(Received for publication, June 9, 1995)
From the
Several different oncogenes and growth factors promote G phase progression. Cyclin D1, the regulatory subunit of several
cyclin-dependent kinases, is required for, and capable of shortening,
the G
phase of the cell cycle. The present study
demonstrates that transforming mutants of p21
(Ras Val-12, Ras Leu-61) induce the cyclin D1 promoter in
human trophoblasts (JEG-3), mink lung epithelial (Mv1.Lu), and in
Chinese hamster ovary fibroblast cell lines. Site-directed mutagenesis
of AP-1-like sequences at -954 abolished
p21
-dependent activation of cyclin D1
expression. The AP-1-like sequences were also required for activation
of the cyclin D1 promoter by c-Jun. In electrophoretic mobility shift
assays using nuclear extracts from cultured cells and primary tissues,
several AP-1 proteins (c-Jun, JunB, JunD, and c-Fos) bound the cyclin
D1 -954 region. Cyclin D1 promoter activity was stimulated by
overexpression of mitogen-activated protein kinase
(p41
) or c-Ets-2 through the proximal 22 base
pairs. Expression of plasmids encoding either dominant negative MAPK
(p41
) or dominant negatives of ETS activation
(Ets-LacZ), antagonized MAPK-dependent induction of cyclin D1 promoter
activity. Epidermal growth factor induction of cyclin D1 transcription,
through the proximal promoter region, was antagonized by either
p41
or Ets-LacZ, suggesting that ETS functions
downstream of epidermal growth factor and MAPK in the context of the
cyclin D1 promoter. The activation of cyclin D1 transcription by
p21
provides evidence for cross-talk between the
p21
and cell cycle regulatory pathways.
The cyclin-dependent kinases are a family of serine/threonine
kinases that play a pivotal role in controlling progression through the
cell cycle(1, 2, 3, 4) . The
regulatory subunits of the cyclin-dependent kinases, known as cyclins,
form complexes with their catalytic partners to function as kinases of
specific proteins at different phases of the cell
cycle(3, 4) . Cyclin D1 is induced in late G following the treatment of growth-arrested macrophage cell lines
with colony-stimulating factor 1 (5) . Cyclin D1 is required
for progression of the G
phase (6) and is,
therefore, a critical target for proliferative signals in
G
. Cyclin D1 is capable of shortening the G
phase of the cell cycle, suggesting that cyclin D1 may be rate
limiting in G
progression(7, 8, 9) . The induction of
cyclin D1 mRNA in response to cell cycle progression and the addition
of growth factor is very rapid (6, 10, 11, 12) and is likely
regulated at the level of
transcription(5, 13, 14, 15) .
The ras gene, which is highly conserved in evolution, plays
an essential role in cellular proliferation(16) , cellular
development (17) , and cellular
differentiation(18, 19, 20, 21) .
The normal ras gene, activated by point mutations at amino
acids 12, 13, or 61, is also capable of inducing cellular
proliferation, development, and
differentiation(18, 19, 20, 21) .
Dominant negative mutants of p21block cellular
proliferation of NIH3T3 cells (22, 23) and the
induction of DNA synthesis and gene expression induced by
serum(22) . p21
acts at several distinct
phases of the cell cycle including early G
, the
G
/S boundary(24) , and at
G
/M(25) . In evidence for a role of p21
in early G
, ras activity is required
soon after the release of cells from quiescence(24) . After the
addition of serum, the proportion of Ras-GTP increases within 5 min,
and the induction of immediate-early gene expression can be at least
partially inhibited by previous injection of anti-Ras
antibody(26) . In addition, anti-Ras antibody or the dominant
Ras inhibitor proteins (27) efficiently inhibit DNA synthesis
within the recipient cell as long as the injection occurs prior to the
onset of the S phase. These findings suggest that p21
is also required late in the G
phase of the cell
cycle, presumably at the G
/S boundary.
Recent studies
have suggested functional interactions between p21 and cyclin D1. Cyclin D1 collaborates with p21
in primary rat kidney (28) or rat embryo fibroblasts
transformation assays(29) . In cell lines overexpressing
p21
, cyclin D1 mRNA levels were
induced(30) . In yeast, Ras activates transcription of the CLN genes(31) , which are analogous regulators of
G
phase progression(32) . p21
has the capacity to phosphorylate and/or activate target
transcription factors including
c-Jun(33, 34, 35) . The ability of
p21
to augment the transactivation by
c-Jun(36, 37) , likely involves a Jun kinase pathway
distinct from p42
(
)(36, 37, 38) . c-Jun,
in conjunction with several related AP-1 proteins, promotes G
phase progression (39, 40) and DNA
synthesis(41) . For example, inhibition of Jun expression with
antisense RNA (42) or microinjection of antibodies (40, 43) inhibits cell cycle progression induced by
the addition of serum to G
-arrested cells. Thus, several
lines of evidence suggest that p21
activates
c-Jun and that members of the c-Jun/AP-1 family are involved in
promoting cellular proliferation(44) .
In addition to the Jun kinase pathway, a distinct MAPK pathway is involved in the intracellular transmission of growth factor signals (34) . Protein kinases including the MAPK or extracellular signal-regulated kinases (ERKs) (45) are induced in response to EGF. Induction of the MAPK pathway is associated with activation of several different transcription factors including the ETS family proteins(17, 46, 47, 48) . The synthesis of the relatively ubiquitous c-Ets-2 is induced upon growth factor stimulation, and its phosphorylation is increased in response to mitogenic stimulation(48) .
The target DNA sequences of the ETS proteins includes a core motif with extensive variation at both the 5` and 3` sides of the invariant GGA core(48) . In a variety of promoters that lack TATA sequences, ETS binding sites have been localized close to the initiation site(48, 49) . Several mechanisms are likely to restrict or condition the activity of these sites as effective ETS-responsive elements. The binding of ETS family members to select promoter/enhancer sequences and their full transcriptional activity may require additional nuclear factors including Sp-1(50) . Sequences resembling the core motif (GGA) required for ETS protein binding (48) are located within the proximal cyclin D1 promoter.
We hypothesized that the recently
identified pathways linking tyrosine kinase receptor growth factors,
p21, and MAPK (18, 45) may be
involved in transcriptional regulation of the cyclin D1 gene and
thereby cell cycle progression. Cyclin D1 promoter fragments linked to
the luciferase reporter gene were examined in the presence of
activating or dominant negative, p21
, MAPK,
c-Jun, and c-Ets mutants, to determine their role in cyclin D1
transcription. Two distinct regions of the cyclin D1 promoter were
identified as the site of activation by either
p21
/c-Jun, or EGF,
p41
/c-Ets-2. The identification of
distinguishable mitogenic pathways regulating cyclin D1 transcription
provides a mechanism for specificity in signal transduction cross-talk
between proliferative pathways and a cell cycle regulatory pathway.
Cell culture, DNA transfection, and luciferase
assays were performed as described previously(58) . The
trophoblast cell line JEG-3, the fibroblast cell line COS, and the mink
lung epithelial cell line Mv1.Lu (CCl-64) were maintained in
Dulbecco's modified Eagle's medium with 10% fetal calf
serum and 1% penicillin/streptomycin. Chinese hamster ovary (CHO) cells
were maintained in -minimum essential medium with 10% fetal 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 h.
In cotransfection experiments, comparison was made between the effect of transfecting active expression vector with the effect of an equal amount of the parental empty expression vector. At least three different plasmid preparations of each construct were used. In cotransfection experiments, a dose response was determined in each experiment with 40, 60, 80, 100, and 200 ng of expression vector and the cyclin D1 promoter reporter plasmids (1.6 µg). The fold effect was determined for 100 ng of expression vector. Luciferase assays were performed at room temperature using an Autolumat LB 953 (EG&, Berthold). Luciferase content was measured by calculating the light emitted during the initial 30 s of the reaction, and the values are expressed in arbitrary light units(58) . Background activity from cell extracts was typically < 150 arbitrary light units/30 s. Statistical analyses were performed using the Mann Whitney U-test. Significant differences were established as p < 0.05. EGF treatment was performed for 6-24 h at doses from 2 to 20 ng/ml to determine maximal responses. Subsequent experiments were conducted using EGF at 2.5 ng/ml for 24 h.
Flow cytometric analysis was carried out in a fluorescence-activated cell sorter (FACStar plus; Beckton Dickonson). DNA synthesis of the synchronized cells was determined by detection of 5-bromodeoxyuridine incorporation into DNA essentially as described(68) . The cells were grown in six-well culture dishes, and 5-bromodeoxyuridine was added with serum. All nuclei were counterstained with Hoechst 33258 (Sigma).
Figure 1:
Activating p21 mutants stimulate cyclin D1 transcription. Panel A,
the -1745CD1LUC reporter was transfected in conjunction with
either activating (Ras Val-12 pMTEj, Ras Leu-61, Ras Val-12), dominant
negative (Ras N17), or antisense (Rev3) p21
expression vectors into JEG-3 cells as described under
``Materials and Methods.'' The mutant Ras Leu-61,Ser-186, is
incapable of inserting in the plasma membrane. The Ras Val-12 pMTEj or
Rev3 vector was transfected into Mv.1Lu or CHO cells (panel
B). Cells transfected with the p21
expression vectors pMTEj or Rev3 (300 ng) and reporter plasmid
(4.8 µg) were treated with Zn
for 48 h. The mean
data ± S.E. of either (panel A) n separate
transfections indicated in figure or (panel B) n = three separate transfections are shown. Results are shown
as the percent relative activity for the effect of the transfected
expression vector on the reporter plasmid. Inset, Western blot
analysis of whole cell extracts from JEG-3 cells either (left
lane) mock transfected, or transfected with (right lane)
the p21
antisense plasmid (Rev3). The blot was
probed with the p21
antibody H-Ras (259) (Santa
Cruz Biotechnology Inc.) as described under ``Materials and
Methods.''
The effect of several transforming p21 mutants on cyclin D1 promoter activity was examined in JEG-3
cells. The magnitude of cyclin D1 reporter induction by the activating
p21
mutant expression vector was dependent
upon the amount of transfected expression vector and increased from 24
to 48 h. The activating p21
mutants (Ras Val-12 and Ras
Leu-61) and pMTEj stimulated the cyclin D1 promoter in several
different cell lines. The induction by the pMTEj Ras vector was
dependent upon the addition of Zn
(not shown). The
constitutively active p21
mutants induced cyclin D1
reporter activity 4-5-fold in JEG-3 cells (Fig. 1A). The expression vector encoding the double
mutant Ras Leu-61,Ser-186, which is incapable of insertion in the
plasma membrane, did not affect cyclin D1 promoter activity (Fig. 1A). The expression vector cassettes driving Ras
Val-12 and Ras Leu-61 did not affect cyclin D1 promoter activity (not
shown).
The constitutively active p21 mutant induced cyclin D1 reporter activity 22-fold
in Mv1.Lu cells and 5-fold in CHO cells (Fig. 1B). The
induction by the pMTEj vector was dependent upon the addition of
Zn
(not shown). Rev3 reduced basal -1745CD1
reporter activity in CHO cells but not in Mv1.Lu cells (Fig. 1B).
To determine the region of the cyclin D1
promoter required for regulation by p21, the cyclin D1 5`
promoter deletions (Fig. 2A) were transfected in the
presence of either the sense or antisense p21
expression
vectors (Fig. 2B). Mutation of sequences resembling an
AP-1 site at -954 abolished transcriptional activation by
p21
(pMTEj) (Fig. 2B) and the
-964mtDCLUC reporter was repressed to less than 40% by the
activating p21
mutant.
Figure 2:
p21 activation of
the cyclin D1 promoter requires the -954 region in JEG-3 cells. Panel A, schematic representation of the series of cyclin D1
5` promoter constructs in the vector pA
LUC. The area
homologous to the AP-1 (striped oval), and E2F elements (black oval), and the INR (gray square) are
represented schematically. Panel B, p21
expression vector (pMTEj 300 ng), was transfected with 4.8
µg of -1745CD1LUC or an equal molar amount of the other 5`
promoter constructs including the wild type -964CD1 promoter or
the site-directed mutant of the AP-1 site (-964 mtCD1LUC), into
JEG-3 cells. The data are shown as the mean ± S.E. for n separate transfections as indicated in parentheses adjacent to the designated construct. Dose-response curves using
100-300 ng demonstrated similar trends (not shown). * represents
a significant difference from the adjacent 5` deletion construct for p < 0.05.
Figure 3:
c-Jun activation of the cyclin D1 promoter
requires the -964 region in JEG-3 cells. Expression vectors
encoding wild type or c-Jun mutant DNA (300 ng) were
transfected with 4.8 µg of -1745CD1LUC into JEG-3 or Mv.1Lu
cells. For comparison with the effect of c-Jun on -1745CDLUC, the
canonical AP-1 reporter p
TP-LUX was transfected into JEG-3
cells with c-Jun. Panel B, cotransfection experiments were
conducted using the -1745CD1LUC reporter construct (or an equal
molar amount of the other 5` promoter constructs including the wild
type -964CD1 promoter or the site-directed mutant of the AP-1
site (AP-1mtCD1LUC)) with the RSV c-Jun expression vector in JEG-3
cells. The mean data ± S.E. of 7-14 separate
transfections, are shown. * represents a significant difference from
the adjacent 5` deletion construct for p < 0.05. Panel
C, expression vectors encoding wild type or mutant c-Jun proteins
(300 ng) were transfected with 4.8 µg of -1745CD1LUC into
JEG-3 cells. The data are shown as the mean ± S.E. for six
separate transfections. Dose-response curves using 100-300 ng
demonstrated similar trends (not shown).
To determine the minimal region of the cyclin D1 promoter activated by c-Jun, the series of 5` promoter deletion constructs were transfected into JEG-3 cells with either wild type or mutant expression vectors. The region of the cyclin D1 promoter activated by c-Jun was localized to the region between -964 and -485. Site-directed mutagenesis of the AP-1 site at -954 abolished activation by c-Jun in JEG-3 cells (Fig. 3B).
The domains of c-Jun required for activation of cyclin D1
transcription were determined using a series of expression vectors
encoding wild type and mutant c-Jun
proteins(58, 66, 74) . Previous studies
demonstrated that the DNA binding activity of these mutants, other than
DNA, were similar to wild type(66) . Western
blot analyses (74) demonstrated that all the mutant proteins
used in these studies were expressed similarly in transfected cells.
Deletion of the amino-terminal 91 amino acids, deletion of the A2
activation domain, or mutation of the leucine zipper reduced activation
of the cyclin D1 promoter by c-Jun to less than 20% of wild type (Fig. 3C).
Figure 4:
Binding of AP-1 to the cyclin D1
-954 region characterized using EMSA. EMSA were performed with
nuclear extracts from either Mv1.Lu cells (panels A and B), JEG-3 cells (panel C), or rat adrenal medulla
cells (panels D and E). Panels A and B, EMSA were performed with in vitro translated c-Fos
with bacterially expressed c-Jun (Promega) (lane 1) and
comparison made with the mobility of the complexes binding the cyclin
D1 AP-1 site or wild type (collagenase) AP-1 site in Mv1.Lu cells. The
specific band binding the cyclin D1 AP-1 site or the collagenase AP-1
site probe is marked with an arrow. Panel C, the
-
P-labeled cyclin D1 -954 region (CD1 AP-1)
probe was incubated with (lane 1) cellular nuclear extracts
alone or (lane 2) with the addition of 100-fold excess of cold
self competitor, (lane 3) 100-fold excess of mutant cyclin D1
AP-1 cold competitor. Comparison was made with the binding of cellular
nuclear extracts with the
-
P-labeled wild type
collagenase AP-1 site probe. The complexes binding the cyclin D1 AP-1
site and wild type AP-1 site is labeled 1. Panels D and E, nuclear extracts prepared from the adrenal medulla
of rats were incubated with competitor or antibodies as indicated in
the figure. The specific complex binding the
-
P-labeled cyclin D1 -954 region is indicated
by 1. The complex shifted by the c-Fos or c-Jun antibodies is
indicated by *. Details of the antibodies are outlined under
``Materials and Methods.''
To determine whether the cyclin D1 AP-1 site was capable of binding AP-1 proteins in nontransformed cells, nuclear extracts were prepared from rat tissues and the abundance of AP-1 proteins binding these sequences examined. The c-Jun antibody (Jun Ab2) inhibited binding of most of the complex binding the cyclin D1 AP-1 site, and the JunB and JunD antibodies (40, 67) induced a minor supershift of the complex binding either the cyclin D1 AP-1 site (Fig. 4D) or the wild type AP-1 site (Fig. 4E). The c-Fos antibody shifted most of the complex binding either AP-1 site (indicated by an asterisk in Fig. 4, D and E). These studies demonstrate that the cyclin D1 AP-1 site is capable of binding AP-1 proteins from cultured cells and primary tissue.
Figure 5: AP-1 proteins have distinct transcriptional effects on the cyclin D1 promoter. Panels A and B, cotransfection experiments were conducted using the -1745CD1LUC reporter construct with the designated AP-1 expression vectors in JEG-3 and COS cells. Data are shown as the mean ± S.E. for n separate transfections as indicated in parentheses. Dose-response curves using 100-300 ng demonstrated similar trends (not shown). * represents a significant difference from the adjacent 5` deletion construct for p < 0.05.
Figure 6:
MAPK
stimulates cyclin D1 reporter activity through the proximal promoter. Panel A, expression plasmids encoding either p41 or the dominant negative mutant p41
(400 ng) were transfected with reporter plasmid (4.8 µg)
into JEG-3 (panel A) or Mv1.Lu cells (panel B). The
reporter plasmids were (panel A) the -1745CD1LUC
reporter, or (panel B) the cyclin D1 5` promoter deletion
constructs (-1745CD1LUC, -141CD1LUC, -22CD1LUC, or
-22RevCD1LUC reporter). The basal level activity of -22
CD1LUC was >1,000 relative light units/s with background 3-6
relative light units/s). The mean data ± S.E. of (panel
A) JEG-3, n = 6, Mv.1Lu, n = 3 (panel B). n = 5 separate transfections are
shown. Panel C, MAPK activity was measured in JEG-3 cells
treated with EGF (10 ng/ml) for the time points as indicated.
Several growth factors, including EGF, stimulate
MAPK activity in a cell type-specific manner. Previous studies have
demonstrated that EGF induced cyclin D1 mRNA levels in fibroblast cell
lines(10) . Trophoblast cell lines, such as JEG-3 cells,
express EGF receptors, and EGF promotes trophoblast outgrowth and
blastocoel expansion(57) . The effect of EGF on MAPK activity
was therefore determined in JEG-3 cells. EGF (10 ng/ml) was used to
treat JEG-3 cells, and cells were harvested at time points from 30 min
to 24 h. MAPK activity was induced 6-fold within 30 min (Fig. 6C). Using fluorescence-activated cell sorter
analysis EGF stimulated a 12-15% increase in the proportion of
JEG-3 cells moving from G/G
to S phase (not
shown).
Figure 7:
EGF
activates the cyclin D1 promoter. Panel A, the
-1745CD1LUC reporter was transfected into JEG-3 cells, and the
cells were treated with EGF (0.125-20 ng/ml) for 12 h. The data
of a representative experiment determined by comparison with untreated
cells are shown as fold induction. Panel B, JEG-3 cells were
transfected with a variety of native or synthetic promoters in the
pALUC plasmid, and EGF treatment (2.5 ng/ml) was conducted
for 24 h. The data are shown for the mean ± S.E. of at least
four separate transfections. Panel C, JEG-3 cells transfected
with the series of cyclin D1 5` promoter constructs were treated with
EGF (2.5 ng/ml). The mean data ± S.E. of six separate
transfections are shown. * represent significant differences from the
adjacent 5` promoter construct (p <
0.05).
The DNA sequence requirements for EGF-induced transcription of the cyclin D1 promoter were determined using a series of 5` promoter fragments. Cells were treated for 24 h with EGF (2.5 ng/ml), and the effect was compared with untreated cells. The induction by EGF (3-4-fold) was conveyed by the minimal -22-bp promoter fragment. The -22 bp in the inverted orientation was not activated by EGF (Fig. 7C).
Figure 8:
c-Ets-2 specifically activates the minimal
cyclin D1 promoter. The -1745 CD1LUC reporter and a variety of
native or synthetic promoters in the pALUC plasmid were
transfected into JEG-3 cells with either c-Ets-2 or ets-LacZ expression
plasmids. The data are shown for the mean ± S.E. of at least
four separate transfections. Panel B, the 5` promoter
deletions of the cyclin D1 promoter were transfected with the c-Ets-2
expression vector into JEG-3 cells. Expression vector (300 ng) was
transfected with 4.8 µg of -1745CD1LUC (or an equal molar
amount of the other 5` promoter constructs). The data are shown as the
mean ± S.E. for five separate transfections. The effect of
c-Ets-2 on the -1745CD1LUC reporter was also determined in (panel C) Mv.1Lu and (panel D) COS cells. The mean
data ± S.E. of n = four separate transfections
are shown.
The series of cyclin D1 5` promoter deletions was transfected into JEG-3 cells with c-Ets-2 to determine the minimal ETS-responsive region of the cyclin D1 promoter. The 5` promoter deletion constructs were activated 7-10-fold by c-Ets-2. The minimal c-Ets-2-responsive region was located within 22 bp of the transcriptional start site in JEG-3 cells (Fig. 7B). To determine whether regulation of the cyclin D1 promoter by c-Ets-2 was observed in other cell types, transient expression studies were performed in Mv1.Lu and COS cells. c-Ets-2 activated -1745CD1LUC 18-fold in Mv1.Lu cells and 4-fold in COS cells (Fig. 8, C and D).
As the regions activated by EGF, MAPK, and
c-Ets-2 colocalized within the proximal promoter region, studies were
performed with activating or dominant negative expression vectors to
determine whether sequential or parallel pathways linked EGF,
p41, and c-Ets-2 in the context of the cyclin D1
promoter. Overexpression of p41
induced the -141
cyclin D1 promoter fragment 3-6-fold (Fig. 9A).
EGF activated -141CD1LUC 3-5-fold. MAPKi or ets-LacZ
reduced the induction of the cyclin D1 promoter by EGF 60-80%,
suggesting MAPK and ETS functions downstream of EGF in the context of
the cyclin D1 promoter (Fig. 9, A and B). The
induction of -141CDLUC by p41
was reduced or
abolished by ets-LacZ in JEG-3 (Fig. 9C). Activation by
c-Ets-2 was increased only 25% by the overexpression of MAPK,
suggesting sequential rather than parallel pathways link MAPK and ETS
signaling in JEG-3 cells (Fig. 9C).
Figure 9:
EGF activation of the proximal cyclin D1
promoter is antagonized by dominant negative MAPK and ETS expression
vectors. Panels A-D, the -141 cyclin D1 reporter
was transfected into JEG-3 cells with expression vectors encoding
proteins for MAPK (p42 or
p42
), p21
(activating
(pMTEj) or p21
antisense (Rev3)), or ETS (the
activating (c-Ets-2) or dominant negative (ets-LacZ)). Expression
vector (300 ng) was transfected with 4.8 µg of -141CD1LUC. In panel A the effect of MAPKi and ets-LacZ on EGF-induced
-141CDLUC activity is shown as a percent of wild type. The data
from a representative experiment from at least three separate
transfections are shown. In panel C the induction of the
-141CDLUC reporter by MAPK overexpression (3-4-fold) was
normalized to 1 for the purpose of showing in the same figure the
additional effect of c-Ets-2. In panel D the induction by EGF
was normalized to 1 for comparison with the additional effects of
c-Ets-2.
In these experiments, activating p21 mutants
stimulated cyclin D1 promoter activity in a DNA sequence-dependent
manner. The activation of cyclin D1 transcription by p21
is consistent with recent observations in which the abundance of
cyclin D1 mRNA was increased in v-Ha-Ras-transformed pre-B cell
lines(30) . p21
promotes cellular proliferation
and early G
phase
progression(16, 23, 24, 77) . As
cyclin D1 may be rate-limiting in promoting G
phase
progression, the induction of cyclin D1 transcription by p21
may provide, in part, a mechanism by which p21
promotes G
phase progression. The observation that
distinguishable regions of the cyclin D1 promoter were targeted by
either p21
and c-Jun through one region and by MAPK and
ETS through another are consistent with recent observations suggesting
that a specific Jun kinase, distinguishable from MAPK, modulates
activity of c-Jun(37) .
Several lines of evidence indicate a
role for c-Jun and the AP-1 proteins in promoting G phase
progression(40, 67, 78) . In these studies,
c-Jun activated the cyclin D1 promoter in all cell lines examined.
Mutation of the cyclin D1 AP-1 site abolished the induction by c-Jun in
JEG-3 cells, consistent with a role for this site in mediating
transactivation by c-Jun. The DNA binding domain of c-Jun was required
for induction of the cyclin D1 promoter. In vitro translated
c-Fos with c-Jun bound the cyclin D1 AP-1 site in EMSA. The cyclin D1
AP-1 site bound nuclear complexes from JEG-3 and Mv1.Lu cells, and the
mobility of these complexes was similar to the complexes binding the
wild type AP-1 site in EMSA. Together these findings suggest that the
cyclin D1 -954 region is capable of binding AP-1 proteins and
conveys c-Jun-induced transcription in the cell line examined. The
signal transduction pathway modulating c-Jun-induced transcription of
cyclin D1 is currently unknown; however, the recently identified Jun
kinase(38) , which phosphorylates the amino terminus of c-Jun,
is a likely candidate. Recent studies suggested a role for the
-58 sequences (``cAMP response element'' TGAGGTAA) in
c-Jun-mediated transactivation of cyclin D1 in fibroblast cells using
the pOLUC vector (79) . A residual 5-fold induction by c-Jun
was observed by Herber et al.(79) . after mutation of
the cAMP response element site, and no deletion or mutation abolished
transactivation by c-Jun. Cell type-specific differences may be
responsible for the different localization of the site of c-Jun action.
It remains to be determined whether the effect of c-Ets-2 to activate the cyclin D1 promoter is mediated directly through binding target sequences within the minimal promoter or is mediated indirectly through interaction with the basal transcription apparatus. The ETS protein, PU.1, binds the basal level transcription factor TFIID(86) , which in turn binds either TATAA or Inr sequences(87) . Recent studies demonstrated that the cyclin D1 Inr is sufficient for negative regulation by c-Myc(88) , and that the Inr may also function to convey regulation by several other factors including ETS.
These studies demonstrate that EGF, activating p21 mutants, p42
, c-Jun, and c-Ets-2 augment cyclin D1
promoter activity. These findings suggest that the cyclin D1 promoter
may be an important target for several distinct signal transduction
pathways involved in conveying proliferative signals during
G
. c-Jun and c-Ets-2 target distinct regions of the
promoter providing a mechanism for collaborative interactions between
these two proto-oncogenes in stimulating abundance of this cell cycle
regulatory kinase subunit. These studies demonstrate an avenue for
signal transduction cross-talk between the MAPK and cell cycle
regulatory pathways and a mechanism by which different mitogenic and
transforming factors may interact to promote cellular proliferation.