Growth Factor-specific Signaling Pathway Stimulation and Gene Expression Mediated by ErbB Receptors*,

Colleen SweeneyDagger §, Douglas Fambrough||, Christine Huard, A. John DiamontiDagger **, Eric S. LanderDagger Dagger , Lewis C. CantleyDagger , and Kermit L. Carraway IIIDagger **

From the Dagger  Department of Cell Biology, Harvard Medical School and Division of Signal Transduction, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215, the  Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, Massachusetts 02142, and the Dagger Dagger  Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

Received for publication, January 22, 2001, and in revised form, March 19, 2001

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The mechanisms by which receptor tyrosine kinases (RTKs) utilize intracellular signaling pathways to direct gene expression and cellular response remain unclear. A current question is whether different RTKs within a single cell target similar or different sets of genes. In this study we have used the ErbB receptor network to explore the relationship between RTK activation and gene expression. We profiled growth factor-stimulated signaling pathway usage and broad gene expression patterns in two human mammary tumor cell lines expressing different complements of ErbB receptors. Although the growth factors epidermal growth factor (EGF) and neuregulin (NRG) 1 similarly stimulated Erk1/2 in MDA-MB-361 cells, EGF acting through an EGF receptor/ErbB2 heterodimer preferentially stimulated protein kinase C, and NRG1beta acting through an ErbB2/ErbB3 heterodimer preferentially stimulated Akt. The two growth factors regulated partially overlapping yet distinct sets of genes in these cells. In MDA-MB-453 cells, NRG1beta acting through an ErbB2/ErbB3 heterodimer stimulated prolonged signaling of all pathways examined relative to NRG2beta acting through the same heterodimeric receptor species. Surprisingly, NRG1beta and NRG2beta also regulated partially overlapping but distinct sets of genes in these cells. These results demonstrate that the activation of different RTKs, or activation of the same RTKs with different ligands, can lead to distinct profiles of gene regulation within a single cell type. Our observations also suggest that the identity and kinetics of signaling pathway usage by RTKs may play a role in the selection of regulated genes.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Polypeptide growth factor hormones act on individual cells within tissues or on pluripotent stem cells to induce responses that contribute to development, tissue maintenance and repair, or disease state. Depending on cell type and the identity of the growth factor presented to the cell, a variety of responses are possible, including proliferation, differentiation, apoptosis, survival, migration, and fate specification. A fundamental question in growth factor signaling concerns the mechanisms by which specificity is generated: how do different growth factors elicit different responses within a single cell type, and how do different cell types respond differently to a single growth factor?

Cellular responses to growth factors are mediated by cell surface receptor tyrosine kinases (RTKs)1 that possess an intrinsic protein tyrosine kinase activity. Growth factor binding stimulates receptor dimerization and autophosphorylation on several tyrosine residues, and phosphorylated tyrosines provide docking sites for intracellular signaling proteins that contain Src homology 2 or protein tyrosine binding domains (1, 2). The sequence-specific recruitment of signaling proteins couples activated RTKs to intracellular signaling cascades that propagate signals to the nucleus to elicit initial changes in gene expression. Growth factor-stimulated expression of immediate early genes (IEGs), or genes whose induction does not require protein synthesis, then lays the foundation for the ultimate cellular response (3-5).

Over the past decade, several signaling cascades connecting activated RTKs to the nucleus have been characterized (6). For example, activation of the Erk serine/threonine kinases follows the recruitment of the Grb2/SOS complex to receptors and the stimulation of the Ras GTPase. Erk phosphorylation of ternary complex factors leads to the stimulation of genes containing serum response elements in their promoters. Phosphoinositide 3'-kinase (PI3K) activation by growth factors leads to the phosphorylation of the forkhead and CREB transcription factors through stimulation of the serine/threonine kinase Akt, as well as the activation of nuclear factor kappa B through regulation of Ikappa B kinase. These factors are thought to play a prominent role in the regulation of genes involved in cellular survival and apoptosis. Members of the STAT family of transcription factors may be phosphorylated directly by growth factor receptors to regulate cytokine-inducible genes, and phospholipase C-gamma 1 can mediate the response of calcium-sensitive genes and protein kinase C (PKC) targets to growth factor stimulation.

Through the phosphorylation of multiple tyrosine residues, each RTK has the capacity to stimulate several different signaling cascades. By independently targeting different subsets of genes or by acting in a combinatorial manner to regulate gene expression, the different signaling pathways have the potential to mediate a variety of cellular responses. However, many different RTKs utilize identical signaling pathways in mediating diverse responses to growth factors. Hence, a major question has become how signaling cascades couple growth factor receptors with specific gene expression patterns to mediate a diverse array of cellular responses.

One model suggests that RTKs send general signals through a limited number of pathways, and that these signals are interpreted in the target cell by context-specific transcription factors. A series of recent studies with invertebrate model systems support this view. Analysis of the regulatory regions of marker genes for specific late stage developmental events in Drosophila melanogaster suggests that gene expression is markedly dependent on the immediate environment and developmental history of the target cell (7-9). In addition, a single RTK in Caenorhabditis elegans uses a different signaling pathway to mediate the development of two different tissues (10), suggesting that some pathways may be dispensable depending on cellular context. In a mammalian system utilizing immortalized mouse fibroblasts, activation of fibroblast growth factor receptor-1 and the PDGFbeta receptor led to identical profiles of gene expression (11). These observations point to a model whereby pathways emanating from growth factor-activated RTKs are generic and simply provide a "go" signal to pre-primed tissue precursor cells to alter gene expression and initiate tissue development (12).

However, this model does not easily explain how multipotent stem cells differentially respond to different RTK-binding growth factors, a common theme in mammalian systems. For example, epidermal growth factor (EGF) receptor ligands act as survival/proliferation factors for many neural precursor cells whereas other growth factors such as nerve growth factor, brain-derived neurotrophic factor, and the neuregulins stimulate their differentiation into glial cells or neurons (13). Cultured rat PC12 pheochromacytoma cells are a biochemical model for neural differential RTK signaling. It has been suggested that a quantitative difference in signaling pathway usage, the prolonged activation of the Erk1/2 pathway by nerve growth factor relative to EGF, accounts for the differentiation activity of this factor toward these cells (14).

To begin to explore the link between receptor tyrosine kinase activation and gene expression in mammalian cells, we have focused on the ErbB RTK signaling network, a model for the generation of specificity and diversity in growth factor signaling. Signaling through ErbB receptor family members has been observed to play roles in a variety of developmental processes (15), including cardiac and neural development (16-19), glial cell development (20-22), the remodeling of mammary tissue during pregnancy (23, 24), and the development of the neuromuscular junction (25-27). The EGF-like ligand NRG has been demonstrated to promote cellular proliferation (28), differentiation (29), migration (30), apoptosis (31), survival (32), and fate (33), depending on cell type and the NRG isoform used in stimulation. The broad range of cellular responses to EGF-like growth factors makes the ErbB network uniquely suited for addressing fundamental questions pertaining to RTK signaling specificity and cellular response.

The network consists of four known RTKs (EGF receptor, ErbB2, ErbB3, and ErbB4) and more than a dozen EGF-like ligands. Each of the receptors is predicted to couple to unique complements of signaling cascades (34-36). This, together with their ability to undergo an extensive array of ligand-induced receptor homo- and heterodimerization events, has been proposed to underlie the diverse array of cellular and developmental responses attributed to the EGF-like growth factors in mammals (15, 36, 37). In this study we use different EGF-like ligands to differentially stimulate ErbB receptors. We observe that that different ErbB dimeric pairs differentially stimulate intracellular signaling pathways and gene expression. Interestingly, the same dimeric receptor pair, when activated by two different growth factors, also leads to differences in gene expression.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Culture and Immunoprecipitation Experiments-- MDA-MB-361 and MDA-MB-453 cells were from ATCC and maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Immunoprecipitation experiments were carried out as described previously (38, 39). Briefly, cells at 50-60% confluence were serum-starved overnight in Dulbecco's modified Eagle's medium, 0.1% fetal bovine serum, and then treated with 30 nM growth factor for 2 min at 37 °C. After rinsing twice with ice-cold phosphate-buffered saline, cells were lysed in co-immunoprecipitation buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 1 mM MgCl2, 1% Nonidet P-40, 10% glycerol, 1 mM Na3VO4, 1 mM NaF, 1 mM ZnCl2, 10 mM beta -glycerophosphate, 5 mM tetrasodium pyrophosphate, 1 mM phenylmethylsulfonyl fluoride, and 4 µg/ml each aprotinin, leupeptin, and pepstatin), and cleared lysates were immunoprecipitated with 1.5 µg of antibodies. Precipitating receptor monoclonal antibodies used were: anti-epidermal growth factor receptor Ab-1 (clone 528), anti-ErbB2 Ab-4, anti-ErbB3 Ab-4, and anti-ErbB4 Ab-1, all from NeoMarkers. Precipitating signaling protein antibodies used were: rabbit anti-Grb2 and rabbit anti-Cbl from Santa Cruz, rabbit anti-Shc and mouse anti-SHP2 from Transduction Labs, and rabbit anti-p85 described previously (40). Precipitates were washed three times in wash buffer (20 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 1 mM Na3VO4, 1 mM NaF, 10 mM beta -glycerophosphate, 5 mM tetrasodium pyrophosphate, 0.2 mM phenylmethylsulfonyl fluoride, and 4 µg/ml each aprotinin, leupeptin, and pepstatin). Precipitated proteins were resolved by 6-10% gradient SDS-polyacrylamide gel electrophoresis and immunoblotted using enhanced chemiluminescence for detection. Blotting antibodies used were: anti-phosphotyrosine RC20, mouse anti-SHP2, and mouse anti-Shc from Transduction Laboratories, rabbit anti-Grb2 and rabbit anti-Cbl from Santa Cruz, and rabbit anti-p85 described previously (40).

Signaling Pathway Activation-- Cells in 60-mm dishes were grown to 50-60% confluence, serum-starved overnight, and then treated with 30 nM growth factor for various times at 37 °C. Cells were rinsed twice in ice-cold phosphate-buffered saline and lysed in 1 ml of 1× sample buffer. 100-µl lysates were resolved by 6-10% gradient SDS-polyacrylamide gel electrophoresis and blotted with antibodies to phosphorylated kinases, kinase targets, and transcription factors. All antibodies to phosphoproteins were from New England Biolabs and Cell Signaling Technology, and those used were: rabbit anti-pErk1/2(Thr-202,Tyr-204), rabbit anti-pAkt(Ser-473), rabbit anti-pp70(Thr-389,Thr-421,Ser-424), rabbit anti-pp90(Ser-381), rabbit anti-pPKC(pan), rabbit anti-phospho-c-Myc(Thr-58, Ser-62), rabbit anti-phospho-c-Jun(Ser-63,Ser-73), and rabbit anti-pCREB(Ser-133). Filters were stripped and re-probed with antibodies to actin (Sigma).

Gene Expression Analysis-- Cells in 100-mm dishes were grown to 50-60% confluence, serum-starved overnight, and treated for 1 h with 30 nM growth factor. RNA was harvested from treated cells using Tri Reagent (Molecular Research Center) according to the directions of the manufacturer. cRNA target was prepared from 20 µg of total RNA. Preparation of cRNA target and hybridization to HuGeneFL chips (Affymetrix) has been described previously (11, 41), and is detailed at the Whitehead/MIT Genome Center's Molecular Pattern Recognition web site. Fluorescence intensities were obtained with a laser confocal scanner (Hewlett Packard), and analyzed using GeneChip software (Affymetrix).

Each gene sequence is represented on the chips by 20 25-mer oligonucleotides identical to the cDNA of interest and a corresponding control 20 25-mer oligonucleotides containing a mismatch at the thirteenth residue. Expression of each gene sequence is reflected in the fluorescence of the identical series relative to the mismatch series, and is reported by GeneChip software as an "average difference" (AD) value (for a further discussion, see Ref. 11). AD values reported for genes on chips corresponding to growth factor-treated samples were normalized to control sample chips by scaling to equivalent total average differences as described previously (41). A threshold value of 30 was assigned to genes on all chips with a reported AD value of less than 30. Genes were then sorted for growth factor response according to these normalized, thresholded AD values. Genes that exhibited a difference in AD values between growth factor treatment and control of less than 100 in at least one of duplicate experiments were discarded as either not expressed or not reproducibly regulated by growth factor. To be considered regulated by growth factor, the AD value for a gene must have been 2.5-fold higher or lower with growth factor treatment relative to control in both of duplicate experiments. To be considered preferentially regulated by a given growth factor, the -fold stimulation or suppression by one growth factor must have been 1.5-fold greater or lesser than by the other in both of duplicate experiments.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

To begin to explore the relationship between activation of RTKs and gene expression, we assessed growth factor-stimulated gene expression in two different cultured human mammary tumor cell lines. MDA-MB-361 cells express modest levels of three of the four ErbB receptor family members. In these cells the activation of different receptor dimeric pairs may be achieved by treating cells with different EGF-like growth factors. MDA-MB-453 cells express abundant ErbB2 and ErbB3, but very little EGF receptor or ErbB4. The stimulation of these cells with the ligands NRG1 and NRG2 leads to the activation of the same receptor dimeric pair, the ErbB2/ErbB3 heterodimer.

Differential Signaling Pathway Usage by EGF and NRG1beta in MDA-MB-361 Cells-- Numerous studies using model transfected cell systems suggest that different EGF-like growth factors preferentially induce the formation and activation of different ErbB receptor dimeric pairs (42-44). Two ligands that exhibit markedly different receptor-activating properties are EGF and NRG1beta . While EGF binds directly to EGF receptor to stimulate EGF receptor homodimers and EGF receptor/ErbB2 heterodimers, NRG1beta binds to either ErbB3 or ErbB4 to induce ErbB2/ErbB3 and ErbB2/ErbB4 heterodimers. In MDA-MB-361 cells EGF strongly stimulated the tyrosine phosphorylation of the 170-kDa EGF receptor and the 185-kDa ErbB2 protein, and had little effect on ErbB3. NRG1beta strongly stimulated the tyrosine phosphorylation of ErbB2 and ErbB3 (185 kDa) and had little effect on EGF receptor (Fig. 1A, upper panel). These cells express very low levels of ErbB4, which with prolonged exposure was observed to be stimulated exclusively by NRG1beta (data not shown).


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Fig. 1.   Differential signaling through ErbB receptors in MDA-MB-361 cells. A, ErbB receptor activation profile. Lysates from EGF- and NRG1beta -treated cells were immunoprecipitated with anti-receptor antibodies. Precipitates were blotted as indicated with anti-phosphotyrosine (pY), anti-p85, and anti-Grb2. B, association of signaling proteins with tyrosine-phosphorylated proteins. Lysates were immunoprecipitated with antibodies to the indicated signaling proteins. Precipitates were blotted first with anti-phosphotyrosine (upper panels) and then re-probed with antibodies to the respective precipitated signaling proteins (lower panels).

The initial impact of differential receptor activation is on the first step in signal transduction, the recruitment of Src homology 2 and protein tyrosine binding domain-containing proteins to activated receptors. We examined the association of signaling proteins with activated receptors first by blotting receptor immunoprecipitates with antibodies to signaling proteins (Fig. 1A, lower two panels). As expected, EGF preferentially stimulated the recruitment of the adaptor protein Grb2 to EGF receptor whereas NRG1beta preferentially stimulated the recruitment of this protein to ErbB3. NRG1beta also preferentially stimulated the association of p85, the 85-kDa subunit of PI3K, with ErbB3, as demonstrated previously (40).

We also examined the growth factor-stimulated recruitment of tyrosine-phosphorylated proteins to complexes with the adaptor proteins Grb2 and Shc, the protein-tyrosine phosphatase SHP2, p85, and the negative regulatory protein Cbl (Fig. 1B). In general each of the signaling proteins associated with the EGF receptor upon EGF treatment, and with 185-kDa ErbB receptors upon NRG1beta treatment. The exception was Cbl, which, as suggested previously (45), responded preferentially to EGF stimulation. However, other ligand-dependent differences in recruitment were also observed. A notable example was p85, which associated similarly with tyrosine-phosphorylated proteins at 47, 55, and 185 kDa after EGF and NRG1beta treatment, but preferentially associated with other tyrosine-phosphorylated proteins in the 90-130-kDa range in response to EGF. These observations confirm that a major outcome of differential ErbB receptor activation by different EGF-like growth factors is the differential recruitment of intracellular signaling proteins into complexes with activated receptors and other tyrosine-phosphorylated proteins.

As expected, differential ErbB receptor stimulation by EGF and NRG1beta in MDA-MB-361 cells resulted in the differential stimulation of intracellular serine/threonine kinase cascades, as determined by the phosphorylation of these enzymes in response to growth factor treatment (Fig. 2). The extent and kinetics of activation of Erk1 and Erk2 were similar for the two growth factors, perhaps reflecting the similar levels of Grb2 recruitment to activated receptors. EGF more potently stimulated the phosphorylation of two PKC isoforms, consistent with its preferential recruitment of phospholipase Cgamma (46). In contrast, NRG1beta was reproducibly stronger in stimulating the phosphorylation of Akt at serine 473, consistent with the stronger recruitment of p85 to ErbB3 in response to this factor. Interestingly, EGF preferentially stimulated the phosphorylation of p70S6 kinase. Although Akt and p70S6 kinase phosphorylation have both been demonstrated to depend on PI3K, the regulation of p70S6K is complex and not yet fully understood (47).


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Fig. 2.   Differential stimulation of kinase cascades in MDA-MB-361 cells. Cells were treated with 30 nM EGF or NRG1beta for the indicated times, and lysates were blotted with antibodies specific for phosphorylated kinases (right panels). Filters were re-probed with anti-actin, and the intensities of the phosphokinase bands were normalized to the intensities of the corresponding actin bands. Normalized intensities were plotted as a function of time (left two sets of panels).

The ultimate outcome of RTK stimulation by growth factors is the phosphorylation of nuclear factors to elicit changes in transcriptional regulation. To determine whether differential signaling through ErbB receptors might influence transcription factor regulation, we examined the phosphorylation state of three transcription factors, Myc, Jun, and CREB, in response to EGF and NRG1beta in MDA-MB-361 cells. Although the phosphorylation of these factors was stimulated by both growth factors, EGF reproducibly elicited a stronger response than did NRG1beta (Fig. 3A).


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Fig. 3.   Differential stimulation of nuclear events in MDA-MB-361 cells. A, transcription factor phosphorylation. Lysates from growth factor-treated cells were blotted with antibodies specific for phosphorylated transcription factors (right panels) and the normalized signals were plotted (left two sets of panels). B, ligand-stimulated message abundance. Transcript abundance was measured using gene microarray chips for untreated cells, and cells treated with either EGF or NRG1beta . The -fold stimulation by EGF and NRG1beta of the indicated messages is plotted. Asterisks indicate previously reported IEGs.

Differential Gene Expression in MDA-MB-361 Cells-- Since EGF and NRG1beta stimulated overlapping yet distinct patterns of intracellular signaling events in MDA-MB-361 cells, we examined whether the two growth factors also stimulated differences in gene expression. To test this we used Affymetrix HuGeneFL Array chips to simultaneously assess changes in transcript levels of ~5600 genes after 1 h of growth factor treatment. We observed that, although the levels of a host of mRNAs were similarly elevated by both EGF and NRG1beta , many were preferentially elevated by one or the other growth factor. The -fold stimulations observed for a subset of genes is plotted in Fig. 3B.

Our analysis revealed 133 genes whose transcript abundance reproducibly changed in response to one or both growth factors. 92 mRNAs were enhanced at least 2.5-fold with growth factor treatment, whereas in two separate experiments 41 mRNAs were suppressed by at least 2.5-fold. Interestingly, despite its weaker potency in stimulating several signaling cascades, NRG1beta was the stronger factor in regulating mRNA levels. 44 mRNAs were preferentially elevated by NRG1beta , where the -fold induction by this growth factor was at least 1.5-fold greater than that of EGF, compared with 18 mRNAs that were preferentially elevated by EGF. Likewise, 20 mRNAs were preferentially suppressed by NRG1beta by at least a 1.5-fold margin compared with 10 mRNAs that were preferentially suppressed by EGF.

Table I shows an abbreviated list of genes regulated by growth factors in MDA-MB-361 cells. (A full list of growth factor-responsive genes is provided as supplementary material in the on-line edition of this article.) Particularly noteworthy is the distribution of known IEGs, indicated in Table I and in Fig. 3B by an asterisk. mRNA levels of most IEGs were similarly stimulated by both EGF and NRG1beta , suggesting that overlapping pathways elicited by the two growth factors may be responsible for the regulation of these transcript levels. However, EGF preferentially elevated transcripts of four IEGs and NRG1beta preferentially stimulated six, indicating that differential signaling through ErbB receptors can result in differential immediate early gene expression.

                              
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Table I
Growth factor-induced mRNA changes in MDA-MB-361 cells
The gene name, its -fold stimulation by EGF and NRG1beta in each of duplicate experiments, its general function, and its potential role in breast cancer are indicated. Known IEGs are indicated by an asterisk.

Differential Signaling in MDA-MB-453 Cells-- Our previous studies indicate that the EGF-like growth factors NRG1beta and NRG2beta exhibit markedly different biological potencies in MDA-MB-453 cells, which express abundant ErbB2 and ErbB3 but very little EGF receptor or ErbB4. In these cells, both NRG1beta and NRG2beta signal through an ErbB2/ErbB3 receptor heterodimer (38). Although NRG1beta very efficiently induced morphological changes in these cells consistent with their differentiation (48), NRG2beta had negligible activity in this assay (38, 49). Paradoxically, the difference in the biological activities could not be explained by differences in receptor occupation or gross receptor tyrosine phosphorylation; NRG2 stimulated the tyrosine phosphorylation of the ErbB2 and ErbB3 receptors to the same extent as NRG1. However, in examining individual signaling protein recruitment to activated receptors, differences became apparent. NRG1beta was more potent than NRG2beta in stimulating the recruitment of Grb2, Shc, SHP2, and p85 to ErbB2 and in stimulating the recruitment of Grb2 to ErbB3. The results of those previous studies are summarized in Table II.

                              
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Table II
ErbB receptor expression and recruitment of signaling proteins in MDA-MB-453 cells

To further confirm differential signaling by NRG1beta and NRG2beta , we examined the association of a broad range of tyrosine-phosphorylated proteins with individual signaling proteins (Fig. 4). Although the two growth factors similarly stimulated the association of 130- and 60-kDa tyrosine-phosphorylated proteins and the 185-kDa ErbB receptors with SHP2, NRG1beta preferentially stimulated the recruitment of ErbB receptors and multiple tyrosine-phosphorylated proteins to Grb2. Additionally, NRG1beta more strongly stimulated the recruitment of the ErbB receptors and 150- and 105-kDa tyrosine-phosphorylated proteins to p85. These observations are consistent with our previous studies (summarized in Table II) indicating that different EGF-like growth factors can differentially signal through a single ErbB receptor dimeric species (38), perhaps by inducing differential tyrosine phosphorylation site usage (39).


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Fig. 4.   Differential recruitment and activation of intracellular signaling proteins in MDA-MB-453 cells. Lysates from growth factor-treated cells were immunoprecipitated with antibodies to the indicated signaling proteins. Precipitates were blotted first with anti-phosphotyrosine (upper panels) and then re-probed with antibodies to the respective precipitated signaling proteins (lower panels).

The impact of differential signaling protein recruitment to activated receptors on downstream pathways in the MDA-MB-453 cells is shown in Fig. 5. The phosphorylation of each of the downstream kinases examined, Erk1, Erk2, Akt, p70S6k, p90Rsk, and PKCs, was augmented or prolonged in response to NRG1beta relative to NRG2beta . The final outcome of the differences in signaling duration was reflected in the phosphorylation of the transcription factors Myc, Jun, and CREB. Myc and CREB exhibited an abbreviated response to NRG2beta relative to NRG1beta (Fig. 6A), whereas the extent of Jun phosphorylation was suppressed with NRG2beta stimulation. The prolonged kinetics of signaling pathway activation by NRG1beta may underlie its ability to promote MDA-MB-453 cell differentiation (14).


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Fig. 5.   Abbreviated stimulation of kinase cascades by NRG2beta in MDA-MB-453 cells. Lysates from NRG1beta - or NRG2beta -treated cells were blotted with antibodies specific for the indicated phosphorylated kinases, and the normalized signals plotted as described in the Fig. 2 legend.


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Fig. 6.   Suppressed stimulation of transcription factor phosphorylation by NRG2beta and differential gene expression induced by NRGs in MDA-MB-453 cells. A, lysates from growth factor-treated cells were blotted with antibodies specific for phosphorylated transcription factors and normalized intensities plotted. B, microarray chips were used to assess transcript abundance before and after growth factor treatment. The -fold stimulation of the indicated messages by NRG1beta and NRG2beta are plotted. Asterisks indicate previously reported IEGs.

Because of the attenuated response of signaling pathways to NRG2beta compared with NRG1beta , one might expect NRG2beta to regulate fewer genes or to cause smaller responses in the same set of genes. Indeed, a subset of mRNAs, many of which were previously identified as immediate early genes, were preferentially elevated in response to NRG1beta (Fig. 6B). However, a significant number of mRNAs were preferentially elevated in response to NRG2beta compared with NRG1beta , and some of these have also been previously identified as IEGs. A more complete tabulation of expression profiles is presented in Table III and in the supplementary material available on-line. These results suggest that the duration of activation of signaling pathways may play a complex role in both positive and negative regulation of mRNA levels. Alternatively, the results might be explained if NRG2beta activates as yet unidentified pathways that are not responsive to NRG1beta . In any event, these results indicate that a single receptor dimeric species can respond uniquely to different growth factors, both in activation of intracellular signaling pathways and in gene expression.

                              
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Table III
Growth factor-induced mRNA changes in MDA-MB-453 cells
The gene name, its -fold stimulation by NRG1beta and NRG2beta in each of duplicate experiments, its general function, and its potential role in breast cancer are indicated. Known IEGs are indicated by an asterisk.

Effect of Cell Type on ErbB-mediated Transcript Abundance-- A comparison of the genes whose transcript levels are changed by growth factors in the two cell lines reveals that cell type has a dramatic impact on growth factor response. Of 133 responsive genes in the MDA-MB-361 cells and 107 in the MDA-MB-453 cells, only 19 overlap between the two cell lines (Table IV). Moreover, the response of a given gene to a single growth factor can be quite disparate depending on cellular context. Since in each case cells were treated with 30 nM NRG1beta for 1 h, and in each case signaling is mediated predominantly by an ErbB2/ErbB3 heterodimer, the response of genes to this growth factor may be directly compared. Ten of the 19 overlapping mRNAs were elevated in response to NRG1beta in both cell lines. These mRNAs were also responsive to the other growth factors, and 9 of these 10 have been identified previously as IEGs. In contrast, several mRNAs showed an opposite response to NRG1beta in the two cell lines. For example, ROM1 was enhanced 8.4-fold in MDA-MB-361 cells but reduced 5-fold in the MDA-MB-453 cells. Interestingly, none of the genes that showed opposite regulation in the two cell lines were immediate early genes.

                              
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Table IV
Comparison of growth factor-regulated transcripts in MDA-MB-361 and MDA-MB-453 cells
The -fold stimulation of mRNA levels of the indicated genes observed in experiment 1 are given for each growth factor. Known IEGs are indicated by an asterisk.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this study we have used the ErbB receptor network to address the question of whether activation of different RTKs leads to identical or distinct profiles of gene expression. We first provide a surface-to-nucleus survey of signaling events triggered in response to two different EGF-like growth factors over the course of 1 h after presentation to cultured human mammary tumor cells. Then, using DNA microarray technology, we provide a broad comparison of the gene transcripts that are induced or suppressed in the cells after 1 h of growth factor treatment. It should be noted that, although our expression studies focus on genes that respond with immediate early kinetics, we have not limited our analyses to cycloheximide-insensitive growth factor-induced genes. These relaxed criteria more fully reflect the breadth of the response to different growth factors, and also allow for an analysis of genes that are suppressed in response to growth factor treatment. It should also be noted that this is not intended to be a comprehensive analysis of genes that are regulated by EGF-like growth factors in mammary tumor cells, but rather a survey of genes that uncover trends in cellular responses to RTK activation.

We have utilized the MDA-MB-361 cells to activate distinct ErbB receptor heterodimers and downstream signaling pathways. We confirm that stimulation with different EGF-like growth factors in these cells results in different patterns of receptor activation, which in turn leads to the differential recruitment of Src homology 2 and protein tyrosine binding domain-containing proteins to receptors and other tyrosine phosphorylated proteins. In these cells EGF stimulates the tyrosine phosphorylation of the EGF receptor and ErbB2, whereas NRG1beta stimulates the tyrosine phosphorylation of ErbB2 and ErbB3. The similar level of recruitment of Grb2 to EGF receptor in response to EGF and to ErbB3 in response to NRG1beta likely underlies the similar activation of the Erk1 and Erk2 kinases by the two growth factors. On the other hand, the preferential recruitment of p85 to ErbB3 in response to NRG1beta translates into an increased activation of the Akt kinase by this factor. The preferential stimulation of PKC by EGF may be related to previous observations that phospholipase C-gamma is more strongly stimulated by EGF receptor activation than by ErbB3 activation (46, 50, 51). Although both growth factors stimulated the phosphorylation of the Myc, Jun, and CREB transcription factors, EGF appeared to be reproducibly stronger in doing so. An analysis of the phosphorylation of other transcription factors such as the STATs and forkhead family members could uncover NRG1beta -specific nuclear factors.

Most of the known IEGs were similarly stimulated by both growth factors in these cells, suggesting that these genes may be downstream of a common pathway. A strong candidate is Erk1/2-mediated transcriptional regulation through the serum response promoter element. Other genes that are similarly stimulated by both factors could be IEGs that have not been reported previously, or genes whose expression is regulated in response to IEG induction. The IEGs that preferentially respond to one of the growth factors may contain additional promoter elements sensitive to pathways specific to signaling by that factor. Although more kinase cascades were preferentially stimulated with EGF, NRG1beta was the stronger growth factor in regulating gene expression. This might be explained by the preferential stimulation of Akt by NRG1beta . Akt-regulated genes are of acute interest because of the known role for PI3K/Akt signaling in mediating cell survival (52, 53). More work is needed to determine which pathways preferentially affect specific mRNA levels. These issues are currently being addressed using pharmacological and mutagenesis methods to suppress specific pathways.

Our findings contrast those of a previous study that concluded that in immortalized mouse fibroblasts, different RTKs and diverse RTK-stimulated signaling pathways mediate the expression of primarily overlapping sets of immediate early genes (11). Whether the differences in conclusions arise from the complexity inherent in the ErbB signaling network, or whether fibroblasts are pre-programmed to respond to a variety of stimuli with the expression of a defined set of genes remains to be resolved.

We have utilized the MDA-MB-453 cells to activate the same ErbB receptor heterodimer with different growth factors, and the results point to another mechanism by which early gene expression may be regulated. In these cells, we found no major differences in the identity of the signaling cascades activated by the growth factors NRG1beta and NRG2beta . Instead, we observed that each of the pathways was induced with abbreviated kinetics with NRG2beta relative to NRG1beta , possibly resulting from the suppressed ability of NRG2beta to stimulate the recruitment of signaling proteins to ErbB2 (38). This in turn resulted in marked differences in gene expression, which may underlie the dramatic difference in biological potencies between the two factors (38, 49). This interpretation is consistent with results from other systems where signaling strength or duration is thought to impact biological response. For example, overexpression of some RTKs in PC12 pheochromacytoma cells is sufficient to confer ligand-dependent differentiation (54, 55). Interestingly, despite its dominance in stimulating signaling pathways and differentiation, NRG1beta is not necessarily dominant over NRG2beta in regulating gene expression. With a few exceptions NRG1beta by far dominates the induction of the known IEGs. However, as many genes respond preferentially to NRG2beta as NRG1beta , suggesting that signaling strength may not proportionally translate into gene expression. Again, effects of pathways preferentially stimulated by NRG2beta overlooked in our analysis, or NRG2beta -induced stabilization of messages independent of transcriptional regulation cannot be ruled out.

Comparison of genes in common between the two cell lines indicates that ligand-regulated gene expression is exquisitely cell type-dependent. Less than 20% of the genes that respond to growth factor treatment in one cell line also respond in the other, and roughly half of those are known IEGs. This discrepancy is undoubtedly due to the very different lineages of the two cell lines. MDA-MB-453 cells were cultured from the breast effusion of a patient with metastatic disease. These cells partially retain their mammary epithelial characteristics, in that ligand treatment induces the production of milk components (29). MDA-MB-361 cells were cultured from the brain metastasis of a breast cancer patient. The morphology of these cells is quite different from the MDA-MB-453 cells, and they exhibit no propensity to differentiate upon growth factor treatment. Changes in the expression of nuclear factors that accompany metastasis could account for cell type differences in growth factor-regulated transcript abundance.

In addition to highlighting the behavior of the known immediate early genes, Tables I and III summarize other genes that may play roles in the survival, growth, progression, or invasiveness of breast cancer cells. These genes may be regulated in vivo by autocrine EGF-like growth factors produced by the tumors themselves, or by paracrine factors received from the immediate environment. A number of examples of genes implicated in cell growth deregulation were modulated by growth factors. Transcripts encoding cyclins D1 and D3 were stimulated in response to growth factors, as was the E2F transcription factor involved in the G1 to S phase transition. On the other hand, putative tumor suppressor genes such as the transcription factor AP2, the homeobox protein Cdx2, amphiphysin II, and CGM2 were suppressed with growth factor stimulation, along with genes implicated in cell growth inhibition such as the USF2 transcription factors, the calcium-regulated protease calpain, the Ras/mitogen-activated protein kinase pathway inhibitor Gps2, cadherin 4, and Tob2. Other identified genes have been implicated in epithelial cell migration or invasion such as the growth factor MST1, urokinase plasminogen activator and plasminogen, the actin bundling protein fascin, and the protease inhibitor nexin. Particularly noteworthy is the number of stimulated genes involved in angiogenic processes. Connective tissue growth factor, interleukin 8, urocortin, atrial natriuretic factor, Gdf5, PDGFA, IGF-2, and vascular endothelial growth factor are all secreted factors that have been implicated in angiogenesis or vascular remodeling.

It will be of interest to compare profiles of genes induced by EGF-like growth factors in cultured mammary tumor cells with gene expression patterns in primary and metastatic tumors taken from patients. Ultimately, patient tumor gene expression profiles similar to those observed with ErbB receptor activation could point to the utility of ErbB signaling-targeted anti-tumor therapeutic strategies, such as the use of herceptin (56).

In summary, using pathway profiling and global gene expression analysis methods, we demonstrate that differences in signal transduction pathway usage by the ErbB RTKs precede differences in gene regulation by growth factors (Fig. 7). These results indicate that RTK signaling is not generic and that specific pathways or combinations of pathways may target specific genes. Hence, EGF-like growth factors have the potential to serve as more than a "go" signal in cells of pre-determined fate; through differential pathway utilization, growth factors can influence cellular response to stimulation.


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Fig. 7.   Differential gene induction by EGF-like growth factors. In MDA-MB-361 cells, EGF (E) signaling through an EGF receptor/ErbB2 heterodimer efficiently stimulates the Erk1/2 and PKC pathways, while NRG1beta (N1) signaling through an ErbB2/ErbB3 heterodimer efficiently stimulates the Erk1/2 and Akt pathways. The two growth factors induce overlapping but distinct sets of genes. In MDA-MB-453 cells NRG1beta more strongly stimulates Erk1/2, Akt, and PKC pathways than does NRG2beta (N2), but the two growth factors induce overlapping yet distinct sets of genes.

Our results also indicate that, consistent with the results from invertebrate models, cell history plays a marked role in RTK-mediated gene regulation. We envision a model where tissue-specific nuclear factors are expressed in cells according to their developmental histories. These factors mediate the expression of a relatively narrow set of genes in cells that exhibit a limited range of possible fates. This is illustrated by the preferential induction of genes involved in cellular proliferation and wound healing by serum in cultured human fibroblasts (57). In this case a single RTK-stimulated pathway may be sufficient to provide a "go" signal to induce a largely pre-programmed response. Multipotent stem and precursor cells instead express nuclear factors that can mediate multiple transcriptional programs, and the program selected depends on RTK-mediated signaling pathway usage and kinetics.

    FOOTNOTES

* This work was supported in part by grants from Bristol Myers Squibb, Millenium Pharmaceuticals, and Affymetrix (to E. S. L.), and by National Institutes of Health Grants GM41890 (to L. C. C.) and CA71702 (to K. L. C.).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.

The on-line version of this article (available at http://www.jbc.org) contains a full list of growth factor-responsive genes.

§ Supported by a grant from the Massachusetts Department of Public Health Breast Cancer Research Program. To whom correspondence should be addressed. Present address: University of California at Davis Cancer Center, Research Bldg. III, Rm. 1400, 4645 2nd Ave., Sacramento, CA 95817. Tel.: 916-734-0726; Fax: 916-734-0190; E-mail: casweeney@ucdavis.edu.

|| Fellow of the Jane Coffin Childs Memorial Fund for Medical Research. Present address: Oxford Bioscience Partners, Westport, CT 06880.

** Present address: University of California at Davis Cancer Center, Sacramento, CA 95817.

Published, JBC Papers in Press, April 10, 2001, DOI 10.1074/jbc.M100602200

    ABBREVIATIONS

The abbreviations used are: RTK, receptor tyrosine kinase; IEG, immediate early gene; AD, average difference; NRG, neuregulin; CREB, cAMP-responsive element-binding protein; PKC, protein kinase C; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; STAT, signal transducers and activators of transcription; PI3K, phosphoinositide 3-kinase; MAPK, mitogen-activated protein kinase; JNK, c-Jun N-terminal kinase; TNF, tumor necrosis factor; PAF, platelet-activating factor; BC, breast cancer.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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