EGF receptor function is required in late G1 for cell cycle progression induced by bombesin and bradykinin

Chintda Santiskulvong, James Sinnett-Smith, and Enrique Rozengurt

Department of Medicine, School of Medicine and Molecular Biology Institute, University of California, Los Angeles, California 90095-1786


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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We examined the role of epidermal growth factor (EGF) receptor (EGFR) tyrosine kinase activation in G protein-coupled receptor (GPCR) agonist-induced mitogenesis in Swiss 3T3 and Rat-1 cells. Addition of EGFR tyrosine kinase inhibitors (e.g., tyrphostin AG-1478) abrogated bombesin-induced extracellular signal-regulated kinase (ERK) activation in Rat-1 cells but not in Swiss 3T3 cells, indicating the importance of cell context in determining the role of EGFR in ERK activation. In striking contrast, treatment with tyrphostin AG-1478 markedly (~70%) inhibited DNA synthesis induced by bombesin in both Swiss 3T3 and Rat-1 cells. Similar inhibition of bombesin-induced DNA synthesis in Swiss 3T3 cells was obtained using four structurally different inhibitors of EGFR tyrosine kinase. Furthermore, kinetic analysis indicates that EGFR function is necessary for bombesin-induced mitogenesis in mid-late G1 in both Swiss 3T3 and Rat-1 cells. Our results indicate that EGFR kinase activity is necessary in mid-late G1 for promoting the accumulation of cyclins D1 and E and implicate EGFR function in the coupling of GPCR signaling to the activation of the cell cycle.

epidermal growth factor receptor transactivation; tyrphostin AG-1478; G protein-coupled receptors; Swiss 3T3 cells; mitogen-activated protein kinases


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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NEUROPEPTIDES STIMULATE DNA synthesis and cell proliferation in cultured cells and are implicated as growth factors in a variety of fundamental processes, including development, inflammation, tissue regeneration, and neoplastic transformation (40-42). In particular, bombesin and its mammalian counterpart gastrin-releasing peptide (GRP) bind to a G protein-coupled receptor (GPCR; see Refs. 1 and 64) that promotes Galpha q-mediated activation of beta -isoforms of phospholipase C (20, 25, 33) to produce the following two second messengers: inositol 1,4,5-trisphosphate that mobilizes Ca2+ from internal stores and diacylglycerol that activates protein kinase C (PKC; see Refs. 5, 13, 31, 63). The bombesin/GRP GPCR also interacts with members of the G12 family leading to Rho-dependent actin remodeling and tyrosine phosphorylation of focal adhesion proteins, including focal adhesion kinase and paxillin (3, 5, 32, 38, 39, 46, 52, 65). Subsequently, bombesin induces striking activation of serine phosphorylation cascades involving p42mapk [extracellular signal-regulated kinase (ERK)-2]/p44mapk(ERK-1) and p70S6K (30, 47, 50, 60), leading to increased expression of immediate early response genes, stimulation of cell cycle events including induction of cyclin D1, and subsequent cell proliferation (5, 28, 43, 44, 53). The mechanism(s) linking the early signaling pathways to the subsequent stimulation of cell cycle events remains incompletely understood.

The epidermal growth factor (EGF) receptor (EGFR) is a single-pass transmembrane tyrosine kinase that is activated by direct binding of at least six EGF-related ligands that are synthesized as transmembrane precursors (4, 18). Recently, it has been shown that a variety of GPCR agonists also induce a rapid increase in EGFR tyrosine autophosphorylation in several cell types (7-9, 22, 57), a receptor cross-talk mediated by rapid proteolytic generation of EGFR ligands at the cell surface (36) termed transactivation (4, 36). In Rat-1 cells and in COS-7 cells, inhibition of EGFR tyrosine kinase activity has been shown to prevent ERK activation in response to GPCR agonists (8). EGFR transactivation is thought to induce ERK activation via a well-defined mechanism involving SOS-Grb2-mediated accumulation of Ras-GTP, which then recruits Raf-1 to the plasma membrane and activates a kinase cascade comprising Raf, mitogen/extracellular signal-regulated kinase, and the ERKs (29, 48). It is widely thought that EGFR contributes to GPCR mitogenic signaling via activation of the ERK cascade (36). However, it is increasingly recognized that GPCR stimulation of ERK-1/2 is mediated by multiple pathways, and cell context has emerged as a critical determinant of the mechanism(s) involved (10, 42, 45). For example, bombesin stimulation of Swiss 3T3 cells neither increased the fraction of Ras bound to GTP (30) nor stimulated Raf-1 activity (50) but promoted ERK activation via a PKC-dependent pathway (35, 50). In contrast, the same agonist induced Ras, Raf, and ERK activation through a PKC-independent pathway in Rat-1 cells (6). It is also relevant that the role of the ERK cascade in EGF-induced DNA synthesis has been questioned in at least some cell types (54, 58). It is not known, therefore, whether the contribution of EGFR to cell cycle progression and DNA synthesis induced by GPCR activation is mediated exclusively through the ERK cascade and if it is also dependent on cell context.

The experiments presented here were designed to elucidate the role of EGFR function in GPCR-induced mitogenesis in both Swiss 3T3 and Rat-1 cells. Our results demonstrate that bombesin stimulates ERK activation via EGFR in Rat-1 cells but through an EGFR-independent pathway in Swiss 3T3 cells, confirming the importance of cell context in determining the pathway leading to ERK activation. In striking contrast, we found that EGFR tyrosine kinase is required for bombesin-induced DNA synthesis in both Swiss 3T3 cells and Rat-1 cells. Kinetic analysis indicates that EGFR function is required in mid-late G1, i.e., after ERK activity returned to baseline levels. An attractive model is that transactivated EGFR function is necessary to promote the accumulation of cyclins D1 and E, thus implicating EGFR in the coupling of GPCR signaling to the activation of the cell cycle.


    MATERIALS AND METHODS
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Cell culture. Stock cultures of Swiss 3T3 cells were maintained at 37°C in DMEM, supplemented with 10% FBS in a humidified atmosphere containing 10% CO2 and 90% air. For experimental purposes, cells were plated in 100-mm dishes at 6 × 105 cells/dish or 35-mm dishes at 1 × 105 cells/dish and were grown in DMEM containing 10% FBS for 7-9 days until they became confluent and quiescent (49).

Rat-1 cells transfected with the bombesin/GRP receptor (5, 6) were maintained at 37°C in DMEM, supplemented with 5% FBS and 0.5 mg/ml G-418 in a humidified atmosphere containing 10% CO2 and 90% air. For experimental purposes, cells were plated in 35-mm dishes at 1 × 105 cells/dish and were grown in DMEM containing 5% FBS for 6 days until confluent. The cultures were then switched to serum-free DMEM for 24 h before use.

DNA synthesis measurements. Confluent and quiescent cultures of Swiss 3T3 cells were washed two times with DMEM and incubated with DMEM-Waymouth's medium (1:1 vol/vol) containing 25 ng/ml insulin, (0.2 µCi/ml, 1 µM) [3H]thymidine, and various additions as described in the legends for Figs. 1-7. Insulin, at the concentration used in these experiments, does not induce any significant increase of DNA synthesis in Swiss 3T3 cells. However, the addition of this hormone enhanced the mitogenic response induced by bombesin in these cells, as documented in previous studies (43) and further confirmed in the course of the experiments presented in this study. After 40 h of incubation at 37°C, cultures were washed two times with PBS and incubated in 5% TCA at 4°C for 20 min to remove acid-soluble radioactivity, washed with ethanol, and solubilized in 1 ml of 2% Na2CO3 and 0.1 M NaOH, as previously described (11, 43). The acid-insoluble radioactivity was determined by scintillation counting in 6 ml of Beckman Readysafe.


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Fig. 1.   A and B: effect of tyrphostin AG-1478, compound 56, GF-109203X, and Ro-31-8220 on bombesin-induced p42mapk [extracellular signal-regulated kinase (ERK)-2] and p44mapk (ERK-1) phosphorylation in Rat-1 (A) and Swiss 3T3 (B) cells. Confluent and quiescent Rat-1 cells and Swiss 3T3 cells were washed two times with DMEM and preincubated for 1 h in this medium, either without or with 250 nM tyrphostin AG-1478 (AG), 250 nM compound 56 (56), or the PKC inhibitors 3.5 µM GF-109203X (GF1), or 2.5 µM Ro-31-8220 (Ro), as indicated. Cultures were subsequently stimulated with either 10 nM bombesin or 5 ng/ml epidermal growth factor (EGF) for 5 min, lysed in 2× SDS-PAGE sample buffer, and analyzed by SDS-PAGE and immunoblotting with phospho (p)-ERK antibody as described in MATERIALS AND METHODS. The results presented are typical of 4 independent experiments. Arrows indicate the positions of ERK-1 and ERK-2. C and D: tyrphostin AG-1478 inhibits bombesin-induced mitogenesis in Rat-1 cells. C: effect of AG-1478 on [3H]thymidine incorporation induced by increasing concentrations of bombesin. Confluent and quiescent cultures of Rat-1 cells were washed and incubated at 37°C in 2 ml DMEM-Waymouth's medium containing increasing concentrations of bombesin either without (open circle ) or with () 250 nM tyrphostin AG-1478. After 18 h, 1 µCi/ml [3H]thymidine was added to the cultures, and 4 h later DNA synthesis was assessed by measuring the [3H]thymidine incorporated in acid-precipitable material. Results are expressed as a percentage of the maximal incorporation induced by bombesin (achieved at either 100 or 300 nM) and are means ± SE (n = 3). Data shown are typical of 3 independent experiments performed in triplicate. D: tyrphostin AG-1478 inhibits bombesin-stimulated DNA synthesis in Rat-1 cells in a dose-dependent manner. Confluent and quiescent cultures of Rat-1 cells were washed and incubated at 37°C in 2 ml of DMEM-Waymouth's medium containing 10 nM bombesin and increasing concentrations of AG-1478, as indicated. After 18 h, [3H]thymidine was added to the cultures, and 4 h later DNA synthesis was assessed by measuring the [3H]thymidine incorporated into acid-precipitable material. Results are representative of 3 independent experiments and are expressed as a percentage of the incorporation induced by 10 nM bombesin. Data are shown as the means ± SE (n = 3). In these experiments, the addition of medium containing 10 nM bombesin resulted in 25,316 ± 7,064 counts/min (cpm) incorporated for each dish.



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Fig. 2.   Tyrphostin AG-1478 inhibits bombesin (Bom)-induced mitogenesis in Swiss 3T3 cells. A: tyrphostin AG-1478 inhibits bombesin-stimulated DNA synthesis in Swiss 3T3 cells in a dose-dependent manner. Confluent and quiescent cultures of Swiss 3T3 cells were washed and incubated at 37°C in 2 ml of DMEM-Waymouth's medium containing 10 nM bombesin, 25 ng/ml insulin, [3H]thymidine, and increasing concentrations of tyrphostin AG-1478, as indicated. After 40 h of incubation, DNA synthesis was assessed by measuring the [3H]thymidine incorporated in acid-precipitable material. Results are representative of 3 independent experiments and are expressed as a percentage of the incorporation induced by 10 nM bombesin. Data are shown as means ± SE (n = 3). In this experiment, addition of medium containing 10 nM bombesin and 25 ng/ml insulin resulted in 32,967 ± 2,612 cpm incorporated/dish. B: time course of DNA synthesis stimulated by bombesin without (open circle ) and with () AG-1478. Confluent and quiescent cultures of Swiss 3T3 cells were washed and incubated at 37°C in 2 ml DMEM-Waymouth's medium containing 10 nM bombesin, 25 ng/ml insulin, and [3H]thymidine without (open circle ) or with () 250 nM tyrphostin AG-1478. Cumulative DNA synthesis was assessed at various times by measuring the [3H]thymidine incorporated into acid-precipitable material. The results presented here are typical of 3 independent experiments. Results are expressed as a percentage of the incorporation induced by 10 nM bombesin with 25 ng/ml insulin at 48 h, and data are shown as means ± SE (n = 3). In this experiment, the addition of medium containing 10 nM bombesin and 25 ng/ml insulin resulted in 35,209 ± 1,303 cpm incorporated/dish. C: effect of AG-1478 on [3H]thymidine incorporation induced by increasing concentrations of bombesin. Confluent and quiescent cultures of Swiss 3T3 cells were washed and incubated at 37°C in 2 ml DMEM-Waymouth's medium containing increasing concentrations of bombesin, 25 ng/ml insulin, and [3H]thymidine either without (open circle ) or with () 250 nM tyrphostin AG-1478. After 40 h, DNA synthesis was assessed by measuring the [3H]thymidine incorporated into acid-precipitable material. Results are expressed as a percentage of the incorporation induced by 100 nM bombesin with 25 ng/ml insulin, and data are shown as means ± SE (n = 3). Data shown are typical of 3 independent experiments. In this experiment, the addition of medium containing 100 nM bombesin and 25 ng/ml insulin resulted in 38,856 ± 2,312 cpm incorporated/dish. D: tyrphostin AG-1478 prevents movement of cells from Go/G1 to S and G2 + M. Confluent and quiescent cultures of Swiss 3T3 cells were washed and incubated at 37°C in 2 ml of DMEM-Waymouth's medium containing 10 nM bombesin and 25 ng/ml insulin in the absence (Bom) or presence (Bom + AG-1478) of 250 nM tyrphostin AG-1478. Cultures incubated in DMEM-Waymouth's medium in the absence of any other addition (control) or in the presence of 250 nM tyrphostin AG-1478 but without agonists (AG-1478) are also presented for comparison. After 40 h of incubation, all cultures were subjected to flow cytometric analysis as described in MATERIALS AND METHODS. Graphs represent the no. of cells (y-axis) as a function of the amount of DNA (fluorescence intensity) on the x-axis and are typical of 3 independent experiments. All other details were as described in MATERIALS AND METHODS.



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Fig. 3.   The EGF receptor (EGFR) tyrosine kinase inhibitors compound 56, PD-153035, and PD-158780 inhibit bombesin-induced DNA synthesis in a dose-dependent manner. A, C, and D: confluent and quiescent cultures of Swiss 3T3 cells were washed and incubated at 37°C in 2 ml of DMEM-Waymouth's medium containing 10 nM bombesin with 25 ng/ml insulin, [3H]thymidine, and increasing concentrations of the inhibitors, as indicated. After 40 h, DNA synthesis was assessed by measuring the [3H]thymidine incorporated into acid-precipitable material. Results are expressed as a percentage of the incorporation induced by 10 nM bombesin with 25 ng/ml insulin, and data are shown as means ± SE (n = 3). In these experiments, the addition of medium containing 10 nM bombesin and 25 ng/ml insulin resulted in 44,781 ± 1,169 cpm incorporated/dish in A and C and 50,451 ± 2,644 cpm in E. B: time course of DNA synthesis stimulated by bombesin without (open circle ) and with () compound 56. Confluent and quiescent cultures of Swiss 3T3 cells were washed and incubated at 37°C in 2 ml DMEM-Waymouth's medium containing 10 nM bombesin with 25 ng/ml insulin without (open circle ) or with () 250 nM compound 56 and [3H]thymidine. DNA synthesis was assessed at various times by measuring the [3H]thymidine incorporated into acid-precipitable material. The results presented are typical of 3 independent experiments. Results are expressed as a percentage of the incorporation induced by 10 nM bombesin with 25 ng/ml insulin at 48 h, and data are means ± SE (n = 3). In this experiment, the addition of medium containing 10 nM bombesin and 25 ng/ml insulin resulted in 43,531 ± 611 cpm incorporated/dish. E: tyrphostin AG-1478 blocks bombesin-induced autophosphorylation of the EGFR. Quiescent Swiss 3T3 cells were washed two times with DMEM and incubated without (-) or with (+) 250 nM tyrphostin AG-1478 for 1 h. Cells were then incubated with 10 nM bombesin or 1 ng/ml EGF for 1 min and lysed. The cell lysates were immunoprecipitated (IP) with anti-EGFR polyclonal antibody followed by Western blotting (WB) with anti-Tyr(P) mAb. Results shown are typical of 2 independent experiments. Arrow indicates the position of the EGFR. All other details were as described in MATERIALS AND METHODS. F: insulin does not induce tyrosine phosphorylation of the EGFR. Quiescent Swiss 3T3 cells were washed and treated with or without 10 nM bombesin or 1 µg/ml of insulin at 37°C for 1 min. Cells were then lysed, and the lysates were immunoprecipitated with anti-EGFR polyclonal antibody followed by Western blotting with anti-Tyr(P) mAb. PY, phosphotyrosine. The autoradiogram shown is representative of 2 independent experiments.



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Fig. 4.   EGFR tyrosine kinase activity late in G1 is essential for bombesin-stimulated entry into DNA synthesis in Swiss 3T3 cells. A: time course of ERK-1 and ERK-2 phosphorylation in Swiss 3T3 cells stimulated with bombesin. Confluent and quiescent cells were washed two times with DMEM and stimulated with 10 nM bombesin and 25 ng/ml insulin for the indicated times. Cells were then lysed in 2× SDS-PAGE sample buffer and analyzed by SDS-PAGE and immunoblotting with phospho-ERK antibody. The results presented are typical of 2 independent experiments. Arrows indicate the positions of ERK-1 and ERK-2. B: time course of bombesin-induced autophosphorylation of the EGFR. Confluent and quiescent cells were washed two times with DMEM and stimulated with 10 nM bombesin for the indicated times. Cells were then lysed, and the lysates were immunoprecipitated with anti-EGFR polyclonal antibody followed by Western blotting with anti-Tyr(P) mAb. The autoradiogram shown is representative of 2 independent experiments. Equal EGFR levels in the immunoprecipitates were verified by stripping the membranes and reprobing with anti-EGFR polyclonal antibody (B, bottom). C and D: EGFR tyrosine kinase activity late in G1 is essential for bombesin-induced entry into DNA synthesis. Confluent and quiescent cultures of Swiss 3T3 cells were washed and incubated at 37°C in 2 ml DMEM-Waymouth's medium containing 10 nM bombesin with 25 ng/ml insulin and [3H]thymidine. Either 250 nM tyrphostin AG-1478 (B) or 250 nM compound 56 (C) was added at various times after the addition of bombesin, as indicated. In all cases, DNA synthesis was assessed by measuring the [3H]thymidine incorporated into acid-precipitable material after 40 h of incubation. The results presented are typical of 3 independent experiments. Results are expressed as a percentage of the incorporation induced by 10 nM bombesin with 25 ng/ml insulin (open bar), and data are means ± SE (n = 3). In these experiments, the addition of medium containing 10 nM bombesin and 25 ng/ml insulin resulted in 35,755 ± 1,842 cpm incorporated/dish. All other details were as described in MATERIALS AND METHODS.



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Fig. 5.   EGFR tyrosine kinase activity late in G1 is essential for bombesin-stimulated entry into DNA synthesis in Rat-1 cells. A: time course of ERK-1 and ERK-2 phosphorylation in Rat-1 cells stimulated with bombesin. Confluent and quiescent cells were washed two times with DMEM and were stimulated with 10 nM bombesin for the indicated times. Cells were then lysed in 2× SDS-PAGE sample buffer and analyzed by SDS-PAGE and immunoblotting with phospho-ERK antibody. The results presented are typical of 2 independent experiments. Arrows indicate the positions of ERK-1 and ERK-2. B: EGFR tyrosine kinase activity late in G1 is essential for bombesin-induced entry into DNA synthesis. Confluent and quiescent cultures of Rat-1 cells were washed and incubated at 37°C in 2 ml DMEM-Waymouth's medium containing 10 nM bombesin. Tyrphostin AG-1478 at 250 nM was added at various times after the addition of bombesin, as indicated. In all cases, DNA synthesis was assessed by adding [3H]thymidine at 18 h and by measuring the [3H]thymidine incorporated into acid-precipitable material after an additional 4 h of incubation. The results presented here are typical of 3 independent experiments. Results are expressed as a percentage of the incorporation induced by 10 nM bombesin, and data are means ± SE (n = 3). In this experiment, the addition of medium containing 10 nM bombesin and 25 ng/ml insulin resulted in 19,473 ± 1,854 cpm incorporated/dish. All other details were as described in MATERIALS AND METHODS.



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Fig. 6.   Role of EGFR in bradykinin-induced DNA synthesis. A: time course of ERK-1 and ERK-2 phosphorylation in Swiss 3T3 cells stimulated with bradykinin. Confluent and quiescent cells were washed two times with DMEM, stimulated with 50 nM bradykinin and 250 ng/ml insulin for the indicated times, and then lysed in 2× SDS-PAGE sample buffer. B: tyrphostin AG-1478 and compound 56 do not inhibit bradykinin-stimulated ERK-1 and ERK-2 activation in Swiss 3T3 cells. Confluent and quiescent Swiss 3T3 cells were washed two times with DMEM and preincubated for 1 h in this medium, either without or with 250 nM tyrphostin AG-1478 or 250 nM compound 56, as indicated. Cultures were subsequently stimulated with either 50 nM bradykinin and 250 ng/ml insulin or 5 ng/ml EGF for 5 min and then lysed in 2× SDS-PAGE sample buffer. In both A and B, the samples were analyzed by SDS-PAGE and immunoblotting with phospho-ERK antibody as described in MATERIALS AND METHODS. The results presented are typical of 4 independent experiments. Arrows indicate the positions of ERK-2 and ERK-1. C: tyrphostin AG-1478 inhibits bradykinin-stimulated [3H]thymidine uptake in Swiss 3T3 cells in a dose-dependent manner. Confluent and quiescent cultures of Swiss 3T3 cells were washed and incubated at 37°C in 2 ml of DMEM-Waymouth's medium containing 50 nM bradykinin with 250 ng/ml insulin, [3H]thymidine, and increasing concentrations of tyrphostin AG-1478, as indicated. After 40 h, DNA synthesis was assessed by measuring the [3H]thymidine incorporated into acid-precipitable material. Results are representative of 3 independent experiments and are expressed as a percentage of the incorporation induced by 50 nM bradykinin with 250 ng/ml insulin. Data are means ± SE (n = 3). In this experiment, the addition of medium containing 50 nM bradykinin and 250 ng/ml insulin resulted in 37,552 ± 1,180 cpm incorporated/dish. D: EGFR tyrosine kinase activity late in G1 is essential for bradykinin-induced entry into DNA synthesis. Confluent and quiescent cultures of Swiss 3T3 cells were washed and incubated at 37°C in 2 ml DMEM-Waymouth's medium containing 50 nM bradykinin, 250 ng/ml insulin, and [3H]thymidine. Tyrphostin AG-1478 (250 nM) was added at various times after bradykinin addition, as indicated. In all cases, DNA synthesis was assessed by measuring the [3H]thymidine incorporated into acid-precipitable material after 40 h of incubation. Results are expressed as a percentage of the incorporation induced by 50 nM bradykinin with 250 ng/ml insulin, and data are means ± SE (n = 3). In this experiment, the addition of medium containing 50 nM bradykinin and 250 ng/ml insulin resulted in 29,196 ± 3,085 cpm incorporated/dish. All other details were as described in MATERIALS AND METHODS.



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Fig. 7.   Role of EGFR in G protein-coupled receptor (GPCR)-stimulated cyclin D1 and E accumulation. A: effect of AG-1478 or compound 56 on bombesin-stimulated cyclin D1 and cyclin E protein accumulation. Quiescent Swiss 3T3 cells were washed two times and incubated for 18 h with 10 nM bombesin and 25 ng/ml insulin in the absence or in the presence of 250 nM tyrphostin AG-1478 or 250 nM compound 56 added either concomitantly or 6 h after the agonists, as indicated. Cells were lysed in 2× SDS-PAGE sample buffer and analyzed by SDS-PAGE and immunoblotting with either cyclin D1 or cyclin E mAbs as described in MATERIALS AND METHODS. The autoradiograms shown are representative of 3 independent experiments. The bars represent the quantification of the levels of cyclin D1 and cyclin E expression analyzed by scanning densitometry. Results are expressed as a percentage of the maximum increase in expression and are the means ± SE of 3 independent experiments. B: tyrphostin AG-1478 and compound 56 inhibit bradykinin-stimulated cyclin D1 and cyclin E accumulation. Cells were treated with 50 nM bradykinin and 250 ng/ml insulin in the absence or in the presence of 250 nM tyrphostin AG-1478 or 250 nM compound 56 added either concomitantly or 6 h after the agonists. Cells were lysed in 2× SDS-PAGE sample buffer and were analyzed by SDS-PAGE and immunoblotting with either cyclin D1 or cyclin E mAbs. The autoradiograms shown are representative of 2 independent experiments.

Confluent and quiescent cultures of Rat-1 cells were washed two times with DMEM and incubated with DMEM-Waymouth's medium (1:1 vol/vol) and various additions as described in the legends for Figs. 1-7. After 18 h of incubation at 37°C, [3H]thymidine (0.2 µCi/ml, 1 µM) was added, and the cultures were incubated for a further 4 h at 37°C, as previously described (5). Incorporated [3H]thymidine was determined as described above for Swiss 3T3 cells. The difference in the protocols used to determine [3H]thymidine incorporation in Swiss 3T3 and Rat-1 cells reflects differences in cell cycle kinetics between these cell lines.

Flow cytometric analysis. The proportion of cells in the G0/G1, S, G2, and M phases of the cell cycle was determined by flow cytometric analysis. Confluent and quiescent cultures of Swiss 3T3 cells were washed two times with DMEM and incubated with DMEM-Waymouth's medium (1:1 vol/vol) containing various additions as described in the legends for Figs. 1-7. After 18 h of incubation at 37°C, 1 µM colchicine was added. After an additional 22 h of incubation, the cultures were washed three times with PBS containing 4 mM EDTA. Cells were detached by treatment with trypsin (0.025%), suspended in DMEM containing 10% FBS, centrifuged at 1,000 g for 5 min, and resuspended and washed three times in PBS. Cells (106) in a volume of 200 µl were stained by adding 800 µl of a solution containing propidium iodide (50 µg/ml), sodium citrate (1 mg/ml), and Triton X-100 (0.1%). The stained chromosomal DNA was kept on ice for 15 min and analyzed on a FACScalabar (Becton-Dickinson).

Assay of p42mapk (ERK-2) and p44mapk (ERK-1) activation. Quiescent cultures of Swiss 3T3 cells grown on 35-mm dishes were washed two times with DMEM and incubated for 1 h at 37°C with inhibitors, as indicated. The cultures were then treated with the appropriate agonists as indicated and incubated for a further 5 min at 37°C. The stimulation was terminated on ice by aspirating the medium and solubilizing the cells with 200 µl of 2× SDS-PAGE sample buffer. The samples were boiled for 10 min, resolved by 10% SDS-PAGE, and transferred to Immobilon-P membranes. Activation of ERK-1 and ERK-2 occurs through phosphorylation of specific threonine and tyrosine residues (48), resulting in slower migrating forms in SDS-PAGE. These activated forms were monitored by using a specific anti-phospho-ERK-1/ERK-2 monoclonal antibody (mAb; New England Biolabs) that recognizes only the activated forms phosphorylated on Thr202 and Tyr204.

EGFR transactivation. Quiescent cultures of Swiss 3T3 cells grown in 100-mm dishes (1-2 × 106 cells) were washed two times with serum-free DMEM, equilibrated at 37°C for at least 10 min, and then treated with bombesin with or without inhibitors, as indicated. The stimulation was terminated on ice by aspirating the medium and solubilizing the cells in 1 ml of ice-cold lysis buffer (10 mM Tris · HCl, pH 7.6, 5 mM EDTA, 50 mM NaCl, 1% Triton X-100, 30 mM Na2H2P2O7, 50 mM NaF, 0.1 mM Na3VO4, and 1 mM Pefablock sc). Cell lysates were clarified by centrifugation at 14,000 rpm for 10 min. The supernatants were transferred to new microtubes, and proteins were immunoprecipitated at 4°C for at least 4 h using anti-EGFR polyclonal antibody linked to protein A-agarose. The precipitates were washed three times with ice-cold lysis buffer and subsequently solubilized in 2× SDS-PAGE sample buffer (200 mM Tris · HCl, pH 6.8, 1 mM EDTA, 6% SDS, 2 mM EDTA, 4% 2-mercaptoethanol, and 10% glycerol). Samples were boiled and resolved by 8% SDS-PAGE and then transferred to Immobilon-P membranes. Tyrosine-phosphorylated EGFR was detected using 4G10 anti-Tyr(P) mAb.

Assay of cyclin D1 and cyclin E accumulation. Quiescent cultures of Swiss 3T3 cells grown on 100-mm dishes were washed two times with DMEM and incubated with DMEM-Waymouth's medium (1:1 vol/vol) containing various additions as described in the legends for Figs. 1-7. The cultures were then incubated at 37°C for 18 h, and the stimulation was then terminated on ice by aspirating the medium and solubilizing the cells with 500 µl of 2× SDS-PAGE sample buffer. The samples were boiled for 10 min, resolved by 10% SDS-PAGE, and transferred to Immobilon-P membranes. Cyclin D1 and cyclin E were detected using specific mAbs to cyclin D1 and cyclin E (1 µg/ml).

Immunoblotting. After SDS-PAGE, proteins were transferred to Immobilon-P membranes. For detection of proteins, membranes were blocked using 5% nonfat dried milk in PBS (pH 7.2) and then incubated for at least 2 h with the desired antibodies diluted in PBS (pH 7.2) containing 3% nonfat dried milk. Bound primary antibodies to immunoreactive bands were visualized by enhanced chemiluminescence detection with horseradish peroxidase-conjugated anti-mouse, anti-rabbit, or anti-goat antibodies.

Materials. Bombesin, bradykinin, EGF, phorbol 12,13-dibutyrate, insulin, and tyrphostin AG-1478 were obtained from Sigma (St. Louis, MO). Platelet-derived growth factor and [3H]thymidine were from Amersham Pharmacia Biotech (Piscataway, NJ). Protein A-agarose was from Boehringer Mannheim (Indianapolis, IN). The monoclonal anti-cyclin D1, anti-cyclin E, and polyclonal anti-EGFR were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). 4G10 anti-Tyr(P) mAb was from Upstate Biotechnology (Lake Placid, NY). Anti-phospho-p44/p42mapk mAb was obtained from New England Biolabs (Beverly, MA). PD-153035, compound 56, GF-109203X, and Ro-31-8220 were purchased from Calbiochem (San Diego, CA). PD-158780 was a kind gift from Dr. W. Leopold (Parke-Davis Pharmaceutical Research). All other materials were of the highest grade available.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Role of EGFR in bombesin-induced ERK activation in Swiss 3T3 cells and Rat-1 cells. GPCR stimulation of ERK-1/2 is mediated by multiple pathways, and cell context has emerged as a critical determinant of the mechanism(s) involved (10, 42). For example, our previous results demonstrated that bombesin induces ERK activation through a PKC-dependent pathway in Swiss 3T3 cells (50) but via a PKC-independent pathway in Rat-1 cells (6). Here, we examined the contribution of EGFR function to the activation of the ERK pathway induced by bombesin in quiescent cultures of these cell lines. After 5 min of agonist stimulation, the cells were lysed, and the active forms of ERK-1 (p44mapk) and ERK-2 (p42mapk) were detected by Western blotting using an antibody that recognizes the dually phosphorylated forms of these enzymes.

As shown in Fig. 1A, ERK activation induced by bombesin in Rat-1 cells transfected with the bombesin/GRP GPCR was virtually abolished by treatment with the selective EGFR tyrosine kinase inhibitors tyrphostin AG-1478 (26) or compound 56 (2, 14, 19), at concentrations that completely blocked EGF-induced ERK activation in these cells. Conversely, addition of the PKC inhibitors GF-109203X (55) or Ro-31-8220 (62) did not affect bombesin-induced ERK activation in Rat-1 cells, in agreement with our previous results indicating that PKC is not a major pathway leading to ERK activation in these cells (6).

In striking contrast to the results obtained with Rat-1 cells, treatment of Swiss 3T3 cells with either tyrphostin AG-1478 or compound 56, at concentrations that abolished ERK activation induced by addition of EGF to these cells, did not prevent ERK activation in response to bombesin. In addition, GF-109203X or Ro-31-8220 almost entirely suppressed bombesin-induced ERK activation in Swiss 3T3 cells (Fig. 1B), also in agreement with previous results (35, 50).

Thus bombesin stimulates ERK activation via an EGFR-dependent (but PKC-independent) pathway in Rat-1 cells, whereas the same agonist promotes ERK activation through an EGFR-independent (but PKC-dependent) pathway in Swiss 3T3 cells.

Effect of tyrphostin AG-1478 on bombesin-induced DNA synthesis in Rat-1 and Swiss 3T3 cells. If EGFR contributes to cell cycle progression in response to GPCR agonists primarily via ERK activation, suppression of EGFR tyrosine kinase activity should prevent bombesin-induced DNA synthesis in Rat-1 cells but not in Swiss 3T3 cells. As shown in Fig. 1C, bombesin-induced [3H]thymidine incorporation into DNA in Rat-1 cells transfected with the bombesin/GRP receptor was strikingly inhibited by addition of tyrphostin AG-1478 (250 nM), even at the higher concentration of bombesin tested (300 nM). Tyrphostin AG-1478 inhibited bombesin-induced DNA synthesis in a concentration-dependent manner; maximal inhibition (~70%) was obtained at 250 nM (Fig. 1D). These results indicate that EGFR function is essential for bombesin-induced mitogenesis in Rat-1 cells.

Next, we determined whether EGFR function is also required for bombesin-induced mitogenesis in Swiss 3T3 cells. In contrast to the results obtained with ERK activation in these cells, treatment with tyrphostin AG-1478 (10-500 nM) markedly inhibited DNA synthesis induced by bombesin in a concentration-dependent fashion (Fig. 2A). Maximal inhibition (~70%) was achieved at 250-500 nM. We verified that tyrphostin AG-1478 at 250 nM suppressed EGF-stimulated DNA synthesis but did not interfere with the mitogenic response induced by platelet-derived growth factor in Swiss 3T3 cells (results not shown).

Stimulation of DNA synthesis in response to bombesin started after a lag period (G1) of 12-14 h and reached a maximal level after 40-48 h of incubation (Fig. 2B). Tyrphostin AG-1478 markedly inhibited bombesin-induced DNA synthesis at all times examined, up to 48 h of incubation. We also determined [3H]thymidine incorporation into Swiss 3T3 cells stimulated with increasing concentrations of bombesin (0.1-100 nM) in the absence or in the presence of tyrphostin AG-1478 (250 nM). As shown in Fig. 2C, tyrphostin AG-1478 inhibited the reinitiation of DNA synthesis, even at the highest concentration of bombesin tested (100 nM).

Bombesin induces stimulation of DNA synthesis in Swiss 3T3 cells in the absence of other factors, but its mitogenic activity is, however, strikingly potentiated by addition of insulin (43). A significant increase in bombesin-stimulated DNA synthesis can be obtained with insulin at a concentration as low as 25 ng/ml, as used in our assays. At this concentration, insulin alone did not stimulate any significant ERK activation (59) or DNA synthesis (43) in Swiss 3T3 cells. We verified that addition of tyrphostin AG-1478 markedly inhibited DNA synthesis induced by bombesin in the absence of insulin (results not shown).

To corroborate that the inhibitory effect of tyrphostin AG-1478 on [3H]thymidine incorporation reflects a decrease in DNA replication through the S phase of the cell cycle rather than a decrease in the transport and/or phosphorylation of [3H]thymidine, we used flow cytometric analysis to determine the proportion of cells in the various phases of the cell cycle. As shown in Fig. 2D, addition of tyrphostin AG-1478 strikingly reduced the movement from G1 to S and G2 plus M induced by bombesin in Swiss 3T3 cells. Thus tyrphostin AG-1478 markedly inhibits bombesin-induced progression through the cell cycle in Swiss 3T3 cells.

Multiple EGFR tyrosine kinase inhibitors prevent bombesin-induced DNA synthesis in Swiss 3T3 cells. To further test the requirement of EGFR tyrosine kinase in bombesin-mediated mitogenesis in Swiss 3T3 cells, we examined the effect of other potent and selective inhibitors of EGFR tyrosine kinase. The results, shown in Fig. 3A demonstrate that compound 56, at nanomolar concentrations, was as effective as tyrphostin AG-1478 in decreasing bombesin-induced DNA synthesis (~70% inhibition) in Swiss 3T3 cells. Addition of compound 56 markedly inhibited bombesin-induced DNA synthesis at all times examined, up to 48 h of incubation (Fig. 3B).

Treatment with increasing concentrations (5-1,000 nM) of other selective EGFR tyrosine kinase inhibitors, including PD-153035 (compound 32) and PD-158780 (2, 7, 14, 15, 19, 37), also prevented bombesin stimulation of DNA synthesis in a concentration-dependent fashion (Fig. 3, C and D) but did not interfere with bombesin-induced ERK activation in parallel cultures (results not shown). Thus the use of four structurally different inhibitors of EGFR tyrosine kinase establishes a requirement for EGFR tyrosine kinase activity in bombesin signaling of mitogenesis.

Bombesin induces EGFR transactivation in Swiss 3T3 cells. The results presented in Figs. 1-3 indicating that EGFR function is necessary for bombesin-induced cell cycle progression but not ERK activation in Swiss 3T3 cells prompted us to verify that bombesin stimulates EGFR tyrosine phosphorylation in Swiss 3T3 cells. Lysates of these cells treated with 10 nM bombesin were immunoprecipitated with anti-EGFR polyclonal antibody (Fig. 3E). The immunocomplexes were analyzed by SDS-PAGE followed by Western blotting using anti-Tyr(P) mAb. As shown in Fig. 3E, addition of bombesin to Swiss 3T3 cells induced a marked increase in the tyrosine phosphorylation of the EGFR. Treatment of these cells with 250 nM tyrphostin AG-1478 completely prevented the increase in tyrosine phosphorylation of EGFR induced by either bombesin or EGF. Western blotting with anti-EGFR antibody of the same samples (after stripping) confirmed that similar amounts of EGFR protein were recovered after bombesin stimulation (results not shown). The results presented in Fig. 3E demonstrate that bombesin stimulates EGFR tyrosine kinase activity in Swiss 3T3 cells. In contrast to bombesin, addition of insulin at a concentration as high as 1 µg/l did not induce any detectable increase in EGFR tyrosine phosphorylation in Swiss 3T3 cells (Fig. 3F).

EGFR tyrosine kinase activity is necessary in late G1. Subsequently, we performed a kinetic analysis to determine at which point in G1 phase the EGFR pathway is required for bombesin-induced mitogenesis in Swiss 3T3 cells. Cultures of these cells stimulated with bombesin received tyrphostin AG-1478 (Fig. 4C) or compound 56 (Fig. 4D) either simultaneously with the peptides (time 0) or at various times after stimulation, as indicated in Fig. 4, C and D (i.e., 1, 2, 3, 4, 5, 6, or 14 h after bombesin). In all cases, DNA synthesis was determined by measuring [3H]thymidine incorporation after 40 h of incubation. The level of DNA synthesis in the absence of either tyrphostin AG-1478 or compound 56 is shown in Fig. 4.

We verified, in parallel cultures, that bombesin-induced ERK activation gradually declined to baseline values after 4 h of exposure to the peptide (Fig. 4A). We also examined bombesin stimulation of EGFR tyrosine phosphorylation as a function of time. Lysates of Swiss 3T3 cells treated for various lengths of time with 10 nM bombesin were immunoprecipitated with anti-EGFR polyclonal antibody. The immunocomplexes were analyzed by SDS-PAGE followed by Western blotting using anti-Tyr(P) mAb. An increased level of EGFR tyrosine phosphorylation could be detected as early as 1 min but, in contrast to the time course of ERK activation, EGFR tyrosine phosphorylation remained elevated, even after 6 h of bombesin addition (Fig. 4B). Western blotting with anti-EGFR antibody of the same samples (after stripping) confirmed that similar amounts of EGFR protein were recovered after bombesin stimulation (Fig. 4B).

In agreement with the preceding results, addition of either tyrphostin AG-1478 or compound 56 together with bombesin at time 0 markedly inhibited DNA synthesis (Fig. 4, C and D). If EGFR activity is required for bombesin-induced mitogenesis during the first 6 h of stimulation, a gradual decrease in the degree of inhibition of DNA synthesis produced by either AG-1478 or compound 56 should be observed when these compounds are added to the cultures 1, 2, 3, 4, 5, or 6 h postbombesin. Conversely, if EGFR function is required after the first 6 h of bombesin stimulation (e.g., in mid-late G1), a similar degree of inhibition of DNA synthesis produced by AG-1478 and compound 56 should be seen when these compounds are added together with bombesin or 6 h after bombesin. As shown in Fig. 4, C and D, addition of EGFR tyrosine kinase inhibitors (tyrphostin AG-1478 or compound 56) as late as 6 h after bombesin stimulation (i.e., after ERK activity returned to basal levels) prevented bombesin-dependent DNA synthesis almost as effectively as when the inhibitors were added concomitantly with bombesin. Addition of the EGFR tyrosine kinase inhibitors as late as 14 h after bombesin (i.e., at the end of G1, see Fig. 2B) produced a smaller but significant inhibitory effect. These results indicate that EGFR tyrosine kinase activity is essential in mid-late G1 for bombesin-stimulated DNA synthesis in Swiss 3T3 cells.

We also examined at which point in G1 EGFR function is necessary for bombesin-induced mitogenesis in Rat-1 cells transfected with the bombesin/GRP receptor. The experimental design was similar to that depicted in Fig. 4 for Swiss 3T3 cells. We verified that bombesin-induced ERK activation in Rat-1 cells gradually declined to baseline values after 2 h of exposure to the peptide (Fig. 5A). Addition of tyrphostin AG-1478 after ERK activity returned to basal levels (i.e., 3-6 h postbombesin stimulation) also prevented bombesin-induced DNA synthesis in Rat-1 cells almost as effectively as when the inhibitor was added concomitantly with bombesin (Fig. 5B).

The results presented in Figs. 4 and 5 indicate that EGFR tyrosine kinase activity is essential in mid-late G1 for bombesin-stimulated DNA synthesis in both Swiss 3T3 cells and Rat-1 cells.

Role of EGFR in bradykinin-induced ERK activation and DNA synthesis. We next examined the role of EGFR in ERK-1/2 activation and stimulation of DNA synthesis induced by activation of a different GPCR in Swiss 3T3 cells. Addition of bradykinin in the presence of insulin, a mitogenic combination for Swiss 3T3 cells (23, 61), induced a rapid and striking ERK activation that peaked within 4 min of incubation and thereafter gradually declined, reaching baseline values after 3 h of exposure to the peptides (Fig. 6A). Treatment with either tyrphostin AG-1478 or compound 56, at concentrations that abolished EGF-induced ERK-1/2 activation, had little inhibitory effect on ERK activation stimulated by bradykinin (Fig. 6B). These results indicate that bradykinin, like bombesin, induces ERK activation in Swiss 3T3 cells primarily through an EGFR-independent pathway.

Bradykinin acts synergistically with insulin to induce DNA synthesis in Swiss 3T3 cells (23, 61). As shown in Fig. 6C, addition of tyrphostin AG-1478 inhibited bradykinin-induced DNA synthesis in a concentration-dependent manner. Maximal inhibition (~85%) was achieved at a concentration of 62.5 nM.

We also determined whether tyrphostin AG-1478 inhibits DNA synthesis induced by bradykinin and insulin in Swiss 3T3 cells in mid-late G1. Cultures of these cells received tyrphostin AG-1478 either simultaneously or at various times after bradykinin and insulin. As indicated in Fig. 6D, addition of tyrphostin AG-1478 after bradykinin-induced ERK activation returned to baseline levels (i.e., 3-6 h poststimulation) prevented bradykinin-induced DNA synthesis almost as effectively as when the inhibitor was added concomitantly with the stimuli. Addition of the EGFR tyrosine kinase inhibitor as late as 14 h after bradykinin and insulin, i.e., well after bradykinin-induced ERK activation returned to basal levels, inhibited DNA synthesis by ~60% (Fig. 6D). The results obtained with bradykinin-stimulated Swiss 3T3 cells substantiate the notion that EGFR function is required in mid-late G1 for GPCR-induced DNA synthesis.

Effect of inhibitors of EGFR tyrosine kinase activity on cyclin D1 and E expression in GPCR-stimulated cells. One of the critical events elicited by mitogenic stimulation leading to cell cycle activation in fibroblasts is the increase in the level of cyclin D1 (51). Our previous studies demonstrated that bombesin induces accumulation of cyclin D1 in mid-G1 in Swiss 3T3 cells (28, 60), and the results presented above indicate that EGFR tyrosine kinase activity is required in mid-late G1, a time that correlates with cyclin D1 expression. Consequently, we examined whether suppression of EGFR tyrosine kinase activity interferes with the accumulation of cyclin D1 protein induced by bombesin in these cells.

As shown in Fig. 7A, left, treatment with either AG-1478 or compound 56 strikingly inhibited (~75% inhibition) the increase in the level of cyclin D1 protein induced by addition of bombesin. In view of the kinetic analysis shown in Fig. 4, we also tested whether AG-1478 or compound 56 could prevent the induction of cyclin D1 when they were added 6 h after bombesin and insulin. The results presented in Fig. 7A, left, demonstrate that this is in fact the case; when these inhibitors of EGFR tyrosine kinase activity were added 6 h after agonist stimulation, they reduced cyclin D1 expression as effectively as when they were added concomitantly with bombesin and insulin.

To corroborate that EGFR contributes to cyclin D1 protein accumulation in GPCR-induced mitogenesis, we determined the effect of the inhibitors of EGFR tyrosine kinase on cyclin D1 expression promoted by bradykinin and insulin. As shown in Fig. 7B, left, addition of either tyrphostin AG-1478 or compound 56 dramatically inhibited cyclin D1 expression induced by bradykinin and insulin. These inhibitors of EGFR tyrosine kinase activity also prevented cyclin D1 expression when added 6 h after peptide stimulation. These findings with bradykinin are in agreement with those obtained with bombesin and provide further support to the conclusion that transactivated EGFR function contributes to GPCR-stimulated progression of the cell cycle in mid-late G1.

Cyclin D1 is known to promote cyclin E expression by inducing E2F-dependent cyclin E gene transcription, and recent findings indicate that cyclin E is the major downstream target of cyclin D1 (16). Accordingly, addition of either tyrphostin AG-1478 or compound 56 markedly inhibited cyclin E accumulation in Swiss 3T3 cells stimulated by either bombesin or bradykinin (Fig. 7, A and B, right). When these inhibitors were added 6 h after stimulation, they reduced cyclin E accumulation almost as effectively as when they were added concomitantly with the agonists. Taken together, these findings identify the EGFR as an important element in coupling GPCR mitogenic signaling to the expression of cyclins D1 and E.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Activation of a variety of GPCRs has been shown to stimulate an increase in the tyrosine kinase activity of the EGFR, a process termed transactivation (4, 36). The EGFR has been proposed to mediate Ras/ERK-1/2 activation in response to GPCR activation (36), and the ERK pathway has been implicated in mitogenic signal transduction by many stimuli (56). Therefore, it is widely assumed that EGFR transactivation contributes to GPCR-stimulated mitogenesis through ERK activation.

It is also increasingly recognized that the contribution of EGFR transactivation to ERK activation depends on cellular context (9, 12, 24, 27). In agreement with this notion, we show here that bombesin induces rapid ERK activation via an EGFR-dependent mechanism in Rat-1 cells, whereas the same agonist promotes EGFR-independent ERK activation in Swiss 3T3 cells. We reason that, if EGFR contributes to cell cycle progression in response to GPCR agonists primarily via ERK activation, suppression of EGFR tyrosine kinase activity should prevent bombesin-induced DNA synthesis in Rat-1 cells but not in Swiss 3T3 cells. In the present study, we used these cellular model systems to test this prediction and thus clarify the role of EGFR in GPCR signaling of DNA synthesis.

Our results produced several lines of evidence indicating that a functional EGFR pathway is necessary for the stimulation of cell cycle progression induced by bombesin in both Rat-1 cells and Swiss 3T3 cells. Specifically, tyrphostin AG-1478, a selective inhibitor of EGFR tyrosine kinase activity, markedly and selectively inhibited DNA synthesis in Swiss 3T3 cells monitored either by [3H]thymidine incorporation or by flow cytometric analysis. Furthermore, we demonstrated that four structurally different inhibitors of EGFR tyrosine kinase decreased DNA synthesis induced via the bombesin/GRP receptor to the same degree, supporting the specificity of these inhibitory effects. In addition, we demonstrated that the EGFR pathway is also required for stimulation of DNA synthesis in Swiss 3T3 cells via a different endogenously expressed GPCR, namely the bradykinin receptor. Interestingly, treatment with inhibitors of EGFR tyrosine kinase activity, at concentrations that profoundly reduced agonist-stimulated mitogenesis, did not prevent ERK activation in response to either bombesin or bradykinin in these cells. The results presented here indicate that EGFR function contributes to GPCR-induced mitogenesis through an ERK-independent mechanism.

To further examine the role of functional EGFR in G1 cell cycle progression, we performed a kinetic analysis in which the EGFR tyrosine kinase was chemically inhibited at various times after bombesin stimulation. We found that addition of either tyrphostin AG-1478 or compound 56 after ERK activity returned to basal levels still prevented bombesin-induced DNA synthesis in either Swiss 3T3 cells or in Rat-1 cells almost as effectively as when these inhibitors were added concomitantly with bombesin. These results demonstrate that EGFR tyrosine kinase activity late in G1 is essential for entry into DNA synthesis stimulated by bombesin and thus further separate the ERKs from the contribution of EGFR function to cell cycle progression induced by GPCR agonists.

Progression from G1 to the S phase of the cell cycle depends on the periodic expression of cyclins that regulate the activity of the cyclin-dependent kinases (51). One of the crucial events elicited by mitogenic stimulation leading to cell cycle activation in fibroblasts is the increase in the level of the G1 cyclins, cyclin D1 and E. Our previous studies demonstrated that bombesin induces the accumulation of cyclin D1 in mid-late G1 (28, 60), and the results presented above indicate that EGFR tyrosine kinase activity is required in mid-late G1. Consequently, we hypothesized that EGFR contributes to agonist-induced mitogenesis by promoting the expression of cyclins, and we focused on the accumulation of cyclin D1 because this is a critical early event in the progression from G1 to the S phase of the cell cycle (34, 51). In agreement with this hypothesis, inhibition of EGFR tyrosine kinase activity prevented bombesin-induced induction of cyclin D1 in Swiss 3T3 cells. Cyclin D1 accumulation was also prevented when the inhibitors of EGFR tyrosine kinase were added in mid-G1, in accord with the kinetic analysis positioning the requirement of EGFR for GPCR-induced DNA synthesis also in mid-G1.

Cyclin E has emerged as a major downstream target of cyclin D1 (16). Accordingly, we found that inhibitors of EGFR tyrosine kinase also prevented the increase in the expression of cyclin E induced by the GPCR agonists bombesin and bradykinin. Thus our findings identify the EGFR as an important element in coupling GPCR mitogenic signaling to the expression of cyclins D1 and E, a key event in the activation of the cell cycle. Recently, ERK-independent pathways initiated by activation of phosphatidylinositol 3-kinase and Ral GTPases have been implicated in transcriptional activation of the cyclin D1 promoter activity (17, 21). These pathways could be potential mediators of transactivated EGFR function in mid-G1, a proposition that warrants further experimental work.

In conclusion, the results presented here have several important implications for our understanding of the role of the EGFR pathway in GPCR-induced mitogenesis. Specifically, we propose that EGFR contributes to GPCR mitogenesis in mid-late G1. An attractive model is that transactivated EGFR function is required for inducing the accumulation of cyclins D1 and E, implicating the EGFR as an important element in coupling GPCR signaling to activation of the cell cycle.


    ACKNOWLEDGEMENTS

C. Santiskulvong and J. Sinnett-Smith contributed equally to this work.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-56930 and DK-55003.

Address for reprint requests and other correspondence: E. Rozengurt, 900 Veteran Ave., Warren Hall, Rm. 11-124, Dept. of Medicine, School of Medicine, Univ. of California, Los Angeles, CA 90095-1786 (E-mail: erozengurt{at}mednet.ucla.edu).

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.

Received 11 January 2001; accepted in final form 4 April 2001.


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