Epidermal Growth Factor Induces Fibroblast Contractility and Motility via a Protein Kinase C δ-dependent Pathway
2004; Elsevier BV; Volume: 279; Issue: 15 Linguagem: Inglês
10.1074/jbc.m311981200
ISSN1083-351X
AutoresAkihiro Iwabu, Kirsty D. Smith, Fred Allen, Douglas A. Lauffenburger, Alan Wells,
Tópico(s)Cell Adhesion Molecules Research
ResumoMyosin-based cell contractile force is considered to be a critical process in cell motility. However, for epidermal growth factor (EGF)-induced fibroblast migration, molecular links between EGF receptor (EGFR) activation and force generation have not been clarified. Herein, we demonstrate that EGF stimulation increases myosin light chain (MLC) phosphorylation, a marker for contractile force, concomitant with protein kinase C (PKC) activity in mouse fibroblasts expressing human EGFR constructs. Interestingly, PKCδ is the most strongly phosphorylated isoform, and the preferential PKCδ inhibitor rottlerin largely prevented EGF-induced phosphorylation of PKC substrates and MARCKS. The pathway through which EGFR activates PKCδ is suggested by the fact that the MEK-1 inhibitor U0126 and the phosphatidylinositol 3-kinase inhibitor LY294002 had no effect on PKCδ activation, whereas lack of PLCγ signaling resulted in delayed PKCδ activation. EGF-enhanced MLC phosphorylation was prevented by a specific MLC kinase inhibitor ML-7 and the PKC inhibitors chelerythrine chloride and rottlerin. Further indicating that PKCδ is required, a dominant-negative PKCδ construct or RNAi-mediated PKCδ depletion also prevented MLC phosphorylation. In the absence of PLC signaling, MLC phosphorylation and cell force generation were delayed similarly to PKCδ activation. All of the interventions that blocked PKCδ activation or MLC phosphorylation abrogated EGF-induced cell contractile force generation and motility. Our results suggest that PKCδ activation is responsible for a major part of EGF-induced fibroblast contractile force generation. Hence, we identify here a new pathway helping to govern cell motility, with PLC signaling playing a role in activation of PKCδ to promote the acute phase of EGF-induced MLC activation. Myosin-based cell contractile force is considered to be a critical process in cell motility. However, for epidermal growth factor (EGF)-induced fibroblast migration, molecular links between EGF receptor (EGFR) activation and force generation have not been clarified. Herein, we demonstrate that EGF stimulation increases myosin light chain (MLC) phosphorylation, a marker for contractile force, concomitant with protein kinase C (PKC) activity in mouse fibroblasts expressing human EGFR constructs. Interestingly, PKCδ is the most strongly phosphorylated isoform, and the preferential PKCδ inhibitor rottlerin largely prevented EGF-induced phosphorylation of PKC substrates and MARCKS. The pathway through which EGFR activates PKCδ is suggested by the fact that the MEK-1 inhibitor U0126 and the phosphatidylinositol 3-kinase inhibitor LY294002 had no effect on PKCδ activation, whereas lack of PLCγ signaling resulted in delayed PKCδ activation. EGF-enhanced MLC phosphorylation was prevented by a specific MLC kinase inhibitor ML-7 and the PKC inhibitors chelerythrine chloride and rottlerin. Further indicating that PKCδ is required, a dominant-negative PKCδ construct or RNAi-mediated PKCδ depletion also prevented MLC phosphorylation. In the absence of PLC signaling, MLC phosphorylation and cell force generation were delayed similarly to PKCδ activation. All of the interventions that blocked PKCδ activation or MLC phosphorylation abrogated EGF-induced cell contractile force generation and motility. Our results suggest that PKCδ activation is responsible for a major part of EGF-induced fibroblast contractile force generation. Hence, we identify here a new pathway helping to govern cell motility, with PLC signaling playing a role in activation of PKCδ to promote the acute phase of EGF-induced MLC activation. Cell motility induced by activation of epidermal growth factor receptor (EGFR), 1The abbreviations used are: EGFR, epidermal growth factor receptor; AP, alkaline phosphatase; CMV, cytomegalovirus; DAG, diacylglycerol; DN, dominant-negative; EGF, epidermal growth factor; ERK, extracellular signal-related kinase; FPCL, fibroblast populated collagen lattices; MARCKS, myristoylated alanine-rich protein kinase C substrate; MEK, mitogen-activated protein kinase kinase; MEM, minimum essential medium; MLC, myosin light chain; MLCK, myosin light chain kinase; MMTVp, mouse mammary tumor virus promoter; PDK1, 3-phosphoinositide-dependent protein kinase-1; PKC, protein kinase C; PLC, phospholipase C; PI, phosphatidylinositol; PI3K, PI 3-kinase; WT, wild type. and related receptor tyrosine kinases, can be deconstructed into a series of orchestrated events: lamellipodial extension, formation of forward adhesions, exertion of contractile forces to pull the cell body forward, and detachment of the rear (1Lauffenburger D.A. Horwitz A.F. Cell. 1996; 84: 359-369Abstract Full Text Full Text PDF PubMed Scopus (3312) Google Scholar). While each process is required for net cell locomotion, it is not necessarily the case that signals downstream of receptor activation must concomitantly be involved in triggering all of the processes. Despite longstanding anecdotal indications, only recently have formal demonstrations emerged that signaling via EGFR actually elicits cell contractile force generation (2Allen F.D. Asnes C.F. Chang P. Elson E.L. Lauffenburger D.A. Wells A. Wound Repair Regen. 2002; 10: 67-761Crossref PubMed Scopus (34) Google Scholar, 3Haase I. Evans R. Pofahl R. Watt F.M. J. Cell Sci. 2003; 116: 3227-3238Crossref PubMed Scopus (189) Google Scholar) along with the other biophysical processes (4Glading A. Lauffenburger D.A. Wells A. Trends Cell Biol. 2002; 12: 46-54Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar, 5Ware M.F. Wells A. Lauffenburger D.A. J. Cell Sci. 1998; 111: 2423-2432PubMed Google Scholar, 6Wells A. Gupta K. Chang P. Swindle S. Glading A. Shiraha H. Micros. Res. Tech. 1998; 43: 395-411Crossref PubMed Scopus (89) Google Scholar). Because of the central importance of growth factor-induced cell motility in physiological and pathological applications, such as organogenesis, wound repair, and tumor invasion, determination of key pathways involved in connecting EGFR activity to contractile force generation, as well as the other processes underlying motility, is a crucial undertaking. Myosin motors operating on cytoskeletal actin filaments are presumed to be involved in growth factor-induced cell motility in manner similar to the roles they play in integrin-mediated migration (7Mitchison T.J. Cramer L.P. Cell. 1996; 84: 371-379Abstract Full Text Full Text PDF PubMed Scopus (1315) Google Scholar). Myosin II is a prominent actor in this context. Myosin II is localized along with actin fibers in the protrusive anterior region and at posterior regions of motile cells, where it is thought to generate contractility, in organizing and breaking cell-substratum adhesion, and/or in reorganizing the actin cytoskeleton (8Conrad A. Jaffredo T. Conrad G. Cell Motil. Cytoskeleton. 1995; 31: 93-112Crossref PubMed Scopus (29) Google Scholar, 9Gough A. Taylor D.L. J. Cell Biol. 1993; 121: 1095-1107Crossref PubMed Scopus (79) Google Scholar). A recent report finds EGF to induce myosin II heavy chain phosphorylation (at least indirectly), with implications for subcellular localization of the active motor and consequent chemotactic cell movement (10Ben-Ya'acov A. Ravid S. J. Biol. Chem. 2003; 278: 40032-40040Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Phosphorylation at a regulatory serine 19 of the 20-kDa myosin light chain (MLC) subunit of myosin II promotes cell contractility in a variety of cell types responding to diverse stimuli. In addition, serine 19-phosphorylated MLC is enriched near membrane protrusion and retraction areas in motile fibroblast (11Matsumura F. Ono S. Yamakita Y. Totsukawa G. Yamashiro S. J. Cell Biol. 1998; 140: 119-129Crossref PubMed Scopus (194) Google Scholar). The extent of MLC phosphorylation is regulated not only by protein kinases, such as Ca2+/calmodulin-dependent MLC kinase (MLCK) and Ca2+/calmodulin-independent Rho-kinase, but also by myosin phosphatase (MLCP) (12Hartshorne D. Ito M. Erdodi F. J. Muscle Res. Cell Motil. 1998; 19: 325-341Crossref PubMed Scopus (347) Google Scholar, 13Kamm K. Stull J. J. Biol. Chem. 2001; 276: 4527-4530Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar, 14Somlyo A.P. Somlyo A.V. J. Physiol. 2000; 522: 177-185Crossref PubMed Scopus (1085) Google Scholar). During haptotactic migration, mitogen-activated protein (MAP) kinase ERK has been shown to mediate MLC phosphorylation through MLCK (15Klemke R.L. Cai S. Giannini A. Gallagher P. deLanerolle P. Cheresh D.A. J. Cell Biol. 1997; 137: 481-492Crossref PubMed Scopus (1107) Google Scholar). Thus, the final myosin II contractile force generation machinery is engaged during both chemotactic and haptotactic motility. However, key signaling pathways through which EGFR actuates myosin-based contractility have yet to be identified. Possible links between EGFR and myosin-based contractility are suggested by a few prior reports. Although ERK activity is required for MLC phosphorylation in haptotactic cell migration (15Klemke R.L. Cai S. Giannini A. Gallagher P. deLanerolle P. Cheresh D.A. J. Cell Biol. 1997; 137: 481-492Crossref PubMed Scopus (1107) Google Scholar), ERK activity promotes the extension of membrane protrusions rather than retraction (16Brahmbhatt A. Klemke R.L. J. Biol. Chem. 2003; 278: 13016-13025Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). A fair inference from these findings is that transcellular contractility is not solely driven by an ERK "master switch." EGF-induced myosin heavy chain phosphorylation and localization requires a protein kinase C (PKC) intermediary (17Straussman S. Even L. Ravid. S. J. Cell Sci. 2001; 114: 3047-3057Crossref PubMed Google Scholar). The PKC family of molecules is an attractive candidate for connecting EGFR-elicited signals to myosin-mediated force generation, which is implicated in the contraction of muscle and non-muscle cells (18Bresnick A. Curr. Opin. Cell Biol. 1999; 11: 26-33Crossref PubMed Scopus (316) Google Scholar). PKC consists of a family of at least 11 isoforms. Specific isoforms of PKC are activated by phospholipids, diacylglycerol (DAG) generated by phospholipase C (PLC) or phospholipase D (PLD) from phosphatidylinositol 4,5-bisphosphate (PIP2), fatty acids generated by PLA2, and calcium, depending on isoforms. Based on their structural and biochemical properties these PKC isoforms can be divided into three major groups: (i) the classical PKC (cPKC; α, βI, βII, and γ), which are activated by DAG and are Ca2+-dependent; (ii) the novel PKC (nPKC; δ, ϵ, θ, η, and μ), which are activated by DAG but Ca2+-independent, and (iii) the atypical PKC (aPKC; ζ and λ), which do not respond to either DAG or calcium. Importantly, EGFR triggers PKC activity (19Welsh J. Gill G.N. Rosenfeld M.G. Wells A. J. Cell Biol. 1991; 114: 533-543Crossref PubMed Scopus (75) Google Scholar) at least in part downstream of phospholipase signaling (20Chen P. Xie H. Wells A. Mol. Biol. Cell. 1996; 7: 871-881Crossref PubMed Scopus (100) Google Scholar). These data support the hypothesis that one or more PKC isoform, in the classical or novel groups, contributes to EGFR-mediated cell contractility during motility. A picture of how PKC isoforms are regulated, in addition to final activation by lipid-containing molecules, has recently emerged in which direct phosphorylations play a major role (21Newton A.C. Biochem. J. 2003; 370: 361-371Crossref PubMed Scopus (669) Google Scholar, 22Kikkawa U. Matsuzaki H. Yamamoto T. J. Biochem. 2002; 132: 831-839Crossref PubMed Scopus (200) Google Scholar). cPKC and nPKC isoforms contain three conserved serine/threonine phosphorylation motifs of serine or threonine residues in the catalytic domain; a threonine in the activation loop (Thr-505 in PKCδ) and serines in the hydrophobic (Ser-643 in PKCδ) and C-terminal (Ser-662 in PKCδ) regions (21Newton A.C. Biochem. J. 2003; 370: 361-371Crossref PubMed Scopus (669) Google Scholar). An upstream kinase, 3-phosphoinositide-dependent protein kinase-1 (PDK1), phosphorylates the activation loop (23Le Good J.A. Ziegler W.H. Parekh D.B. Alessi D.R. Cohen P. Parker P.J. Science. 1998; 281: 2042-2045Crossref PubMed Scopus (977) Google Scholar, 24Balendran A. Hare G.R. Kieloch A. Williams M.R. Alessi D.R. FEBS Lett. 2000; 484: 217-223Crossref PubMed Scopus (182) Google Scholar), which is necessary for the catalytic activity of cPKC isoforms (25Dutil E.M. Toker A. Newton A.C. Curr. Biol. 1998; 8: 1366-1375Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar, 26Bornancin F. Parker P.J. Curr. Biol. 1996; 6: 1114-1123Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). The turn motif and hydrophobic sites then undergo autophosphorylation. The regulation of these sites in nPKC isoforms may mirror those of cPKC isoforms (27Cenni V. Doppler H. Sonnenburg E.D. Maraldi N. Newton A.C. Toker A. Biochem. J. 2002; 363: 537-545Crossref PubMed Scopus (143) Google Scholar), though the existence of a heterologous upstream kinase has been inferred (28Ziegler W.H. Parekh D.B. Le Good J.A. Whelan R.D. Kelly J.J. Frech M. Hemmings B.A. Parker P.J. Curr. Biol. 1999; 9: 522-529Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). These sequential phosphorylations render cPKC isoforms stable and ready for activation by DAG (29Parekh D.B. Ziegler W. Parker P.J. EMBO J. 2000; 19: 496-503Crossref PubMed Scopus (514) Google Scholar). However, PKCδ differs from cPKC in its regulation by phosphorylation (22Kikkawa U. Matsuzaki H. Yamamoto T. J. Biochem. 2002; 132: 831-839Crossref PubMed Scopus (200) Google Scholar). A study suggests that phosphorylation of the activation loop Thr-505 is not essential for subsequent activation; the PKCδ-specific acidic Glu-500 may assume at least some of the role of threonine phosphorylation (30Stempka L. Schnolzer M. Radke S. Rincke G. Marks F. Gschwendt M. J. Biol. Chem. 1999; 274: 8886-8892Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). On the other hand, phosphorylation at Thr-505 by PDK1 is thought to be required for the stability of the enzyme (24Balendran A. Hare G.R. Kieloch A. Williams M.R. Alessi D.R. FEBS Lett. 2000; 484: 217-223Crossref PubMed Scopus (182) Google Scholar). The functional consequence of phosphorylation of other sites is also not settled, Li et al. (31Li W. Zhang J. Bottaro D.P. Li W. Pierce J.H. J. Biol. Chem. 1997; 272: 24550-24555Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) demonstrated that the mutation of Ser-643 markedly decreases PKCδ activity but Stempka et al. (30Stempka L. Schnolzer M. Radke S. Rincke G. Marks F. Gschwendt M. J. Biol. Chem. 1999; 274: 8886-8892Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) showed that the same mutation has no effect on PKCδ catalytic function. Interestingly, the upstream PDK1 appears itself dependent on phospholipids, PIP3 in particular, for activation (32Alessi D.R. James S.R. Downes C.P. Holmes A.B. Gaffney P.R. Reese C.B. Cohen P. Curr. Biol. 1997; 7: 261-269Abstract Full Text Full Text PDF PubMed Google Scholar). In the present model, PI 3-kinase generates PIP3 after activation by receptors with tyrosine kinase, such as EGFR. These investigations, even with the uncertainties, point to many PKC isoforms as potential downstream effectors of growth factor receptor signaling. In this present contribution, we show that EGFR signaling leads to MLC phosphorylation at serine 19 and consequently to cell contractile force generation and motility in fibroblasts. The pathway to myosin activation required activity of the novel PKCδ isoform. Moreover, we find that there appear to be two distinct phases of PKCδ activation, with PLCγ signaling required only for the acute phase. We have thus identified a novel pathway connection downstream of EGFR activation to one of the crucial biophysical processes underlying cell migration. Reagents and Antibodies—Rottlerin, selective PKCδ inhibitor (33Gschwendt M. Muller H. Kielbassa K. Zang R. Kittstein W. Rincke G. Marks F. Biochem. Biophys. Res. Commun. 1994; 199: 93-98Crossref PubMed Scopus (766) Google Scholar), Gö 6976, PKCα/β selective inhibitor, U73122, PLC inhibitor, U73343, inactive analogue of U73122, LY294002, PI 3-kinase inhibitor, and ML-7, MLCK inhibitor were obtained from Biomol (Plymouth Meeting, PA). PD153035, EGFR tyrosine kinase inhibitor, U0126, mitogen-activated protein kinase kinase-1 (MEK-1) inhibitor, and chelerythrine chloride, pan-PKC inhibitor were obtained from Calbiochem (La Jolla, CA). Human recombinant EGF was obtained from BD Biosciences (San Jose, CA). Type I collagen was from Upstate Biotechnology (Lake Placid, NY). The rabbit polyclonal antibodies against phosphorylated PKC (pan), phosphorylated (Ser) PKC substrate, phosphorylated MARCKS (myristoylated alanine-rich protein C kinase substrate) (Ser-152/156), phosphorylated PKCδ (Ser-643), phospho-ERK (p44/42), phospho-Akt (Ser-473; 587F11), and phosphorylated MLC (Ser-19) were obtained from Cell Signaling Technology (Beverly, MA). The rabbit polyclonal antibody against MLC (FL-172), nPKCδ (C-20), and cPKCα (C-20) were from Santa Cruz Biotechnology. The rabbit polyclonal antibody against α-actin was from Sigma-Aldrich. All cell culture reagents were obtained from CellGro (Herndon, VA) or Invitrogen. Cell Culture—The establishment and maintenance of the NR6 WT, expressing full-length wild-type EGFR, or NR6 c′973, expressing signaling-restricted EGFR lacking all autophosphorylation motifs and fails to activate PLCγ, cell lines have been described (19Welsh J. Gill G.N. Rosenfeld M.G. Wells A. J. Cell Biol. 1991; 114: 533-543Crossref PubMed Scopus (75) Google Scholar, 34Chen P. Xie H. Sekar M.C. Gupta K. Wells A. J. Cell Biol. 1994; 127: 847-857Crossref PubMed Scopus (287) Google Scholar, 35Wells A. Welsh J. Lazar C. Wiley H.S. Gill G.N. Rosenfeld M.G. Science. 1990; 247: 962-996Crossref PubMed Scopus (343) Google Scholar). Briefly, these constructs were retrovirally transduced in NR6 cells, Swiss 3T3-derived fibroblasts, which lack endogenous EGFRs (35Wells A. Welsh J. Lazar C. Wiley H.S. Gill G.N. Rosenfeld M.G. Science. 1990; 247: 962-996Crossref PubMed Scopus (343) Google Scholar). Cells were cultured in minimum essential medium (MEM)-α with 7.5% fetal calf serum plus 2 mm l-glutamine, 1 mm sodium pyruvate, 0.1 mm MEM, nonessential amino acids, and the antibiotics penicillin (100 units/ml), streptomycin (100 μg/ml), and G418 (350 μg/ml) as the growth medium. Subconfluent cells were passaged with a 1:8 split ratio at 3-day intervals using 0.25% trypsin with 0.25 mm EDTA. Cells were quiesced using restricted or no serum conditions without G418 prior to experiments. Plasmid Construction and Transfection—The dominant-negative (DN) PKCδ construct (36Murakami M. Horowitz A. Tang S. Ware J.A. Simons M. J. Biol. Chem. 2002; 277: 20367-20371Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar) was a generous gift from Dr. Michael Simons (Dartmouth Medical School). The DN construct was generated by replacing the conserved lysine 376 in the ATP binding domain with tryptophan. For stable expression, DN PKCδ cDNA was subcloned into pCEP4 (Invitrogen) hygromycin resistant vector downstream from a cytomegalovirus promoter (CMVp). CMVp was replaced with a mouse mammary tumor virus promoter (MMTVp) for inducible expression (20Chen P. Xie H. Wells A. Mol. Biol. Cell. 1996; 7: 871-881Crossref PubMed Scopus (100) Google Scholar). The empty pCEP4 vector, CMVp of which was replaced with MMTVp, was used as control. The construct was stably transfected into NR6 WT cells by electroporation. Cells were trypsinized, pelleted, and resuspended in Opti-MEM medium (Invitrogen) in electroporation cuvette, and the plasmid was added to a total of 30 μg. The cells were electroporated at 0.300 kV and 950 μF (Gene Pulser, Bio-Rad). 48 h after electroporation, cells were selected in the growth medium containing hygromycin B (Calbiochem) (100 μg/ml). Polyclonal cell lines consisting of more than 20 colonies were established. Two independent electroporations and stably transfected cell lines were established and tested. Dexamethasone (2 μm for 24 h) was used to induce MMTV-driven DN PKCδ expression at the same time with quiescence. siRNA Transfections—siRNA duplexes (siRNAs) were synthesized and purified by IDT (Coralville, IA). The siRNA sequence for targeting PKCδ (GenBank™ accession number NM_011103) was PKCδ siRNA (5′-AGUACUUGGCAAAGGCAGCTT-3′). The siRNA sequence for targeting PKCα (GenBank™ accession number NM_011101) was PKCα siRNA (5′-ACAACCUGGACAGAGUGAATT-3′). GFPsiRNA (5′-GACCCGCGCCGAGGUGAAGTT-3′) was used as a negative control (37Hirai I. Wang H.G. J. Biol. Chem. 2002; 277: 25722-25727Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Transfection of siRNAs was performed using the manufacturer's protocol for LipofectAMINE™ 2000 (Invitrogen). Briefly, 4 μlof20 μm siRNA was mixed with 200 μl of Opti-MEM. 4 μl of LipofectAMINE™ 2000 was diluted in 200 μl of Opti-MEM and incubated at room temperature for 5 min. After the incubation, the diluted LipofectAMINE™ 2000 was combined with the diluted siRNA and then incubated for an additional 20 min at room temperature. Total 400 μl of siRNA- LipofectAMINE™ 2000 complexes was applied to each well of cultured NR6 WT fibroblasts at ∼70% confluence in a 6-well plate. Immunoblotting and Immunoprecipitation—Cells were grown to confluence in 6-well tissue culture plastic plates. After 24 h of quiescence in MEMα with 0.5% dialyzed fetal calf serum, cells were treated. Cells were lysed with sodium dodecyl sulfate (SDS)-sample buffer containing 0.1 m Tris-HCl, 4% SDS, 0.2% bromphenol blue, and 5% β-mercaptoethanol. Cell lysates were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Immobilon-P; Millipore, Bedford, MA). Blots were probed by primary antibodies before visualizing with alkaline phosphatase-conjugated or horseradish peroxidase-conjugated secondary antibodies (Promega, Madison, WI) followed by development with a colorimetric method (Promega) or an enhanced chemiluminescence kit (ECL™; Amersham Biosciences). For immunoprecipitation, cells were prepared and treated as described above. Cells were lysed with radioimmune precipitation lysis buffer (50 mm Tris, pH 8.0, 150 mm NaCl, 0.1% SDS, 1% Nonidet P-40, and 0.5% deoxycholate) containing 1 mm EDTA, 2 μg/ml aprotinin, 2 μg/ml leupeptin and 1 mm phenylmethylsulfonyl fluoride. Cell lysates were cleared by centrifugation at 13,000 × g for 15 min. Soluble proteins were incubated with anti-PKCδ antibody for 4–6 h at 4 °C. Immunocomplexes were incubated with protein G-agarose and centrifuged. The pellets were washed three times with radioimmune precipitation lysis buffer containing protease inhibitors. Precipitated proteins were subjected to analyze by immunoblotting as described above. Motility Assay—EGF-induced migration was assessed by the ability of the cells to move into an acellular area (38Chen P. Gupta K. Wells A. J. Cell Biol. 1994; 124: 547-555Crossref PubMed Scopus (201) Google Scholar). Cells were plated on 6-well plastic dishes and grown to confluence in MEM-α with 7.5% fetal bovine serum. After 24 h quiescence, an area was denuded by a rubber policeman. Cells were then treated with or without EGF (10 nm) and incubated at 37 °C; inhibitors or diluent alone were added at the same time as EGF. Photographs were taken at 0 and 24 h, and the relative distance traveled by the cells was determined. Isometric Force Measurement—The preparation of three-dimensional fibroblast populated collagen lattices (FPCL) has been described previously (2Allen F.D. Asnes C.F. Chang P. Elson E.L. Lauffenburger D.A. Wells A. Wound Repair Regen. 2002; 10: 67-761Crossref PubMed Scopus (34) Google Scholar). Briefly, FPCL containing 7.5 × 105 cells were prepared with modifications of Kolodney and Elson (39Kolodney M. Elson E. J. Biol. Chem. 1993; 268: 23850-23855Abstract Full Text PDF PubMed Google Scholar). A mixture of cells and 0.75 mg/ml type I collagen was poured into the annular space of cylindrical Teflon molds (1 ml/mold) and gelled at 37 °C in 5% CO2 for 1 h. Ring-shaped FPCL gels were removed from the molds and placed in tissue culture medium for ∼40 h, maintaining the original diameter (15 mm) of the FPCL with sterile parallel spacing rods. This configuration established a tensed FPCL at the time of force measurement. Contractile force measurements of the WT NR6 and c'973 fibroblasts were performed with an isometric force transduction system. The system uses force transducers (model 52-9545, Harvard Apparatus, South Natick, MA) and mounts one FPCL per transducer vertically. The mounted tissue was placed in an organ bath containing 50 ml HEPES-buffered, serum-free Dulbecco's MEM (37 °C). Each FPCL was permitted to reach an equilibrium force over a period of 30 to 60 min prior to addition of 10 nm EGF. The contraction force response of the FPCL was monitored by computer data acquisition. The strain level of the FPCL was maintained throughout so as to measure both the initial contractile force and the persistence of contractile force as a function of time. Gel Compaction Assay—NR6 WT fibroblasts were grown in floating collagen type I matrices using a modification of described methods (40Grinnell F. Ho C. Skuta G. J. Biol. Chem. 1999; 274: 918-923Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Cells were harvested from monolayer culture using 0.25% trypsin/EDTA and resuspended in quiescent media (serum-free medium containing 1 mg/ml bovine serum albumin). Cells were incubated with inhibitors for 30 min prior to matrix preparation. Neutralized collagen solutions (1 mg/ml) containing 106 cells/ml, ± EGF (1 μg/ml) and inhibitors (or diluent alone) were dispensed into 24-well culture plates (0.5 ml solution/well). Collagen solutions were left to polymerize for 60 min at 37 °C in a humidified incubator with 5% CO2 and then each matrix was overlaid with 1 ml of quiescent medium ± EGF (1 μg/ml) and inhibitors. The matrices were gently released from the surface and sides of each well using a scalpel and incubated for 24 h. Compaction was determined by weighing the matrices after the incubation period. Data are shown as the percentage of the control matrix weight. Cell viability was assessed following compaction by removal using collagenase digestion. Matrices were washed in phosphate-buffered saline and then incubated with 0.05% trypsin for 10 min followed by collagenase (0.25 mg/ml) for a further 10 min. Fetal bovine serum (20%) was added to quench the collagenase activity and cell viability was determined by trypan blue staining. Cell viability was not significantly affected (<10%) by ML-7 at 20 mm. Statistical Analyses—All data are expressed as means ± S.E. of separate experiments. Differences between means were determined by Student's t test for unpaired samples, and those at p < 0.05 were considered significant. EGF Induces Phosphorylation of Myosin Light Chain—Exposure of fibroblasts to EGF leads to cell contractility and motility (2Allen F.D. Asnes C.F. Chang P. Elson E.L. Lauffenburger D.A. Wells A. Wound Repair Regen. 2002; 10: 67-761Crossref PubMed Scopus (34) Google Scholar, 38Chen P. Gupta K. Wells A. J. Cell Biol. 1994; 124: 547-555Crossref PubMed Scopus (201) Google Scholar); both of which are proposed to require actomyosin-based contraction (1Lauffenburger D.A. Horwitz A.F. Cell. 1996; 84: 359-369Abstract Full Text Full Text PDF PubMed Scopus (3312) Google Scholar). To verify this, we found that EGF leads to phosphorylation of MLC at the activation-specific site serine 19 (Fig. 1). This occurred rapidly in cells expressing the wild type EGFR, but dissipated by more than half over the ensuing 2 h see below). As expected, EGF-induced phosphorylation was blocked by the EGFR-selective inhibitor PD153035 and the MLCK-selective inhibitor ML-7. Similarly, EGFR-mediated compaction and motility was blunted by ML-7 (Fig. 2). While these data were anticipated, they establish directly for the first time that EGF leads to MLC phosphorylation and that motility depends on this contractile mechanism.Fig. 2EGF-induced compaction (A) and motility (B) are dependent on the MLCK. Inhibition of MLCK by ML-7 abrogates cell compaction and motility in the NR6 WT cell line. Cell motility and compaction assays were performed as described under "Experimental Procedures." MLCK selective inhibitor ML-7 (20 μm) was added at the same time as EGF (1 μg/ml). A, gel compaction values are shown as the percentage of nontreated gels. The data in the graph are the mean ± S.E. of three independent experiments, with each experiment was performed in triplicate. Statistical analysis was performed by Student's t test: *, p < 0.05; **, p < 0.01. B, cell motility was calculated as fold increase over basal traveling distance of nontreated cells. The data in the graph are the mean ± S.E. of three independent experiments, with each experiment was performed in triplicate. Statistical analysis was performed by Student's t test: **, p < 0.01.View Large Image Figure ViewerDownload Hi-res image Download (PPT) EGF Induces Activation of PKC with PKCδ as a Major Isoform Activated—To determine the key pathway(s) from EGFR to MLC, we focused on PKC. EGF has been shown to induce PKC activities (19Welsh J. Gill G.N. Rosenfeld M.G. Wells A. J. Cell Biol. 1991; 114: 533-543Crossref PubMed Scopus (75) Google Scholar). We confirmed that EGF exposure of mouse fibroblasts leads to phosphorylation of PKC targets MARCKS and other target sites (Fig. 3). Th
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