title: MRTF-A signaling regulates the acquisition of the contractile phenotype in dedifferentiated chondrocytes.
abstract: Chondrocyte culture as a monolayer for cell number expansion results in dedifferentiation whereby expanded cells acquire contractile features and increased actin polymerization status. This study determined whether the actin polymerization based signaling pathway, myocardin-related transcription factor-a (MRTF-A) is involved in regulating this contractile phenotype. Serial passaging of chondrocytes in monolayer culture to passage 2 resulted in increased gene and protein expression of the contractile molecules alpha-smooth muscle actin, transgelin and vinculin compared to non-passaged, primary cells. This resulted in a functional change as passaged 2, but not primary, chondrocytes were capable of contracting type I collagen gels in a stress-relaxed contraction assay. These changes were associated with increased actin polymerization and MRTF-A nuclear localization. The involvement of actin was demonstrated by latrunculin B depolymerization of actin which reversed these changes. Alternatively cytochalasin D which activates MRTF-A increased gene and protein expression of α-smooth muscle actin, transgelin and vinculin, whereas CCG1423 which deactivates MRTF-A decreased these molecules. The involvement of MRTF-A signaling was confirmed by gene silencing of MRTF or its co-factor serum response factor. Knockdown experiments revealed downregulation of α-smooth muscle actin and transgelin gene and protein expression, and inhibition of gel contraction. These findings demonstrate that passaged chondrocytes acquire a contractile phenotype and that this change is modulated by the actin-MRTF-A-serum response factor signaling pathway.
authors.affiliation: BioEngineering of Skeletal Tissues Team, Mount Sinai Hospital, University of Toronto, Toronto, Canada; Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Canada; Department of Laboratory Medicine and Pathobiology, University of Toronto, Canada.
authors.forename: Justin
authors.initials: J
authors.lastname: Parreno
authors.affiliation: BioEngineering of Skeletal Tissues Team, Mount Sinai Hospital, University of Toronto, Toronto, Canada; Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Canada.
authors.forename: Sneha
authors.initials: S
authors.lastname: Raju
authors.affiliation: BioEngineering of Skeletal Tissues Team, Mount Sinai Hospital, University of Toronto, Toronto, Canada; Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Canada.
authors.forename: Po-Han
authors.initials: PH
authors.lastname: Wu
authors.affiliation: BioEngineering of Skeletal Tissues Team, Mount Sinai Hospital, University of Toronto, Toronto, Canada; Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Canada; Department of Laboratory Medicine and Pathobiology, University of Toronto, Canada; Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Canada. Electronic address: rita.kandel@sinaihealthsystem.ca.
authors.forename: Rita A
authors.initials: RA
authors.lastname: Kandel
mesh_terms: D000199:Actins / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D054337:Cell Dedifferentiation; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology* / Q000378:metabolism; D008840:Microfilament Proteins / Q000378:metabolism*; D009124:Muscle Proteins / Q000378:metabolism*; D010641:Phenotype; D015398:Signal Transduction; D015534:Trans-Activators / Q000378:metabolism*; D016596:Vinculin / Q000378:metabolism*
chemical_list: D000199:Actins; D008840:Microfilament Proteins; D009124:Muscle Proteins; D015534:Trans-Activators; C051615:transgelin; D016596:Vinculin
keywords: Actin; Cartilage; Chondrocyte; Contraction; Dedifferentiation; MRTF