKK3/6 (Ad-Ca-MKK3/6), kinase-deficient ILK (Ad-Kd-ILK), or wild type ILK(Ad-Wt-ILK) with multiplicity of infection of 100(MOI = 100). Following overnight incubation at 37°C,SMCs were replenished with fresh medium. Transfec-tion efficiency was measured by fluorescence microscopyand Western blot analysis at 36 hours post transfection.For all experiments, transfected SMCs were used at36 hours post transfection. For ILK RNA inhibitionexperiments, SMCs were transfected with 20 nM of a21-base pair double-stranded small interfering RNA(siRNA) molecule targeting the PH domain of ILK or acontrol nonspecific siRNA. ILK siRNA was introducedto cells using 2.5 μl of siLentFect Transfection Reagentaccording to the manufacturer’s protocol (Bio-RadLaboratories, Mississauga, ON, Canada). Twenty fourhours post-transfection cells were replenished with freshserum-containing medium, incubated for 96 hours, andthen used for various experiments. ILK silencing wasdetermined by Western blot of transfected lysates withan anti-ILK antibody.Western Blot AnalysisCells either untreated or treated with various experi-mental reagents were washed twice with ice-cold PBS,and kept on ice for 15 min in lysis buffer containing 50mM pyrophosphate, 50 mM, NaF, 50 mM NaCl, 5 mMEDTA, 5 mM EGTA, 100 μM Na3VO4, 10 mM HEPES(pH 7.4), 0.1% Triton X-100, 10 μg/ml leupeptin, and 1mM phenylmethylsulfonyl fluoride. Cell lysates were col-lected by scraping and protein concentration was deter-mined using Bradford assay. Extracted protein (40-80μg) was fractionated by electrophoresis in 7% to 9%sodium dodecyl sulfate-polyacrylamide gels, transferredto nitrocellulose membranes, and blocked with PBS con-taining 0.1% Tween-20 and 5% non-fat dry milk for1 hour. Afterward, the membrane was incubated withspecific primary antibody overnight at 4°C, followed bysecondary antibody for one hour at room temperature.Immunoblots were visualized with an enhanced chemi-luminescence detection system according to the pro-tocol of the manufacturer (Pierce Biotechnology,Rockford, IL, USA). Densitometry analysis wasperformed by using the National Institutes of HealthImageJ software (version 1.27z). Density values for pro-teins were normalized to the level for control groups(arbitrarily set to 1.0-fold).ILK Kinase AssayKinase assay was performed using 250 μg of proteinlysates immunoprecipitated with 5 μg ILK antibody andprotein A Sepharose beads overnight at 4°C, while shak-ing. The immunocomplex was washed twice with highsalt NP-40 lysis buffer (containing 750 mM NaCl), andthen three times with wash buffer containing 50 mMHEPES pH 7.5, 85 mM KCl, 10 mM ethyleneglycol tetra-acetate (EGTA), 0.1% Tween 80, 1 mM Na3VO4, 10 mMMg2Cl. The kinase assay was performed in the reactionbuffer containing 0.5 μg ATP (250 μM ATP and 5 μCi[g -32P] ATP) and 5 μg of myelin basic protein (MBP) assubstrate at 30°C for 20 minutes in a shaker-incubator.Samples were fractionated by electrophoresis in a 12%sodium dodecyl sulfate-polyacrylamide gel, and phos-phorylation of substrate by ILK was measured by a scin-tillation counter autoradiography.Cell Migration AssayCell migration was measured using QCM™ TranswellColorimetric Cell Migration Assay according to themanufacturer’s protocol (Chemicon International,Temecula, CA, USA). Briefly, 1 × 105 serum-starvedSMCs were loaded onto the upper well of the chamberin the presence or absence of inhibitors while lowerwells were filled with culture medium with or withoutPDGF-BB (25 ng/ml). Following 12 hours incubation,non-migrating cells on the upper side of membraneswere removed by wiping and rinsing, and migrated cellswere counted using colorimetric assay. Data are repre-sented as fold change in cell migration where migratoryrates for control groups are arbitrarily set to 1.0 fold.Wound Healing AssaySub-confluent transfected or non-transfected SMCswere grown on glass coverslips. Following overnightserum starvation, cell cultures were scratched with asterile pipette tip to form a wound, washed with pre-warmed sterile PBS, and incubated with medium in thepresence or absence of inhibitors for 18-24 hours. Atdesired timepoints post injury cells were fixed and sub-jected to imaging using Nikon inverted microscope andSpot digital camera.Cell Viability AssaySub-confluent SMCs were plated in a 24-well cultureplate and treated with various doses of PDGF for 24hours. The CellTiter 96® AQueous Non-Radioactive CellProliferation Assay (MTS) was used to measure cellEsfandiarei et al. BMC Cell Biology 2010, 11:16http://www.biomedcentral.com/1471-2121/11/16Page 10 of 12viability according to the manufacturer’s protocol (Pro-mega, Madison, WI).Actin Cytoskeleton StainingCells were grown on glass slides and subjected to var-ious treatments. At desired timepoints cells were fixed(3.7% formaldehyde for 15 min & 70% ethanol for 2min). Following permeabilization in 0.1% Triton X-100,cells were incubated with AlexaFluor 488-labelled phal-loidin (Molecular Probes, Invitrogen Detection Technol-ogies) for 1 hour in room temparature. Glass slides weremounted and sealed using ProLong® Gold anti-fadereagent (Molecular Probes, Invitrogen Detection Tech-nologies), and then imaged with Olmpus invertedmicroscope and Spot digital camera.Statistical AnalysisTwo-way analysis of variance (ANOVA) with multiplecomparisons andpaired Student’s t tests were performed.Values shown are the mean ± standard deviation. Avalue of P < 0.05 was considered significant.Additional file 1: Figure 1S. Effect of PDGF treatment on mouseaortic SMCs migration and proliferation. Mouse SMCs were treatedwith increasing doses of PDGF-BB for 24 hours and cell migration andproliferation were measured. Cells treated with 10% serum were used asthe positive control. As shown, 25 ng/ml of PDGF-BB significantlyincreased SMCs migration (3 fold increase) with no effect on cellproliferation indicating that the observed increase in cell migration is notdue to the proliferatory effect of PDGF-BB in SMC culture. Data isrepresentative of three independent experiments (n = 3 of each culturecondition).Click here for file[ http://www.biomedcentral.com/content/supplementary/1471-2121-11-16-S1.PDF ]Additional file 2: Figure 2S. Kinetics of MAPKs activation in mouseaortic SMC culture. PDGF-BB treatment resulted in phosphorylation andactivation of all three members of MAPKs family in mouse aortic SMCs.Data represents three independent experiments.Click here for file[ http://www.biomedcentral.com/content/supplementary/1471-2121-11-16-S2.PDF ]Additional file 3: Figure 3S. PDGF induces LIMK and cofilinphosphorylation in mouse aortic SMCs. Aortic SMCs were serumstarved overnight and then treated with 25 ng/ml of PDGF-BB. Celllysates were collected in various timepoints and phosphorylation of LIMKand cofilin was measured. Data represents three independentexperiments.Click here for file[ http://www.biomedcentral.com/content/supplementary/1471-2121-11-16-S3.PDF ]AcknowledgementsThis study was supported by a grant-in-aid from the Canadian Institutes ofHealth Research (CV); and fellowships from the Heart & Stroke Foundation ofCanada/AstraZeneca Canada (ME), Michael Smith Foundation for HealthResearch (ME), and Canadian Diabetes Association (ME).Author details1Child & Family Research Institute, Department of Anesthesiology,Pharmacology, and Therapeutics, University of British Columbia, 950 West28th Avenue, Vancouver, BC, V5Z 4H4, Canada. 2Department of CancerGenetics, British Columbia Cancer Research Centre, University of BritishColumbia, 675 West 10th Avenue, Vancouver, BC, V5Z 1L3, Canada.Authors’ contributionsThe scientific idea and experimental design were developed by ME and CVB.Experiments were performed by ME, SAY. ILK kinase assay was performed byVG. 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