Metalloprotease-mediated ligand release regulates autocrine signaling through the epidermal growth factor receptor

Abstract

Ligands that activate the epidermal growth factor receptor (EGFR) are synthesized as membrane-anchored precursors that appear to be proteolytically released by members of the ADAM family of metalloproteases. Because membrane-anchored EGFR ligands are thought to be biologically active, the role of ligand release in the regulation of EGFR signaling is unclear. To investigate this question, we used metalloprotease inhibitors to block EGFR ligand release from human mammary epithelial cells. These cells express both transforming growth factor alpha and amphiregulin and require autocrine signaling through the EGFR for proliferation and migration. We found that metalloprotease inhibitors reduced cell proliferation in direct proportion to their effect on transforming growth factor alpha release. Metalloprotease inhibitors also reduced growth of EGF-responsive tumorigenic cell lines and were synergistic with the inhibitory effects of antagonistic EGFR antibodies. Blocking release of EGFR ligands also strongly inhibited autocrine activation of the EGFR and reduced both the rate and persistence of cell migration. The effects of metalloprotease inhibitors could be reversed by either adding exogenous EGF or by expressing an artificial gene for EGF that lacked a membrane-anchoring domain. Our results indicate that soluble rather than membrane-anchored forms of the ligands mediate most of the biological effects of EGFR ligands. Metalloprotease inhibitors have shown promise in preventing spread of metastatic disease. Many of their antimetastatic effects could be the result of their ability to inhibit autocrine signaling through the EGFR.

Figure 1

Batimastat (Bat) inhibits proliferation of EGF-dependent cells. (A) HMEC strain 184A1 were plated on coverslips and treated with mAb 225 (67 nM), batimastat (10 μM), or EGF (50 ng/ml) for 48 hr. Cells were labeled during the last 18 hr with BrdUrd. The results are the average of two independent experiments. (B) The indicated cells were grown for 5 days in growth medium alone (control), the presence of mAb 225 (67 nM), batimastat (7.5 μM), or a combination of mAb 225 and batimastat or batimastat and EGF (4 nM). Media were changed on days 1 and 3.

Figure 2

Inhibition of cell proliferation is correlated with inhibition of TGFα release. (Upper) HMEC strain 184A1 were grown to near confluency and changed to medium containing the indicated concentration of BB-2116 and 20 μg/ml of mAb 225 to prevent ligand uptake by the cells. After 18 hr, the medium was collected and evaluated for TGFα concentration, normalized to 10⁶ cells and ± SD. As a control, cells were also incubated with 50 μM matrix metalloproteinase 3 inhibitor (□) or with no inhibitor (○). (Lower) Cells were split 1:10 into 12-well dishes, and 18 hr later were changed to medium containing the indicated concentration of BB-2116. The medium was changed every 2 days and cells were counted on day 6. Shown are the results of duplicate wells ± SD. Controls are same as Upper as well as 20 μg/ml mAb 225 (▵).

Figure 3

Batimastat inhibits both release and mitogenic activity of a membrane-anchored, but not a soluble form of EGF. (A) Map of the artificial EGF genes expressed in HMEC. (Upper) The native EGF gene from which the two artificial genes were derived. (B) Cells expressing either the EGF-Ct or sEGF constructs were incubated with 67 nM mAb 225 (to prevent ligand uptake) for 18 hr either without (empty bar) or with (filled bar) 5 μM batimastat. The results are the average of two independent experiments. (C) Parental cells and those expressing the indicated construct were plated at a 1:400 dilution and grown for 2 weeks either with or without 10 μM batimastat (Bat). The medium was changed every 2 days. Cells then were stained with Giemsa.

Figure 4

Batimastat blocks autocrine signaling of cleavage-dependent EGFR ligands. Confluent cultures of either parental HMEC or cells expressing EGF-Ct or sEGF were preincubated for 24 hr with either 67 nM mAb 225 or 10 μM batimastat. Treatment with EGF (100 ng/ml) was for 20 min. Total EGFR was immunoprecipitated (IP) and visualized by Western blot (WB) using anti-phosphotyrosine (anti-PY) antibodies. The blots were then stripped and reprobed with anti-EGFR antibodies. The numbers under the lanes are the relative densities of the bands normalized to the untreated controls.

Figure 5

Inhibition of cell migration by batimastat. HMEC expressing the indicated ligand were followed for 15 hr by time-lapse video microscopy using 10-min intervals. The tracks of eight random cells from each plate are plotted as flower plots (32) with the origin of each cell set to 0,0. Batimastat was used at 10 μM, and EGF was used at 50 ng/ml.