FIGURE 1.

Collagen XVII shedding is stimulated by ionomycin but not by PMA or pervanadate. A, COS-7 cells transfected with full-length human collagen XVII were treated with DMSO as vehicle control (Co) or with 20 ng/ml PMA or 2.5 μm IM for 25 min. Representative immunoblots of the 180-kDa full-length human collagen XVII (CXVII) and the 120-kDa shed ectodomain (ECTO) from lysates and media probed with the polyclonal rabbit anti-human NC16A antibody are shown. ERK-2 was used as lysate loading control. Collagen XVII shedding was significantly stimulated by ionomycin but not PMA. The graph summarizes the densitometric analysis of collagen XVII ectodomain shedding in three independent experiments (mean ± S.D.; *, p < 0.05). B, COS-7 cells were either transfected with a cDNA vector coding for an AP-tagged murine collagen (Col) XVII construct or an AP-tagged construct for Kit ligand 2 (Kitl2; Ref. 19), which was used as a representative substrate of ADAM17. The C-terminally truncated collagen XVII construct consists of its cytoplasmic domain, transmembrane domain, and the extracellular domains NC14A, Col13, and NC13, which were fused in-frame with a C-terminal AP tag. A Western blot probed with the MO-NC14A monoclonal antibodies on the left shows the 145-kDa membrane-bound form in the cell lysate and the shed 90-kDa form in the supernatant. Twenty-four hours after transfection the cells were washed, and fresh medium with DMSO vehicle control (Co), 20 ng/ml PMA, or 100 μm pervanadate (PV) was added. After 60 min, 100 μl of culture media and 10 μl of cell lysate were used for the AP assay. Treatment with either PMA or pervanadate failed to stimulate collagen XVII shedding in contrast to the significant stimulation of shedding of the ADAM17 substrate Kit ligand 2. Data are represented as mean ± S.D. (n = 3; **, p < 0.01; ****, p < 0.0001). C, murine keratinocytes were treated with DMSO vehicle as control (Co), 20 ng/ml PMA, or 100 μm pervanadate (PV) for 60 min or with 1 μm IM for 20 min, and the cell lysates and the concentrated supernatants were subjected to Western blot analysis with the polyclonal rabbit anti-mouse MO-NC14A antibody with ERK-2 levels serving as loading control. The graph summarizes the analysis of immunoblots of three independent experiments (one representative blot is shown) as the ratio of released ectodomain from treated cells versus control cells (mean ± S.D.). As in transfected COS-7 cells, shedding of endogenously expressed collagen XVII from murine keratinocytes was stimulated by ionomycin (*, p < 0.05) but not by PMA or pervanadate. Error bars indicate S.D.