In this experiment, the phosphorylation selleck screening library of AKT was in fact reduced by the suppression of 5 or B1 integrin expression. These results consistently support our proposed hypothesis that phosphoinositide 3 kinase AKT signaling is promoted in dedifferentiating chondrocytes via 5B1 integrin, which induces the expression of noncartilaginous procollagens. AKT has three isoforms in human. Thus, we finally attempted to clarify which isoform is most involved in the induction of noncartilaginous procollagen gene ex pression during dedifferentiation. From the results of the RNAi experiment, AKT1 was considered to play the most critical role in the induction among the three isoforms, where AKT2 might be the most abundant isoform in human articular chondrocytes.

Small GTPase RRAS regulates 5B1 integrin activity and promotes noncartilaginous procollagen gene expression in dedifferentiating chondrocytes In the previous study we have shown that in dedifferen tiating chondrocytes the activity of vB5 integrin, or the avidity and affinity of the integrin Inhibitors,Modulators,Libraries to ligands, is regulated by Inhibitors,Modulators,Libraries a small GTPase RRAS. During the course of de differentiation, RRAS is gradually activated, which pro motes dedifferentiation process by activating vB5 integrin. In light of this finding, we investigated whether the activity of 5B1 integrin is also regulated by RRAS in monolayer cultured chondrocytes. To this end, we first conducted a cell attachment assay. Human articular chondrocytes were cultured in a monolayer for 2 or 7 days, and cell attachment was eval uated using noncoated plates or plates coated with BSA or fibronectin, a known ligand to 5B1 integrin.

The re sult of this experiment showed that the attachment of chondrocytes to fibronectin coated plates was obviously increased between Inhibitors,Modulators,Libraries 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 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 Inhibitors,Modulators,Libraries 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 Inhibitors,Modulators,Libraries observed increase in integrin ac tivity. In the experiment, chondrocytes cultured in monolayers for 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 was evaluated selleck chemicals Trichostatin A 3 days later. These five small GTPases are known to be involved in the regulation of integrin activity in certain types of cells.