Figure 2.

Ectodomain of soluble low‐density lipoprotein receptor–related protein 1 (sLRP‐1) prevents endocytosis of ADAMTS‐5 and matrix metalloproteinase 13 (MMP‐13) without interfering with their activities. A and B, Normal human chondrocytes (n = 3 donors) were cultured with Dulbecco's modified Eagle's medium containing 10 nM ADAMTS‐5 (A) or 10 nM MMP‐13 (B) with no additional treatment (Control) or in the presence of 2 nM sLRP‐1, 10 nM sLRP‐1, or 500 nM receptor‐associated protein (RAP) for 0–4 hours. ADAMTS‐5 and MMP‐13 in the medium were detected by Western blotting using anti–FLAG M2 antibody. Top, Representative Western blotting. Bottom, Quantification of findings in Western blotting. C, Bovine aggrecan (0.5 mg/ml) was incubated with 0.05 nM ADAMTS‐5 alone (1:0) or in the presence of 0.05 nM sLRP‐1 (1:1) or 0.25 nM sLRP‐1 (1:5) for 1–4 hours at 37°C. The reactions were stopped with 10 mM EDTA, and the reaction products were deglycosylated and subjected to Western blotting using antibody against the aggrecan neoepitope ³⁷⁴ARGSV (ARGS). Top, Representative Western blotting. Bottom, Quantification of findings in Western blotting. D, Type II collagen (1 mg/ml) was incubated with 5 nM MMP‐13 alone (1:0) or in the presence of 5 nM sLRP‐1 (1:1) or 25 nM sLRP‐1 (1:5) for 1–3 hours at 25°C. The reactions were stopped with 10 mM EDTA, and the reaction products were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‐PAGE) with Coomassie brilliant blue staining. Top, Representative SDS‐PAGE. Bottom, Quantification of findings in SDS‐PAGE. In C and D, the mean values after 1 hour of incubation without sLRP‐1 were set at 1 (dashed lines). Values are the mean ± SD. ∗ = P < 0.05; ∗∗∗∗ = P < 0.0001, by Student's 2‐tailed t‐test (A and B) or one‐way analysis of variance followed by Dunnett's multiple comparison test (C).