From the 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
Department of
Biology, Massachusetts Institute of Technology,
Cambridge, Massachusetts 02139
Received for publication, January 22, 2001, and in revised form, March 19, 2001
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ABSTRACT |
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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 NRG1 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 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 PDGF 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.
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
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.
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 NRG1
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 NRG1
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 NRG1
As expected, differential ErbB receptor stimulation by EGF and NRG1
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 NRG1 Differential Gene Expression in MDA-MB-361 Cells--
Since EGF
and NRG1
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, NRG1
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 NRG1 Differential Signaling in MDA-MB-453 Cells--
Our previous
studies indicate that the EGF-like growth factors NRG1
To further confirm differential signaling by NRG1
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 NRG1
Because of the attenuated response of signaling pathways to NRG2 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 NRG1 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 NRG1 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, NRG1 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
NRG1 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.
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, NRG1
acting through an ErbB2/ErbB3
heterodimer stimulated prolonged signaling of all pathways examined
relative to NRG2
acting through the same heterodimeric receptor
species. Surprisingly, NRG1
and NRG2
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
B through regulation of I
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-
1 can mediate the
response of calcium-sensitive genes and protein kinase C (PKC) targets
to growth factor stimulation.
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).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-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
-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).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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 NRG1
. While EGF binds
directly to EGF receptor to stimulate EGF receptor homodimers and EGF
receptor/ErbB2 heterodimers, NRG1
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. NRG1
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
NRG1
(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 NRG1 -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).
preferentially stimulated the recruitment of this
protein to ErbB3. NRG1
also preferentially stimulated the
association of p85, the 85-kDa subunit of PI3K, with ErbB3, as
demonstrated previously (40).
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
NRG1
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.
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
C
(46). In contrast, NRG1
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 NRG1 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).
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 NRG1
(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 NRG1 . The -fold stimulation by EGF and NRG1
of the
indicated messages is plotted. Asterisks indicate previously
reported IEGs.
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 NRG1
, 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.
was the stronger factor in regulating
mRNA levels. 44 mRNAs were preferentially elevated by NRG1
,
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 NRG1
by at least a 1.5-fold margin
compared with 10 mRNAs that were preferentially suppressed by
EGF.
, 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 NRG1
preferentially stimulated six, indicating that differential signaling
through ErbB receptors can result in differential immediate early gene
expression.
Growth factor-induced mRNA changes in MDA-MB-361 cells
in each of
duplicate experiments, its general function, and its potential role in
breast cancer are indicated. Known IEGs are indicated by an asterisk.
and NRG2
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 NRG1
and NRG2
signal through an
ErbB2/ErbB3 receptor heterodimer (38). Although NRG1
very
efficiently induced morphological changes in these cells consistent
with their differentiation (48), NRG2
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.
NRG1
was more potent than NRG2
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.
ErbB receptor expression and recruitment of signaling proteins in
MDA-MB-453 cells
and NRG2
, 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, NRG1
preferentially stimulated the recruitment of ErbB
receptors and multiple tyrosine-phosphorylated proteins to Grb2.
Additionally, NRG1
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).
View larger version (84K):
[in a new window]
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).
relative to
NRG2
. 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 NRG2
relative to NRG1
(Fig. 6A),
whereas the extent of Jun phosphorylation was suppressed with NRG2
stimulation. The prolonged kinetics of signaling pathway activation by
NRG1
may underlie its ability to promote MDA-MB-453 cell
differentiation (14).
View larger version (63K):
[in a new window]
Fig. 5.
Abbreviated stimulation of kinase cascades by
NRG2 in MDA-MB-453 cells. Lysates from
NRG1
- or NRG2
-treated cells were blotted with antibodies specific
for the indicated phosphorylated kinases, and the normalized signals
plotted as described in the Fig. 2 legend.
View larger version (53K):
[in a new window]
Fig. 6.
Suppressed stimulation of transcription
factor phosphorylation by NRG2 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 NRG1
and NRG2
are plotted. Asterisks indicate previously
reported IEGs.
compared with NRG1
, one might expect NRG2
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
NRG1
(Fig. 6B). However, a significant number of
mRNAs were preferentially elevated in response to NRG2
compared
with NRG1
, 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 NRG2
activates as yet unidentified
pathways that are not responsive to NRG1
. 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.
Growth factor-induced mRNA changes in MDA-MB-453 cells
and NRG2
in each of
duplicate experiments, its general function, and its potential role in
breast cancer are indicated. Known IEGs are indicated by an asterisk.
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
NRG1
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
NRG1
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.
Comparison of growth factor-regulated transcripts in MDA-MB-361 and
MDA-MB-453 cells
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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 NRG1
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 NRG1
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-
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 NRG1
-specific nuclear factors.
was the stronger growth factor in regulating gene expression. This
might be explained by the preferential stimulation of Akt by NRG1
.
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.
and NRG2
. Instead, we observed that each of the pathways was
induced with abbreviated kinetics with NRG2
relative to NRG1
,
possibly resulting from the suppressed ability of NRG2
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, NRG1
is not necessarily dominant over NRG2
in
regulating gene expression. With a few exceptions NRG1
by far
dominates the induction of the known IEGs. However, as many genes
respond preferentially to NRG2
as NRG1
, suggesting that signaling
strength may not proportionally translate into gene expression. Again,
effects of pathways preferentially stimulated by NRG2
overlooked in
our analysis, or NRG2
-induced stabilization of messages independent of transcriptional regulation cannot be ruled out.
View larger version (88K):
[in a new window]
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 NRG1 (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 NRG1
more strongly
stimulates Erk1/2, Akt, and PKC pathways than does NRG2
(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.
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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.
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