(Received for publication, May 25, 1995; and in revised form, August 8, 1995)
From the
There exist two distinct isozymes of prostaglandin-endoperoxide
synthase (PES). PES-2 mRNA is synergistically induced by
lipopolysaccharide (LPS) and
12-O-tetradecanoylphorbol-13-acetate (TPA) in bovine arterial
endothelial cells. On the other hand, PES-1 mRNA is constitutively
expressed under these conditions. Therefore, the promoter activities of
the human genes for PES-1 and -2 in bovine arterial endothelial cells
were examined. The 5`-flanking region of the human PES-2 gene
(nucleotides -327 to +59) showed promoter activity inducible
by LPS and TPA using transient transfection analysis, whereas that of
the PES-1 gene (nucleotides -1010 to +69) showed
constitutive promoter activity. Destruction of both consensus sequences
for the nuclear factor responsible for the interleukin-6 expression
(NF-IL6) site (nucleotides -132 to -124) and the cyclic AMP
response element (CRE) (nucleotides -59 to -53) of the
human PES-2 gene markedly reduced the promoter activity (25%) of the
PES-2 gene after combined treatment with LPS and TPA, although single
destruction of the NF-IL6 site or the CRE slightly reduced the promoter
activity (60 or 90%, respectively). Moreover, cotransfection
experiments showed that a trans-acting factor, CCAAT enhancer binding
protein (C/EBP
), which binds to both the NF-IL6 site and the
CRE, increased the promoter activity of the PES-2 gene mainly through
the CRE. C/EBP
mRNA was rapidly induced by LPS. Collectively,
these results suggest that transcription of the PES-2 gene in vascular
endothelial cells is regulated through combination of the NF-IL6 site
and the CRE and that C/EBP
functions as one of the trans-acting
factors.
Prostaglandin-endoperoxide synthase (PES) ()catalyzes
the first step of the biosynthesis of prostanoids such as
prostaglandins, thromboxane and prostacyclin(1, 2) ,
and is an effective pharmacological target for nonsteroidal
antiinflammatory drugs such as aspirin(3) . There exist two
distinct isozymes for PES, PES-1 and PES-2. The human PES-1 gene,
mapped to chromosome 9q32-q33.3, is about 22 kilobase pairs (kb) in
size with 11 exons(4, 5) , whereas the human PES-2
gene, mapped to chromosome 1q25.2-q25.3, is about 8.3 kb in size with
10 exons(5) . PES-2 mRNA is induced by a variety of factors, i.e. inflammatory mediators, growth factors, mitogens, and
hormones(6, 7, 8, 9, 10, 11, 12, 13) ,
whereas expression of PES-1 mRNA is generally
constitutive(14) . From these findings, it seems likely that
PES-2 and PES-1 play distinct roles in the production of prostanoids.
Studies on the transcriptional regulation of eukaryotic genes have
led to identification of a number of transcription factors that are
mediated through specific cis-acting elements. Transcriptional
activation in response to extracellular signals involves the regulated
assembly of multiprotein complexes on enhancers and
promoters(15) . In the human PES-2 gene, the nucleotide
sequence of the 5`-flanking region with a canonical TATA box (5) does not show similarity to that in the human PES-1 gene
with no TATA box(16) , which likely reflects the distinct
expression patterns of the two genes. Consensus sequences of the
nuclear factor-B (NF-
B) site, the nuclear factor for
interleukin-6 expression (NF-IL6) site and the cyclic AMP response
element (CRE) are found in the 275-bp region upstream from the
transcriptional start site in the human PES-2 gene(5) . The
sequences homologous to the consensus NF-IL6 site and CRE are also
found in the corresponding regions of the mouse (17) and rat (13) PES-2 genes.
Trans-acting factors binding to the NF-IL6
site have several isoforms with a leucine zipper motif for dimer
formation(18, 19, 20, 21) , i.e. C/EBP, C/EBP
(NF-IL6), and C/EBP
(NF-IL6
).
Sirois and Richards (22) have reported that C/EBP
may play
a key role in regulating induction of the PES-2 gene in rat granulosa
cells (22) . On the other hand, we have reported that the CRE
is essential for expression of the human PES-2 gene in
monocytic-differentiated U937 cells(23) . Xie et al. have also reported that v-src induction of the PES-2 gene
is mediated by the CRE in NIH 3T3 cells(24) . Several factors
that specifically recognize the CRE have also been identified as
trans-acting factors with a leucine zipper motif for dimer
formation(25) . Interestingly, rat C/EBP
cDNA has been
isolated as a trans-acting factor that binds to the CRE of the
substance P precursor gene by expression cloning(26) .
Moreover, heterodimer formation of NF-IL6 (C/EBP
) with CRE-binding
protein has been reported in the composite NF-IL6-CRE binding site of
the human prointerleukin 1
gene(27) . These reports have
raised the possibility of complex formation among distinct trans-acting
factors binding to several cis-acting elements in the PES-2 gene.
In
the present study, we first investigated the promoter activities of
both the human PES-2 and PES-1 genes in bovine arterial endothelial
cells (BAEC) using a transient transfection method and found that the
expression patterns of these genes induced by LPS and TPA were
primarily ascribable to the transcriptional activities of their
5`-flanking regions. Secondly, we found that the NF-IL6 site and the
CRE (nucleotides -132 to -124 and -59 to -53)
are involved cooperatively in the promoter activity of the PES-2 gene
and that C/EBP, which binds to both the NF-IL6 site and the CRE,
is suggested to function as a trans-acting factor in the human PES-2
gene.
Figure 1:
Expression of PES-2 (A) or PES-1 (B) mRNA in BAEC. BAEC were incubated
for 5 h with no stimulant (Control), 100 nM TPA, 10
µg/ml LPS, or a combination of both. Total RNA was isolated, and 24
µg was fractionated through formaldehyde-containing agarose gels.
The fractionated RNAs were transferred to a nylon membrane and
hybridized with a P-labeled human PES-2 probe. After
analysis of the hybridization signals, the blot was stripped in boiling
0.1% SDS and rehybridized with a
P-labeled human PES-1
probe and then with a
P-labeled glyceraldehyde-3-phosphate
dehydrogenase probe. Hybridization signals were visualized and measured
by a BAS2000 imaging analyzer. Results are expressed as values relative
to the control as 1 (means ± S.D. of triplicate cultures).
Expression levels of glyceraldehyde-3-phosphate dehydrogenase mRNA
relative to the control (100 ± 4%) were 92 ± 5, 96
± 3, and 68 ± 8% after treatments with TPA, LPS, and a
combination of both, respectively.
Figure 2:
Promoter activity of the 5`-flanking
region of the human PES-2 (A) or PES-1 (B) gene. BAEC
were transiently transfected with each reporter plasmid together with
pCMV-gal used as an internal control for the transfections.
Following transfection, the cells were incubated for 5 h with no
stimulant (Control), 100 nM TPA, 10 µg/ml LPS, or
a combination of both. The cells were then harvested, lysed, and
assayed for both luciferase and
-galactosidase activities. Results
are represented as relative luciferase activities obtained by dividing
the normalized luciferase activity from the reporter vector
phPES2(-327/+59) (A) or from the control reporter
vector pGV-C (B). Experiments were carried out in triplicate.
The data are presented as means ±
S.D.
Figure 3: Schematic representation of the 5`-flanking region of the human PES-2 (A) or PES-1 (B) gene. Potential cis-acting elements in nucleotides -327/+69 of the human PES-2 gene (A) and nucleotides -1010/+59 of the human PES-1 gene (B). The sequences of up to -1681 bp of the human PES-2 gene (5) and up to -898 bp of the human PES-1 gene (16) reported previously. We determined nucleotide sequence -1010/+69 of the human PES-1 gene in this study. The diagrams show the potential response elements based on sequence similarities to consensus response elements. Distances are given as nucleotide positions relative to the transcriptional start site as +1.
Figure 4:
Identification of regions responsible for
inducible promoter activity of the human PES-2 gene. Reporter plasmids
containing the 5`-flanking region of the human PES-2 gene with deletion
or site-specific mutation are represented schematically on the left. Relative positions and sequences of the NF-B site,
NF-IL6 site, and CRE are indicated. Mutated sequences of the CRE and
NF-IL6 site are shown in Fig. 5A. BAEC were transiently
transfected with each reporter plasmid together with pCMV-
gal used
as an internal control for the transfections. Following transfection,
the cells were incubated for 5 h with no stimulant (Control),
100 nM TPA, 10 µg/ml LPS, or a combination of both. The
cells were then harvested, lysed, and assayed for both luciferase and
-galactosidase activities. Results are represented as relative
luciferase activities obtained by dividing the normalized luciferase
activity from the reporter vector phPES2(-327/+59).
Experiments were carried out in triplicate. The data are presented as
means ± S.D.
Figure 5:
Ability of C/EBP,
, or
to
binding to the CRE and the NF-IL6 site in the PES-2 gene. A,
the sequences (sense-strand) of oligonucleotides obtained using
electrophoretic mobility shift assays. PES2CRE (nucleotides -69
to -43) and PES2NFIL6 (nucleotides -140 to -116)
contain consensus sequences (indicated by underline) for the
CRE and NF-IL6 site, respectively. PES2CRM and PES2NFILM contain point
mutations (indicated by lowercaseletters) within the
consensus sequences, respectively. PES2CRM and PES2NFILM have sequences
identical to the reporter vectors phPES2(CRM) and phPES2(ILM) (Fig. 4), respectively. B, gel retardation assays were
performed as described under ``Materials and Methods.'' The
nuclear extract (0.5 µg) from BAEC transfected with pGV-B (Control) or the expression vector for C/EBP
, -
, or
-
was incubated with the
P-labeled PES-2CRE. C, the nuclear extract (0.5 µg) from BAEC transfected with
the expression vector for C/EBP
was incubated with
P-labeled PES2CRE or PES2NFIL6. Cold chase experiments
were performed with a 50-fold molar excess of competitor
oligonucleotide as indicated.
Figure 6:
Effect of C/EBP, -
, or -
on
the human PES-2 promoter in BAEC. phPES2(-327/+59) as a
reporter vector was transfected into BAEC with the indicated amount of
effector plasmids expressing the C/EBP family members and with 0.1
µg of pCMV-
gal as an internal control for the transfections.
pGV-B DNA, a promoterless reporter vector, was used instead of effector
plasmids for the control and used to make the total amount of plasmid
constant (0.6 µg/well) for each transfection. 48 h after
transfection, cells were harvested, lysed, and assayed for both
luciferase and
-galactosidase activities. Results are represented
as relative luciferase activities obtained by dividing the normalized
luciferase activity by the control. Experiments were carried out in
triplicate. The data are presented as means ±
S.D.
Figure 7:
Involvement of the CRE and the NF-IL6 site
in activation of the human PES-2 promoter by C/EBP. Each reporter
plasmid (0.3 µg), phPES2(-327/+59), phPES2(CRM),
phPES2(ILM), or phPES2(CRM,ILM), was transfected into BAEC with 0.2
µg of pGV-B DNA (promoterless reporter vector) as the control or
with 0.2 µg of an effector plasmid expressing C/EBP
.
pCMV-
gal (0.1 µg) was used as an internal control for the
transfections in each experiment. 48 h after transfection, the cells
were harvested, lysed, and assayed for both luciferase and
-galactosidase activities. Results are represented as relative
luciferase activities obtained by dividing the normalized luciferase
activity of the control by that with phPES2(-327/+59).
Experiments were carried out in triplicate. The data are presented as
means ± S.D.
Figure 8:
Expression of C/EBP (A) or
C/EBP
(B) mRNA in BAEC. BAEC were incubated for 4 h with
no stimulant (Control), 100 nM TPA, 10 µg/ml LPS,
or a combination of both. Ten µg of each poly(A)
RNA was fractionated through formaldehyde-containing agarose gels
electrophoretically. The fractionated RNAs were transferred to a nylon
membrane and hybridized with a
P-labeled C/EBP
probe
as described under ``Materials and Methods.'' The blot was
stripped in boiling 0.1% SDS and rehybridized with a
P-labeled C/EBP
probe and then with a
P-labeled glyceraldehyde-3-phosphate dehydrogenase probe.
The expression level of glyceraldehyde-3-phosphate dehydrogenase mRNA
relative to the control was 76, 70, or 44% after treatment with TPA,
LPS, or a combination of both, respectively. The positions of 28 and 18
S ribosomal RNAs are indicated on the right.
Figure 9:
Time course of PES-2 mRNA induction by LPS
in BAEC (A) and effect of cycloheximide in its induction (B). Total RNAs were isolated from BAEC at the indicated times
after LPS (10 µg/ml) stimulation (A) or at 5 h with no
stimulant (Control), LPS, or a combination of LPS and TPA (100
nM) in the presence or absence with cycloheximide (10
µg/ml) (B). RNAs (24 µg) fractionated through
formaldehyde-containing agarose gel electrophoretically were
transferred to a nylon membrane and hybridized with a P-labeled PES-2 probe and then with a
P-labeled glyceraldehyde-3-phosphate dehydrogenase probe.
The positions of 28 and 18 S ribosomal RNAs are indicated on the right.
The promoter activity obtained with the human PES-1 gene using the nucleotides -1010 to +69 in BAEC was 8% of that of SV40 promoter/enhancer, and this activity was similar to that reported previously using nucleotides -898 to +12 of the human PES-1 gene in murine neuroblastoma NS-20 cells (5% of the activity of the SV40 early promoter)(16) . On the other hand, the promoter activity of the human PES-2 gene obtained using nucleotides -1432 to +59 was 15-fold higher than that of the PES-1 gene in BAEC (Fig. 2). This result was different from the finding (Fig. 1) that the amount of PES-1 mRNA was greater than that of PES-2 mRNA in the control BAEC. This difference may be due to instability of the PES-2 mRNA conferred by 17 copies of Shaw-Kamen sequence (AUUUA) in the 3`-untranslated region(5, 6, 13) , which is found in many immediate-early genes and have been shown to enhance mRNA degradation(32) . Ristimäk et al. (33) have reported that post-transcriptional mechanisms are also important in the sustained induction of PES-2 mRNA(33) . However, the possibility remains that transcriptional inhibition of the PES-2 gene occurs in regions other than nucleotides -1432 to +59.
Recently, it was suggested that transcriptional regulation
may be conducted through the cooperation of more than one trans-acting
factor with regulated assembly of multiprotein complexes on enhancers
and promoters. The complex nature of these processes is considered to
result in an elaborate fail-safe mechanism for controlling gene
expression(15) . In this study, we showed that combination of
the CRE and the NF-IL6 site in the human PES-2 gene was involved in
promoter activity, although single destruction of the CRE or the NF-IL6
site reduced the activity slightly (Fig. 4). A similar result
has also been obtained in transfection experiments using human
umbilical vein endothelial cells (data not shown). On the other hand,
destruction of both the CRE and the NF-IL6 site could not completely
eliminate the inducible promoter activity (Fig. 4), suggesting
the need to examine the contribution of other cis-acting element(s),
although we could not exclude from the possibility that the CRE and the
NF-IL6 site modulate the basal promoter activity not the inducible
promoter activity. These results can be explained by the fail-safe
mechanism of gene expression resulting from complex formation between
transcription factors through the CRE, the NF-IL6 site, and other
cis-acting element(s). In fact, the promoter activity with phPES2(CRM)
was increased as little as 2-fold by expression of C/EBP, although
that with the wild-type phPES2(-327/+59) was increased about
7-fold as shown in Fig. 7. From these results, we interpreted
that C/EBP
was a component of transcription factor, which
increased the PES-2 promoter activity mainly through the CRE, but in
the induction by LPS or TPA/LPS, the increased promoter activity by
C/EBP
through the CRE was likely to be complemented by other
transcription factor(s) through NF-IL6 site or other cis-acting
element(s). The NF-
B site may be a candidate of such cis-acting
elements, since NF-IL6 (C/EBP
) cDNA has been isolated as a
trans-acting factor that binds to the p50 subunit of
NF-
B(34) .
C/EBP, a DNA-binding protein with
affinity for both the CRE and the NF-IL6 site (Fig. 5), was
demonstrated to activate the PES-2 promoter mainly through the CRE (Fig. 7). CRE-binding proteins have been reported to bind to two
classes of CRE: (i) symmetrical CRE (5`-TGACGTCA-3`), consisting of two
overlapping CGTCA palindromic half-sites such as those found in the
somatostatin (35) and
-chorionic gonadotropin (36) genes, and (ii) asymmetrical CRE, consisting of the
sequence CGTCA, representing a single perfect half-site such as that
present in the human PES-2 gene, as shown in Fig. 5A.
Very recently, the asymmetrical CRE recognized by the heterodimer of
NF-IL6 (C/EBP
) with CRE-binding protein was found in the human
prointerleukin 1
gene as the LPS-responsive enhancer (27) . Electrophoretic mobility shift assays showed that the
C/EBP
expressed in BAEC recognized both PES2CRE and PES2NFIL6 (Fig. 5, A and C) but did not recognize the
symmetrical CRE of somatostatin (data not shown), suggesting the
possibility of heterodimer formation of C/EBP
with CRE-binding
protein on the CRE of the human PES-2 gene. On the other hand,
expression of C/EBP
mRNA was rapidly induced by LPS but not by TPA (Fig. 8). As shown in Fig. 1, LPS and TPA synergistically
induced PES-2 mRNA in BAEC. In fact, the amount of PES-2 mRNA was
increased by LPS in a concentration-dependent manner up to 10
µg/ml, but it was less than 50% of that induced with a combination
of 10 µg/ml LPS and 100 nM TPA (data not shown).
Interestingly, induction of PES-2 mRNA by LPS in BAEC was not transient
but sustained for at least 24 h, which essentially agreed with the
induction pattern by interleukin-1
in human umbilical vein
endothelial cells(33) , but was distinct from the transient
induction by TPA in Swiss 3T3 cells(6) . Furthermore, as shown
in Fig. 9B, cycloheximide did not potentiate the induction
of PES-2 mRNA by LPS, although cycloheximide alone induced PES-2 mRNA.
Taken together, it is suggested that C/EBP
plays a role, at least,
in sustained induction of PES2 mRNA by treatment with LPS. It is also
possible that C/EBP
participates in the initial step of induction
of PES-2 mRNA since phosphorylation of C/EBP family members was
reported to participate in transcriptional regulation of several
genes(27) . However, further study is necessary for
understanding the function of C/EBP
in the expression in vivo of PES-2 gene.
It has been reported that C/EBP may play a
key role in regulating the induction of the PES-2 gene in rat granulosa
cells prior to ovulation(22) . This contradicts our finding
that coexpression of C/EBP
decreased the promoter activity of the
PES-2 gene in BAEC (Fig. 6). On the other hand, we previously
reported that the promoter activity obtained with phPES2(CRM) or
phPES2(ILM) was about 20 or 85% of that with
phPES2(-327/+59) in differentiated U937 monocytic cells,
respectively(23) . This was also different from our finding
that the promoter activity obtained with phPES2(CRM) or phPES2(ILM) was
about 58% of that with phPES2(-327/+59) in BAEC (Fig. 4). These differences may reflect distinct complex
formation by trans-acting factors in distinct cells (BAEC, U937 cells,
and granulosa cells).
In summary, we have first shown that the
expression patterns of the human PES-2 and PES-1 genes are primarily
accounted for by the transcriptional activities of their 5`-flanking
regions. Second, the NF-IL6 site and the CRE were shown to be
cooperatively involved in the promoter activity of the PES-2 gene
inducible by LPS and TPA, although other cis-acting element(s) will
also be necessary to explain the inducible promoter activity. Third, we
have suggested that C/EBP, as a trans-acting factor, is involved
in activation of the PES-2 promoter through the CRE by LPS.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) D64068[GenBank].