Abstract
The molecular mechanisms controlling differentiation of mesenchymal precursor cells into chondrocytes (chondrogenesis) are not completely understood. We have recently shown that the small GTPase RhoA inhibits this process. Here we demonstrate that a different Rho GTPase family member, Rac1, promotes chondrogenesis. Pharmacological inhibition of Rac1 expression in micromass culture resulted in reduced mRNA levels of the chondrogenic markers collagen II and aggrecan, and decreased accumulation of glycosaminoglycans. Expression of the essential chondrogenic transcription factors Sox9, Sox5, and Sox6 was also reduced upon inhibition of Rac1 signaling. In contrast, overexpression of Rac1 in the chondrogenic ATDC5 cell line increased mRNA transcripts of Sox9, 5, and 6, collagen II, and aggrecan. Inhibition of Rac1 resulted in a reduction in the number, size, and organization of cellular condensations and decreased expression of N-cadherin. Overexpression of Rac1 resulted in an increase in N-cadherin expression levels. Furthermore, genetic ablation of Rac1 in primary micromass cultures resulted in reduced expression of chondrogenic markers. Additionally, we provide evidence that Cdc42 also promotes chondrogenesis. Overexpression of Cdc42 in ATDC5 cells resulted in increased expression of Sox5, Sox9, and collagen II but not Sox6, aggrecan, or N-cadherin. Therefore, we demonstrate that Rac1 and Cdc42 are positive regulators of chondrogenesis, but act at least in part through different cellular and molecular mechanisms.
Highlights
Adhesion molecules N-cadherin and N-CAM, and increased cell-cell interactions [5,6,7]. The expression of these adhesion molecules is decreased [8]. Cells within these condensations commit to the chondrogenic lineage, acquire a spherical cell morphology and induce expression of the essential chondrogenic transcription factor Sox9 [9, 10]
The production of glycosaminoglycans are partially regulated by the enzymes chondroitin 4-sulfotransferase 11 (Chst 11) and chondroitin 6-sulfotransferase 3 (Chst 3), which have been determined to be important for proper cartilage and bone formation [16, 17]
Levels of Sox5, 6, and 9—We examined the effects of Rac1 inhibition on the expression of Sox9, Sox5, and Sox6 to investigate whether down-regulation of these transcription factors could be responsible for the observed attenuation of chondrogenesis
Summary
Time-impregnated CD1 mice were purchased from Charles River Laboratories. All medium components were purchased from Invitrogen or Sigma unless otherwise stated. The Rac inhibitor NSC23766 [45] and the ROCK inhibitor Y27632 were purchased from Calbiochem. The following antibodies were used: N-cadherin, cat.
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