Abstract
The number of distinct signaling pathways that can transactivate the epidermal growth factor receptor (EGFR) in a single cell type is unclear. Using a single strain of human mammary epithelial cells, we found that a wide variety of agonists, such as lysophosphatidic acid (LPA), uridine triphosphate, growth hormone, vascular endothelial growth factor, insulin-like growth factor-1 (IGF-1), and tumor necrosis factor-alpha, require EGFR activity to induce ERK phosphorylation. In contrast, hepatocyte growth factor can stimulate ERK phosphorylation independent of the EGFR. EGFR transactivation also correlated with an increase in cell proliferation and could be inhibited with metalloprotease inhibitors. However, there were significant differences with respect to transactivation kinetics and sensitivity to different inhibitors. In particular, IGF-1 displayed relatively slow transactivation kinetics and was resistant to inhibition by the selective ADAM-17 inhibitor WAY-022 compared with LPA-induced transactivation. Studies using anti-ligand antibodies showed that IGF-1 transactivation required amphiregulin production, whereas LPA was dependent on multiple ligands. Direct measurement of ligand shedding confirmed that LPA treatment stimulated shedding of multiple EGFR ligands, but paradoxically, IGF-1 had little effect on the shedding rate of any ligand, including amphiregulin. Instead, IGF-1 appeared to work by enhancing EGFR activation of Ras in response to constitutively produced amphiregulin. This enhancement of EGFR signaling was independent of both receptor phosphorylation and PI-3-kinase activity, suggestive of a novel mechanism. Our studies demonstrate that within a single cell type, the EGFR autocrine system can couple multiple signaling pathways to ERK activation and that this modulation of EGFR autocrine signaling can be accomplished at multiple regulatory steps.
Highlights
The extracellular milieu is a rich mixture of small molecules, each conveying a particular type of information
To characterize the range of transactivating signals operative in human mammary epithelial cells (HMEC), we selected a panel of agonists that have previously been shown to induce epidermal growth factor receptor (EGFR) transactivation in other cell types
Coupling of Proliferation to EGFR Transactivation—Since EGFR activation is required for the proliferation of the 184A1 and 184A1–1 HMEC lines [33, 34], we investigated whether those extracellular stimuli that transactivate the EGFR in HMEC could induce a proliferative response in the absence of exogenous EGF and, if so, whether this response was dependent on EGFR kinase activity
Summary
Antibodies and Materials—Rabbit polyclonal anti-EGFR (1005) was obtained from Santa Cruz Biotechnology. AntiAKT (Pan), anti-phospho-AKT (Ser473), anti-ERK, and antiphospho-p44/42 MAPK (Thr202/Tyr204) (E10) antibodies were obtained from Cell Signaling Technology. Immunoblot Analysis of p42/44 ERK and AKT Phosphorylation—HMEC 184A1-1 cells were seeded into 100-mm dishes in DFCI-1 medium, cultured to ϳ80% confluence, and serum-deprived for 18 h. 20 g of total protein of each sample was loaded onto a 10% BisTris gel, transferred to polyvinylidene difluoride membrane, and probed for phospho-ERK. AKT phosphorylation was determined using an anti-phosphoAKT antibody purchased from Cell Signaling Technology that was used at a 1:1000 dilution. The beads were rinsed with a wash buffer twice, suspended in SDS sample buffer, heated to 95 °C for 5 min, and run on a 12% SDS gel and transferred to polyvinylidene difluoride membrane and probed with an anti-Ras antibody. A significance level of 0.05 was used in all cases
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