Figure 2

Effects mediated by FGFR1 upon FGF-2 binding. (A, B, E) Human articular chondrocytes in monolayer were treated with FGF-2 (100 ng/mL), in the absence or presence of FGFR1 neutralization antibody (1:1,000, 1:200) or SU5402 (5 μM), for 24 hours. In parallel, chondrocytes were transfected with siRNA targeting FGFR1 or FGFR3, and subjected to 24-hour FGF-2 (100 ng/mL) stimulation. Conditioned media were collected for immunoblotting analyses of matrix metalloproteinase-13 (MMP-13) and a disintegrin and metalloproteinase with a thrombospondin type 1 motif 5 (ADAMTS5). Total RNA was extracted for cDNA synthesis and qPCR quantification of gene expression. * P < 0.05. (C) Chondrocyte lysates were prepared 48 hours after siRNA transfection. The samples were resolved by SDS-PAGE, and immunoblotted for FGFR1 and FGFR3 protein expression. GAPDH was used as a loading control. (D) -1600MMP-13 Luciferase-promoter construct was transiently transfected into human articular chondrocytes. The transfected cells were pre-incubated with neutralizing antibody (1:1,000, 1:200) or 5 μM SU5402 for 1 hour, and then stimulated with FGF-2 (100 ng/mL) for 24 hours. The luciferase activity representing MMP-13 promoter activity was measured. A Renilla vector was co-transfected as an internal control for normalization. * P < 0.05. ADAMTS, a disintegrin and metalloproteinase with a thrombospondin type 1 motif; FGF, fibroblast growth factor; FGFR, FGF receptor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MMP, matrix metalloproteinase; qPCR, quantative polymerase chain reaction; siRNA, small interfering RNA.