FIGURE 1:

(A–D) TNF-α activates TACE. Confluent LLC-PK₁ (A, B) or NRK (C, D) cells were treated with 50 ng/ml TNF-α for 30 min, and MMP activity was measured in the cell lysates using a fluorigenic peptide substrate, as described in Materials and Methods. In A and C cells were pretreated for 30 min with 10 or 20 μM TAPI-1 as indicated. In B and D cells were transfected with NR or TACE-specific siRNA 48 h before TNF-α addition. After subtraction of the background fluorescence, the control fluorescence values in each experiment were taken as unity, and the fluorescence in the treated samples was expressed as fold increase. The graphs represent mean ± SEM from three independent experiments performed in triplicate. (E, F) TACE mediates TNF-α–induced ERK activation. LLC-PK₁ (E) or NRK (F) cells were transfected with nonrelated siRNA or siRNA directed against pig (E) or rat (F) TACE. Forty-eight hours later the cells were treated with 10 ng/ml TNF-α for 10 min, and the levels of phospho-ERK, total ERK, and TACE were detected by Western blotting. The graphs show quantification of the blots using densitometry. The amount of phospho-ERK was normalized to total ERK in the corresponding cell lysates. The results in each experiment were expressed as percentage compared with the TNF–α–treated sample, taken as 100%. The graphs show mean ± SE from n = 3 independent experiments. Statistical analysis is described in Materials and Methods.