Abstract
IntroductionArticular chondrocytes undergo an obvious phenotypic change when cultured in monolayers. During this change, or dedifferentiation, the expression of type I and type III procollagen is induced where normal chondrocytes express little type I and type III procollagen. In this study, we attempted to determine the mechanism(s) for the induction of such procollagen expression in dedifferentiating chondrocytes.MethodsAll experiments were performed using primary-cultured human articular chondrocytes under approval of institutional review boards. Integrin(s) responsible for the induction of type I and type III procollagen expression were specified by RNAi experiments. The signal pathway(s) involved in the induction were determined by specific inhibitors and RNAi experiments. Adenovirus-mediated experiments were performed to identify a small GTPase regulating the activity of integrins in dedifferentiating chondrocytes. The effect of inhibition of integrins on dedifferentiation was investigated by experiments using echistatin, a potent disintegrin. The effect of echistatin was investigated first with monolayer-cultured chondrocytes, and then with pellet-cultured chondrocytes.ResultsIn dedifferentiating chondrocytes, α5β1 integrin was found to be involved in the induction of type I and type III procollagen expression. The induction was known to be mediated by v-akt murine thymoma viral oncogene homolog (AKT) signaling. Among the three AKT isoforms, AKT1 seemed to be most involved in the signaling. Elated RAS viral (r-ras) oncogene homolog (RRAS) was considered to regulate the progression of dedifferentiation by modulating the affinity and avidity of α5β1 integrin to ligands. Echistatin inhibited dedifferentiation of monolayer-cultured chondrocytes. Furthermore, the matrix formed by pellet-cultured chondrocytes more closely resembled that of normal cartilage compared with the controls.ConclusionsThe result of this study has shown, for the first time, that α5β1 integrin may be responsible for the induction of non-cartilaginous collagen expression in chondrocytes undergoing dedifferentiation. Again, this study has shown that the inhibition of ligand ligation to integrins may be an effective strategy to inhibit phenotypic change of cultured chondrocytes, and to improve the quality of matrix synthesized by primary cultured chondrocytes.
Highlights
Articular chondrocytes undergo an obvious phenotypic change when cultured in monolayers
We found that the inhibition of ligand ligation to integrins prevented dedifferentiation of chondrocytes cultured in a monolayer, and improved the quality of matrix generated by pellet-cultured chondrocytes
The results of this study indicated that α5β1 integrin could play a pivotal role in the induction of noncartilaginous procollagen expression in dedifferentiating chondrocytes
Summary
Articular chondrocytes undergo an obvious phenotypic change when cultured in monolayers During this change, or dedifferentiation, the expression of type I and type III procollagen is induced where normal chondrocytes express little type I and type III procollagen. Articular chondrocytes undergo an obvious phenotypic change when isolated from cartilage matrix and cultured in a monolayer. With the initiation of dedifferentiation, the expression of type II collagen and aggrecan declines gradually, and the expression of type I and type III procollagens is induced instead. In parallel with this metabolic change, the cell shape changes dramatically from the original spherical form to flattened elongated forms resembling those of fibroblasts [1]. The mechanism for the appearance of these noncartilaginous procollagens remains unknown
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