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Multiple Mechanisms Are Responsible for Transactivation of the Epidermal Growth Factor Receptor in Mammary Epithelial Cells

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Multiple Mechanisms Are Responsible for Transactivation of the Epidermal Growth Factor Receptor in Mammary Epithelial Cells

Karin D Rodland et al. J Biol Chem.

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.

Figures

FIGURE 1.
FIGURE 1.
Dependence of EGFR activity and proteolytic activity for activation of ERK by multiple stimuli. HMEC 184A1–1 cells were treated with the indicated agonists for 15 min: TNFα (10 ng/ml), EGF (1 ng/ml), CaCl2 (2.0 mm), insulin (10 nm), sorbitol (Srbtol) (0.5 m), anisomycin (50 ng/ml), VEGF (100 ng/ml), LPA (20 μm), HGF (20 ng/ml), and GH (500 ng/ml) in the absence or presence of inhibitors, as indicated below. Cell lysates (normalized to 20 μgof protein) were separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and phosphorylated ERK visualized using anti-phospho-ERK1/2 antibodies, as described under “Experimental Procedures.” Top, effect of the EGFR kinase inhibitor AG1517. Cells were pretreated with the specific EGFR inhibitor AG1517 at a concentration of 500 nm for 15 min prior to agonist addition. The concentration of AG1517 was based on preliminary experiments showing it to be the minimum concentration that completely blocked EGF-induced EGFR phosphorylation (data not shown). Results are typical of triplicate samples from three independent experiments. Bottom, effect of the metalloprotease inhibitor Galardin (also known as Ilomastat or GM6001). The inhibitor was added at a final concentration of 10 μm 15 min prior to the agonist addition. Results are typical of triplicate samples from three independent experiments.
FIGURE 2.
FIGURE 2.
Kinetics of ERK phosphorylation in response to multiple agonists. A, cells were serum-deprived for 18 h and then treated with the indicated agonists: control (DMSO alone; ○), IGF-1 (1 nm; ▪), LPA (20 μm; ▴) UTP (100 μm; □), EGF (1 ng/ml; •), and HGF (20 ng/ml; ▵). Cell lysates were harvested on ice at the indicated times after agonist addition. Changes in the level of ERK phosphorylation were quantified by ELISA as described under “Experimental Procedures.” Results are presented as ng/ml pERK/40 μgof total protein ± S.D. (n = 3). B, same as A, with the addition of the tyrosine kinase inhibitor, AG 1517, at 500 nm for 1 h prior to the addition of agonists.
FIGURE 3.
FIGURE 3.
HMEC proliferation in response to multiple agonists is dependent on EGFR kinase activity. HMEC were plated at 30% confluence in serum-free DFCI medium supplemented with the indicated agonists or 0.1% DMSO (control), in the absence (light gray bars) or presence (dark gray bars) of the EGFR inhibitor AG1517. Cell number was estimated by MTT assay on day 1 and 3 after stimulation, as described under “Experimental Procedures.” Results are presented as the ratio of MTT absorbance on day 3/day 1 (mean ± S.D., n = 3). Vertical lines indicate the values of control cells as a basis for comparisons.
FIGURE 4.
FIGURE 4.
Effect of metalloprotease inhibitors on transactivation. HMEC in serum-free medium were treated with either the metalloprotease inhibitor Batimastat (10 μm) or the selective inhibitor of TACE/ADAM-17 WAY-022 (1 μm) for 1 h and then stimulated with IGF-1 (1 nm), LPA (20 μm), or UTP (100 μm) for 5 min. An ELISA was then used to quantify ERK phosphorylation, as described under “Experimental Procedures.” Values are mean ± S.D., n = 3. The asterisks denote statistically significant differences between control and inhibitor treatment within each agonist group, as determined by one-tailed Student's t test. *, p ≤ 0.05; **, p ≤ 0.01. The concentrations of inhibitors used have previously been shown to significantly inhibit the shedding of EGFR ligands by cells (37, 38).
FIGURE 5.
FIGURE 5.
Effect of antagonistic antibodies against either the EGFR or its ligands on stimulation of ERK phosphorylation. A, following overnight incubation in serum-free medium, HMEC were pretreated with the following inhibitors at a concentration of 10 μg/ml for 1 h: anti-EGFR mAb 225 (which blocks all ligand binding); anti-EGFR mAb 13A9 (which blocks TGFα and AR but not EGF or HB-EGF); CRM-197 (a specific inhibitor of HB-EGF binding), and specific neutralizing antibodies for either TGFα or epiregulin. The cells were then stimulated with IGF-1 (1 nm), LPA (20 μm), UTP (100 μm), or EGF (1 ng/ml) for 5 min. ELISAs were used to quantify ERK phosphorylation, as described under “Experimental Procedures.” Values are mean ± S.D. (n = 3). The asterisks denote statistically significant differences between control and inhibitor treatment within each agonist group, as determined by one-tailed Student's t test. *, p ≤ 0.05; **, p ≤ 0.01. B, combinations of neutralizing antibodies to AR, TGFα, and EPR were used, each at a concentration of 10 μg/ml. All other conditions are the same as in A. The phospho-ERK levels were below the ELISA limits of detection for all control samples (not shown) and for IGF-1-stimulated cells treated with anti-AR and a second antibody.
FIGURE 6.
FIGURE 6.
Stimulation of amphiregulin release from HMEC by different agonists. A, cells were seeded at ∼300,000 cells/35-mm dish and then serum-deprived the following day. Following an 18-h incubation in low serum medium, the cells were treated with IGF-1, LPA, UTP, HGF, or PMA for 2 h. Medium was collected from each dish and then analyzed for amphiregulin release by ELISA. B, HMEC were incubated overnight in serum-free medium and then changed to fresh medium containing IGF-1 at concentrations ranging from 0 to 10 nm for 2 h. The conditioned medium was collected and analyzed for amphiregulin release using an ELISA, as described under “Experimental Procedures.” C, HMEC were incubated overnight in serum-free medium before adding IGF-1 (10 nm), LPA (20 μm), or EGF (10 ng/ml) for 5 min (white bars). Alternately, the medium was changed before agonist addition for 1 h (gray bar) or 2 h (dark gray bar). The results are means from three samples ± S.D.
FIGURE 7.
FIGURE 7.
Sensitization of the response of HMEC to EGFR activation by treatment with IGF-1. HMEC were incubated overnight in serum-free medium and treated with (closed circle) or without (open circle) IGF-1 (10 nm) and EGF at the indicated final concentration, and cells were harvested 5 min later. ELISA assays were used to quantify EGFR phosphorylation (top) or ERK phosphorylation (bottom) as described under “Experimental Procedures.” Alternately, cells were lysed, and activated Ras was isolated using RBD-glutathione S-transferase bead affinity isolation. The amount of isolated Ras was then quantified by Western blot analysis, followed by densitometry. The amount of Ras from each sample is expressed in arbitrary densitometry units.
FIGURE 8.
FIGURE 8.
Sensitization of the EGFR response by IGF-1 is independent of PI3K activity. A, HMEC were pretreated with LY294002 (30 μm) or mAb 225 (10 μg/ml) or vehicle (DMSO) for 1 h and then stimulated with or without 0.1 ng/ml of EGF for 10 min in either the absence (left) or presence (right) of 10 nm IGF-1. The cells were then solubilized, and the extract was evaluated by Western blot (WB) analysis using antibodies against AKT or phospho-AKT as indicated. B, same as A, except the antibodies used were specific for ERK and phospho-ERK. C, HMEC were treated with the indicated concentrations of LY294002 for 1 h prior to stimulation with the indicated concentration of EGF for 5 min in the presence or absence of IGF-1. Cells were then extracted, and the levels of phospho-ERK were quantified by ELISA. Shown are averages of duplicate samples.
FIGURE 9.
FIGURE 9.
Summary of the multiple pathways for EGFR transactivation and ERK activation in HMEC. Modulators of G-protein-coupled receptors, such as LPA, stimulate the shedding of EGFR ligands, such as AR or TGFα, which in turn activate the EGFR. Alternately, IGF1 working through its receptor stimulates the activity of the EGFR through an unknown mechanism upstream of Ras. HGF operating through its own receptor can activate Ras and ERK independently of the EGFR.

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