(Received for publication, July 24, 1995; and in revised form, September 15, 1995)
From the
A consensus cyclic AMP response element (CRE) in the murine
prostaglandin synthase-2 (PGS2) promoter is essential for pgs2 gene expression induced by
pp60, the v-src oncogene
product. In this study, we investigate (i) the transcription factors
active at the PGS2 ``CRE site'' in response to v-src activation and (ii) the signal transduction pathways by which
pp60
activates these transcription
factors. Transient transfection assays with pgs2 promoter/luciferase reporter chimeric genes suggest that c-Jun
mediates v-src-induced pgs2 gene expression. Antibody
supershift experiments demonstrate that c-Jun can participate in a
complex with the pgs2 promoter CRE site. Moreover, in
vitro immunocomplex assays demonstrate that
pp60
expression strongly activates
c-Jun N-terminal kinase (JNK1) enzyme activity. Serines 63 and 73, the
sites of c-Jun phosphorylation by JNK, are essential for
v-src-induced, pgs2 promoter-mediated luciferase
expression. Cotransfection studies with plasmids expressing wild-type
JNK, dominant-negative JNK, and dominant-negative MEKK-1 confirm that
activation of the Ras/MEKK-1/JNK/c-Jun pathway is required for
v-src-induced pgs2 gene expression. Overexpression of
either wild-type ERK-1 or ERK-2 proteins also potentiate
v-src-mediated luciferase expression driven by the pgs2 promoter, and expression of dominant-negative mutants of ERK-1,
ERK-2, or Raf-1 attenuate this response. Thus, in response to v-src expression, a Ras/MEKK-1/JNK signal transduction pathway
activating c-Jun and a Ras/Raf-1/ERK pathway converge to mediate pgs2 gene expression via the CRE site in the pgs2 promoter.
The prostaglandins play key roles in a variety of biological
processes, including cell division, blood pressure regulation, immune
responses, ovulation, bone development, wound healing, and water
balance. Altered prostaglandin production is associated with several
pathophysiological states, including bone resorption, cardiovascular
disease, acute inflammation, atherosclerosis, and colon
cancer(1) . Prostaglandin synthase (PGS), ()also
known as cyclooxygenase, is the key enzyme in the conversion of free
arachidonic acid to PGH
, the common precursor to all the
prostaglandins, prostacyclins, and thromboxanes (2) . Several
laboratories have recently described the cloning of a second, inducible
form of PGS. This gene, now referred to as pgs2 or cox2, was identified in differential library screens in
fibroblasts transformed by the v-src oncogene(3) , in
fibroblasts treated with the tumor promoter
12-O-tetradecanoylphorbol-13-acetate(4) , and in
fibroblasts treated with platelet-derived growth factor(5) .
Subsequent studies have shown that the pgs2 gene can be
induced in a variety of cells, including macrophages, mast cells,
epithelial cells, endothelial cells, neurons, smooth muscle cells, and
ovarian granulosa cells. A variety of stimuli, including agents that
act via G protein-mediated mechanisms, as well as the protein kinase
C-mediated pathway activated by
12-O-tetradecanoylphorbol-13-acetate and the tyrosine
kinase-mediated pathways activated by both growth factor receptors and
pp60
, the product of the v-src oncogene, can stimulate the expression of the pgs2 gene
(reviewed in (6) ). However, the signal transduction
mechanisms, the transcription factors, and the regulatory regions of
the pgs2 gene necessary for PGS2 induction are not well
understood.
pp60 encodes a
non-receptor tyrosine kinase(7) . Several different approaches
have been taken to try to elucidate how v-src transforms
cells. These include identifying the substrates of the
pp60
tyrosine kinase, isolating
v-src inducible genes, and studying the transcriptional
regulation of known genes whose mRNA levels are elevated in response to
v-src expression. Because of (i) the wide range of cells that
express PGS2 and the diverse stimuli that modulate pgs2 gene
expression, (ii) the great interest in therapeutic modulation of
prostaglandin synthesis, and (iii) the great interest in the mechanisms
of v-src-mediated gene expression and transformation, we have
been investigating the cis-acting DNA elements, the transcription
factors, and the signal transduction pathways that mediate v-src activation of the pgs2 gene. By using promoter deletions
and site-directed mutagenesis in pgs2-luciferase chimeric
reporter genes, we show that a CRE site located between nucleotides
-56 and -52 of the pgs2 gene is essential for
v-src induction(8) . A dominant-negative cyclic AMP
binding protein (CREB) mutant, M1, blocked v-src-mediated
expression, supporting the conclusion that this CRE site is necessary
for pgs2 expression induced by
pp60
(8) . Finally,
cotransfection of a dominant-negative Ras mutant blocked
v-src-mediated induction of the pgs2-luciferase
chimeric gene, suggesting that the
pp60
-initiated pathway of PGS2
activation is mediated by Ras(8) . In this report, we identify
c-Jun as a transcription factor active at the PGS2 CRE site following
v-src expression and determine the signal transduction pathway
that mediates the activation of this factor.
We first tested whether increasing the
levels of wild-type CREB in cells could enhance v-src-mediated
induction from the pgs2 promoter of the
pTIS10LUC chimeric reporter gene. This construct
contains nucleotides -80 to +3 of the pgs2 promoter
(we previously referred to PGS2 as TIS10(4, 9) ; our
promoter constructs thus historically have the TIS10
designation(8, 9) ). However, overexpression of CREB,
like overexpression of the dominant-negative CREB-M1 mutant, inhibits v-src-induced luciferase expression from the
PGS2 reporter (Fig. 1). Because we were concerned that high
levels of CREB might ``squelch'' (17) CREB-dependent
expression from the pgs2 promoter, we also used lower
concentrations of wild-type CREB expression vector (data not shown).
Enhancement of luciferase expression from the pgs2 promoter by
CREB was never observed. We conclude that some transcription factor
other than CREB is activated by pp60
expression and elevates pgs2 gene expression by
transcriptional activation from the PGS2 CRE site.
Figure 1:
CREB overexpression blocks
v-src-mediated induction from the pgs2 promoter. We
previously referred to PGS2 as TIS10(4) . Our promoter
constructs thus historically have the TIS10 designation(8) . 3
µg of the pgs2 luciferase reporter
pTIS10LUC, which utilizes nucleotides from
-80 to +3 of the murine tis10/pgs2 promoter to drive them to the luciferase reporter
gene(8) , were transfected into NIH 3T3 cells in 60-mm culture
dishes, along with 1.5 µg of the v-src expression vector
pMV-src (+ symbols in the v-src row) or corresponding
empty vector pEVX (- symbols in the v-src row). As
indicated in the figure, 1 µg of dominant-negative CREB (DN-CREB)
expression vector, 1 µg of CREB expression vector, or 3 µg of
CREB expression vector were cotransfected with the v-src and
luciferase reporter vectors. The total amount of DNA in each reaction
was kept constant by using the corresponding empty expression vectors.
Three plates of NIH 3T3 cells were used for each transfection
condition. Data are expressed as averages ±
S.D.
We next tested whether overexpression of other ATF transcription factors might modulate v-src induction from the pgs2 promoter. Overexpression of both ATF-2 and ATF-3, like CREB or CREB-M1 overexpression, also inhibited, rather than augmented, v-src-induced expression from the PGS2 CRE site (data not shown). We conclude that the ATF family members CREB, ATF-2, and ATF-3 do not mediate v-src induction of the pgs2 gene. The data presented in this section demonstrating that occupation of the PGS2 CRE site by CREB, DN-CREB, ATF-2, and ATF-3 block v-src-mediated pgs2 gene expression suggest (i) that this site is necessary for v-src-mediated induction of the pgs2 gene and (ii) that some transcription factor(s) other than these molecules must mediate this induction.
An E-box overlaps the CRE site of the pgs2 gene and acts as an alternative site for binding of nuclear proteins(8) . By adding E-box oligonucleotide competitor, we were able to reduce binding of proteins in nuclear extracts to the E-box sequence of the pgs2 promoter and enhance the binding of CREB and the additional nuclear factor(s) that binds to the PGS2 CRE site(8) . The gel shift experiment shown in Fig. 2was performed in the presence of E-box competitor to reduce (but not eliminate) the E-box complexes. The faster moving CRE site complex was previously identified, by supershift with anti-CREB antibody, as a CREB-containing complex(8) . c-Jun antibody can recognize and supershift one of the slower moving complexes of the PGS2 CRE site (Fig. 2). In contrast, antibody to another protein (CREB binding protein) does not cause a supershift. c-Jun antibody alone does not form any complex with the CRE probe. The data demonstrate that the c-Jun protein is able to participate in a binding complex at the PGS2 CRE site.
Figure 2:
c-Jun
binds to the CRE site of the pgs2 promoter. A P
probe from nucleotides -65 to -39 of the pgs2 promoter was used for EMGS assay. E-box competitor (75-fold
excess) was used in lanes 2-4 to reduce the contribution
of the E-box complexes. The E-box binding complexes and the CREB
binding complexes indicated in the figure have been identified
previously, both by competition with consensus E-box and CRE sequences,
and by EMGS-antibody supershift experiments(8) . The
c-Jun-containing complex is identified on the basis of the c-Jun
antibody-dependent supershift.
Figure 3:
v-src activates JNK. NIH 3T3
cells in 100-mm dishes were mock transfected (lane 1),
transfected with six µg of Flag-tagged JNK1 expression vector and 4
µg of empty expression vector pEVX (lane 2), or
transfected with 6 µg of Flag-tagged JNK1 expression vector and 4
µg of pp60 expression vector
pMV-src (lane 3). The immunocomplex kinase assay was performed
as described under ``Experimental Procedures.'' Equal amounts
of protein from each lysate were used in the immunoprecipitation/kinase
assay.
Figure 4:
v-src activation of the pgs2 promoter is mediated by the MEKK1/JNK signal transduction pathway. Left panel, pTIS10LUC (3 µg) was
cotransfected with the pp60
expression vector pMV-src (1.5 µg) and with expression
vectors encoding wild-type JNK (1 µg) or kinase-defective JNK (2
and 4 µg), as shown in the figure. DNA concentrations for
transfection were held constant by including DNAs for appropriate empty
expression vectors. Right panel, pTIS10
LUC
(3 µg) was cotransfected the pp60
expression vector pMV-src (1.5 µg) and increasing
amounts of dominant-negative MEKK-1 expression vector. The total amount
of DNA in each transfection was kept constant by using appropriate
empty expression vectors. Three plates were used for each transfection
condition. Data are expressed as averages ±
S.D.
We previously demonstrated that
v-src-induced pgs2 gene expression required mediation
by Ras(8) . Ras activates several signal transduction pathways,
each pathway leading to phosphorylation of distinct subsets of
transcription factors(25) . The downstream effector of Ras
leading to activation of JNK enzyme activity and phosphorylation of
c-Jun is the MAP kinase kinase kinase MEKK-1 (15) . In
contrast, Ras activation of the Raf-1 MAP kinase kinase kinase leads to
phosphorylation of transcription factors such as TCF/Elk-1 and
c-Myc(25) . Expression of kinase-defective, dominant-negative
MEKK-1 blocks v-src-mediated expression of luciferase from the
pTIS10LUC luciferase reporter gene (Fig. 4, right panel). We conclude that activated transcription from
the pgs2 gene following expression of
pp60
requires Ras activation of MEKK-1 and
JNK, leading to phosphorylation of c-Jun, and subsequent increased
transcription mediated by the CRE site of the pgs2 promoter.
Figure 5:
The
c-Jun activation domain is required for v-srcmediated
luciferase expression from the pGal4TIS10LUC
chimeric promoter. Top panel, the reporter construct
pGal4TIS10
LUC, consisting of five GAL4 DNA binding
sites ligated to nucleotides -40 to +3 of the tis10/pgs2 promoter and driving the luciferase
reporter gene. Middle panel, pGal4TIS10
LUC
(3 µg) was cotransfected with 1 µg of the vectors expressing
either the GAL4 DNA binding domain (GAL4-DB) or the GAL4-DB fused to
the c-Jun activation domain (GAL4-JUN). Lower panel, 3 µg
of pGal4TIS10
LUC was cotransfected with 1 µg of
the vectors expressing either GAL4-DB or GAL4-DB fused to the indicated
transcription factors. Three plates were used for each transfection
condition. Data are expressed as averages ±
S.D.
A luciferase reporter gene
containing only the first 40 nucleotides of the pgs2 promoter,
TIS10LUC, expresses only minimal luciferase
activity. This plasmid does not contain the overlapping CRE and E-box
sites of the pgs2 promoter and is not responsive to v-src expression(8) . If five GAL4 binding sites are added to
this promoter, this gal4-pgs2 luciferase construct similarly
expresses only minimal luciferase activity (Fig. 5, middle
panel, lane 1) and does not respond to v-src expression (Fig. 5, middle panel, lane
3). Expression of the GAL4 DNA binding domain alone (GAL4-DB) does
not enhance luciferase expression from the gal4-pgs2 luciferase chimeric reporter vector (Fig. 5, middle
panel, lane 2; lower panel, lane 1) or
mediate v-src induction from this reporter (Fig. 5, middle panel, lane 4). Expression of a fusion protein
containing the GAL4 DNA binding domain and the c-Jun activation domain
(amino acids 5-200; GAL4-JUN) can activate basal expression from
the Gal4-TIS10
luciferase chimeric reporter vector (Fig. 5, middle panel, lane 5; lower
panel, lane 2). However, when pp60
is also expressed, along with the GAL4-JUN protein, expression
from the GAL4-TIS10
reporter vector is
substantially enhanced (Fig. 5, middle panel, lane
6; lower panel, lane 3). We conclude that the
c-Jun activation domain, if positioned upstream of the minimal pgs2 promoter, can drive v-src-induced gene expression. In
contrast, if the activation domains of ATF-2 or CREB are positioned
adjacent to the minimal pgs2 promoter by the GAL4 DNA binding
domain of GAL4-DB chimeric proteins, these activation domains are
unable to mediate either basal or v-src-induced luciferase
expression from the pgs2 promoter (Fig. 5, lower
panel, lanes 5 and 6).
Phosphorylation of two serine residues, Ser-63 and Ser-73, is required for c-Jun activation and transcription from AP-1 sites(26, 27) . To determine if phosphorylation of these two sites is essential for v-src-induced expression from the pgs2 reporter, we used an expression vector in which the serines at these two sites have been mutated to leucines. GAL4-JUN 63/73, in which these sites of phosphorylation have been altered, is unable to mediate v-src activation of the gal4-pgs2 luciferase reporter gene (Fig. 5, lower panel, lane 4). An intact c-Jun activation domain, with sites of phosphorylation available, is necessary for v-src-mediated transcriptional activation of the pgs2 promoter.
Figure 6:
Activation of ERK-1 and/or ERK-2 is also
required for PGS2 induction by v-src. Left panel,
pTIS10LUC (3 µg) was cotransfected with the
pMV-src pp60
expression vector (1.5
µg) and with expression vectors encoding wild-type ERK-1 or ERK-2
proteins (1 µg) or with expression vectors encoding
dominant-negative ERK-1 or ERK-2 proteins (2 µg). Right
panel, pTIS10
LUC (3 µg) was cotransfected
the pMV-src pp60
expression vector
(1.5 µg) and increasing amounts of the expression vector encoding a
dominant-negative Raf-1 protein. The total amount of DNA in each
reaction was kept constant by using corresponding empty expression
vectors. Three plates were used for each transfection condition. Data
are expressed as averages ± S.D.
Raf-1 is the MAP kinase kinase kinase that mediates activation of the ERK enzymes by Ras(25) . If the Ras/Raf-1/MEK/ERK pathway plays a role in v-src induction of gene expression from the pgs2 promoter, one would expect that a dominant-negative Raf-1 mutation should also block this induction. This is the case; increased inhibition of v-src-mediated luciferase induction from the pgs2 promoter is observed as increasing amounts of a dominant-negative Raf-1 protein are expressed (Fig. 6, right panel).
Figure 7:
Overexpression of c-Jun and c-Fos augments
PGS2 induction by v-src. Cells were transfected with the
v-src expression vector pMV-src or the empty vector pEVX,
along with the pTIS10LUC luciferase expression
vector and expression vectors for c-Jun (2 µg), c-Fos (2 µg),
ATF-3 (2 µg), CREB (2 µg), or a combination of c-Jun and c-Fos
(1 µg of each). The total amount of DNA in each transfection was
kept constant by using corresponding empty expression vectors. Three
plates were used for each transfection condition. Data are expressed as
averages ± S.D.
Elevated prostaglandin production is a characteristic of Rous
sarcoma virus-transformed fibroblasts(30) . Persistent
elevation of pgs2 gene expression in
v-src-transformed cells is responsible for this increased
prostaglandin production(3, 31) . A CRE at nucleotides
-56 to -52 of the pgs2 promoter is the critical
element in v-src-mediated activation of the pgs2 gene(8) . pp60 has been
reported to modulate immediate-early gene expression through the serum
response element of the egr1/tis8 gene(32) , at a dyad
symmetry element and a Sis-inducible factor responsive element in the
c-fos gene(33) , through the CCAAT and TATAA elements
of the junB gene(34) , and via a
v-src-responsive element of the 9E3/CEF-4 gene(35) .
However, v-src modulation of gene expression via a CRE has
only been observed for the pgs2 gene(8) .
Figure 8: v-src-mediated pgs2 expression. v-src mediates Ras activation via the Shc/Grb2/Sos pathway(36) . Ras activation of MEKK1, resulting in phosphorylation of JNK, and Ras activation of Raf-1, resulting in phosphorylation of Elk-1, are discussed in the text.
The c-Jun transactivation domain, when fused to a GAL4 DNA binding domain, can drive expression from a pgs2 promoter in which the CRE is replaced by GAL4 DNA binding sites. In contrast, ATF transactivation domains placed at this site are unable to mediate pgs2 gene expression. Moreover, this response is eliminated when the sites of JNK phosphorylation are mutationally altered in the c-Jun transactivation domain of the GAL4-JUN chimeric transactivator, clearly demonstrating that phosphorylation of the c-Jun transactivation domain by JNK plays a critical role in v-src-induced transcription from the pgs2 gene.
c-Jun is also phosphorylated following exposure of cells to
inflammatory mediators such as tumor necrosis factor (24) and interleukin 1(21) . Tumor necrosis factor
and interleukin 1 are also potent activators of prostaglandin
production and induce the expression of the pgs2 gene in rat
mesangial cells(38) . Thus, c-Jun activation and the CRE of the pgs2 promoter may also play a role in mediating PGS2 induction
by these inflammatory cytokines. UV irradiation is among the strongest
JNK activators(21) . It will be of great interest to determine
whether UV radiation can induce pgs2 gene expression.
It is possible that another member(s) of the ATF family may act as a heterodimer with c-Jun to mediate v-src-mediated induction of the pgs2 gene. Protein X in Fig. 8might thus be an as yet untested member of the ATF transcription factor family, forming a heterodimer with activated c-Jun. This possibility could be explored by similar transfection experiments with other members of the ATF family. Antisense experiments to inhibit the expression of members of the ATF family could also be used to further explore this question, as described by Du et al.(39) .