The re sult of this experiment showed that the attachment of chondrocytes to fibronectin coated plates was obviously increased between 2 and 7 days after plating. Next, to determine the significance of 5B1 integrin in cell attachment, 7 day cultured chondrocytes, once harvested, were incubated with a function blocking anti selleckchem Bosutinib 5B1 integrin antibody or control IgG for 90 mi nutes at room temperature, and were then plated onto fibronectin coated plates. This experiment confirmed that the attachment of chondrocytes to fibronectin coated plates was primarily mediated by 5B1 integrin. Since the level of expression of 5BB1 integ rin changed little within that culture period, this result was considered to indicate an increase in the activity of 5B1 integrin.

Given this result, we next examined whether RRAS is indeed involved in the observed increase in integrin ac tivity. In the experiment, chondrocytes cultured in monolayers for Inhibitors,Modulators,Libraries 7 days were infected with the adenovi ruses carrying CA or DN mutants of five small GTPases, and the attachment of the cells to fibronectin coated plates Inhibitors,Modulators,Libraries was evaluated 3 days later. Inhibitors,Modulators,Libraries These five small GTPases are known to be involved in the regulation of integrin activity in certain types of cells. In this experiment, cell attachment was significantly increased by the overexpression of a CA mutant of RRAS, and tended to be reduced by that of a DN mu tant. Such a change in cell at tachment was not observed with any other small GTPases.

Induction of type I and type III procollagen expression and AKT phosphorylation was indeed regulated by RRAS in monolayer cultured chondrocytes The following experiments were performed to confirm the involvement of RRAS in the induction Inhibitors,Modulators,Libraries of type I and type III procollagen expression and AKT phosphory lation. If our above presumption is correct, phosphory lation of AKT should be modulated by RRAS through the change in the activity of 5B1 integrin. To examine this hypothesis, CA RRAS or DN Inhibitors,Modulators,Libraries RRAS was over expressed in monolayer cultured chondrocytes by means of adenoviral transduction, and phosphorylation of AKT was evaluated. As anticipated, the phosphorylation was enhanced by the overexpression of CA RRAS, and tended to be reduced by that of DN RRAS. Consistently, in those chondrocytes, the expression of type I and type III procollagen was significantly ele vated by the overexpression of CA RRAS. For further confirmation, we suppressed the expression of RRAS by RNAi and observed whether any changes occurred in AKT phosphorylation and noncartilaginous procollagen expression. In this experiment, AKT phos phorylation and procollagen expression were reduced, selleck bio as predicted, by the suppression of RRAS expression.