FIGURE 5.

Collagen XVII shedding is stimulated by H₂O₂ in cells and skin from wild type but not Adam9^−/− mice. A, Adam9^−/−, Adam10^−/−, and wild type (WT) mEFs were transfected with murine AP-tagged collagen XVII cDNA and treated with increasing concentrations of H₂O₂ for 5.5 h. AP assays revealed a dose-dependent increase in collagen XVII shedding in Adam10^−/− and wild type but not in Adam9^−/− cells. Data are shown as mean ± S.D. (n = 3; **, p < 0.01). B, epidermis sheets were prepared from 3-day-old wild type or Adam9^−/− pups as described under “Experimental Procedures” and incubated with 0, 250, and 500 μm H₂O₂ for 5.5 h. Lysates of epidermis sheets from three different experiments, each using tissue from a different animal, were analyzed by Western blotting for the presence of full-length collagen XVII (CXVII) and the collagen XVII ectodomain (ECTO), ADAM9 (AD9), and ERK-2 as loading control. Densitometric analysis of immunoblots for the collagen XVII ectodomain and for ADAM9 content revealed a dose-dependent increase of the collagen XVII ectodomain concomitant with increased ADAM9 expression in epidermis sheets from wild type but not from Adam9^−/− mice (data represent the mean ± S.D.; n = 3; *, p < 0.05). C, wild type and Adam9^−/− keratinocytes were treated with 300 μm H₂O₂ with or without 3 μm marimastat (MM) for 8.5 h. Cell lysates and methanol-chloroform-precipitated media of three different experiments were analyzed by Western blotting for the presence of full-length collagen XVII (CXVII; in lysates) or ectodomain (ECTO; in medium) and ERK-2 as lysate loading control. Densitometric analysis of immunoblots for collagen XVII ectodomain revealed a nearly 2-fold increase in H₂O₂-treated wild type but not Adam9^−/− keratinocytes, and the H₂O₂-stimulated shedding was sensitive to marimastat (data represent the mean ± S.D.; n = 3; **, p < 0.01; #, p < 0.05; ##, p < 0.01). D, in vivo application of H₂O₂ on shaved skin on the back of wild type mice (details under “Experimental Procedures”) increased the level of the collagen XVII ectodomain as well as of ADAM9 (AD9) in skin lysates. These effects were not seen in skin lysates of Adam9^−/− animals. Representative immunoblots of three independent experiments are shown. Co, control; 9KO, ADAM9 knock-out.