Rac1 Signaling Stimulates N-cadherin Expression, Mesenchymal Condensation, and Chondrogenesis

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.Chondrocytes are the cellular component of cartilage, responsible for generating and maintaining its extracellular environment (1). Cartilage has multiple functions such as providing a cushion on the articular surfaces of joints (2), providing a template for the formation of endochondral bone (3), and contributing to fracture repair (4). The formation of cartilage templates in endochondral ossification begins with condensation of mesenchymal cells, increased expression of the cell adhesion molecules N-cadherin and N-CAM, and therefore increased cell-cell interactions (5-7). As cells become chondrogenic, 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). Sox5 and Sox6 cooperate with Sox9 to control chondrogenesis and are themselves under the transcriptional control of Sox9 (9,11,12). Together, these transcription factors activate transcription of the major chondrogenic matrix genes, collagen II and aggrecan (11,(13)(14)(15). Furthermore, increased glycosaminoglycan (GAG) 3 content is another marker of the chondrogenic extracellular matrix (1). 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).Although many...

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

“…Promotion of actin polymerisation using jasplakinolide treatment (which stabilizes existing actin filaments and thus blocks actin dynamics), enhances expression of chondrocyte marker genes in both monolayer and micromass culture (Woods and Beier 2006). While RhoA may inhibit chondrogenesis, overexpression of another small GTPase, Rac1, leads to increased N-cadherin (involved in cell-cell contacts) expression and mRNA transcripts of Sox5, SOX6, and SOX9, collagen II, and aggrecan (Woods et al, 2007). MSC differentiation is also directed by the local matrix stiffness (Engler et al, 2006).…”

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

“…[19] In contrast, Rac-1 null limb micromass cultures have lowered proteoglycan content and levels of collagen type II and aggrecan mRNA. [20,21] However, the experiment using limb micromass cultures monitors chondrogenic induction, while our experimental system recapitulates chondrocyte maturation. Thus, these two findings indicate that Rac-1 diversely regulates chondrocyte matrix metabolism at the initiation versus late maturation stages.…”

“…Unlike previous reports, a loss of Rac-1 function in our cultures does not strongly inhibit cell spreading, which could be due to the different levels of Rac-1 activity in the chondrocytes used for the experiments. [19][20][21] We use immature chondrocytes, whereas the cultures in the previous study likely contained immature and mature chondrocytes. Since we use immature chondrocytes, which have low Rac-1 activity, a further decrease in Rac-1 function might not induce a strong effect.…”

“…[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.…”

Modelling condensation and the initiation of chondrogenesis in chick wing bud mesenchymal cells levitated in an ultrasound trap

“…Each step of chondrogenesis can be classified by the expression of a different set of transcription factors, cell adhesion molecules and extracellular matrix components. An expanded knowledge of the signalling pathways and mechanisms that lead to chondrogenic differentiation is of interest as it would aid technological progression in being able to repair damaged cartilage (Woods et al, 2007).…”