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Experimental Cell Research 257, 190–197 (2000)doi:10.1006/excr.2000.4866, available online at http://www.idealibrary.com on

Activation of ERK and p38 MAP Kinases in Human Fibroblastsduring Collagen Matrix Contraction

David J. Lee, Hans Rosenfeldt, and Frederick Grinnell1

Department of Cell Biology and Neuroscience, UT Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75235

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Studies were carried out to characterize changes inMAP kinase activation during contraction of collagenmatrices by fibroblasts under isometric tension. Wefound that both ERK and p38 MAP kinases were acti-vated during contraction, as determined by immuno-blotting and in vitro kinase assays. ERK activation

as biphasic, with peaks at 10 min and 2 h; whereas38 activation was monophasic, with a single peak at0 min. Activation of ERK, but not p38, appeared toepend at least in part on the Gi class of heterotri-

meric G proteins. The results show that ERK and p38cooperate in contraction-stimulated activation of c-fostranscription. © 2000 Academic Press

Key Words: mechanoregulation; signaling; wound; Gprotein; c-fos.

INTRODUCTION

Wound repair in skin occurs through integrated cel-lular and molecular events leading to healing of thewound defect by a combination of epithelization, con-traction, and extracellular matrix biosynthesis [1, 2].The wound contraction process depends on fibroblast-like cells known as myofibroblasts [3]. These a-smoothmuscle actin containing cells are characterized by elon-gated shape, prominent stress fibers, and fibronexusjunctions [4]. Their development is promoted by trans-forming growth factor-b (TGF-b) [5, 6] and the ED-Adomain of fibronectin [7]. At the end of the repairprocess, myofibroblasts in wound tissue regressthrough apoptosis [8].

Studies with fibroblasts cultured in collagen matri-ces have been used to model repair in vitro [9, 10]. In

atrices attached to culture dishes, cells develop theppearance of myofibroblasts, including polarized mor-hology, prominent stress fibers, fibronexus junctions,nd increased expression of a-smooth muscle actin un-

der the influence of TGF-b [11–13]. These cells exertisometric tension on the matrix similar to that in heal-

1 To whom correspondence and reprint requests should be ad-ressed. Fax: (214) 648-8694. E-mail: frederick.grinnell@email.

wmed.edu.

1900014-4827/00 $35.00Copyright © 2000 by Academic PressAll rights of reproduction in any form reserved.

ing wounds [14, 15]. Release of the matrices from un-derlying attachment sites results in rapid contractionfollowed by transcriptional activation of c-fos and othermmediate early genes [16], after which the cells begino regress through apoptosis [17].

Previously, we showed that contraction of collagenatrices by fibroblasts under isometric tension re-

ulted in activation of extracellular signal regulatedinase (ERK) [16]. Mitogen-activated protein (MAP)inase cascades have been implicated in a broad rangef cellular responses to stimuli that result in cell pro-iferation, differentiation, and apoptosis [18, 19].herefore, experiments were carried out to more fullyharacterize the activation of ERK and other MAPinases during collagen matrix contraction. Here wehow that both ERK and p38 MAP kinases undergoctivation when human fibroblasts under isometricension contract collagen matrices. Activation of ERK,ut not p38, occurred in a biphasic manner and de-ended in part on the Gi class of heterotrimeric G

proteins. ERK and p38 also cooperated in the contrac-tion-stimulated activation of c-fos transcription butwere not required for collagen matrix contraction perse.

METHODS

Materials. Dulbecco’s modified Eagle’s medium (DME), reversetranscriptase, and deoxynucleotides were purchased from GIBCO/BRL (Gaithersburg, MD). Fetal bovine serum (FBS) was purchasedfrom Intergen Co. (Purchase, NY). Type I collagen (Vitrogen) waspurchased from Collagen Corp. (Palo Alto, CA). MEK inhibitorPD98059, p38 inhibitor SB202190, and pertussis toxin were fromCalbiochem (San Diego, CA). Anti-active ERK1/2 antibodies wereobtained from Promega Corp. (Madison, WI). Phosphospecific p38MAP kinase antibodies were from New England Biolabs (Beverly,MA). Anti-ERK2 and p38 antibodies were from Santa Cruz Biotech-nology, Inc. (Santa Cruz, CA). Nylon (Nytran) membranes for South-ern hybridization were purchased from Schleicher & Schuell (Keene,NH). Guanidinium thiocyanate for Solution D was purchased fromFluka Chemical Co. (Ronkonkoma, NY). Fatty acid free bovine se-rum albumin (BSA) and lysophosphatidic acid (LPA) were obtainedfrom Sigma. [3H]Arachidonic acid was from New England Nuclear.Budgetsolve was from Research Products International Corp. (Mt.Prospect, IL).

Culture of human fibroblasts in collagen matrices. Fibroblasts

from human foreskin specimens (up to 10 passages) were maintained

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in Falcon 75-cm2 tissue culture flasks in DME supplemented with0% FBS. Hydrated collagen matrices were prepared from Vitrogen00 collagen. Neutralized collagen solutions (1.5 mg/ml) containedbroblasts in DME but no serum. Cell/collagen mixtures were pre-armed to 37°C for 3–4 min, and aliquots (0.2 ml) were placed inorning 24-well culture plates. Each aliquot occupied an area out-

ined by a 12-mm-diameter circular score within a well. Polymeriza-ion of collagen matrices required 60 min at 37°C in a humidifiedncubator with 5% CO2.

The protocol for collagen matrix contraction has been describedreviously [16]. After polymerization, 1.0 ml of DMEM with 10%BS and 50 mg/ml ascorbic acid was added to each well. Cultures

were incubated for 24 h during which isometric tension develops andthen for an additional 24 h with medium containing 1% FBS unlessindicated otherwise. To release isometric tension, matrices wererinsed three times for 5 min with serum-free DMEM, gently releasedfrom the underlying culture dish with a spatula, and incubated at37°C in serum-free DMEM containing other additions as indicated.To determine the extent of contraction, matrices were fixed with 3%paraformaldehyde in DPBS [150 mM NaCl, 3 mM KCl, 6 mMNa2HPO4, 1 mM KH2PO4, 0.5 mM MgCl2, 1.0 mM CaCl2, pH 7.2] for10 min at 22°C, washed and placed on a glass slide, and measuredwith a ruler. Contraction data are presented as the difference be-tween final matrix diameter and the diameter of a cell-free matrix(10.5 mm) measured in millimeters [20].

All figures present representative results from two or more sepa-rate experiments.

SDS–PAGE and immunoblotting. Collagen matrices to be ex-tracted (two matrices per sample) were placed into 200 ml of ice-coldlysis buffer (0.5% NP40 in DPBS with 1 mg/ml leupeptin, 1 mg/ml

epstatin A, 1 mM AEBSF, 50 mM NaF, 1 mM Na3VO4, and 1 mMa2MoO4, pH 7.4) and homogenized (50 strokes) using a Dounce

homogenizer (pestle A, Wheaton Scientific, Millville, NJ). Sampleswere clarified by centrifugation for 10 min, 4°C at 16,000g (Beck-man Microfuge 11), and the supernatants were dissolved in 43reducing sample buffer (250 mM Tris, 4% (w/v) SDS, 40% glycerol,20% mercaptoethanol, 0.04% (w/v) bromophenol blue) and boiled for3.5 min. Sample volumes, adjusted for equivalent lactate dehydro-genase (LDH) activities with the LDH diagnostic kit (Sigma), weresubjected to SDS–PAGE using 10% acrylamide minislab gels andtransferred to PVDF membranes (Millipore). Blots were then probedwith the indicated antibodies.

In vitro kinase assays. Cell extracts were prepared as for immu-noblotting, with 6–14 matrices per sample. Equivalent LDH activi-ties (approximately 360 ml) were immunoprecipitated overnight at°C with 30 ml of Protein A–agarose (Boehringer Mannheim) and 1

mg of anti-ERK2 antibody (C-14, Santa Cruz) or anti-p38 antibody(C-20, Santa Cruz). Beads were washed twice in 0.25 M Tris–HCl,0.5 M NaCl, pH 7.5, once in 0.25 M Tris–HCl, 0.1 M NaCl, pH 7.5,and once in kinase buffer (10 mM Hepes, 10 mM MgCl2, 1 mMdithiothreitol, 1 mM benzamidine, pH 8.0). Kinase reactions wereinitiated by resuspending beads in kinase buffer with 100 mM ATP,0.5 mg/ml myelin basic protein (for ERK2) or 2 mg of substrateGST-ATF2D (for p38), and 10 mCi of [g-32P]ATP (Amersham) andincubating 15 min at 30°C (for ERK2) or 1 h at 30°C (for p38), 30 mlf mixture per reaction. Reactions were terminated by adding reduc-ng SDS sample buffer. Samples were heated at 100°C for 3 min andnalyzed by SDS–PAGE on polyacrylamide minislab gels. Gels wereoomassie stained and dried. Radioactivity was visualized by auto-adiography, and gels were quantified by scintillation counting ofxcised bands. Bacterial expression construct for glutathione-transferase (GST)-ATF2D (amino acids 1–254) was a gift of Dr.elanie Cobb, UT Southwestern [21].Reverse transcriptase (RT) PCR/Southern blotting. RNA (one ma-

rix per sample) was isolated as described previously [16], exceptNA was dissolved in 10 ml of autoclaved water prior to first-strand

synthesis. First-strand synthesis reactions were carried out with

M-MLV reverse transcriptase (GIBCO/BRL) and oligo-dT(15) primeraccording to the manufacturer’s protocol. After incubation for 3 h at37°C, M-MLV RT enzyme was heat killed by incubation at 70°C for10 min. PCR reactions were then carried out using 1 ml of cDNApreparation per sample. DNA was denatured at 95°C for 4 minfollowed by five cycles of 30-s denaturation at 94°C, 30-s annealing at57°C, 60-s elongation at 72°C, and a final elongation phase for 10 minat 72°C. Reaction mixtures (12 ml) contained 20 mM Tris–HCl (pH.4), 0.5 U of Taq polymerase, 50 mM KCl, 1.5 mM MgCl2, 200 mMNTPs, and 1 mM of each primer. After PCR, 7.5 ml from each sampleas removed and 1.5 ml of 63 loading buffer II [22] was added, and

he mixture was loaded onto 2% agarose gels containing 13 Tris–cetate–EDTA (TAE) [22]. Samples were subjected to electrophoresisor 30 min at 100 V. The gel was then soaked in 0.5 M NaOH, 1.5 MaCl for 45 min to denature the DNA, and then pH was neutralized

n 203 SSPE (41). After transfer, DNA was cross linked with atratalinker UV light source (Stratagene) at a setting of 1200 mJ of

radiation per cm2. c-fos and GAPDH PCR products were then de-tected under aqueous conditions at 65°C according to standard pro-tocols [22]. DNA probes for hybridization were generated with aBoehringer Mannheim random primed labeling kit. Gel-purifiedPCR products using c-fos and GAPDH primers served as templates.-fos primers were synthesized by the UT Southwestern Moleculariology Core Facility as follows: 59-atgatgttctcgggcttcaacgcagcag-39nd 59-tctggagataactgttccaccttg-39. GAPDH primers were purchasedrom Clontech (Palo Alto, CA).

RESULTS

Contraction of human fibroblasts cultured under iso-metric tension results in rapid activation of ERK andp38 MAP kinases. Placing fibroblasts in collagen ma-trices results in activation of ERK and p38 MAP ki-nases [23, 24]. After culture of the matrices attached toculture dishes for 48 h, isometric tension develops [25].Over the same time period, the MAP kinases returnedto the nonphosphorylated state. When the collagenmatrices were released from the culture dishes to ini-tiate contraction, however, we found that activation ofERK and p38 MAP kinases occurred within 10 min.

Figures 1A and 1B show the time course of ERKactivation detected by immunoblotting with antibodiesspecific for active ERK. The level of active ERK in-creased rapidly and peaked within 10 min, and a sec-ond peak of activity occurred about 2 h later. Over thesame time, there was no change in the level of activeERK in cells in matrices that remained attached afterbeing subjected to the same medium changes (Fig. 1B).

Results of the immunoblotting experiments wereconfirmed by in vitro kinase assays. Figure 1C showskinase activity of immunoprecipitated ERK2 at timepoints selected based on the immunoblotting analyses.A 3-fold increase in ERK activity occurred 10 min afterinitiation of contraction, and a second increase wasdetected at 150 min.

The biphasic pattern of ERK activation was unex-pected, perhaps a feature specific to contraction of hu-man fibroblasts in collagen matrices. To test this pos-sibility, cells in attached matrices were stimulated

with lysophosphatidic acid (LPA). Figure 2 shows typ-

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192 LEE, ROSENFELDT, AND GRINNELL

ical results, demonstrating a single peak of ERK acti-vation around 10 min after LPA addition. A single peakof ERK activation also was detected following cell stim-ulation with serum, platelet-derived growth factor, andphorbol ester (not shown). Therefore, the biphasic ac-tivation of ERK appeared to be unique to fibroblasts

FIG. 1. ERK activation during contraction. (A) At the timesindicated after releasing matrices, cell extracts were prepared andimmunoblotted for active ERK1/2 and total ERK2 as shown. (B)Same as A except a parallel set of matrices remained attached duringthe incubation. (C) Following release of attached matrices, cell ex-tracts were prepared at the times indicated and used to test kinaseactivity of immunoprecipitated ERK2 using myelin basic protein(MBP) as a substrate. Top: Autoradiograph of kinase reaction. Bot-tom: Quantitation of 32P incorporation into MBP.

FIG. 2. Monophasic ERK activation by LPA. Cells in attachedmatrices were incubated with 10 mM LPA. At the times indicated,cell extracts were prepared and immunoblotted for active ERK1/2

and total ERK2.

contracting collagen matrices and not the presence ofthe collagen matrix per se.

Figures 3A and 3B show the time course of activationof p38 MAP kinase. Based on immunoblotting, a singlepeak of activity occurred around 10 min. Unlike ERK,however, no second peak of activation was detected.Also, no activation of p38 occurred in fibroblasts inattached matrices subjected to identical mediumchanges (Fig. 3B). Activation of p38 also was confirmedusing an in vitro kinase assay. As shown in Fig. 3C, a2-fold increase in enzyme activity could be detected 10min after initiation of contraction.

Role of Gi class of heterotrimeric G proteins in acti-ation of ERK but not p38. The experiments de-cribed above demonstrated that activation of ERKnd p38 MAP kinases occurred when fibroblasts inollagen matrices under isometric tension were al-owed to contract. In endothelial cells and cardiomyo-ytes, the Gi class of heterotrimeric G proteins has

FIG. 3. p38 activation during contraction. (A) At the times indi-cated after releasing matrices, cell extracts were prepared and im-munoblotted for active p38 and total p38 as shown. (B) Same as Aexcept a parallel set of matrices remained attached during the incu-bation. (C) Following release of attached matrices, cell extracts wereprepared at the times indicated and used to test kinase activity ofimmunoprecipitated p38 using GST-ATF2D (ATF2) as a substrate.Top: Autoradiograph of kinase reaction. Bottom: Quantitation of 32Pncorporation into ATF2.

been implicated in mechanostimulated ERK activation

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based on inhibition of activation by pertussis toxin(PTx) [26, 27]. Figure 4A shows a dose–response exper-iment indicating that PTx markedly diminished ERKactivation during contraction, even at the lowest dosetested, 6.25 ng/ml. On the other hand, as shown in Fig.4B, p38 activation during contraction was insensitiveto PTx treatment at the highest dose tested, 100 ng/ml.Parallel control experiments demonstrated that PTxcompletely blocked LPA stimulation of ERK activationin cells in attached collagen matrices (data not shown).These results suggested that activation of ERK andp38 MAP kinase during contraction were regulateddifferently and that the ERK response depended on theGi class of heterotrimeric G proteins.

Inhibition of MAP kinase activation by PD98059 andSB202190. Specific pharmacologic inhibitors of theMAP kinase cascades have been used to explore down-stream regulatory functions of the signaling pathways.The reagent PD98059 prevents activation of ERK byblocking activation of its immediate upstream activa-tors MEK1 and MEK2 [28], whereas the reagentSB202190 directly binds to and inhibits the a and bhomologues of p38 MAP kinase [29–31].

Figures 5A and 5B show the effects of the inhibitorson fibroblasts in contracting collagen matrices. Addi-tion of 30 mM PD98059 completely blocked ERK acti-vation (Fig. 5A) but did not alter p38 activation (Fig.5B). Concentrations of PD98059 lower than 30 mM onlypartially blocked ERK activation (not shown). On theother hand, addition of 50 mM SB202190 not onlylocked p38 activation (Fig. 5B) but also substantiallyeduced the first peak of ERK activation (Fig. 5A).ddition of SB202190 to fibroblasts in attached matri-

es also caused a slight increase in the level of active

FIG. 4. ERK, but not p38, activation is inhibited by pertussisoxin. Cells in attached matrices were incubated 18 h with PTx at theoncentrations indicated. Subsequently, matrices were harvestedATT) or released for 10 min (REL). Cell extracts were prepared andmmunoblotted for active and total ERK (A) and p38 (B).

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MAP kinases are not required for matrix contraction.Using the inhibitors described above, we carried outexperiments to learn whether activation of MAP ki-nases was necessary for matrix contraction. Fifty mi-cromolar PD98059 has been reported to reduce colla-gen matrix contraction by COS-7 cells [32]. Figures 6Aand 6B show, however, that neither 50 mM PD98059nor 50 mM SB202190 affected the rate or extent ofcontraction.

Activation of the MAP kinases is required for contrac-tion-stimulated immediate early gene transcription.Studies also were carried out to learn the relationshipbetween MAP kinase stimulation and the increasedtranscription of c-fos that follows contraction [16]. Fig-ure 7 shows that addition of 50 mM PD98059 reducedsubstantially, but did not completely inhibit, the c-fosesponse, even though ERK activation was completelylocked. SB202190 alone, in marked contrast, had nonhibitory effect on the c-fos response. When SB202190

FIG. 5. Effect of MEK inhibitor PD98059 and p38 inhibitorSB202190 on ERK and p38 activation. Cells in attached matriceswere treated for 30 min with DMSO, 30 mM PD98059, or 50 mMSB202190 as indicated. Matrices were then harvested (0) or releasedor left attached for the indicated times. Cell extracts were prepared

and immunoblotted for active and total ERK (A) and p38 (B).

194 LEE, ROSENFELDT, AND GRINNELL

was combined with PD98059, however, c-fos mRNAtranscription was completely prevented. These find-ings suggested that ERK and p38 MAP kinases coop-erated in c-fos activation during contraction.

DISCUSSION

The current studies were carried out to characterizethe activation of MAP kinases during contraction ofcollagen matrices by fibroblasts under isometric ten-sion. Activation of ERK and p38 was demonstratedboth by immunoblotting with specific antibodies and byin vitro kinase assays. Previous studies showed thatERK and p38 MAP kinase activation occurs upon ini-tial fibroblast attachment to collagen matrices [23, 24],but during the 48-h culture over which isometric ten-sion developed, the MAP kinases had returned to the

FIG. 6. MAP kinase activity is not required for matrix contrac-tion. Matrices were treated for 30 min with 50 mM PD98059 (A) or 50mM SB202190 (B). Subsequently, the matrices were released and theextent of contraction measured at the times indicated. DMSO wasadded to control samples at the same concentration as present in thesamples containing inhibitor.

basal state.

The pattern of ERK activation was biphasic, withpeaks at 10 min and 2 h. Biphasic activation of ERK isatypical but has been observed [33, 34]. The signifi-cance of biphasic ERK activation during contraction ofcollagen matrices is currently unknown, but the re-sponse was highly specific. That is, treatment of fibro-blasts in mechanically loaded collagen matrices withLPA or other agonists caused only monophasic activa-tion of ERK. Moreover, although p38 MAP kinase alsowas activated by contraction, the p38 response wasmonophasic and coincided with the first peak of ERKactivation.

Other studies with a variety of cell types, includingmuscle and endothelium, have implicated ERK andp38 activation in the cellular response to mechanicalstimulation [26, 35–42]. In these other studies, ERKactivation accompanied increased mechanical loading.In the experimental system that we are using, how-ever, ERK and p38 activation accompanied contractionof fibroblasts under isometric tension, which leads todecreased mechanical loading [17]. Consequently, MAPkinase activation might be an important general re-sponse to mechanical changes in the cell environment.

Treatment of cells with pertussis toxin inhibited con-traction-stimulated activation of ERK but not p38, sug-gesting that the two MAP kinase cascades respondedto contraction by different upstream regulatory mech-anisms. Stimulation of the sphingosine 1-phosphatereceptor was recently shown to be coupled to activationof ERK and p38 by PTx-sensitive and -insensitivemechanisms, respectively [43]. Inhibition by PTx sug-gested that the Gi class of heterotrimeric G proteinswas involved in coupling of contraction to the ERKcascade. PTx-sensitive G proteins also have been im-plicated in activation of ERK in cardiac and endothelialcells subjected to changes in mechanical force [26, 27].How changes in mechanical force are transmitted to Gproteins remains to be determined. One possibility isthat some G proteins can act as mechanoreceptors. Forinstance, G proteins in liposomes were shown to in-crease their GTPase activity in response to shear stress

FIG. 7. Inhibition of c-fos mRNA upregulation by MEK inhibitorPD98059 and p38 inhibitor SB202190. Cells in attached matriceswere incubated for 30 min with PD98059 and SB202190 as indicated.Subsequently, the matrices were released, and RNA was isolatedafter 50 min. c-fos and GAPDH mRNAs were analyzed by RT-PCRusing five cycles of PCR amplification, followed by Southern hybrid-

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[44]. Since inhibition of ERK activation by pertussistoxin was not complete, a second mechanoregulatedmechanism may also be involved.

Our studies also revealed possible cross-regulation ofthe ERK cascade by p38 since the p38 inhibitorSB202190 decreased ERK activation in contractingmatrices and increased basal levels of active ERK inattached matrices. Previously, the p38 inhibitor wasreported to inhibit activation of ERK following arsenitetreatment of several types of cells [45] and to increasebasal levels of active ERK in unstimulated HepG2 cells[46]. Our data need to be interpreted with caution,however, since the related p38 kinase inhibitorSB203580 also has been shown to inhibit Raf [47, 48],so the effect might be indirect. It should be emphasizedthat the p38 inhibitor SB202190 also blocked contrac-tion-stimulated activation of p38, consistent with therecent report that SB202190 binds to the inactive formof the enzyme and prevents its phosphorylation [49].

Under conditions in which ERK or p38 was inhib-ited, there was no apparent change in the rate orextent of collagen matrix contraction. Therefore, wecould not confirm the recent observation that 50 mMPD98059 reduced collagen matrix contraction byCOS-7 cells [32]. In the latter study, the extent ofreduction was quite modest, however. Also, it recentlyhas been shown that contraction of collagen matricesby fibroblasts under isometric tension and stimulatedwith serum or lysophosphatidic acid occurs by Rho-dependent myosin light chain phosphorylation [50],which is independent of ERK. Moreover, ERK activityis not required for cell contraction of floating collagenmatrices (i.e., matrices that contract before they de-velop isometric tension) [51]. Taken together, theseresults suggest that the mechanism of collagen matrixcontraction probably has at most a minimal depen-dence on ERK activation.

Contraction of fibroblasts in collagen matrices underisometric tension is followed within 1 h by transcrip-ion of immediate early genes such as c-fos [16]. We

found that the c-fos response to contraction could beblocked by simultaneously inhibiting the ERK and p38MAP kinase signaling pathways. Inhibiting ERK acti-vation alone partly blocked the c-fos response, whereasinhibiting the p38 pathway alone had no effect or evenslightly increased c-fos expression. Based on these

ndings, we suggest that during contraction, the ERKnd p38 MAP kinase pathways cooperate in c-fos tran-criptional activation, which is consistent with previ-us research demonstrating that ERK and p38 canegulate immediate early genes through different ter-ary complex transcription factors [52–56].During wound repair in vivo, fibroblasts undergo

roliferation, differentiation into myofibroblasts, con-raction, and apoptosis [1, 2, 8]. Since MAP kinase

ascades participate in a broad range of cellular re-

ponses to stimuli that result in cell proliferation, dif-erentiation, and apoptosis [18, 19], they are likely tolay important roles in diverse aspects of wound re-air. Consistent with this possibility, activation ofAP kinases has been observed following wounding at

oth the cellular [57, 58] and tissue levels [59, 60]. Ourtudies suggest that during wound repair, MAP ki-ases may regulate contraction-stimulated changes inene expression, although not contraction itself.

We are indebted to William Snell, Melanie Cobb, Michael White,Joseph Albanesi, and Chin-Han Ho for their advice and assistance.This research was supported by Grant GM31321 from NIGMS, NIH,and by the UT Southwestern Skin Diseases Research Center(AR41940).

REFERENCES

1. Clark, R. A. F. (1996). Wound repair: Overview and generalconsiderations. In “The Molecular and Cellular Basis of WoundRepair” (R. A. F. Clark, Ed.), pp. 3–50, Plenum, New York.

2. Martin, P. (1997). Wound healing: Aiming for perfect skin re-generation. Science 276, 75–81.

3. Gabbiani, G., Hirschel, B. J., Ryan, G. B., Statkov, P. R., andMajno, G. (1972). Granulation tissue as a contractile organ. Astudy of structure and function. J. Exp. Med. 135, 719–734.

4. Singer, I. I., Kawka, D. W., Kazazis, D. M., and Clark, R. A.(1984). In vivo co-distribution of fibronectin and actin fibers ingranulation tissue: Immunofluorescence and electron micro-scope studies of the fibronexus at the myofibroblast surface.J. Cell Biol. 98, 2091–2106.

5. Desmouliere, A., Geinoz, A., Gabbiani, F., and Gabbiani, G.(1993). Transforming growth factor-beta 1 induces alpha-smooth muscle actin expression in quiescent and growing cul-tured fibroblasts. J. Cell Biol. 122, 103–111.

6. Rønnov-Jessen, L., and Petersen, O. W. (1993). Induction ofalpha-smooth muscle actin by transforming growth factor-b1 inquiescent human breast gland fibroblasts. Lab. Invest. 68, 696–707.

7. Serini, G., Bochaton-Piallat, M. L., Ropraz, P., Geinoz, A., Borsi,L., Zardi, L., and Gabbiani, G. (1998). The fibronectin domainED-A is crucial for myofibroblastic phenotype induction bytransforming growth factor-beta1. J. Cell Biol. 142, 873–881.

8. Desmouliere, A., and Gabbiani, G. (1996). The role of the myo-fibroblast in wound healing and fibrocontractive disease. In“The Molecular and Cellular Basis of Wound Repair” (R. A. F.Clark, Ed.), pp. 391–423, Plenum, New York.

9. Bell, E., Ivarsson, B., and Merrill, C. (1979). Production of atissue-like structure by contraction of collagen lattices by hu-man fibroblasts of different proliferative potential in vitro. Proc.Natl. Acad. Sci. USA 76, 1274–1278.

0. Grinnell, F. (1994). Fibroblasts, myofibroblasts, and wound con-traction. J. Cell Biol. 124, 401–404.

1. Mochitate, K., Pawelek, P., and Grinnell, F. (1991). Stress re-laxation of contracted collagen gels: Disruption of actin filamentbundles, release of cell surface fibronectin, and down regulationof DNA and protein synthesis. Exp. Cell Res. 193, 198–207.

2. Tomasek, J. J., Haaksma, C. J., Eddy, R. J., and Vaughan, M. B.(1992). Fibroblast contraction occurs on release of tension inattached collagen lattices: Dependency on an organized actin

cytoskeleton and serum. Anat. Rec. 232, 359–368.

1

2

2

2

2

2

2

2

2

2

2

3

3

3

3

3

3

3

3

3

3

4

4

4

4

4

196 LEE, ROSENFELDT, AND GRINNELL

13. Arora, P. D., Narani, N., and McCulloch, C. A. (1999). Thecompliance of collagen gels regulates transforming growth fac-tor-beta induction of alpha-smooth muscle actin in fibroblasts.Am. J. Pathol. 154, 871–882.

14. Kolodney, M. S., and Wysolmerski, R. B. (1992). Isometric con-traction by fibroblasts and endothelial cells in tissue culture.J. Cell Biol. 117, 73–82.

15. Eastwood, M., McGrouther, D. A., and Brown, R. A. (1994). Aculture force monitor for measurement of contraction forcesgenerated in human dermal fibroblast cultures: Evidence forcell–matrix mechanical signalling. Biochim. Biophys. Acta.1201, 186–192.

16. Rosenfeldt, H., Lee, D. J., and Grinnell, F. (1998). Increasedc-fos mRNA expression by human fibroblasts contractingstressed collagen matrices. Mol. Cell. Biol. 18, 2659–2667.

17. Grinnell, F., Zhu, M., Carlson, M. A., and Abrams, J. A. (1999).Release of mechanical tension triggers apoptosis of human fi-broblasts in a model of regressing granulation tissue. Exp. CellRes. 248, 608–619.

18. Cobb, M. H. (1999). MAP kinase pathways. Prog. Biophys. Mol.Biol. 71, 479–500.

9. Garrington, T. P., and Johnson, G. L. (1999). Organization andregulation of mitogen-activated protein kinase signaling path-ways. Curr. Opin. Cell Biol. 11, 211–218.

0. Grinnell, F., Ho, C. H., Lin, Y. C., and Skuta, G. (1999). Differ-ences in the regulation of fibroblast contraction of floating ver-sus stressed collagen matrices. J. Biol. Chem. 274, 918–923.

1. Frost, J. A., Xu, S., Hutchison, M. R., Marcus, S., and Cobb,M. H. (1996). Actions of Rho family small G proteins and p21-activated protein kinases on mitogen-activated protein kinasefamily members. Mol. Cell. Biol. 16, 3707–3713.

2. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). “Molecu-lar Cloning: A Laboratory Manual,” Cold Spring Harbor Press,Cold Spring Harbor, NY.

3. Langholz, O., Roeckel, D., Petersohn, D., Broermann, E., Eckes,B., and Krieg, T. (1997). Cell–matrix interactions induce ty-rosine phosphorylation of MAP kinases ERK1 and ERK2 andPLCgamma-1 in two-dimensional and three-dimensional cul-tures of human fibroblasts. Exp. Cell Res. 235, 22–27.

4. Ravanti, L., Heino, J., Lopez-Otin, C., and Kahari, V. M. (1999).Induction of collagenase-3 (MMP-13) expression in human skinfibroblasts by three-dimensional collagen is mediated by p38mitogen-activated protein kinase. J. Biol. Chem. 274, 2446–2455.

5. Lin, Y. C., Ho, C. H., and Grinnell, F. (1997). Fibroblasts con-tracting collagen matrices form transient plasma membranepassages through which the cells take up fluorescein isothio-cyanate-dextran and Ca21. Mol. Biol. Cell 8, 59–71.

6. Jo, H., Sipos, K., Go, Y. M., Law, R., Rong, J., and McDonald,J. M. (1997). Differential effect of shear stress on extracellularsignal-regulated kinase and N-terminal Jun kinase in endothe-lial cells. Gi2- and Gbeta/gamma-dependent signaling path-ways. J. Biol. Chem. 272, 1395–1401.

7. Yamazaki, T., Komuro, I., and Yazaki, Y. (1998). Signallingpathways for cardiac hypertrophy. Cell. Signal. 10, 693–698.

8. Dudley, D. T., Pang, L., Decker, S. J., Bridges, A. J., and Saltiel,A. R. (1995). A synthetic inhibitor of the mitogen-activatedprotein kinase cascade. Proc. Natl. Acad. Sci. USA 92, 7686–7689.

9. Lee, J. C., Laydon, J. T., McDonnell, P. C., Gallagher, T. F.,Kumar, S., Green, D., McNulty, D., Blumenthal, M. J., Heys,J. R., Landvatter, S. W., et al. (1994). A protein kinase involvedin the regulation of inflammatory cytokine biosynthesis. Nature

372, 739–746.

0. Cuenda, A., Cohen, P., Buee-Scherrer, V., and Goedert, M.(1997). Activation of stress-activated protein kinase-3 (SAPK3)by cytokines and cellular stresses is mediated via SAPKK3(MKK6): Comparison of the specificities of SAPK3 and SAPK2(RK/p38). EMBO J. 16, 295–305.

1. Kumar, S., McDonnell, P. C., Gum, R. J., Hand, A. T., Lee, J. C.,and Young, P. R. (1997). Novel homologues of CSBP/p38 MAPkinase: Activation, substrate specificity and sensitivity to inhi-bition by pyridinyl imidazoles. Biochem. Biophys. Res. Com-mun. 235, 533–538.

2. Cheresh, D. A., Leng, J., and Klemke, R. L. (1999). Regulationof cell contraction and membrane ruffling by distinct signals inmigratory cells. J. Cell. Biol. 146, 1107–1116.

3. Kahan, C., Seuwen, K., Meloche, S., and Pouyssegur, J. (1992).Coordinate, biphasic activation of p44 mitogen-activated pro-tein kinase and S6 kinase by growth factors in hamster fibro-blasts. Evidence for thrombin-induced signals different fromphosphoinositide turnover and adenylylcyclase inhibition.J. Biol. Chem. 267, 13369–13375.

4. Wen, Y., Nadler, J. L., Gonzales, N., Scott, S., Clauser, E., andNatarajan, R. (1996). Mechanisms of ANG II-induced mitogenicresponses: Role of 12- lipoxygenase and biphasic MAP kinase.Am. J. Physiol. 271, C1212–C1220.

5. Sadoshima, J., and Izumo, S. (1993). Mechanical stretch rapidlyactivates multiple signal transduction pathways in cardiacmyocytes: Potential involvement of an autocrine/paracrinemechanism. EMBO J. 12, 1681–1692.

6. Komuro, I., and Yazaki, Y. (1993). Control of cardiac geneexpression by mechanical stress. Annu. Rev. Physiol. 55, 55–75.

7. Yamazaki, T., Komuro, I., Kudoh, S., Zou, Y., Shiojima, I.,Mizuno, T., Takano, H., Hiroi, Y., Ueki, K., Tobe, K., et al.(1995). Mechanical stress activates protein kinase cascade ofphosphorylation in neonatal rat cardiac myocytes. J. Clin. In-vest. 96, 438–446.

8. Traub, O., Monia, B. P., Dean, N. M., and Berk, B. C. (1997).PKC-epsilon is required for mechano-sensitive activation ofERK1/2 in endothelial cells. J. Biol. Chem. 272, 31251–31257.

9. Kudoh, S., Komuro, I., Hiroi, Y., Zou, Y., Harada, K., Sugaya,T., Takekoshi, N., Murakami, K., Kadowaki, T., and Yazaki, Y.(1998). Mechanical stretch induces hypertrophic responses incardiac myocytes of angiotensin II type 1a receptor knockoutmice. J. Biol. Chem. 273, 24037–24043.

0. Lew, A. M., Glogauer, M., and McUlloch, C. A. (1999). Specificinhibition of skeletal alpha-actin gene transcription by appliedmechanical forces through integrins and actin. Biochem. J. 341,647–653.

1. Muller, E., Burger-Kentischer, A., Neuhofer, W., Fraek, M. L.,Marz, J., Thurau, K., and Beck, F. X. (1999). Possible involve-ment of heat shock protein 25 in the angiotensin II-inducedglomerular mesangial cell contraction via p38 MAP kinase.J. Cell. Physiol. 181, 462–469.

2. Hines, W. A., Thorburn, J., and Thorburn, A. (1999). Cell den-sity and contraction regulate p38 MAP kinase-dependent re-sponses in neonatal rat cardiac myocytes. Am. J. Physiol. 277,H331–H341.

3. Gonda, K., Okamoto, H., Takuwa, N., Yatomi, Y., Okazaki, H.,Sakurai, T., Kimura, S., Sillard, R., Harii, K., and Takuwa, Y.(1999). The novel sphingosine 1-phosphate receptor AGR16 iscoupled via pertussis toxin-sensitive and -insensitive G-pro-teins to multiple signalling pathways. Biochem. J. 337, 67–75.

4. Gudi, S., Nolan, J. P., and Frangos, J. A. (1998). Modulation ofGTPase activity of G proteins by fluid shear stress and phos-pholipid composition. Proc. Natl. Acad. Sci. USA 95, 2515–

2519.

4

4

4

4

5

5

5

5

197FIBROBLAST CONTRACTION ACTIVATES ERK AND p38

45. Ludwig, S., Hoffmeyer, A., Goebeler, M., Kilian, K., Hafner, H.,Neufeld, B., Han, J., and Rapp, U. R. (1998). The stress inducerarsenite activates mitogen-activated protein kinases extracel-lular signal-regulated kinases 1 and 2 via a MAPK kinase6/p38-dependent pathway. J. Biol. Chem. 273, 1917–1922.

6. Singh, R. P., Dhawan, P., Golden, C., Kapoor, G. S., and Mehta,K. D. (1999). One-way cross-talk between p38(MAPK) and p42/44(MAPK). Inhibition of p38(MAPK) induces low density li-poprotein receptor expression through activation of the p42/44(MAPK) cascade. J. Biol. Chem. 274, 19593–19600.

7. Hall-Jackson, C. A., Eyers, P. A., Cohen, P., Goedert, M., Boyle,F. T., Hewitt, N., Plant, H., and Hedge, P. (1999). Paradoxicalactivation of Raf by a novel Raf inhibitor. Chem. Biol. 6, 559–568.

8. Hall-Jackson, C. A., Goedert, M., Hedge, P., and Cohen, P.(1999). Effect of SB 203580 on the activity of c-Raf in vitro andin vivo. Oncogene 18, 2047–2054.

9. Frantz, B., Klatt, T., Pang, M., Parsons, J., Rolando, A., Wil-liams, H., Tocci, M. J., et al. (1998). The activation state of p38mitogen-activated protein kinase determines the efficiency ofATP competition for pyridinylimidazole inhibitor binding. Bio-chemistry 37, 13846–13853.

50. Parizi, M., Howard, E. W., and Tomasek, J. J. (2000). Regula-tion of LPA-promoted myofibroblast contraction: Role of Rho,myosin light chain kinase, and myosin light chain phosphatase.Exp. Cell Res. 254, 210–220.

51. Zent, R., Ailenberg, M., and Silverman, M. (1998). Tyrosinekinase cell signaling pathways of rat mesangial cells in 3-di-mensional cultures: Response to fetal bovine serum and plate-let-derived growth factor-b. Exp. Cell Res. 240, 134–143.

2. Janknecht, R., and Hunter, T. (1997). Convergence of MAPkinase pathways on the ternary complex factor Sap-1a. EMBO

J. 16, 1620–1627.

3. Strahl, T., Gille, H., and Shaw, P. E. (1996). Selective responseof ternary complex factor Sap1a to different mitogen-activatedprotein kinase subgroups. Proc. Natl. Acad. Sci. USA 93,11563–11568.

4. Hazzalin, C. A., Cano, E., Cuenda, A., Barratt, M. J., Cohan, P.,and Mahadevan, L. C. (1996). p38/Rk is essential for stress-induced nuclear responses: Jnk/Sapks and c-Jun/ATF-2 phos-phorylation are insufficient. Curr. Biol. 6, 1028–1031.

5. Hazzalin, C. A., Cuenda, A., Cano, E., Cohen, P., and Ma-hadevan, L. C. (1997). Effects of the inhibition of p38/RK MAPkinase on induction of five fos and jun genes by diverse stimuli.Oncogene 15, 2321–2331.

56. Whitmarsh, A. J., Yang, S. H., Su, M. S., Sharrocks, A. D., andDavis, R. J. (1997). Role of p38 and JNK mitogen-activatedprotein kinases in the activation of ternary complex factors.Mol. Cell. Biol. 17, 2360–2371.

57. Dieckgraefe, B. K., Weems, D. M., Santoro, S. A., and Alpers,D. H. (1997). ERK and p38 MAP kinase pathways are media-tors of intestinal epithelial wound-induced signal transduction.Biochem. Biophys. Res. Commun. 233, 389–394.

58. Goke, M., Kanai, M., Lynch-Devaney, K., and Podolsky, D. K.(1998). Rapid mitogen-activated protein kinase activation bytransforming growth factor alpha in wounded rat intestinalepithelial cells. Gastroenterology 114, 697–705.

59. Seo, S., Okamoto, M., Seto, H., Ishizuka, K., Sano, H., andOhashi, Y. (1995). Tobacco MAP kinase: A possible mediator inwound signal transduction pathways. Science 270, 1988–1992.

60. Aronson, D., Wojtaszewski, J. F., Thorell, A., Nygren, J., Zan-gen, D., Richter, E. A., Ljungqvist, O., Fielding, R. A., andGoodyear, L. J. (1998). Extracellular-regulated protein kinasecascades are activated in response to injury in human skeletal

muscle. Am. J. Physiol. 275, C555–C561.

Received December 9, 1999Revised version received February 14, 2000