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J. Biol. Chem., Vol. 275, Issue 34, 26545-26550, August 25, 2000

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Epidermal Growth Factor Receptor Transactivation Mediates Substance P-induced Mitogenic Responses in U-373 MG Cells^*

Ignazio Castagliuolo, Leyla Valenick, Jennifer Liu, and Charalabos Pothoulakis

From the Gastroenterology Division, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215

Received for publication, May 10, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Ligand-induced activation of G protein-coupled receptors is emerging as an important pathway leading to the activation of certain receptors with intrinsic tyrosine kinase activity, such as the epidermal growth factor receptor (EGFR). Substance P (SP) exerts many effects via activation of its G protein-coupled receptor (neurokinin-1, NK-1). SP participates in acute inflammation and activates key proteins involved in mitogenic pathways, such mitogen-activated protein kinases (MAPKs), stimulating DNA synthesis. We tested the hypothesis that SP-induced MAPK activation and DNA synthesis require activation of the EGFR. In U-373 MG cells, which express functional NK-1, SP induced tyrosine phosphorylation of several proteins including EGFR. SP induced formation of an activated EGFR complex containing the adapter proteins SHC and Grb2, but not c-Src. SP activated the MAPK pathway as shown by increased Erk2 kinase activity. SP induced Erk2 activation, and DNA synthesis was inhibited in cells transfected with a dominant negative EGFR plasmid lacking kinase activity, as well as in cells treated with a specific EGFR inhibitor. In addition, pertussis toxin, an inhibitor of G protein subunits, prevented SP-induced EGFR transactivation and subsequent DNA synthesis. Our results implicate EGFR as an essential regulator in SP/NK-1-induced activation of the MAPK pathway and cell proliferation in U-373 MG cells, and these events are mediated by a pertussis toxin-sensitive G protein. We suggest that this mechanism by which SP controls cell proliferation is an important pathway in tissue restoration and healing.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Substance P (SP),¹ an 11-amino acid peptide member of the tachykinin family, is distributed widely in the central and peripheral nervous systems (1). This peptide exerts its effects by binding to its high affinity NK-1 receptor subtype (SPR) (2). SP has been implicated in the regulation of several physiologic and pathophysiologic processes such as pain, smooth muscle contraction, and intestinal secretion (1). A large body of in vitro and in vivo studies suggests a role for SP as a proinflammatory agent. For example, SP is a chemoattractant for monocytes (3, 4) and neutrophils (4), a stimulator of macrophage phagocytosis (5), and mast cell degranulation (6). SP induces the secretion of interleukin-1, interleukin-6, and tumor necrosis factor  from monocytes and macrophages (6, 7). In vivo studies also showed that SP plays a pro-inflammatory role in acute intestinal inflammation (8), acute pancreatitis (9), and lung disease (10).

Besides acting as a pro-inflammatory mediator and a neurotransmitter, SP induces mitogenesis of several cell types including T lymphocytes (11), skin fibroblasts (12), smooth muscle cells (12), and synoviocytes (13). In addition, SP acts synergistically with growth factors to stimulate skin fibroblast proliferation (14) and corneal epithelial cell migration (15). Induction of mitogenesis and migration by SP suggests a possible beneficial role for this peptide in tissue healing during chronic inflammation. Indeed, several in vivo studies show that SP and SPR are up-regulated during chronic inflammatory disorders characterized by diffuse tissue damage (16, 17). Furthermore, SP depletion by the neurotoxin capsaicin (18) or administration of a SP antagonist (19) delay wound healing in experimental animals. However, little is known about the mechanisms by which SP exerts its effects on cell proliferation during wound healing and tissue restoration.

SP binding sites have been identified in the nervous system as well as peripheral tissues such as in immune (3), epithelial (20), and endothelial cells (16). Sequence analysis of the cloned NK-1 has demonstrated that it belongs to the G protein-coupled receptor (GPCR) family, and displays a typical 7-transmembrane-spanning domain structure (21). The SPR has been shown to associate with a variety of G protein isotypes, such as G_0/11, G_i, and G_s (22, 23)_. Thus, SP binding to SPR and consequent activation of G protein subunits results in the activation of a variety of second messengers, including inositol-3-phosphate kinase resulting in hydrolysis of phosphoinositides, cAMP formation (24), and arachidonic acid release (25). These events lead to mobilization of intracellular calcium and enhancement of protein kinase activity, including protein kinase C (24).

Mitogenic pathways are tightly regulated by a variety of molecular events, most notably the phosphorylation/dephosphorylation of tyrosine residues on several key effector proteins. Growth factors, such EGF and platelet-derived growth factor, bind specifically to membrane receptors with intrinsic tyrosine kinase activity, resulting in autophosphorylation of the receptor. Once activated, such receptor tyrosine kinases (RTKs) recruit adapter proteins to the cell plasma membrane, forming an activated complex that initiates a signaling cascade leading to nuclear translocation of transcription factors and cell proliferation (26). Mitogenic mediators such as lysophosphatidic acid (27), thrombin (27), angiotensin II (28), and SP (29), which bind to GPCRs, ultimately activate similar signaling pathways. Because GPCRs lack intrinsic kinase activity, transactivation of growth factor receptors is a possible mechanism by which ligand binding to GPCRs can activate mitogenic signal transduction pathways. For example, the EGFR has been recently identified as an essential element in the GPCR-mediated activation of MAPK-dependent pathways in cells treated with various GPCR agonists (27). Moreover, angiotensin II (30) and thrombin (31) stimulate phosphorylation of different growth factor RTKs, suggesting that transactivation of distinct RTKs may contribute to GPCR-mediated mitogenic signaling.

In this study, we sought to characterize the role of transactivation of growth factor RTKs in SP-induced cell proliferation. Specifically, we determined whether SP-induced EGFR activation can activate the MAPK cascade and induce cell proliferation. Using U-373 MG cells, an astroglioma cell line that expresses functional NK-1 receptors (31-33), we found that nanomolar concentrations of SP cause transphosphorylation of EGFR and the formation of an activated EGFR complex containing specific adapter proteins. We demonstrate that SP-induced MAPK activation and cell proliferation require the presence of a functional EGFR kinase domain and involve a pertussis toxin (PTX)-sensitive G protein. To our knowledge, these results represent the first demonstration that transactivation of the EGFR is involved in SP-induced cell proliferation.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cell Culture-- U-373 MG cells (ATCC, Rockville, MD) were cultured in minimum essential medium containing 10% FCS, 1% penicillin/streptomycin solution, 25 mM sodium bicarbonate, and 10 mM sodium pyruvate (Sigma). CHO cells (ATCC) were cultured in F-12 nutrient mixture containing 10% FCS, 1% penicillin/streptomycin solution (Sigma).

Immunoprecipitation and Western Blotting-- U-373 MG cells were cultured in complete medium, and when 75% confluent, they were incubated for 18 h in medium containing 0% FCS. Cells were then incubated with SP (10 nM) (29) or EGF (20 ng/ml) for the indicated times. In some experiments, cells were preincubated (18 h) with PTX (100 ng/ml) (Calbiochem). Monolayers were then washed twice with ice-cold PBS and lysed (45 min on ice) using nondenaturing RIPA buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.4, 0.25% sodium deoxycholate, 0.1% Nonidet P-40, 100 µM NaVO₄, 1 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin). Particulate material was removed by centrifugation (15,000 × g for 5 min), and the supernatant was collected. Protein concentrations were determined by the bicinichonic acid method (Pierce). Lysates (2 mg/ml) were incubated with the appropriate antibody (10 µg/mg cell lysate) for 2 h at 4 °C. EGFR was precipitated with a mouse monoclonal antibody, whereas SHC and c-Src were precipitated with rabbit polyclonal antibodies (1 µg of antibody/200 µg of whole cell lysate for all above immunoprecipitates) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Protein A-agarose or protein G Plus-agarose (50 µl, Santa Cruz Biotechnology, Inc.) was added to the mixture and incubated for 1 h at 4 °C. Beads were washed twice by centrifugation (20 s, 12,000 × g) with ice-cold RIPA buffer followed by one wash with ice-cold PBS and then boiled (5 min) in 30 µl of sample loading buffer (62.5 mM Tris, pH 6.8, 10% glycerol, 2% SDS, 5% -mercaptoethanol, and 0.1% bromphenol blue). Immunoprecipitated proteins were fractionated on a SDS-PAGE gel and then transferred and immobilized onto a nitrocellulose membrane. Membranes were blocked overnight at 4 °C in 5% skim milk in phosphate-buffered saline (pH 7.4) containing 0.05% Tween-20 and then incubated with the appropriate antibodies. Phosphorylated tyrosine residues were identified using the PY99 antibody (1:1000 dilution), and SHC proteins were identified using a rabbit polyclonal antibody (1:1000 dilution) (Santa Cruz Biotechnology, Inc.). Grb2 proteins were identified using a mouse monoclonal antibody (1:5000 dilution) (Transduction Laboratories; Lexington, KY). Immunocomplexes were visualized using the ECL Western blotting detection reagents (Amersham Pharmacia Biotech).

Immunocytochemistry on Cultured Cells-- Cells were plated on sterile coverslips at a density of 100,000 cells/ml and cultured in complete media. After 24 h, medium containing 0.5% FCS was placed on the cells overnight. Cells were stimulated with SP (10 nM) for the indicated time periods, followed by two washes with ice-cold PBS. The following procedures were done at room temperature using PBS at a pH of 7.4 unless otherwise stated. Cells were then fixed on the coverslip using 4% paraformaldehyde in PBS for 10 min and then washed (three times for 10 min with PBS). Cells were then permeabilized by incubation with 0.2% Triton X-100 in PBS for 5 min. The Triton X mixture was removed, and free aldehyde groups were inactivated by the addition of 0.5 mg/ml sodium borohydride in PBS for 10 min and then washed (three times for 5 min with PBS). Cells were then incubated in 3.0% bovine serum albumin in PBS for 1 h to block any nonspecific binding. Cells were then probed with 4 ng/ml antiphosphotyrosine antibody (Santa Cruz Biotechnology, Inc.) in PBS containing 1% bovine serum albumin for 40 min, followed by washing (three times for 5 min with PBS). Antiphosphotyrosine antibodies were then detected by the addition of donkey anti-mouse rhodamine-labeled antibody for 40 min (Jackson Immunoresearch Laboratories, West Grove, PA). Excess rhodamine-labeled antibody was removed by washing (three times for 5 min with PBS). Coverslips were then mounted, and proteins phosphorylated on tyrosine residues were visualized using a confocal microscope (Model MRC1024, Bio-Rad) with Plan-Neofluar objective (100×). Images were digitally stored in Bio-Rad COMOS software.

Transfection Experiments-- U-373 MG cells were transfected using a modified CaPO₄/DNA precipitation procedure (34). Briefly, cells were plated at a density of 25,000 cells/ml and cultured for 24 h. Two hours prior to transfection, the medium was replaced with fresh medium. Then, a total of 2.5 µg of plasmid DNA/ml of medium was introduced to the cells. After 18 h, the medium was removed, and cells were washed twice with PBS and cultured in complete medium for the indicated period of time. Preliminary experiments showed that the transfection efficiency (evaluated by expression of green fluorescent protein (In vitrogen)) in cells transfected with pCHER (35) and selected with methotrexate (10 mM), was ~30-40%, whereas the efficiency was improved to over 90% by co-transfection with pSP73 (Promega) a plasmid carrying a neomycin resistance gene (data not shown).

In Vitro Kinase Assays-- Erk2 in vitro kinase activity was determined as described previously by Daub et al. (36). Briefly, mock and pCHER transfected U-373 cells (75% confluent) were treated with SP (10 nM) for 7 min or EGF (10 ng/ml) for 3 min. Erk2 proteins were immunoprecipitated from the RIPA soluble lysates using rabbit polyclonal antibodies (Santa Cruz Biotechnology, Inc.). The immunoprecipitates were then washed twice in RIPA and once in kinase buffer (20 mM Hepes, pH 7.5, 10 mM MgCl₂, 1 mM dithiothreitol, 200 µM sodium orthovanidate). Kinase reactions were performed immediately in a 30-µl volume of kinase buffer containing 0.5 mg/ml myelin basic protein (Sigma), 50 µM unlabeled ATP, and 1 µCi/ml [-³²P]ATP (NEN Life Science Products). After 10 min at 37 °C, reactions were stopped by the addition of 30 µl of 2× sample buffer. Reactions were then subjected to electrophoresis on a 12% SDS-PAGE gel. Gels were dried, and phosphorylated myelin basic protein was identified by audoradiography.

Proliferation Assay-- DNA proliferation assays were performed as described previously (37). Briefly, U-373 MG cells were plated at 25,000/cm² in 6-well plates, and after 24 h cells were either mock-transfected or transfected with pCHER (2.0 µg/well) and pSP73 (0.5 µg/well), which contains a neomycin resistance gene. Cells were washed twice with PBS after 18 h and then cultured in complete medium containing geneticin (1 mg/ml) to select for transfected cells. After 48 h, medium was removed, and cells were cultured overnight in medium containing no FCS. In some experiments mock-transfected cells were incubated in the presence of 1 µM tyrphostin AG1478 (Calbiochem) for 15 min before stimulation. Cells were then stimulated (6 h at 37 °C) with SP (10 nM) or EGF (10 ng/ml) in the presence of [³H]thymidine (0.5 µCi/well). Cells were washed (three times) with ice-cold PBS and then lysed with 0.3 M NaOH, and [³H]thymidine incorporation was determined by scintillation counting. DNA synthesis was expressed as counts/min.

In separate experiments, control CHO cells, a cell line that does not express functional EGFR, and CHO cells stably expressing the human NK-1 receptor (a kind gift of Dr. Nick Boyd, Boston University Medical School, Boston, MA) were transfected with pEGFR (a kind gift of Dr. David Levy, New York University) using the same procedure as stated above. DNA proliferation assays in response to SP were performed as described above.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Substance P Induces Tyrosine Phosphorylation of Distinct Proteins in U-373 MG Cells-- U-373 MG cells express functional NK-1 (32, 33) and release inflammatory cytokines when exposed to SP (38). In basal conditions, few proteins in U-373 MG cells appear to be tyrosine-phosphorylated (Fig. 1). Upon treatment with SP (10 nM), at least four distinct protein bands showed increased tyrosine phosphorylation by Western blot (Fig. 1). Increased protein tyrosine phosphorylation was detected as early as 2 min after exposure to SP, and several proteins remained phosphorylated for up to 1 h. Similarly, immunocytochemical analysis demonstrated that only a few proteins were tyrosine-phosphorylated in unstimulated cells (Fig. 2A). However, at 30, 60, and 90 min, there was a dramatic increase of phosphorylated tyrosine residues localized in the nucleus, as well as on the cell's outer membrane (Fig. 2, B-D). These data, taken together, demonstrate the ability of SP to induce phosphorylation of proteins on tyrosine residues, even though NK-1 lacks intrinsic kinase activity (2).

View larger version (30K): [in this window] [in a new window]   Fig. 1.   SP induces tyrosine phosphorylation of multiple proteins in U-373 MG cells. SP (10 nM) was incubated with cells for the indicated time periods. Cells were then lysed with RIPA buffer, and equal amounts of protein were fractionated on a 7.5% SDS-PAGE gel. Proteins phosphorylated on tyrosine residues were identified using an antiphosphotyrosine antibody and detected using ECL reagents. Data presented are representative of three different experiments.

View larger version (143K): [in this window] [in a new window]   Fig. 2.   SP induces phosphorylation of tyrosine residues in U-373 MG cells. Cells were cultured on coverslips and then incubated without (A) or with SP (10 nM) for 30 (B), 60 (C), or 90 (D) min. Cells were fixed and subject to immunocytochemistry. Proteins phosphorylated on tyrosine residues were identified using an antiphosphotyrosine antibody and visualized using confocal microscopy. The amount of phosphorylated tyrosine residues increases dramatically and time dependently in response to SP. Phosphorylated proteins appear to be localized mainly in focal regions of the plasma membrane and in the nucleus.

Substance P Transactivates EGFR-- Because the NK-1 is a GPCR and recent studies have indicated that GPCRs can transactivate RTKs, we examined the effect of SP on EGFR. As shown in Fig. 3A, SP at nanomolar concentrations induced a transient and biphasic increase in tyrosine phosphorylation of EGFR in U-373 MG cells. EGFR tyrosine phosphorylation was detectable as early as 2 min after SP exposure and returned to control levels at 5-7 min. EGFR tyrosine phosphorylation was elevated again at 10-15 min and began to diminish at 30 min (Fig. 3A).

View larger version (33K): [in this window] [in a new window]   Fig. 3.   SP induces tyrosine phosphorylation of EGFR and the formation of an activated EGFR complex containing the adapter proteins SHC and Grb2. U-373 MG cells incubated with SP (10 nM) for the indicated time periods were lysed with RIPA buffer. Soluble proteins were immunoprecipitated with either an anti-EGFR antibody (A, B, and D) or an anti-SHC antibody (C). Proteins were fractionated on a 7.5% SDS-PAGE gel, and proteins were detected using either an antiphosphotyrosine antibody (A, B, and C) or an anti-Grb2 antibody (D). A, SP induces increased tyrosine phosphorylation of EGFR and multiple co-precipitating proteins in a time-dependent manner. B, SP induces increased association of the adapter protein SHC with the EGFR in a time-dependent manner. C, SP induces tyrosine phosphorylation of the three isoforms of the protein SHC. D, SP induces the association of the adapter protein Grb2 with the EGFR in a time-dependent manner. Data presented are representative of four different experiments that give similar results.

Ligand-induced autophosphorylation of EGFR causes recruitment of adapter proteins such as SHC, Grb2, Sos, and Src family kinases to the cytoplasmic domain of EGFR (26). As shown in Fig. 3, SP induces tyrosine phosphorylation of several proteins that co-precipitated with EGFR. To identify the proteins co-precipitating with EGFR, blots were stripped and reprobed with anti-SHC, Grb2, and c-Src antibodies. Increased association with activated EGFR (Fig. 3B) and tyrosine phosphorylation (Fig. 3C) of SHC in response to SP was detected. Furthermore, the adapter protein Grb2 was also associated with the activated EGFR complex in response to SP (Fig. 3D). However, there was no increase in the amount of c-Src associated with the EGFR complex, and no tyrosine phosphorylation of c-Src in response to SP was evident (data not shown).

SP Induces Kinase Activity of Erk2 via Transactivation of the EGFR-- The association between Grb2 and EGFR is an essential step in the activation of the Ras/mitogen-activated protein kinase pathway, ultimately initiating DNA synthesis. Because SP triggers both EGFR transactivation and MAPK activation, we tested the functional role of EGFR in SP-induced activation of the MAPK Erk2. As determined by increased phosphorylation of myelin basic protein in vitro, Erk2 was activated following U-373 MG stimulation with SP and with the RTK ligand EGF (Fig. 4). In cells transfected with a dominant negative EGFR plasmid lacking kinase activity (pCHER), however, Erk2 activation by both SP and EGF was considerably inhibited (Fig. 4).

View larger version (20K): [in this window] [in a new window]   Fig. 4.   SP- induced MAPK activity requires transactivation of the EGFR. U-373 MG cells were either mock-transfected or transfected with pCHER, serum-starved overnight, and treated without or with either SP (10 nM) for 10 min or EGF (10 ng/ml) for 3 min. Cells were lysed with RIPA buffer, and Erk2 proteins were precipitated using an anti-Erk2 antibody. Kinase reactions were performed using the immunoprecipitated proteins in the presence of myelin basic protein (MBP) as substrate for Erk2 kinases and [32P]ATP. Reactions were loaded onto a 12% SDS-PAGE gel, and Erk2 activity was determined by autoradiography. In mock-transfected cells (lanes 1-3), SP (lane 2), and EGF (lane 3) induced Erk2 kinase activity when compared with control (lane 1). In cells transfected with pCHER (lanes 4-6), SP (lane 5), and EGF (lane 6) induced Erk2 kinase activity is greatly inhibited when compared with respective nontransfected cells (lanes 2 and 3). This suggests that the presence of a dominant negative EGFR lacking kinase activity partially inhibits SP- and EGF-induced MAPK activity. Data presented are representative of three different experiments.

SP-induced DNA Synthesis Is Mediated through EGFR Transactivation-- To further determine the functional role of SP-induced EGFR tyrosine phosphorylation and activation, we tested the effect of pCHER and tyrphostin AG1478 (a specific inhibitor of EGFR) on SP-induced DNA synthesis. As shown in Fig. 5A, SP and EGF induced DNA synthesis in mock-transfected U-373 MG cells as indicated by increased [³H]thymidine incorporation. SP- and EGF-induced DNA synthesis was significantly inhibited in pCHER-transfected U-373 MG cells. Furthermore, incubation of cells with tyrphostin AG1478 almost completely inhibited both SP- and EGF-induced DNA synthesis (Fig. 5A). The requirement for EGFR in SP-induced DNA synthesis was also tested in CHO cells that do not normally express either NK-1 and EGF receptors. In these experiments CHO cells were mock-transfected, transfected with NK-1 alone, or with NK-1 and EGFR together, and then SP-induced DNA synthesis was determined. As shown in Fig. 5B, SP and EGF did not stimulate DNA synthesis in either normal CHO cells or CHO cells expressing NK-1. However, in CHO cells expressing NK-1 and EGFR, exposure to SP significantly increased DNA synthesis (Fig. 5B). These data indicate that the kinase activity of the EGFR is required for SP to induce DNA synthesis.

View larger version (29K): [in this window] [in a new window]   Fig. 5.   SP-induced DNA synthesis requires transactivation of the EGFR. A, mock- or pCHER-transfected U-373 MG cells were serum-starved and, where indicated, incubated for 15 min with tyrphostin AG1478 (T-1478) (1 µg/ml), before exposure to SP (10 nM) or EGF (10 ng/ml). Cells were incubated in the presence of [3H]thymidine (0.5 µCi/well) for 6 h. Cells were collected and placed in scintillation vials to determine [3H]thymidine incorporation, and the results were expressed as counts/min (cpm). As expected, SP and EGF significantly increased DNA synthesis in mock-transfected cells. SP- and EGF-induced DNA synthesis in mock-transfected cells was significantly inhibited in the presence of tyrphostin. In cells transfected with pCHER, SP- and EGF- induced DNA synthesis was significantly prevented. This suggests that inhibition of EGFR kinase activity prevents SP- and EGF- induced DNA synthesis. Data are expressed as mean ± S.E. and are representative of four different experiments each with triplicate determinations. * indicates p < 0.01 versus control in mock-transfected cells, + indicates p < 0.01 versus SP-treated mock transfected cells. B, mock or pEGFR-transfected control and NK-1-transfected CHO cells were serum-starved for 18 h and then exposed to SP (10 nM) or EGF (10 ng/ml) in the presence of [3H]thymidine (0.5 µCi/well). [3H]Thymidine incorporation was determined after 6 h, and results were expressed as counts/min (cpm). SP and EGF failed to stimulate DNA synthesis in CHO cells expressing the NK-1 receptor, whereas SP significantly increased DNA synthesis in CHO cells co-expressing NK-1 and EGFR. Data are expressed as mean ± S.E. and are representative of four different experiments each with triplicate determinations. * indicates p < 0.01 versus control in mock- and EGFR-transfected CHO cells.

A PTX-sensitive G Subunit Is Involved in SP-induced Transactivation of EGFR-- NK-1 associates with several G protein  subunits such as G_q/11, G_o, and G_s (22, 23). We next determined whether SP-mediated EGFR activation and subsequent DNA synthesis was mediated through a PTX-sensitive G subunit. As shown in Fig. 6, pretreatment of cells with PTX completely prevented SP-induced EGFR tyrosine phosphorylation. In addition, SP-induced DNA synthesis was inhibited by pretreatment of cells with PTX (Fig. 7), whereas PTX had no effect on EGF-induced DNA synthesis. These results suggest that SP-induced activation of EGFR involves a PTX-sensitive G subunit.

View larger version (12K): [in this window] [in a new window]   Fig. 6.   SP-induced EGFR phosphorylation requires a PTX-sensitive G subunit. U-373 MG cells were serum-starved and incubated with PTX overnight. Cells were then exposed to either SP (10 nM) or EGF (10 ng/ml) for 10 or 7 min, respectively. Cells were then lysed with RIPA buffer, and soluble proteins were subjected to immunoprecipitation with an anti-EGFR. Proteins were then fractionated on a 7.5% SDS-PAGE gel and detected using an antiphosphotyrosine antibody. SP (lane 4) and EGF (lane 2) both increased EGFR phosphorylation as compared with control (lane 1), whereas pretreatment of cells with PTX completely prevented SP-induced EGFR tyrosine phosphorylation (lane 5). PTX had no effect on EGF-induced EGFR phosphorylation (lane 3). Data presented are representative of three different experiments that give similar results.

View larger version (18K): [in this window] [in a new window]   Fig. 7.   SP-induced DNA synthesis requires a PTX-sensitive G subunit. U-373 MG cells were serum-starved and incubated with PTX overnight. Then they were incubated with SP (10 nM) or EGF (10 ng/ml) in the presence of [3H]thymidine (0.5 µCi/well). After 6 h, cells were washed, scraped, and placed in scintillation vials to determine [3H]thymidine incorporation. Results were expressed as counts/min (cpm). SP and EGF significantly increased DNA synthesis over control. SP-induced DNA synthesis was completely prevented by PTX treatment, whereas PTX had no significant effect on EGF induced DNA synthesis. Data are expressed as mean ± S.E. and are representative of three different experiments each with triplicate determinations. * indicates p < 0.01 versus SP-treated control cells.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The rapid phosphorylation and consequent activation or inactivation of proteins leading to DNA synthesis generally characterizes growth factor-mediated cell proliferation (26). The peptide SP stimulates mitogenesis in various cell types including human astrocytoma cell lines (29, 32) and activates pathways involving protein kinase signaling such as MAPKs (29). Here we report for the first time that SP binding to its NK-1 receptor induces transactivation of EGFR. We also present evidence that SP-induced MAPK activation and mitogenesis involves the formation of an activated EGFR signaling complex.

Upon ligand binding, the dimerized EGFR activates a sequence of phosphorylation events initiated by autophosphorylation. Activated EGFR creates a docking area for adapter proteins such as Grb2, Sos, and SHC (11). Formation of such a complex on the intracellular domain of EGFR activates the Ras/Raf signaling pathway, resulting in MAPK activity (39). Recently, several GPCR agonists have been shown to transactivate RTKs, such as the EGFR, by inducing tyrosine phosphorylation and association of adapter proteins (40). For example, Daub et al. (27) recently reported that lysophosphatidic acid or thrombin binding to their GPCRs induce EGFR tyrosine phosphorylation and its association with activated Grb2. We show that SP causes a biphasic tyrosine phosphorylation of EGFR (Fig. 3A) and its association with SHC and Grb2 (Fig. 3, B and D, respectively). Recent studies also suggest that EGF-induced EGFR activation may complex with Src tyrosine kinase and that this interaction is required for MAPK activation (41). Although we did not demonstrate any association of c-Src with EGFR following SP-mediated transactivation, we cannot exclude that other Src family kinases (i.e. fyn, lck, yes) are involved.

Besides its well known effects as a neurotransmitter and inflammatory mediator, SP has been recently recognized as a mitogenic factor. SP-induced mitogenesis has been described in several cell types, suggesting that this effect may be relevant in physiological or pathological conditions. The functional role of SP-induced EGFR transactivation was investigated using a dominant-negative EGFR construct lacking kinase activity (pCHER) (35) and by co-expressing EGF and NK-1 receptors in CHO cells. Previous studies indicate that SP is capable of inducing MAPK activation (29). MAPKs such as Erk1 and Erk2 are key components of signaling pathways involved in the regulation of cell growth and differentiation, mediating such events as c-fos induction and DNA synthesis (42, 43). In our system, SP-induced Erk2 kinase activity and cell proliferation were drastically inhibited in U-373 MG cells transfected with pCHER or treated with an EGFR pharmacological inhibitor. Previous studies also reported that SP-induced cell proliferation was substantially inhibited in the presence of tyrosine kinase inhibitors (29). The inability of pCHER to completely prevent SP or EGF-induced Erk2 kinase activity in our experiments is probably because of a transfection efficiency of less than 100% in the U-373 MG cells (data not shown). Furthermore, previous studies indicated that LPA- and thrombin-induced DNA synthesis was only partially inhibited by a specific EGFR inhibitor (27). It is possible that different signaling pathways activated by SP lead to MAPK activation and cell proliferation, because SP-induced EGFR phosphorylation in U-373 MG cells in our experiments was biphasic (Fig. 3A). Several pathways may be involved in the mechanism of NK-1 receptor and EGFR trans-activation. For example, stimulation of G protein-coupled receptors can induce transactivation of EGFR trough activation of transmembrane metalloproteinases leading to the extracellular processing of a transmembrane growth factor precursor and release of the mature growth factor, which interacts with the binding domain of the EGFR (44). Furthermore GPCR agonists (LPA, thrombin, and SP) may regulate proliferative responses via activation of different RTKs, ion channels, or regulation of second messenger systems (45).

When GPCR ligands bind to their receptor, G proteins associated with the cytoplasmic portion of the receptor become activated (46). In the inactive form, three G protein subunits (, , and ) associate with each other, and the  subunit is constitutively bound to GDP. Once activated, the  subunit exchanges GDP for GTP and the subunit dissociates, allowing the monomer  subunit and the dimer subunit to initiate various metabolic pathways by activating specific second messengers. There are four subfamilies of  subunits grouped together based on sequence homology: _o, _i, _q, and ₁₂. An increasing number of reports have indicated that certain isoforms of G protein subunits can activate the MAPK pathway. For example, Van Brunsen et al. (47) demonstrated that G subunits activate MAPKs by a pathway common to RTKs containing an activated Shc-Grb2-Sos complex, whereas Daub et al. (36) reported that both G_q and G_i proteins coupled to GPCRs can promote activation of the MAPK pathway. The exact nature of the G subunit(s) involved in SP/SPR signal transduction is not completely lucid. So far, G_q/ll, G_s, and G_o protein subunits have been shown to associate with the SPR (23). Because PTX pretreatment completely abolished SP-induced EGFR phosphorylation and subsequent DNA synthesis in U-373 MG cells, our results suggest that a PTX-sensitive G_i subunit mediates these events. Activated G_i subunits have been implicated in SPR-mediated increases in arachidonic acid levels (48). Indeed, SP stimulates arachidonic acid metabolism into prostaglandins primarily of the PGE2 family that have been implicated in mitogenic activities such as fibroblast growth (25) and angiogenesis (49). However, it is unknown whether the pathways by which SP induces arachidonic acid metabolism and EGFR tyrosine phosphorylation converge to stimulate DNA synthesis and cell proliferation.

Cell attachment and migration, proliferation, and differentiation are the three steps involved in wound healing. Although SP is a potent positive regulator of cell proliferation, SP activity has also been correlated with cell attachment and migration, and, more loosely, with cellular differentiation. SP, in synergy with insulin-like growth factor-1, has been shown to induce rabbit corneal epithelial cell attachment to fibronectin matrix, promote subsequent migration, and increase levels of the integrins ₅ and ₁ mRNA (50, 51). Furthermore, SP has been implicated in angiogenesis by inducing the formation of capillary-like structures from endothelial cells placed on a Matrigel matrix (52). Thus, SP may play an important role in many processes associated with wound healing. Our observations demonstrating a "cross-talk" between NK-1 receptor and EGFR in cell proliferation may be pathophysiologically relevant because up-regulation of both NK-1 and EGFR occurs during diseases characterized by ulcer formations. For example, NK-1 and EGFR protein and mRNA levels are up-regulated in the colonic mucosa of patients with chronic inflammatory disorders such as inflammatory bowel disease (16, 53).

Although a cross-talk between NK-1 and EGFR during chronic inflammatory processes may be an important mechanism in stimulating tissue healing, enhanced sensitivity to SP-induced proliferative effects may eventually lead to aberrant growth and neoplasia formation. Indeed, many chronic inflammatory diseases, such as ulcerative colitis, are associated with a high incidence of cancer (54). In addition, blood vessels surrounding human colorectal cancers overexpress NK-1 (55). Furthermore, target genes of SP have been shown to include certain oncogenes such as c-myc, a well documented promoter of cell growth (29). As demonstrated by Luo et al. (29), SP is able to increase expression of c-myc mRNA and protein levels in U-373 MG cells. Understanding the mechanisms by which SP and NK-1 activate genes and proteins involved in cell growth and proliferation may provide insights into the possible involvement of SP/NK-1 in carcinogenesis.

In conclusion, we report for the first time that SP, acting via the NK-1, is able to induce formation of an activated EGFR complex containing the adapter proteins SHC and Grb2. SP-induced kinase activity of the MAPK Erk2, as well as SP-induced DNA synthesis, require functional EGFR kinase activity. Finally, SP-induced EGFR transactivation and subsequent DNA synthesis occurs via activation of a PTX-sensitive G_i subunit. These data suggest that the signaling pathways between SP receptor and growth factor RTKs converge at a very upstream point, increasing the possible pathways by which SP may tightly control the intracellular mechanisms leading to cell proliferation, and eventually, tissue restoration.

	FOOTNOTES

^* 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.

To whom correspondence should be addressed: Beth Israel Deaconess Medical Center, Division of Gastroenterology, Dana 501, 330 Brookline Ave., Boston, MA 02215. Tel.: 617-667-1259; Fax: 617-667-2767; E-mail: icastagl@ux1.unipd.it.

Published, JBC Papers in Press, June 8, 2000, DOI 10.1074/jbc.M003990200

	ABBREVIATIONS

The abbreviations used are: SP, substance P; GPCR, G protein-coupled receptor; EGF, epidermal growth factor; RTK, receptor tyrosine kinases; PTX, pertussis toxin; FCS, fetal calf serum; PBS, phosphate-buffered saline; RIPA, radioimmune precipitation buffer; PAGE, polyacrylamide gel electrophoresis; Erk, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; CHO, Chinese hamster ovary; NK-1, neurokinin-1; SPR, substance P receptor.

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