EGF activates ErbB-2 and stimulates phosphatidylinositol 3-kinase in human airway smooth muscle cells

Vera P. Krymskaya, Rebecca Hoffman, Andrew Eszterhas, Sibyl Kane, Vincenzo Ciocca, and Reynold A. Panettieri Jr.

Pulmonary and Critical Care Division, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104


    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

The epidermal growth factor (EGF)-receptor (EGFR) family includes four homologous transmembrane receptor protein tyrosine kinases, EGFR, ErbB-2, ErbB-3, and ErbB-4. This receptor family plays a pivotal role in regulating cell proliferation, differentiation, and transformation. Ligand-induced activation of these receptors results in formation of homo- and heterodimers, which undergo transphosphorylation and transactivation. The aim of this study was to characterize the role of EGFR family members in signaling EGF-induced human airway smooth muscle (HASM) cell proliferation. Here, we show that EGF stimulates activation of EGFR and transactivation of ErbB-2 in quiescent HASM cells. Phosphatidylinositol (PI) 3-kinase, a critical signaling enzyme that modulates HASM cell growth, is preferentially associated with ErbB-2, and EGF-stimulated transactivation of ErbB-2 induces PI 3-kinase activation. ErbB-3 and ErbB-4 are present in HASM cells; however, EGF has no effect on their activation. Betacellulin, a ligand for EGFR, ErbB-3, and ErbB-4, induced DNA synthesis of HASM cells and stimulated signaling through the activation of EGFR and ErbB-2 but not of ErbB-3 and ErbB-4. Heregulin, a specific ligand for ErbB-3 and ErbB-4, did not effect DNA synthesis and did not activate its specific receptors. These data suggest that EGF imparts signals that involve activation of ErbB-2 and are associated with ErbB-2 PI 3-kinase activation. Despite the mRNA and protein expression of all members of the EGFR family, ligand-stimulated signaling induced activation of EGFR and ErbB-2 but not of ErbB-3 and ErbB-4.

epidermal growth factor; asthma; airway remodeling; heregulin; betacellulin; ErbB-3; ErbB-4


    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

HYPERTROPHY AND HYPERPLASIA of airway smooth muscle are well-described features of chronic severe asthma. These changes may alter myocyte contractility and may induce irreversible airway obstruction (13, 18). The structural remodeling of airways may be due to the frequent stimulation of airway smooth muscle by contractile agonists, inflammatory mediators, and growth factors. The cellular mechanisms that regulate and promote increases in myocyte number and myocyte size are not well understood.

Polypeptide growth factors induce cell proliferation, differentiation, or transformation. Growth factors signal by binding to receptors, and these receptors have intrinsic protein tyrosine kinase activity. The epidermal growth factor (EGF)-receptor (EGFR) family of receptor protein tyrosine kinases (RPTKs) includes EGFR (ErbB, HER), ErbB-2 (Neu, HER2), ErbB-3 (HER3), and ErbB-4 (HER4), which share a similar structural topology and close amino acid sequence homology (14, 44). Ligand binding to RPTKs induces receptor homo- and heterodimerization followed by receptor transphosphorylation and activation (16, 24, 43). Autophosphorylated RPTKs with enhanced protein tyrosine kinase activities transduce signals by recruitment and activation of intracellular substrates (44).

EGFR and the EGFR ligands EGF and transforming growth factor-alpha are necessary for normal growth and development. Knockout experiments of the EGFR gene in mice results in epithelial cell immaturity, organ failure, and postnatal lethality (27). Neuregulin (NRG), Neu differentiation factor (NDF), heregulin (HRG), glial growth factor, and acetylcholine receptor-inducing activity, which are differently spliced forms of the NRG gene, comprise the family of ligands for ErbB-3 and ErbB-4 (5, 8, 10, 45). Although a specific ligand for ErbB-2 has not been described, ErbB-2 plays an essential role in ErbB-3 and ErbB-4 signaling by forming heterodimeric complexes (6, 7, 16, 37, 42). Knockout studies of genes encoding ErbB-2, ErbB-3, ErbB-4, and NRG revealed their critical role in neural and cardiac development (17, 23, 26, 33). Besides the fundamental role of the EGFR family of RPTKs in the regulation of cell proliferation and differentiation (4), aberrant activation or overexpression of the EGFR family may play a role in the carcinogenesis of various human tumors and is correlated with poor patient prognosis (31). To date, the repertoire of the EGFR family on human airway smooth muscle (HASM) cells remains unknown.

Phosphatidylinositol (PI) 3-kinase is one of the intracellular targets in EGF-induced signaling pathways and is essential for EGF-stimulated DNA synthesis (34). PI 3-kinase is a cytosolic heterodimer composed of an 85-kDa (p85) regulatory subunit and a 110-kDa (p110) catalytic subunit (20). In response to growth factor stimulation, PI 3-kinase binds directly through SH2 domains of the p85 regulatory subunit to activated receptors at domains that are autophosphorylated on tyrosine or associates with the phosphorylated substrate of RPTKs (20). Activated PI 3-kinase specifically phosphorylates PI and other phosphoinositides (PI 4-phosphate and PI 4,5-bisphosphate) on the D-3 position of the inositol ring. Although EGFR lacks a tyrosine-phosphorylated Tyr-X-X-Met motif, which is necessary for PI 3-kinase association, EGF stimulates PI 3-kinase activity (19, 40). Previous studies (21, 38, 39) showed that EGF-induced PI 3-kinase activation may be due to PI 3-kinase association with ErbB-2 and ErbB-3 transactivated by EGFR. Also in response to EGF stimulation, the non-RPTK Src can phosphorylate EGFR, generating new docking sites for p85 PI 3-kinase (41). Naturally occurring mutants of EGFR promoting the development of numerous human tumors were found constitutively associated with PI 3-kinase, suggesting an essential role for PI 3-kinase in cell transformation (28). However, PI 3-kinase activation by the EGFR family appears to be cell-type specific (21, 38, 39).

Previously, Cohen et al. (12) demonstrated that EGF is a potent and effective HASM cell mitogen; EGF also markedly enhanced PI 3-kinase activity (22). We postulate that EGF-induced HASM cell growth is mediated by heterodimerization of EGFRs and requires transactivation of this receptor family. We now demonstrate that EGF-induced signaling involves activation of ErbB-2, and EGF-stimulated PI 3-kinase activity is associated with ErbB-2 in HASM cells. In addition, we characterized the specific EGFR members expressed on HASM cells and make the novel observation that HASM mitogenesis is mediated via ErbB-2 activation.


    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Materials. Ham's F-12 medium, Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin, and trypsin were obtained from Life Technologies (Grand Island, NY). [gamma -32P]ATP (5,000 Ci/mmol) and [methyl-3H]thymidine (70-86 Ci/mmol) were purchased from Amersham (Arlington Heights, IL). EGF, HRG-alpha (EGF-like domain), betacellulin, normal rabbit IgG control, and mouse IgG2B isotype control were obtained from R&D Systems (Minneapolis, MN). Tyrphostin AG-1478 was purchased from Calbiochem (San Diego, CA). Anti-EGFR, anti-phosphotyrosine, and anti-PI 3-kinase antibodies and lysates of the human Jurkat T cells and EGF-stimulated A431 cells were obtained from Upstate Biotechnology (Lake Placid, NY). Anti-Neu (ErbB-2; C-18), anti-ErbB-3 (C-17), and anti-ErbB-4 (C-18) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). All other reagents were purchased from Sigma (St. Louis, MO).

Cell culture. Human tracheae were obtained from lung transplant donors in accordance with procedures approved by the University of Pennsylvania (Philadelphia) Committee on Studies Involving Human Beings. A segment of trachea just proximal to the carina was removed under sterile conditions, and the trachealis muscle was isolated. With this technique, ~0.5 g of wet tissue was obtained, minced, centrifuged, and resuspended in 10 ml of buffer containing 0.2 mM CaCl2, 640 U/ml of collagenase, 1 mg/ml of soybean trypsin inhibitor, and 10 U/ml of elastase. Enzymatic dissociation of the tissue was performed for 90 min in a shaking water bath at 37°C. The cell suspension was filtered through 105-µm Nytex mesh, and the filtrate was washed with an equal volume of cold Ham's F-12 medium supplemented with 10% FBS (HyClone, Logan, UT). Aliquots of the cell suspension were plated at a density of 1.0 × 104 cells/cm2. The cells were cultured in Ham's F-12 medium supplemented with 10% FBS, 100 U/ml of penicillin, 0.1 mg/ml of streptomycin, and 2.5 µg/ml of amphotericin B, and this was replaced every 72 h. Cell counts were obtained from triplicate wells with 0.5% trypsin in a 1 mM EDTA solution.

HASM cells were grown in 100-mm diameter dishes and were maintained in Ham's F-12 medium supplemented with 10% FBS, 100 U/ml of penicillin, and 100 mg/ml of streptomycin. Details regarding the characterization of this cell line by indirect immunofluorescence of smooth muscle-specific actin have been previously reported by our laboratory (29). Confluent HASM cells were growth arrested in serum-free Ham's F-12 medium supplemented with 0.1% bovine serum albumin (BSA) for 48 h before the experiments. In our studies, third- and fourth-passage HASM cells were used.

MDA-MB-453 and T-47D cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA) and maintained in DMEM supplemented with 10% FBS and 5 µg/ml of insulin. Before the experiments, the cells were growth arrested in serum-free DMEM supplemented with 5 µg/ml of insulin and 10 µg/ml of transferrin for 24 h.

Cell proliferation assay. Cell proliferation was monitored by [3H]thymidine incorporation as previously described (29). Briefly, confluent, growth-arrested HASM cells were stimulated with either EGF (10 ng/ml), HRG (100 ng/ml), or betacellulin (10 ng/ml). After 16 h of mitogen stimulation, HASM cells were labeled with 3.0 µCi of [methyl-3H]thymidine. The cells were then incubated for 24 h at 37°C, washed with phosphate-buffered saline (PBS), harvested with 0.05% trypsin in a 1 mM EDTA solution, and lysed with 20% trichloracetic acid. The precipitate was aspirated onto filter paper and counted in scintillation vials. All experiments were performed in triplicate with a minimum of three different cell lines. Data are means ± SE. Statistical analyses were performed with analysis of variance (Bonferroni-Dunn).

Preparation of cell lysates and immunoprecipitation. EGF, tyrphostin AG-1478, HRG, and betacellulin were added to the cells for the indicated times at 37°C. For the PI 3-kinase activity assay, the cells were washed twice with ice-cold wash buffer (137 mM NaCl, 20 mM Tris · HCl, pH 7.5, 1 mM MgCl2, 1 mM CaCl2, and 0.2 mM vanadate) and lysed in lysis buffer [wash buffer plus 10% (vol/vol) glycerol, 1% (vol/vol) Nonidet P-40 (NP-40), 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml of aprotinin, and 10 µg/ml of leupeptin] (39). The lysates were centrifuged at 13,200 g for 10 min. The supernatants were normalized for protein content with Bio-Rad protein assays (Bio-Rad Laboratories, Hercules, CA) and then incubated with either anti-phosphotyrosine (3 µg/ml), anti-EGFR (4 µg/ml), anti-ErbB-2 (5 µg/ml), anti-ErbB-3 (5 µg/ml), or anti-ErbB-4 (5 µg/ml) antibodies overnight. Protein A-Sepharose (80 µl, 1:1 slurry in PBS; Pharmacia Biotech, Uppsala, Sweden) was then added to the lysates for 2 h at 4°C. The immunoprecipitates were washed three times with PBS with 1% NP-40, three times with 0.1 M Tris · HCl (pH 7.5) and 0.5 M LiCl, and two times with 10 mM Tris · HCl, 100 mM NaCl, and 1 mM EDTA, pH 7.5. All solutions contained 0.2 mM vanadate.

Immunoprecipitation for SDS-PAGE and immunoblotting was performed as described above except that the cells were washed twice with ice-cold PBS and lysed in radioimmunoprecipitation assay (RIPA) buffer [20 mM Tris · HCl, pH 7.4, 150 mM NaCl, 0.5% (wt/vol) sodium deoxycholate, 1.0% (vol/vol) NP-40, 0.1% (vol/vol) SDS, 1 mM EGTA, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml of aprotinin, 10 µg/ml of leupeptin, and 0.2 mM vanadate]. The immunoprecipitates were washed once with RIPA buffer with 0.5 M NaCl and 1 mM EDTA, twice with RIPA buffer with 1 mM EDTA, and once with PBS, and then SDS-PAGE sample buffer was added.

PI 3-kinase activity assay. PI 3-kinase activity assays were performed as previously described (39). Briefly, sonicated PI (final concentration 0.2 mg/ml) in 10 mM Tris (pH 7.4) in 1 mM EGTA was added to the immunoprecipitates. The phosphorylation reactions were started by the addition of MgCl2, ATP, and [gamma -32P]ATP (30 µCi/sample) to a final concentration of 4 mM MgCl2-50 mM ATP for 10 min at room temperature. The reactions were stopped by addition of 20 µl of 6 N HCl and extracted with 160 µl of chloroform-methanol (1:1). The lipids were separated on oxalate-coated thin-layer chromatography (TLC) plates (Silica Gel 60, Merck, Darmstadt, Germany) by using a chloroform-methanol-water-ammonium hydroxide (60:40:11.3:2) solvent system. The position of [32P]PI monophosphate was determined by the position of standard PI monophosphate subsequently separated by TLC in parallel and developed in iodine vapor. The lipids were then visualized by autoradiography. Radioactivity of ATP and related products was present in the samples, and they developed as a dark area on the autoradiographs at the origin of TLC.

Identification of proteins by immunoblotting assay. Immunoprecipitated proteins were subjected to 8% SDS-PAGE and immunoblot assays as previously described (39). The blots were exposed to 1 µg/ml of either anti-phosphotyrosine, anti-PI 3-kinase, anti-EGFR, anti-Neu, anti-ErbB-3, or anti-ErbB-4 in Tris-buffered saline-0.5% Tween 20 (TBS-T) overnight at 4°C. After three washes in TBS-T, the nitrocellulose filters were exposed to matched a primary antibody isotype horseradish peroxide-conjugated secondary antibody [anti-rabbit, anti-mouse (Boehringer Mannheim, Indianapolis, IN); anti-sheep (Rockland, Gilbertsville, PA)] at a 1:3,000 dilution. The filters were washed five times in TBS-T and were visualized with a chemiluminescence system (ECL, Amersham).

RT-PCR analysis. RT-PCR analysis was performed as previously described (11). Briefly, total RNA was extracted from HASM cells growth arrested for 48 h in medium supplemented with 0.1% BSA by the acid-phenol method (11). cDNA was produced from this RNA by reverse transcription with oligo(dT) primers (Promega, Madison, WI) and Superscript II reverse transcriptase (Life Technologies). Specific cDNAs for the EGFR family were amplified with primer pairs (see Table 1) by PCR for 30 cycles, denaturing at 94°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 30 s. The primer pairs yielded the appropriate sizes of PCR products as determined by electrophoresis on 1% agarose gels (EGFR 304 bp, ErbB-2 450 bp, ErbB-3 496 bp, and ErbB-4 1,588 bp). The PCR products were subcloned into Bluescript SK(+) (Stratagene, La Jolla, CA), and their nucleotide sequences were confirmed by dideoxy sequencing with Sequenase (United States Biological or Amersham).

                              
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Table 1.   EGFR family PCR primer pairs


    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

EGF stimulates tyrosine phosphorylation of EGFR and ErbB-2. To determine whether EGF induces activation of EGFR family members, HASM cells were growth arrested for 48 h with Ham's F-12 medium supplemented with 0.1% BSA and then were stimulated with 10 ng/ml of EGF for 0-20 min. This dose of EGF was used because Cohen et al. (12) previously determined that 10 ng/ml of EGF maximally stimulates DNA synthesis in HASM cells. After incubation of HASM cells with EGF, immunoprecipitation and immunoblot with anti-phosphotyrosine antibody revealed robust stimulation of tyrosine-phosphorylated proteins at 180 kDa, a molecular mass consistent with members of the EGFR family, as shown in Fig. 1A. A whole cell lysate of EGF-stimulated human A431 carcinoma cells, which express a high density of EGFR, was used as a positive control. To determine the effects of EGF on EGFR, ErbB-2, ErbB-3, and ErbB-4 activation, immunoprecipitation with antibodies specific for EGFR, ErbB-2, ErbB-3, and ErbB-4 was performed. The immunoprecipitates were then examined with immunoblot analysis with an anti-phosphotyrosine antibody. As shown in Fig. 1B, EGF induced a sustained tyrosine phosphorylation of EGFR. Stimulation of HASM cells with EGF showed time-dependent activation of ErbB-2, which reached a maximum in the first minute and declined over 20 min as shown in Fig. 1C. To examine whether EGF-induced activation of ErbB-2 is mediated by transactivation by activated EGFR, HASM cells were pretreated for 30 min with 2 µM tyrphostin AG-1478, a selective EGFR inhibitor (16), before stimulation with 10 ng/ml of EGF. As shown on Fig. 1D, tyrphostin AG-1478 completely inhibited tyrosine phosphorylation of EGFR and ErbB-2 in response to EGF stimulation. These data suggest that inhibition of EGFR activation abrogates activation of ErbB-2. ErbB-3 and ErbB-4 in EGF-stimulated cells did not appear to be activated (data not shown). Negative controls to support specificity of immunoprecipitation were performed with isotype nonimmune IgG for each antibody used. In some experiments, however, we observed that specific antibodies also recognized an additional band with a higher molecular mass than the protein of interest, which may represent the glycosylated form of the receptor. Together, these data demonstrate that EGF stimulates tyrosine phosphorylation of EGFR and ErbB-2; however, EGF has no effect on ErbB-3 and ErbB-4 activation in quiescent HASM cells.


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Fig. 1.   Analysis of epidermal growth factor (EGF)-induced tyrosine phosphorylation in human airway smooth muscle (HASM) cells. Growth-arrested HASM cells were stimulated with EGF (10 ng/ml) for 0.5, 1, 2, 5, 10, and 20 min and compared with those treated with diluent alone. Cell lysates, equalized for protein content, were prepared and subjected to immunoprecipitation (IP) followed by SDS-PAGE and immunoblot analysis (IB) with the indicated antibodies. A: tyrosine-phosphorylated proteins were immunoprecipitated with anti-phosphotyrosine antibody and analyzed by immunoblotting with the same antibody. EGF-stimulated A431 carcinoma cells (A431) were used as a positive control. Nos. at left, molecular mass. Arrow, EGF-receptor (EGFR) family. This is a representative immunoblot of 4 experiments. B: EGF-induced activation of EGFR was analyzed. EGFR was immunoprecipitated with a specific anti-EGFR antibody (alpha -EGFR) followed by immunoblotting with anti-phosphotyrosine antibody (alpha -PY) and alpha -EGFR. This is a representative immunoblot of 3 experiments. C: EGF-induced activation of ErbB-2 was analyzed. ErbB-2 was immunoprecipitated with a specific anti-ErbB-2 antibody (alpha -ErbB-2) and examined by immunoblotting with alpha -PY and alpha -ErbB-2. This is a representative immunoblot of 4 experiments. D: growth-arrested cells were pretreated with tyrphostin AG-1478 (AG 1478) for 30 min, stimulated with EGF for 1 min, and compared with those treated with diluent alone. Protein-equalized cell lysates were prepared and subjected to IP with alpha -EGFR and alpha -ErbB-2. IB was performed with alpha -PY. This is a representative experiment of 2 replicates with different HASM cell lines. +, Presence; -, absence. Nos. at left, molecular mass.

EGF-induced PI 3-kinase activation associates with ErbB-2. As described in EGF stimulates tyrosine phosphorylation of EGFR and ErbB-2, EGF stimulation of HASM cells induced robust phosphorylation on tyrosine residues of cellular proteins. In many cell types, investigators (20) have reported that in response to growth factor stimulation, PI 3-kinase, through SH2 domains of its adaptor p85 subunit, associates with proteins phosphorylated on tyrosine residues. To determine the effect of EGF on PI 3-kinase in HASM cells, we examined whether EGF stimulation induced PI 3-kinase association with tyrosine-phosphorylated proteins and whether EGF activated PI 3-kinase. HASM cells were growth arrested for 48 h and then stimulated with 10 ng/ml of EGF for 1 min. After stimulation, the cells were lysed, and immunoprecipitation was performed with an anti-phosphotyrosine antibody. Immunoprecipitates were subjected to SDS-PAGE, and immunoblot analysis was performed with a polyclonal antibody against the p85 regulatory subunit of PI 3-kinase. As shown in Fig. 2A, EGF stimulation significantly induced association of PI 3-kinase with tyrosine-phosphorylated proteins. We further measured PI 3-kinase activation in anti-phosphotyrosine immunoprecipitates from cells stimulated with EGF. As shown in Fig. 2B, EGF stimulated PI 3-kinase activation. The immunoblot analysis of cell lysates from Jurkat T cells, which abundantly express PI 3-kinase, was used as a positive control; negative controls included using isotype-matched nonimmune IgG in the PI 3-kinase activity assays (data not shown).


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Fig. 2.   Effect of EGF on phosphatidylinositol (PI) 3-kinase (PI3K). Growth-arrested HASM cells were stimulated with EGF (10 ng/ml) for 1 min and compared with those treated with diluent alone. Cell lysates, equalized for protein content, were prepared and subjected to IP with alpha -PY. A: IB was performed with an anti-p85 PI3K antibody (p85 PI3K). Left: representative immunoblot. Right: densitometric analysis of immunoblots. Nos. at left, molecular mass. Data are means ± SE of 3 separate experiments. * P < 0.0002. B: PI3K activity was measured in alpha -PY immunoprecipitates. Immunoprecipitates were incubated with PI and [gamma -32P]ATP, and in vitro phosphorylation of PI was examined by TLC and autoradiography. Left: representative autoradiograph. PIP, [32P]PI monophosphate; Origin, radioactivity at origin that represents ATP or related products. Right: densitometric analysis of 3 separate experiments. Data are means ± SE from 3 separate experiments.

In response to growth factor stimulation, PI 3-kinase also associated with an activated growth factor receptor (20). Interestingly, however, EGFR does not possess the binding sites necessary for association with PI 3-kinase (6). We thus postulated that EGF-induced PI 3-kinase activation likely occurs via transactivation of other EGFR family members. To test this hypothesis, we next studied whether EGF stimulation induced PI 3-kinase association with ErbB-2, ErbB-3, or ErbB-4. Immunoprecipitation of these receptors from EGF-stimulated HASM cells was performed. After SDS-PAGE, immunoblot analysis of these immunoprecipitates was performed with an anti-p85 PI 3-kinase antibody. As shown in Fig. 3A, PI 3-kinase was associated preferentially with ErbB-2. In parallel experiments, an in vitro PI 3-kinase activity assay in anti-EGFR and anti-ErbB-2 immunoprecipitates was performed. PI 3-kinase activity was associated with ErbB-2, and EGF stimulation of HASM cells enhanced activation of PI 3-kinase (Fig. 3, B and C, bottom). Quantitative analysis of these experiments revealed that PI 3-kinase association with ErbB-2 was increased 215 ± 29% in cells stimulated with EGF (Fig. 3C, top). The specificity of PI 3-kinase association with ErbB-2 was confirmed by showing that immunoprecipitation performed with nonimmune rabbit IgG had no PI 3-kinase activity. EGFR immunoprecipitates from EGF-stimulated HASM cells and cells treated with diluent alone had no associated PI 3-kinase, and no PI 3-kinase activity was associated with EGFR (Fig. 3, A and B). ErbB-3 and ErbB-4 immunoprecipitates also did not contain PI 3-kinase (data not shown). Together, these data suggest that in HASM cells PI 3-kinase associates with ErbB-2 and that EGF stimulation augments ErbB-2-associated PI 3-kinase activity compared with that obtained in diluent-treated cells.


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Fig. 3.   PI3K activity associated with ErbB-2 after EGF stimulation. Growth-arrested HASM cells were stimulated with EGF (10 ng/ml) for 1 min and compared with those treated with diluent alone. Cell lysates, equalized for protein content, were prepared and subjected to IP with alpha -EGFR or alpha -ErbB-2. A: EGFR and ErbB-2 immunoprecipitates were immunoblotted with p85 PI3K. This is a representative immunoblot from 4 separate experiments. B: PI3K activity was measured in EGFR and ErbB-2 immunoprecipitates that were incubated with PI and [gamma -32P]ATP, and in vitro phosphorylation of PI was examined by TLC and autoradiography. C: EGF-induced PI3K association with ErbB-2 (top) and PI3K activity associated with ErbB-2 (bottom) were quantitated by densitometric analysis. Results are means ± SE expressed as percent increase in PI3K association with ErbB-2 or PI3K activation in ErbB-2 immunoprecipitates over unstimulated (control) values of 3 separate experiments.

ErbB-3 and ErbB-4 are expressed in quiescent HASM cells but are functionally inactive. Evidence suggests that ErbB-3 plays a role in EGF-induced signaling in some cell types (1, 21, 39). EGF stimulation of A431, 32D, and MDA-MB-468 carcinoma cells induces activation of ErbB-3, which then associates with PI 3-kinase. Although EGF did not activate ErbB-3 and ErbB-4 in HASM cells, to determine the functional state of ErbB-3 and ErbB-4 in quiescent HASM cells, cells were stimulated with HRG and betacellulin, ligands to ErbB-3 and ErbB-4 receptors. HRG, a member of EGF-like factors termed NRGs, is a ligand for ErbB-3 and ErbB-4 (8, 10, 45); betacellulin is a ligand for EGFR, ErbB-3, and ErbB-4 (1, 2). Quiescent HASM cells were stimulated for 10 min with 100 ng/ml of HRG or 10 ng/ml of betacellulin. These doses of HRG and betacellulin were used because studies previously showed that these concentrations maximally stimulated DNA synthesis in human breast carcinoma (25) and murine fibroblast cell lines (36). Stimulated HASM cells were then lysed, and immunoprecipitation and immunoblot analysis were performed with an anti-phosphotyrosine antibody. As shown in Fig. 4A, betacellulin induced tyrosine phosphorylation of 180-kDa proteins, a molecular mass that is similar to that of members of the EGFR family. Interestingly, HRG had little effect on the stimulation of quiescent HASM cells (Fig. 4A).


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Fig. 4.   Betacellulin (BTC) activated EGFR and ErbB-2 and induced HASM cell proliferation. A: effects of BTC and heregulin (HRG) on tyrosine phosphorylation in HASM cells. Cells, growth arrested for 48 h in serum-deprived medium, were stimulated with EGF (10 ng/ml) for 1 min and HRG (100 ng/ml) or BTC (10 ng/ml) for 10 min and compared with those treated with diluent alone. Cell lysates, equalized for protein content, were prepared and subjected to IP with alpha -PY. Immunoprecipitated proteins were separated by SDS-PAGE, and IB was performed with alpha -PY. This is a representative immunoblot from 4 separate experiments. B: effects of EGF, HRG, and BTC on HASM cell growth. Confluent, growth-arrested HASM cells were stimulated with EGF (10 ng/ml), HRG (100 ng/ml), BTC (10 ng/ml), or diluent alone (control). [3H]thymidine incorporation was then measured as described in METHODS. Data are means ± SE of 6 replicates from 3 separate experiments. * P < 0.001 vs. control. C: BTC induced tyrosine phosphorylation of EGFR and ErbB-2. Growth-arrested HASM cells were stimulated with BTC (10 ng/ml) for 10 min. Protein-equalized cell lysates were prepared and subjected to IP with alpha -EGFR or alpha -ErbB-2. Immunoprecipitated proteins were separated by SDS-PAGE, and IB was performed with either alpha -PY, alpha -EGFR, or alpha -ErbB-2. This is a representative immunoblot of 3 separate experiments.

To address the role of EGFR, ErbB-3, and ErbB-4 in modulating HASM cell growth, DNA synthesis as measured by [3H]thymidine incorporation was measured in quiescent HASM cells stimulated with betacellulin (10 ng/ml), EGF (10 ng/ml), HRG (100 ng/ml), or diluent alone. Compared with diluent-treated cells, betacellulin was as effective as EGF in stimulating [3H]thymidine incorporation in HASM cells; interestingly, HRG had no effect (Fig. 4B). To identify the receptor subtype involved in betacellulin-induced signaling, we next performed immunoprecipitation of EGFR, ErbB-2, ErbB-3, and ErbB-4 from betacellulin-stimulated quiescent HASM cells and immunoblot analysis with anti-phosphotyrosine antibody. As shown in Fig. 4C, betacellulin stimulated EGFR and ErbB-2 activation. Betacellulin had little effect on ErbB-3 and ErbB-4 activation despite the expression of ErbB-3 and ErbB-4 proteins in HASM cells as shown in Fig. 5, A and B. As a positive control, human breast carcinoma cell lines were used because these cell lines constitutively express abundant levels of ErbB-3 and ErbB-4. Stimulation of MDA-MB-453 and T-47D cells with HRG (100 ng/ml, 10 min) and betacellulin (10 ng/ml, 10 min) induced activation of ErbB-3 and ErbB-4, respectively (Fig. 5, A and B).


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Fig. 5.   ErbB-3 and ErbB-4 were expressed in HASM cells but were functionally inactive. A: BTC did not stimulate ErbB-3 in HASM cells. Growth-arrested HASM cells were stimulated with BTC (10 ng/ml) for 10 min. Serum-deprived MDA-MB-453 cells were stimulated with HRG (100 ng/ml) or BTC (10 ng/ml) for 10 min. Cell lysates, equalized for protein content, were prepared and subjected to IP with an anti-ErbB-3 antibody (alpha -ErbB-3). Immunoprecipitated proteins were separated by SDS-PAGE, and IB was performed with either alpha -PY or alpha -ErbB-3. Data are from a representative experiment that was repeated in triplicate. B: ErbB-4 was not activated by BTC in HASM cells. HASM cells were stimulated with BTC (10 ng/ml) for 10 min. Serum-deprived T-47D cells were stimulated with HRG (100 ng/ml) or BTC (10 ng/ml) for 10 min. Protein-equalized cell lysates were prepared and subjected to IP with an anti-ErbB-4 antibody (alpha -ErbB-4). Immunoprecipitated proteins were separated by SDS-PAGE, and IB was performed with either alpha -PY or alpha -ErbB-4. These data represent an experiment that was repeated on 3 occasions. C: expression of EGFR, ErbB-2, ErbB-3, and ErbB-4 in HASM cells. Total RNA from cultured HASM and MDA-MB-453 cells was reverse transcribed, and resulting cDNA was amplified with primers specific for EGFR family. PCR products were size fractionated on a 1% agarose gel. Lane 1, 304-bp PCR product from EGFR; lane 2, 450-bp PCR product from ErbB-2; lanes 3 and 5, 496-bp PCR product from ErbB-3; lanes 4 and 6, 1,588-bp PCR product from ErbB-4. This is a representative experiment from 6 replicate experiments.

To determine whether ErbB-3 and ErbB-4 receptors were expressed in quiescent HASM cells, although they were not activated by HRG and betacellulin, analysis of mRNA expression of EGFR, ErbB-2, ErbB-3, and ErbB-4 RNAs was also performed with RT-PCR analysis. Specific oligonucleotide primers for human EGFR-, ErbB-2-, ErbB-3-, and ErbB-4-amplified cDNA products of 308 bp from EGFR, 432 bp from ErbB-2, 496 bp from ErbB-3, and 1,588 bp from ErbB-4 were detected in HASM cells (Fig. 5C). These data suggest that ErbB-3 and ErbB-4 are expressed in HASM cells; however, the ligands betacellulin and HRG do not activate them.


    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

EGF as well as other polypeptide growth factors are potent and effective HASM cell mitogens (12). The precise signaling mechanisms that regulate growth of HASM cells remain unknown. In the present study, we show that EGF signaling involves activation of EGFR and ErbB-2, which then stimulates DNA synthesis in HASM cells. EGF stimulation induces tyrosine phosphorylation of ErbB-2, and PI 3-kinase associates with ErbB-2 and is activated in response to EGF stimulation. Although all EGFR family members are expressed in quiescent HASM cells, ErbB-3 and ErbB-4 are functionally inactive. Betacellulin, an agonist of EGFR, ErbB-3, and ErbB-4, appears to activate EGFR and ErbB-2 but has little effect on ErbB-3 and ErbB-4. HRG, a specific ligand for ErbB-3 and ErbB-4, does not activate these receptors and does not involve DNA synthesis in quiescent HASM cells.

Signal transduction of EGFR family members represents a network of interreceptor interactions (43). On ligand binding, receptors form homo- and heteromeric complexes followed by transactivation. EGF is a ligand for EGFR; HRG is a ligand for ErbB-3 and ErbB-4 (7, 8, 10, 24, 37, 45). Currently, ErbB-2 remains the only receptor without a known ligand. ErbB-2, however, plays a role in interreceptor interactions and in the hierarchical network of EGFR family members (42). Selective overexpression of different EGFR family members in Chinese hamster ovary cells revealed that ErbB-2 is a preferential candidate to form heterodimeric complexes with ligand-activated EGFR, ErbB-3, and ErbB-4, and ErbB-2 plays a critical role in mitogenesis (43). Formation of heterodimeric complexes with ErbB-2 is due to the low-affinity site on the COOH-terminal domain of EGFR, ErbB-3, and ErbB-4 (42). Interreceptor interactions of the EGFR family were observed in vivo on coimmunoprecipitation, but these experiments were often performed on cell lines overexpressing EGFR receptors or on cells that were purposefully transfected with receptor constructs. To our knowledge, direct in vivo interreceptor interactions have not been demonstrated in primary cultured cells. Here, we demonstrate that stimulation of HASM cells with EGF induces activation of EGFR and ErbB-2. Activated EGFR transactivates only ErbB-2, which then results in tyrosine phosphorylation of ErbB-2 but not of ErbB-3 and ErbB-4. In our experimental conditions, using the selective EGFR inhibitor tyrphostin AG-1478, we demonstrate that inhibition of EGFR activation inhibits ErbB-2. Our conclusion that ErbB-2 activation is a result of transactivation by EGFR is based on the notion that EGF does not bind ErbB-2 and could act only through EGFR. Our data suggest that EGF signaling involves EGFR and ErbB-2 activation and that these two receptors play a role in EGF-induced mitogenesis in HASM cells. Whether ErbB-2 directly associates with EGFR in HASM cells remains unknown.

PI 3-kinase activation is one of the key enzymes involved in the transduction of growth factor-induced mitogenesis in many cell types (3). Roche et al. (34) demonstrated that PI 3-kinase is essential for EGF-stimulated DNA synthesis in NIH/3T3 cells. Recent studies have shown that PI 3-kinase is necessary for HASM and bovine airway smooth muscle cell proliferation (30, 35). In response to growth factor stimulation, PI 3-kinase associates with activated growth factor receptors or other phosphorylated proteins (46). In HASM cells, EGF induced tyrosine phosphorylation of 180-kDa proteins, which are consistent with proteins of the EGFR family. EGF stimulation also induced PI 3-kinase association with other tyrosine-phosphorylated proteins. In parallel experiments, immunoblot analysis with an anti-p85 PI 3-kinase antibody and an in vitro PI 3-kinase activity assay performed on EGFR immunoprecipitates did not show EGFR-associated PI 3-kinase or PI 3-kinase activation in response to EGF stimulation. These data are consistent with those reported by Songyang et al. (40), who showed that activated EGFR does not have potential binding sites that are necessary for association with PI 3-kinase. EGF-induced stimulation of PI 3-kinase in HASM cells, however, suggested that PI 3-kinase may associate with another EGFR family member. Interestingly, separate experiments showed that EGF induces ErbB-2 activation in HASM cells. Immunoblot analysis of ErbB-2 immunoprecipitates with an anti-p85 antibody also revealed the association of PI 3-kinase with ErbB-2. The PI 3-kinase activity assay performed on anti-ErbB-2 immunoprecipitates confirmed EGF-induced activation of PI 3-kinase in HASM cells. In some cell types, PI 3-kinase activity is associated with EGF-dependent activation of the EGFR family. In A431 cells but not in PC12 or A549 cells, EGF-stimulated PI 3-kinase activity was also found to be associated with the ErbB-3 receptor (39). EGF stimulation of MDA-MB-468 cells induced activation of ErbB-3 followed by PI 3-kinase association with ErbB-3 and activation of PI 3-kinase (21). To the best of our knowledge, this is the first report demonstrating that ErbB-2 is the only receptor involved in stimulating PI 3-kinase in response to EGF. Published data (1, 21, 39) and our findings suggest that EGF signaling induces PI 3-kinase association with ErbB-2 in HASM cells or in other cell types with ErbB-3. Stimulation of PI 3-kinase via transactivation of members of the EGFR family appears to be cell-type specific.

To characterize the interreceptor interactions and transactivation of EGFR family members in HASM cells, experiments were performed to determine the functional role of ErbB-3 and ErbB-4 in airway myocytes. Curiously, in some cell types, ErbB-3 plays an important role in modulating EGF-induced signaling (1, 21, 39). RT-PCR analysis of endogenous RNA transcripts revealed that all members of the EGFR family are expressed in HASM cells. To determine the role of ErbB-3 and ErbB-4 signaling in quiescent HASM cells, the effects of HRG, a specific agonist for ErbB-3 and ErbB-4 (2, 7, 9, 15, 37), on tyrosine phosphorylation were studied. HRG had little effect on tyrosine phosphorylation in cell lysates obtained from quiescent HASM cells and no effect on DNA synthesis. Betacellulin, a specific agonist for ErbB-3, ErbB-4, and EGFR (1, 2, 32), induced tyrosine phosphorylation of proteins in the region of 180 kDa and stimulated DNA synthesis in quiescent HASM cells. To identify the EGFR family involved in betacellulin-induced signaling, immunoprecipitation of EGFR, ErbB-2, ErbB-3, and ErbB-4 receptors and immunoblotting with anti-phosphotyrosine antibody were performed. EGFR and ErbB-2 were activated by betacellulin but not ErbB-3 or ErbB-4. Although all EGFR family members are expressed in quiescent HASM cells, our data suggest that ligand-induced signaling involves only EGFR and ErbB-2 activation.

EGF regulation of HASM cell growth has potential physiological significance. Increased airway smooth muscle mass, which has been attributed to increases in myocyte number, is a well-documented pathological finding in the airways of patients with chronic severe asthma (13, 18). These alterations may have important consequences in determining airway caliber and airway smooth muscle contractility. Structural changes in the airway wall may be due to the frequent stimulation of airway smooth muscle by growth factors, inflammatory mediators, and contractile agonists. EGF is a potent and effective HASM cell mitogen (12). Previously, Panettieri et al. (30) demonstrated that wortmannin, an inhibitor of PI 3-kinase, abrogated EGF-induced HASM cell proliferation. Here, we now show that ErbB-2 plays a critical role in EGF-induced signaling and associates with PI 3-kinase in response to EGF stimulation. Further studies are needed to identify the functional roles of ErbB-3 and ErbB-4 in airway smooth muscle and to characterize whether therapies directed at inhibiting ErbB-2 may be valuable in abrogating or reversing the airway smooth muscle hyperplasia that is seen in the bronchi of patients with chronic severe asthma.


    ACKNOWLEDGEMENTS

We thank Mary McNichol for expert assistance in preparing the manuscript.


    FOOTNOTES

These studies were supported by National Heart, Lung, and Blood Institute Grant R01-HL-55301; National Aeronautics and Space Administration Grant NRA-94-OLMSA-02; and a Career Investigator Award from the American Lung Association (all to R. A. Panettieri, Jr.).

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. §1734 solely to indicate this fact.

Address for reprint requests: V. P. Krymskaya, Pulmonary and Critical Care Division, Rm. 816 East Gates Bldg., Hospital of the Univ. of Pennsylvania, 3400 Spruce St., Philadelphia, PA 19104-4283.

Received 8 April 1998; accepted in final form 14 October 1998.


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