Published online before print December 20, 2007, doi: 10.1161/CIRCRESAHA.107.158923
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© 2008 American Heart Association, Inc.
Molecular Medicine |
Dual Regulation of Cofilin Activity by LIM Kinase and Slingshot-1L Phosphatase Controls Platelet-Derived Growth Factor–Induced Migration of Human Aortic Smooth Muscle Cells
From the Division of Cardiology (A.S.M., M.Y.L., H.C.W., B.L., K.K.G.), Emory University, Atlanta, Ga; and Department of Biomolecular Sciences (K.M.), Tohoku University, Aramaki-aza-aoba, Aoba-ku, Sendai, Japan.
Correspondence to Kathy K. Griendling, Division of Cardiology, Emory University, 1639 Pierce Dr, 319 WMB, Atlanta, GA 30322. E-mail kgriend{at}emory.edu
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Abstract |
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Platelet-derived growth factor (PDGF) plays a central role in vascular healing, atherosclerosis, and restenosis, partly by stimulating vascular smooth muscle cell (VSMC) migration. Migration requires rapid turnover of actin filaments, which is partially controlled by cofilin. Although cofilin is negatively regulated by Ser3 phosphorylation, the upstream signaling pathways have not been defined, nor has its role in VSMC migration been studied. We hypothesized that PDGF-induced migration of VSMCs involves cofilin activation and that this is regulated by the serine kinase LIM kinase (LIMK) and the novel phosphatase Slingshot (SSH)1L. In human VSMCs, stimulation with PDGF increased G-actin incorporation into the actin cytoskeleton. PDGF transiently activated the cofilin kinase, LIMK, with a peak at 5 minutes. However, cofilin was dephosphorylated between 5 and 45 minutes, with a maximum of 43±5% dephosphorylation at 30 minutes, suggesting that PDGF also activates a cofilin phosphatase. We found that VSMCs express SSH1L, which is induced and activated (564±73 versus 1021±141 picomoles of PO4; P=0.015) by PDGF. Of importance, small interfering RNA directed against SSH1L blocked cofilin dephosphorylation and decreased migration (528±33 versus 318±25 cells/field; P<0.01). Taken together, our results suggest that PDGF participates in actin dynamics by dual regulation of cofilin activity via LIMK and SSH1L.
Key Words: vascular smooth muscle • slingshot phosphatase • platelet-derived growth factor • migration • LIMK
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Introduction |
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Directed cell migration is an integrated, dynamic, and cyclical process that plays a key role in mounting immune responses and repairing injured tissues, as well as in guiding the morphogenesis of the embryo during development. In addition, migration contributes to pathologies including chronic inflammatory diseases, postangioplasty restenosis, atherosclerosis, and cancer. Vascular smooth muscle cell (VSMC) migration is influenced by many factors but in vivo is largely a consequence of stimulation of the platelet-derived growth factor (PDGF)-β receptors by PDGF.1 However, the signaling pathways by which PDGF controls VSMC migration are still not completely understood.
Regardless of the cell type, a fast and efficient reorganization of the actin cytoskeleton is absolutely essential for migration. Different proteins, including actin-related proteins 2 and 3 (Arp2/3), Wiscott–Aldrich syndrome protein (WASP), profilin, mammalian diaphanous proteins, and cofilin, have been shown to regulate the assembly and disassembly of the actin cytoskeleton in response to multiple extracellular stimuli.2 Of these, cofilin, a protein belonging to the actin depolarizing factor (ADF) family of proteins, is capable of stimulating disassembly and severance of actin filaments at or near the pointed ends, thereby continuously supplying actin monomers for polymerization and rapid turnover of actin filaments.3 Thus, the activation of cofilin plays an essential role in the protrusion of lamellipodia at the leading edge of migrating cells. The activity of cofilin is negatively regulated by the LIM kinase (LIMK) family of serine/threonine kinases through phosphorylation at Ser3 of cofilin.4–6 Suppression of cofilin activity by LIMK overexpression abolishes lamellipodium formation and polarized cell migration, which implicates cofilin in cell polarity and migration.7,8
Previous studies have revealed that phospho-cofilin is dephosphorylated, and therefore activated, in response to various extracellular stimuli.9–11 Several proteins have been shown to be cofilin phosphatases, including chronophin and the Slingshot (SSH) family of protein phosphatases.12,13 Chronophin is highly expressed in brain, heart, and liver but is nearly absent in smooth muscle–containing tissues,13 whereas SSH family members are widely expressed in mouse tissues.12 Of the SSH family of proteins, SSH1L, the long form of SSH1 that has an actin-binding site near the C terminus, has been shown to be important in chemotaxis.14 SSH1L colocalizes with F-actin and its phosphatase activity correlates with the level of cofilin dephosphorylation.10
Based on these observations, we hypothesized that PDGF-induced changes in VSMC actin polymerization and migration involve cofilin activation, and that this activation is the result of regulation of LIMK and SSH1L phosphatase. We found that a net dephosphorylation of cofilin results from simultaneous activation of both enzymes, thus providing a mechanism for fine control of VSMC migration.
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Materials and Methods |
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Recombinant human PDGF-BB was purchased from R&D Systems Inc (Minneapolis, Minn) and was used in all the experiments.
Cell Culture
Human aortic smooth muscle cells were purchased from Clonetics (San Diego, Calif). The cells were cultured in Clonetics SmGM-2 medium containing human epidermal growth factor 0.5 ng/mL, insulin 5 µg/mL, hFGF 2 ng/mL, 5% FBS, gentamicin 50 µg/mL, and amphotericin B 50 ng/mL. Cell cultures were incubated at 37°C in a humidified atmosphere of 5% CO2. VSMCs between passages 6 and 12 were used for the experiments. Cultures at 70% to 80% confluence were made quiescent by incubation in serum free media for 24 to 48 hours before the experiments.
Western Blotting
After treatment, cells were lysed in 1% Triton–containing lysis buffer and analyzed by Western blotting as described previously.15 Membranes were incubated with primary antibodies against phospho-cofilin(Ser3), cofilin, phospho-LIMK1(Thr508)/LIMK2(Thr505), LIMK1 (Cell Signaling), or β-tubulin (Sigma). Rabbit polyclonal antibody against SSH1L has been described previously.14 After incubation with horseradish peroxidase-conjugated secondary antibody, proteins were detected by ECL chemiluminescence. The films were analyzed by densitometry with ImageJ software (NIH).
Small Interfering RNA and Plasmid Transfection Experiments
Cells were transfected by electroporation using a Nucleofector (Amaxa Biosystems) set to the U25 program with 2 µg of plasmid enhanced green fluorescent protein (pEGFP)-KS (encoding a kinase-dead mutant, which acts as a dominant negative16; a kind gift of Dr Condeelis, Albert Einstein College of Medicine, NY) or pEGFP (Clontech) plasmids per 1x106 cells, and/or with 1.5 µg of annealed small interfering (si)RNA duplexes for SSH1L or nonsilencing control sequence no. 1 from Qiagen per 1.6x106 cells. siRNA duplexes were synthesized using a previously published sequence17 (sense: 5'-UCG UCA CCC AAG AAA GAU AUU-3'; antisense: 5'-UAU CUU UCU UGG GUG ACG AUU-3'). The transfection was made three days before a 24-hour serum deprivation before the experiments.
Real-Time Quantitative RT-PCR
Total RNA was purified from VSMCs using the RNeasy kit (Qiagen), as recommended by the manufacturer. RNA was reverse-transcribed with Superscript II enzyme (Invitrogen) using random primers. cDNA samples were additionally purified using microbiospin 30 columns (Bio-Rad) and amplified in the LightCycler (Roche) real-time thermocycler using recombinant Platinum Taq DNA polymerase (Invitrogen) with SYBR green dye. Amplification conditions were for SSH1L, 150 nmol/L primers (upstream primer, 5'-GATCAAAACCTGCTCAACTCGGAGAACCT-3'; downstream primer, 5'-CTGGAGCCTGCTGGTGGTAGGAAC-3'), 4 mmol/L MgCl2, 5% DMSO, annealing at 72°C; for 18S, 50 nmol/L Alternate II 18S rRNA primers (Ambion), 4 mmol/L MgCl2, and annealing at 60°C. Standard curves were generated using 10-fold serial dilutions of a plasmid containing cloned cDNA (either SSH1L or 18S) and measured in each run at the same time as unknown samples. Copy numbers were calculated by the software provided with the instrument from a linear regression of CT versus Log (copy number). Finally copy numbers of SSH1L were normalized to copy numbers of 18S measured in duplicate samples.
VSMC Migration Assay
Migration was assayed using a modified Boyden chamber assay as previously described.15 This method has previously been shown to measure directed cell migration.18 VSMCs were grown to 85% confluence and then made quiescent in serum-free media for 24 hours before migration. VSMCs (5x104 cells/well) were added to the upper chamber of a Transwell dish on a 6.5-mm insert with a collagen-coated polycarbonate membrane containing 8-µm pores (Costar). VSMCs were then exposed to PDGF (10 ng/mL) in the lower chamber for 3 hours, after which nonmigrated cells were removed from the upper chamber using a cotton swab. The cells remaining on the inserts were fluorescently stained with 4',6-diamidino-2-phenylindole (DAPI) (1 µg/mL) and visualized using a Zeiss Axioskop microscope. Migrated cells per membrane were quantified using ImageJ software.
G-Actin Incorporation
Incorporation of G-actin into the cytoskeleton was measured in vivo in permeabilized cells using a previously published protocol19 with slight modifications. VSMCs were seeded in Laboratory-Tek Chamber Slides (Nunc) covered with collagen (5 ng/mL). After stimulation with PDGF (10 ng/mL) for 30 minutes, cells were incubated with permeabilization buffer (20 mmol/L HEPES [pH 7.5], 138 mmol/L KCl, 4 mmol/L MgCl2, 3 mmol/L EGTA, 1% BSA) containing 40 µg/mL saponin, 0.45 µmol/L biotinylated nonmuscle G-actin (Cytoskeleton Inc), and 1 mmol/L ATP for 3 minutes. After fixation, incorporated G actin was visualized with an anti-biotin antibody coupled to Rhodamine Red-X (The Jackson Laboratory). Actin filaments were stained with phalloidin Alexa-488 (Molecular Probes).
Phosphatase Assay
Phosphatase activity was measured in cell lysates or immunoprecipitates with a commercial kit (Promega) with modifications. VSMCs were lysed in 20 mmol/L imidazole–HCl (pH 7.2) containing 1 mmol/L benzamidine, 1 mmol/L dithiothreitol, 0.05% NP40, and protease inhibitors and were centrifuged at 100 000g for 1 hour at 4°C. The supernatant was passed through a Sephadex G-25 column and incubated with a phosphopeptide mimicking the N terminus of cofilin [MAS(PO4)GVA] at a concentration of 100 µmol/L in reaction buffer (50 mmol/L imidazole, 0.02% β-mercaptoethanol, 0.1 mg/mL BSA) for 15 minutes at 37°C. The reaction was stopped with a molybdate/malachite complex and the absorbance was measured at 600 nm. For immunoprecipitation experiments, VSMCs were lysed in Tris buffer (50 mmol/L Tris [pH 7.5], 150 mmol/L NaCl, 1% Nonidet P-40, 5% glycerol, 1 mmol/L dithiothreitol, 10 µg/mL leupeptin, 1 mmol/L PMSF). Lysates (300 µg per reaction) were immunoprecipitated with anti-SSH1L antibody for 2 hours at 4°C. Then 20 µL of Protein A/G-Agarose Plus (Santa Cruz Biotechnology) were added and incubated at 4°C for 1 hour. The pellet was collected by centrifugation at 3000 rpm for 3 minutes at 4°C and washed 4 times with lysis buffer. Immunoprecipitates were subjected to the phosphatase assay described above.
Statistics
Results are expressed as means±SEM. Differences among groups were analyzed using 1-way ANOVA, with post hoc contrasts adjusted according to the Duncan correction using SPSS 14.0 for Windows. A value of P<0.05 was considered to be statistically significant.
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Actin filament dynamics and reorganization are essential for cell shape change, polarity formation, cytokinesis, and migration.20 Because PDGF induces VSMC migration, we evaluated its capacity to induce changes in the actin cytoskeleton. Actin remodeling is dependent on the equilibrium between depolymerization at the pointed end and polymerization at the barbed end of the actin filament.21 Thus, integration of G-actin can be used to assess actin filament remodeling.19 Using permeabilized VSMCs, we examined the incorporation of biotinylated G-actin into the actin cytoskeleton. As expected, PDGF stimulation (30 minutes, 10 ng/mL) induced an increased incorporation of G-actin in cell protrusions (Figure 1).
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Cofilin is a key protein in the regulation of actin cytoskeleton dynamics. It has been shown that its activation generates protrusions and determines the direction of cell migration.22 Therefore, we hypothesized that PDGF induces cofilin activation in VSMCs. Indeed, we observed that PDGF-treated VSMCs undergo a rapid activation/dephosphorylation of cofilin, with significant activation occurring between 5 and 45 minutes after PDGF addition, and a maximum 43±5% dephosphorylation at 30 minutes (Figure 2).
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These results suggest that PDGF activates a cofilin phosphatase. As noted above, the SSH family of phosphatases has been shown to target cofilin. Using real-time quantitative PCR, we found that human VSMCs express SSH1, SSH2, and SSH3 (data not shown). Because the long form of SSH1 (SSH1L) has an actin binding site and has been implicated in chemotaxis, we examined its role in PDGF-induced cofilin activation. As shown in Figure 3A, PDGF acutely stimulates SSH1L activity by approximately 2-fold at 30 minutes. Using an in vitro immunoprecipitation assay, we observed a detectable increase in its activity as early as 1 minute (Figure 3A, inset), before cofilin dephosphorylation begins. Moreover, PDGF induces SSH1L mRNA and protein over 12 to 48 hours (Figure 3B and 3C), suggesting a need for additional protein over the long term.
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To determine whether SSH1L phosphatase is responsible for cofilin dephosphorylation in VSMCs treated with PDGF, the expression of SSH1L was downregulated using siRNA. We were able to significantly reduce SSH1L mRNA (Figure 4A), protein (Figure 4B), or PDGF-induced activity (Figure 4C) without affecting SSH2 and SSH3 mRNA (not shown). Most importantly, when SSH1L was downregulated, PDGF-induced dephosphorylation of cofilin was blocked (Figure 5), confirming the participation of this phosphatase in PDGF-stimulated cofilin activation.
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In other cells, activation of SSH1L occurs in conjunction with activation of LIMKs,17 a family of proteins that are unique kinases for cofilin.4 To determine whether a similar mechanism occurs in VSMCs, we tested the effect of PDGF on LIMK1/2 activity. Using a phosphospecific antibody against the conserved Thr508 or Thr505 residues in the activation loop, we found that PDGF induced a robust activation of LIMK, as early as 1 minute, with a maximum at 5 minutes and a return to near basal levels by 30 minutes (Figure 6). No further change in LIMK activity was observed for up to 4 hours (data not shown). This activation of LIMK is directly responsible for cofilin phosphorylation, because PDGF phosphorylates cofilin when SSH1L activity is blocked, and a dominant negative form of LIMK (LIMK kinase-dead domain or KS)16 blocks this phosphorylation (Figure 7). In support of this interpretation, in control cells, where cofilin phosphorylation is clearly detectable and SSH1L activity is negligible, KS-LIMK also reduces basal cofilin phosphorylation (Figure I in the online data supplement, available at http://circres.ahajournals.org).
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Finally, to address the role of SSH1L phosphatase in VSMC migration, the impact of SSH1L downregulation on the ability of VSMCs to pass through a 8-µm membrane in a Boyden chamber was evaluated. As shown in Figure 8, downregulation of SSH1L phosphatase induced a significant inhibition (38.2±4.9%) of the number of cells able to migrate through the chamber membrane in response to PDGF. The downregulation of SSH1L was not associated with changes in cell adhesion (76.3±4.1% after scrambled control treatment versus 76.2±1.0% after siSSH1L treatment; P=0.99 after 30 minutes), confirming a role for SSH1L/cofilin dephosphorylation in PDGF-induced migration of VSMCs.
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Discussion |
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In this report, we show for the first time that PDGF stimulation of VSMCs activates the LIMK/cofilin/SSH1L pathway to regulate migration. The regulation of cofilin phosphorylation is complex, with PDGF activating both a cofilin kinase and a cofilin phosphatase. The net effect of these events is dephosphorylation of cofilin, leading to remodeling of the actin cytoskeleton and ultimately migration. These results point to a central role for SSH1L in regulating VSMC migration.
Actin remodeling is central to the migration process. All actin polymerization and branching in vivo is dependent on the generation of free barbed ends23 by actin-severing molecules, the removal of capping proteins, and de novo nucleation of actin filaments. Multiple actin-binding proteins regulate these processes. For instance, the Arp2/3 complex (controlled by WAVE/Scar, WASP, and N-WASP proteins) control nucleation and branching, whereas the formins, mammalian diaphanous proteins mDia1 and mDia2 (regulated by RhoA and Cdc42), are involved in extension of new actin filaments.24 Profilin must be liberated from the capped end of actin filaments, after which it promotes G-actin nucleotide exchange.25 Cofilin stimulates disassembly and severance of actin filaments at or near the pointed ends and thereby continuously supplies actin monomers for polymerization and rapid turnover of actin filaments.3 One of the major effects of cofilin activation is to generate free barbed ends on actin filaments. Even though PDGF is one of the major migratory stimuli for VSMC migration, virtually nothing is known about its ability to regulate these pathways.
In this study, we focused on the ability of PDGF to regulate cofilin because it has been shown to be critically important for migration. In human erythroleukemia cells, expression of either constitutively active or inactive cofilin inhibits migration, highlighting the importance of cofilin-mediated actin turnover.26 Furthermore, the up- or downregulation of cofilin activity has been shown to correlate with proportional increases or decreases in motility of tumor cells, respectively.16 We found that PDGF stimulation of VSMC migration was accompanied by cofilin activation (Figure 2). Moreover, this activity was required for cell movement, because preventing dephosphorylation by siSSH1L inhibited migration (Figure 8). This suggests that the mechanisms controlling cofilin activation are critical to the regulation of PDGF-induced VSMC motility.
The apparently simultaneous dual regulation of cofilin by LIMK and SSH1L by PDGF in VSMCs may represent a fine-tuned control of directional movement. It is surprising that we were unable to detect cofilin phosphorylation even at early times after PDGF stimulation when LIMK was activated. The appearance of cofilin phosphorylation at 5 minutes, when SSH1L is inhibited, and the ability of dominant negative LIMK to block this phosphorylation (Figure 7), confirm that LIMK is in fact targeting cofilin. Consistent with this, at 30 minutes, when LIMK is no longer active (Figure 6), downregulation of SSH1L no longer unmasks increased cofilin phosphorylation (Figure 5). In Jurkat T cells stimulated with stromal cell derived factor-1
, LIMK was found diffusely in the cytoplasm, whereas SSH1L was localized to lamellipodia, but activation of both proteins was necessary for chemotaxis.17 This suggests that the spatial relationship between LIMK/cofilin/SSH1L is crucial for the polarization of cell movement. Alternatively, it may be the temporal relationship between LIMK and SSH1L activation that dictates the migratory response. After 30 minutes, LIMK activation has returned to baseline, whereas SSH1L activity is still robust (Figures 3 and 4
). It has been shown that SSH1L can dephosphorylate LIMK,27 which may explain the transient nature of LIMK activation, contrasting with a long-term activation of the phosphatase in PDGF-stimulated cells, as we deduce from the sustained SSH1L upregulation at messenger and protein level. Ultimately, it is the dynamic ratio of active LIMK to active SSH1L that is important. In VSMCs, the predominant effect of PDGF appears to be stimulation of SSH1L activity and dephosphorylation of cofilin.
SSH1L is a member of the slingshot family of phosphatases that includes SSH1L, SSH2L, and SSH3L. Each of these proteins has distinct specific activities and subcellular distributions.12,14 Our results show that although all 3 SSH isoforms are expressed in VSMCs, SSH1L is activated by PDGF (Figure 3), and, for the first time, clearly demonstrate that it is required for PDGF-mediated migration (Figure 8). The mechanisms that regulate SSH1L activity remain to be elucidated. It has been shown that ATP or histamine-induced SSH1L activation is mediated by calcium-dependent stimulation of calcineurin28 and that dephosphorylation of SSH1L on Ser978 increases its activity.11 However, calcineurin does not mediate PDGF-induced migration in fibroblasts.29 In contrast, insulin stimulation of SSH1L activity is dependent on phosphatidylinositol 3-kinase,30 a signaling pathway proposed to be important in PDGF-induced VSMC migration in some types of smooth muscle cells.31 Determining the upstream signaling mechanisms responsible for SSH1L activation in migrating VSMC will require further study.
In summary, we find that one of the key factors regulating PDGF-induced migration is the phosphorylation state of cofilin. PDGF activates both the cofilin kinase LIMK and the novel phosphatase SSH1L, the latter of which predominates to dephosphorylate cofilin. In accordance with these findings, blocking SSH1L significantly inhibits PDGF-induced migration. These results suggest that PDGF-induced migration requires fine control of cofilin phosphorylation and that SSH1L plays a critical role in maintaining cofilin activity during PDGF-induced migration of VSMCs.
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Acknowledgments |
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We are grateful to Dr John Condeelis for providing the KS-LIMK plasmid.
Sources of Funding
This work was supported by NIH grants HL38206 and HL058863 (to K.K.G.) and American Heart Association Fellowship 0525465B (to A.S.M.).
Disclosures
None.
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Footnotes |
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This manuscript was sent to Mark Taubman, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Original received June 28, 2007; revision received November 20, 2007; accepted December 7, 2007.
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