Article

Inhibition of RhoA but not ROCK induces chondrogenesis of chick limb mesenchymal cells

Department of Biology, College of Natural Sciences, Kyungpook National University, Daegu 702-701, South Korea.
Biochemical and Biophysical Research Communications (Impact Factor: 2.28). 02/2012; 418(3):500-5. DOI: 10.1016/j.bbrc.2012.01.053
Source: PubMed

ABSTRACT Cell shape change and cytoskeletal reorganization are known to be involved in the chondrogenesis. Negative role of RhoA, a cytoskeleton-regulating protein, and its downstream target, Rho-associated protein kinase (ROCK) in the chondrogenesis has been studied in many different culture systems including primary chondrocytes, chondrogenic cell lines, dedifferentiated chondrocytes, and micromass culture of mesenchymal cells. To further investigate the role of RhoA and ROCK in the chondrogenesis, we examined the RhoA-ROCK-myosin light chains (MLC) pathway in low density culture of chick limb bud mesenchymal cells. We observed for the first time that inhibition of RhoA by C3 cell-permeable transferase, CT04, induced chondrogenesis of undifferentiated mesenchymal single cells following dissolution of actin stress fibers. Inhibition of RhoA activity by CT04 was confirmed by pull down assay using the Rho-GTP binding domain of Rhotekin. CT04 also inhibited ROCK activity. In contrast, inhibition of ROCK by Y27632 neither altered the actin stress fibers nor induced chondrogenesis. In addition, inhibition of RhoA or ROCK did not affect the phosphorylation of MLC. Inhibition of myosin light chain kinase (MLCK) by ML-7 or inhibition of myosin ATPase with blebbistatin dissolved actin stress fibers and induced chondrogenesis. ML-7 reduced the MLC phosphorylation. Taken together, our current study suggests that RhoA uses other pathway than ROCK/MLC in the modulation of actin stress fibers and chondrogenesis. Our data also imply that, irrespective of mechanisms, dissolution of actin stress fibers is crucial for chondrogenesis.

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    • "Recently, we have shown that RhoA regulates ROCK activity but inhibition of ROCK activity neither affects MLC phosphorylation nor induces chondrogenesis and suggested that RhoA uses other pathway than ROCK for the chondrogenic differentiation (Kim et al., 2012). In accordance with these results, staurosporine affects neither the phosphorylation of MLC nor the phosphorylation of cofilin supporting the hypothesis that RhoA does not utilize ROCK pathway in the chondrogenic differentiation as suggested by Kim et al. (2012). In the present study, cytochalasin D reduced phosphorylation of cofilin. "
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