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Differential roles of Rho-kinase and myosin light chain kinase in regulating shape, adhesion, and migration of HT1080 fibrosarcoma cells
BBRC Biochemical and Biophysical Research Communications 343 (2006) 602–608 www.elsevier.com/locate/ybbrc
Differential roles of Rho-kinase and myosin light chain kinase in regulating shape, adhesion, and migration of HT1080 fibrosarcoma cells Verena Niggli *, Manuela Schmid, Alexandra Nievergelt Department of Pathology, University of Bern, Murtenstr. 31, CH-3010 Bern, Switzerland Received 2 March 2006 Available online 15 March 2006
Abstract We present evidence for differential roles of Rho-kinase and myosin light chain kinase (MLCK) in regulating shape, adhesion, migration, and chemotaxis of human fibrosarcoma HT1080 cells on laminin-coated surfaces. Pharmacological inhibition of Rho-kinase by Y-27632 or inhibition of MLCK by W-7 or ML-7 resulted in significant attenuation of constitutive myosin light chain phosphorylation. Rho-kinase inhibition resulted in sickle-shaped cells featuring long, thin F-actin-rich protrusions. These cells adhered more strongly to laminin and migrated faster. Inhibition of MLCK in contrast resulted in spherical cells and marked impairment of adhesion and migration. Inhibition of myosin II activation with blebbistatin resulted in a morphology similar to that induced by Y-27632 and enhanced migration and adhesion. Cells treated first with blebbistatin and then with ML-7 also rounded up, suggesting that effects of MLCK inhibition on HT1080 cell shape and motility are independent of inhibition of myosin activity. 2006 Elsevier Inc. All rights reserved. Keywords: HT1080 cells; Cell migration; Chemotaxis; Myosin light chain kinase; Rho-kinase; Myosin light chain phosphorylation
Cancer progression from a primary tumor to secondary metastasis is a highly complex process involving altered gene expression, the acquisition of cell motility, interaction with the extracellular matrix (ECM), changes in cell adhesion, and expression of ECM degrading proteases [1,2]. For these processes, cells need to activate intracellular signaling pathways. The Rho-GTPase proteins Rho, Rac, and CDC42 are key modulators of a variety of signaling cascades involved in actin cytoskeleton reorganization, cell shape changes, and cell migration [3,4]. Overactivation of these pathways may contribute to tumor progression and metastasis [5]. A number of Rho effectors have been identified which associate specifically with the GTP-bound forms of Rho GTPases. One of them, Rho-kinase (also called ROCK, Rho-associated coiled-coil forming protein kinase) [6,7], is an important regulator of myosin function. Targets of
*
Corresponding author. Fax: +41 31 381 34 12. E-mail address: [email protected] (V. Niggli).
0006-291X/$ - see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.03.022
Rho-kinase have been shown to be myosin light chain (MLC) and myosin binding subunit (MBS) of myosin phosphatase [8]. Non-muscle myosin II activity is mainly controlled through the phosphorylation of MLC, which is regulated by myosin light chain kinase (MLCK), Rho-kinase, and myosin phosphatase. An increase in intracellular Ca2+ concentration leads to activation of Ca2+/calmodulin-dependent myosin light chain kinase (MLCK), which then phosphorylates Thr-18 and Ser-19 on MLC [9]. Rho-kinase can increase MLC phosphorylation in two ways: on one hand by phosphorylating the myosin binding subunit (MBS) of myosin phosphatase and thereby inhibiting myosin phosphatase activity [10] and on the other hand by direct phosphorylation of MLC [11]. Rhokinase activity can be suppressed with Y-27632, an inhibitor with some specificity for this enzyme [12], whereas MLCK activity can be targeted by two inhibitors with different mechanisms of action; ML-7 [13] or W-7 [14], the first competing with ATP, the latter acting by inhibiting calmodulin whose activity is required for activation of MLCK. Depending on the cell type, Rho-kinase is required
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for migration or is dispensible for this function, as shown for leukocytes, endothelial cells, epithelial cells, fibroblasts, and different types of tumor cells [15–18]. Interestingly, MLCK and Rho-kinase have been shown to have spatially different roles in controlling MLC phosphorylation and actin organization. In fibroblasts for example, Rho-kinase appears to be involved in maintaining the central actin stress fibers, whereas MLCK is required for peripheral stress fibers. In gerbil fibroma cells, Y-27632 reduces MLC phosphorylation and focal contacts in the center of the cells, whereas a peptide interfering with MLCK reduces peripheral phosphorylated MLC and focal contacts at the leading edge [19,20]. Here, we have compared the roles of Rho-kinase, myosin light chain kinase (MLCK), and myosin in regulating migration and adhesion of the human fibrosarcoma cell line HT1080 on an important component of the basal membrane, that is, laminin. These cells, of fibroblastic origin, are highly invasive and able to migrate spontaneously on extracellular matrix components. In contrast to normal fibroblasts, they are poorly adherent and lack stress fibers. This appears to be due to a mutated constitutively active n-ras allele, as deletion of this allele resulted in more adherent cells with stress fibers [21]. We previously provided evidence for a role of protein kinase C in regulating HT1080 cell migration [22]. HT1080 cells express a number of integrin isoforms, among them the laminin receptors a3b1 and a6b1 [23,24]. HT1080 cells constitutively synthesize and secrete PDGF which binds to cell surface PDGF-receptors, resulting in constitutively activated PI 3-kinase. Furthermore, RhoA is also constitutively activated downstream of activated n-ras [21,25]. Data on the role of the Rho target Rho-kinase in HT1080 cell migration are somewhat contradictory. The ROCK inhibitor Y-27632 suppresses the plasminogen activator-induced increase in migration of HT1080 cells through filters coated with vitronectin but does not affect migration in the absence of plasminogen activator [26] or on laminin-coated surfaces [27]. In contrast, experiments using ribozymes that selectively cleave mRNA of Rho-kinase I suggest a specific requirement of this isoform for migration of HT1080 cells across filters in response to fetal bovine serum (FBS) [28]. We now provide new data on differential roles of Rhokinase and MLCK in regulating shape, adhesion, migration, and chemotaxis of HT1080 cells on laminin-coated surfaces. Inhibition of both enzymes resulted in a similar marked inhibition of constitutive myosin light chain phosphorylation. However, cells treated with Rho-kinase inhibitor or with blebbistatin, which inhibits the actin-activated Mg2+-ATPase activity of muscle and non-muscle myosin II with some specificity [29], were sickle shaped with long, thin F-actin-rich protrusions and showed increased adhesion and migration. In contrast, inhibition of MLCK resulted in spherical cells and markedly reduced adhesion and migration. We moreover show that the effects of the MLCK inhibitors were obtained also in cells where myosin activity was inhibited first with blebbistatin.
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Materials and methods Materials. Cell dissociation solution (non-enzymatic), laminin purified from basement membrane of Engelbreth-Holm-Swarm mouse sarcoma, Hoechst Dye (Bisbenzimide trihydrochloride), and dimethyl sulfoxide (DMSO) were from Sigma (St. Louis, MO). Bovine serum albumin (BSA) was from Serva (Heidelberg, Germany); fetal bovine serum (FBS) from Animed (Allschwil, Switzerland); plasminogen from Chromogenix (Milano, Italy). Alexa 488-phalloidin was from Molecular Probes (Junction City, OR). Y-27632 was from Calbiochem/VWR International AG (Lucern, Switzerland). A 10 mM stock solution of Y-27632 was prepared in H2O and stored at 20 C. ML-7 and W-7 were from Alexis Biochemicals (Lausen, Switzerland). Blebbistatin (+/ ) was from Tocris (Avonmouth, UK). Stock solutions of ML-7, blebbistatin, and W-7 were prepared in DMSO and stored at 20 C. DMSO alone had no effect at the final concentrations used. Antibodies. A monoclonal murine anti-myosin light chain antibody was from Sigma, St. Louis, MO, USA (clone MY-21, Cat. Nr. M4401). A polyclonal anti-phospho MLC2 (Thr-18/Ser-19) antibody (Cat. Nr. 3674) was from Cell Signaling, Beverly, MA, USA. Horseradish peroxidaselabeled goat anti-mouse IgG and horseradish peroxidase-labeled goat anti-rabbit antibodies were from Bio-Rad (Hercules, CA). The rhodamine-coupled goat-anti-mouse IgM antibody was from Bio-Science Products AG (Emmenbru¨cke, Switzerland). Cell culture. HT1080 cells (American Type Culture Collection, ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Sigma D-5671) supplemented with 2 mM stable glutamine, 10% heat-inactivated FBS, 40 mg/L streptomycin, and 40,000 U/L penicillin. Migration assays. Two-dimensional migration assays (videomicroscopy): Wells of a 12-well plate (Costar, Cambridge, MA) were coated overnight with 5 lg/well laminin (in 0.1 M Tris, pH 8.0), followed by washing with DMEM with 10% FBS and drying at room temperature. Thirty-five thousand HT1080 cells were added per well and incubated overnight in DMEM containing 10% FBS at 37 C and 5% CO2. The medium was then removed and cells were incubated for 30 min in serumfree DMEM containing plasminogen (5 lg/ml), followed by preincubation without or with inhibitors (see Results). Migration was subsequently recorded at 37 C and 5% CO2 on videotape for 2 h and evaluated as reported previously [22]. Chemotaxis assays. Twenty-four-transwell plates with inserts (6.5 mm diameter, 8.0 lm pore size, Costar) were laminated as described above, followed by washing with serum-free DMEM and drying at room temperature. 200,000 cells, resuspended in 200 ll DMEM with 0.1% BSA, were added per insert. The lower wells contained either 600 ll DMEM with 0.1% BSA as a control or DMEM with 10% FBS as a chemoattractant. Inhibitors were added to upper and lower wells (see Results). Then the cells were incubated for 4 h at 37 C and 5% CO2. Cells on the upper side of the filter were subsequently removed with a cotton swab. After washing with phosphate-buffered saline (PBS), the filters were fixed with 3.7% paraformaldehyde (PFA) in PBS for 15 min at 37 C followed by staining with 10 lM Hoechst Dye (in PBS) for 30 min at room temperature. The nuclei of migrated cells on the lower side of the filter were counted in six fields using a Nikon Eclipse TE300 fluorescence microscope (20· objective). Adhesion assays. For adhesion assays, cells (35,000 per well) were incubated in laminin-coated 12-well plates in DMEM containing 10% FBS at 37 C and 5% CO2, followed by treatment with the different inhibitors (see Results), washing with DMEM lacking FBS, and incubation on a rotating shaker at 200 rpm for 3 min at room temperature after resuspension in DMEM with 10% FBS. Cells were washed twice afterwards and fresh DMEM with 10% FBS was added. The number of adherent cells in the wells was evaluated by counting the cells in 10 different standardized frames per sample. The value obtained for the control cells was set to 100%. Detection of phosphorylated myosin light chain by immunoblotting. Cells (200,000 cells per well) were seeded into laminin-coated 12-well plates as described above and incubated for 4 h in DMEM containing 10% FBS at 37 C and 5% CO2, followed by removal of the medium, addition of
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DMEM lacking FBS (0.5 ml per well), and a further incubation for 30 min at 37 C. Cells were then incubated without or with inhibitors (see Results) for 30 min at 37 C, followed by harvesting of the cells in ice-cold 12.5% TCA (trichloroacetic acid), 40 mM NaF, and 10 mM Na2HPO4, as described [22]. The protein pellets were solubilized in a buffer containing 60 mM Tris, pH 7.5, 1% SDS, and 1 mM EDTA (15 min at 95 C), and subjected to immunoblotting (15% SDS–polyacrylamide gel) using a polyclonal rabbit anti-phospho-MLC2 (Thr-18/Ser-19) antibody (diluted 1:500). In order to control for protein loading, blots were stripped and reprobed with an antibody reacting with MLC independent of its state of phosphorylation (diluted 1:750). Assessment of morphology; visualization of F-actin and MLC. Cells (20,000 cells per well) were seeded into laminin-coated wells of Permanox 8-well slide flasks, incubated for 6 h in DMEM containing 10% FBS, followed by removal of the medium, addition of DMEM lacking FBS, and a further incubation for 30 min at 37 C. Cells were then incubated without or with inhibitors (see Results) for 30 min at 37 C and fixed with 1.25% glutaraldehyde in PBS (pH 7.4) for studies of morphology. For immunofluorescence staining, cells were fixed in 4% PFA in PBS (pH 7.4) for 15 min at 37 C and washed with PBS. Cells were permeabilized with 0.5% Triton X-100 in PBS for 10 min at room temperature followed by blocking with TBST (50 mM Tris, pH 7.4, 150 mM NaCl, and 0.05% Tween) containing 5% defatted milk powder and 10% normal goat serum. Cells were incubated first with monoclonal anti-MLC antibody, diluted 1:50 in TBST containing 0.2% BSA, followed by rhodamine-coupled goatanti-mouse diluted 1:500 in TBST containing 0.2% BSA (1 hr at R.T. each). Subsequently F-actin staining was performed as described [22] using Alexa 488-phalloidin. Pictures were taken with a Zeiss MC 80 Axioplan microscope (10· or 40· objective) connected to a Nikon digital camera DXM1200F. Cell viability assays. Cells (35,000 per well) were seeded into laminated 12-well plates as described for the chemotaxis assays. After washing with prewarmed DMEM lacking FBS, the different inhibitors, diluted in 450 ll DMEM + 0.1% BSA, were added (100 lM blebbistatin, 20 lM ML-7, 20 lM W-7 or 20 lM Y-27632), followed by incubation at 37 C for 4 h, corresponding to the time of migration in the chemotaxis assays.
Subsequently Trypan blue was added to the medium (0.2% final concentration) and the fraction of cells taking up the dye was determined after a 5 min incubation. Control cell cultures contained 99 ± 1% (n = 3) viable cells. None of the inhibitors tested reduced cell viability by more than 6% (data not shown). Statistical analysis of data. Differences between the data were assessed using Student’s t test for paired data with a p value <0.05 considered significant. Data correspond to means ± SEM.
Results Comparison of impact of inhibition of Rho-kinase, MLCK or myosin II on adhesion, morphology, migration, and chemotaxis of HT1080 cells 31 ± 2% (n = 3) of untreated cells adhering to laminincoated surfaces showed a clearly polarized morphology with F-actin-rich ruffles on one side and a contracted tail on the other side. Myosin light chain was located diffusely in the cytosol but also showed some enrichment in tips of ruffles and at the cell margins (controls in Figs. 1 and 2). Inhibition of ROCK by Y-27632 in HT1080 cells resulted in formation of long thin F-actin-rich protrusions and sickle-shaped cells with F-actin-rich ruffles at one side in approximately 70% of the cells. Myosin light chain was also located in the ruffles as well as in the protrusions and the cytosol. These protrusions formed contacts with those of neighboring cells. A similar morphology was obtained by exposing cells to 50 lM of the myosin II inhibitor blebbistatin (Figs. 1 and 2). In contrast, treatment of cells with two different inhibitors of MLCK (20 lM
Fig. 1. Impact of inhibition of ROCK, MLCK or myosin on morphology of HT1080 cells. Cells grown on laminin were incubated with medium (control) or with either 20 lM Y-27632 or 50 lM blebbistatin or 20 lM ML-7 or 20 lM W-7 for 30 min at 37 C, or with 50 lM blebbistatin for 30 min followed by the addition of 20 lM ML-7 and a further incubation for 30 min at 37 C, as indicated, followed by fixation with glutaraldehyde. Bar, 50 lm.
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Fig. 2. Impact of inhibition of ROCK, MLCK or myosin on localization of F-actin and MLC in HT1080 cells. Cells grown on laminin were incubated with medium (control) or with either 20 lM Y-27632 or 50 lM blebbistatin or 20 lM ML-7 or 20 lM W-7 for 30 min at 37 C, or with 50 lM blebbistatin for 30 min followed by the addition of 20 lM ML-7 and a further incubation for 30 min at 37 C, as indicated, followed by fixation of the cells and doublestaining for F-actin and MLC. Bar, 50 lm.
ML-7 or W-7) resulted in 100% of completely spherical cells lacking any protrusions with diffusely distributed F-actin and myosin light chain (Figs. 1 and 2). Rounding up of the cells induced by these MLCK inhibitors was independent of myosin activity, as it could also be observed when the cells were first treated with blebbistatin, followed by a further incubation with ML-7. However, in contrast to the cells treated with ML-7 alone, the latter rounded cells still displayed multiple protrusions (Figs. 1 and 2). We then compared the impact of these inhibitors on adhesion, migration, and chemotaxis. Similar to the findings on morphology, inhibition of ROCK or myosin on one hand and of MLCK on the other hand differed markedly in their impact on adhesion, 2-dimensional migration, and chemotaxis. Adhesion was significantly increased by a factor of 1.5 ± 0.1 (n = 4, p < 0.0025) by Rho-kinase inhibition or by a factor of 1.8 ± 0.1 (n = 3, p < 0.005) by myosin inhibition whereas MLCK inhibition by ML-7 or W-7 almost completely suppressed firm adhesion (Fig. 3). Similarly, preincubation of cells with 20 lM Y-27632 significantly increased speed of migration 1.7 ± 0.2-fold (n = 5, p < 0.001) and % of migrating cells 1.4 ± 0.1-fold (n = 5,
p < 0.0005) in a 2-D assay system on laminin-coated surfaces (Table 1). Blebbistatin similarly enhanced the speed of migration 1.9 ± 0.1-fold (n = 5, p < 0.005, Table 1). In contrast, ML-7 and W-7 completely suppressed migration (Table 1). We also assessed the impact of inhibiting Rho-kinase, MLCK, and myosin on chemotaxis of HT1080 cells through laminin-coated 8 lm filters towards 10% FBS in the lower wells. Chemotaxis of HT1080 cells to the lower side of the filter was markedly increased 4 ± 1-fold (p < 0.001, n = 9) by 10% FBS (Fig. 4A). Migration was attenuated when including 10% FBS in the upper and lower wells by 67 ± 11% (p < 0.025, n = 3), indicating that approximately 70% of the total migratory activity corresponds to chemotaxis as compared to stimulated undirected migration (chemokinesis) (not shown). Similar to their impact on 2-D migration, ML-7 and W7 also negatively affected chemotaxis. W-7 suppressed the FBS-induced increase completely, whereas ML-7 inhibited this increase by 72 ± 9% (p < 0.0005, n = 6) (Figs. 4C and D). Again in agreement with the results obtained by videomicroscopy, inhibition of myosin activity by blebbistatin Table 1 Impact of inhibition of ROCK, MLCK or myosin on 2-D migration of HT1080 cells
Fig. 3. Impact of inhibition of Rho-kinase, MLCK or myosin on adhesion of HT1080 cells to laminin. Cells were grown in laminin-coated wells. After incubation with medium (control) or the different inhibitors as indicated for 30 min at 37 C, cells were washed and the number of adherent cells as % of untreated controls was determined. Concentrations of the inhibitors were 20 lM for ML-7 and Y-27632, and 50 lM for blebbistatin (blebb.). Means ± SEM of 3–4 experiments.
Addition
Mean speed of migrating cells (lm/min)
Migrated cells (%)
Medium Y-27632 (20 lM)
50 ± 6 (n = 5) 81 ± 4 (n = 5)
68 ± 9 (n = 5) 90 ± 4 (n = 5)
DMSO (0.1%) Blebbistatin (50 lM)
35 ± 3 (n = 5) 64 ± 1 (n = 5)
79 ± 3 (n = 5) 88 ± 3 (n = 5)
DMSO (0.1%) ML-7 (20 lM)
41± 4 (n = 4) 0 ± 0 (n = 4)
80 ± 2 (n = 4) 0 ± 0 (n = 4)
DMSO (0.1%) W-7 (20 lM)
38 ± 4 (n = 3) 0 ± 0 (n = 4)
82 ± 2 (n = 3) 0 ± 0 (n = 4)
HT1080 cells were preincubated with the indicated concentrations of inhibitors or DMSO for 30 min at 37 C and 5% CO2. Migration was subsequently recorded on videotape for 2 h and the percentage of migration cells and the mean speed of migration was evaluated (means ± SEM of 3–5 independent experiments).
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Fig. 4. Impact of inhibition of Rho-kinase, MLCK or myosin on chemotaxis of HT1080 cells. 200,000 cells in DMEM with 0.1% BSA were placed in 8 lm filter inserts. Lower wells contained either DMEM with 0.1% BSA or DMEM with 10% FBS as a chemoattractant, as indicated. For part of the samples inserts and lower wells contained in addition (A) Y-27632 (n = 9), (B) blebbistatin (n = 7), (C) ML-7 (n = 6) or (D) W-7 (n = 3), as indicated. Migration through 8 lm transwell filters was determined for 4 h at 37 C. The number of cells detected on the lower side of the filter obtained with 10% FBS as chemoattractant was set at 100%. Means ± SEM of 3–9 experiments.
significantly increased migration of cells through filters in the absence of chemoattractant 1.7 ± 0.2-fold (p < 0.005, n = 7) and also that in the presence of FBS 1.5 ± 0.1-fold (p < 0.025, n = 7) (Fig. 4B). The only discrepancy between data obtained with the two assay systems was observed for Y-27632 (Fig. 4A), where we observed a small but statistically not significant reduction in FBS-induced chemotaxis, in contrast to the increase observed in 2-D migration (Table 1). Comparison of impact of inhibition of Rho-kinase and MLCK on MLC phosphorylation MLCK and Rho-kinase are major enzymes controlling MLC phosphorylation [9]. The above results could be explained by differential roles of ROCK and MLCK in controlling myosin phosphorylation in HT1080 cells. We therefore compared effects of Y-27632, W-7, and ML-7 on phosphorylation of MLC on Thr18 and Ser 19. As shown in Fig. 5, all three inhibitors significantly (p < 0.05) reduced constitutive MLC phosphorylation in HT1080 cells by 40–60%, Y-27632 being most effective. The differential impact of these inhibitors on cell shape and migration cannot thus be explained by marked differences in their impact on MLC phosphorylation.
Fig. 5. Impact of inhibition of Rho-kinase, MLCK or myosin II on phosphorylation of myosin light chain (MLC) in HT1080 cells. Cells grown on laminin were incubated with medium (control) or the different inhibitors (20 lM each) for 30 min at 37 C, followed by harvesting of the cells with TCA and immunoblotting using a polyclonal rabbit antiphospho-MLC (Thr-18/Ser-19) antibody. Controls for ML-7 and W-7 contained 0.4% DMSO. PP-MLC indicates MLC phosphorylated on Thr-18 and Ser-19. The blots were stripped and reprobed with an antibody reacting with MLC independent of its state of phosphorylation. (A) Blot of a representative experiment; (B) quantitative evaluation, means ± SEM of 3–4 experiments.
Discussion We provide novel data documenting differential roles of Rho-kinase and MLCK in regulating shape, adhesion, and
migration of HT1080 cells. Inhibition of Rho-kinase with Y-27632 results in sickle-shaped HT1080 cells with long fine F-actin-rich protrusions, which adhere more strongly
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to laminin and migrate faster in 2-D systems and whose chemotaxis to FBS is not significantly impaired. The specificity of the Rho-kinase inhibitor Y-27632 has been tested on a large panel of protein kinases. Y-27632 was inactive on 21 tested enzymes and inhibited only one (PRK; protein kinase C-related protein kinase) with similar potency as the Rho-kinase isoforms [12]. Our data could be confirmed by transfection with dominant-negative Rho-kinase [30] which induced a comparable morphology as Y-27632 (data not shown). These findings indicate that the pool of myosin II controlled by Rho-kinase specifically restricts formation of lateral protrusions and limits migration and adhesion. Invasion of rat MM1 hepatoma cells studied in mouse models could be inhibited by administration of Rho-kinase inhibitor [16]. Our data now suggest that this may not be the case for all types of tumors and that in some cases metastasis and invasion could even be stimulated by Rho-kinase inhibition. Intriguingly cells treated with either ML-7 or W-7, inhibitors of MLCK, showed a completely different spherical morphology without any protrusions, as well as markedly decreased adhesion, migration, and chemotaxis. ML-7, as noted by Bain et al. [13], is a relatively specific, ATP-competitive MLCK inhibitor which did not significantly inhibit activity of 28 tested other enzymes. Only the activity of the mitogen- and stress-activated protein kinase 1 (MSK1) was reduced albeit at much higher concentrations than that of MLCK. W-7 acts mainly as a calmodulin antagonist and inhibits activity of a calciumcalmodulin-independent fragment of MLCK much less potently than ML-7 [14]. As we obtained the same findings with these two differentially acting inhibitors, we think it unlikely that they can be explained by effects on proteins other than MLCK. Shoemaker et al. [31] observed similarly that chicken embryo fibroblasts treated with an antisense probe directed against MLCK assumed a spherical morphology without protrusions. Our attempts to down-regulate MLCK expression by incubating HT1080 cells with two different siRNAs targeting MLCK up to 4 days failed, possibly due to very slow turnover of this protein, as we could successfully prevent EGFP expression by EGFP siRNA (data not shown). Our findings obtained with inhibitors cannot be explained by a more important role of MLCK in regulating MLC phosphorylation and activation, as Y-27632 was in our hands even somewhat more effective than ML-7 or W-7 in attenuating constitutive MLC phosphorylation in HT1080 cells (Fig. 5). Our data suggest that Rho-kinase limits HT1080 migration whereas MLCK activity is absolutely required for this process, despite the fact that both enzymes promote myosin activation by enhancing its phosphorylation. This is different from our previous findings for Walker carcinosarcoma cells, where inhibition of both Rho-kinase and MLCK abrogated MLC phosphorylation, polarization, and migration [32]. One explanation of our data would be differences in the spatial control of myosin II activation by these two enzymes, as shown in fibroblasts [19,20].
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In order to clarify this issue, we studied the impact of abrogation of myosin II activity by blebbistatin, an inhibitor with some selectivity for non-muscle myosin IIA and IIB [29], on morphology, adhesion, and migration of HT1080 cells. Treatment with blebbistatin mimicked effects of Rho-kinase inhibition on cell morphology, adhesion, and 2-D migration. Similarly, fibroblasts derived from myosin IIB null mice exhibit a sickle-shaped morphology with long processes and migrate faster than wild-type cells [33]. The only difference we observed between effects of Y-27632 and blebbistatin concerned chemotaxis, where blebbistatin clearly increased migration through filters in the absence or presence of chemoattractant, whereas Y-27632 did not markedly affect these processes. Possibly the advantage which myosin inhibition confers to chemotaxis is abrogated by inhibition of activity of Rho-kinase targets other than myosin. Importantly, treatment of cells first exposed to blebbistatin with ML-7 still resulted in rounding up of cells, albeit the fine protrusions induced by myosin inhibition were conserved. This finding strongly suggests that MLCK has cellular functions unrelated to its capacity to phosphorylate and activate myosin II. Indeed, MLCK may have actin-bundling activity [34], and has been reported to control calcium-dependent signaling via MLC-independent mechanisms [35]. Further investigations will focus on downstream targets of MLCK different from myosin. In summary, both Rho-kinase and MLCK are involved in controlling MLC phosphorylation in HT1080 cells, but whereas Rho-kinase limits adhesion and migration, possibly by preventing protrusion formation via inhibition of myosin activation, MLCK may have additional roles in controlling actin organization required for HT1080 cell migration and adhesion. Acknowledgments This work was supported by the Bernese Cancer League and the Swiss National Science Foundation (Grant No. 3100A0-103708/1). We are grateful to Christoph Oppliger, Tamara Locher, Maddalena Lis, and Dominique Schlicht for expert technical assistance. We thank S. Narumiya (Department of Pharmacology, Kyoto University, Faculty of Medicine, Kyoto, Japan) for the kind gift of pCAGmyc-WT-ROCKI and pCAG-myc-KD-IA-ROCKI, and E. Sigel for critical reading of the manuscript. References [1] J. Kassis, D.A. Lauffenburger, T. Turner, A. Wells, Tumor invasion as dysregulated cell motility, Semin. Cancer Biol. 11 (2001) 105–117. [2] J. Condeelis, J.E. Segall, Intravital imaging of cell movement in tumours, Nat. Rev. Cancer 3 (2003) 921–930. [3] S. Etienne-Manneville, A. Hall, Rho GTPases in cell biology, Nature 420 (2002) 629–635. [4] A.P. Wheeler, A.J. Ridley, Why three Rho proteins? RhoA, RhoB, RhoC, and cell motility, Exp. Cell Res. 301 (2004) 43–49.
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Differential roles of Rho-kinase and myosin light chain kinase in regulating shape, adhesion, and migration of HT1080 fibrosarcoma cells Verena Niggli *, Manuela Schmid, Alexandra Nievergelt Department of Pathology, University of Bern, Murtenstr. 31, CH-3010 Bern, Switzerland Received 2 March 2006 Available online 15 March 2006
Abstract We present evidence for differential roles of Rho-kinase and myosin light chain kinase (MLCK) in regulating shape, adhesion, migration, and chemotaxis of human fibrosarcoma HT1080 cells on laminin-coated surfaces. Pharmacological inhibition of Rho-kinase by Y-27632 or inhibition of MLCK by W-7 or ML-7 resulted in significant attenuation of constitutive myosin light chain phosphorylation. Rho-kinase inhibition resulted in sickle-shaped cells featuring long, thin F-actin-rich protrusions. These cells adhered more strongly to laminin and migrated faster. Inhibition of MLCK in contrast resulted in spherical cells and marked impairment of adhesion and migration. Inhibition of myosin II activation with blebbistatin resulted in a morphology similar to that induced by Y-27632 and enhanced migration and adhesion. Cells treated first with blebbistatin and then with ML-7 also rounded up, suggesting that effects of MLCK inhibition on HT1080 cell shape and motility are independent of inhibition of myosin activity. 2006 Elsevier Inc. All rights reserved. Keywords: HT1080 cells; Cell migration; Chemotaxis; Myosin light chain kinase; Rho-kinase; Myosin light chain phosphorylation
Cancer progression from a primary tumor to secondary metastasis is a highly complex process involving altered gene expression, the acquisition of cell motility, interaction with the extracellular matrix (ECM), changes in cell adhesion, and expression of ECM degrading proteases [1,2]. For these processes, cells need to activate intracellular signaling pathways. The Rho-GTPase proteins Rho, Rac, and CDC42 are key modulators of a variety of signaling cascades involved in actin cytoskeleton reorganization, cell shape changes, and cell migration [3,4]. Overactivation of these pathways may contribute to tumor progression and metastasis [5]. A number of Rho effectors have been identified which associate specifically with the GTP-bound forms of Rho GTPases. One of them, Rho-kinase (also called ROCK, Rho-associated coiled-coil forming protein kinase) [6,7], is an important regulator of myosin function. Targets of
*
Corresponding author. Fax: +41 31 381 34 12. E-mail address: [email protected] (V. Niggli).
0006-291X/$ - see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.03.022
Rho-kinase have been shown to be myosin light chain (MLC) and myosin binding subunit (MBS) of myosin phosphatase [8]. Non-muscle myosin II activity is mainly controlled through the phosphorylation of MLC, which is regulated by myosin light chain kinase (MLCK), Rho-kinase, and myosin phosphatase. An increase in intracellular Ca2+ concentration leads to activation of Ca2+/calmodulin-dependent myosin light chain kinase (MLCK), which then phosphorylates Thr-18 and Ser-19 on MLC [9]. Rho-kinase can increase MLC phosphorylation in two ways: on one hand by phosphorylating the myosin binding subunit (MBS) of myosin phosphatase and thereby inhibiting myosin phosphatase activity [10] and on the other hand by direct phosphorylation of MLC [11]. Rhokinase activity can be suppressed with Y-27632, an inhibitor with some specificity for this enzyme [12], whereas MLCK activity can be targeted by two inhibitors with different mechanisms of action; ML-7 [13] or W-7 [14], the first competing with ATP, the latter acting by inhibiting calmodulin whose activity is required for activation of MLCK. Depending on the cell type, Rho-kinase is required
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for migration or is dispensible for this function, as shown for leukocytes, endothelial cells, epithelial cells, fibroblasts, and different types of tumor cells [15–18]. Interestingly, MLCK and Rho-kinase have been shown to have spatially different roles in controlling MLC phosphorylation and actin organization. In fibroblasts for example, Rho-kinase appears to be involved in maintaining the central actin stress fibers, whereas MLCK is required for peripheral stress fibers. In gerbil fibroma cells, Y-27632 reduces MLC phosphorylation and focal contacts in the center of the cells, whereas a peptide interfering with MLCK reduces peripheral phosphorylated MLC and focal contacts at the leading edge [19,20]. Here, we have compared the roles of Rho-kinase, myosin light chain kinase (MLCK), and myosin in regulating migration and adhesion of the human fibrosarcoma cell line HT1080 on an important component of the basal membrane, that is, laminin. These cells, of fibroblastic origin, are highly invasive and able to migrate spontaneously on extracellular matrix components. In contrast to normal fibroblasts, they are poorly adherent and lack stress fibers. This appears to be due to a mutated constitutively active n-ras allele, as deletion of this allele resulted in more adherent cells with stress fibers [21]. We previously provided evidence for a role of protein kinase C in regulating HT1080 cell migration [22]. HT1080 cells express a number of integrin isoforms, among them the laminin receptors a3b1 and a6b1 [23,24]. HT1080 cells constitutively synthesize and secrete PDGF which binds to cell surface PDGF-receptors, resulting in constitutively activated PI 3-kinase. Furthermore, RhoA is also constitutively activated downstream of activated n-ras [21,25]. Data on the role of the Rho target Rho-kinase in HT1080 cell migration are somewhat contradictory. The ROCK inhibitor Y-27632 suppresses the plasminogen activator-induced increase in migration of HT1080 cells through filters coated with vitronectin but does not affect migration in the absence of plasminogen activator [26] or on laminin-coated surfaces [27]. In contrast, experiments using ribozymes that selectively cleave mRNA of Rho-kinase I suggest a specific requirement of this isoform for migration of HT1080 cells across filters in response to fetal bovine serum (FBS) [28]. We now provide new data on differential roles of Rhokinase and MLCK in regulating shape, adhesion, migration, and chemotaxis of HT1080 cells on laminin-coated surfaces. Inhibition of both enzymes resulted in a similar marked inhibition of constitutive myosin light chain phosphorylation. However, cells treated with Rho-kinase inhibitor or with blebbistatin, which inhibits the actin-activated Mg2+-ATPase activity of muscle and non-muscle myosin II with some specificity [29], were sickle shaped with long, thin F-actin-rich protrusions and showed increased adhesion and migration. In contrast, inhibition of MLCK resulted in spherical cells and markedly reduced adhesion and migration. We moreover show that the effects of the MLCK inhibitors were obtained also in cells where myosin activity was inhibited first with blebbistatin.
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Materials and methods Materials. Cell dissociation solution (non-enzymatic), laminin purified from basement membrane of Engelbreth-Holm-Swarm mouse sarcoma, Hoechst Dye (Bisbenzimide trihydrochloride), and dimethyl sulfoxide (DMSO) were from Sigma (St. Louis, MO). Bovine serum albumin (BSA) was from Serva (Heidelberg, Germany); fetal bovine serum (FBS) from Animed (Allschwil, Switzerland); plasminogen from Chromogenix (Milano, Italy). Alexa 488-phalloidin was from Molecular Probes (Junction City, OR). Y-27632 was from Calbiochem/VWR International AG (Lucern, Switzerland). A 10 mM stock solution of Y-27632 was prepared in H2O and stored at 20 C. ML-7 and W-7 were from Alexis Biochemicals (Lausen, Switzerland). Blebbistatin (+/ ) was from Tocris (Avonmouth, UK). Stock solutions of ML-7, blebbistatin, and W-7 were prepared in DMSO and stored at 20 C. DMSO alone had no effect at the final concentrations used. Antibodies. A monoclonal murine anti-myosin light chain antibody was from Sigma, St. Louis, MO, USA (clone MY-21, Cat. Nr. M4401). A polyclonal anti-phospho MLC2 (Thr-18/Ser-19) antibody (Cat. Nr. 3674) was from Cell Signaling, Beverly, MA, USA. Horseradish peroxidaselabeled goat anti-mouse IgG and horseradish peroxidase-labeled goat anti-rabbit antibodies were from Bio-Rad (Hercules, CA). The rhodamine-coupled goat-anti-mouse IgM antibody was from Bio-Science Products AG (Emmenbru¨cke, Switzerland). Cell culture. HT1080 cells (American Type Culture Collection, ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Sigma D-5671) supplemented with 2 mM stable glutamine, 10% heat-inactivated FBS, 40 mg/L streptomycin, and 40,000 U/L penicillin. Migration assays. Two-dimensional migration assays (videomicroscopy): Wells of a 12-well plate (Costar, Cambridge, MA) were coated overnight with 5 lg/well laminin (in 0.1 M Tris, pH 8.0), followed by washing with DMEM with 10% FBS and drying at room temperature. Thirty-five thousand HT1080 cells were added per well and incubated overnight in DMEM containing 10% FBS at 37 C and 5% CO2. The medium was then removed and cells were incubated for 30 min in serumfree DMEM containing plasminogen (5 lg/ml), followed by preincubation without or with inhibitors (see Results). Migration was subsequently recorded at 37 C and 5% CO2 on videotape for 2 h and evaluated as reported previously [22]. Chemotaxis assays. Twenty-four-transwell plates with inserts (6.5 mm diameter, 8.0 lm pore size, Costar) were laminated as described above, followed by washing with serum-free DMEM and drying at room temperature. 200,000 cells, resuspended in 200 ll DMEM with 0.1% BSA, were added per insert. The lower wells contained either 600 ll DMEM with 0.1% BSA as a control or DMEM with 10% FBS as a chemoattractant. Inhibitors were added to upper and lower wells (see Results). Then the cells were incubated for 4 h at 37 C and 5% CO2. Cells on the upper side of the filter were subsequently removed with a cotton swab. After washing with phosphate-buffered saline (PBS), the filters were fixed with 3.7% paraformaldehyde (PFA) in PBS for 15 min at 37 C followed by staining with 10 lM Hoechst Dye (in PBS) for 30 min at room temperature. The nuclei of migrated cells on the lower side of the filter were counted in six fields using a Nikon Eclipse TE300 fluorescence microscope (20· objective). Adhesion assays. For adhesion assays, cells (35,000 per well) were incubated in laminin-coated 12-well plates in DMEM containing 10% FBS at 37 C and 5% CO2, followed by treatment with the different inhibitors (see Results), washing with DMEM lacking FBS, and incubation on a rotating shaker at 200 rpm for 3 min at room temperature after resuspension in DMEM with 10% FBS. Cells were washed twice afterwards and fresh DMEM with 10% FBS was added. The number of adherent cells in the wells was evaluated by counting the cells in 10 different standardized frames per sample. The value obtained for the control cells was set to 100%. Detection of phosphorylated myosin light chain by immunoblotting. Cells (200,000 cells per well) were seeded into laminin-coated 12-well plates as described above and incubated for 4 h in DMEM containing 10% FBS at 37 C and 5% CO2, followed by removal of the medium, addition of
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DMEM lacking FBS (0.5 ml per well), and a further incubation for 30 min at 37 C. Cells were then incubated without or with inhibitors (see Results) for 30 min at 37 C, followed by harvesting of the cells in ice-cold 12.5% TCA (trichloroacetic acid), 40 mM NaF, and 10 mM Na2HPO4, as described [22]. The protein pellets were solubilized in a buffer containing 60 mM Tris, pH 7.5, 1% SDS, and 1 mM EDTA (15 min at 95 C), and subjected to immunoblotting (15% SDS–polyacrylamide gel) using a polyclonal rabbit anti-phospho-MLC2 (Thr-18/Ser-19) antibody (diluted 1:500). In order to control for protein loading, blots were stripped and reprobed with an antibody reacting with MLC independent of its state of phosphorylation (diluted 1:750). Assessment of morphology; visualization of F-actin and MLC. Cells (20,000 cells per well) were seeded into laminin-coated wells of Permanox 8-well slide flasks, incubated for 6 h in DMEM containing 10% FBS, followed by removal of the medium, addition of DMEM lacking FBS, and a further incubation for 30 min at 37 C. Cells were then incubated without or with inhibitors (see Results) for 30 min at 37 C and fixed with 1.25% glutaraldehyde in PBS (pH 7.4) for studies of morphology. For immunofluorescence staining, cells were fixed in 4% PFA in PBS (pH 7.4) for 15 min at 37 C and washed with PBS. Cells were permeabilized with 0.5% Triton X-100 in PBS for 10 min at room temperature followed by blocking with TBST (50 mM Tris, pH 7.4, 150 mM NaCl, and 0.05% Tween) containing 5% defatted milk powder and 10% normal goat serum. Cells were incubated first with monoclonal anti-MLC antibody, diluted 1:50 in TBST containing 0.2% BSA, followed by rhodamine-coupled goatanti-mouse diluted 1:500 in TBST containing 0.2% BSA (1 hr at R.T. each). Subsequently F-actin staining was performed as described [22] using Alexa 488-phalloidin. Pictures were taken with a Zeiss MC 80 Axioplan microscope (10· or 40· objective) connected to a Nikon digital camera DXM1200F. Cell viability assays. Cells (35,000 per well) were seeded into laminated 12-well plates as described for the chemotaxis assays. After washing with prewarmed DMEM lacking FBS, the different inhibitors, diluted in 450 ll DMEM + 0.1% BSA, were added (100 lM blebbistatin, 20 lM ML-7, 20 lM W-7 or 20 lM Y-27632), followed by incubation at 37 C for 4 h, corresponding to the time of migration in the chemotaxis assays.
Subsequently Trypan blue was added to the medium (0.2% final concentration) and the fraction of cells taking up the dye was determined after a 5 min incubation. Control cell cultures contained 99 ± 1% (n = 3) viable cells. None of the inhibitors tested reduced cell viability by more than 6% (data not shown). Statistical analysis of data. Differences between the data were assessed using Student’s t test for paired data with a p value <0.05 considered significant. Data correspond to means ± SEM.
Results Comparison of impact of inhibition of Rho-kinase, MLCK or myosin II on adhesion, morphology, migration, and chemotaxis of HT1080 cells 31 ± 2% (n = 3) of untreated cells adhering to laminincoated surfaces showed a clearly polarized morphology with F-actin-rich ruffles on one side and a contracted tail on the other side. Myosin light chain was located diffusely in the cytosol but also showed some enrichment in tips of ruffles and at the cell margins (controls in Figs. 1 and 2). Inhibition of ROCK by Y-27632 in HT1080 cells resulted in formation of long thin F-actin-rich protrusions and sickle-shaped cells with F-actin-rich ruffles at one side in approximately 70% of the cells. Myosin light chain was also located in the ruffles as well as in the protrusions and the cytosol. These protrusions formed contacts with those of neighboring cells. A similar morphology was obtained by exposing cells to 50 lM of the myosin II inhibitor blebbistatin (Figs. 1 and 2). In contrast, treatment of cells with two different inhibitors of MLCK (20 lM
Fig. 1. Impact of inhibition of ROCK, MLCK or myosin on morphology of HT1080 cells. Cells grown on laminin were incubated with medium (control) or with either 20 lM Y-27632 or 50 lM blebbistatin or 20 lM ML-7 or 20 lM W-7 for 30 min at 37 C, or with 50 lM blebbistatin for 30 min followed by the addition of 20 lM ML-7 and a further incubation for 30 min at 37 C, as indicated, followed by fixation with glutaraldehyde. Bar, 50 lm.
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Fig. 2. Impact of inhibition of ROCK, MLCK or myosin on localization of F-actin and MLC in HT1080 cells. Cells grown on laminin were incubated with medium (control) or with either 20 lM Y-27632 or 50 lM blebbistatin or 20 lM ML-7 or 20 lM W-7 for 30 min at 37 C, or with 50 lM blebbistatin for 30 min followed by the addition of 20 lM ML-7 and a further incubation for 30 min at 37 C, as indicated, followed by fixation of the cells and doublestaining for F-actin and MLC. Bar, 50 lm.
ML-7 or W-7) resulted in 100% of completely spherical cells lacking any protrusions with diffusely distributed F-actin and myosin light chain (Figs. 1 and 2). Rounding up of the cells induced by these MLCK inhibitors was independent of myosin activity, as it could also be observed when the cells were first treated with blebbistatin, followed by a further incubation with ML-7. However, in contrast to the cells treated with ML-7 alone, the latter rounded cells still displayed multiple protrusions (Figs. 1 and 2). We then compared the impact of these inhibitors on adhesion, migration, and chemotaxis. Similar to the findings on morphology, inhibition of ROCK or myosin on one hand and of MLCK on the other hand differed markedly in their impact on adhesion, 2-dimensional migration, and chemotaxis. Adhesion was significantly increased by a factor of 1.5 ± 0.1 (n = 4, p < 0.0025) by Rho-kinase inhibition or by a factor of 1.8 ± 0.1 (n = 3, p < 0.005) by myosin inhibition whereas MLCK inhibition by ML-7 or W-7 almost completely suppressed firm adhesion (Fig. 3). Similarly, preincubation of cells with 20 lM Y-27632 significantly increased speed of migration 1.7 ± 0.2-fold (n = 5, p < 0.001) and % of migrating cells 1.4 ± 0.1-fold (n = 5,
p < 0.0005) in a 2-D assay system on laminin-coated surfaces (Table 1). Blebbistatin similarly enhanced the speed of migration 1.9 ± 0.1-fold (n = 5, p < 0.005, Table 1). In contrast, ML-7 and W-7 completely suppressed migration (Table 1). We also assessed the impact of inhibiting Rho-kinase, MLCK, and myosin on chemotaxis of HT1080 cells through laminin-coated 8 lm filters towards 10% FBS in the lower wells. Chemotaxis of HT1080 cells to the lower side of the filter was markedly increased 4 ± 1-fold (p < 0.001, n = 9) by 10% FBS (Fig. 4A). Migration was attenuated when including 10% FBS in the upper and lower wells by 67 ± 11% (p < 0.025, n = 3), indicating that approximately 70% of the total migratory activity corresponds to chemotaxis as compared to stimulated undirected migration (chemokinesis) (not shown). Similar to their impact on 2-D migration, ML-7 and W7 also negatively affected chemotaxis. W-7 suppressed the FBS-induced increase completely, whereas ML-7 inhibited this increase by 72 ± 9% (p < 0.0005, n = 6) (Figs. 4C and D). Again in agreement with the results obtained by videomicroscopy, inhibition of myosin activity by blebbistatin Table 1 Impact of inhibition of ROCK, MLCK or myosin on 2-D migration of HT1080 cells
Fig. 3. Impact of inhibition of Rho-kinase, MLCK or myosin on adhesion of HT1080 cells to laminin. Cells were grown in laminin-coated wells. After incubation with medium (control) or the different inhibitors as indicated for 30 min at 37 C, cells were washed and the number of adherent cells as % of untreated controls was determined. Concentrations of the inhibitors were 20 lM for ML-7 and Y-27632, and 50 lM for blebbistatin (blebb.). Means ± SEM of 3–4 experiments.
Addition
Mean speed of migrating cells (lm/min)
Migrated cells (%)
Medium Y-27632 (20 lM)
50 ± 6 (n = 5) 81 ± 4 (n = 5)
68 ± 9 (n = 5) 90 ± 4 (n = 5)
DMSO (0.1%) Blebbistatin (50 lM)
35 ± 3 (n = 5) 64 ± 1 (n = 5)
79 ± 3 (n = 5) 88 ± 3 (n = 5)
DMSO (0.1%) ML-7 (20 lM)
41± 4 (n = 4) 0 ± 0 (n = 4)
80 ± 2 (n = 4) 0 ± 0 (n = 4)
DMSO (0.1%) W-7 (20 lM)
38 ± 4 (n = 3) 0 ± 0 (n = 4)
82 ± 2 (n = 3) 0 ± 0 (n = 4)
HT1080 cells were preincubated with the indicated concentrations of inhibitors or DMSO for 30 min at 37 C and 5% CO2. Migration was subsequently recorded on videotape for 2 h and the percentage of migration cells and the mean speed of migration was evaluated (means ± SEM of 3–5 independent experiments).
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Fig. 4. Impact of inhibition of Rho-kinase, MLCK or myosin on chemotaxis of HT1080 cells. 200,000 cells in DMEM with 0.1% BSA were placed in 8 lm filter inserts. Lower wells contained either DMEM with 0.1% BSA or DMEM with 10% FBS as a chemoattractant, as indicated. For part of the samples inserts and lower wells contained in addition (A) Y-27632 (n = 9), (B) blebbistatin (n = 7), (C) ML-7 (n = 6) or (D) W-7 (n = 3), as indicated. Migration through 8 lm transwell filters was determined for 4 h at 37 C. The number of cells detected on the lower side of the filter obtained with 10% FBS as chemoattractant was set at 100%. Means ± SEM of 3–9 experiments.
significantly increased migration of cells through filters in the absence of chemoattractant 1.7 ± 0.2-fold (p < 0.005, n = 7) and also that in the presence of FBS 1.5 ± 0.1-fold (p < 0.025, n = 7) (Fig. 4B). The only discrepancy between data obtained with the two assay systems was observed for Y-27632 (Fig. 4A), where we observed a small but statistically not significant reduction in FBS-induced chemotaxis, in contrast to the increase observed in 2-D migration (Table 1). Comparison of impact of inhibition of Rho-kinase and MLCK on MLC phosphorylation MLCK and Rho-kinase are major enzymes controlling MLC phosphorylation [9]. The above results could be explained by differential roles of ROCK and MLCK in controlling myosin phosphorylation in HT1080 cells. We therefore compared effects of Y-27632, W-7, and ML-7 on phosphorylation of MLC on Thr18 and Ser 19. As shown in Fig. 5, all three inhibitors significantly (p < 0.05) reduced constitutive MLC phosphorylation in HT1080 cells by 40–60%, Y-27632 being most effective. The differential impact of these inhibitors on cell shape and migration cannot thus be explained by marked differences in their impact on MLC phosphorylation.
Fig. 5. Impact of inhibition of Rho-kinase, MLCK or myosin II on phosphorylation of myosin light chain (MLC) in HT1080 cells. Cells grown on laminin were incubated with medium (control) or the different inhibitors (20 lM each) for 30 min at 37 C, followed by harvesting of the cells with TCA and immunoblotting using a polyclonal rabbit antiphospho-MLC (Thr-18/Ser-19) antibody. Controls for ML-7 and W-7 contained 0.4% DMSO. PP-MLC indicates MLC phosphorylated on Thr-18 and Ser-19. The blots were stripped and reprobed with an antibody reacting with MLC independent of its state of phosphorylation. (A) Blot of a representative experiment; (B) quantitative evaluation, means ± SEM of 3–4 experiments.
Discussion We provide novel data documenting differential roles of Rho-kinase and MLCK in regulating shape, adhesion, and
migration of HT1080 cells. Inhibition of Rho-kinase with Y-27632 results in sickle-shaped HT1080 cells with long fine F-actin-rich protrusions, which adhere more strongly
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to laminin and migrate faster in 2-D systems and whose chemotaxis to FBS is not significantly impaired. The specificity of the Rho-kinase inhibitor Y-27632 has been tested on a large panel of protein kinases. Y-27632 was inactive on 21 tested enzymes and inhibited only one (PRK; protein kinase C-related protein kinase) with similar potency as the Rho-kinase isoforms [12]. Our data could be confirmed by transfection with dominant-negative Rho-kinase [30] which induced a comparable morphology as Y-27632 (data not shown). These findings indicate that the pool of myosin II controlled by Rho-kinase specifically restricts formation of lateral protrusions and limits migration and adhesion. Invasion of rat MM1 hepatoma cells studied in mouse models could be inhibited by administration of Rho-kinase inhibitor [16]. Our data now suggest that this may not be the case for all types of tumors and that in some cases metastasis and invasion could even be stimulated by Rho-kinase inhibition. Intriguingly cells treated with either ML-7 or W-7, inhibitors of MLCK, showed a completely different spherical morphology without any protrusions, as well as markedly decreased adhesion, migration, and chemotaxis. ML-7, as noted by Bain et al. [13], is a relatively specific, ATP-competitive MLCK inhibitor which did not significantly inhibit activity of 28 tested other enzymes. Only the activity of the mitogen- and stress-activated protein kinase 1 (MSK1) was reduced albeit at much higher concentrations than that of MLCK. W-7 acts mainly as a calmodulin antagonist and inhibits activity of a calciumcalmodulin-independent fragment of MLCK much less potently than ML-7 [14]. As we obtained the same findings with these two differentially acting inhibitors, we think it unlikely that they can be explained by effects on proteins other than MLCK. Shoemaker et al. [31] observed similarly that chicken embryo fibroblasts treated with an antisense probe directed against MLCK assumed a spherical morphology without protrusions. Our attempts to down-regulate MLCK expression by incubating HT1080 cells with two different siRNAs targeting MLCK up to 4 days failed, possibly due to very slow turnover of this protein, as we could successfully prevent EGFP expression by EGFP siRNA (data not shown). Our findings obtained with inhibitors cannot be explained by a more important role of MLCK in regulating MLC phosphorylation and activation, as Y-27632 was in our hands even somewhat more effective than ML-7 or W-7 in attenuating constitutive MLC phosphorylation in HT1080 cells (Fig. 5). Our data suggest that Rho-kinase limits HT1080 migration whereas MLCK activity is absolutely required for this process, despite the fact that both enzymes promote myosin activation by enhancing its phosphorylation. This is different from our previous findings for Walker carcinosarcoma cells, where inhibition of both Rho-kinase and MLCK abrogated MLC phosphorylation, polarization, and migration [32]. One explanation of our data would be differences in the spatial control of myosin II activation by these two enzymes, as shown in fibroblasts [19,20].
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In order to clarify this issue, we studied the impact of abrogation of myosin II activity by blebbistatin, an inhibitor with some selectivity for non-muscle myosin IIA and IIB [29], on morphology, adhesion, and migration of HT1080 cells. Treatment with blebbistatin mimicked effects of Rho-kinase inhibition on cell morphology, adhesion, and 2-D migration. Similarly, fibroblasts derived from myosin IIB null mice exhibit a sickle-shaped morphology with long processes and migrate faster than wild-type cells [33]. The only difference we observed between effects of Y-27632 and blebbistatin concerned chemotaxis, where blebbistatin clearly increased migration through filters in the absence or presence of chemoattractant, whereas Y-27632 did not markedly affect these processes. Possibly the advantage which myosin inhibition confers to chemotaxis is abrogated by inhibition of activity of Rho-kinase targets other than myosin. Importantly, treatment of cells first exposed to blebbistatin with ML-7 still resulted in rounding up of cells, albeit the fine protrusions induced by myosin inhibition were conserved. This finding strongly suggests that MLCK has cellular functions unrelated to its capacity to phosphorylate and activate myosin II. Indeed, MLCK may have actin-bundling activity [34], and has been reported to control calcium-dependent signaling via MLC-independent mechanisms [35]. Further investigations will focus on downstream targets of MLCK different from myosin. In summary, both Rho-kinase and MLCK are involved in controlling MLC phosphorylation in HT1080 cells, but whereas Rho-kinase limits adhesion and migration, possibly by preventing protrusion formation via inhibition of myosin activation, MLCK may have additional roles in controlling actin organization required for HT1080 cell migration and adhesion. Acknowledgments This work was supported by the Bernese Cancer League and the Swiss National Science Foundation (Grant No. 3100A0-103708/1). We are grateful to Christoph Oppliger, Tamara Locher, Maddalena Lis, and Dominique Schlicht for expert technical assistance. We thank S. Narumiya (Department of Pharmacology, Kyoto University, Faculty of Medicine, Kyoto, Japan) for the kind gift of pCAGmyc-WT-ROCKI and pCAG-myc-KD-IA-ROCKI, and E. Sigel for critical reading of the manuscript. References [1] J. Kassis, D.A. Lauffenburger, T. Turner, A. Wells, Tumor invasion as dysregulated cell motility, Semin. Cancer Biol. 11 (2001) 105–117. [2] J. Condeelis, J.E. Segall, Intravital imaging of cell movement in tumours, Nat. Rev. Cancer 3 (2003) 921–930. [3] S. Etienne-Manneville, A. Hall, Rho GTPases in cell biology, Nature 420 (2002) 629–635. [4] A.P. Wheeler, A.J. Ridley, Why three Rho proteins? RhoA, RhoB, RhoC, and cell motility, Exp. Cell Res. 301 (2004) 43–49.
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