Catabolic regulation of LRP5 mediates via β-catenin-Tcf/Lef signaling. (A) Chondrocytes were transfected with 1 μg of empty vector (EV) or pSPORT6-Lrp5 plus the TOPflash or FOPflash reporter constructs. After 24 hours, transcriptional activation by β-catenin was determined by luciferase reporter gene assays (n = 7 independent experiments). (B) Chondrocytes obtained from wild-type (WT) and Lrp5-/- mice were treated with 1 ng/ml interleukin 1β (IL-1β), 50 ng/ml Wnt3a or 500 ng/ml Wnt7a, and transcriptional activation by β-catenin was evaluated by luciferase reporter gene assays (n = 6). Values are expressed as means ± SEM (*P < 0.005). (C) β-catenin and LRP5 expression levels in cartilage after sham operation or destabilization of the medial meniscus (DMM) surgery were determined by immunofluorescence microscopy. 4′,6-diamidino-2-phenylindole (DAPI) staining was used for visualization of nuclei. Scale bar: 20 μm. (D) β-catenin, matrix metalloproteinase 3 (MMP3) and MMP13 expression levels in undamaged (Normal) and damaged (osteoarthritis; OA) human osteoarthritic cartilage were examined by immunostaining. (E) and (F) β-catenin, MMP3 and MMP13 expression levels in spontaneous (aging-induced) osteoarthritic cartilage and DMM-induced osteoarthritic cartilage from Lrp5-/- mice and their WT littermates were examined by immunohistochemistry. Scale bar: 50 μm.