title: Regulation of cellular contractile force in response to mechanical stretch by diphosphorylation of myosin regulatory light chain via RhoA signaling cascade.
abstract: Fibroblasts regulate their contractile force in response to external stretch; however, the detailed mechanism by which the force is regulated is unclear. Here, we show that diphosphorylation and dephosphorylation of myosin regulatory light chain (MRLC) are involved in the stretch-induced force response. Cellular stiffness, which reflects the cellular contractile force, under external stretch was measured by mechanical-scanning probe microscopy. Fibroblasts (NIH-3T3) expressing green fluorescent protein (GFP)-tagged mutant-type MRLC (MRLC(T18A)-GFP), which cannot be diphosphorylated, did not show any stretch-induced stiffness response, whereas the stiffness in cells expressing GFP-tagged wild-type MRLC (MRLC(WT)-GFP) increased immediately after the stretch and subsequently decreased after 1-2 h. Urea-PAGE western blot analysis showed that the proportion of diphosphorylated MRLC (PP-MRLC) transiently increased after the stretch and decreased after 1-2 h. Dominant-negative RhoA (RhoA(N19))-expressing cells did not show the stiffness response to the stretch, whereas wild-type RhoA-expressing cells did. It was concluded that the cellular force response originates in the stretch-induced diphosphorylation and dephosphorylation of MRLC and is regulated via the RhoA signaling cascade.
authors.affiliation: Division of Biological Sciences, Graduate School of Science, Hokkaido University, Kita-ku, Sapporo, Japan. mizutani@sci.hokudai.ac.jp
authors.forename: Takeomi
authors.initials: T
authors.lastname: Mizutani
authors.forename: Kazushige
authors.initials: K
authors.lastname: Kawabata
authors.forename: Yoshikazu
authors.initials: Y
authors.lastname: Koyama
authors.forename: Masayuki
authors.initials: M
authors.lastname: Takahashi
authors.forename: Hisashi
authors.initials: H
authors.lastname: Haga
mesh_terms: D000818:Animals; D002460:Cell Line; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology; D051379:Mice; D009119:Muscle Contraction / Q000502:physiology*; D050505:Mutant Proteins / Q000378:metabolism*; D018994:Myosin Light Chains / Q000235:genetics / Q000378:metabolism*; D041681:NIH 3T3 Cells; D010766:Phosphorylation / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology; D013314:Stress, Mechanical; D020741:rho GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein
chemical_list: D050505:Mutant Proteins; D018994:Myosin Light Chains; C526380:RhoA protein, mouse; D020741:rho GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein