Ectopic expression of LRP5 regulates type II collagen expression in mouse articular chondrocytes. (A) Mouse articular chondrocytes were transfected with empty vector (EV; 1 μg) or the indicated amounts of pSPORT6-Lrp5 for 24 hours. Downregulation of Col2a1 expression in Lrp5-overexpressing chondrocytes was determined by RT-PCR and measured by quantitative RT-PCR. The protein levels of type II collagen and LRP5 were examined by Western blot analysis. Values are presented as means ± SEM (*P < 0.05 and **P < 0.01 versus EV-transfected cells; n = 8). (B) Articular chondrocytes cultured on glass coverslips were transfected with pSPORT6-Lrp5 and immunostained with antibodies against type II collagen and LRP5. Scale bar: 20 μm. (C) through (E) Chondrocytes were transfected with 100 nM control small interfering RNA (C-si) or the indicated nanomolar concentrations of Lrp5 siRNA (Lrp5-si) and treated with 1 ng/ml recombinant human IL-1β (C), 50 ng/ml recombinant mouse Wnt3a (D) or 500 ng/ml recombinant human Wnt7a (E). The mRNA expression levels of Lrp5, Col2a1 and various catabolic factors (Mmp3, Mmp13, Ptgs2 and Nos2) were assessed by RT-PCR.