Epidermal growth factor induces fibroblast contractility and motility via a protein kinase C delta-dependent pathway.
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, PKCdelta is the most strongly phosphorylated isoform, and the preferential PKCdelta inhibitor rottlerin largely prevented EGF-induced phosphorylation of PKC substrates and MARCKS. The pathway through which EGFR activates PKCdelta is suggested by the fact that the MEK-1 inhibitor U0126 and the phosphatidylinositol 3-kinase inhibitor LY294002 had no effect on PKCdelta activation, whereas lack of PLCgamma signaling resulted in delayed PKCdelta 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 PKCdelta is required, a dominant-negative PKCdelta construct or RNAi-mediated PKCdelta depletion also prevented MLC phosphorylation. In the absence of PLC signaling, MLC phosphorylation and cell force generation were delayed similarly to PKCdelta activation. All of the interventions that blocked PKCdelta activation or MLC phosphorylation abrogated EGF-induced cell contractile force generation and motility. Our results suggest that PKCdelta 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 PKCdelta to promote the acute phase of EGF-induced MLC activation.
Iwabu
Akihiro
A
Department of Pathology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.
Smith
Kirsty
K
Allen
Fred D
FD
Lauffenburger
Douglas A
DA
Wells
Alan
A
D000098:Acetophenones / Q000737:chemistry / Q000494:pharmacology; D000470:Alkaloids; D000818:Animals; D053119:Benzophenanthridines; D001578:Benzopyrans / Q000737:chemistry / Q000494:pharmacology; D002070:Butadienes / Q000494:pharmacology; D002460:Cell Line; D002465:Cell Movement; D002867:Chromones / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000378:metabolism / Q000502:physiology*; D066246:ErbB Receptors / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism*; D005799:Genes, Dominant; D005822:Genetic Vectors; D015151:Immunoblotting; D007537:Isometric Contraction; D051379:Mice; D009025:Morpholines / Q000494:pharmacology; D018994:Myosin Light Chains / Q000737:chemistry / Q000378:metabolism; D009570:Nitriles / Q000494:pharmacology; D010617:Phenanthridines / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000737:chemistry / Q000378:metabolism*; D051745:Protein Kinase C-delta; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection
D000098:Acetophenones; D000470:Alkaloids; D053119:Benzophenanthridines; D001578:Benzopyrans; D002070:Butadienes; D002867:Chromones; D004791:Enzyme Inhibitors; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D009025:Morpholines; D018994:Myosin Light Chains; D009570:Nitriles; D010617:Phenanthridines; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D020033:Protein Isoforms; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; D034741:RNA, Small Interfering; C113580:U 0126; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C085746:rottlerin; C016299:chelerythrine; C497259:Prkcd protein, mouse