Fig. 2.

LRP1 is the primary endocytic receptor for MMP-13 in human normal chondrocytes. A, Confocal microscopy analysis of proMMP-13 endocytosis by human normal chondrocytes. Cells were incubated with 20 nM proMMP-13 (FLAG tag at N-terminus) in the presence or absence of 500 nM RAP for 1 h. Endocytosed proMMP-13, EEA1, cytoskeleton, and nucleus were visualized as described under “Experimental procedures”. B and C, Human chondrocytes (n = 3) transfected with non-targeting siRNA (siCtrl) or LRP1 targeting siRNA (siLRP1) were cultured for 2 days in DMEM containing 10% FCS. B, left panel, Representative Western blotting for LRP1 α-chain (515 kDa) and β-chain (85 kDa) in cell lysate using anti-LRP1 α-chain (8G1) and β-chain (EPR3724) antibodies, respectively. Right panel, Densitometric analysis of immunoreactive LRP1 bands detected was then carried out and the amount of LRP1 was expressed as a % of the amount of LRP1 in untransfected cells (None). C, Human chondrocytes (n = 3) were further incubated with 10 nM proMMP-13 in the absence or presence of 500 nM RAP for 0–4 h and proMMP-13 in the medium was detected by Western blotting using an MMP-13 specific antibody (H-230). Upper panel, Representative Western blotting. Lower panel, Densitometric analysis of immunoreactive proMMP-13 bands detected in the medium was carried out and the amount of proMMP-13 was expressed as a % of the amount of proMMP-13 at 0 h. D, Human chondrocytes (n = 3) were incubated with 10 nM proMMP-13 in the absence or presence of 500 nM RAP for 1 h at 37 °C or 4 °C, and proMMP-13 in the medium and the cell lysate was detected as in C. Upper panel, Representative Western blotting. Lower panel, Densitometric analysis of immunoreactive proMMP-13 bands was carried out as in C. Bars and points represent the means ± S.D. *, p < 0.05, **, p < 0.01; unpaired t test.