RhoA/ROCK Signaling Regulates Sox9 Expression and Actin Organization during Chondrogenesis

Abstract

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.

The role of mechanical signals in regulating chondrogenesis and osteogenesis of mesenchymal stem cells

“…Similar to previous findings (Woods et al, 2005), the authors of this study demonstrated that inhibiting tension within the actin cytoskeleton increased SOX9 expression. In addition, flow-induced SOX9 upregulation was abrogated by ROCKII inhibition, myosin II inhibition, actin polymerisation inhibition or actin stabilization, suggesting an intact and dynamic cytoskeleton is necessary for flow-induced chondrogenesis of MSCs .…”

“…For such reasons, in vitro models, as described in the proceeding sections, play a key role in improving our understanding of MSC mechanobiology. shown increase expression of SOX9 and its target genes collagen II and aggrecan (Woods et al, 2005;Woods and Beier 2006). In addition, treatment with cytochalasin, a cytoskeleton-interrupting reagent, promotes chondrogenesis of embryonic mesenchymal cells (Zanetti and Solursh 1984) and bone marrow stem cells (Lim et al, 2000).…”

SOX9 determines RUNX2 transactivity by directing intracellular degradation

“…2B), and the low level of SOX9 may be part of the reason during the initiation of the chondrogenesis of ATDC5. (39,40) SOX9-induced RUNX2 degradation is ubiquitin-proteasome-independent Since RUNX2 is degraded through the ubiquitin-proteasome pathway, (22,25,41) we hypothesized that SOX9 promoted RUNX2 degradation through this mechanism. To test this hypothesis, the proteasome inhibitor MG132 (42) was applied to HEK293 cells in which we overexpressed RUNX2, SOX9, or both.…”

Small GTPase protein Rac-1 is activated with maturation and regulates cell morphology and function in chondrocytes

“…In gain-of-function experiments using the chondrogenic cell line ATDC5, RhoA over-expression was found to induce actin filament organization and stress fibers, to increase proliferation and proteoglycan production, and to suppress maturation and hypertrophy. [16,17] Conversely, overexpression of Rac-1 and Cdc42 in ATDC5 cells induces collagen type X expression, alkaline phosphatase activity, and matrix mineralization, which are associated with mature chondrocytes. [18] A recent study has indicated that genetic ablation of Rac-1 in developing cartilage leads to growth retardation with irregular growth plate organization, suppression of chondrocyte proliferation and a decreased area of collagen type X expression, indicating a delay in chondrocyte differentiation.…”

“…[16,17,[19][20][21] In this study, we monitor the status of Rho GTPases in primary chondrocytes by a biochemical pull down assay and live cell imaging analysis. We find that Rac-1 and Cdc42, but not RhoA, activation increases with chondrocyte maturation, and that Rac-1 distribution corresponds to changes in chondrocyte morphology.…”