05. RESULTS EGF and TGF�� Are Shed by Meprin�� To investigate the role of meprin�� in ectodomain shedding of EGF and TGF��, a cell-based assay using AP-tagged EGFR ligands was used. Shedding of EGF and to a lesser extent, TGF�� was significantly stimulated in MDCK cells (p < 0.001; p < 0.05; Fig. 1, A and B) as well as in Caco-2 cells selleckbio (p < 0.001; p < 0.01; Fig. 1, D and E) by meprin��. Addition of the meprin�� inhibitor actinonin showed a significant decrease in ligand shedding in MDCK cells (p < 0.01; p < 0.05; Fig. 1, A and B) and in Caco-2 cells (p < 0.01; p < 0.01; Fig. 1, D and E). Actinonin did not influence constitutive shedding, suggesting that this is catalyzed by a different metalloprotease (data not shown).

Results obtained with the spectrophotometric assay were confirmed by in-gel detection of AP-tagged ligands, using NBT/BCIP as a substrate for the alkaline phosphatase (Fig. 1, C and F). FIGURE 1. EGF and TGF�� are shed by meprin��. MDCK or Caco-2 cells, transiently transfected with AP-EGF or AP-TGF��, were stimulated for 1 h with serum-free medium, recombinant active meprin��, or recombinant inhibited meprin��. … Endogenous EGF released from Caco-2 cells into the medium was quantified by EGF ELISA (Fig. 1G). Stimulation of Caco-2 cells with recombinant active meprin�� resulted in a significant increase of soluble EGF compared with control values (p < 0.001) and recombinant pro-meprin��, the inactive form of meprin�� (p < 0.001). Quantification of soluble TGF�� upon meprin�� activation by ELISA has been shown before (22).

Together these data suggest that meprin�� acts as a sheddase for EGF and TGF��. Meprin�� Induces EGFR and ERK1/2 Phosphorylation EGFR ligands, once released from the plasma membrane bind to the EGFR, which in turn is phosphorylated at several amino acid positions (Tyr-992, Tyr-1068, Tyr-1086, Tyr-1148, and Tyr-1173) (45). To investigate the effect of meprin�� on the phosphorylation of EGFR via EGF or TGF�� shedding, Caco-2 cells were stimulated for various periods of time with either medium alone, recombinant active meprin��, recombinant pro-meprin��, or with EGF (positive control). Fig. 2A shows representative Western blots of the phosphorylation experiment, and Fig. 2B shows the densitometric analysis of four individual experiments. In the latter, control values were subtracted and the values were normalized against total EGFR.

After 5 min of treatment with meprin�� (Fig. 2A, lane 2), an increase in phosphorylation Entinostat was observed. A maximum activation of EGFR was achieved between 15 min and 30 min of treatment (Fig. 2A, lanes 3 and 4), followed by a decrease (Fig. 2A, lane 5). Non-active pro-meprin�� showed only a very slight increase in phosphorylation of the EGFR. Minor amounts of pro-meprin�� might be activated over time, for instance by plasmin or kallikreins (KLKs) (40, 46).