Abstract
The dysregulation of EGF family ligand cleavage has severe consequences for the developing as well as the adult organism. Therefore, their production is highly regulated. The limiting step is the ectodomain cleavage of membrane-bound precursors by one of several a disintegrin and metalloprotease (ADAM) metalloproteases, and understanding the regulation of cleavage is an important goal of current research. We have previously reported that in mouse lung epithelial cells, the pro-EGF ligands TGFα, neuregulin 1β (NRG), and heparin-binding EGF are differentially cleaved depending on the cleavage stimulus (Herrlich, A., Klinman, E., Fu, J., Sadegh, C., and Lodish, H. (2008) FASEB J.). In this study in mouse embryonic fibroblasts that lack different ADAMs, we show that induced cleavage of EGF ligands can involve the same substrate-specific metalloprotease but does require different stimulus-dependent signaling pathways. Cleavage was stimulated by phorbol ester (12-O-tetradecanoylphorbol-13-acetate (TPA), a mimic of diacylglycerol and PKC activator), hypertonic stress, lysophosphatidic acid (LPA)-induced G protein-coupled receptor activation, or by ionomycin-induced intracellular calcium release. Although ADAMs showed substrate preference (ADAM17, TGFα and heparin-binding EGF; and ADAM9, NRG), substrate cleavage differed substantially with the stimulus, and cleavage of the same substrate depended on the presence of different, sometimes multiple, PKC isoforms. For instance, classical PKC was required for TPA-induced but not hypertonic stress-induced cleavage of all EGF family ligands. Inhibition of PKCζ enhanced NRG release upon TPA stimulation, but it blocked NRG release in response to hypertonic stress. Our results suggest a model in which substantial regulation of ectodomain cleavage occurs not only on the metalloprotease level but also on the level of the substrate or of a third protein.
Highlights
Maximal cleavage of TGF-␣ and NRG occurred at approximately 30 min, and maximal cleavage of the HB-EGF substrate was observed at approximately 15 min
Hypertonic Stress-induced TGF␣ Cleavage Depends on a Metalloprotease but Not ADAM9, -10, or -17—In contrast to the results observed with TPA, LPA, and IM (Fig. 4, E–H, M–P, and Q–T, solid lines; Table 3), hypertonic stress-induced cleavage of TGF␣ (70 – 80% of control-treated cells) was independent of ADAM9, -10, and -17 (Fig. 4, I–L, solid lines; Table 3) but could be blocked by the broad spectrum metalloprotease inhibitor, BB94 (Fig. 2E)
Hypertonic Stress-induced NRG Cleavage Depends on ADAM9 and -17—When stimulated with hypertonic stress, 50% of the NRG substrate was cleaved in wild type MEF cells and ADAM10 knockouts (Fig. 4, I and K, dashed line; Table 3)
Summary
Antibodies—The following antibodies and dilutions were used: monoclonal anti-FLAG antibody M2 for FACS (1:100), immunoprecipitation (1:100), and Western blot (1:1000); monoclonal antiMYC 9E10 for FACS (1:100 dilution; Covance); monoclonal anti-HA11.1 antibody for FACS (1:100 dilution; Covance); allophycocyanin-coupled goat anti-mouse antibody for FACS (1:150 dilution; BD Biosciences); and polyclonal anti-phospho-specific PKC substrate antibody (1:1000 dilution; Cell Signaling). Stimulation was stopped by adding 1 ml of a cold PBS-based enzyme-free proprietary cell dissociation solution (Millipore S-014-B) and by placing cells on ice. Cells were dissociated, resuspended, washed with cold PBS, 3% FCS, and subsequently incubated at 4 °C for 1 h with the respective anti-epitope primary antibody. After washing three times with cold PBS, 3% FCS, cells were incubated at 4 °C for 1 h with anti-mouse allophycocyanin-coupled secondary antibody. Cells were again washed three times with cold PBS, 3% FCS and incubated with a PBS-based enzyme-free proprietary cell dissociation solution (Millipore S-014-B) containing 2 g/ml propidium iodine. Cells were washed with cold PBS and incubated on ice with anti-FLAG M2 antibody (1:100) in PBS, FCS 3% to capture only cell-surface located reporter. Membranes were washed three times with TBST and incubated for 1 h at room temperature with secondary antibody in 5% milk/TBST
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