LPA-stimulated fibroblast contraction of floating collagen matrices doesnot require Rho kinase activity or retraction of fibroblast extensions

David J. Lee,1 Chin-Han Ho, and Frederick Grinnell*Department of Cell Biology, UT Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9039, USA

Received 3 March 2003, revised version received 3 April 2003

Abstract

Fibroblasts synthesize, organize, and maintain connective tissues during development and in response to injury and fibrotic disease.These morphogenetic processes depend on cell-matrix remodeling, which has been investigated using cells cultured in three-dimensionalcollagen matrices. The current studies were carried out to test the role of Rho kinase activity and retraction of fibroblast extensions on thematrix remodeling process. We found that remodeling (contraction) of floating collagen matrices stimulated by lysophosphatidic acid (LPA)did not require Rho kinase activity or retraction of fibroblast extensions. On the other hand, LPA-stimulated contraction of restrainedmatrices became Rho kinase dependent after the matrices were allowed to develop mechanical loading for 2–4 h, suggesting that theremodeling process itself was able to feed back to modulate cell behavior in an iterative process. Modulation was specific for LPA sincefibroblast-collagen matrix contraction stimulated by platelet-derived growth factor was Rho kinase dependent before or after mechanicalloading developed.© 2003 Elsevier Science (USA). All rights reserved.

Keywords: Extracellular matrix; Growth factors; G proteins; Mechanical loading; Focal adhesion kinase; Microtubules

Introduction

Fibroblasts synthesize, organize, and maintain connec-tive tissues during development and in response to injuryand fibrotic disease. These morphogenetic processes dependon cell-matrix remodeling, which has been studied usingcells cultured in three dimensional collagen matrices [1–4].

As fibroblasts remodel three-dimensional matrices, col-lagen concentration around the cells increases, and the cor-responding decrease in matrix volume (contraction) resultsin a decrease in the diameter of free matrices (floating inculture medium) or a decrease in height of restrained ma-trices (i.e., attached to culture dishes). Under the latterconditions, collagen fibrils become oriented in the sameplane as restraint, and mechanical loading develops. Con-versely, in floating matrices, remodeling occurs without

collagen fibrils developing a particular orientation, and thematrix remains mechanically unloaded. These differences inmechanical loading are important because they determinecellular biosynthetic and proliferative phenotype [5–7].

The small G protein Rho and Rho effector Rho kinasehave been implicated in the ability of fibroblasts to exertcontractile forces on two-dimensional surfaces [8–11]. Sim-ilarly, Rho and Rho kinase activity are required for fibro-blasts to contract restrained collagen matrices that havebeen allowed to develop mechanical load for several daysand subsequently released in the presence of lysophospha-tidic acid (LPA) [12,13]. In addition to stimulating fibro-blast-collagen matrix contraction, LPA also causes retrac-tion of the dendritic network of extensions formed byfibroblasts in newly polymerized collagen matrices, andretraction can be blocked by inhibiting Rho kinase [14].

The current studies were carried out to test the possibilitythat fibroblast contraction of floating collagen matrices alsodepends on Rho kinase and, more specifically, on retractionof fibroblast extensions. Instead, we found that LPA-stim-ulated fibroblast-contraction of floating collagen matrices

* Corresponding author. Fax: �1-214-648-8694.E-mail address: frederick.grinnell@utsouthwestern.edu (F. Grinnell).1 Current address: Physicians’ Education Resource, 3535 Worth Street,

Dallas, TX 75246, USA.

R

Available online at www.sciencedirect.com

Experimental Cell Research 289 (2003) 86–94 www.elsevier.com/locate/yexcr

0014-4827/03/$ – see front matter © 2003 Elsevier Science (USA). All rights reserved.doi:10.1016/S0014-4827(03)00254-4

required neither Rho kinase activity nor retraction of fibro-blast extensions. On the other hand, LPA-stimulated con-traction of restrained matrices became Rho kinase depen-dent after the matrices were allowed to develop mechanicalloading for 2–4 h, suggesting that the remodeling processitself was able to feed back to modulate cell behavior in aniterative process. Details are reported herein.

Materials and methods

Materials

Type I collagen (Vitrogen) was purchased from Cohe-sion (Palo Alto, CA, USA). Dulbecco’s modified Eaglemedium (DMEM) was purchased from GIBCO/BRL(Gaithersburg, MD, USA). Fetal Bovine Serum (FBS) waspurchased from Intergen Co. (Purchase, NY, USA). Pertus-sis toxin (PTx) and Rho kinase inhibitor Y-27632 wereobtained from Calbiochem (San Diego, CA, USA). Platelet-derived growth factor BB isotype (PDGF) was obtainedfrom Upstate Biotechnology, Inc (Lake Placid, NY, USA).Fatty acid-free bovine serum albumin (BSA), lysophospha-tidic acid, nocodazole, and mouse anti-� tubulin antibodywere obtained from Sigma. Antibodies to FAK and phos-pho-FAK were from BD Transduction Lab, CA and Bio-source International, CA respectively. HRP conjugatedgoat-anti-rabbit or mouse antibodies were from ICN Bio-medicals INC., (Aurora, OH, USA). ECL Western BlottingDetection Reagent was from Amersham Biosciences (Pis-cataway, NJ, USA).

Incubation of human fibroblasts in collagen matrices

Fibroblasts from human foreskin specimens (up to 10passages) were maintained in Falcon 75-cm2 tissue cultureflasks in DMEM supplemented with 10% FBS. Hydratedcollagen matrices were prepared from Vitrogen 100 colla-gen. Neutralized collagen solutions (1.5 mg/ml) containedfibroblasts in DMEM but no serum. Cell/collagen mixtureswere prewarmed to 37°C for 4 min, and aliquots (200 �l)were placed on Corning 24-well culture plates. Unless in-dicated otherwise, matrices contained 106 cells/ml. Eachaliquot occupied an area outlined by a 12-mm-diametercircular score within a well. Polymerization of collagenmatrices required 60 min at 37°C in a humidified incubatorwith 5% CO2. Subsequently, the polymerized matrices wereused immediately or preincubated with 1.0 ml of DMEMand specified agonists for the indicated times.

Matrices were gently released from the underlying cul-ture dish with a spatula into 1 ml of serum-free DMEMcontaining 5 mg/ml BSA and growth factors as indicated,after which the matrices were incubated at 37°C. Contrac-tion was quantified by determining the difference betweenthe starting diameter (12 mm) and the final diameter. Beforemeasuring the diameter, matrices were fixed 10 min at 22°C

in 3% formaldehyde in PBS (150 mM NaCl, 3 mM KCl, 6mM Na2HPO4, 1 mM KH2PO4, pH 7.2). All measurementswere made on duplicate samples. The results shown are theaverages �/� standard deviations.

Immunofluorescence microscopy

Collagen matrices were fixed for 10–15 min at 22°Cwith 3% formaldehyde in PBS. To block nonspecific stain-ing, the samples were treated with 1% glycine and 2%bovine serum albumin (Fraction V, ICN Biomedicals, Au-rora, OH, USA) in DPBS (PBS � 1 mM CaCl2 � 0.5 mMMgCl2, pH 7.2) for 30 min. The matrices were permeabil-ized with 0.5% Triton X100 in DPBS for 20 min after whichthey were incubated at 37°C for 30 min with DPBS con-taining 1% bovine serum albumin and 0.8 U/ml of rhodam-ine-conjugated phalloidin (Molecular Probes, Eugene, OR,USA). To visualize tubulin and actin, samples were blockedand permeabilized as above, incubated with mouse anti-�tubulin in 1% bovine serum albumin at 37°C for 30 minfollowed by Alexafluor 488 goat anti-mouse IgG (molecularprobes, Eugene, OR, USA) for 37°C for 30 min and thenwith rhodamine conjugated phalloidin as above. After ex-tensive washing, samples were mounted with FluoromountG (Southern Biotechnology Associates, Birmingham, AL,USA), examined using a Nikon Elipse 400 FluorescentMicroscope, and digital images were collected using a Pho-tometrics SenSys camera and MetaView (Universal Imag-ing Corporation).

SDS-PAGE and immunoblotting

RIPA extraction buffer (150 mM NaCl, 6 mM Na2HPO4,4 mM NaH2PO4, 2 mM EDTA. 1% NaDOC, 1% NP40, 1mM sodium orthovanadate, pH 7.0 containing 1 �g/mlleupeptin and pepstatin A, 1 mM AEBSF, 50 mM NaF, and1 mM ammonium molybdate) was added as follows: to cellsin monolayer culture (logarithmically growing or newlyconfluent), cell pellets after trypsinization, or cells in ma-trices (3/sample). Homogenization was accomplished by 50strokes with a Dounce homogenizer (pestle B; WheatonScientific, Millville, NJ, USA). After homogenization, sam-ples were clarified by centrifugation for 10 min at 4°C and16,000 g (Eppendorf microfuge). Aliquots of the superna-tants (equalized by measuring lactic dehydrogenase activ-ity) were boiled reducing sample buffer (62.5 mM Tris-HCl,2% SDS, 10% glycerol, 0.01% bromophenol blue, 5% mer-captoethanol, pH 6.8) and subjected to SDS-PAGE electro-phoresis using 5% acrylamide mini-slab gels. After transferto PVDF membranes (Millipore), samples were immuno-blotted with anti-FAK and phospho-FAK antibodies. Sec-ondary antibodies were HRP conjugated goat-anti-rabbit ormouse antibodies. The membranes were developed by theECL system (Amersham) according to the manufacturer’sprotocol.

87D.J. Lee et al. / Experimental Cell Research 289 (2003) 86–94

Results

Effect of blocking Rho kinase on LPA stimulatedcontraction of floating collagen matrices and retraction ofthe fibroblast dendritic network

Fibroblasts were polymerized in collagen matrices for 1hr after which the matrices were released and allowed tocontract in the presence or absence of LPA and the Rhokinase inhibitor Y27632. Fig. 1A shows that LPA stimu-lated the time course of contraction over a 4-hr periodcompared to basal conditions (DMEM containing 5 mg/mlBSA), and that addition of the Rho kinase inhibitor had littleeffect. Therefore, LPA-stimulated fibroblast contraction offloating matrices did not require Rho kinase.

During the 1-hr polymerization period, fibroblasts incollagen matrices projected a dendritic network of processesas shown in Fig. 2A. Subsequent stimulation of cells byLPA but not incubation in basal medium caused retractionof these extensions after an additional 1 hr incubation (Fig.2C compared to Fig. 2B). Retraction was completelyblocked by inhibiting Rho kinase as shown by Fig. 2D.Taken together, these results and those in Fig. 1A demon-strate that LPA stimulation of fibroblasts in newly polymer-ized collagen matrices caused not only contraction of col-lagen matrices, but also retraction of dendritic extensions.Retraction of processes was not required for matrix contrac-

tion, however, since blocking the former did not prevent thelatter.

Switch in the mechanism of LPA but not PDGFstimulated matrix contraction

As mentioned in the Introduction and in contrast to theresults in Fig. 1A, Rho kinase activity was reported to berequired for fibroblasts to contract restrained collagen ma-trices that had developed mechanical load for several days[12,13]. Therefore, fibroblasts in polymerized collagen ma-trices were preincubated various times in serum-containingmedium after which the matrices were released and allowedto contract in the presence or absence of LPA and the Rhokinase inhibitor Y27632.

Instead of several days, an initial preincubation period asshort as 2–4 h after polymerization was found to be suffi-cient for a switch in regulatory mechanisms to occur. Forinstance, Fig. 1B shows results from an experiment in whichpolymerized matrices were preincubated 2 h in DMEM/10% FBS before release. In this case, addition of the Rhokinase inhibitor blocked the LPA-stimulated component ofcontraction. (See also Fig. 5).

Studies using PDGF instead of LPA as the agonist forcontraction demonstrated that the Rho kinase-independentmechanism of contraction as well as the switch in mecha-nisms were unique to LPA. That is, Figs. 3A and B showthat PDGF-stimulated contraction was blocked by inhibitingRho kinase regardless whether the matrices were releasedimmediately after polymerization (Fig. 3A) or after thepolymerized matrices were preincubated 2 h in serum-con-taining medium (Fig. 3B).

Switch in Gi dependence of LPA-stimulated fibroblast-collagen matrix contraction

The heterotrimeric G protein Gi has been implicated inLPA-stimulated contraction of floating collagen matrices[15–17], but not contraction of restrained matrices. Wetested the possibility, therefore, that in parallel to the re-quirement for Rho kinase, the ability of cells to use the Gidependent pathway was lost. Fig. 4A shows that treatmentof cells with pertussis toxin inhibited LPA but not PDGF-stimulated contraction of newly polymerized matrices. If,however, matrices were preincubated 2 h in serum-contain-ing medium before release, then pertussis toxin had muchless effect on contraction (Fig. 4B). These results suggestedthat concomitant with onset of Rho kinase dependence ofLPA-stimulated contraction, fibroblasts lost the alternativemechanism.

Development of mechanical loading in restrainedcollagen matrices

Other experiments were carried out to explore whatchanges occurred during the 2–4 hr preincubation period

Fig. 1. Switch in Rho kinase dependence of LPA-stimulated fibroblastcollagen matrix contraction. Collagen matrices containing fibroblasts werereleased from culture dishes to initiate contraction either (A) immediatelyafter polymerization or (B) after preincubation for 2 hr in DMEM/10%FBS. Contraction medium was DMEM/BSA with 10 �M LPA and 10 �MY27632 added as indicated, and the extent of contraction was measured atthe times shown after release.

88 D.J. Lee et al. / Experimental Cell Research 289 (2003) 86–94

that might account for the switch in regulation of LPA-dependent contraction. As shown by Fig. 5, if the preincu-bation was carried out in the absence of serum (Fig. 5B)instead of serum-containing medium (Fig. 5C), then subse-quent LPA-stimulated contraction of released matrices didnot become Rho kinase dependent even after 4 h (compareto Fig. 5A).

During the preincubation period, restrained collagen ma-trices in serum-containing medium underwent a greater de-crease in matrix height, i.e., increase in collagen density,compared with matrices in serum-free medium (not shown).As matrix density increases, stiffness increases as well [18],potentially allowing cells to form focal adhesions [19].

Consistent with this possibility, the different preincubationconditions—with and without serum in the medium—hadmarkedly different consequences for cell morphology asrevealed by immunofluorescence staining for tubulin andactin. Fig. 6D shows that in the presence of serum relativeto serum-free conditions (Fig. 6A), there was an increase incell spreading. In addition, the corresponding images ofactin shown in Fig. 6B and 6E as well as higher resolutionimages shown in Fig. 6C and 6F showed an increase in actinstress fibers in the cytoplasm of fibroblasts in matrices inserum-containing medium. In counts made on randomlyselected fields, 95 out of 105 cells in matrices that had beenincubated in serum-containing medium contained stress fi-

Fig. 2. Rho kinase-dependent fibroblast dendrite retraction. Polymerized collagen matrices containing fibroblasts (105/ml) (A) were released and incubatedfor 1 h in DMEM/BSA (B) or in medium containing 10 �M LPA (C) or 10 �M LPA and 10�M Y27632 (D). Samples were fixed and stained for actin. Bar� 40 �m.

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bers compared to 5 out of 113 cells in matrices that had beenincubated in serum-free medium.

Cell spreading and development of actin stress fibersdepends on integrin clustering at focal adhesions [20]. Focaladhesion kinase (FAK) is then recruited to these sites and

activated by autophosphorylation at tyrosine-397 [21,22].Extracts prepared from fibroblast monolayer cultures beforeand after trypsinization, from cells in newly polymerizedcollagen matrices, or from cells in matrices preincubated 2 hwith or without FBS were subjected to immunoblotting withantibodies specific for FAK and phosphorylated FAK[pY397]. Fig. 7 shows that phosphorylated FAK was de-tected in cells in monolayer culture but disappeared aftertrypsinization. Subsequently, FAK phosphorylation was ob-served in fibroblasts in matrices incubated in serum-con-taining medium consistent with the idea that these cellswere forming focal adhesions.

Effect of nocodazole on fibroblast collagen-matrixcontraction

Taken together, the foregoing results suggested that theswitch in cell regulation of LPA but not PDGF-stimulatedmatrix contraction correlated with preincubation conditions

Fig. 3. Rho kinase dependence of PDGF-stimulated fibroblast collagenmatrix contraction. Collagen matrices containing fibroblasts were releasedfrom culture dishes to initiate contraction either (A) immediately afterpolymerization or (B) after preincubation for 2 hr in DMEM/10% FBS.Contraction medium was DMEM/BSA with 50 ng/ml PDGF and 10 �MY27632 added as indicated, and the extent of contraction was measured atthe times shown after release.

Fig. 4. Switch in Gi dependence of LPA-stimulated fibroblast collagen matrixcontraction. (A) Collagen matrices prepared containing fibroblasts previouslytreated 2 h with DMEM/10% FBS and 200 ng/ml pertussis toxin as indicatedwere released immediately after polymerization to initiate contraction inDMEM/BSA with 10 �M LPA or 50 ng/ml PDGF added as shown. (B)Fibroblasts in polymerized collagen matrices were preincubated 2 hr withDMEM/10% FBS containing 200 ng/ml pertussis toxin as indicated at whichtime matrices were released to initiate contraction in DMEM/BSA with 10 �MLPA added as shown. The extent of matrix contraction was measured 4 h afterrelease. Results shown are averages and standard deviations from two separateexperiments each carried out in duplicate.

Fig. 5. Switch in Rho kinase dependence of LPA-stimulated fibroblastcollagen matrix contraction after incubation in serum-containing but notbasal medium. Collagen matrices containing fibroblasts were released fromculture dishes to initiate contraction immediately after polymerization (A)or after preincubation for 4 h in DMEM/BSA without (B) or with (C) 10%FBS. Contraction medium was DMEM/BSA with 10 �M LPA and 10 �MY27632 added as indicated. At the times shown after release, matrixcontraction was measured.

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under which fibroblasts spread and begin to develop stressfibers and focal adhesions. The idea that a change in theextent of cell spreading could change how cells respond to

agonists and inhibitors might also have explained the dif-ferent effects of the microtubule disrupting agent nocoda-zole on fibroblast contraction.

Fig. 6. Tubulin and actin organization in fibroblasts in collagen matrices. Polymerized collagen matrices containing fibroblasts were incubated for 3 hr inDMEM/BSA without (A–C) or with (D–F) 10% FBS. At the end of the incubations, samples were fixed and stained for tubulin (A, D) and actin (B, E) oractin alone (C, F). Bar � 80 �m (A, B, D, E), 40 �m (C, F).

91D.J. Lee et al. / Experimental Cell Research 289 (2003) 86–94

On one hand, nocodazole has been reported to inhibitserum-dependent contraction of newly polymerized colla-gen matrices [23]. On the other hand, disrupting microtu-bules also has been reported to stimulate exertion of con-tractile force by fibroblasts in restrained collagen matrices[24,25], which has been attributed to activation of Rho andRho kinase [26–28]. Therefore, experiments were carriedout to learn if the switch in nocodazole from an inhibitor ofcontraction to an activator occurred over the same timeinterval as the switch in the regulatory mechanism of LPA-stimulated contraction.

Figure 8 shows that the addition of nocodazole to fibro-blasts in newly polymerized collagen matrices inhibitedboth PDGF and LPA-stimulated contraction but had nostimulatory effect on basal (BSA) contraction up to 4 h. If,on the other hand, polymerized matrices were preincubatedfor 3 h before release, then nocodazole stimulated matrixcontraction. Under these conditions, the switch occurredregardless whether the matrices had been preincubation inserum-containing or serum-free medium, and blocking Rhokinase activity markedly decreased nocodazole-stimulatedcontraction in either case.

Discussion

In this study we demonstrate that LPA can stimulatefibroblast-collagen matrix contraction by two different G-protein regulated mechanisms. Fibroblast contraction ofnewly polymerized matrices was Rho kinase-independentand Gi-dependent. If the restrained matrices were preincu-bated 2–4 h in serum-containing medium before release,then LPA-stimulated contraction became Rho kinase-de-pendent and Gi-independent, suggesting that signals fromthe remodeled matrix were able to feed back to modulatecell behavior in an iterative process.

The change in regulatory mechanisms might have re-sulted if cells in newly polymerized collagen matrices ini-tially lacked a Rho kinase-dependent contractile mecha-nism. This was not the case, however as shown by thedependence of PDGF-stimulated contraction of Rho kinase.Moreover, LPA also stimulated Rho kinase-dependent re-

traction of fibroblast dendritic processes. Since inhibition ofRho kinase blocked retraction of the processes withoutaffecting floating matrix contraction, it could be concludedthat the two events were independent of each other. That is,LPA-stimulated contraction of floating collagen matriceswas not caused by LPA-stimulated retraction of cellularextensions.

Fibroblasts respond to LPA stimulation through severalEdg receptors that have multiple downstream effectors in-cluding Rho (via G12/13)/Rho kinase and Gi [29–32]. Oneexplanation for our findings might have been a switch inLPA receptors [33] used by cells in newly polymerized vs2–4 h preincubated matrices. Since, however, Rho/Rhokinase was activated by LPA stimulation in newly polymer-ized or preincubated matrices, our results indicated thatrather than a switch in receptor coupling, what changed wascellular regulation of contraction itself.

Significantly, the switch in Rho kinase dependence ofcontraction occurred when cells were preincubated in se-rum-containing medium more so than in serum-free me-dium. In the presence of serum, there was increased cellspreading and formation of actin stress fibers. Moreover,FAK activation (phosphorylation at Y397) occurred, consis-tent with cellular development of focal adhesions [20–22].Fibroblast spreading and development of stress fibers wasobserved concomitant with the increase in collagen density,which took place to a greater extent when serum waspresent during the 2–4 hr preincubation period. As matrixdensity increases, matrix stiffness increases as well [18],and it is in part this increase in matrix stiffness that makesit possible for cells to form stress fibers and focal adhesions[19].

The switch in the mechanism of collagen matrix contrac-

Fig. 8. Switch in nocodazole inhibition/dependence of collagen matrixcontraction. (A) Collagen matrices containing fibroblasts and polymerized1 h were released from culture dishes to initiate contraction in DMEM/BSA with 10 �M LPA, 50 ng/ml PDGF, and 2 �M nocodazole added asindicated. The extent of contraction was measured after 1 h or 4 h asindicated. (B) Collagen matrices containing fibroblasts were polymerized1 h, preincubated for 3 h in DMEM/10% without or with FBS, and thenreleased from culture dishes in DMEM/BSA with 2 �M nocodazole and 10�M Y27632 added as indicated. The extent of contraction was measuredafter 1 h.

Fig. 7. Activation of FAK during incubation in collagen matrices Cellsfrom monolayer culture (Mon), harvested by trypsinization (Try), in ma-trices polymerized 1 h (Pol) or in matrices incubated 2 h in DMEM/BSAwith or without 10% FBS were extracted and subjected to immunoblottingwith antibodies directed against phospho(Y397)-FAK and total FAK.

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tion can be understood by drawing an analogy to cellsattempting to spread on two dimensional surfaces and cellsthat already have spread. For cells attempting to spread,activation of Rho by the combination of integrin occupancy[34] and agonist stimulation will impede spreading by an-tagonizing Rac-dependent formation of cell protrusions[35,36]. Under these conditions, therefore, feedback mech-anisms that inactivate Rho actually enhance spreading andmigration [35,37,38]. Moreover, in the case of LPA stimu-lation, Gi is required because LPA activates Rac and cellprotrusion in a Gi-dependent fashion [36]. On the otherhand, in cells that have already attached and spread, agoniststimulation of contractile force leads to a Rho/Rho kinase-dependent increase in isometric tension as shown by ap-pearance of cellular stress fibers and focal adhesions[9,11,39,40], which is independent of Gi [41].

The experiments examining the effect of nocodazole onmatrix contraction provided additional support for the ideathat a change in the extent of cell spreading can influencehow cells respond to agonists and inhibitors. Disruptingmicrotubules of fibroblasts in newly polymerized matricescauses retraction of the cell’s dendritic extensions [14], butthis retraction was not sufficient to cause matrix contraction.Indeed, disrupting microtubules inhibited contraction ofnewly polymerized matrices that depended on LPA orPDGF. After cells had spread in the matrices for severalhours, however, then the disruption of microtubules wasitself able to stimulate contraction even under conditionswhen the cells did not become mechanically loaded. Exer-tion of force dependent on microtubule disruption waslargely Rho kinase dependent as described by others [26–28].

The LPA-stimulated, Rho kinase-independent mecha-nism by which fibroblasts contract floating collagen matri-ces requires further investigation. Preliminary evidencesuggests that neither myosin light chain kinase nor phos-phorylation of myosin regulatory light chain is required (M.Abe, K. Kamm and F. Grinnell, unpublished observations).Previously, fibroblasts have been shown to bend and trans-locate collagen fibrils along the cell surface in an integrin-dependent fashion [42,43], but the force generating mech-anisms for moving the collagen fibrils have not beendetermined. In smooth muscle cells, an LPA-stimulated,Rho kinase-independent, Gi-dependent mechanism of con-traction has been reported, which involves phosphorylationof caldesmon by p21-activated kinase [44–46]. Whetherthis regulatory pathway functions in fibroblasts has yet to betested.

Studies on the mechanisms regulating collagen matrixremodeling have important implications for our understand-ing of matrix remodeling during wound repair. As repairprogresses, wound fibroblasts differentiate into myofibro-blasts in response to mechanical loading in the wound andmediate contraction through a process that requires Rho/Rho kinase, myosin light chain phosphorylation, and�-smooth muscle actin [4,47,48]. Initially, however, sub-

stantial wound matrix remodeling can occur without theappearance of myofibroblasts [49,50]. The molecular andregulatory bases for the early wound remodeling/contrac-tion process remain to be determined, but our researchsuggests a role for a Rho kinase-independent mechanism.

Acknowledgments

This research was supported by a grant from the NIH,GM31321. We are grateful to Dr. William Snell for helpfulcomments and suggestions.

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