Cardiovascular Research Institute, Cancer Center, and Departments of Medicine and Physiology, University of California San Francisco, San Francisco, California
Submitted 20 October 2004 ; accepted in final form 27 July 2005
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ABSTRACT |
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epidermal growth factor receptor; airway epithelium
Biological responses to EGFR signaling are pleiotropic and include proliferation, differentiation, and apoptosis (reviewed in Ref. 19). In multicellular organisms, cell neighbors influence cell growth and differentiation. When normal epithelial cells and some immortalized epithelial cells are in close contact, they undergo contact-dependent cell cycle arrest in the G1 phase (a phenomenon known as "contact inhibition") and cease proliferating in response to further EGFR stimulation (54, 55, 59). The reduction in EGFR-dependent cell proliferation characteristic of dense cultures of normal human breast epithelial cells (59) and of three-dimensional cultures of colon carcinoma cells (55) is mediated, in part, via "outside-in" signals from the cell surface adhesion molecule, E-cadherin (reviewed in Ref. 12). E-cadherin-mediated cell-cell contacts also promote intestinal epithelial cell differentiation (21, 28) and survival of prostate and breast epithelial cells (13).
How does EGFR govern diverse cell outcomes such as proliferation and differentiation? Marshall (36) proposed that the temporal organization of MAPK activity downstream of receptor tyrosine kinase activation plays a role in the generation of specific biological responses. More recently, the amplitude of EGFR tyrosine phosphorylation has also been reported to influence cell outcome. For example, increased cell-cell contact has been shown to decrease EGFR tyrosine phosphorylation, leading to cell cycle arrest (54, 55, 59). The decrease in EGFR phosphotyrosine levels by cell-cell contact also suggests that an outside-in signal from the cell surface modulates EGFR activation. Indeed, E-cadherin-mediated adhesion has been shown to decrease EGFR tyrosine phosphorylation in dense cultures of breast, colon, and kidney epithelial cells (45, 55, 59). However, except for its role in contact-dependent growth inhibition, the effect of E-cadherin on biological responses to EGFR activation has not been studied previously.
How do E-cadherin-mediated cell-cell contacts modulate EGFR tyrosine phosphorylation? Present evidence suggests that E-cadherin-containing complexes interact with EGFR at the cell membrane (4, 20, 23, 42, 45, 55, 59). In lung epithelial cells, increased cell density decreases EGFR phosphotyrosine levels via increased EGFR-directed protein tyrosine phosphatase activity (54). Similarly, in human breast carcinoma cells, E-cadherin inhibits EGFR activation via protein tyrosine phosphatase activation (55). EGFR and several protein tyrosine phosphatases reported to dephosphorylate EGFR (27, 34, 46, 57) colocalize to E-cadherin-containing adherens junctions (1, 47, 58, 65), suggesting that E-cadherin may modulate EGFR phosphotyrosine levels via recruitment of an EGFR-directed protein tyrosine phosphatase to adherens junctions.
The above findings led us to examine whether cell density affects EGFR-dependent mucin production in human NCI-H292 airway epithelial cells and whether mucin production occurs via cell differentiation. Because E-cadherin-mediated adhesion is reported to promote contact-dependent growth inhibition and cell differentiation, and because E-cadherin interacts with EGFR, we examined the effect of E-cadherin-mediated cell-cell contacts on mucin production and on cell proliferation caused by EGFR activation. Because E-cadherin is reported to decrease EGFR phosphotyrosine levels in other epithelia via EGFR-directed protein tyrosine phosphatase activity, we examined whether E-cadherin affects the amplitude of EGFR tyrosine phosphorylation in NCI-H292 airway epithelial cells and whether this occurs via a protein tyrosine phosphatase. Our findings are the first to implicate E-cadherin in the regulation of EGFR-dependent airway epithelial (goblet) cell differentiation and mucin production.
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MATERIALS AND METHODS |
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To measure mucin production in sparse and dense cultures, cells were seeded to yield a density of <5 x 104 cells/cm2 (sparse cultures) and >4 x 105 cells/cm2 (dense cultures) 34 days after start of culture. After reaching the desired cell densities, cells were washed with PBS and incubated with fresh serum-free medium containing transforming growth factor (TGF)- (human recombinant TGF-
, 5 ng/ml; Calbiochem, La Jolla, CA) or with serum-free medium alone for 24 h. In experiments examining bromodeoxyuridine (BrdU) incorporation, cultures were labeled with BrdU (10 µM; Sigma, St. Louis, MO) for the last 6 of 24 h of incubation.
To measure EGFR tyrosine phosphorylation in sparse and dense cultures, cells were serum starved overnight and then stimulated with TGF- (5 ng/ml) for 30 min. In confluent cultures of NCI-H292 cells, TGF-
-induced EGFR tyrosine phosphorylation is maximal at 1530 min (26). To detect EGFR-directed protein tyrosine phosphatase activity in dense cultures, before being stimulated with TGF-
, cells were incubated with the protein tyrosine phosphatase inhibitor sodium orthovanadate (100 µM; Sigma) for 30 min at 37°C.
Proliferating normal human bronchial epithelial (NHBE) cells were cultured as described previously (49). In brief, NHBE cells were seeded at 2 x 104 cells/cm2 onto semipermeable Transwell polyester inserts and grown in immersed culture in bronchial epithelial growth medium (Cambrex, Walkersville, MD) until confluent (7 days). After reaching confluence, NHBE cells were grown in air-liquid interface for 23 wk before being used in E-cadherin blockade experiments. Confluent NHBE cells at air-liquid interface have been shown to express E-cadherin at the cell surface (9) and to form tight junctions (11, 35).
E-cadherin blockade.
To disrupt E-cadherin-mediated cell-cell contacts in NCI-H292 cells, dense cultures were washed twice with Ca2+-free PBS and then incubated with RPMI 1640 medium containing 4 mM EGTA for 45 min at 37°C. Subsequently, intercellular contacts were allowed to reform in Ca2+-containing serum-free RPMI 1640 medium for 2 h at 37°C (42). TGF--induced mucin production and EGFR tyrosine phosphorylation were equivalent in naïve dense cultures and in dense cultures allowed to reestablish intercellular contacts for 2 h. Alternatively, to prevent reformation of E-cadherin-containing adherens junctions, a mouse monoclonal E-cadherin blocking antibody (HECD-1, 2 µg/ml; Calbiochem) was included in the serum-free medium. In NCI-H292 cells, this concentration (2 µg/ml) of E-cadherin blocking antibody inhibited E-cadherin-mediated adhesion maximally in cell adhesion assays performed as described previously (55). As an antibody control, cells were incubated with a mouse monoclonal antibody to CD68 (IgG1; Dako, Carpinteria, CA). The control antibody did not affect cell adhesion, mucin production, EGFR tyrosine phosphorylation, or cell proliferation. Cells were then stimulated with TGF-
(24 h for mucin and BrdU measurements, 30 min for EGFR tyrosine phosphorylation). E-cadherin blockade in NHBE cells was performed as described above except that bronchial epithelial growth medium lacking recombinant EGF was used as medium. In inhibition studies, cells were pretreated with a selective EGFR tyrosine kinase inhibitor, BIBX1522 (5 µg/ml), or with a selective platelet-derived growth factor receptor (PDGFR) tyrosine kinase inhibitor, tyrphostin AG-1295 (10 µm, Calbiochem), 30 min before the addition of TGF-
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EGFR tyrosine dephosphorylation was measured as described by Böhmer et al. (6). After E-cadherin blockade and stimulation with TGF- for 15 min, BIBX1522 (5 µg/ml) was added to block further EGFR tyrosine phosphorylation. Dephosphorylation was stopped by lysis of the cells at various time points after BIBX1522 administration. Immunoblotting for phosphorylated EGFR was performed as described below.
Immunocytochemical staining. Cells grown on eight-chamber slides were stained as described previously (60) using mouse monoclonal antibodies to MUC5AC (clone 45M1 or clone 1-13M1, 1:500; NeoMarkers, Fremont, CA), E-cadherin (HECD-1, 2 µg/ml), or BrdU (Ab-2, 1:100; Oncogene Science, San Diego, CA), or rabbit polyclonal antibodies to EGFR (1:500; Research Diagnostics, Flanders, NJ), or EGFR phosphotyrosine (pY1068, 1:500; Biosource, Camarillo, CA). Before staining for incorporated BrdU, DNA was partially denatured with 2 N HCl for 30 min. As a control for specific staining, cells were also incubated with assay buffer but the primary antibody was omitted. To stain for both MUC5AC and BrdU, cells were serially stained: bound MUC5AC antibody was visualized using the avidin-biotin-alkaline phosphatase complex method (blue color, ABC kit; Vector Laboratories, Burlingame, CA). Bound biotinylated anti-BrdU antibody (Ab-3; Oncogene Science) was visualized using 3,3'-diaminobenzidine (brown color) as a peroxidase substrate.
Of the 11 monoclonal antibodies developed against human gastric mucin by the Mucin Immunochemistry Laboratory (3), the 45M1 antibody has been extensively used by our research group and others in studies of MUC5AC mucin expression. In recent studies, the 45M1 antibody did not react with peptides encoded by the 3' (40) or 5' ends of the MUC5AC gene (39), suggesting that this antibody reacts with an undescribed MUC5AC epitope or that it reacts with a mucin closely related to MUC5AC. Therefore, we also used another MUC5AC monoclonal antibody, clone 1-13M1 that reacts with the Cys2 and Cys4 domains of the 5' end of MUC5AC (39), to confirm findings from immunocytochemical studies and immunoassays using antibody 45M1.
Immunoassay of MUC5AC protein.
MUC5AC protein in cell lysates and in cell culture supernatants was measured by ELISA as described previously (61). Total MUC5AC protein was normalized to total protein in cell lysate and expressed as µg/mg of cell protein. In experiments comparing mucin production in sparse and dense cultures treated with TGF-, results were expressed as percent above MUC5AC/total protein of cultures incubated with serum-free medium alone (baseline). In experiments using the E-cadherin blocking antibody HECD-1, the standard MUC5AC ELISA protocol was modified. Because the goat anti-mouse IgG (secondary antibody) horseradish peroxidase conjugate recognized both antibodies to MUC5AC (primary antibody) and to E-cadherin (blocking antibody used as reagent), the absorbance from MUC5AC was calculated by subtracting the absorbance from E-cadherin antibody (measured by performing ELISA without MUC5AC primary antibody) from the total absorbance. A control IgG antibody (anti-CD68) did not affect calculated MUC5AC absorbances, thus validating this method. The amount of E-cadherin-blocking antibody added was <0.5% of the total protein in the cell lysate and did not change MUC5AC/total protein values significantly. The effect of E-cadherin blockade on mucin production was confirmed by using a rat monoclonal E-cadherin blocking antibody (DECMA-1; Sigma) that was not recognized by the goat anti-mouse secondary antibody.
Immunoblotting for phosphorylated EGFR. After various treatments, cells were lysed on ice in PBS lysis buffer containing 1% Triton X-100, 1% deoxycholic acid, 50 mM NaF, 1 mM sodium orthovanadate, and protease inhibitors (Complete Mini; Roche, Mannheim, Germany). Lysates were precleared by centrifugation at 14,000 rpm for 5 min at 4°C. Protein concentration was measured using a bicinchoninic acid-based protein assay kit (Pierce, Rockford, IL). For immunoprecipitation, aliquots of cell lysates from 1 x 105 cells were immunoprecipitated with 0.5 µg of anti-EGFR antibody (Ab 5, Calbiochem) and 20 µl of protein A/G PLUS-agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA) for 16 h at 4°C. Immunoprecipitates were washed with PBS, suspended in SDS sample buffer, boiled for 5 min, and separated by electrophoresis on an SDS/7.5% polyacrylamide gel. For immunoblotting, proteins were transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Richmond, CA), which were blocked with Tris-buffered saline containing 1% fatfree skim milk and 1% BSA overnight at 4°C and then incubated with monoclonal antibodies to either phosphotyrosine (PY99 or PY20, Santa Cruz Biotechnology) or EGFR (Ab-5, Calbiochem). Bound antibody was visualized using a standard protocol for the avidin-biotin-alkaline phosphatase complex method (Vector Laboratories). Immunoblots were scanned, and band intensities were quantified using NIH Image 1.63 software (developed at the National Institutes of Health and available for free download at rsbweb.nih.gov).
Flow cytometry. NCI-H292 cells were detached from dishes using Ca+2-free PBS containing 0.02% EDTA and were fixed, washed, and resuspended in PBS containing 0.3% saponin, 2% FBS, and 0.1% sodium azide. Aliquots of 5 x 105 cells were incubated with antibody to MUC5AC or with an isotype-matched control (anti-CD68) for 30 min at 4°C, washed twice, and then incubated with a goat anti-mouse FITC-conjugated secondary antibody (Dako) for 30 min at 4°C. Cells were then stained with propidium iodide and analyzed on an FACSCalibur (Becton Dickinson, San Jose, CA). Green FITC fluorescence (MUC5AC) was measured in the fluorescence 1 (FL1) photomultiplier tube; red fluorescence of propidium iodide-stained nuclei was measured in the fluorescence 3 (FL3) photomultiplier tube. Twenty thousand events were collected for each sample. Cells were considered to be MUC5AC positive if their FL1 fluorescence was greater than a gate set to exclude all cells in the FL1 peak from an isotype-matched control antibody (anti-CD68). The percentages of G0/1, S, G2/M, and sub-G1 cells were obtained by deconvolution of the DNA histograms of integrated red fluorescence using Modfit software (Verity Software House, Topsham, ME). For bivariate analysis of MUC5AC vs. DNA content, percentages of cells within each quadrant were calculated using Cellquest software (Becton Dickinson).
Statistical analysis. All data are expressed as means ± SE. One-way ANOVA was used to determine statistically significant differences between groups (P < 0.05 for the null hypothesis).
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RESULTS |
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Next, we examined the effect of E-cadherin on cell proliferation in dense cultures. In the absence of E-cadherin blocking antibody, TGF- increased the percentage of BrdU-positive cells slightly compared with control (Fig. 4D). Incubation of dense cultures with E-cadherin blocking antibody plus TGF-
increased the percentage of BrdU-positive cells markedly compared with control and with TGF-
alone (Fig. 4D). An EGFR-selective tyrosine kinase inhibitor, BIBX1522, blocked this increase in BrdU incorporation, whereas a PDGFR-selective tyrosine kinase inhibitor, AG-1295, had no effect (Fig. 4D), indicating that increased cell proliferation due to E-cadherin blockade is EGFR dependent. From these results, we conclude that E-cadherin-mediated cell-cell contacts promote EGFR-dependent MUC5AC production and inhibit EGFR-dependent cell proliferation.
E-cadherin-dependent mucin production is associated with decreased EGFR tyrosine phosphorylation.
Because E-cadherin-mediated cell-cell contacts have been shown to decrease EGFR tyrosine phosphorylation in epithelial cells (45, 59), we hypothesized that E-cadherin may promote mucin production and inhibit cell proliferation via modulation of EGFR tyrosine phosphorylation. To test this hypothesis, first we measured EGFR tyrosine phosphorylation in sparse and dense cultures. Cells were harvested 30 min after addition of TGF-, the time point at which EGFR tyrosine phosphorylation in confluent cultures of NCI-H292 cells is maximal (26). Very low levels of EGFR phosphotyrosine were detected in the absence of added TGF-
in both sparse and in dense cultures (Fig. 5A). As expected, TGF-
induced EGFR tyrosine phosphorylation in both sparse and in dense cultures (Fig. 5A). After TGF-
treatment, dense cultures contained less phosphorylated EGFR relative to total EGFR than sparse cultures (Fig. 5A). Quantitative measurement of phosphorylated EGFR and total EGFR by ELISA confirmed that, after TGF-
treatment, dense cultures contain twofold less phosphorylated EGFR/total EGFR per cell than sparse cultures (data not shown). To examine whether increased cell-cell contact mediated by E-cadherin decreases TGF-
-induced EGFR phosphotyrosine in dense cultures, EGFR tyrosine phosphorylation was measured in the presence of E-cadherin blockade. Incubation with an E-cadherin-blocking antibody increased EGFR tyrosine phosphorylation in dense cultures treated with TGF-
without affecting levels of total EGFR (Fig. 5B). In the absence of added TGF-
, E-cadherin blockade did not increase EGFR phosphotyrosine (Fig. 5B). To confirm that E-cadherin-mediated cell-cell contacts decrease EGFR tyrosine phosphorylation in the subset of cells that produce MUC5AC in response to TGF-
, we stained cells in three-dimensional aggregates (those that make mucin) for EGFR phosphotyrosine and EGFR. As expected, TGF-
increased staining for EGFR phosphotyrosine at sites of cell-cell contact in cell aggregates compared with medium alone (Fig. 5C). Coincubation of TGF-
with an E-cadherin blocking antibody further increased staining for EGFR phosphotyrosine at sites of cell-cell contact in cell aggregates (Fig. 5C), suggesting that the levels of EGFR phosphotyrosine measured by immunoblot (Fig. 5B) are representative of cells that will either produce mucin (intermediate phosphorylation) or proliferate (high phosphorylation). EGFR staining in cell aggregates was not affected by either TGF-
or an E-cadherin blocking antibody.
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E-cadherin decreases EGFR phosphorylation via protein tyrosine phosphatase activity.
Because protein tyrosine phosphatases and EGFR colocalize to E-cadherin-containing cell-cell contacts in human epidermoid carcinoma cells (1, 57) and in breast cancer cells (58), we examined whether E-cadherin decreases EGFR phosphorylation via a pathway involving a protein tyrosine phosphatase. A protein tyrosine phosphatase inhibitor, sodium orthovanadate, increased EGFR tyrosine phosphorylation markedly in dense cultures treated with TGF- without affecting total levels of EGFR (Fig. 6A), indicating that one or more protein tyrosine phosphatases dephosphorylate EGFR in dense cultures. To demonstrate E-cadherin-dependent tyrosine dephosphorylation of EGFR, we employed a method described by Böhmer et al. (6) to monitor the dephosphorylation rate of receptor tyrosine kinases. After stimulation with TGF-
for 15 min, we blocked further EGFR tyrosine kinase activity and monitored the rate of dephosphorylation by analyzing the amount of phosphorylated EGFR at various time points after EGFR inhibition with an EGFR-selective tyrosine kinase inhibitor. In the absence of E-cadherin blockade, EGFR phosphotyrosine was decreased after 2 min of EGFR inhibition (Fig. 6B). After 5 min of EGFR inhibition, EGFR dephosphorylation was almost complete (Fig. 6B). However, with E-cadherin blockade, the rate of EGFR dephosphorylation was decreased markedly. In the presence of E-cadherin blocking antibody, EGFR phosphotyrosine was not decreased significantly after 2 or 5 min of EGFR inhibition (Fig. 6B). From these results, we conclude that E-cadherin decreases EGFR tyrosine phosphorylation via a pathway involving protein tyrosine phosphatase-dependent EGFR dephosphorylation.
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DISCUSSION |
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Our findings suggest that, in dense cultures of NCI-H292 cells, EGFR activation induces mucin production in cells arrested in G1 phase. Therefore, we examined the cell cycle status of mucin-positive cells. EGFR activation induced mucin production in cells that did not incorporate BrdU and that contained 2 N DNA content, indicating that mucin-positive airway epithelial cells develop via differentiation. Consistent with this finding, present evidence suggests that mucin-containing goblet cells arise via the differentiation, and not proliferation, of precursor epithelial cells. This evidence derives from several sources. First, goblet cells show no evidence of DNA synthesis (5, 18, 66) and contain unphosphorylated retinoblastoma protein and decreased levels of cyclin D1 and cyclin-dependent kinase 2 (10), consistent with cell cycle arrest in the G1 phase. Second, in the endotoxin-treated rat nose, goblet cell production proceeds in the presence of metaphase blockade, indicating that goblet cell metaplasia can occur in the absence of cell proliferation (53). Third, in pathogen-free rats, the total number of airway epithelial cells remains constant during TGF--induced goblet cell production, whereas the number of Clara cells decreases, suggesting that goblet cells form via differentiation of Clara cells (61). Indeed, some Clara cells can serve as stem cells (16, 22), and some airway epithelial cells express both Clara cell secretory protein and mucin (5, 14, 25), suggesting that Clara cells are goblet cell precursors. However, whether goblet cells arise via cell differentiation has been questioned recently. In rat airways exposed to endotoxin, 50% of goblet cells were reported to stain for BrdU, leading the authors to conclude that goblet cells can arise via cell proliferation (63). We suggest that the BrdU-positive goblet cells described by these authors may have arisen via proliferation of precursor cells before differentiation into mucin-containing goblet cells for the following reasons: 1) precursor epithelial cells decide to differentiate while in their most rapid state of proliferation (8); and 2) BrdU was administered continuously from 24 h before to 48 h after endotoxin exposure (63), a time period during which goblet cell precursor cell proliferation and subsequent goblet cell differentiation could occur.
Because EGFR-dependent mucin production was cell density and cell cycle dependent, we hypothesized that an outside-in signal from the cell surface adhesion molecule, E-cadherin, promotes EGFR-dependent cell differentiation (mucin production) and G1 cell cycle arrest. To test this hypothesis, first we characterized E-cadherin expression in NCI-H292 cells. Consistent with a previous observation in NCI-H292 cells (67) and with confluent cultures of NHBE cells at air-liquid interface (9), E-cadherin was localized to surfaces of cells in close contact in dense cultures of NCI-H292 cells. In sparse cultures, E-cadherin accumulated on cell extensions at focal contacts between cells; in the absence of cell-cell contacts, E-cadherin accumulated in the cytoplasm. -Catenin was localized to the cell surface in cells in close contact and appeared nuclear, or perinuclear, in dividing cells in sparse cultures (data not shown). Both E-cadherin and
-catenin accumulated at the surface of cells in close contact that produced mucin, findings consistent with involvement of E-cadherin-mediated cell-cell contacts in EGFR-dependent mucin production and in contact-dependent growth inhibition.
Next, we examined the effects of E-cadherin-mediated cell-cell contacts on EGFR-dependent mucin production and cell proliferation. In dense cultures of NCI-H292 cells and in confluent cultures of NHBE cells grown at air-liquid interface, E-cadherin blockade decreased EGFR-dependent mucin production dose dependently. E-cadherin blockade did not remove cell aggregates from dense cultures, indicating that the decrease in EGFR-dependent mucin production was not caused by removal of mucin-producing cell aggregates from monolayer culture and implicating the contribution of other molecules to intercellular adhesion. E-cadherin blockade inhibited EGFR-dependent mucin production only partially, suggesting that surface molecules besides E-cadherin may also promote mucin production in cells in close contact. In addition, E-cadherin blockade increased EGFR-dependent BrdU incorporation (cell proliferation) in dense cultures markedly, consistent with studies showing the role of E-cadherin in contact-dependent growth inhibition (55, 56). Importantly, selective inhibition of EGFR tyrosine kinase activity prevented the E-cadherin blockade-induced increase in BrdU incorporation completely, implicating EGFR activation in this effect. Together, these results show that E-cadherin-mediated cell-cell contacts promote EGFR-dependent cell differentiation (mucin production) and inhibit EGFR-dependent cell proliferation and are consistent with other studies that show the importance of E-cadherin in epithelial cell differentiation (21, 2830, 64).
EGFR activation has been shown to induce mucin production in NHBE cells (2, 37, 49). Although the role of E-cadherin in mucin production has not been examined previously in normal cells, disruption of E-cadherin-mediated cell-cell contacts has been shown to increase adenovirus infection (35) and transfection efficiency (11) in NHBE cells. In our experience, NCI-H292 cells and NHBE cells at air-liquid interface produce similar amounts of mucin in response to EGFR activation. Both NCI-H292 and NHBE cells share key components of the signaling pathways upstream and downstream of EGFR responsible for mucin production (49), suggesting that NCI-H292 cells are a valid model of mucin production in normal cells.
Next, we examined how E-cadherin-mediated adhesion influences the biological response to EGFR activation (celldifferentiation vs. cell proliferation). In dense cultures,E-cadherin blockade increased the amplitude, but not the duration, of EGFR tyrosine phosphorylation in response to TGF- without affecting levels of total EGFR. In contrast, in sparse cultures, E-cadherin blockade had no effect on the amplitude of EGFR phosphotyrosine and had an insignificant effect on EGFR-dependent mucin production and cell proliferation (data not shown), suggesting that E-cadherin plays a limited role in modulation of EGFR signaling when not present on the surface of cells in close contact. Consistent with a previous study of lung epithelial cells (54), we confirmed that increased cell density decreases EGFR tyrosine phosphorylation in NCI-H292 cells independent of total EGFR. Decreased EGFR phosphotyrosine in dense cultures was associated with production of mucin in G1-arrested cells. In contrast, increased EGFR phosphotyrosine in sparse cultures was associated with EGFR-dependent cell proliferation. These findings are in agreement with previous reports showing decreased levels of EGFR phosphotyrosine in other epithelial cells undergoing contact-dependent growth inhibition (54, 55, 59) and support the idea that the amplitude of tyrosine phosphorylation is a critical determinant of cell outcome in response to receptor tyrosine kinase activation (reviewed in Refs. 36 and 48). The results of the present study show that E-cadherin-mediated adhesion decreases the amplitude of EGFR tyrosine phosphorylation and, in concert with previous findings, suggest that the amount of EGFR phosphotyrosine determines whether cells differentiate (make mucin) or divide in response to EGFR activation.
Because E-cadherin is reported to decrease EGFR tyrosine phosphorylation in other epithelia via a protein tyrosine phosphatase (55), we examined whether the E-cadherin-dependent decrease in EGFR phosphotyrosine in NCI-H292 cells occurs via an EGFR-directed protein tyrosine phosphatase. We found that, in the absence of further EGFR tyrosine phosphorylation, EGFR-directed protein tyrosine phosphatase activity reduces TGF--induced EGFR phosphotyrosine to control levels within 5 min, consistent with the rapid EGFR tyrosine dephosphorylation (halftime < 2 min) reported in other cells (6, 15). E-cadherin blockade decreased the rate of EGFR tyrosine dephosphorylation markedly, indicating that EGFR-directed protein tyrosine phosphatase activity requires intact E-cadherin-mediated cell-cell contacts. Similarly, in endothelial cells, vascular endothelial cadherin has been shown to attenuate vascular endothelial growth factor (VEGF)-induced VEGF receptor tyrosine phosphorylation via a protein tyrosine phosphatase-dependent mechanism, leading to contact-dependent growth inhibition (17). In the present study, a protein tyrosine phosphatase inhibitor, sodium orthovanadate, caused a greater increase in EGFR phosphotyrosine than did E-cadherin blockade, suggesting either that E-cadherin blockade was incomplete or that E-cadherin-independent mechanisms of EGFR dephosphorylation also exist. These results suggest that, in cells in close contact, an EGFR-directed protein tyrosine phosphatase dephosphorylates EGFR in an E-cadherin-dependent fashion.
In a recent study, Qian et al. (45) showed that E-cadherin inhibits EGFR activation in dense cultures of kidney epithelial cells by reducing EGFR mobility in the plasma membrane and by decreasing the number of high-affinity EGFR. These findings are consistent with cell density-dependent decreases in both EGFR dimerization (59) and in high-affinity EGFR (33) reported previously in dense cultures of epithelial cells. The results of the present study do not exclude these alternative mechanisms of inhibition of EGFR activation by E-cadherin. In addition, we examined the outcome of EGFR activation in cells with intact vs. disrupted E-cadherin-mediated cell-cell contacts. The conditions used in the present study are different from those used in studies that report transient EGFR activation upon formation of nascent E-cadherin-mediated cell-cell contacts (4, 43). Perhaps transient EGFR tyrosine phosphorylation by E-cadherin-mediated adhesion (via an unknown mechanism) is made possible by the later recruitment of EGFR-directed protein tyrosine phosphatases to maturing adherens junctions.
In conclusion, we show that E-cadherin-mediated cell-cell contacts promote EGFR-dependent mucin production in airway epithelial cells and that this occurs via cell differentiation. In addition, we show that E-cadherin decreases the amplitude of EGFR tyrosine phosphorylation in part via a protein tyrosine phosphatase-dependent mechanism. Together with previous findings, our results suggest that E-cadherin-dependent modulation of EGFR tyrosine phosphorylation determines the biological response (cell differentiation vs. cell proliferation) to EGFR activation. The decreased EGFR phosphotyrosine in cells with intact E-cadherin-mediated cell-cell contacts probably reflects dephosphorylation of specific tyrosine residues. Because the signaling molecules recruited to activated EGFR are defined by the pattern of phosphorylated tyrosines, site-selective dephosphorylation of specific tyrosines will generate different downstream signals (reviewed in Ref. 41). Identification of the protein tyrosine phosphatase(s) that dephosphorylate(s) EGFR and characterization of signals that lead to either EGFR-dependent mucin production or cell proliferation will be an important subject of future studies. Such studies will increase our understanding of the mechanisms involved in mucus hypersecretion and may lead to the identification of new therapeutic targets in the EGFR signaling pathway.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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