title: Differing contributions of LIMK and ROCK to TGFβ-induced transcription, motility and invasion.
abstract: The ability of transforming growth factor β (TGFβ) to induce epithelial-mesenchymal transition (EMT) is mediated by SMAD-dependent and SMAD-independent pathways such as the activation of Rho GTPase signalling. Upon activation, GTP-bound Rho stimulates the ROCK kinases, which in turn phosphorylate numerous substrates including the LIM kinases (LIMK). The net result of ROCK activation is increased actin-myosin contractile force generation, with a contribution from LIMK-induced actin filament stabilisation. In this study, we made use of siRNA-mediated knockdown and selective inhibitors to determine the contributions of ROCK and LIMK to TGFβ-induced responses. We find that both ROCK and LIMK are required for TGFβ stimulation of serum-response factor (SRF) transcriptional activity and actin stress fibre formation during EMT. In contrast, although LIMK inhibition had little effect on cell motility in scratch wound and Transwell migration assays, ROCK inhibition actually promoted TGFβ-induced cell motility by helping individual cells to break free from the epithelial sheet. Furthermore, we demonstrate that selective inhibition of LIMK, but not ROCK, effectively blocked TGFβ driven invasion through a layer of matrigel extracellular matrix protein. These results indicate that the roles of LIMK and ROCK in the Rho signalling pathway downstream of TGFβ are not identical and suggest that LIMK represents an attractive therapeutic target in TGFβ driven organ fibrosis and metastatic cancer spread.
authors.affiliation: Beatson Institute for Cancer Research, Garscube Estate, Switchback Road, Glasgow G61 1BD, UK.
authors.forename: Pierre
authors.initials: P
authors.lastname: Morin
authors.forename: Grant
authors.initials: G
authors.lastname: Wickman
authors.forename: June
authors.initials: J
authors.lastname: Munro
authors.forename: Gareth J
authors.initials: GJ
authors.lastname: Inman
authors.forename: Michael F
authors.initials: MF
authors.lastname: Olson
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002465:Cell Movement; D058750:Epithelial-Mesenchymal Transition / Q000502:physiology; D016326:Extracellular Matrix Proteins / Q000502:physiology; D005455:Fluorescent Antibody Technique; D055785:Gene Knockdown Techniques; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D054461:Lim Kinases / Q000378:metabolism*; D051379:Mice; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D034741:RNA, Small Interfering; D015398:Signal Transduction; D014158:Transcription, Genetic; D016212:Transforming Growth Factor beta / Q000378:metabolism* / Q000494:pharmacology; D050808:ets-Domain Protein Elk-4 / Q000235:genetics / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000378:metabolism; D054460:rho-Associated Kinases / Q000378:metabolism*
chemical_list: D000199:Actins; D016326:Extracellular Matrix Proteins; D047908:Intracellular Signaling Peptides and Proteins; D034741:RNA, Small Interfering; D016212:Transforming Growth Factor beta; D050808:ets-Domain Protein Elk-4; D054461:Lim Kinases; D054460:rho-Associated Kinases; D020741:rho GTP-Binding Proteins