Tài liệu Báo cáo khoa học: Characterization of promoter 3 of the human thromboxane A2 receptor gene A functional AP-1 and octamer motif are required for basal promoter activity docx

Characterization of promoter 3 of the human thromboxane
A2 receptor gene
A functional AP-1 and octamer motif are required for basal
promoter activity
Adrian T. Coyle and B. Therese Kinsella
Department of Biochemistry, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin, Ireland

Keywords
AP-1; gene expression; isoforms; Oct;
promoter; splicing; thromboxane receptor
Correspondence
B. T. Kinsella, Department of Biochemistry,
Conway Institute of Biomolecular and
Biomedical Research, University College
Dublin, Belfield, Dublin 4, Ireland
Fax: +353 1 2837211
Tel: +353 1 7166727
E-mail:
(Received 18 October 2004, revised 8
December 2004, accepted 20 December
2004)
doi:10.1111/j.1742-4658.2004.04538.x

The TPa and TPb isoforms of the human thromboxane A2 receptor (TP)
arise by differential splicing but are under the transcriptional control of
two distinct promoters, termed Prm1 and Prm3, respectively (Coyle et al.
2002 Eur J Biochem 269, 4058–4073). The aim of the current study was to
determine the key factors regulating TPb expression by functionally characterizing Prm3, identifying the core promoter and the cis-acting elements
regulating basal Prm3 activity. Hence, the ability of Prm3 and a series of
Prm3 deleted ⁄ mutated subfragments to direct reporter gene expression in
human erythroleukemia 92.1.7 and human embryonic kidney 293 cells was

investigated. It was established that nucleotides )118 to +1 are critical for
core Prm3 activity in both cell types. Furthermore, three distinct regulatory
regions comprising of an upstream repressor sequence, located between
)404 to )320, and two positive regulatory regions required for efficient
basal gene expression, located between )154 to )106 and )50 to +1, were
identified within the core Prm3. Deletion and site-directed mutagenesis of
consensus Oct-1 ⁄ 2 and AP-1 elements within the latter )154 to )106 and
)50 to +1 regions, respectively, substantially reduced Prm3 activity while
mutation of both elements abolished Prm3 activity. Electromobility shift
and supershift assays confirmed the specificity of nuclear factor binding to
the latter Oct-1 ⁄ 2 and AP-1 elements. Moreover, herein it was established
that the core AP-1 element mediates phorbol myristic acid-induction of
Prm3 activity hence providing a mechanistic explanation of phorbol ester
up-regulation of TPb mRNA expression.

The prostanoid thromboxane (TX)A2 induces activation and aggregation of platelets, constriction of vascular (V) and bronchial smooth muscle (SM) and of
renal mesangial cells [1–4], and may induce other
diverse cellular responses including mitogenic and ⁄ or
hypertrophic growth of VSM [5,6], inhibition of angiogenesis ⁄ neo-vascularization [7] and apoptosis of
+⁄
CD4 ⁄ CD8+ – thymocytes [8]. Alterations in the level

of this potent autocoid, or of its specific synthase or
its receptor (TP) are widely implicated in a variety of
vascular diseases including thrombosis, unstable angina,
asthma, systemic and pregnancy-induced hypertension, and glomerulonephritis [9–12]. Moreover, mice
deficient in the TXA2 receptor (TP– ⁄ –) display
increased bleeding and altered hemodynamic properties, highlighting the essential role of TXA2 and its

Abbreviations

AP-1, activator protein-1; EMSA, electromobility shift assay; FBS, fetal bovine serum; HEK, human embryonic kidney; HEL, human
erythroleukemia; I, intron; NT, nucleotide; PMA, phorbol myristic acid; Prm, promoter; RLU, relative luciferase units; TP, thromboxane
receptor; TI, transcription initiation; TXA2, thromboxane A2; UAS, upstream activation sequence; URS, upstream repressor sequence;
UTR, untranslated region.

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FEBS Journal 272 (2005) 1036–1053 ª 2005 FEBS


A. T. Coyle and B. T. Kinsella

receptor in the dynamic regulation of haemostasis
[13,14].
As a member of the G protein coupled receptor
(GPCR) superfamily, the TXA2 receptor or TP is primarily coupled to Gq-dependent activation of phospholipase (PLC) Cb isoforms [1,3]. In humans, but not
in nonprimates, TXA2 signals through two TP isoforms
referred to as TPa and TPb that are encoded by a single
TP gene located on chromosome 19p13.3 and that arise
through a novel differential splicing mechanism involving retention of bifunctional intronic ⁄ exonic sequences
within the TPa mRNA [15–17]. TPa and TPb are identical for their N-terminal 328 amino acids but differ
exclusively in their C-terminal domains [16,17]. Whilst
TPa and TPb mediate almost identical PLCb effector
activation, they differentially couple to adenylyl cyclase
via Gas and Gai, respectively [18], and TPa, but not
TPb, couples to the novel high molecular weight G-protein Gh [19]. TPa and TPb also undergo differential
homologous (agonist-dependent) and heterologous
desensitization. For example, TPb, as opposed to TPa,
undergoes tonic and agonist induced-internalization
[20,21]. On the other hand, TPa, but not TPb,

undergoes inhibitory cross-talk or heterologous
desensitization of its signaling in response to the potent
anti-aggregatory ⁄ vasodilatory autocoids prostaglandin
(PG) I2 (prostacyclin) and nitric oxide (NO) through
mechanisms involving direct cAMP- and cGMPdependent protein kinase phosphorylation, respectively,
of TPa within its unique C-tail domain [22,23].
Hence, whilst the biological significance for the
existence of two TP receptors in humans is indeed
unclear, there is mounting evidence that they undergo
differential signaling and regulation, strengthening the
viewpoint that TPa and TPb may have distinct physiologic ⁄ pathophysiologic roles. Consistent with this, TPa
and TPb are also subject to differential expression and
gene regulation [24,25]. Whilst TPa and TPb mRNAs
are coexpressed in a range of cell ⁄ tissue types of relevance to TXA2 biology, there are extensive differences
in the relative levels of expression of TPa: TPb mRNA
in several tissues [24]. Moreover, recent studies have
confirmed that TPa and TPb expression are under the
genetic control of distinct promoters within the single
human TP gene located on chromosome 19 [16,25].
Whilst the originally identified promoter (Prm) 1
directs TPa expression, a novel promoter (Prm3) was
identified within the human TP gene that exclusively
directs TPb expression [25]. Similar to that of the previously characterized Prm1 and Prm2, Prm3 lacks a
consensus TATA box or initiator element and, hence,
the transcription factor elements directing basal Prm3activity remain to be identified [25].
FEBS Journal 272 (2005) 1036–1053 ª 2005 FEBS

Thromboxane A2 receptor gene expression

The aim of the current study was to define the core

promoter and to identify the cis-acting elements regulating basal Prm3 activity with the view to determining
the key factors that regulate TPb expression in human
subjects. Through 5¢- and 3¢-deletion analyses it was
found that the nucleotides between )118 to +1 were
required for core Prm3 activity, where +1 represents
the translational ATG start codon and the transcription initiation of the TPb mRNA was previously
identified at )12 [25]. Furthermore, three distinct regulatory regions were identified, the first of which was an
upstream repressor sequence (URS) located between
)404 and )320 and was found to have a repressive
effect on the basal Prm3 activity in both cell types.
Two additional regions that positively regulate or are
required for efficient basal Prm3-directed gene expression were also identified within the core Prm3.
Detailed characterization of the consensus transcription factor elements within these latter sites revealed a
crucial role for both an Oct-1 ⁄ 2 and an activator protein-1 (AP-1) element in the regulation of basal Prm3
activity. It is anticipated that the functional characterization of Prm3 reported herein should provide critical
knowledge of the modes of regulation of TPb
expression and hence may shed further insights as to
the physiologic requirement for two TP receptors,
namely TPa and TPb, in humans.

Results
Functional analysis of promoter 3 of the human
TXA2 receptor (TP) gene
We have previously identified a novel promoter (Prm)3
within the human TXA2 receptor (TP) gene that
directs expression of TPb in HEL92.1.7 and HEK293
cells [25]. A schematic of the human TP gene highlighting the positions of the previously identified Prm1,
Prm2 [16,25] and the novel Prm3 [25] relative to its
translational start site (ATG, designated +1) is
presented in Fig. 1. In order to gain further insights

into the modes of regulation of TPb, the aim of the
current study was to map the minimal transcriptional
unit and to identify the key regulatory elements within
Prm3 directing basal gene expression.
The recombinant pGL3Basic encoding Prm3 directed 3.65 ± 0.23 RLU and 3.0 ± 0.25 RLU of luciferase activity in HEL (Fig. 1A) and HEK293
(Fig. 1C) cells, respectively, whilst the empty pGL3
Basic vector directed minimal activity in either cell
type (Fig. 1A,C). Progressive 5¢ deletion of Prm3
sequences in pGL3Basic to generate )975 and )404
subfragments did not significantly affect luciferase
1037


Thromboxane A2 receptor gene expression

A. T. Coyle and B. T. Kinsella

B

A
Prm1

E1
-5895

-8500

Prm2 E1b

Prm3

-1979 -1394

-3308

E2

Prm1
+786

+1

+1 Luc

-1394

-106
-50

+786

+1 Luc

-404

+1 Luc

-320

+1 Luc

-154

E2
+1

+1 Luc

-975

+1 Luc

-320

Prm3

-1979 -1394

+1 Luc

+1 Luc

-404

Prm2 E1b
-3308

-1394

+1 Luc

-975

E1
-5895

-8500

+1 Luc

-154

+1 Luc

-106

+1 Luc

-50

pGL3Basic +1 Luc

+1 Luc
+1 Luc

pGL3Enhancer +1 Luc
0

2

4

6

8

0

2

4

Luciferase Activity (RLU)

C

D
Prm1

-8500

Prm2 E1b

E1
-5895

Prm3

-1979 -1394

-3308

-106
-50
pGL3Basic

10

12

E2
+786

+1

+1 Luc
+1 Luc

-404

+1 Luc

-320

+1 Luc

-154

Luc

-154

8

+1 Luc

+1 Luc

-320

Prm3

-1979 -1394

-975

+1 Luc

-404

Prm2 E1b
-3308

-1394

+1 Luc

-975

E1

-5895

-8500

+786

+1 Luc

-1394

Prm1

E2
+1

6

Luciferase Activity (RLU)

+1 Luc

-106
-50

+1 Luc

Luc

+1 Luc

pGL3Enhancer +1 Luc

+1 Luc
0

1

2

3

4

5

Luciferase Activity (RLU)

6

7

0

5

10

15

20

25

Luciferase Activity (RLU)

Fig. 1. Effect of 5¢-deletion mutagenesis on Prm3-directed luciferase expression. (A–D) A schematic figure of the human TP genomic region
spanning nucleotides )8500 to +786 encoding promoter (Prm) 1, Prm2 and Prm3, in addition to exon (E) 1, E1b and E2, which are illustrated
above each panel. Nucleotide +1 corresponds to the translational start site (ATG) and nucleotides 5¢ of that site are given a – designation.
DNA fragments corresponding to Prm3 ()1394 to +1) and its successive deletion fragments Prm3b ()975 to +1), Prm3a ()404 to +1),
Prm3ab ()320 to +1), Prm3aa ()154 to +1), Prm3aab ()106 to +1) and 3aaa ()50 to +1) were subcloned into pGL3Basic (A and C) or
pGL3Enhancer (B and D). Resulting recombinant plasmids or, as controls, pGL2Basic ⁄ pGL3Enhancer empty vectors were cotransfected with
pRL-TK into HEL92.1.7 (A and B) and HEK293 (C and D) cells. Firefly and renilla luciferase activity was assayed 48 h post-transfection;
results are presented as mean firefly relative to renilla luciferase activity, expressed in arbitrary relative luciferase units (RLU ± SEM; n ¼ 5).

expression in either HEL (Fig. 1A) or HEK293
(Fig. 1C) cells. Similarly, whilst the overall levels of
Prm3-luciferase activity directed by the pGL3Enhancer
plasmids, containing an SV40 enhancer element downstream of the luciferase gene, were generally two- to
three-fold higher than by the equivalent pGL3Basic
plasmids in both HEL (7.57 ± 0.53 RLU; Fig. 1B)
and HEK (9.60 ± 0.41 RLU; Fig. 1D) cells, there was
no significant difference in luciferase expression directed by the corresponding )1394, )975 or )404 fragments cloned into pGL3Enhancer in either HEL
(Fig. 1C) or HEK293 (Fig. 1D) cells. Moreover, the
empty pGL3Enhancer vector yielded minimal luciferase activity in either cell type (Fig. 1B,D). Hence,
deletion of sequences between )1394 and )404 of
Prm3 does not affect Prm3-directed basal gene expression.
1038

However, further 5¢ deletion of Prm3 from a )404 bp
to a )320 bp fragment expressed in either pGL3Basic

or pGL3Enhancer yielded approximately two-fold
increases in luciferase activity in both HEL (Fig. 1A,B)
and HEK293 (Fig. 1C,D) cells. Moreover, further 5¢
deletion of the )320 bp to a )154 bp fragment did not
significantly affect the level of luciferase expression in
either pGL3Basic or pGL3Enhancer vectors expressed
in either HEL (Fig. 1A,B) or in HEK293 (Fig. 1C,D)
cells. Hence, deletion of nucleotides between )404 and
)320 removes a gene segment that has a repressive
effect on Prm3 activity in both HEL and HEK293 cells
whilst further deletion of nucleotides between )320 and
)154 had no additional effect on luciferase expression
in either cell type.
Further 5¢ deletion of nucleotides )154 to )106
resulted in between two- and eight-fold decreases in
FEBS Journal 272 (2005) 1036–1053 ª 2005 FEBS


A. T. Coyle and B. T. Kinsella

Thromboxane A2 receptor gene expression

luciferase expression in HEL (Fig. 1A,B) and HEK293
(Fig. 1C,D) cells indicating that the )154 to )106
sequence contains positive regulatory element(s)
required for efficient basal Prm3 activity. Moreover, 5¢
deletion of the )106 bp to a )50 bp fragment did not
further reduce the level of luciferase expression in
either pGL3Basic or pGL3Enhancer plasmids in either
HEL (Fig. 1A,B) or in HEK293 (Fig. 1C,D) cells. It

was noteworthy that plasmids containing the )50 to
+1 bp subfragment of Prm3 retained low though
significant promoter activity relative to the empty
pGL3Basic (P £ 0.05) and pGL3Enhancer (P £ 0.05)
vectors, respectively, when expressed in both HEL
(Fig. 1A,B) and HEK293 (Fig. 1C,D) cells. Hence,
these data suggest that Prm3 contains positive regulatory DNA sequences between )50 and +1 in addition
to sequences between )154 and )106 required for efficient basal Prm3 activity.
To test this hypothesis, 3¢ deletion of nucleotides
)118 to +1 abolished Prm3-directed luciferase activity
when expressed in HEL cells (Fig. 2A,B) such that the
level of luciferase activity directed by the respective
recombinant plasmids was not substantially different
from that of the corresponding empty pGL3Basic
(compare 0.35 ± 0.08 RLU vs. 0.11 ± 0.03 RLU)
or pGL3Enhancer (compare 0.84 ± 0.11 RLU vs.
0.32 ± 0.01 RLU) vectors. Similar data were observed
in HEK293 cells (data not shown). Moreover, the
possible requirement for regulatory DNA sequences 3¢
of the +1 translational start site was investigated by

A
-1394

-404

comparing luciferase activity of the previously characterized )404 to +1 fragment to that of a )404 to
+119 fragment, containing an additional 119 bp of TP
genomic sequence downstream of the translational
start site (Fig. 2A,B). However, the level of )404 to

+
119 directed luciferase activity was not significantly
different from that of the Prm3-directed luciferase
activity (e.g. )404 to +1) expressed in HEL cells irrespective of whether recombinant pGL3Basic (Fig. 2A)
or pGL3Enhancer (Fig. 2B) based-vectors were used.
Similar data were observed in HEK293 cells (data not
shown). In summary, we have identified three regulatory regions within Prm3 that contribute to basal promoter activity, one that negatively ()404 to )320)
regulates the action of Prm3 while two of which positively ()154 to )106, )50 to +1) regulate basal Prm3
activity. Moreover, we have confirmed that nucleotides
)118 to +1 are essential for the core Prm3.
Identification of a functional Oct-1 ⁄ 2 element
within promoter 3
In order to further localize and identify the positive
regulatory element(s) positioned between )154 to )106
of Prm3, additional 5¢ deletions were generated in
pGL3Basic. Successive 5¢ deletion of nucleotides
between )154 and )119 did not significantly affect the
level of luciferase activity in HEL cells (Fig. 3A) suggesting that the latter gene segment is not required for
efficient basal Prm3 activity. In contrast, further 5¢

-118

+1

+ 119

+786

E2

+1 Luc

-118 Luc

+119 Luc
0

1

2

3

4

5

8

10

Luciferase Activity (RLU)

B
-1394

-404

-118

+1

+1 19

+786

E2

+1 Luc

****

FEBS Journal 272 (2005) 1036–1053 ª 2005 FEBS

****
****
****

Fig. 2. Localization of the core Prm3 by
5¢- and 3¢-deletion analysis. (A and B) The
TP genomic region spanning nucleotides
)1394 to +786, and encoding Prm3 ()1394
to +1) in addition to exon (E) 2, is illustrated
above each panel. Recombinant pGL3Basic
(A) or pGL3Enhancer (B) plasmids encoding
Prm3a ()404 to +1), Prm3f ()404 to )118)
and Prm3e ()404 to +119) were cotransfected with pRL-TK into HEL92.1.7 cells. Firefly
and renilla luciferase activity was assayed
48 h post-transfection; mean firefly relative
to renilla luciferase activity are expressed in

arbitrary relative luciferase units (RLU ±
SEM; n ¼ 5). The asterisks (*) indicate that
the level of Prm3f-directed luciferase activity
was significantly reduced relative to
Prm3a-directed luciferase expression, where
****P £ 0.0001.

-118 Luc

+119 Luc
0

2

4

6

Luciferase Activity (RLU)

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Thromboxane A2 receptor gene expression

A

Oct1
(-123)
Oct1 /2

(-105)

A. T. Coyle and B. T. Kinsella

AP-1
(-27)
+1

-1394

-154

+1 Luc
-140

+1 Luc
-119
-106

+1 Luc
+1 Luc
0

2

4

6

8

Luciferase Activity (RLU)

B

Oct1
(-123)
AP-1
Oct1 /2 (-27)
(-105)
+1

-1394

+1 Luc

-404

+1 Luc

-404

+1 Luc
+1 Luc

-320

+1 Luc

-320

+1 Luc

****

-320

****
****
****
****
****

-404

0

2

4

Luciferase Activity (RLU)

deletion of a 13 bp gene segment between )119 and
)106 led to a 2.5-fold decrease in luciferase expression
(Fig. 3A), confirming that this sequence contains
positive regulatory element(s) required for basal Prm3
activity.
Bioinformatic analysis of Prm3, using the matinspectorTM program [26], for transcription factor elements between )154 and )106 identified three
consensus transcription factor binding sites including a

putative Oct-1 site centered at )123, a Oct-1 ⁄ 2 site at
)105 and an adjacent AP-1 element at )27 (Fig. 3).
Hence, to investigate the role of these elements in regulating basal Prm3 activity, site-directed mutagenesis
was used to disrupt the putative Oct elements located
between )154 and )106 (Fig. 3B). Mutation of the
consensus Oct-1 site (GCATTTCA to GCTTCCCA)
had no effect on luciferase activity directed by the
)404 or )320 subfragments of Prm3 suggesting that
the putative Oct-1 site centered at )123 is not required
for basal Prm3 activity (Fig. 3B). Conversely, mutation
of the consensus Oct-1 ⁄ 2 site (AAGCAAAT to
AAGCAAGT) centered at )105 significantly reduced
1040

6

8

Fig. 3. Identification of a functional Oct-1 ⁄ 2
site within Prm3. (A and B) Scheme of the
TP genomic region spanning Prm3 ()1394 to
+1) in addition to the relative positions of
putative Oct-1, Oct-1 ⁄ 2 and AP-1 elements
is illustrated above each panel. Recombinant
pGL3Basic plasmids encoding Prm3aa ()154
to +1), Prm3ax ()140 to +1), Prm3ac ()119
to +1) and Prm3aab ()330 to +1) (A) or
Prm3a ()404 to +1) or Prm3ab ()320 to +1)
and their site-directed variants Prm3aOct-1*
and Prm3abOct-1*, Prm3aOct-1 ⁄ 2* and

Prm3aOct-1 ⁄ 2* (B) were cotransfected with
pRL-TK into HEL92.1.7. Firefly and renilla
luciferase activity was assayed 48 h posttransfection; mean firefly relative to renilla
luciferase activity are expressed in arbitrary
relative luciferase units (RLU ± SEM; n ¼ 5).
The star symbol indicates mutated transcription factor elements. The asterisks (*)
indicate that either deletion or site-directed
mutagenesis of Prm3 sequences significantly reduced luciferase expression in HEL
cells, where **** indicates P £ 0.0001.

luciferase activity directed by the )404 and )320 subfragments of Prm3 approximately 2.5- to threefold
(Fig. 3B). Hence, these data suggest that the latter
putative Oct-1 ⁄ 2 element at )105 may be critical for
basal Prm3 activity in HEL cells. Similar data were
observed in HEK293 cells (data not shown).
To confirm the presence of nuclear ⁄ transcription
factors capable of binding to the latter Oct-1 ⁄ 2 site
centered at )105, electromobility shift assays (EMSAs)
were carried out using a radiolabeled double-stranded
DNA probe spanning nucleotides )115 to )92 (Oct1 ⁄ 2WT; Kin195) and nuclear extract prepared from
HEL cells. Incubation of the radiolabeled Oct-1 ⁄ 2WT
probe with HEL nuclear extract resulted in the appearance of a single-labeled DNA–protein band (Fig. 4A,
lane 2) that was efficiently inhibited by an excess of
the corresponding nonlabeled double-stranded Oct1 ⁄ 2WT oligonucleotide (Fig. 4A, lane 3) or by a double-stranded oligonucleotide containing a recognized
consensus Oct-1 ⁄ 2 (Fig. 4A, lane 5). The specificity of
nuclear factor binding to the latter Oct-1 ⁄ 2 site was
also verified by the failure of excess double-stranded
FEBS Journal 272 (2005) 1036–1053 ª 2005 FEBS

A. T. Coyle and B. T. Kinsella

A

Thromboxane A2 receptor gene expression

B

C

D

E

Fig. 4. Demonstration of nuclear factor binding to an Oct-1 ⁄ 2 Site within Prm3. A 32P-labeled double-stranded Oct-1 ⁄ 2WT DNA probe
(Kin195 and its complement corresponding to nucleotides )115 to )92 of Prm3) was used in EMSAs (A) or in supershift assays (B) using
nuclear extracts from HEL92.1.7 cells. (A) 32P-labeled Oct-1 ⁄ 2WT probe was incubated: without nuclear extract (lane 1); with nuclear extract
alone (lane 2); with nuclear extract in the presence of excess nonlabeled double-stranded specific competitor Oct-1 ⁄ 2WT oligonucleotide
(Kin195 and its complement, lane 3); with nuclear extract in the presence of excess nonlabeled double-stranded noncompetitor Oct-1 ⁄ 2*
oligonucleotide (Kin193 and its complement, lane 4); with nuclear extract in the presence of excess nonlabeled double-stranded consensus
Oct-1 ⁄ 2 oligonucleotide (Kin340 and its complement, lane 5); with nuclear extract in the presence of excess nonlabeled double-stranded
Ap-1 noncompetitor oligonucleotide (Kin189 and its complement, lane 6). (B) 32P-labeled Oct-1 ⁄ 2WT probe was incubated without nuclear
extract (lane 1); with nuclear extract alone (lane 2); with nuclear extract preincubated for 30 min with anti-(Oct-1) IgG (sc-232x; lane 3); with
nuclear extract preincubated for 30 min with anti-(Oct-1) IgG in the presence of excess nonlabeled double-stranded consensus Oct-1 ⁄ 2WT
oligonucleotide (Kin340 and its complement, lane 4); with nuclear extract preincubated for 30 min with anti-(Oct-2) IgG (sc-233x; lane 5); with
nuclear extract preincubated for 30 min with anti-(Oct-2) IgG in the presence of excess nonlabeled double-stranded consensus Oct-1 ⁄ 2*
oligonucleotide (Kin340 and its complement, lane 6). The arrow indicates the supershifted transcription factor: DNA complex detected in the
presence of the anti-(Oct-2) IgG (lane 5). DNA–protein complexes were subject to PAGE followed by autoradiography, as outlined. (C and D)
Western blot analysis of Oct-1 (C) and Oct-2 (D) expression in whole cell protein (60 lgỈ lane)1) prepared from HEL (C and D; lane 1) and
HEK293 (C and D; lane 2) cells. The positions of the molecular size markers (kDa) are indicated to the left and right of the (C) and (D),

respectively, whilst the position of the Oct-1 (98 kDa approximately) and the two major forms of Oct-2 (75–80 and 55–60 kDa) detected in
HEK293 and HEL cells, respectively, are indicated by arrows in (C) and (D). (E) To investigate the effect of Oct-1 or Oct-2 on Prm3-directed
luciferase gene expression, HEK293 cells were transiently cotransfected with pGL3b:Prm3ab plus pRL TK in the presence of pcDNA3:
HaOct-1 (Oct-1), pcDNA3:HaOct-2 (Oct-2) or, as a control, with pcDNA3 (Control). Firefly and renilla luciferase activity was assayed 48 h
post-transfection; mean firefly relative to renilla luciferase activity are expressed in arbitrary relative luciferase units (RLU ± SEM; n ¼ 6). The
asterisks (*) indicate that over-expression of Oct-1 and Oct-2 significantly increased Prm3ab-directed luciferase expression in HEK293 cells,
where * and ** indicate P £ 0.05 and 0.01, respectively.

oligonucleotides containing a mutated Oct-1 ⁄ 2*
sequence (Kin193) or an AP-1 consensus sequence to
effectively inhibit nuclear factor- DNA complex
FEBS Journal 272 (2005) 1036–1053 ª 2005 FEBS

formation (Fig. 4A, lanes 4 and 6, respectively).
Western blot analysis of whole cell lysates confirmed
the presence of Oct-2, but not Oct-1, in HEL cells
1041


Thromboxane A2 receptor gene expression

(Fig. 4C,D, lane 1) while Oct-1, but not Oct-2,
expression was readily detected in HEK293 cells
(Fig. 4C,D, lane 2). Moreover, electromobility supershift assays employing anti-Oct selective antibodies
demonstrated the direct binding of Oct-2, but not
Oct-1, to the Oct-1 ⁄ 2 element of Prm3 within HEL
cells (Fig. 4B, lane 5). Oct nuclear factor–DNA complexes (Fig. 4B, lanes 4 and 6) and anti-Oct-2 supershifted complexes (Fig. 4B, lane 6) were efficiently
competed by an excess of a non-labeled doublestranded oligonucleotide containing a consensus Oct1 ⁄ 2 site. Failure to observe an anti Oct-1 supershift
is consistent with the absence of Oct-1 in HEL cells
but did not exclude the possibility that Oct-1 may

regulate Prm3 activity in cell types where Oct-1 is
abundantly expressed. Consistent with this, heterologous over-expression of both Oct-1 and Oct-2 significantly increased Prm3-directed luciferase activity in
HEK293 (Fig. 4E) and HEL (data not shown)
cells. Hence, we have identified a consensus Oct-1 ⁄ 2
transcription factor site centered at )105 that is critical for efficient basal Prm3-directed gene expression
and have confirmed the ability of both Oct-1 and
Oct-2 to bind and regulate Prm3-directed gene
expression.
Identification of a functional AP-1 element within
promoter 3
To further investigate the positive regulatory element(s)
located between )50 to +1 of Prm3 that directs low,
though significant, luciferase activity in both HEL
and HEK293 cells (Fig. 1), matinspectorTM analysis
[26] of Prm3 revealed the presence of a high consensus AP-1 element centered at )27 (Fig. 4) located
some 15 bp 5¢ of the previously identified transcription initiation site within the TPb mRNA [25]. Hence,
to ascertain the functional role of this AP-1 site in
mediating basal Prm3 activity, its consensus core
sequence was disrupted by site directed mutagenesis
(GTGACT to GATCCT) in a range of 5¢-deletion
subfragments and the ability of the mutated AP-1
(AP-1*) relative to the AP-1WT Prm3 subfragments to
direct luciferase activity in HEL (Fig. 5A,B) and
HEK293 (Fig. 5C,D) cells was investigated. Following
transfection into HEL cells, in general the 5¢-deletion
fragments containing the mutated AP-1* site yielded
approximately 2.5-fold reductions in luciferase activity
relative to that of the corresponding subfragments
containing an intact AP-1 site in either pGL3Basic
(Fig. 5A) or pGL3Enhancer (Fig. 5B). Consistent

with this, mutation of the AP-1* element within the
smallest )50 bp fragment almost abolished luciferase
1042

A. T. Coyle and B. T. Kinsella

activity indicating an essential role for the AP-1
element in mediating basal Prm3 gene expression.
Similarly, transfection of HEK293 cells with the various 5¢-deletion fragments containing the mutated
AP-1* site yielded between four- and five-fold reductions in luciferase activity relative to that of the corresponding subfragments containing an intact AP-1
site in either pGL3Basic (Fig. 5C) or pGL3Enhancer
(Fig. 5D) whilst mutation of the AP-1* element
within the smallest )50 bp fragment almost completely abolished luciferase activity also, similar to
that observed in HEL cells. Hence, whilst disruption
of the AP-1 element centered at )27 significantly
reduces basal Prm3 activity and this effect appears to
be independent of the presence or absence of the previously identified negative regulatory element located
between )404 and )320 in either cell type, Prm3directed gene expression shows a greater sensitivity to
AP-1 disruption in HEK293 cells than in HEL cells.
Moreover, the absence of any discernable Prm3 activity directed by the )50 bp subfragment containing the
AP-1* mutation confirms that there are no other regulatory elements within the )50 to +1 bp region
required for basal Prm3 activity.
To confirm the presence of nuclear ⁄ transcription
factors capable of binding to the latter AP-1 site
centered at )27, EMSAs were carried out using a
radiolabeled double-stranded oligonucleotide probe
spanning nucleotides )32 to )10 (AP-1WT) of Prm3
and nuclear extracts prepared from HEL92.1.7 cells.
Incubation of the radiolabeled AP-1WT probe (Kin189;
Fig. 6) with HEL nuclear extract resulted in the formation of a single radiolabeled nuclear factor–DNA complex (Fig. 6, lane 2) that was efficiently competed by

an excess of the corresponding nonlabeled doublestranded AP-1WT oligonucleotide (Fig. 6, lane 3) or
by a double-stranded oligonucleotide containing a
recognized consensus AP-1 sequence (Fig. 6, lane 5).
The specificity of nuclear factor binding to the radiolabeled AP-1WT probe was further confirmed by the
failure of both a double-stranded oligonucleotide spanning nucleotides )32 to )10 but containing a mutated
AP-1* site (Kin162; Fig. 6, lane 4) and a double-stranded oligonucleotide based on the previously identified
Oct-1 ⁄ 2 (Oct-1 ⁄ 2WT, Kin195, Fig. 6, lane 6) to interfere with nuclear factor: DNA complex formation.
Similar data were generated in HEK293 cells (data not
shown). Hence, we have identified a consensus AP-1
transcription factor site centered at )27 that is critical
for efficient basal Prm3-directed gene expression and
have confirmed the presence and specificity of nuclear
factors in HEL and HEK293 cells that specifically bind
to the latter AP-1 site.
FEBS Journal 272 (2005) 1036–1053 ª 2005 FEBS


A. T. Coyle and B. T. Kinsella

Thromboxane A2 receptor gene expression

A

B

AP-1
-1394

Promoter 3

AP-1
A

+1

+786

-1394

E2

+786

E2
+1 Luc
+1 Luc

***

+1 Luc

+1 Luc

-404

+1 Luc

**

-404

***

+1 Luc

-404

+1

-1394

+1 Luc
-404

1

-1394

+1 Luc

-1394

-

***

-1394

P

Promoter 3

+1 Luc

+1 Luc
+1 Luc

+1 Luc

-154

-50

+1 Luc

+1 Luc
+1 Luc

-50

1

2

+1 Luc

**
**

0

+1 Luc

-50

***

-50

****

+1 Luc

-154

+1 Luc

-154

***

-154

****

+1 Luc

-320

-320

-320

***

-320

3

4

5

6

7

0

Luciferase Activity (RLU)

C

6

8

10

12

D
AP-1

+1

Promoter 3

4

Luciferase Activity (RLU)

AP-1
-1394

2

+786

-1394

E2

-1394

+1 Luc

-1394

+1 Luc

-1394

+1 Luc

****

+1 Luc

+786

E2

***

-1394

+1

Promoter 3

-404

+1 Luc

+1 Luc

-154

+1 Luc

+1 Luc

-154

+1 Luc

-154

+1 Luc

+1 Luc

-50

+1 Luc

-50

+1 Luc

****
****

-50

+1 Luc

***
***
***

-50

+1 Luc

****
****

+1 Luc

-320

-320

****

-154

+1 Luc

****
****

-320

+1 Luc

-404

Luc

****

-320

-404

****

+1 Luc

****

-404

0

1

2

3

4

5

6

Luciferase Activity (RLU)

7

0

5

10

15

20

Luciferase Activity (RLU)

Fig. 5. Identification of a functional AP-1 element within Prm3. (A–D) The TP genomic region spanning nucleotides )1394 to +786 encoding
Prm3 ()1394 to +1), a putative AP-1 element in addition to exon (E) 2 are illustrated above each panel. Recombinant pGL3Basic (A and C) or
pGL3Enhancer (B and D) plasmids encoding Prm3 ()1394 to +1), Prm3a ()404 to +1), Prm3ab ()320 to +1), Prm3aa ()154 to +1), Prm3aaa
()50 to +1) or their respective site-directed variants Prm3AP)1*, Prm3aAP)1*, Prm3abAP)1*, Prm3aaAP)1* and Prm3aaaAP)1*, where the AP-1
element centered at )27 was mutated, were cotransfected with pRL-TK into HEL92.1.7 (A and B) and HEK293 (C and D) cells. Firefly and
renilla luciferase activity was assayed 48 h post-transfection; results are presented as mean firefly relative to renilla luciferase activity,
expressed in arbitrary relative luciferase units (RLU ± SEM; n ¼ 5). The asterisks (*) indicate that mutation of the AP-1 element significantly
reduced Prm3-directed luciferase activity in HEL and HEK293 cells, where *, **, ***, **** indicate P £ 0.05, P £ 0.02, P £ 0.001,
P £ 0.0001, respectively.

Examination of the coordinate regulation of promoter 3 basal activity by the AP-1 and Oct-1/2
transcription factors
To determine the combined contribution of the AP-1
and Oct-1 ⁄ 2 cis-acting elements in directing basal
Prm3 activity, the effect of collectively mutating the

FEBS Journal 272 (2005) 1036–1053 ª 2005 FEBS

latter sites (AP-1*, Oct-1 ⁄ 2*) within the )320 bp
Prm3 subfragment subcloned into pGL3Basic
(Fig. 7A) or pGL3Enhancer (Fig. 7B) on luciferase
activity was compared to the corresponding subfragment containing the wild-type AP-1 and Oct-1 ⁄ 2 elements. Following transfection into HEL92.1.7 cells,
recombinant pGL3Basic (Fig. 7A) or pGL3Enhancer
1043


Thromboxane A2 receptor gene expression

A. T. Coyle and B. T. Kinsella

level of luciferase activity directed by the AP-1*,
Oct-1 ⁄ 2* subfragment in either pGL3Basic or
pGL3 Enhancer was not substantially greater than
that level found in cells transfected with the equivalent promoter-less empty vectors (Fig. 7A,B). Similar
data were generated in HEK293 cells (data not
shown). These data strongly indicate that the Oct-1 ⁄ 2
and AP-1 elements independently regulate Prm3 activity and that disruption of both sites obliterates basal
Prm3 activity.
Investigation of the role of the AP-1 site at -27
in phorbol myristic acid (PMA) induction of
promoter 3

Fig. 6. Demonstration of nuclear factor binding to a putative AP-1
element within Prm3 by electromobility shift assay. A 32P-labeled
double-stranded AP-1WT DNA probe (Kin189 and its complement)
was used in electromobility shift assays (EMSAs) using nuclear

extracts prepared from HEL92.1.7 cells. 32P-labeled AP-1WT probe
was incubated: without nuclear extract (lane 1); with nuclear extract
(lane 2); with nuclear extract in the presence of excess of nonlabeled specific double-stranded competitor AP-1WT oligonucleotide
(Kin189 and its complement; lane 3); with nuclear extract in the
presence of excess nonlabeled double-stranded AP-1* noncompetitor oligonucleotide (Kin162 and its complement where the putative
AP-1 element centered at )27 was mutated, lane 4); with nuclear
extract in the presence of excess nonlabeled consensus doublestranded AP-1 oligonucleotide (Kin338 and its complement, lane 5);
with nuclear extract in the presence of excess nonlabeled doublestranded Oct-1 ⁄ 2 noncompetitor oligonucleotide (Kin195 and its
complement, lane 6). DNA–protein complexes were subject to
PAGE followed by autoradiography, as outlined in Experimental
procedures.

(Fig. 7B) plasmids containing these mutations resulted
in a complete loss in Prm3-directed luciferase expression. More specifically, there was a 10- to 16-fold
reduction in luciferase activity directed by the Prm3
subfragment containing the mutated AP-1*, Oct-1 ⁄ 2*
sites relative to the corresponding subfragments containing the wild-type sequences in either pGL3Basic
(Fig. 7A) or pGL3Enhancer (Fig. 7B). In fact, the
1044

Previous studies have shown that TPb mRNA and
Prm3-directed luciferase activity in HEL92.1.7 cells is
up-regulated in response to phorbol myristic acid
(PMA) [25]. Moreover, AP-1 elements have a
well-established role in the transduction of PMAmediated gene expression and mitotic signaling in a
number of cell models [27]. Hence, in the current
study, we examined the effect of PMA on luciferase
expression directed by Prm3 containing either the
wild-type AP-1 element or its mutated AP-1* equivalent. Following transfection into HEL92.1.7 cells,
consistent with previous data, Prm3 containing the

mutated AP-1* site yielded approximately 2.0-fold
reductions in basal luciferase activity relative to that
of the corresponding Prm3 subfragments containing
the wild-type AP-1 sequence in pGL3Basic (Fig. 8A).
Whilst preincubation of cells with PMA (100 nm,
16 h) yielded a 1.5-fold increase in Prm3 directedluciferase activity, PMA did not significantly increase
luciferase activity directed by Prm3 subfragments
containing the AP-1* mutation. Moreover, preincubation of HEL cells with PMA resulted in a 2.3-fold
increase in nuclear factor binding to the consensus
AP-1 element centered at )27 within Prm3 relative
to vehicle-treated cells, as determined in EMSAs
(Fig. 8B). Nuclear factor–DNA complex formation
was efficiently competed by an excess of a doublestranded oligonucleotide containing a consensus AP-1
site, regardless of preincubation of cells with PMA
or not (Fig. 8B). Hence, to conclude, Prm3 contains
an AP-1 and Oct-1 ⁄ 2 element centered at )27 and
)105 within the core promoter, respectively, that are
critical for basal Prm3 activity and the AP-1 element
mediates PMA-induction of Prm3 expression. In
addition, we have identified the presence of a negative regulatory region between )404 and )320
upstream of the core promoter that acts as an
upstream repressor sequence.
FEBS Journal 272 (2005) 1036–1053 ª 2005 FEBS


A. T. Coyle and B. T. Kinsella

Thromboxane A2 receptor gene expression

A

-1394

-404

-320

-320

+1 Luc

-320

+1 Luc

****

pGL3Basic +1 Luc
0

Luciferase Activity (RLU)

B

-1394

-404

-320

Oct 1
(-123)
AP-1
Oct 1/2 (-27)
(-105)
+1

-320

+1 Luc

-320

+1 Luc

Discussion
In humans, TXA2 signals through two receptor isoforms termed TPa and TPb. Although the physiologic
requirement for two TXA2 receptors in humans is
unclear, alterations in TP expression are implicated in
a range of vascular diseases [9–12]. Whether TPa
and ⁄ or TPb independently or differentially contribute
to those disease processes in human subjects is currently unknown but in view of the extensive differences
between the TP isoforms in terms of their mechanisms
of signaling [18,19], modes of regulation ⁄ desensitization [20–23] and patterns of expression [24], this represents a question of potentially immense importance.
The fact that TPa and TPb are differentially expressed
and are under the transcriptional control of two distinct promoters, Prm1 and Prm3, respectively [25],
greatly adds to the complexity of TXA2 signaling and
provides an additional critical mechanism whereby the
effects of TXA2 can be modulated in an isoform
and ⁄ or cell ⁄ tissue specific manner. The overall aim of

the current study was to carry out a detailed functional
characterization of Prm3, identifying the cis-acting elements regulating basal Prm3 activity with a view to
defining the key factors that direct TPb expression
under normal cellular conditions.
Similar to that of the previously characterized Prm1,
Prm3 belongs to the class of TATA-less promoters
FEBS Journal 272 (2005) 1036–1053 ª 2005 FEBS

2

****

Fig. 7. Effect of mutation of the Oct-1 ⁄ 2
and AP-1 sites on Prm3-directed luciferase
expression. (A and B) The TP genomic
region spanning Prm3 ()1394 to +1) in addition to the relative positions of putative Oct1, Oct-1 ⁄ 2 and AP-1 elements are illustrated
above each panel. Recombinant pGL3Basic
(A) or pGL3Enhancer (B) plasmids encoding
Prm3ab ()320 to +1) or its respective sitedirected variant Prm3abOct1 ⁄ 2*,AP)1*, where
both the Oct-1 ⁄ 2 and AP-1 elements centered at )105 and )27 of Prm3, respectively, was disrupted by site-directed
mutagenesis were cotransfected with
pRL-TK into HEL92.1.7. Firefly and renilla
luciferase activity was assayed 48 h posttransfection; results are presented as mean
firefly relative to renilla luciferase activity,
expressed in arbitrary relative luciferase
units (RLU ± SEM; n ¼ 5). The asterisks (*)
indicate that either deletion or site-directed
mutagenesis of Prm3 sequences significantly reduced luciferase expression in HEL
cells, where **** indicates P £ 0.0001.

Oct 1
(-123)
AP-1
Oct 1/2 (-27)
(-105)
+1

pGL3Enhancer

+1 Luc
0

2

4

6

8

10

12

Luciferase Activity (RLU)

[16,25]. In TATA-less promoters, assembly of the preinitiation complex relies on binding of multiple general
transcription factors, such as SP-1, in proximity to the
transcription initiation site [28]. Herein, successive
5¢-deletion of Prm3 to either 106 bp ()106 to +1) or

50 bp ()50 to +1) yielded a subfragment that retained
a significant, albeit reduced, ability to direct reporter
gene expression in both HEL and HEK293 cells whilst
deletion of the 3¢-terminal 118 bp of Prm3 ()118
to +1) led to a complete loss of promoter activity in
both cell types. Collectively these data established that
the critical core element(s) are located within the )118
to +1 region of Prm3.
Upstream activation sequences (UAS) and upstream
repressor sequences (URS) are gene-specific sequences
controlling the rate of transcription initiation [29].
Negative regulatory elements in particular have been
identified in a number of TATA-less promoters [30,31].
Consistent with this, successive 5¢-deletion of nucleotides between )404 and )320 removed a URS that has
a repressive effect (two-fold) on Prm3 activity in both
HEL and HEK293 cells. Further deletion of nucleotides between )320 and )154 had no additional effect
in either cell type suggesting that )320 to )154 region
does not contribute to basal Prm3 activity. The identity of the transcription factors element(s) regulating
the URS is unknown but will be a subject of further
characterization of Prm3.
1045


Thromboxane A2 receptor gene expression

A. T. Coyle and B. T. Kinsella

A

AP-1

+1
-1394

+786

E2

Luc

-1394

**

-1394

+1 Luc

Vehicle
PMA

+1

-404

***

-404

+1 Luc

Luc

0

2

Luciferase Activity (RLU)

B

1 2 3 4 5 6 7

Fig. 8. Effect of PMA on Prm3-directed luciferase expression and nuclear factor binding. (A) The TP genomic region spanning nucleotides
)1394 to +786 encoding Prm3 ()1394 to +1) in addition to an AP-1 element and exon (E) 2 are illustrated above the panel. Recombinant
pGL3Basic plasmids encoding Prm3 ()1394 to +1), Prm3a ()404 to +1) or their respective site-directed variants Prm3AP)1* Prm3aAP)1*,
where the AP-1 element centered at )27 of Prm3 was mutated, were cotransfected with pRL-TK into HEL92.1.7 cells. Thirty-six hours posttransfection, cells were incubated with either 100 nM PMA or the vehicle (0.1% dimethylsulfoxide) for 16 h. Thereafter, firefly and renilla
luciferase activity was assayed; results are presented as mean firefly relative to renilla luciferase activity, expressed in arbitrary relative
luciferase units (RLU ± SEM; n ¼ 4). The asterisks (*) indicate that luciferase expression in HEL cells was significantly altered in PMAtreated cells relative to vehicle treated cells, where **, *** indicates P £ 0.02, P £ 0.001, respectively. (B) A 32P-labeled double-stranded
AP-1WT DNA probe (Kin189 and its complement) was used in EMSAs using nuclear extracts prepared from vehicle- (lanes 2–4) or PMA(lanes 5–7) treated HEL92.1.7 cells. 32P-labeled AP-1WT probe was incubated: without nuclear extract (lane 1); with nuclear extract (lanes 2
and 5); with nuclear extract in the presence of excess nonlabeled consensus double-stranded AP-1 oligonucleotide (Kin338 and its complement, lanes 3 and 5); with nuclear extract in the presence of excess nonlabeled double-stranded AP-1*noncompetitor oligonucleotide
(Kin162 and its complement where the AP-1 sequence centered at )27 was mutated, lanes 4 and 6). DNA–protein complexes were subject
to PAGE followed by autoradiography, as outlined in Experimental procedures.

The octamer sequence element (consensus 5¢-ATGC
AAAT-3¢) present in promoters of immunoglobulin
and numerous ubiquitously expressed genes [32–35]
can facilitate functional preinitiation complex assembly
[36]. Amongst the key members, Oct-1 is ubiquitous
[37,38] while Oct-2 is mainly expressed in B lymphocytes, neuronal cells [39–42], and in megakaryocytes
[43,44]. In the current study, 5¢-deletion of nucleotides

)154 to )106 within Prm3 yielded two to eightfold
reductions in luciferase expression in HEL and
HEK293 cells indicating that the latter region contains
positive regulatory element(s) required for efficient
1046

basal Prm3 activity. Whilst successive 5¢-deletion of
nucleotides between )154 and )119 did not significantly affect luciferase expression, further 5¢-deletion of
the 13 bp gene segment between )119 and )106 led to
a 2.5-fold decrease in luciferase expression confirming
that this sequence contains the positive regulatory elements. Bioinformatic analysis revealed the presence of
a putative Oct-1 site centered at )123 and an adjacent
Oct-1 ⁄ 2 element at )105 within this sequence. Whilst
the putative Oct-1 site at )154 does not closely resemble a consensus octamer element, its complementary
sequence (5¢-TGAAATGC-3¢) shows some homology
FEBS Journal 272 (2005) 1036–1053 ª 2005 FEBS


A. T. Coyle and B. T. Kinsella

to known Oct elements, for example to that of the
bovine alpha s2-casein gene [45], and hence was predicted by the matinspector program [26] to potentially
serve as an Oct element. While deletion or mutation of
the former Oct-1 site at )154 did not affect Prm3
activity, site-directed mutagenesis of the putative Oct1 ⁄ 2 site at )105 resulted in 2.5- to three-fold reductions in luciferase expression in both HEL and
HEK293 cells. Hence, these data indicate that the Oct1 ⁄ 2 element at )105 may be critical for basal Prm3
activity. Noteworthy, our mutagenesis data are in full
agreement with previous studies demonstrating the
critical role of adenosine (A) at position 7 within the
octamer motif (AAGCAAAT to AAGCAAGT)

whereby an A ⁄ G mutation severely affected Oct-1 or
Oct-2 binding in vitro [46].
Furthermore, EMSAs confirmed the presence of
transcription factors capable of binding to a doublestranded DNA probe ()115 to )92; Oct-1 ⁄ 2WT) spanning the Oct-1 ⁄ 2 site at )105 in nuclear extracts from
HEL92.1.7 cells. Nuclear factor ⁄ DNA complex formation was efficiently competed by an excess of the
corresponding nonlabeled double-stranded Oct-1 ⁄ 2WT
oligonucleotide but was not competed by the equivalent double-stranded oligonucleotide harboring the
A ⁄ G mutation within the core Oct-1 ⁄ 2* site. Western
blot analysis confirmed abundant expression of Oct-2,
but not Oct-1, in HEL cells and supershift assays
employing anti-(Oct-2) IgG further confirmed Oct-2
binding to the Oct-1 ⁄ 2 site of Prm3. Owing to the
absence of Oct-1 expression in HEL cells, these data
did not rule out the possibility that Oct-1 may regulate
Prm3-directed gene expression in other cell types, such
as in HEK293 cells where it is abundantly expressed.
Consistent with this, heterologous over-expression of
both Oct-1 and Oct-2 significantly increased Prm3directed luciferase activity in HEK293 and HEL cells.
Hence, it is evident that Oct-2 can function as a transacting element capable of regulating Prm3 and TPb
expression in megakaryocytic HEL92.1.7 cells. In addition Oct-1 can also regulate Prm3 activity in other
cell types, such as HEK293 cells, where Oct-1 is also
abundantly expressed.
The AP-1 transcription factor complex participates
in the control of cellular responses to stimuli that
regulate proliferation, differentiation, immunity, cell
death and stress but may also play a critical role in
the assembly of the preinitiation complex within
TATA-less promoters [47]. The AP-1 complex is comprised of a group of proteins encoded by the jun
(c-Jun, JunB, JunD) and fos (c-Fos, FosB, Fra1 and
Fra2) gene families which can bind the AP-1 consensus sequence either as homo- or heterodimers [27]. In

FEBS Journal 272 (2005) 1036–1053 ª 2005 FEBS

Thromboxane A2 receptor gene expression

the current study, the role of the consensus AP-1 site,
located at )27, in mediating basal Prm3 activity was
investigated by mutating its core sequence (GTGACT
to GATCCT) in a range of 5¢-deletion subfragments
of Prm3 site. In general, Prm3 subfragments harboring the mutated AP-1* site yielded approximately 2.5to five-fold reductions in luciferase activity in HEL
and HEK293 cells relative to corresponding subfragments containing the intact AP-1 element whilst mutation of the AP-1* element within the smallest )50 bp
fragment practically abolished luciferase activity. The
ability of nuclear factors to bind to this consensus
AP-1 element within Prm3 was demonstrated by
EMSAs using nuclear extract from HEL cells. Nuclear factor–DNA complex formation was sequence
specific requiring the presence of the ‘core AP-1 element’ and was efficiently competed by an excess of
the corresponding nonlabeled double-stranded AP-1
oligonucleotide but not by the corresponding doublestranded oligonucleotide containing the mutated
AP-1* element. Moreover, EMSAs employing a panJun antibody confirmed binding of Jun protein to the
AP-1 element that was specifically competed by
the nonlabeled double-stranded AP-1wt but not by the
AP-1* oligonucleotide (data not shown). The combined contribution of the AP-1 and Oct-1 ⁄ 2 cis-acting
elements were confirmed whereby mutation of both
the AP-1* and Oct-1 ⁄ 2* elements yielded 10- to
16-fold reductions in luciferase expression, effectively
abolishing Prm3 activity in both HEL and HEK293
cells. Consistent with our observations, both AP-1
and Oct members can bind and direct transcription
from a number of TATA-less promoters [48–51]. In
addition, although both the AP-1 complex and certain
Oct family members are ubiquitously expressed, they

can also regulate transcription in a cell type-specific
manner through the recruitment of cell specific transcription factors and coregulators [52].
The AP-1 binding sequence was originally identified
as a tetradecanoyl phorbol myristic acid (TPA) ⁄ phorbol myristate acetate (PMA) response element (TRE)
and treatment of cultured cells with PMA results in a
strong increase in AP-1 binding to TREs [53]. Prm1and Prm3- reporter gene expression and TPa and TPb
mRNA expression are up-regulated by PMA in HEL
cells [25]. While the site of action of PMA within
Prm1 has been localized to an SP-1 element [54], the
PMA-responsive element(s) within Prm3 has, as yet, to
be identified. Hence, herein, we investigated whether
the AP-1 site at )27 mediates PMA increases in Prm3
activity. Consistent with previous reports [25], stimulation of HEL cells with PMA led to a 1.5-fold increase
in Prm3-directed gene expression. In contrast, Prm3
1047


Thromboxane A2 receptor gene expression

subfragments containing the mutated AP-1* element
yielded 2.0-fold reductions in basal luciferase activity
relative to corresponding Prm3 subfragments containing the wild-type AP-1 and PMA did not significantly
increase luciferase activity by those Prm3 subfragments
containing the AP-1* mutation. Moreover, EMSAs
confirmed that PMA significantly increased nuclear
factor binding to the AP-1 element relative to vehicle
treated HEL cells. Similar data were generated in
HEK293 cells (data not shown). Hence, the AP-1 element mediates PMA induction of Prm3 and thereby
provides a mechanism, at least in part, to account for
the previously reported PMA up-regulation of TPb

expression in HEL cells [25]. Taken together, these
latter observations point to further differences in the
modes of regulation of TPa and TPb expression
through SP-1 [54] and AP-1 elements within Prm1 and
Prm3, respectively, such as during megakaryocyte differentiation for example, that can be readily induced
experimentally in response to PMA.
In conclusion, Prm3 contains an AP-1 and Oct-1 ⁄ 2
core promoter elements that are critical for basal Prm3
activity and the AP-1 element mediates PMA-induction of Prm3 activity. In addition, we have identified a
negative URS between )404 and )320. Collectively,
these data provide valuable insights into the factors
determining both the basal and PMA up-regulation of
TPb expression and may provide a platform to determine the relative contributions of differentially regulated expression of TPa and TPb to haemostasis and
possibly to vascular disease.

Experimental procedures
Materials
pGL3Basic, pGL3Enhancer, pRL-Thymidine Kinase
(pRL-TK) and Dual LuciferaseÒ Reporter Assay System
were obtained from Promega Corporation (Madison, WI,
USA). DMRIE-CÒ was from Invitrogen Life Technologies (CA, USA). EffecteneÒ was from Qiagen Ltd
(Crawley, UK). [32P]ATP[cP] (6000 CiỈmmol)1 at 10 mCiỈ
mL)1) was from Valeant Pharmaceuticals (ICN; Costa
Mesa, USA). All other reagents were molecular biology
grade. Anti-(Oct-1) (sc-232x), anti-(Oct-2) (sc-233x), anti-(cjun) (sc-44x) Igs were obtained from Santa Cruz Biotechnology.

Construction of luciferase-based genetic reporter
plasmids
To identify sequence elements required for promoter (Prm)
3 activity, a range of 5¢- and 3¢-deletion fragments were

1048

A. T. Coyle and B. T. Kinsella

subcloned into pGL3Basic and ⁄ or pGL3Enhancer genetic
reporter vectors. Gene fragments were amplified by the
polymerase chain reaction (PCR) using as template
pGL3b:Prm3 [25] containing Prm3 (1394 bp) cloned into
pGL3Basic. Specifically, for all 5¢ deletions, PCR fragments were generated using the antisense primer Kin113
(5¢-dAGAGACGCGTGGCTCCGGAGCCCTGAGGGA
TC-3¢, complementary to nucleotides )19 to +1 where the
underlined sequence corresponds to the Mlu1 cloning site)
in combination with specific sense primers designed to
amplify progressively shorter regions of Prm3. The following lists the identities of the Prm3 gene fragments and corresponding plasmids generated in either pGL3Basic
(pGL3b) or pGL3Enhancer (pGL3e) vectors and the identity of the specific sense oligonucleotide primer, its sequence
and corresponding nucleotides (NTs) where, in each case,
the – designation indicates NTs 5¢ of the translational ATG
start codon (designated +1) and underlined sequences represent the Kpn1 cloning site.
(1) Prm3b; pGL3b:Prm3b & pGL3e:Prm3b. (Primer
Kin142; 5¢-dGAGAGGTACCACTTCACTCATCACACC
TGGCCC-3¢, corresponding to NTs )975 to )952).
(2) Prm3a; pGL3b:Prm3a & pGL3e:Prm3a. (Primer
Kin143; 5¢-dGAGAGGTACCCTCACGCCTGTAATCCC
AG-3¢, corresponding to NTs )404 to )386).
(3) Prm3ab, pGL3b:Prm3ab & pGL3e:Prm3ab. (Primer
Kin146; 5¢-dGAGAGGTACCTGGGAGGCTGAGATGG3¢, corresponding to NTs )320–304).
(4) Prm3aa, pGL3b:Prm3aa & pGL3e:Prm3aa. (Primer
Kin145;
5¢-dGAGAGGTACCTAGGAGTTCACCAGA

GC-3¢, corresponding to NTs )154 to )137).
(5) Prm3ax; pGL3b:Prm3ax & pGL3e:Prm3ax. (Primer
Kin177; 5¢-dGAGAGGTACCAGCTACTTACACTGAAA
TGCAG-3¢, corresponding to NTs )140 to )118).
(6) Prm3ac; pGL3b:Prm3ac & pGL3e:Prm3ac. (Primer
Kin188; 5¢-dGAGAGGTACCGAATTAATCACAAGCAA
ATCTTCTC-3¢, corresponding to NTs )119 to )94).
(7) Prm3aab; pGL3b:Prm3aab & pGL3e:Prm3aab. (Primer
Kin160; 5¢-dGAGAGGTACCGCAAATCTTCTCTCGCC
TCC-3¢, corresponding to NTs )106 to )86).
(8) Prm3aaa; pGL3b:Prm3aaa & pGL3e:Prm3aaa. (Primer
Kin161, 5¢-dGAGAGGTACCGCAGCATCGGCCTGATG
GG-3¢, corresponding to NTs )50 to )31).
The gene fragment Prm3e ()404 to +119 of Prm3) was
amplified by PCR using pWE15:TXR [55] as template and
primers Kin143 (5¢-dGAGAGGTACCCTCACGCCTGTA
ATCCCAG-3¢, corresponding to NTs )404 to )385) and
Kin245 (5¢-dAGAGACGCGTGCCAGGCCCACCACGC
AG-3¢, corresponding to NTs +1 to +119) and was subcloned into the Kpn1–Mlu1 sites of pGL3Basic to generate
the plasmid pGL3b:Prm3e.
The 3¢-deletion Prm3f (nucleotide )404 to )118) was
amplified by PCR using pGL3b:Prm3 as template and the
primers Kin143 (5Â-dGAGAGGTACCCTCACGCCTGTA

FEBS Journal 272 (2005) 10361053 ê 2005 FEBS


A. T. Coyle and B. T. Kinsella

ATCCCAG-3¢, corresponding to NTs )404 to )385) and

Kin263 (5¢dAGAGACGCGTGAGAAGATTTGCTTGTG
ATTAATTC-3¢, corresponding to NTs )143 to )118) and
was subcloned into the Kpn1–Mlu1 sites of pGL3Basic to
generate the plasmid pGL3b:Prm3f.
The identity and fidelity of all Prm3 gene fragments in
the latter recombinant plasmids was verified by doublestranded DNA sequencing.

Site-directed mutagenesis
Site-directed mutagenesis was carried out using the QuikChangeTM (Stratagene, Amsterdam, the Netherlands)
method. Specifically, mutation of the AP-1 element with
the sequence ggTGACtg to ggATCCtg (core bases shown
in uppercase) centered at )27 within Prm3 was performed
using mutator primers Kin162 (5¢-dCGGCCTGATGGG
GTGGATCCTGATCCCTCAGGGCTC-3¢; sense primer)
vs. its complement generating pGL3b:Prm3AP)1*, pGL3e:
Prm3AP)1*, pGL3b: Prm3aAP)1*, pGL3e:Prm3aAP)1*,
pGL3b:Prm3abAP)1*, pGL3e: Prm3abAP)1*, pGL3b:
Prm3aaAP)1*, pGL3e:Prm3aaAP)1*, pGL3b:Prm3aaaAP)1*
and pGL3e:Prm3aaaAP)1*.
Mutation of the Oct-1 element with the sequence aaA
TGCa to aaTTCCa (core bases shown in uppercase letters)
centered at )123 within Prm3 was performed using the
mutator primers Kin175 (5¢-CACCAGAGCTACTTACA
CTGAATTCCAGAATAATCACAAGCAAATC-3¢; sense
primer) vs. its complement generating pGL3b:Prm3aOCT)1*,
pGL3b:Prm3abOCT)1*
and
pGL3e:Prm3aOCT)1*,
OCT)1
*.

pGL3e:Prm3ab
Mutation of the Oct-1 ⁄ 2 element with the sequence
aGCAAAtc to aGCAAGtc (core bases shown in uppercase
letters) centered at )105 within Prm3 was performed using
the mutator primers Kin193 (5¢-dGAATTAATCACAAGC
AAGTCTTCTCTCGCCTCCCAG-3¢; sense primer) vs. its
complement generating pGL3b:Prm3aOCT1 ⁄ 2*, pGL3e:Prm3aOCT1 ⁄ 2*, pGL3b:Prm3aOCT1 ⁄ 2* and pGL3e:Prm3aOCT1 ⁄ 2*.
Mutation of both the consensus Oct-1 ⁄ 2 and AP-1 elements centered at )105 and )27, respectively, was performed using the mutator primers Kin162 (5¢-dCGGCCTG
ATGGGGTGGATCCTGATCCCTCAGGGCTC-3¢; sense
primer)
vs.
its
complement
generating
pGL3b:
Prm3abOct1 ⁄ 2*,AP)1* and pGL3e:Prm3ab Oct1 ⁄ 2*,AP)1*. In
each case, mutated bases within the mutator primers are
highlighted in bold type.

Cell culture
All mammalian cells were grown at 37 °C in a humid environment with 5% CO2. Human erythroleukemic (HEL)
92.1.7 cells and human embryonic kidney (HEK) 293 cells
were cultured in RPMI 1640, 10% fetal bovine serum and
in Eagle’s minimal essential medium (MEM), 10% fetal
bovine serum, respectively.

FEBS Journal 272 (2005) 1036–1053 ª 2005 FEBS

Thromboxane A2 receptor gene expression

Assay of luciferase activity
HEK293 cells were plated in MEM, 10% FBS in six well
dishes at 1 · 105 cells per well. At 70–80% confluence, cells
were cotransfected with control ⁄ recombinant pGL3Basic or
pGL3Enhancer vectors (0.4 lg per well), encoding firefly
luciferase, along with pRL TK (50 ng per well), encoding
renilla luciferase, using effectene as recommended by the
supplier. To investigate the effect of Oct-1 or Oct-2 on
Prm3-directed luciferase expression, the previously described pcDNA3:HaOct-1 or pcDNA3:HaOct-2 plasmids
[46] encoding Oct-1 and Oct-2, respectively, were transiently
over-expressed in HEK293 cells along with recombinant
pGL3Basic vectors encoding Prm3ab. Briefly, pGL3b:Prm3ab (0.2 lg per well) plus pRL TK (50 ng per well) plasmids were transiently cotransfected along with either
pcDNA3:HaOct-1 (0.2 lg per well), pcDNA3:HaOct-2
(0.2 lg per well) or, as a negative control, pcDNA3 (0.2 lg
per well) using Effectene. Forty-eight hours post transfection, cells were washed in phosphate-buffered saline
(NaCl ⁄ Pi), were lysed and harvested by scraping in 350 lL
Reporter Lysis Buffer (Promega) and centrifuged at
14 000 g for 1 min at room temperature.
HEL92.1.7 cells were transfected using DMRIE-C.
Briefly, per transfection, 0.5 mL of serum free RPMI 1640
medium was dispensed into a six-well dish and 6 lL of
DMRIE-C reagent was added. Thereafter, 0.5 mL of
serum-free RPMI 1640 medium containing 2 lg of recombinant ⁄ control pGL3Basic or pGL3Enhancer plasmids and
200 ng of pRL-TK was added and DNA ⁄ DMRIE-C reagent was complexed at room temperature for 30 min.
Thereafter, 0.2 mL of serum free RPMI 1640 medium containing 2 · 106 HEL cells were added and incubated for
4 h at 37 °C in a CO2 incubator, after which 2 mL of
RPMI 1640 containing 15% fetal bovine serum was
added. Forty-eight hours after transfection, cells were
washed in ice-cold NaCl ⁄ Pi and harvested at 1200 g for
5 min at 4 °C. Where relevant, 36 h prior post-transfection

HEL cells with incubated with PMA (100 nm) or, as controls, with an equivalent volume of the vehicle (0.1%
dimethylsulfoxide) and cells were further incubated for
16 h prior to harvesting. Cell pellets were resuspended in
reporter lysis buffer (100 lL) and were lysed by repeated
trituration. Cell lysates were prepared by centrifugation at
14 000 g for 1 min at room temperature. To investigate
the effect of Oct-1 or Oct-2 on Prm3-directed luciferase
expression, pcDNA3:HaOct-1 or pcDNA3:HaOct-2 plasmids [46] were transiently over-expressed in HEL cells
along with recombinant pGL3Basic vectors encoding Prm3
or its subfragments. Briefly, recombinant pGL3b:Prm3ab
(1.0 lg per well) plus pRL TK (200 ng per well) plasmids
were transiently cotransfected along with either pcDNA3:
HaOct-1 (1.0 lg per well), pcDNA3:HaOct-2 (1.0 lg per
well) or, as a negative control, pcDNA3 (1.0 lg per well)
using DMRIE-C. Cells were harvested 48 h post-transfec-

1049


Thromboxane A2 receptor gene expression

tion and cells lysed and prepared for luciferase assays as
described above.
HEK293 and HEL cell supernatants were assayed for
both firefly and renilla luciferase activity using the Dual
Luciferase Assay SystemTM, essentially as previously described [25]. Relative firefly to renilla luciferase activities
(arbitrary units) were calculated as a ratio and were
expressed in relative luciferase units (RLU).

Preparation of nuclear extracts

Nuclear extracts were prepared from both untreated and
phorbol myristic acid (PMA) treated (100 nm, 16 h)
HEL92.1.7 cells (as previously described with minor modifications [56]. Specifically, HEL cells (1.6–2 · 106) were
pelleted at 717 g for 5 min at 4 °C and lysed by triturating
6–8 times on ice in 1.5 mL cell lysis buffer (1.5 mm Hepes,
pH 7.9., 8 mm KCl, 0.2 mm EDTA, 50 lm spermine, 1%
glycerol, 0.5 mm dithiothreitol, 1% NP40, 10 lm sodium
orthovandadate, 40 mm NaFl, 0.1 mm phenylmethylsulfonyl fluoride, 1 mm leupeptin, 0.7 mgỈmL)1 pepstatin) using
21 and 26 gauge needles, respectively. Nuclei were isolated
and lysed [56] and nuclear extracts were dialysed vs. 20 mm
Hepes, pH 7.9, 20% glycerol, 100 mm KCl, 0.4 mm PMSF,
0.5 mm EDTA, 0.2 mm EGTA and 0.2 mm EGTA. Following dialysis, nuclear debris was pelleted at 16 000 g for
10 min and the protein concentration of nuclear extracts
were determined using the Bradford assay [57]. For electrophoretic mobility supershift assays, nuclear extracts were
prepared under identical conditions except that dithiothreitol was excluded from the various isolation buffers.

Electrophoretic mobility shift and supershift
assays
Oligonucleotides corresponding to the sense and antisense
strands of each probe (0.35 lm of each) were annealed in
1· T4 polynucleotide kinase (PNK) buffer (70 mm
Tris ⁄ HCl, pH 7.6.,10 mm MgCl2, 5 mm dithiothreitol;
8 lL) by heating at 95 °C for 2 min followed by slow cooling to room temperature for 30 min. The resulting doublestranded probes were then radiolabeled in 10 lL reactions
containing 0.35 lm double-stranded oligonucleotide, 1 lL
[32P]ATP[cP] (6000 CiỈmmol)1 at 10 mCiỈmL)1; ICN) and
1 lL T4 PNK (10 lL)1) at 37 °C for 30 min. Following
labeling, the reactions were diluted 1 : 10 with 10 mm
Tris ⁄ HCl, pH 8.0.,1 mm EDTA (TE, pH 8.0) buffer to
achieve a final concentration of 32P-radiolabeled labeled of
0.035 lm. The unincorporated [32P]ATP[cP] was removed

by gel filtration chromatography using preprepared Sephadex G25 spin columns (Roche).
Nuclear extract (4 lg total protein) was incubated for
15 min at room temperature with ⁄ without a 57-fold molar
excess of unlabeled double-stranded competitor ⁄ noncompetitor oligonucleotide (2 lm) in 1· Binding Buffer (20 mm

1050

A. T. Coyle and B. T. Kinsella

Hepes, pH 7.9., 50 mm KCl, 0.1 mm EDTA, 0.1 mm
EGTA, 0.5 mm dithiothreitol, 4% Ficoll, 50 lgỈmL)1 poly
(dI-dC; Sigma). The appropriate 32P-radiolabeled doublestranded oligonucleotide (0.035 lm; 1 lL per reaction) was
then added and reactions were incubated for 20 min at
room temperature. Following incubation, binding reactions
were subjected to electrophoresis through a 4% polyacrylamide gel (20 cm · 20 cm) in 89 mm Tris borate, 2 mm
EDTA buffer for 3 h at room temperature; thereafter, gels
were dried and analyzed by autoradiography.
The sequences of the competitor ⁄ noncompetitor oligonucleotides used were as follows: (a) Oct-1 ⁄ 2WT (Kin195;
5¢-dTAATCACAAGCAAATCTTCTCTC-3¢; corresponding
to NTs )115 to )92 of Prm3); (b) mutated Oct-1 ⁄ 2*
(Kin193; 5¢-dGAATTAATCACAAGCAAGTCTTCTCTC
GCCTCCCAGTC-3¢; corresponding to NTs )119 to )83 of
Prm3 where bases mutated from the wild-type Prm3 sequence
are in bold italics); (c) AP-1WT(Kin189; 5¢-dGGTGGTGAC
TGATCCCTCAGGGC-3¢; corresponding to NTs )32 to
)10 of Prm3); (d) mutated AP-1* (Kin162; 5¢-dCGGCCT
GATGGGGTGGATCCTGATCCCTCAGGGC-3¢; corresponding to NTs )46 to )7 of Prm3 where bases mutated
from the wild-type Prm3 sequence are in bold italics); (e) SP1 consensus site (Promega) with the sequence: 5¢-dATTCG
ATCGGGGCGGGGCGAG-3¢; (f) AP-1 consensus site
(Promega; Kin338; 5¢ dCGCTTGATGAGTCAGCCGGAA3¢); (g) octamer (Oct) consensus site (Promega; Kin340;

5¢-dTGTCAGATGCAAATCACTAGAA-3¢). Note, only
forward oligonucleotides are given and sequences of the
corresponding complementary strands are omitted.
For electrophoretic mobility supershift assays, nuclear
extracts (4 lg total protein) was preincubated with 2 lL for
either anti-(Oct-1) (sc-232x; 2 mgỈmL)1 stock concentration), anti-(Oct-2) (sc-233x; 2 mgỈmL)1 stock concentration), anti-(c-jun) (sc-44x; 2 mgỈmL)1 stock concentration)
for 30 min at 4 °C. Thereafter, nuclear extract ⁄ antibody
mixtures were incubated for 15 min at room temperature
with ⁄ without a 57-fold molar excess of unlabeled doublestranded competitor ⁄ noncompetitor oligonucleotide (2 lm)
in 1· binding buffer followed by the addition of the appropriate 32P-radiolabeled double-stranded oligonucleotide
(0.035 lm; 1 lL per reaction), as described above.

Western blot analysis
Levels of Oct-1 and Oct-2 were determined by Western blot
analysis. Briefly, 60 lg of whole cell protein from HEL or
HEK293 cells were resolved by SDS ⁄ PAGE (10% gels)
and transferred to polyvinylidene difluoride membrane
according to standard methodology. Membranes were
screened using anti-(Oct-1) (sc-232x) or anti-(Oct-2)
(sc-233x) at 0.2 lg polyvinylidene difluoride per mL in
5% non fat dried milk in 1· TBST (0.01 m Tris ⁄ HCl,
0.1 m NaCl, 0.1% Tween 20; pH 7.5) for 2 h at room
temperature followed by washing and screening using

FEBS Journal 272 (2005) 1036–1053 ª 2005 FEBS


A. T. Coyle and B. T. Kinsella

goat anti-rabbit horseradish peroxidase (HRP; sc-2204)

and detection using BM Chemiluminesence Western blotting Kit (Roche, E. Sussex, UK) as described by the
manufacturer.

Statistical analysis
Statistical analyses of differences were analyzed using the
two-tailed Student’s unpaired t-test. All values are
expressed as mean ± standard error of the mean (SEM).
P-values £ 0.05 were considered to indicate statistically significance differences.

Thromboxane A2 receptor gene expression

9

10

11

Acknowledgements

12

This work was supported by grants from The Wellcome Trust, The Health Research Board and Enterprise Ireland. We are grateful to Dr Harinder Singh,
Department Molecular Genetics and Cell Biology,
University of Chicago for the gifts of pcDNA3:HaOct1
and pcDNA3:HaOct2.

13

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