From the Departments of Developmental and Molecular
Biology and Medicine, Albert Einstein Cancer Center, Albert Einstein
College of Medicine, Bronx, New York 10461, the
§ Department of Pathology, New York University Medical
Center, New York, NY 10016, the ¶ Department of Pathology,
Northwestern University Medical School, Chicago, Illinois 60611, the
Department of Pathology, McMaster University, West Hamilton,
Ontario L8S 4K1, Canada, the
Departments
of Pathology and Cell Biology, Harvard Medical School,
Boston Massachusetts 02115, and the §§ Howard
Hughes Medical Institute and Program in Molecular Medicine, Department
of Biochemistry and Molecular biology, University of Massachusetts
Medical School, Worcester, Massachusetts 01605
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ABSTRACT |
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The cyclin D1 gene is overexpressed
in breast tumors and encodes a regulatory subunit of
cyclin-dependent kinases that phosphorylate the
retinoblastoma protein. pp60c-src activity is
frequently increased in breast tumors; however, the mechanisms
governing pp60c-src regulation of the cell cycle in
breast epithelium are poorly understood. In these studies,
pp60v-src induced cyclin D1 protein levels and
promoter activity (48-fold) in MCF7 cells. Cyclin D1-associated kinase
activity and protein levels were increased in mammary tumors from
murine mammary tumor virus-pp60c-src527F transgenic mice.
Optimal induction of cyclin D1 by pp60v-src
involved the extracellular signal-regulated kinase, p38, and c-Jun
N-terminal kinase members of the mitogen-activated protein kinase
family. Cyclin D1 promoter activation by pp60v-src
involved a cAMP response element-binding protein (CREB)/activating transcription factor 2 (ATF-2) binding site. Dominant negative mutants
of CREB and ATF-2 but not c-Jun inhibited pp60v-src
induction of cyclin D1. pp60v-src induction of CREB
was blocked by the p38 inhibitor SB203580 or by mutation of CREB at
Ser133. pp60v-src induction of ATF-2
was abolished by the c-Jun N-terminal kinase inhibitor JNK-interacting
protein-1 or by mutation of ATF-2 at Thr69 and
Thr71. CREB and ATF-2, which bind to a common
pp60v-src response element, are transcriptionally
activated by distinct mitogen-activated protein kinases. Induction of
cyclin D1 activity by pp60v-src may contribute to
breast tumorigenesis through phosphorylation and inactivation of the
retinoblastoma protein.
The multistep process of tumorigenesis involves the accumulation
of genetic defects contributing to the tumor phenotype. In addition to
a critical role in the orchestration of orderly cell cycle progression,
components of the G1 phase regulatory apparatus play an
important role in tumorigenesis. The protein kinase complexes regulating the G1 phase include a catalytic subunit, the
cyclin-dependent kinase
(Cdk),1 its regulatory
activating partner, the cyclin, a Cdk-activating kinase, and
cyclin-dependent kinase inhibitors (1). The cyclin D1 gene encodes a regulatory subunit of the Cdk holoenzymes that phosphorylate and thereby inactivate the retinoblastoma tumor suppressor, pRB (2, 3). Immunoneutralization and antisense experiments
have demonstrated that in the breast cancer cell lines MCF7 and T-47D,
the abundance of cyclin D1 is rate-limiting in mitogen-induced
G1 phase progression (4-6).
Cyclin D1 collaborates with oncogenes in cellular transformation and
transgenic overexpression of cyclin D1 in the mouse mammary gland
induced adenocarcinomas (7, 8). Cyclin D1 protein levels are frequently
increased in breast tumor cell lines (9, 10) and human breast cancers
(5, 10, 11). Several different factors may contribute to the increased
cyclin D1 abundance found in breast tumors. Cyclin D1 abundance is
induced transcriptionally, whereas the protein is degraded rapidly upon
the withdrawal of growth factors via the ubiquitin-proteasome pathway
(12). The human breast cancer cell line MCF7 has been used to examine
the regulation of cyclin D1, demonstrating the induction of cyclin D1
protein levels in these cells by estrogens, serum, and epidermal growth
factor (6, 13, 14). Cyclin D1 promoter activity and mRNA levels are
also induced by growth factors that are mitogenic to breast cancer
cells including epidermal growth factor and insulin-like growth
factor-1 (15-17). Activating mutants of p21ras, Rac, and the
dbl family of oncogenes induce cyclin D1 promoter activity
and protein abundance (16, 18-20). Recent studies suggest that cyclin
D1 plays an important role in Ras-induced NIH3T3 cell transformation
(21) and in Ras-induced skin tumor formation in vivo
(22).
In contrast, relatively little is known of the molecular
mechanisms by which activating Src mutations directly affect
components of the cell cycle regulatory apparatus (23). The
pp60c-src proto-oncogene encodes a 60-kDa
cytoplasmic nonreceptor tyrosine kinase (24) that is sufficient to both
initiate and maintain cellular transformation (25). In previous
studies, performed in fibroblast cell lines, overexpression of
pp60v-src enhanced the rate of G1 phase
progression in association with an induction of cyclin D1 protein
levels in NIH3T3 cells, implicating cyclin D1 in
pp60v-src action (21). In Rat-1 cells, however,
pp60v-src overexpression did not induce cyclin D1
expression, and a reduction in p27Kip1 levels was observed,
suggesting that p27Kip1 transcriptional repression may play
a role in pp60v-src signaling (26). Activation of
the pp60c-src tyrosine kinase has been observed in
a large proportion of human breast malignancies (27). Overexpression of
a constitutively active mutant of pp60c-src under
control of the murine mammary tumor virus (MMTV) long terminal repeat
in transgenic mice induced mammary gland tumor formation (28). The cell
cycle regulatory targets of pp60src in mammary epithelial cells
are virtually unknown, and the intracellular kinase pathways by which
pp60src regulates cell cycle regulatory pathways also remain to
be determined.
In fibroblasts and fibroblast-derived cell lines,
pp60v-src induces several downstream signaling
pathways including members of the mitogen-activated protein kinase
(MAPK) family, the p42 and p44 extracellular signal-regulated kinases
(ERKs) (29, 30) and c-Jun N-terminal kinase (JNK) (31). In addition,
the phosphatidylinositol 3'-kinase (32) and the signal transducers and
activators of transcription (33) play a role in signaling induced by
pp60v-src. The role of MAPKs in
pp60v-src signaling requires reinvestigation as
some ten MAPK family members have now been identified in mammalian
cells, increasing the potential complexity and allowing for specificity
in signaling (34-36). Two of these MAPKs, (MAPK1/ERK1 and MAPK2/ERK2)
are strongly activated by polypeptides and growth factors but are
poorly induced by stress stimuli. In contrast, the other MAPK family
members are strongly activated by stress signals and are frequently
referred to as stress-activated protein kinases (SAPKs). Chemical
inhibitors of specific MAPKs have aided in identification of downstream
signaling pathways. The drug PD98059 suppresses the activation of the
MAPK/ERK pathway by preventing activation of the upstream activator
MAPK kinase 1 (MKK1 or MEK1). Recent studies have identified distinct members of the SAPK family, with MAP kinase kinase 4 (MKK4, also known
as SEK1) and MAP kinase kinase 7 (MKK7) activating JNK, whereas MAP
kinase kinase 3 (MKK3) and MAP kinase kinase 6 (MKK6) activate members
of the p38 MAPK group (34, 36). The drug SB203580, a specific inhibitor
of p38 The induction of gene expression by pp60v-src
involves several different transcription factors and DNA regulatory
sequences, including the activator protein-1 (AP-1) site (38-40), the
serum response element (41), the TATA box (42), a CRE/activating
transcription factor (ATF) site (43) and a unique
pp60v-src-response element (44). A CRE/ATF site
that binds members of the ATF/cAMP response element-binding protein
(CREB) family is required for
pp60v-src-dependent induction of the
prostaglandin synthase (psg2) gene (43). As the ERKs, JNK,
and p38 are capable of phosphorylating ATF-2, ATF-2 may play a role in
pp60v-src signaling (45-48). The CREB protein is
also a distal target of the MAPK/ERK pathway (49), the p38 pathway
(50), or both ERK and p38 pathways (51). These studies suggest that
cell type-specific factors mediate the MAPK pathways activating CREB
transactivation and raise the possibility that CREB may be a target of
pp60v-src signaling.
Because relatively little is known of the molecular mechanisms by which
pp60src engages components of the cell cycle regulatory
apparatus in breast cells, we have examined the regulation of cyclin D1
by pp60v-src in MCF7 cells and determined the
expression and activity of cyclin D1 in mammary tumors induced by
mammary-targeted overexpression of an activating
pp60c-src mutant in transgenic mice. In these
studies, optimal pp60v-src induction of cyclin D1
required collaboration between the MAPK/ERK, p38, and JNK pathways. We
provide evidence that CREB and ATF-2 bind a common DNA element and
serve as distal transcriptional targets of
pp60v-src induction of the cyclin D1
gene in MCF7 cells. The induction of CREB by
pp60v-src required a p38 pathway, and induction of
ATF-2 by pp60v-src required the JNK pathway.
pp60v-src activation of distinct transcription
factors that bind common DNA sequences of the cyclin D1 promoter occurs
through specific MAPK modules.
Western Blots and Immune Complex Kinase Assays--
Western
blotting analysis was performed as described previously (16, 52). To
determine the abundance of cyclin D1 protein the monoclonal cyclin D1
antibody HD-11 (Santa Cruz Biotechnology, Santa Cruz, CA) or DCS-6
(Neomarkers, Fremont, CA) was used; for
Cyclin D1-immunoprecipitation kinase assays were performed essentially
as described previously (52) using saturating amounts of the cyclin D1
antibody, DCS-11 (NeoMarkers, Fremont, CA). The pRB substrate was
prepared by transforming Escherichia coli with the vector
pGEX-Rb (52) (a gift from Dr. E. Harlow).
p42ERK1, p44ERK2, and SAPK/JNK immune complex
assays were performed as described previously (52) on cell extracts
derived from mammary gland tumor tissue. Staphylococcal protein
A-agarose beads (Boehringer Mannheim) were incubated with anti-ERK
antibody (C14) (Santa Cruz Biotechnology) or polyclonal SAPK antibody
(a gift from Dr. J. Kyriakis) for 1 h at 4 °C. The samples were
analyzed by SDS-polyacrylamide gel electrophoresis upon termination of
the reaction by boiling in SDS sample buffer. The phosphorylation of
myelin basic protein or glutathione S-transferase c-Jun
substrates was quantified by densitometry after exposure to
autoradiographic film (Labscientific Inc., Livingston, NJ) using
ImageQuant, version 1.11 (Molecular Dynamics Computing Densitometer,
Sunnyvale, CA).
Immunohistochemistry--
Tissues were processed and analyzed as
described previously (17) using a monoclonal cyclin D1 antibody DCS-6
(Vector Laboratories, Burlingame, CA) (53). Immunohistochemical
analyses were carried out using a biotinylated secondary antibody and
an avidin/biotin-linked horseradish peroxidase (the Vectastain ABC
system) (Vector Laboratories). The complex was stained with
diaminobenzidine tetrahydrochloride (DAB) substrate obtained from
Kirkegaard & Perry Laboratories Inc. (Gaithersburg, MD). Nuclear
staining for cyclin D1 was determined by counting 500 cells within each
tumor sample. Tumor samples from four separate animals were examined.
Reporter Genes and Expression Vectors--
A series of 5'
promoter constructions in which the human cyclin D1 promoter was
subcloned into the vector pA3LUC was described previously
(16, 52). The constructions Cell Culture, DNA Transfection, Luciferase Assays, and
Chemicals--
Cell culture, DNA transfection, and luciferase assays
were performed as described previously (16). The MCF7 cells were
maintained in DME with 10% calf serum and 1% penicillin/streptomycin.
Cells were transfected by calcium phosphate precipitation, the medium was changed after 6 h, and luciferase activity was determined after a further 24-36 h. Comparison was made between the effect of
transfecting active expression vector encoding the protein of interest
with the effect of an equal amount of vector cassette. In each
experiment, a dose response was determined with 300 and 600 ng of
expression vector and the cyclin D1 promoter reporter plasmids (2.4 µg). Luciferase assays were performed at room temperature using an
Autolumat LB 953 (EG&G Berthold), and the initial 10 s of the
reaction were used to assess luciferase content with the values
expressed in arbitrary light units (62). Background activity from cell
extracts was typically <100 arbitrary light units/10 s. Statistical
analyses was performed using the Mann Whitney U test, and
significant differences were established as p < 0.05.
The protein tyrosine kinase inhibitors genistein (Sigma) and herbimycin
A (Calbiochem) were used as Src kinase inhibitors (63). The MEK
inhibitor PD98059 was a gift from Dr. A. Saltiel (Parke Davis) and
SB203580 was a gift from J. Christie (SmithKline Beecham).
Oligodeoxyribonucleotides and Electrophoretic Mobility Shift
Assays--
The wild type CRE/ATF site of the cyclin D1 promoter,
CD1CREwt, and a mutant ATF-2 (CD1CREmut) site were synthesized as
complementary oligodeoxyribonucleotide strands for electrophoretic
mobility gel shift assays (EMSAs) (54). The sequence of the cyclin D1 promoter CRE/ATF site oligodeoxyribonucleotide (CD1CREwt) was 5'-AAC
AAC AGT AAC GTC ACA CGG AC-3'. EMSAs using nuclear extracts or in vitro translated proteins were performed essentially
as described previously (16, 62). The cDNAs were transcribed in vitro and translated using the TNT-coupled reticulocyte
lysate system according to the protocol of the suppliers (Promega,
Madison, WI). Proteins were incubated for 30 min at 4 °C in reaction
buffer (12 mM HEPES, pH 7.9, 0.6 M KCl, 1 mM EDTA, 1 mM dithiothreitol, 12% glycerol)
containing 500 ng of dI·dC. pp60v-src Induction of Cyclin D1--
Previous
studies had shown that cyclin D1 protein levels were increased in
NIH3T3 cells overexpressing pp60v-src (21). To
extend this analysis, we examined the role of
pp60v-src in expression of cyclin D1 in mammary
epithelial cells. Cyclin D1 protein levels are regulated by growth
factors and mitogens in MCF7 cells (6, 13, 14); however, the effect of
pp60src on cyclin D1 abundance in MCF7 cells had not been
determined. MCF7 cells were transfected with the expression vector
encoding pp60v-src, and Western blotting was
performed. Cyclin D1 protein levels were increased 8.1-fold by
overexpression of pp60v-src (Fig.
1A). As a form of internal
control, the blot was probed with the structural protein
In order to determine whether the cyclin D1 gene promoter
was a direct transcriptional target of pp60src, transient
expression studies were conducted with the human cyclin D1 promoter
linked to the luciferase reporter gene ( pp60v-src Induction of Cyclin D1 Involves MEK1,
JNK1, and MKK3 but Not MKK6--
In order to investigate the signal
transduction pathway by which pp60v-src induced
cyclin D1 in MCF7 cells, chemical inhibitors and expression vectors
encoding dominant interfering mutants of intracellular signaling
pathways were employed. The chemical inhibitor genistein was previously
shown to inhibit tyrosine kinases with a preferential inhibition of Src
kinase (65). Genistein inhibited pp60v-src
activation of cyclin D1 promoter activity in a
dose-dependent manner between 36 and 200 µM
(Fig. 2A). Herbimycin A was
also used as a Src kinase inhibitor (63) and blocked cyclin D1 promoter activation by pp60v-src in a
dose-dependent manner (Fig. 2B).
Both the JNK and ERK families of MAPKs have been implicated in
pp60src signaling. In our previous studies, the cyclin D1
promoter was induced by growth factors via the ERK pathway in adrenal
and trophoblast cell lines and in tracheal myocytes (16, 56, 66). The
MEK1/ERK inhibitor PD98059 (10-20 µM) suppresses the
activation of the MAPK/ERK pathway by preventing activation of the
upstream activator MAPK kinase 1 (MEK1) (67).
pp60v-src induction of the cyclin D1 promoter was
reduced 31% by the addition of PD98059 (Fig. 2C). In
previous studies the p38 kinase pathway was identified as an inhibitor
of cyclin D1 expression. The drug SB203580 is a specific inhibitor of
p38
The role of the MKK3 and MKK6 pathways in oncogene signaling or in
regulation of cyclin D1 remained to be determined. To further discriminate the signaling pathways involved in
pp60v-src induction of the cyclin D1
gene, we used plasmids encoding dominant negative mutants of MEK1
(MEKC), MKK3, and MKK6. Dominant negative mutants of MEK1 have been
shown to specifically inhibit induction of the downstream targets, ERK1
and ERK2. MKK3 and MKK6 govern activity of the p38 kinase group of MAP
kinases (48, 68). Expression plasmids encoding dominant negative
mutants of MKK3, MKK6, or MEK1 were co-expressed with
pp60v-src and the
Overexpression of the dominant negative mutant of MKK3 (MKK3 Ala)
reduced pp60v-src induction of
Together, these findings suggest that
pp60v-src-induced cyclin D1 promoter activity
requires several signaling pathways, including the MEK1, JNK, and MKK3
pathways but not the MKK6 pathway. In contrast with previous studies in
which the p38 (69) or JNK pathways (56) inhibited basal cyclin D1
expression, in the presence of the transforming
pp60v-src mutant, the Jun kinase and p38 pathways
are required for optimal activation of cyclin D1.
A CRE/ATF Site Is Required for pp60v-src
Activation of the Cyclin D1 Promoter--
In order to determine the
minimal region of the cyclin D1 promoter required for regulation by
pp60v-src, co-transfection experiments were
conducted with a series of 5' promoter deletion constructions in MCF7
cells. pp60v-src responsiveness of the promoter was
preserved with deletion from
In order to determine whether the CRE/ATF sequences were sufficient to
convey induction in response to overexpression by
pp60v-src, these sequences were linked to an
heterologous reporter to form p(CD1ATF)TKLUC. The p(CD1ATF)TKLUC
reporter was induced 16-fold by pp60v-src compared
with empty expression vector control (Fig. 3D). Thus, the
cyclin D1 CRE/ATF site responds to induction by
pp60v-src.
ATF-2/CREB Proteins Bind the Cyclin D1
We had previously shown that the cyclin D1 promoter AP-1 site at pp60v-src Induction of Cyclin D1 Is Inhibited
by Dominant Negative Expression Plasmids for ATF-2 and CREB but Not
c-Jun--
The EMSA studies indicated that CREB/ATF-2 complexes
bound the cyclin D1 promoter region involved in
pp60v-src induction of the cyclin D1 promoter
in MCF-7 cells. In order to examine the role of CREB and ATF-2 proteins
in pp60v-src induction of the cyclin D1, transient
expression studies were conducted with expression plasmids encoding
dominant negative mutants of these transcription factors. The KCREB
expression plasmid encodes a CREB cDNA that contains a mutation of
a single amino acid in the DNA binding domain and blocks the ability of
wild type CREB to bind to the CRE of the somatostatin promoter (57). The KCREB expression plasmid was previously shown to inhibit
cAMP-induced expression of the somatostatin CRE by 55%. The
pp60v-src-induced cyclin D1 promoter activity was
reduced 70% by the KCREB mutant (Fig.
5A). The ATF-2 dominant
negative expression plasmid ATF-2 M2 contains an alanine to arginine
substitution, which abolishes DNA binding to the CRE/ATF site and
abolished induction of the
Because c-Jun was implicated in pp60v-src signaling
to the psg2 gene (70), and we had previously shown that
c-Jun induced cyclin D1 expression in JEG-3 cells (16), we examined the
effect of the c-Jun dominant negative mutant TAM-67 on
pp60v-src induction of cyclin D1. The TAM-67
mutant, which lacks the transactivation domain of c-Jun, was previously
shown to convey potent dominant negative function and prevented AP-1
mediated transcriptional activation and transformation in breast cancer
cell lines (59). The pp60v-src-induced cyclin D1
promoter activity was further induced 2.4-fold (n = 14)
by TAM-67 (Fig. 5A), although this mutant effectively inhibited Ras induction of the collagenase AP-1 site reporter p3TPLUX (data not shown). Overexpression of TAM-67 also
induced basal cyclin D1 promoter activity 2.2-fold (n = 10) (data not shown). These findings suggest that, in contrast with the
findings in fibroblasts and JEG-3 cells, the cyclin D1 promoter may be under basal repression by c-Jun in MCF7 cells.
pp60v-src Induction of CREB and ATF-2
Transactivation Domains in MCF7 Cells--
These studies suggested
that ATF-2 and CREB were involved in the regulation of cyclin D1 by
pp60v-src in MCF7 cells. We hypothesized that
pp60v-src could either enhance the binding affinity
of proteins at the cyclin D1 CRE/ATF site or increase transactivation
function of transcription factors bound to the site. We examined the
possibility that pp60v-src may directly induce the
activity of CREB or ATF-2. The transactivation domains of these
proteins linked to the GAL4 DNA binding domain were introduced into
MCF7 cells with a heterologous DNA binding site for the GAL4 DNA
binding sequence. The (UAS)5E1BTATALUC reporter consists of
multimeric GAL4 DNA binding sites linked to a luciferase reporter gene
(Fig. 5B). The CREB and ATF-2 transactivation domains conveyed basal enhancer activity in MCF7 cells (Fig. 5B).
Overexpression of pp60v-src enhanced CREB activity
9-fold and point mutation of Ser133 in CREB abolished
induction by pp60v-src (Fig. 5B).
Overexpression of pp60v-src enhanced ATF-2 activity
3-fold. Mutation of the important threonine phosphorylation sites in
the transactivation domain of ATF-2 (Thr69 and
Thr71) to alanine (46) reduced both basal and
pp60v-src-induced activity (Fig.
5B).
The CREB and the ATF-2 proteins have been shown to function at the
distal end of signal transduction pathways involving MKK3 and MKK6 (48,
50, 51, 68, 71). The ERK, p38, and JNK pathways were involved in
pp60v-src induction of the cyclin D1 promoter and
both ATF-2 and CREB bound the pp60v-src response
element. We therefore assessed the independent contribution of the ERK,
p38 and JNK pathways to induction of CREB and ATF-2 transactivation
function in MCF7 cells. pp60v-src induction of CREB
was inhibited 50% by SB203580, whereas JIP-1 and PD98059 did not
affect CREB function (Fig. 5C). The
pp60v-src induction of ATF-2 was inhibited by JIP-1
more than 80%; however, the MEK/ERK inhibitor, PD98059 and the p38
inhibitor, SB203580, did not affect ATF-2 activity (Fig.
5C). These results indicate that
pp60v-src induction of CREB occurs at least in part
through the p38 pathway and pp60v-src induction of
ATF-2 occurs primarily through the JNK pathway.
Kinase Activities in the Mammary Gland Tumors of
MMTV-pp60c-src527F Transgenic
Mice--
We had previously shown that overexpression of
pp60c-src (MMTV-pp60c-SRC527F) under
transcriptional control of the MMTV long terminal repeat induced
mammary gland tumors with increased levels of
pp60c-src activity (28). To determine whether
cyclin D1 levels were induced in these mammary gland tumors, samples
from the MMTV-pp60c-SRC527F transgenic mice were analyzed
and compared with mammary gland tissue from strain-matched nontumorous
mammary tissue. Cyclin D1 protein levels were increased in 14 of 15 mammary gland tumors examined when compared with mammary gland from
nontransgenic mice (Fig.
6A).
Immunostaining of the mammary gland tumors of the transgenic animals
was performed to determine whether the increase in cyclin D1 protein
observed was localized to the nucleus or cytoplasm of the cell. In each
tumor, 500 cells were examined and scored for nuclear cyclin D1
staining. Tumors from four separate animals were examined. Cyclin D1
protein was found in the nucleus and increased nuclear abundance of
cyclin D1 was found in all tumors examined (15, 33, 46, and 47%) (Fig.
6B). Infrequent immunostaining for cyclin D1 was observed in
the adjacent normal mammary gland tissue or mammary tissue from control
animals (2-5%).
In order to examine the activity of cyclin D1 in the mammary gland
tumors, immune-precipitation kinase assays were performed using pRB
protein as substrate. The phosphorylated pRB band in Fig. 6C
was dependent upon the addition of pRB substrate and inhibited by the
addition of p16INK4a protein (data not shown), consistent
with the specificity of the kinase assays. An increase in cyclin
D1-dependent kinase (CD1K) activity was
observed in each of the tumors examined (Fig. 6C). The mean
CD1K activity of the tumors was increased 5.26 ± 0.8-fold (n = 15) compared with kinase activity from
equal amounts of protein derived from the mammary gland tissue of three
virgin mammary glands.
In order to determine the ERK and JNK activity in the mammary tumors of
pp60c-src527F transgenic mice, we
performed immune-precipitation kinase assays. As a substrate for
activation of the ERK pathway, myelin basic protein was used, whereas
for the JNK pathway, a synthetic fusion protein for the N terminus of
c-Jun was used. Equal amounts of protein were used and the induction of
immune-complex kinase activity was normalized to the activity
determined in the mammary gland tissue of virgin nontransgenic animals.
ERK activity was induced in each tumor. The induction varied from 3- to
60-fold (Fig. 6D). JNK activity was either unchanged or
reduced in every tumor. The reduction in JNK activity was between 30 and 50% below basal (Fig. 6D). Although ERK activity and
cyclin D1 protein levels were increased in most mammary gland tumors,
the increase in cyclin D1 protein abundance by Western blotting did not
correlate significantly with ERK activity or JNK activity. These
results are consistent with a model in which additional pathways to
ERK/JNK are involved in the sustained induction of cyclin D1 protein in
these tumors. Alternatively, it may be that heterogeneity within the
tumor confounds interpretation, and analysis of kinase activity at the
single cell level within the tumor may be more informative.
In these studies, we demonstrate that cyclin D1 is induced by
pp60src in mammary tumor cells and identify the intracellular
signaling pathway responsible for induction of the cyclin D1
gene by pp60v-src. Induction of the cyclin
D1 gene by pp60v-src required the MEK1/ERK,
MKK3/p38, and JNK pathways in mammary epithelial cells. Consistent with
our previous results in which ERK induced cyclin D1 promoter activity
and dominant negative mutants of ERK reduced growth factor-regulated
promoter activity (16, 52, 56), we found that the dominant negative
mutants of MEK1 and the MEK inhibitor PD98059 reduced
pp60v-src activation of the cyclin D1 promoter. The
ERKs functioned downstream of pp60c-src signaling
to the vascular endothelial growth factor gene (72). In contrast with
previous studies in which p38 was an inhibitor of cyclin D1 in CCL39
fibroblasts (69), we observed that the p38 inhibitor SB203580 reduced
pp60v-src induction of cyclin D1 by 38%. In
addition, the use of the JIP-1 expression plasmid, which selectively
retains Jun kinase in the cytoplasm thereby inhibiting Jun kinase
signaling, allowed us to identify an important role for Jun kinase in
pp60v-src signaling to cyclin D1. These results are
consistent with a previous study in which pp60v-src
induced JNK/SAPK activity using glutathione
S-transferase-c-Jun as substrate (31). The finding that
cyclin D1 is induced by pp60v-src through a
JNK signaling pathway is consistent with previous correlative analysis
in which activating Rac mutants, which induce JNK activity, also
induced the cyclin D1 promoter in a p21-activated
kinase-dependent manner (18) and that Dbl family
members, which induce transformation and JNK activity, also induced
cyclin D1 expression (20). The current studies are the first to
directly demonstrate the requirement for JNK in oncogene signaling to
cyclin D1.
The cyclin D1 promoter CRE/ATF site was required for optimal induction
by pp60v-src. Additional
pp60v-src-responsive sequences were also present
within the region between The dominant negative mutant of CREB reduced
pp60v-src-induced cyclin D1 expression by 70%.
CREB activity was induced by pp60v-src, and the
induction was abolished by mutation of Ser133. CREB
phosphorylation at Ser133 is induced by protein kinase A
(60), by calcium ions (74), by calmodulin-dependent kinases
II and IV (75), through a growth factor-induced MAPK/ERK cascade (which
was prevented by PD98059 (49)), and by FGF through a p38/MAP
kinase-activated protein pathway (50). In the current studies, SB203580
reduced pp60v-src induction of CREB activity
by 50%, suggesting a role for the p38 pathway. Unlike previous studies
in glial cell progenitors in which growth factor-induced
phosphorylation of CREB was sensitive to PD98059 (49), inhibition of
the MAPK/ERK pathway enhanced pp60v-src induction
of CREB activity in MCF7 cells. As with the induction of CREB
phosphorylation by FGF (50), our studies suggest that CREB activation
by pp60v-src at Ser133 is at
least in part dependent upon the p38 pathway.
ATF-2 binds to and is phosphorylated by JNKs and p38, and both the ERKs
and JNK enhance ATF-2 transcriptional activity (45, 46, 48, 61). ERK
phosphorylates ATF-2 in vitro, promoting ATF-2 DNA binding
(45, 47). JNK phosphorylation of ATF-2 enhances its transcriptional
activity (45, 46) and protects it from JNK-mediated ubiquitination and
degradation, thereby extending its half-life (76). The signaling
pathways regulating ATF-2 function in breast cancer cells were not
known. In the current studies, pp60v-src induced
ATF-2 activity and mutation of threonines 69 and 71 dramatically inhibited both basal and pp60v-src induced
activity. Recently, the JIP-1 protein was identified and shown to bind
JNK in the cytoplasm, interfering with JNK nuclear translocation and
activity (61). JIP-1 binds several components of the JNK signaling
pathway, including hematopoietic progenitor kinase-1 and JNK, thereby
functioning as a scaffold to promote interactions between these
components of the JNK signal transduction module (77). In the present
studies, JIP-1 blocked induction of ATF-2 transactivation by
pp60v-src. Neither the MEK nor p38 inhibitors
reduced pp60v-src-induced ATF-2
activity. These studies provide further evidence that ATF-2 is a target
of pp60v-src through a JNK-dependent
pathway in breast cancer cells.
In the current studies, c-Jun did not contribute to the induction of
cyclin D1 by pp60v-src. The dominant negative of
c-Jun, TAM-67, which efficiently inhibited AP-1 activity in MCF7 cells
(59, 78), further induced the cyclin D1 promoter, consistent with a
role for c-Jun as an inhibitor of cyclin D1 in MCF7 cells.
pp60v-src has been shown to induce components of
the AP-1 signaling pathway in fibroblasts. In the current studies, the
results from the 5' promoter deletion analysis demonstrated the minimal
region required for induction by pp60v-src was
located within the CRE/ATF site at In the current studies, cyclin D1 was induced by
pp60v-src in MCF7 cells. These results contrast
with the findings in fibroblast cells. In Rat-1 cells, cyclin D1
expression was not induced by a temperature-sensitive
pp60v-src (26). We also observed, in a detailed
time course using the Src dominant negative cell line 3T3SrcRF cells
(72) in the presence or absence of
isopropyl-1-thio- The mammary gland tumors from the
pp60c-src527F transgenic mice exhibited
increased cyclin D1 protein levels, CD1K activity, and an induction of ERK activity but unchanged or reduced JNK activity. The
process of tumorigenesis involves multiple events, and cellular heterogeneity within the tumor mass limits detailed correlative analysis; however, the induction of both cyclin D1 levels and ERK
activity in the tumors provides supportive data for a role for cyclin
D1 and ERK in the pathogenesis of the
pp60c-src527F mammary tumors. Cell
lines derived from MMTV-Neu transgenic mice were recently shown to
exhibit increased ERK but unchanged JNK activity (79). Induction of JNK
and p38 kinases play an important role in apoptosis (80, 81). It is
possible, therefore, that an induction of JNK activity in the mammary
gland tumors may not have been detected due to apoptosis of cells
containing activated JNK activity. CD1K activity is
determined by the abundance and subcellular distribution of the cyclin
D1/cdk4 proteins and the cyclin-dependent kinase inhibitor
proteins, including members of the INK4 family and the CIP/KIP family
(3). p27Kip1 increases the association of cyclin D1 with
cdk4 and targets this complex to its nuclear site of action (82, 83).
In contrast with studies in Rat-1 cells in which overexpression of
pp60v-src inhibited p27Kip1 levels
(26), our studies demonstrated that nuclear p27Kip1 was
increased in the majority of mammary gland tumors examined from the
MMTV-pp60c-src527F transgenic mice
(data not shown). p16INK4A protein was not detected in most
of these tumors.2 As deletion
of the CDKI p16 locus occurs in many breast tumors and breast cell
lines (84), and reduced p16INK4A abundance is associated
with increased CD1K activity (85), loss of
p16INK4A may also contribute to the multistep tumorigenesis
in these tumors.
INTRODUCTION
Top
Abstract
Introduction
References
and p38
MAPK, prevents the activation of MAP
kinase-activated protein-K2 and MAP kinase-activated protein-K3
(37).
MATERIALS AND METHODS
-tubulin, the antibody (5H1)
(16, 52) was used; and for the Flag epitope, the M2 monoclonal antibody
(Kodak Scientific Imaging Systems) was used. Cell homogenates were
electrophoresed in an SDS-polyacrylamide gel and transferred
electrophoretically to a nitrocellulose membrane (MSI, Westborough,
MA). After transfer, the gel was stained with Coomassie Blue to assess
transfer efficiency. The blotting membrane was incubated for 2 h
at 25 °C in T-phosphate-buffered saline buffer supplemented with 5%
(w/v) dry milk, and after a 6 h incubation with primary antibody
at a 1:1000 dilution (cyclin D1) or 1:2500 (
-tubulin) in Tween 20 phosphate-buffered saline buffer containing 0.05% (v/v) Tween 20, the
membrane was washed with the same buffer. For detection of cyclin D1
and
-tubulin, the membrane was incubated with goat anti-mouse
horseradish peroxidase second antibody (Santa Cruz Biotechnology) and
washed again. The enhanced chemiluminescence system (Kirkegaard and
Perry Laboratories, Gaithersburg, MD) was used to visualize the cyclin
D1 protein.
66 CD1LUC and
66 CD1ATFmutLUC were
created by polymerase chain reaction from the
141 CD1LUC plasmid
using specific primers and the region including the CRE/ATF site was
mutated from 5'-T AAC GTC ACA CGG ACT-3' to 5'-T cgC
GTC cCc CGG gCc-3' (
66 CD1ATFmutLUC) (54). The sequences
resembling a CRE/ATF site located between
66 and
40 were cloned
into the reporter TK81pA3LUC to form the reporter p(CD1ATF)TKLUC. The reporter p3TPLUX contains a trimeric
collagenase AP-1 site, c-fosLUC contains the
c-fos promoter sequences from
361 to +157, and the serum
response element TATALUC reporter encodes the c-fos serum
response element linked to the luciferase reporter gene (52). The
expression vectors encoding pp60c-src (pMc-src), an
activating mutant of pp60c-src (pSRC527F) and
pp60v-src (pMv-src) (28, 55), the dominant negative
mutants of MKK3 (pRSV-Flag-MKK3 Ala) and MKK6 (pCDNA3-Flag-MKK6
K82A) (48), the vector glutathione S-transferase c-Jun, and
the dominant negative MEK1 (Ala-218/Ala222) were previously described
(56). The CREB dominant negative expression vector KCREB (57) was a
gift from Dr. R. Goodman. The ATF-2 dominant negative mutant ATF-2 M2
(58) was a gift from Dr. L. Glimcher. The ATF-2 dominant negative
pECE-ATF-2 (Ala 69/71) was described previously (46). The c-Jun
dominant negative mutant TAM-67 (59) was a gift from Dr. M. Birrer. The human ATF-2 cDNA was cloned into pGEM3 for in vitro
translation. The GAL4-CREB, GAL4-CREBSer133mut (60),
GAL4-ATF2(1-109), and GAL4-ATF2Ala69,71 constructions (46)
and the expression plasmid encoding JNK-interacting protein-1 (JIP-1),
pCMV-Flag-JIP-1, were previously described (61).
-32P-Labeled
oligonucleotides (50 fmol; 100,000 counts/min.) were added to the
reaction and incubated at room temperature for a further 30 min.
Supershifts were performed using antibodies to ATF-2 (C-19-X), c-Fos
(K-25-X) (Santa Cruz Biotechnology), CREB (HM93) (a generous gift from
Dr. J. Habener) (64), and a Jun antibody that inhibits DNA binding of
c-Jun (c-Jun antibody; Upstate Biotechnology, Inc., Lake Placid, NY)
(54). The protein-DNA complexes were analyzed by electrophoresis
through a 5% polyacrylamide gel, with 0.5× Tris borate, EDTA buffer
(0.045 M Tris borate, 0.001 M EDTA) and 2.5%
glycerol at 180 V for 3-4 h. The gels were dried and exposed to
autoradiographic film (Labscientific Inc.).
RESULTS
-tubulin,
the abundance of which was unchanged by the overexpression of
pp60v-src in the MCF7 cells (Fig. 1A, lower
panel).
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Fig. 1.
pp60v-src induction of
cyclin D1 protein and promoter activity in MCF7 cells.
A, MCF7 cells were transfected with an expression vector
encoding pp60v-src or empty vector cassette, and
Western blotting was performed for cyclin D1 abundance and normalized
by probing of the same blots with an -tubulin antibody.
B, increasing amounts of pp60c-src
(pSRC527F) (representative experiment), or mean data using 300-600 ng
of expression vector for pp60c-src were transfected
with 2.4 µg of
1745 CD1LUC. C and D,
comparison was made with the effect of pp60v-src on
1745 CD1LUC or pA3LUC (C) or
c-fosLUC and serum response element TATALUC reporters
(D). The data are the mean ± S.E. for n
separate transfections as indicated in parentheses.
1745 CD1LUC) and the
pp60c-src527F expression vector.
pp60c-src527F induced the cyclin D1
promoter with a mean of 6.1-fold (± 1.7) in MCF7 cells (Fig.
1B). Comparison was also made with the
pp60v-src expression vector, which induced the
full-length
1745 CD1LUC reporter a mean of 48-fold (± 9.0) (Fig.
1C). In contrast, control plasmids including the
promoterless pA3LUC vector were not regulated by
pp60v-src (Fig. 1C). As a form of
positive control, promoters for genes previously shown to be induced by
pp60c-src in fibroblast cell lines were examined in
MCF7 cells. The c-fosLUC and serum response element TATALUC
reporters were induced 142-fold and 62-fold, respectively (Fig.
1D).
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Fig. 2.
Inhibitors of Src kinase, p38, ERK, and JNK
inhibit pp60v-src induction of the cyclin D1
promoter activity in MCF7 cells. In order to examine the
intracellular signaling pathways involved in
pp60v-src-activation of the cyclin D1 promoter, the
1745 CD1LUC reporter was introduced into MCF7 cells with the
pp60v-src expression vector as described under
"Materials and Methods." The effect of the chemical inhibitors of
the tyrosine kinase pathway induced by Src, genistein (A),
and herbimycin A (B) is shown for concentrations known to
inhibit pp60src kinase activity in several different cell types
(72, 86). The results are shown as the mean ± S.E. of at least
five separate transfections. C, the effect of inhibitors for
the p38 pathway, SB203580 (10-20 µM), and the MEK1/ERK
pathway, PD098059 (10-20 µM), on
pp60v-src induction of the cyclin D1 promoter are
shown as mean data ± S.E. for n separate transfections
as indicated in parentheses. Cyclin D1 promoter-luciferase
reporter activity was measured after 24 h of treatment, and the
effect is shown compared with vehicle-treated cells. D, the
effect of the potent inhibitor of the JNK pathway, JIP-1 (61), on
pp60v-src induction of the
1745 CD1LUC reporter
is shown. In E, the reporter p3TPLUX, which
contains a trimeric collagenase AP-1 site, was used. The effect of
JIP-1 on pp60v-src induction of p3TPLUX
is shown as mean ± S.E. with increasing concentrations of JIP-1.
F, co-transfection experiments were conducted with dominant
negative mutants for MEK1 (MEKC), MKK3 (MKK3
Ala), and MKK6 (MKK6 K82A). Comparison was made between the effect of the
dominant negative mutant and equal amounts of empty expression vector
cassette (control). In G, the effect of
increasing concentration of the MKK6 dominant negative mutant on
pp60v-src-induced cyclin D1 promoter activity was
determined. In the inset, a Western blot is shown of MCF7
cell extracts transfected with increasing amounts of the dominant
negative mutants for MKK3 (MKK3 Ala) or MKK6 (MKK6
K82A). The amount of transfected expression vector is shown
above each lane. Western blotting was performed after
48 h using an antibody to the FLAG epitope. The expression level
of the MKK6 vector was 20% of the MKK3 vector in MCF7 cells.
and p38
MAPK (68). The addition of SB203580 (10-20
µM) reduced pp60v-src induction of
the cyclin D1 promoter activity by 38% (Fig. 2C). To
investigate the role of the Jun kinase pathway in
pp60v-src induction of cyclin D1 promoter activity,
we employed an expression vector encoding JIP-1. Through cytoplasmic
retention of JNK, JIP-1 functions as a powerful dominant negative of
JNK-dependent activation of both c-Jun and ATF-2
transcriptional activities (61). Overexpression of JIP-1 reduced
pp60v-src-induced cyclin D1 promoter activity by
80-90% (Fig. 2D). Inhibition of
pp60v-src-induced cyclin D1 promoter activity by
JIP-1 occurred in a dose-dependent manner (Fig.
2D). Consistent with a role for Jun kinase activity in
pp60v-src induction of an AP-1 reporter, JIP-1
overexpression also inhibited pp60v-src induction
of the collagenase AP-1 reporter, p3TPLUX (Fig.
2E) in a dose-dependent manner.
1745 cyclin D1 promoter
reporter in MCF7 cells. Overexpression of the MEK1 dominant negative
mutant (MEKC) reduced pp60v-src-induced cyclin D1
promoter activity by >50% (Fig. 2F).
1745 CD1LUC
reporter activity by 73%. The dominant negative mutant of MKK6 (MKK6
K82A) did not inhibit pp60v-src induction of cyclin
D1 (Fig. 2F). The expression levels of the MKK3 and MKK6
dominant negative expression plasmids were assessed in transfected 293T
and MCF7 cells. The Flag epitope-tagged MKK3 dominant negative vector
(pRSV-Flag-MKK3 Ala) and MKK6 (pCDNA3-Flag-MKK6 K82A) were
transfected into cells with increasing doses of expression plasmid from
5 to 30 µg. Western blotting was performed of cell extracts using the
Flag antibody (M2). In 293T cells, protein expression was identical by
Western blotting (data not shown). In MCF7 cells, however, in three
separate experiments, the relative abundance of Flag-tagged MKK6
protein was 5-6-fold less than Flag-tagged MKK3 protein when equal
amounts of supercoiled plasmid was transfected (Fig. 2G,
inset). Further comparison was therefore made using up to 10-fold
larger amounts of transfected kinase dead MKK6 expression vector;
however, MKK6 K82A did not inhibit pp60v-src
induction of cyclin D1 (Fig. 2G).
1745 to
141 (Fig.
3B). Deletion from
141 to
66 reduced induction from 46- to 17-fold. Deletion from
66 to
22
reduced induction from 17- to less than 3-fold (Fig. 3B).
Enhancer elements resembling a CRE/ATF site are located within the
pp60v-src-responsive region between
66 and
22
(56). As the minimal pp60v-src-responsive sequences
were located between
66 and
22, the role of the CRE/ATF-like
sequences at
58 were initially assessed. Clustered point mutation of
the ATF site was performed in the context of the
66 bp promoter
fragment. Direct comparison was made between the
66 CD1LUC and the
66 CD1ATFmutLUC reporters in the presence of
pp60v-src. Induction of the
66 bp reporter
fragment was reduced 80% by mutation of the CRE/ATF site (Fig.
3C).
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Fig. 3.
The cyclin D1 promoter CRE/ATF site is
required for induction of the cyclin D1 promoter by
pp60v-src in MCF7 cells. A,
schematic representation of the cyclin D1 promoter deletion constructs.
B, the pp60v-src expression vector or
cassette was transfected with cyclin D1 5' promoter constructs. *
represents significant difference from the adjacent 5' deletion
construct for p < 0.05. Data are shown as the
mean ± S.E. for n separate transfections as indicated
in parentheses, comparing the effect of
pp60v-src expression vector with empty vector
cassette. C, comparison was made between the 66 CD1LUC and
66 CD1ATFmutLUC reporters in the presence of 300-600 ng of
expression vector. Results are shown as percentage of relative activity
for the effect of the transfected pp60v-src
expression vector on
66 CD1ATFmutLUC versus
66 CD1LUC.
D, the pp60v-src expression vector or
empty cassette was transfected with either the heterologous
construction encoding the CRE/ATF region of the cyclin D1 promoter
linked to the minimal TK promoter (P1(CD1ATF) TKLUC) or the
minimal TK promoter (TKLUC).
58 Region in MCF7
Cells--
In previous studies we observed the
58 region bound CREB
proteins in the JEG-3 trophoblast cell line (56) and FOS/CREB proteins
in mouse embryo-derived fibroblasts (MEFs) (54). The transcription
factor complexes binding the cyclin D1 CRE/ATF site were critical for
serum-induced proliferation in MEFs (54). In order to understand the
mechanism by which pp60v-src induced cyclin D1, the
transcription factor complexes binding the CRE/ATF sequences were
assessed in MCF7 cells using EMSAs and specific antibodies to members
of the CREB/ATF protein family. The complex formed in the presence of
the
-32P-radiolabeled cyclin D1 CRE/ATF probe was
specifically competed by a 100-fold molar excess of cold
double-stranded wild type (Fig. 4A,
lane 2) but not mutant competitor (Fig. 4A, lane 3).
The ATF-2 binding complex was super-shifted by the addition of ATF-2
antibody (Fig. 4A, lane 4). The Fos antibody (Fig. 4A,
lane 5) and preimmune serum (Fig. 4A, lane 6) did not
supershift the complex. The same Fos antibody supershifted a complex
binding to the CRE/ATF site in MEF extracts (54). Thus, unlike the
complex found in MEFs, c-Fos did not form part of the complex binding
the cyclin D1 CRE/ATF site in MCF7 cells (Lane 5). In vitro
translated full-length ATF-2 protein was incubated with the cyclin D1
CRE/ATF site probe (Fig. 4A, lane 7). The electrophoretic
mobility of the specific complex binding the cyclin D1 CRE/ATF site was
identical to the mobility of the complex A binding these sequences in
MCF7 cell nuclear extracts (Fig. 4A, lane 1 versus lane 7).
Addition of ATF-2 antibody supershifted the in vitro
translated ATF-2 complex binding the cyclin D1 ATF-CRE site (Fig.
4A, lane 8).
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Fig. 4.
Binding of ATF-2 to the cyclin D1 CRE/ATF
site in MCF7 cells. A, the -32P-labeled
cyclin D1 CRE/ATF probe was incubated with MCF7 nuclear extracts either
alone (lane 1), with 100-fold excess of cold wild type
competitor (lane 2), with an equimolar amount of
double-stranded mutant competitor (lane 3), with ATF-2
antibody (Ab) (lane 4), with c-Fos antibody
(lane 5), or with preimmune serum (lane 6). The
probe was incubated with in vitro translated ATF-2 alone
(lane 7) and in the presence of ATF-2 antibody (lane
8). B, the
-32P-labeled cyclin D1
CRE/ATF probe was incubated either with MCF7 nuclear extracts alone
(lane 1), with preimmune serum (lane 2), or with
antibodies to Jun (lane 3), cAMP response element-binding
protein/cAMP response element modulator (lane 4), or ATF-2
(lane 5).
953
bound c-Jun with similar binding affinity as the canonical AP-1 site
from the collagenase gene (16). The complex A formed in the presence of
MCF7 cell extracts (Fig. 4B, lane 1), was not supershifted
with the addition of the c-Jun antibody, but was shifted by the CREB
antibody (Fig. 4B, lane 4, arrow S/S indicates supershifted
band) and by the ATF-2 antibody (Fig. 4 A, lane 4, and
B, lane 5). These studies indicate that in MCF7 cells the cyclin D1 CRE/ATF site at
58 binds CREB/ATF-2 proteins.
-subunit CRE reporter (58). The
pp60v-src-induced cyclin D1 promoter activity was
reduced 25% by the ATF-2 dominant negative expression plasmid ATF-2 M2
(Fig. 5A).
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Fig. 5.
CREB and ATF-2 function are required for
pp60v-src induction of cyclin D1. CREB and
ATF-2 are transcriptionally induced by
pp60v-src. A, in order to
examine the role of CREB and ATF-2 in pp60v-src
activation of cyclin D1 promoter, dominant negative mutants of CREB
(KCREB), ATF-2 (ATF-2 M2), and c-Jun
(TAM-67) were employed. The 1745 CD1LUC reporter was
introduced into MCF7 cells with the pp60v-src
expression vector and the dominant negative mutants. Comparison was
made between the effect of the dominant negative and equal amounts of
empty expression vector cassette. B, schematic
representation of the GAL4-CREB, GAL4-ATF-2 constructions, and the
heterologous luciferase reporter containing five upstream activator
binding sites for the GAL4 DNA binding domain. The reporter
(UAS)5E1BTATALUC (2.4 µg) was transfected with expression
vectors for GAL4-CREB, GAL4-CREBmut (CREBSer133mut),
GAL4-ATF-2, GAL4-ATF-2Ala69,71mut (46), and either
pp60v-src (300 ng) or empty expression vector
cassette in MCF7 cells. Comparison was made between the effect of the
pp60v-src expression vector and equal amounts of
the empty vector. C, the effect of the inhibitors for the
MEK/ERK pathway (PD98059), the p38 pathway (SB203580), and the JNK
pathway (JIP-1) were assessed for their role in
pp60v-src activation of CREB or ATF-2. The
heterologous reporters GAL4-CREB and GAL4-ATF-2 were transfected with
the reporter (UAS)5E1BTATALUC (2.4 µg) in the presence of
pp60v-src (300 ng). The data are shown for the
percentage of change in activity. The mean induction by
pp60v-src in these experiments was 9-fold for CREB
and 3-fold for ATF-2. The data are shown as mean ± S.E. for
n separate experiments as indicated.
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Fig. 6.
Cyclin D1 abundance and kinase activity in
mammary gland tumors derived from the
pp60c-src527F transgenic animals.
pp60c-src527F transgenic mice mammary
gland tumors protein extracts were analyzed. A, Western
blotting for cyclin D1 protein was performed on mammary gland tumors
derived from transgenic mice overexpressing the
MMTV-pp60c-src (pSRC527F) transgene (lanes
2-8). A representative Western blot is shown with comparison to
nontumorous virgin mammary gland tissue from the same strain of mice
(lane 1). In B, immunohistochemical staining was
performed to examine the presence of cyclin D1 within the mammary gland
tumor cells. Representative example of immunostaining for cyclin D1 in
mammary gland tumors derived from the
pp60c-src527F transgenic animals.
Positive nuclei appear brown (yellow arrow). (500 cells were
counted within the tumors from each of four separate animals. The
percentages of cells scored as positive for nuclear cyclin D1 staining
for the four separate tumors were 33, 46, 15, and 47%). C,
immune-complex kinase assays for cyclin D1 kinase on
pp60c-src527F transgenic mice mammary
gland tumors protein extracted from 15 separate animals. The
phosphorylated pRB band for the cyclin D1 kinase assays is indicated by
the arrow (pRBp). D, ERK and JNK activity were
determined in tumors from separate animals, and activity is shown for each tumor on the y axis,
with cyclin D1 abundance expressed as fold induction compared with wild
type mammary tissue shown on the x axis. In E,
ERK and JNK activity is shown with the CD1K activity for
each tumor.
DISCUSSION
141 and
66; however, the minimal
sequences required for induction remained within the
66 bp fragment.
Mutation of the CRE/ATF site in the context of the native promoter
significantly reduced induction by pp60v-src and
the CRE/ATF sequence was induced 16-fold by
pp60v-src when linked to a heterologous promoter,
consistent with an important role for this sequence in
pp60v-src signaling to the cyclin D1
gene. These studies identified the ATF-2 and CREB transcription factors
as the primary proteins binding these DNA sequences in MCF7 cells, and
overexpression of pp60v-src increased the amount of
total complexes bound at this site (data not shown). We had previously
shown that the cyclin D1 CRE/ATF site binds CREB in human
choriocarcinoma (JEG-3) cells (56). The CRE/ATF site contributed to
induction of the cyclin D1 promoter by the SV40 small t antigen (56)
and was important in serum-induced cyclin D1 expression in MEFs (54).
In MEFs, we observed a serum-inducible complex consisting primarily of
FOS/CREB proteins binding this site (54). In MEFs derived from mice
homozygously deleted of the c-fos and fosB genes
(c-fos
/
/fosB
/
),
the serum-induced proliferation rate was reduced in association with a
loss of serum-induced cyclin D1 mRNA and protein (54). Together
with the observation that cyclin D1
/
MEFs
are reduced in their proliferative response to serum (54), these
studies were consistent with a role for FOS proteins and the cyclin D1
CRE/ATF site in serum responsiveness of the cyclin D1 gene
in MEFs. The cyclin A promoter contains a CRE/ATF binding site that
bound ATF1, CREB1, and ATFa2 in HeLa cell extracts (73). This region of
the cyclin A promoter was important in activation by
TAFII250. Cells mutant in TAFII250 are
defective in cell cycle progression and have reduced cyclin D1 and
cyclin A transcription, suggesting an important link between cellular
proliferation and the CREB/ATF family of proteins (73).
58. Our laboratory previously identified sequences in the cyclin D1 promoter capable of binding AP-1
proteins, located at
953 (16, 52, 56). The induction of cyclin D1 by
the mitogen angiotensin II in human adrenal cells was mediated through
a c-Fos/c-Jun AP-1 binding site located at
953 (52) and the AP-1 site
was also important in activation of the promoter by p21ras in
trophoblast cells (16). The sequences at
953 were not involved in
pp60v-src induction. Furthermore, c-Jun/c-Fos did
not form part of the complex binding to the
pp60v-src response element at
58 in MCF7 cells.
Together these results indicate that distinct components of the
AP-1/CREB protein family can bind either to the
953 or
58 site in
the cyclin D1 promoter to convey signaling by different
mitogenic-oncogenic signaling pathways in a cell-type specific manner.
-D-galactopyranoside (5 mM)
for 24 h, that cyclin D1 levels were unchanged despite a 10-fold
induction of dominant negative Src protein levels (data not shown).
Furthermore, in Rat-1 cells, pp60v-src inhibited
p27Kip1 levels (26), whereas in murine mammary tumors
induced by transgenic overexpression of
pp60c-src527F p27Kip1
protein levels were uniformly increased (see below). These studies support the notion that pp60src utilizes distinct signaling
pathways in mammary compared with fibroblast cells and imply important
differences may exist in the mechanisms by which
pp60v-src regulates the cell cycle in different
cell types.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Drs. J. Avruch, M. Birrer,
R. Goodman, J. Habener, J. Kyriakis, F. McCormick, S. G. McDonald,
and C. Miller for antibodies and A. Saltiel for PD098059. We thank Dr.
Xiaomai Zhou for the
isopropyl-1-thio--D-galactopyranoside-inducible stable
cell line.
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FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants R29CA70897, RO1 CA75503, and P50-HL56399 (to R. G. P.). Work conducted at the Albert Einstein College of Medicine was supported by Cancer Center Core National Institutes of Health Grant 5-P30-CA13330-26 and the Mortimer Harrison foundation (to R. G. P.).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.
** Supported by research grants awarded by the Canadian Breast Cancer Initiative and recipient of a Medical Research Council of Canada Scientist award.
¶¶ Recipient of the Ira T. Hirschl award and an award from the Susan G. Komen Breast Cancer Foundation. To whom correspondence should be addressed: Albert Einstein Cancer Center, Depts. of Medicine and of Developmental and Molecular Biology, Albert Einstein College of Medicine, Chanin 302, 1300 Morris Park Ave., Bronx, NY 10461. Tel.: 718-430-8662; Fax: 718-430-8674; E-mail pestell{at}aecom.yu.edu.
2 R. J. Lee and R. G. Pestell, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: Cdk, cyclin-dependent kinase; pRB, retinoblastoma protein; MAPK, mitogen-activated protein kinase; CREB, cAMP response element-binding protein; MMTV, murine mammary tumor virus; ATF, activating transcription factor; JNK, c-Jun N-terminal kinase; AP, activator protein; JIP, JNK-interacting protein; MEF, mouse embryo fibroblast; SAPK, stress-activated protein kinase; ERK, extracellular signal-regulated kinase; MKK, MAPK kinase; EMSA, electrophoretic mobility gel shift assay.
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REFERENCES |
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