Figure 2

Phosphoinositide 3-kinase/AKT signaling may be involved in induction of noncartilaginous collagen expression in dedifferentiating chondrocytes. (a) Primary cultured chondrocytes maintained in monolayers for 5 days were treated with specific signal inhibitors for 24 hours, and expression of type I procollagen (COL1A1) and type III procollagen (COL3A1) was evaluated by quantitative PCR. For some cells, expression of type II procollagen (COL2A1) and aggrecan (ACAN) was also evaluated. (b) Experiment was repeated using two specific inhibitors for AKT phosphorylation, and expression of the above four genes was evaluated by quantitative PCR. (a), (b) Inhibitors were used at following concentrations: SB202190, 20 μM; SB203580, 20 μM; PD98059, 20 μM; U0126, 10 μM; SP600125, 10 μM; GF109203X, 5 μM; Wortmannin, 0.5 μM; LY294002, 20 μM; Akt inhibitor IV, 5 μM; Akt inhibitor VIII, 5 μM. Results are shown by relative ratios against control cells treated with dimethylsulfoxide (Control; open bars). Bars represent mean ± standard error of the mean (SEM) of three (b), five (SB202190, SB203580, U1026 in (a)) or six (the other inhibitors in (a)) independent experiments. **P <0.01 against control cells. (c) In monolayer-cultured chondrocytes, phosphorylation of AKT at Thr308 and Ser473 was evaluated 2 and 7 days after plating by western blotting, using phosphospecific (pAKT) and then nonphosphospecific anti-AKT antibodies (AKT). (d) Control siRNA, or siRNA for α5 or β1 integrin was introduced into chondrocytes, and the cells were cultured in monolayers. Phosphorylation of AKT at Ser473 was evaluated 3 days after plating. (c), (d) Experiments were repeated five times, and representative results are shown together with results of densitometric measurement. For the latter, results are mean ± SEM. GADPH, glyceraldehyde 3-phosphate dehydrogenase.