Different Molecular Motors Mediate Platelet-derived Growth Factor and Lysophosphatidic Acid-stimulated Floating Collagen Matrix Contraction*

Fibroblast-collagen matrix contraction has been used as a model system to study how cells organize connective tissue. Previous work showed that lysophosphatidic acid (LPA)-stimulated floating collagen matrix contraction is independent of Rho kinase, whereas platelet-derived growth factor (PDGF)-stimulated contraction is Rho kinase-dependent. The current studies were carried out to learn more about the molecular motors responsible for LPA- and PDGF-stimulated contraction. We found that neither PDGF nor LPA-dependent contractile mechanisms require myosin II regulatory light chain kinase or increased phosphorylation of myosin II regulatory light chain (measured as diphosphorylation). Low concentrations of the specific myosin II inhibitor blebbistatin blocked PDGF-stimulated matrix contraction and LPA-stimulated retraction of fibroblast dendritic extensions but not LPA-stimulated matrix contraction. These data suggest that PDGF- and LPA-stimulated floating matrix contraction utilize myosin II-dependent and -independent mechanisms, respectively. LPA-dependent, Rho kinase-independent force generation also was detected during fibroblast spreading on collagen-coated coverslips.

Fibroblast-collagen matrix contraction has been used as a model system to study how cells organize connective tissue. Previous work showed that lysophosphatidic acid (LPA)-stimulated floating collagen matrix contraction is independent of Rho kinase, whereas plateletderived growth factor (PDGF)-stimulated contraction is Rho kinase-dependent. The current studies were carried out to learn more about the molecular motors responsible for LPA-and PDGF-stimulated contraction. We found that neither PDGF nor LPA-dependent contractile mechanisms require myosin II regulatory light chain kinase or increased phosphorylation of myosin II regulatory light chain (measured as diphosphorylation). Low concentrations of the specific myosin II inhibitor blebbistatin blocked PDGF-stimulated matrix contraction and LPA-stimulated retraction of fibroblast dendritic extensions but not LPA-stimulated matrix contraction. These data suggest that PDGF-and LPAstimulated floating matrix contraction utilize myosin II-dependent and -independent mechanisms, respectively. LPA-dependent, Rho kinase-independent force generation also was detected during fibroblast spreading on collagen-coated coverslips.
Form and function of multicellular organisms depend on tissue-specific programs of cell motility. Fibroblasts synthesize, organize, and maintain connective tissues during development and in response to injury and fibrotic disease. The motile mechanisms that fibroblasts use to remodel the extracellular matrix during these morphogenetic processes have been studied by using cells cultured in three-dimensional collagen matrices (1,2).
As fibroblasts exert force on and move collagen fibrils of the matrix, collagen concentration around the cells increases, and the corresponding decrease in matrix volume (typically referred to as contraction) can be measured as a decrease in the diameter of free matrices or a decrease in height of restrained matrices. During contraction of restrained matrices, collagen fibrils become oriented in the same plane as restraint, and mechanical loading develops. In floating matrices, on the other hand, contraction occurs without collagen fibrils developing a particular orientation, and the matrix remains mechanically unloaded (1)(2)(3)(4)(5).
The signaling mechanisms used by fibroblasts to regulate collagen matrix contraction depend on whether the cells are mechanically loaded or unloaded at the time that contraction is initiated as well as on the growth factor used to initiate contraction. For instance, stimulation of fibroblasts by lysophosphatidic acid (LPA) 1 but not by platelet-derived growth factor (PDGF) causes robust force generation in restrained matrices (6), whereas LPA and PDGF stimulate floating matrix contraction equally well (7,8).
Floating matrix contraction has presented something of an enigma because LPA stimulation of fibroblasts in floating matrices causes activation of the small G protein Rho (GTP loading) (9), but blocking Rho kinase with the pharmacological reagent Y27632 does not inhibit contraction (10). Conversely, PDGF stimulation of cells in matrices causes activation of Rac not Rho (9), but blocking Rho kinase inhibits PDGF-stimulated contraction (10).
For fibroblasts migrating on two-dimensional substrata, the ability of cells to exert tractional force on the substratum depends on activation of Rho and Rho kinase (11,12), which is believed to increase cell contraction through increased phosphorylation of myosin II regulatory light chain (MLC) (13,14). In addition to Rho kinase, the spatial and temporal pattern of MLC phosphorylation in fibroblasts is controlled by MLC kinase (15)(16)(17). Other mechanisms of force generation independent of MLC phosphorylation also have been described (18,19), even in smooth muscle cells (20,21) where MLC phosphorylation generally is thought to be the key regulator of contractile activity (22).
The current studies were carried out to learn more about the molecular motors responsible for LPA-and PDGF-stimulated fibroblast-collagen matrix contraction. We found that neither PDGF-nor LPA-dependent contractile mechanisms require MLC kinase or increased phosphorylation of MLC (measured as diphosphorylation). Low concentrations of the specific myosin II inhibitor blebbistatin blocked PDGF-stimulated matrix contraction and LPA-stimulated retraction of fibroblast dendritic extensions but not LPA-stimulated matrix contraction. These data suggest that PDGF-and LPA-stimulated floating matrix contraction utilize myosin II-dependent and -independent mechanisms, respectively. LPA-dependent and Rho kinaseindependent force generation also was detected during fibroblast spreading on collagen-coated coverslips.
Monolayer and Collagen Matrix Culture-Fibroblasts were from human foreskin specimens and cultured up to 10 passages in Falcon 75-cm 2 tissue culture flasks in DMEM supplemented with 10% FBS (DMEM, 10% FBS) at 37°C in a 5% CO 2 humidified incubator. The culture medium was changed every 3-4 days. Cells were harvested by 0.25% trypsin/EDTA for 3 min at 37°C followed by DMEM, 10% FBS. For monolayer culture experiments, harvested cells were seeded at a density of 4 ϫ 10 4 cells on 22-mm square glass coverslips (Fisher) that previously had been collagen-coated (50 g/ml for 30 min) and then cultured in DMEM containing 5 mg/ml BSA, growth factors, and inhibitors as indicated.
Collagen matrix cultures were prepared using Vitrogen 100 collagen as described previously (8,23). Briefly, neutralized collagen solution (1.5 mg/ml) containing harvested cells (10 6 cells/ml unless indicated otherwise) was prewarmed to 37°C for 4 min, and then aliquots (200 l) were placed on an area outlined by a 12-mm-diameter circular score within a well of 24-well culture plates (Greiner Bio-one, Frickenhausen, Germany) and allowed to polymerize for 60 min at 37°C in 5% CO 2humidified incubator. To initiate floating matrix contraction, matrices were gently released from the underlying culture dish with a spatula into 0.8 -1.0 ml of DMEM containing 5 mg/ml BSA (DMEM/BSA), and growth factors and inhibitors were added as indicated. For restrained matrix contraction, polymerized matrices were cultured 24 h in 1.0 ml of DMEM, 10% FBS containing 50 g/ml ascorbic acid before release.
Matrix contraction was carried out for the times shown in the text after which the samples were fixed for 10 min at room temperature with 3% paraformaldehyde in phosphate-buffered saline (150 mM NaCl, 3 mM KCl, 1 mM KH 2 PO 4 , 6 mM Na 2 HPO 4 , pH 7.2). To quantify contraction, fixed matrices were washed and placed on a flat surface, and diameter was measured. Contraction data are presented as the change in diameter (starting-final) in millimeters. All experiments were carried out in duplicate, and every experiment was repeated two or more times. Data points and error bars in the figures represent averages and S.D. Where error bars are not seen, the data points overlapped.
Immunofluorescence Microscopy-Cells in matrices or on coverslips were fixed for 10 min with 3% paraformaldehyde in phosphate-buffered saline at room temperature, blocked with 1% glycine, 2% BSA in DPBS (150 mM NaCl, 3 mM KCl, 1 mM KH 2 PO 4 , 6 mM Na 2 HPO 4 , 0.5 mM MgCl 2 , 1 mM CaCl 2 , pH 7.2) for 30 min, and then permeabilized for 15 min with 0.5% Triton X-100 in DPBS. Subsequently, the samples were washed with DPBS and treated for 10 min with 1% BSA in DPBS. Primary antibody against vinculin was diluted in 1% BSA in DPBS (1:200) and added to cells for 1 h at 37°C. After washing with DPBS, samples were treated for 10 min with 1% BSA in DPBS, and then FITC-conjugated goat anti-mouse IgG in 1% BSA in DPBS (1:300) was added to the cells for 30 min at 37°C. After washing with DPBS, samples were treated for 10 min with 1% BSA in DPBS, and then samples were incubated with rhodamine-conjugated phalloidin (8 units/ ml) in 1% BSA in DPBS for 30 min at 37°C. After additional washes, samples were mounted on glass slides with Fluoromount G. Observations were made and images collected with a Nikon Elipse 400 Fluorescent microscope equipped with a Photometrics SenSys camera and MetaView work station.
Samples for analysis of MLC kinase were rinsed with PHEM buffer (60 mM PIPES, 25 mM HEPES, 10 mM EGTA, 2 mM MgCl 2 , pH 6.9) at 37°C, fixed for 10 min with 2% paraformaldehyde in PHEM at 37°C, and then washed with PHEM at 37°C. Subsequently, the samples were permeabilized for 15 min with 0.2% Triton X-100 in PHEM. Samples were blocked with 3% goat serum in DPBS for 1 h at 37°C and then incubated with mouse anti-MLC kinase in 3% goat serum in DPBS (1:200) for 1 h at 37°C. After washing with DPBS, samples were incubated with FITC-conjugated goat anti-mouse IgG in 3% goat serum in DPBS (1:300) for 1 h at 37°C. Samples were mounted on glass slides with Fluoromount G, and observations were made as above.
Myosin Light Chain Kinase Gene Silencing-Myosin light chain kinase gene silencing was accomplished using siRNA. Oligonucleotide sequences 5Ј-CUAGCCACAUGCCGCAGCATT-3Ј and 5Ј-UGCUGCG-GCAUGUGGCUAGTT-3Ј were obtained from the University of Texas Southwestern siRNA core facility. For annealing, 20 M of each singlestrand 21-nucleotide RNAs was incubated in annealing buffer (100 mM potassium acetate, 30 mM HEPES-KOH, pH 7.4, 2 mM magnesium acetate) for 2 min at 95°C, followed by 2 h at 37°C. To accomplish high efficiency transfection, fibroblast cultures (60 -70% confluent) were first rinsed with antibiotic-free DMEM and then treated with trypsin-EDTA for 1 min to elicit cell rounding but not detachment. Subsequently, antibiotic-free 10% FBS/DMEM was added 4:1 to quench the trypsinization. After cells were rinsed with antibiotic-free DMEM, they were incubated with Opti-MEM I containing 3.5 M siRNA annealed oligonucleotides and 3.5% OligofectAMINE diluted 1:5 with antibioticfree DMEM. After 48 h, transfection medium was removed and replaced with 10% FBS/DMEM containing antibiotics for an additional 24 h at which time cells were subcultured. Fig. 1 shows typical results of experiments measuring the dependence of collagen matrix contraction on Rho kinase activity. Consistent with previous studies (10), addition of the Rho kinase inhibitor Y27632 had little effect on LPA-stimulated contraction of floating matrices although PDGF-stimulated contraction was reduced markedly. If, however, matrices were restrained and allowed to develop mechanical loading overnight, then LPAstimulated contraction became Rho kinase-dependent (10,24,25).

Rho Kinase and Collagen Matrix Contraction-
MLC Kinase and Collagen Matrix Contraction-The foregoing results confirmed that LPA-stimulated floating matrix contraction was Rho kinase-independent. In addition to Rho ki-nase, MLC kinase has been reported to play a role in fibroblastcollagen matrix contraction (24,26,27). To test whether MLC kinase was important for LPA-stimulated collagen matrix contraction, experiments were carried out using fibroblasts in which MLC kinase levels were knocked down using the siRNA method. Fig. 2A shows by immunoblotting with anti-MLC kinase antibodies that cell transfection with MLC kinase-specific oligonucleotides but not mock transfection markedly reduced the levels of the two MLC kinase isotypes in human fibroblasts. Actin was used as a loading control. In corresponding fashion, Fig. 2B shows by immunofluorescence that the cortical staining intensity of MLC kinase was reduced in MLC kinase-silenced cells, although background staining of the cell nucleus was similar in specific and mock transformants as well as controls.
Fibroblasts in which MLC kinase was determined as above to be reduced were used to measure matrix contraction in the presence of LPA or PDGF with or without the Rho kinase inhibitor added. Fig. 3 shows results from a typical experiment. The extent of contraction of MLC kinase-silenced cells was not detectably different from control cells. Moreover, LPA-stimulated floating matrix contraction occurred in MLC kinase-silenced cells even if Rho kinase also was blocked.

MLC Phosphorylation and Collagen Matrix Contraction-
Other experiments were carried out to test the effects of LPA and PDGF on MLC phosphorylation with and without Rho kinase inhibitor. Previously, we showed by using the ureaglycerol method that LPA but not PDGF stimulated both monoand diphosphorylation of fibroblasts on collagen-coated coverslips, but for technical reasons the urea-glycerol method could not be used for fibroblasts in collagen matrices (23). Therefore, MLC phosphorylation in fibroblasts in collagen matrices was assessed using antibodies specific to (di)phosphorylated MLC with total MLC as a loading control. Diphosphorylated MLC as well as the monophosphorylated form has been implicated in cell contractility (28,29). Fig. 4 shows a representative blot and quantification of results from several experiments. After 15 min of growth factor stimulation, levels of diphosphorylated MLC were highest in cells in LPA-containing medium and lowest in PDGF-contain-ing medium. Prior addition of Rho kinase inhibitor markedly reduced phosphorylation in every case. These observations suggested that stimulation of floating collagen matrix contraction did not require growth factor stimulation of MLC phosphorylation (measured as diphosphorylation).
Myosin II and Collagen Matrix Contraction-The foregoing observations suggested that neither MLC kinase nor stimulation of MLC phosphorylation was necessary for collagen matrix  contraction. We could not exclude, however, the possibility that residual MLC kinase or constitutive levels of MLC phosphorylation were sufficient for contraction to occur. Additional experiments were carried out to test more directly the role of myosin II in contraction using the specific myosin II inhibitor blebbistatin (30). Fig. 5 shows a dose-response curve for the effect of blebbistatin on floating matrix contraction. Concentrations of the drug in the range 5-20 M markedly inhibited PDGF-dependent contraction, which is the same concentration range that blocks myosin II ATPase activity (30). In marked contrast, LPA-dependent contraction was only inhibited by blebbistatin at higher drug concentrations.
We also tested the effect of blebbistatin on LPA-stimulated retraction of fibroblast dendritic extensions. As described previously, fibroblasts in collagen matrices project and retract a dendritic network of extensions. PDGF stimulates the size and complexity of this network, whereas LPA stimulates its retraction in a Rho kinase-dependent manner (9). Retraction occurs independently of and is not required for matrix contraction (10). Fig. 6 shows the morphology of fibroblasts with retracted (LPA) and projected (PDGF) extensions. As described previously, addition of the Rho kinase inhibitor Y27632 blocked retraction stimulated by LPA (Fig. 6, LPAϩY27632). Treatment with blebbistatin at 5 (LPAϩBbst5) or 20 M (Fig. 6, LPAϩBbst20) also blocked LPAstimulated retraction but had no effect on the extensions formed in the presence of PDGF (PDGFϩBbst20). These findings show that LPA-dependent retraction of fibroblast dendritic extensions requires myosin II. Taken together, the foregoing results indicated that PDGF and LPA stimulated floating collagen matrix contraction through different molecular motors and that LPA was able to exert force by a Rho kinase-independent, myosin II-independent mechanism.
Rho Kinase and Cell Spreading on Collagen-coated Coverslips-Finally, experiments were carried out to learn if the LPA-stimulated, Rho kinase-independent mechanism of force generation also could be demonstrated during cell spreading on collagen-coated coverslips. Fig. 7 shows that in PDGF-containing medium, fibroblasts spread with prominent stress fibers (actin staining) and focal adhesions (vinculin). In the presence of the Rho kinase inhibitor (Fig. 7, PDGFϩY), focal adhesions and stress fibers were mostly lost. Instead of broad lamellae, cells had long dendritic extensions. Therefore, fibroblasts on collagen-coated surfaces in PDGF-containing medium required Rho kinase to generate tractional force for flattening and formation of stress fibers and focal adhesions.
Fibroblasts incubated on collagen-coated coverslips in LPAcontaining medium also developed stress fibers and focal adhesions but tended to be much less polarized than cells in PDGF-containing medium. Fibroblasts in medium containing LPA and Rho kinase inhibitor became more polarized but, unlike cells in PDGF medium, often retained regions of broad lamellae as well as stress fibers and focal adhesions. These observations indicated that there was a Rho kinase-independent mechanism of force generation. Further evidence for this mechanism was obtained when LPA was added to fibroblasts previously incubated in medium with PDGF ϩ Rho kinase inhibitor (Fig. 7, PDGFϩY/LPA), in which case the dendritic extensions became more lamellar and reformed stress fibers and focal adhesions even in the continuous presence of the Rho kinase inhibitor. Conversely, addition of LPA to cells previously stimulated with PDGF resulted in decreased polarization (not shown).
Addition of 20 M blebbistatin to the incubations in place of Rho kinase inhibitor also caused fibroblasts in PDGF-contain-ing medium to display the dendritic morphology, whereas cells in LPA-containing medium retained regions of broad lamellae as well as stress fibers and focal adhesions (not shown).
Finally, experiments were carried out to assess MLC diphosphorylation in cells on collagen-coated surfaces. Fig. 8 shows that MLC diphosphorylation was elevated in cells in LPAcontaining medium but markedly reduced under all conditions in the presence of the Rho kinase inhibitor.

DISCUSSION
The current studies were carried out to learn more about the molecular motors responsible for LPA-and PDGF-stimulated fibroblast-collagen matrix contraction. Our findings suggest that PDGF-and LPA-stimulated floating matrix contractions utilize myosin II-dependent and -independent mechanisms, respectively.
Previous studies (10) suggested that LPA-stimulated force generation by fibroblasts in floating collagen matrices does not require Rho kinase activity. In the current work, we found that fibroblasts contracted the matrices even when MLC kinase was knocked down using siRNA, and Rho kinase was inhibited. Moreover, in the presence of Rho kinase inhibitor, MLC diphosphorylation was substantially reduced. Although these findings suggested that contraction was independent of MLC phosphorylation, we could not exclude the possibility that residual MLC kinase or constitutive levels of MLC phosphorylation were sufficient for contraction to occur. Monophosphorylated MLC might have been sufficient (28,29).
Additional experiments were carried out, therefore, using the specific myosin II inhibitor blebbistatin. At concentrations sufficient to inhibit myosin II ATPase activity (30), blebbistatin blocked not only PDGF-dependent floating matrix contraction but also LPA-dependent retraction of the dendritic network of cellular extensions. LPA-stimulated retraction of fibroblast dendritic extensions is a Rho kinase-dependent activity independent from and not required for floating matrix contraction (10). Although blebbistatin also blocked LPA-dependent floating matrix contraction, inhibition only occurred at concentrations above those shown to be required to inhibit myosin II.
Taken together, the foregoing results suggested that LPA can stimulate floating matrix contraction through a molecular motor other than myosin II. Myosin Ic recently was shown to play a role in retraction of the neuronal growth cone lamellipodium (31), but preliminary studies using siRNA specific for myosin Ic demonstrated that myosin Ic knockdown did not affect LPA-dependent floating matrix contraction. 2 Also, it has been hypothesized that microtubule dynamics might play a role in exerting force on the cell plasma membrane (32). Nevertheless, because addition of the microtubule disrupting reagents nocodazole or vinblastine or the microtubule-  stabilizing drug taxol at concentrations expected to interfere with microtubule dynamics but not structure had little effect on matrix contraction. 2 Future studies will be required to discover the myosin IIindependent mechanism of LPA-stimulated floating collagen matrix contraction. Because collagen in these matrices is highly compliant, sufficient force to move collagen fibrils bound to the cell surface might be provided by actin polymerization and retrograde flow which can occur independently of myosin II (31,(33)(34)(35).
Whatever the molecular mechanism of LPA-stimulated floating matrix contraction, it is clearly different from LPA-stimulated stressed matrix contraction. Just as cellular adhesions can undergo resistance-induced strengthening (36 -38), the mechanism of force generation by fibroblasts in collagen matrices changes in response to increased stiffness of the matrix (2,10,39). As shown by others and confirmed by us, stressed matrix contraction depends on Rho kinase (24,25) and phosphorylation of myosin II regulatory light chain (6,26,40,41) similar to fibroblasts exerting tractional force on planar surfaces (11)(12)(13)(14)(15)17).
A third molecular mechanism of force generation was observed with floating matrix contraction stimulated by PDGF. PDGF-dependent floating matrix contraction required myosin II judging from its sensitivity to inhibition by blebbistatin and the Rho kinase inhibitor. MLC kinase did not appear to be required, however, and no stimulation of MLC-diphosphorylation was detected, consistent with previous observations of PDGF-treated cells on planar surfaces (21,23). Because PDGF treatment of fibroblasts in collagen matrices activates Rac and not Rho (9), as has been shown for cells on planar surfaces (42,43), Rho activity necessary for PDGF-stimulated contraction likely is provided by a mechanism other than growth factor stimulation, e.g. integrin occupancy (44,45).
One of the implications of our findings was that human fibroblasts spreading on collagen-coated coverslips in the presence of LPA but not PDGF might have a Rho kinase-independent mechanism for exerting force. This was the case, because blocking Rho kinase in cells spreading the presence of PDGF caused loss of most stress fibers and focal adhesions, and the cells became highly dendritic similar to fibroblasts in collagen matrices in the presence of PDGF (9), whereas fibroblasts spreading in the presence of LPA and Rho kinase inhibitor retained broad lamellar regions with residual stress fibers and focal adhesions, even if the LPA was added to cells that already had first become dendritic in the presence of PDGF and Rho kinase inhibitor.
Finally, our studies have important implications for understanding matrix remodeling during wound repair. Myofibroblasts, which utilize Rho/Rho kinase, myosin light chain phosphorylation, and ␣-smooth muscle actin for matrix remodeling and contraction (1,46,47), are not required for initial wound remodeling (48,49). How fibroblasts initially begin to move into and remodel the wound matrix is not well understood. The present research implicates PDGF/Rho kinase-dependent and LPA/Rho kinase-independent mechanisms as potential candidates to explain this phenomenon.