Matrilin-3 MATN3) regulation of type II collagen (COL2A1) and aggrecan (ACAN) gene expression, as well as COL2A1 protein expression requires IL-1Ra. Knocking down IL-1Ra abolishes the ability of recombinant human (rh) MATN3 protein to maintain COL2A1 gene expression in primary human chondrocytes (PHCs) that are challenged with IL-1β (A). A significant reduction in the basal gene expression of COL2A1 is also observed in IL-1Ra knock-down PHCs. PHCs cultured for 48 hours in media supplemented with rh MATN3 protein (200 ng/ml) exhibit increased COL2A1 protein levels; however, upon suppressing IL-1Ra via siRNA-based gene knock-down, MATN3 enhancement of collagen is diminished according to western blot analysis (B). Relative quantification of band intensity is the accumulated result of three individual experiments (n = 3). Knocking down of IL-1Ra also abolishes MATN3 stimulation of ACAN gene expression (C). The concentration of rh MATN3 protein used was 200 ng/ml and of IL-1β was 5.0 ng/ml for all experiments. (A, C) Gene expression analysis was conducted 36 hours post exposure to cell culture treatment conditions. #P ≤ 0.05 for statistically significant differences from the untreated group; *P ≤ 0.05 for statistically significant differences between groups. (B) *P ≤ 0.05 for statistically significant differences from the untreated control group. Individual experiments were done in biological triplicate per patient sample. Data are representative of five individual experiments.