Endochondral ossification is initiated by the differentiation of mesenchymal precursor cells to chondrocytes. This process is characterized by a strong interdependence of cell shape and cytoskeletal organization accompanying the onset of chondrogenic gene expression, but the molecular mechanisms mediating these interactions are not known. In this study, we hypothesized that the activation of matrix metalloproteinase (MMP)-2 would be involved in the reorganization of the actin cytoskeleton and that this would require an Akt-dependent signaling pathway in chick wing bud mesenchymal cells. The pharmacological inhibition of Akt signaling resulted in decreased glycosaminoglycan synthesis and reduced the level of active MMP-2, leading to suppressed cortical actin organization which is characteristic of differentiated chondrocytes. In addition, the exposure of cells to bafilomycin A1 reversed these chondro-inhibitory effects induced by inhibition of Akt signaling. In conclusion, our data indicate that Akt signaling is involved in the activation of MMP-2 and that this Akt-induced activation of MMP-2 is responsible for reorganization of the actin cytoskeleton into a cortical pattern with parallel rounding of chondrogenic competent cells.
Transforming growth factor-beta (TGF-beta) plays crucial roles in controlling cell differentiation and maintaining tissue integrity. Previously we reported that TGF-beta3 treatment decreased the mRNA expression of the gap junction protein, connexin 43 as well as cell number, which lead to the inhibition of chondrogenic condensation in cultured chick leg bud mesenchymal cells. The present study demonstrates that TGF-beta3 can induce cleavage in the ectodomain of neuronal cadherin (N-cadherin) at the initiation stage of chondrogenesis and reduce cell numbers, cellular adhesion and the expression level of connexin 43. Differential displayed PCR (DD-PCR) comparison of adherent- and non-adherent chick leg chondrogenic progenitor cells showed increased expression of the chick ras-responsive element binding transcription factor, cRREB-1, in adherent cells. In chick leg bud mesenchymal cells, cRREB-1 transcription was inhibited by TGF-beta3 at the early stage of chondrogenesis. Small interfering RNA (siRNA)-mediated knockdown of cRREB-1 reduced cell numbers, cellular adhesion, and the expression level of connexin 43 resulting in the inhibition of precartilage condensation. Taken together, these findings indicate that TGF-beta3 mediates the inhibitory signal necessary for precartilage condensation by stimulating N-cadherin shedding and reducing cRREB-1 expression levels.
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