Figure 3.

ADAM‐17 and matrix metalloproteinase 14 (MMP‐14) are the responsible sheddases of low‐density lipoprotein receptor–related protein 1 (LRP‐1) in human chondrocytes. A, Results of culturing human normal chondrocytes (n = 3 donors) with Dulbecco's modified Eagle's medium in the presence or absence of 10 ng/ml interleukin‐1 (IL‐1) or 200 ng/ml tumor necrosis factor (TNF) for 8–48 hours. LRP‐1 proteins in the cell lysate and the medium were analyzed by Western blotting with antibodies against α‐ and β‐chains of LRP‐1. B and C, Quantification of LRP‐1 protein in the cell lysate (B) and soluble LRP‐1 (sLRP‐1) released into the medium (C) with or without IL‐1 or TNF treatment. D, Effect of the metalloproteinase inhibitor CT1746 (20 µM), the serine proteinase inhibitor 4‐(2‐aminoethyl)benzenesulfonyl fluoride (AEBSF) (50 µM), or the cysteine proteinase inhibitor E‐64 (10 µM) on cytokine‐induced LRP‐1 shedding with or without IL‐1 or TNF treatment. E, Effect of tissue inhibitor of metalloproteinases 1 (TIMP‐1) (500 nM), TIMP‐2 (500 nM), or TIMP‐3 (300 nM) on cytokine‐induced LRP‐1 shedding with or without IL‐1 or TNF treatment. F, Effect of small interfering RNA (siRNA)–mediated knockdown of ADAM‐17 and/or MMP‐14 on cytokine‐induced LRP‐1 shedding with or without IL‐1 or TNF treatment. Values in B–F are the mean ± SD. In B–D, the mean values in the cells incubated without cytokine for 8 hours (B) or 24 hours (C and D) were set at 1 (dashed lines). In E and F, the mean values in the cells transfected with nontargeting siRNA were set at 1 (dashed lines). ∗ = P < 0.05; ∗∗ = P < 0.002; ∗∗∗ = P < 0.0002; ∗∗∗∗ = P < 0.0001, by one‐way analysis of variance followed by Dunnett's multiple comparison test.