207398.fig.003a
(a)
207398.fig.003b
(b)
207398.fig.003c
(c)
207398.fig.003d
(d)
Figure 3: TNF-dependent EGFR transactivation requires metalloproteinase activity and results in TGF-α release. Serum-starved cells were treated for 30 min with the metalloproteinase inhibitor BB94 (batimastat, 10 μM) (a), or increasing concentrations of the TNF-converting enzyme (TACE) inhibitor TAPI-1 (b), and stimulated with 10 ng/mL TNF-α for 15 mins. EGFR tyrosine phosphorylation was assessed as described in Section 2. EGFR tyrosine phosphorylation is significantly reduced in the presence of BB94 and to a lesser extent by TAPI-1. (c) shows the effect of BB94 and TAPI-1 pretreatments on TNF-stimulated TGF-α release. Serum-starved cells were pretreated for 30 mins with BB94 or TAPI-1, stimulated with TNF-α for 3 hrs, and TGF-α measured via ELISA. (d) shows total TACE activity as measured using either recombinant TACE or membrane preparations from vehicle and TNF-treated HT-29 cells using a fluorescent substrate. Cells were pretreated with vehicle or TAPI-1 for 30 mins prior to stimulation with TNF-α (10 ng/mL) for 15 mins (see Section 2). Data are representative of at least three experiments.