Multiple signaling pathways mediate compaction of collagen matrices by EGF-stimulated fibroblasts.
Fibroblasts stimulated by EGF within collagen matrices generate contraction forces that are likely of importance to cell migration and matrix compaction during wound healing. We have employed an in vitro fibroblast-embedded collagen matrix compaction assay to ascertain signaling pathway components downstream of EGFR activation leading to generation and transmission of contractile force. EGF compacts this floating collagen matrix to a similar extent as PDGF. We demonstrate that compaction requires EGFR kinase activity, yet is maximal in magnitude at intermediate EGF concentrations. This suggests that transmission of EGFR-induced contractile force to the matrix can be mitigated by consequent anti-adhesive effects of EGFR signaling in a dose-dependent manner. Treatment with pharmacological inhibitors demonstrated involvement of the signaling components extracellular signal-regulated kinase (ERK), Rho kinase, and myosin light chain kinase (MLCK) in the force generation and/or transmission process. Moreover, treatment with the pan-calpain inhibitor ALLN and isoform-specific downregulation of m-calpain (CAPN2) using RNA interference determined m-calpain to be a key component of the EGF-induced force response. ALLN treatment modulated the compaction response in a biphasic manner, enhancing matrix deformation to the greatest extent at intermediate concentrations. Our findings have thus identified key signals downstream of EGFR, which integrate in a complex manner to generate and transmit contractile forces to yield matrix deformation.
Smith
Kirsty D
KD
Biological Engineering Division, 56-341 MIT, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
Wells
Alan
A
Lauffenburger
Douglas A
DA
D000818:Animals; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D005109:Extracellular Matrix / Q000187:drug effects / Q000378:metabolism; D005347:Fibroblasts / Q000187:drug effects* / Q000648:ultrastructure; D006023:Glycoproteins / Q000494:pharmacology; D006801:Humans; D019012:Integrin beta1 / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D007976:Leupeptins / Q000494:pharmacology; D051379:Mice; D048052:Mitogen-Activated Protein Kinase 3 / Q000494:pharmacology; D010455:Peptides / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D054460:rho-Associated Kinases
D006023:Glycoproteins; D019012:Integrin beta1; D047908:Intracellular Signaling Peptides and Proteins; D007976:Leupeptins; D010455:Peptides; D034741:RNA, Small Interfering; C039304:calpain inhibitors; C063261:acetylleucyl-leucyl-norleucinal; C065738:MLCK peptide; D004815:Epidermal Growth Factor; D003094:Collagen; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D048052:Mitogen-Activated Protein Kinase 3