Figure 2

Cathepsin expression by rabbit synovium and inhibition by cysC. (A) Secretion of rabbit catK in vitro. Rabbit primary synoviocytes and chondrocytes were cultured in the absence (U = untreated) and presence of IL-1β (2 ng/ml). Culture media was analyzed for catK protein by Western blot. Purified human catK protein (proform and mature forms), media alone (M) and media with 10% serum (M + S) were run as controls (lanes 1–4 on the left). Protein molecular weight markers (MW) in kDa are shown on the left. Arrows indicate location of catK proform (35 kDa) and mature forms (27 kDa) in the culture media (right). (B) CatK expression in synovium in early rabbit OA. Synovium of normal (sham) and OA joints 4 weeks post ACLT were analyzed for mRNA levels by qPCR. CysC and MMP-13 expression are shown for comparison (n = 2/treatment). (C) Comparison of cathepsin activity in synovium of normal and rabbit OA (4 weeks) joints. Values represent the group mean ± standard deviation (SD) (n = 3) (three independent experiments). Significance by two-tailed Student’s t test. (D) Inhibition of cathepsin activity in rabbit synovium by cysC in vitro. Inhibitors, cysC (mouse) and E-64, were added into 4-week OA homogenates (‘synovium’) and incubated for 1 h followed by cathepsin activity assay. The effect of inhibitors on activity by equal amount of purified catK (50 nM) is shown for comparison. Values represent the group mean ± SD (n = 2). ACLT, anterior cruciate ligament transection; cat, cathepsin; cysC, cystatin C; E-64, trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane; IL-1β, interleukin 1 beta; kDa, kiloDaltons; MMP, matrix metalloproteinase; MW, molecular weight; OA, osteoarthritis.