These data indicate that the proteolytic activity of meprin�� is required for EGFR phosphorylation. FIGURE CHIR99021 2. Meprin�� induces EGFR and ERK1/2 phosphorylation. A, time course (0, 5, 15, 30, and 60 min) of EGFR and ERK1/2 phosphorylation is shown for Caco-2 cells treated with either control media, 1 ��g/ml recombinant active meprin��, 1 ��g/ml … EGFR phosphorylation leads to the activation of intracellular pathways, such as the mitogen-activated protein kinase (MAPK) pathway. The classical MAP kinases, extracellular-signal-regulated kinases 1 and 2 (ERK1/2), are intracellular signaling molecules that are preferentially activated in response to growth factors and phorbol esters (47). To determine whether ERK1/2 are transactivated upon treatment of Caco-2 cells with meprin��, the lysates obtained from the EGFR phosphorylation experiment were also analyzed for phosphorylated ERK1/2 (Fig.

2C). Phosphorylation was calculated by densitometric measurements (Fig. 2D). Control values were subtracted and data were normalized against total ERK1/2. Similar to the treatment with EGF, stimulation with meprin�� led to a peak in phosphorylation after 5 min (Fig. 2C, lane 2), which was attenuated over time. Pro-meprin�� as well as the negative control showed an increase in phosphorylation at time point 5 min, although to a lesser degree (Fig. 2C, lane 2). We assume that this ERK1/2 phosphorylation after 5 min is a transient process that might be caused by the change of culture medium. Taken together, we conclude that active meprin�� leads to the activation and phosphorylation of EGFR and consequently, transactivates the MAPK pathway, which culminates in ERK1/2 phosphorylation.

EGFR and ERK1/2 Phosphorylation Are Meprin��- dependent To analyze whether the EGFR/MAPK signaling pathway is activated by meprin�� via EGF and TGF�� shedding, phosphorylation experiments using neutralizing EGF and TGF�� antibodies were performed (Fig. 3A). Caco-2 cells were stimulated for 5 or 15 min with meprin��, pro-meprin�� or EGF in the absence or presence of the neutralizing antibodies. In the absence of EGF and TGF�� neutralizing antibodies, EGFR and ERK1/2 were phosphorylated when stimulated with meprin�� or EGF but not with pro-meprin��. Cells treated with neutralizing EGF and TGF�� antibodies showed EGFR and ERK1/2 phosphorylation reduced to control levels, after stimulation with meprin��.

After stimulation with EGF, EGFR and ERK1/2 remained phosphorylated to a certain extent in the presence of neutralizing antibodies. This may GSK-3 be the result of ligand excess compared with the amount of antibodies used. EGFR and ERK1/2 phosphorylation remained the same after stimulation with pro-meprin��. Total EGFR and ERK1/2 were not affected by the neutralizing antibodies. We conclude, that EGFR transactivation by meprin�� occurs via shedding of EGF and TGF�� from the plasma membrane by meprin��. FIGURE 3.