RhoA/ROCK signaling regulates Sox9 expression and actin organization during chondrogenesis.
Endochondral ossification is initiated by the differentiation of mesenchymal precursor cells to chondrocytes (chondrogenesis). This process is characterized by a strong interdependence of cell shape, cytoskeletal organization, and the onset of chondrogenic gene expression, but the molecular mechanisms mediating these interactions are not known. Here we investigated the role of the RhoA/ROCK pathway, a well characterized regulator of cytoskeletal organization, in chondrogenesis. We show that pharmacological inhibition of ROCK signaling by Y27632 resulted in increased glycosaminoglycan synthesis and elevated expression of the chondrogenic transcription factor Sox9, whereas overexpression of RhoA in the chondrogenic cell line ATDC5 had the opposite effects. Suppression of Sox9 expression by ROCK signaling was achieved through repression of Sox9 promoter activity. These molecular changes were accompanied by reorganization of the actin cytoskeleton, where RhoA/ROCK signaling suppressed cortical actin organization, a hallmark of differentiated chondrocytes. This led us to analyze the regulation of Sox9 expression by drugs affecting cytoskeletal dynamics. Both inhibition of actin polymerization by cytochalasin D and stabilization of existing actin filaments by jasplakinolide resulted in increased Sox9 mRNA levels, whereas inhibition of microtubule polymerization by colchicine completely blocked Sox9 expression. In conclusion, our data suggest that RhoA/ROCK signaling suppresses chondrogenesis through the control of Sox9 expression and actin organization.
Woods
Anita
A
Canadian Institutes of Health Research Group in Skeletal Development and Remodeling, Department of Physiology and Pharmacology, University of Western Ontario, London, Ontario N6A 5C1, Canada.
Wang
Guoyan
G
Beier
Frank
F
D000199:Actins / Q000378:metabolism*; D000818:Animals; D002478:Cells, Cultured; D020219:Chondrogenesis / Q000502:physiology*; D005786:Gene Expression Regulation; D006025:Glycosaminoglycans / Q000096:biosynthesis; D006609:High Mobility Group Proteins / Q000235:genetics*; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D012333:RNA, Messenger / Q000032:analysis; D055758:SOX9 Transcription Factor; D026362:Serum Response Factor / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology*; D014157:Transcription Factors / Q000235:genetics*; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein / Q000502:physiology*
D000199:Actins; D006025:Glycosaminoglycans; D006609:High Mobility Group Proteins; D047908:Intracellular Signaling Peptides and Proteins; D012333:RNA, Messenger; D055758:SOX9 Transcription Factor; D026362:Serum Response Factor; C527933:Sox9 protein, mouse; D014157:Transcription Factors; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein