15d-PGJ2 suppressed Prm3 transcriptional activity and TPb mRNA expression in the platelet progenitor megakaryocytic human erythroleukemia HEL 92.1.7 cell line but had no effect on Prm1 o
Trang 1by promoter 3 of the human thromboxane A2 receptor
gene through peroxisome proliferator-activated receptor c
in human erythroleukemia cells
Adrian T Coyle, Martina B O’Keeffe and B Therese Kinsella
Department of Biochemistry, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Ireland
The cyclopentanone prostaglandin 15-deoxy-D12,14
-prostaglandin (PG) J2 (15d-PGJ2), a dehydration
prod-uct of cyclooxygenase (COX)-derived PGD2 present in
inflammatory exudates, is elevated during the
resolu-tion phase of inflammaresolu-tion and was initially identified
as a high affinity natural ligand for peroxisome
prolif-erator-activated receptors (PPAR)c [1,2] although a number of PPARc independent effects have recently been reported [3] The nuclear hormone receptor PPARc classically up-regulates gene expression by binding as a heterodimer with the retinoic X receptor (RXR) to specific response elements consisting of one
Keywords
thromboxane receptor; promoter;
peroxisome proliferator-activated receptor c;
15-deoxy D 12,14 -prostaglandin J2; isoforms
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: Therese.Kinsella@ucd.ie
(Received 15 June 2005, revised 28 July
2005, accepted 29 July 2005)
doi:10.1111/j.1742-4658.2005.04890.x
In humans, thromboxane (TX) A2 signals through two receptor isoforms, thromboxane receptor (TP)a and TPb, which are transcriptionally regula-ted by distinct promoters, Prm1 and Prm3, respectively, within the single
TP gene The aim of the current study was to investigate the ability of the endogenous peroxisome proliferator-activated receptor (PPAR)c ligand 15-deoxy-D12,14-prostaglandin J2 (15d-PGJ2) to regulate expression of the human TP gene and to ascertain its potential effects on the individual TPa and TPb isoforms 15d-PGJ2 suppressed Prm3 transcriptional activity and TPb mRNA expression in the platelet progenitor megakaryocytic human erythroleukemia (HEL) 92.1.7 cell line but had no effect on Prm1 or Prm2 activity or on TPa mRNA expression 15d-PGJ2 also resulted in reductions
in the overall level of TP protein expression and TP-mediated intracellular calcium mobilization in HEL cells 15d-PGJ2 suppression of Prm3 tran-scriptional activity and TPb mRNA expression was found to occur through
a novel mechanism involving direct binding of PPARc–retinoic acid X receptor (RXR) heterodimers to a PPARc response element (PPRE) com-posed of two imperfect hexameric direct repeat (DR) sequences centred at )159 and )148, respectively, spaced by five nucleotides (DR5) These data provide direct evidence for the role of PPARc in the regulation of human
TP gene expression within the vasculature and point to further critical dif-ferences in the modes of transcriptional regulation of TPa and TPb in humans Moreover, these data highlight a further link between enhanced risk of cardiovascular disease in diabetes mellitus associated with increased synthesis and action of thromboxane A2(TXA2)
Abbreviations
AP-1, activator protein-1; 15d-PGJ2, 15-deoxy-D 12,14 -prostaglandin J2; CHIP, chromosomal immunoprecipitation; COX, cyclooxygenase;
DR, direct repeat; d ⁄ s, double stranded; EMSA, electromobility shift assay; HEK, human embryonic kidney; HEL, human erythroleukemia;
PG, prostaglandin; PPAR, peroxisome proliferator-activated receptor; PPRE, peroxisome proliferator-activated receptor response element; Prm, promoter; RLU, relative luciferase units; RAR, retinoic acid receptor; RXR, retinoic acid X receptor; TZD, thiazolidinedione;
TP, thromboxane receptor; TXA 2 , thromboxane A 2 ; VSMC, vascular smooth muscle cell.
Trang 2or more copies of the hexameric DNA consensus
sequence AGGTCA arranged as a direct repeat (DR)
spaced by one nucleotide, hence termed DR1 [4,5]
PPARc-transcriptional activation may also involve the
recruitment of various coactivators to specific target
genes such as p300 [6], the SRC-1 coactivators [7–9],
PGC-1 and PGC-2 [8,9], ARA70 [10] and DRIP205
(or TRA220) [11] In addition to activating
trans-cription, PPARc can also negatively regulate gene
expression through mechanisms involving either the
trans-repression (negative cross-talk) of activating
tran-scription factors, e.g NF-jB and activator protein-1
(AP-1) [12,13] or the sequestration of limiting amounts
of coactivator molecules such as CBP [14]
The beneficial insulin-sensitizing actions of 15d-PGJ2
and the thiazolidinediones (TZDs) as PPARc agonists
are widely recognized, such as in the treatment of type
II diabetes [15] Moreover, while the inhibitory effects
of PPARc play a prominent role in the resolution
of inflammation [15], they are also thought to be
important within the vasculature where they offer a
cardioprotective effect during myocardial ischemia,
reperfusion and atherosclerosis and hence it is
rea-soned that PPARc agonists may help to alleviate the
adverse cardiovascular events associated with diabetes
mellitus [16,17] For example, PPARc activators
inhi-bit matrix metalloproteinase-9 expression in vascular
smooth muscle cells (VSMCs) [18] and thrombin
induced endothelin-1 production in endothelial cells
[19] Moreover, it has recently been established that
the expression of a number of other key vascular genes
are suppressed in response to PPARc activation
inclu-ding those encoding the inducible cyclooxgenase
(COX)II [20] and nitric oxide synthase [14], the rat
thromboxane (TX)A2 synthase [21] and the rat
throm-boxane A2 (TXA2) receptor (thromboxane receptor,
TP) [22]
The COX-derived TXA2is a potent biologically
act-ive eicosanoid primarily released from activated
plate-lets, monocytes and damaged vessel walls and plays a
central role in the dynamic regulation of vascular
hae-mostasis [23] Alterations in the level of TXA2, TXA2
synthase or the TXA2 receptor (TP) are widely
implicated in a variety of vascular diseases including
thrombosis, unstable angina, systemic and
pregnancy-induced hypertension [24–26] TXA2 is also known to
play a pathophysiological role in inflammatory diseases
such as in atherosclerosis [27], glomerulonephritis [28]
and diabetic nephropathy [29] TXA2 signals through
the TXA2 receptor, or TP, a G-protein coupled
recep-tor primarily coupled to phospholipase (PL) Cb
activa-tion [23] In humans, but not in other nonprimates,
TXA2 signals through two TP isoforms, namely TPa
and TPb, that arise through differential splicing and differ exclusively within their carboxyl terminal domains [30,31] Whilst the biologic significance for the existence of two TP isoforms in humans has not been fully elucidated, there is extensive evidence that they may be physiologically distinct thereby greatly adding to the complexity of TXA2 signalling in humans [32] For example, while both TPa and TPb identically couple to PLCb, they differentially regulate other secondary effectors including adenylyl cyclase and tissue transglutaminase [33,34]; they undergo dif-ferential homologous and heterologous desensitization [35–38] and are also differentially expressed in a range
of cell⁄ tissue types [39] Moreover, recent studies have established that TPa and TPb expression are actually transcriptionally regulated by distinct promoters within the single human TP gene located on chromosome 19 [40,41] 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 [40]
In view of the observations that PPARc activation
is associated with suppression of a number of key dis-ease-associated genes within the vasculature including that of the rat TP [22] coupled to the fact that there is
no significant sequence homology between the rat TP promoter and human Prm1 or Prm3 sequences [22,41], the aim of the current study was to investigate the effect of 15d-PGJ2 on expression of the human TP gene within the platelet progenitor megakaryocytic human erythroleukemic (HEL) 92.1.7 cell line More-over, in view of the existence of two independently expressed TP isoforms in humans, it was also sought
to investigate whether 15d-PGJ2 regulates TPa and⁄ or TPb expression in an isoform-dependent manner It was established that 15d-PGJ2 suppresses both Prm3-directed luciferase reporter gene expression and TPb mRNA expression without affecting Prm1-directed gene expression or TPa mRNA levels in HEL cells Moreover, we describe a novel mechanism of 15d-PGJ2⁄ PPARc-mediated suppression of gene expression involving the direct binding of the activated PPARc– RXR heterodimer to a PPARc response element (PPRE)–RXR response element within Prm3 resulting
in selective down-regulation of TPb mRNA expression These data provide further evidence for the role of PPARc in the regulation of TP gene expression within the vasculature and point to further critical differences into the modes of regulation of TPa and TPb in humans Moreover, in view of the critical link between the enhanced risk of cardiovascular disease in patients with diabetes mellitus and in animal models of diabetes mellitus associated with increased synthesis and TXA2
Trang 3action [42–45], these data point to an added benefit to
the current use of PPARc agonists in the treatment of
cardiovascular disease-associated diabetes
Results
Analysis of the effect of 15d-PGJ2on Prm1-,
Prm2- and Prm3-directed gene expression
Previous studies have identified the presence of three
promoter regions, designated Prm1, Prm2 and Prm3,
within the single human TXA2 receptor gene located
on chromosome 19p13.3 [40,41] A schematic of the human TP gene highlighting the positions of Prm1, Prm2 and Prm3 relative to its translational start site (ATG, designated +1) is presented in Fig 1 Initially the effect of the endogenous PPARc ligand 15d-PGJ2
on Prm1-, Prm2- and Prm3-directed reporter gene expression in transfected human erythroleukemic 92.1.7 (HEL) cells and, as a negative control, human embryonic kidney (HEK) 293 cells was investigated Consistent with previous reports [39], Prm1, Prm2 and
A
B
Fig 1 Effect of 15d-PGJ 2 on Prm1, Prm2 and Prm3-directed luciferase expression (A and B) A schematic of the human TXA 2 receptor (TP) genomic region spanning nucleotides )8500 to +786 encoding Prm1, Prm2 and Prm3 in addition to exon (E) 1, E1b and E2 are illustrated above each panel where nucleotide +1 corresponds to the translational start site (ATG) and nucleotides 5¢ of that site are given a –designa-tion Recombinant pGL3Basic plasmids encoding Prm1 ( )8500 to )5895), Prm2 ()3308 to )1979), Prm3 ()1394 to +1) or, as a control, pGL2Control were transiently cotransfected along with pRL-TK into HEL 92.1.7 (A) and HEK 293 (B) cells Thirty-six h post-transfection, cells were incubated with either 15d-PGJ2(10 l M ) or the vehicle [0.1% (v ⁄ v) dimethylsulfoxide] for 16 h Mean firefly relative to renilla luciferase activity is expressed in arbitrary relative luciferase units (RLU ± SEM; n ¼ 5) The asterisk (*) indicates that Prm3-directed luciferase activity
in HEL cells was significantly reduced in 15d-PGJ 2 treated cells relative to vehicle treated cells; *P < 0.05.
Trang 4Prm3 each directed luciferase activity in both HEL
and HEK 293 cells, albeit at significantly different
levels relative to each other (Fig 1) Pre-incubation of
HEL cells with 10 lm 15d-PGJ2 for 16 h resulted in a
1.5-fold reduction in Prm3-directed luciferase
expres-sion (P < 0.05) but had no significant effect on either
Prm1- or Prm2-directed luciferase expression (Fig 1A)
Moreover, 15d-PGJ2 suppressed Prm3-directed
luci-ferase expression in a concentration- (Fig 2C) and
time-dependent (Fig 2D) manner but had no
signifi-cant effect on Prm1- (Fig 2A,B) or Prm2-directed
(data not shown) reporter gene expression regardless
of the concentration (0–40 lm) or incubation time
(0–24 h) In control HEK 293 cells, 15d-PGJ2 had no
significant effect on either Prm1-, Prm2- or
Prm3-directed luciferase expression (Fig 1B)
Effect of 15d-PGJ2on TPa and TPb mRNA and
protein expression in HEL cells
As previously stated, the TPa and TPb isoforms of the
human TXA2 receptor (TP) are under the
transcrip-tional control of distinct promoters, namely Prm1 and
Prm3, respectively [40] Hence, in view of the finding
herein that 15d-PGJ2 significantly suppressed Prm3-but not Prm1-directed reporter gene expression the effect of 15d-PGJ2 on TPa and TPb mRNA expression
in HEL cells was investigated Consistent with previ-ous reports [39], RT-PCR followed by Southern blot analysis confirmed expression of TPa, TPb and gly-ceraldehyde 3¢phosphate dehydrogenase (GAPDH) mRNA in HEL cells (Fig 3A,B, lanes 1–3, respect-ively) with an approximately twofold higher level of TPa relative to TPb mRNA expression Pre-incubation with 15d-PGJ2had no significant effect on the levels of either TPa or GAPDH mRNA expression in HEL cells relative to the vehicle-treated cells (Fig 3A–C) In contrast, preincubation with 15d-PGJ2 resulted in a 1.62-fold reduction in TPb mRNA expression in HEL cells compared to vehicle-treated cells (Fig 3A–C) These data correlate well with the observed effect of 15d-PGJ2 on Prm3 activity and provides further evi-dence for a distinct role for Prm3 in the regulation of TPb expression
To assess the affect of 15d-PGJ2on the overall level
of TP protein expression and function, HEL cells were preincubated with 10 lm 15d-PGJ2 for 24 and 48 h and its affect on TP-radioligand binding and on
Fig 2 Concentration- and time-dependent effect of 15d-PGJ2on Prm1 and Prm3-directed luciferase expression HEL 92.1.7 cells were tran-siently cotransfected with pRL-TK along with pGL3b:Prm1 (A and B) or pGL3b:Prm3 (C and D) Thirty-six hours post-transfection, cells were incubated for 16 h with 0–40 l M 15d-PGJ 2 (A and C) or for 0–24 h with 10 l M 15d-PGJ 2 (B and D) Mean firefly relative to renilla luciferase activity is expressed in arbitrary relative luciferase units (RLU ± SEM; n ¼ 5) The asterisks (*) indicate the concentration or time that Prm3-directed luciferase activity was significantly reduced in 15d-PGJ2treated HEL cells relative to vehicle treated cells; ***P £ 0.001.
Trang 5TP-mediated intracellular calcium ([Ca2+]i)
mobiliza-tion, in response to the selective TXA2 mimetic
U46619, was investigated In addition, as a control, we
also investigated the effect of 10 lm 15d-PGJ2 on
signalling by an unrelated receptor, namely the EP1
subtype of the prostaglandin (PG)E2 receptor
Pre-incubation of HEL cells with 15d-PGJ2 for 24 h
signi-ficantly reduced the overall level of TP expression from
58.8 ± 8.2 fmol [3H]SQ29,548Æmg cell protein)1 (n¼
8) to 17.0 ± 4.3 fmol [3H]SQ29,548Æmg cell protein)1
(n¼ 11; P ¼ 0.0001) Moreover, 15d-PGJ2
preincuba-tion significantly reduced the overall level of
U46619-mediated [Ca2+]i mobilization from 23.7 ± 4.2 nm
[Ca2+]i to 8 0 ± 0.82 nm (P¼ 0.01), as illustrated in
Fig 3D,E Similar data was obtained following 48 h
incubation with 15d-PGJ2 (data not shown) On the
other hand, 15d-PGJ2 (10 lm, 48 h) did not
signifi-cantly affect [Ca2+]i mobilization by the control EP1
receptor in response to its agonist 17 phenyl trinor PGE2 (compare D[Ca2+]i¼ 150.9 ± 21.9 nm, n ¼ 3
vs D[Ca2+]i¼ 176.0 ± 9.8 nm, n ¼ 5 in vehicle- and 15d-PGJ2-treated cells, respectively; P¼ 0.255)
Examination of the role of PPARc2, PPARc3 and RXRa in 15d-PGJ2mediated inhibition of Prm3 activity
PPARc⁄ retinoic X receptor (RXR) heterodimerization has been shown to be an important step in mediating the effect of 15d-PGJ2 in a number of cell types Hence, to investigate whether PPARc⁄ RXR hetero-dimers might be involved in regulating Prm3, the effect
of expression of either PPARc2 (Fig 4A) or PPARc3 (Fig 4B) alone or coexpression of PPARc2⁄ PPARc3 along with RXRa on 15d-PGJ2 regulation of Prm3-directed reporter gene expression was investigated
E D
Fig 3 Effect of 15d-PGJ2on TPa and TPb mRNA expression and TP-mediated intracellular signalling (A and B) RT-PCR analysis of RNA iso-lated from HEL cells preincubated for 16 h with the vehicle 0.1% (v ⁄ v) dimethylsulfoxide (lanes 1–3) or 10 l M 15d-PGJ2(lanes 4–6) using primers to amplify TPa (lanes 1 and 4), TPb (lanes 2 and 5) and GAPDH (lanes 3 and 6) mRNA sequences (B) Southern blot analysis of the RT-PCR products (lanes 1–6) coscreened using 32 P-radiolabelled oligonucleotide probes specific for TPa ⁄ TPb mRNA and GAPDH mRNA sequences (C) Mean levels of TPa, TPb and GAPDH mRNA expression in 15d-PGJ2-treated HEL cells were represented as a percentage of their expression in vehicle-treated cells (Relative expression,% ± SEM, n ¼ 4) The asterisks (*) indicate that the level of TPb mRNA expres-sion in HEL cells was significantly reduced in 15d-PGJ2treated cells relative to vehicle treated cells; **P £ 0.02 (D and E) HEL 92.1.3 cells were preincubated for 24 h with the vehicle 0.1% dimethylsulfoxide (D) or with 10 l M 15d-PGJ2(E) prior to stimulation with 1 l M U46619, added at the times indicated by the arrows Data presented are representative profiles from at least four independent experiments and are plotted as changes in intracellular Ca 2+ mobilization (D[Ca 2+ ]i, n M ) as a function of time (second, s) Actual mean changes in U46619-medi-ated [Ca 2+ ]imobilization (n M ± SEM) were as follows: D[Ca 2+ ]i¼ 23.7 ± 4.2 n M for vehicle treated cells (n ¼ 6); D[Ca 2+ ]i¼ 8 0 ± 0.82 n M
for 10 l M 15d-PGJ 2 treated cells (n ¼ 4).
Trang 6(Fig 4) Consistent with previous data, preincubation
of HEL 92.1.7 cells with 15d-PGJ2 resulted in a
1.5-fold suppression of Prm3 activity (Fig 4) Using
anova one way comparisons, it was established that relative to cells transfected with the empty vector, expression of either PPARc2 or RXRa alone (Fig 4A), or PPARc3 or RXRa alone (Fig 4B) had
no significant effect on the ability of 15d-PGJ2to sup-press Prm3-activity (P¼ 0.2196 and p ¼ 0.2235; Fig 4, respectively) However, coexpression of PPARc2 with RXRa significantly augmented 15d-PGJ2-suppression
of Prm3 activity relative to cells cotransfected with the vector alone or vector encoding PPARc2 (P < 0.0014)
or encoding RXRa (P < 0.0014) yielding a 2.2-fold reduction in luciferase expression relative to vehicle treated cells (Fig 4A) Similarly, coexpression of PPARc3 with RXRa augmented 15d-PGJ2-suppression
of Prm3 activity relative to cells cotransfected with the vector alone or vector encoding PPARc2 (P < 0.0009)
or encoding RXRa (P < 0.0005) yielding a 2.4-fold reduction in luciferase expression relative to vehicle treated cells (Fig 4B) Western blot analysis confirmed over-expression of PPARc and RXRa in transfected HEL cells (data no shown) Hence, these data are suggestive that both PPARc and RXRa transcription factors may be required to mediate 15d-PGJ2 -inhibi-tion of Prm3-directed gene expression
Localization of the site of action of 15d-PGJ2 within Prm3 by 5¢ deletion analysis
Thereafter, 5¢ deletion analysis of Prm3 ()1394 to +1) was used to localize the key regulatory domains direct-ing 15d-PGJ2-inhibition of Prm3 within HEL cells Consistent with previous reports [46], 5¢ deletion of Prm3 to generate a )404 subfragment did not signifi-cantly affect the level of basal (nonstimulated) luci-ferase activity in HEL cells (Fig 5A) However, 5¢ deletion of Prm3 from a )404 to a )320 bp fragment yielded an approximately twofold increase in basal lu-ciferase activity while 5¢ deletion of the )320 bp to a )154 bp fragment did not yield a further alteration
in basal luciferase expression These data suggest that the )404 to )320 region contains negative regulatory element(s), the removal of which results in increased basal Prm3 activity whilst nucleotides located between )320 and )154 do not significantly affect basal Prm3 activity [46]
Pre-incubation with 15d-PGJ2 resulted in approxi-mately 1.4–1.6-fold reductions in luciferase activity directed by Prm3 (P < 0.05) and the )404 (P < 0.05) and )320 (P < 0.01) subfragments (Fig 5A) How-ever, 15d-PGJ2 did not significantly affect luciferase activity directed by the )154 subfragment of Prm3 (Fig 5A) Hence, these data indicate that the )320 bp subfragment contains 15d-PGJ2 regulatory domain(s)
A
B
Fig 4 The effect of co-expression of RXRa with either hPPARc2
or hPPARc3 on 15d-PGJ2-mediated inhibition of Prm3-directed
luci-ferase expression HEL 92.1.7 cells were transiently cotransfected
with pGL3b:Prm3 (1 lg) together pSG5-hPPARc2 plus pSG5 (1 lg
each), pSG5-mRXRa plus pSG5 (1 lg each), or pSG5-hPPARc2 plus
pSG5-mRXRa (1 lg each) in the presence of 200 ng pRL-TK (A).
Alternatively, HEL cells were transiently cotransfected with
pGL3b:Prm3 (1 lg) together pcDNA3-hPPARc3 plus pcDNA3 (1 lg
each), pSG5-mRXRa plus pSG5 (1 lg each), or pcDNA3-hPPARc3
plus pSG5-mRXRa (1 lg each) in the presence of 200 ng pRL-TK
(B) Thirty-six hours post-transfection, cells were incubated for 16 h
with 10 l M 15d-PGJ2(Panels A and B) Mean firefly relative to
renil-la luciferase activity is expressed in arbitrary rerenil-lative luciferase units
(RLU ± SEM; n ¼ 5) The asterisks (*) indicate that Prm3-directed
luciferase activity in HEL cells was significantly reduced in
15d-PGJ 2 treated cells relative to vehicle treated cells; *P £ 0.05,
**P £ 0.02, ***P £ 0.001, ****P £ 0.0001, respectively ANOVA one
way analysis was carried out to determine differences due to
over-expression of plasmids encoding hPPARc2 plus RXRa, or hPPARc2
plus RXRa relative to their expression alone or along with the
empty vector.
Trang 7and that the site of action of 15d-PGJ2may be located
between )320 and )154 Consistent with the former,
the effect of 15d-PGJ2on luciferase expression directed
by the )320 bp subfragment was concentration- and
time-dependent (Fig 5B,C)
Bioinformatic analysis of Prm3, using the
matin-spectorTM programme [47], for transcription factor
elements between )320 and )154 identified the
pres-ence of 4 putative retinoic acid X receptor (RXR)
half sites centred at )300, )268, )175 and )148 and
two putative PPARc half sites at )182 and )159,
respectively (Fig 6A) Hence, further 5¢ deletion
ana-lysis was carried out to investigate if any of the latter
sites might be involved in mediating 15d-PGJ2
-inhibi-tion of Prm3-directed gene expression Progressive 5¢
deletion of Prm3 from the )320 bp fragment to yield
)276, )229 and )192 subfragments did not affect
their ability to direct basal luciferase expression in
HEL cells (Fig 6A) While, consistent with previous
data, 15d-PGJ2 did not significantly affect luciferase
activity directed by the )154 subfragment of Prm3
(Figs 5A and 6A), it resulted in approximately 1.6 fold reductions in luciferase activity directed by the )276 (P < 0.0009), )229 (P < 0.006) and )192 (P < 0.007) subfragments (Fig 6A) These data indi-cate that the RXR half sites centred at )300 (RXR I) and )268 (RXR II) do not play a role in 15d-PGJ2-inhibition of Prm3 and that the functional regu-latory element(s) may be located between nucleotides )192 and )154 within Prm3 or the surrounding sequences
To investigate whether PPARc⁄ RXR regulation of Prm3 is mediated by direct nuclear factor binding to cis-acting elements within the )192 to )154 region of Prm3, electromobility shift assays (EMSAs) were car-ried out using a radiolabelled double stranded (ds) DNA probe spanning nucleotides )198 to )150 (PPARc⁄ RXR probe A; Kin242) and nuclear extracts prepared from either vehicle and 15d-PGJ2 treated HEL 92.1.7 cells A diffuse protein:DNA complex was observed following incubation of the c32P-radiolabelled double stranded (ds) PPARc⁄ RXR probe A with either
A
Fig 5 Localization of the site of action of 15d-PGJ2within Prm3 (A) Recombinant pGL3 basic plasmids encoding Prm3 ( )1394 to +1), Prm3a ( )404 to +1), Prm3ab ()320 to +1) and Prm3aa ()154 to +1) were transiently cotransfected along with pRL-TK into HEL 92.1.7 cells Thirty-six h post-transfection, cells were incubated with either 15d-PGJ 2 (10 l M ) or the vehicle (0.1% dimethylsulfoxide) for 16 h Mean fire-fly relative to renilla luciferase activity is expressed in arbitrary relative luciferase units (RLU ± SEM; n ¼ 5) The asterisks (*) indicate that luciferase expression in HEL cells was significantly reduced in 15d-PGJ2treated cells relative to vehicle treated cells; *P £ 0.05, **P £ 0.02, respectively (B and C) HEL 92.1.7 cells were transiently cotransfected with pRL-TK along with pGL3b:Prm3ab Thirty-six hours post-transfec-tion, cells were incubated for 16 h with 0–40 l M 15d-PGJ 2 (B) or for 0–24 h with 10 l M 15d-PGJ 2 (C) Mean firefly relative to renilla luci-ferase activity is expressed in arbitrary relative luciluci-ferase units (RLU ± SEM; n ¼ 5).
Trang 8nuclear extract prepared from vehicle (Fig 6B; lane 2)
or 15d-PGJ2-treated HEL cells (Fig 6B; lane 3) The
formation of the latter nuclear factor-DNA complex
was efficiently competed by an excess of the
corres-ponding nonlabelled ds PPARc⁄ RXR oligonucleotide
using nuclear extracts prepared from both vehicles or
15d-PGJ2 treated-HEL cells (Fig 6B; lanes 4 and 5,
respectively) The specificity of nuclear factor binding
to the latter PPARc⁄ RXR probe A (spanning nucleo-tides )198 to )150) was also verified by the failure of excess ds oligonucleotides based on consensus AP-1, Oct )1 and Sp1 elements to effectively inhibit the formation of the nuclear factor-DNA complex using nuclear extract prepared from either vehicle (Fig 6B;
A
B
Fig 6 Sub-localization of the site of action of 15d-PGJ2within Prm3 (A) A schematic of the TP genomic region encoding Prm3 ( )1394 to +1) in addition to exon (E) 2 spanning nucleotides )1394 to +786 are illustrated In addition, the relative positions of a two putative PPARc-responsive elements (PPREs), designated PPARc(a) and PPARc(b), respectively, and four putative retinoic acid X receptor (RXR) PPARc-responsive elements, designated RXR I–RXR IV, respectively, are also illustrated Recombinant pGL2Basic plasmids encoding Prm3ab ( )320 to +1), Prm3aba ( )276 to +1), Prm3abb ()229 to +1), Prm3abc ()192 to +1) and Prm3aa ()154 to +1) were transiently cotransfected along with pRL-TK into HEL 92.1.7 cells Thirty-six hours post-transfection, cells were incubated with either 15d-PGJ 2 (10 l M ) or the vehicle [0.1% (v ⁄ v) dimethylsulfoxide] for 16 h Mean firefly relative to renilla luciferase activity are expressed in arbitrary relative luciferase units (RLU ± SEM;
n ¼ 5) The asterisks (*) indicate that luciferase expression in HEL cells was significantly reduced in 15d-PGJ 2 treated cells relative to vehicle treated cells, where *P £ 0.05, **P £ 0.02, ***P £ 0.001, ****P £ 0.0001, respectively (B) A c 32
P-radiolabelled ds DNA probe correspond-ing to nucleotides (nucleotides) )198 to )150 of Prm3 (PPARc ⁄ RXR probe A; Kin242 and its complement) was used in EMSAs using nuclear extracts prepared from HEL 92.1.7 cells preincubated with either 15d-PGJ2(10 l M ) or the vehicle (0.1% dimethylsulfoxide) for 16 h, as out-lined in Experimental procedures Lane 1, PPARc ⁄ RXR probe A incubated without nuclear extract; lanes 2 and 3, PPARc ⁄ RXR probe A incu-bated with nuclear extract from vehicle- and 15d-PGJ2-treated HEL cells, respectively; lanes 4 and 5, PPARc ⁄ RXR probe A incubated with nuclear extract from vehicle- and 15d-PGJ2-treated HEL cells, respectively, in the presence of excess nonlabelled specific ds competitor oligonucleotide (Kin242 and its complement); lanes 6 and 7, PPARc ⁄ RXR probe A incubated with nuclear extract from vehicle- and 15d-PGJ2-treated HEL cells, respectively, in the presence of excess nonlabelled ds noncompetitor oligonucleotide (Kin189, corresponding to nucleotides )32 to )10 of Prm3 containing an AP1 consensus element); lanes 8 and 9, PPARc ⁄ RXR probe A incubated with nuclear extract from vehicle- and 15d-PGJ 2 -treated HEL cells, respectively, in the presence of excess nonlabelled ds noncompetitor oligonucleotide (Kin195, corresponding to nucleotides )115 to )92 of Prm3 containing an Oct1 ⁄ 2 consensus element); lanes 10 and 11, PPARc ⁄ RXR probe A incuba-ted with nuclear extract from vehicle- and 15d-PGJ2-treated HEL cells, respectively, in the presence of excess nonlabelled ds noncompetitor oligonucleotide (Sp1 consensus element, Promega) DNAÆprotein complexes were subject to polyacrylamide gel electrophoresis followed by autoradiography, as outlined in Experimental procedures.
Trang 9lanes 6, 8 and 10, respectively) or 15d-PGJ2 treated
HEL cells (Fig 6B; lane 7, 9 and 11, respectively
Taken together these data demonstrate the specific
binding of nuclear factors within HEL cells to the)198
to )150 region of Prm3 and also show that nuclear
factor–DNA complex formation is independent of
15d-PGJ2stimulation
Identification and characterization of a PPARc
response element within Prm3
Detailed analysis of the )192 ⁄ )154 region of Prm3
reveals the presence of two putative PPAR response
elements (PPREs) The first putative PPRE is
com-posed of the PPARc half site centred at )182
[PPARc(a)] and a RXR half site at )175 (RXR III)
while the second corresponds to a PPARc half site
centred at )159 [PPARc(b)] and a RXR IV half site
at )148 Therefore a combination of site-directed and
deletion mutagenesis was employed to investigate the
functional importance of these putative elements in
directing 15d-PGJ2 regulation of Prm3 activity
Ini-tially it was confirmed that neither site-directed
muta-genesis nor deletion of nucleotides between )320 and
)154 significantly affected basal luciferase gene
expres-sion in HEL cells (Fig 7A) Disruption of the PPARc
(a) half site centred at )182 by site-directed
mutagen-esis (mutation of TTGAGC to TTAGGC, mutated
nucleotides in bold) within the )320 bp subfragment
of Prm3 did not significantly affect the level of
15d-PGJ2-inhibition of Prm3 activity (Fig 7A)
More-over, progressive 5¢deletion of nucleotides surrounding
either the PPARc (a) and RXR III half sites to yield
the )186 and )175 subfragments did not affect
15d-PGJ2-suppression of luciferase reporter gene
expression yielding between 1.5- and 1.7-fold
reduc-tions of luciferase expression (Fig 7A) Consistent
with the latter, complete deletion of the PPARc (a)
and RXR III half sites while retaining the PPARc (b)
and RXR IV half sites generated the )170
subfrag-ment that was fully responsive to 15d-PGJ2
-suppres-sion of luciferase expres-suppres-sion On the other hand, either
deletion of the latter PPARc (b) and RXR IV half
sites, such as within the )154 subfragment, or
dis-ruption of the RXR IV half site centred at )148 by
site-directed mutagenesis (sequence AGTTCA to
ATTTTA) within the )320 bp subfragment abolished
15d-PGJ2-suppression of Prm3 activity (Fig 7A)
Thereafter, EMSAs were carried out to investigate
direct nuclear factor binding to the latter cis-acting
PPARc⁄ RXR response elements within the )161 to
)148 region of Prm3 using a radiolabelled ds DNA
probe spanning )168 to )141 (PPARc ⁄ RXR probe B;
Kin281) and nuclear extracts prepared from either vehicle- and 15d-PGJ2-treated HEL 92.1.7 cells A dif-fuse protein:DNA complex was observed following incubation of the c32P-radiolabelled PPARc⁄ RXR probe B with either nuclear extracts prepared from vehicle- (Fig 7B; lane 2) or 15d-PGJ2-treated (Fig 7B; lane 6) HEL cells Nuclear factor-DNA complex formation using nuclear extracts prepared from both vehicle-treated (Fig 7B; lane 3) or 15d-PGJ2 treated-(Fig 7B; lane 7) HEL cells was competed by a 50-fold excess of the corresponding nonlabelled ds PPARc⁄ RXR oligonucleotide, respectively, and by a ds oligo-nucleotide containing a consensus PPARc response element derived from the acyl-CoA oxidase gene (Fig 7B; lanes 4 and 8) On the other hand, nuclear factor binding to the PPARc⁄ RXR probe B (spanning nucleotides )168 to )141) was not competed by an excess of ds oligonucleotides in which both the PPARc(b) plus RXR IV sites were mutated, using nuclear extract prepared from either vehicle- (Fig 7B; lane 5) or 15d-PGJ2-treated (Fig 7B; lane 9) HEL cells, respectively Taken together these data demon-strate that either mutation or deletion of the PPARc (a)⁄ RXR IV half sites centred at )159 and )148, respectively, abolishes 15d-PGJ2 –suppression of Prm3 directed gene expression Moreover, data from EMSAs have confirmed the specific binding of nuclear factors from HEL cells to the )168 to )141 region of Prm3 and, consistent with previous data (Fig 6B), confirm that nuclear factor-DNA complex formation is largely independent of 15d-PGJ2stimulation
Examination of PPARc⁄ RXRa interactions within the PPRE
To further investigate the identity and specificity of the DNA⁄ protein interactions within the PPRE centred at )159 and )148 of Prm3, we examined the ability of recombinant human (h) PPARc2 and⁄ or mouse (m) RXRa to directly bind to the latter cis-acting PPARc⁄ RXR response elements Hence, the PPARc2 and⁄ or RXRa transcription factors were transcribed and translated in vitro in a rabbit reticulocyte lysate cell free system and proteins of approximately 53–54 kDa corresponding to PPARc2 and RXRa were readily detectable by SDS⁄ PAGE following translation
in the presence of [35S]methionine (Fig 8A, lanes 2 and 3) Thereafter, the ability of the translated PPARc2 and⁄ or RXRa factors to bind to the c32 P-radiolabelled ds DNA probe spanning )168 to )141 (PPARc⁄ RXR probe B; Kin281) was investigated The recombinant PPARc2 or RXRa transcription factors exhibited weak, though detectable, binding to
Trang 10the radiolabelled PPARc⁄ RXR probe B (Fig 8B,
lanes 2 and 3, respectively) However, coincubation of
PPARc2 with RXRa significantly augmented
trans-cription factor binding to the PPARc⁄ RXR probe B
(Fig 8B, lane 4) indicating that both PPARc2 and
RXRa are required for efficient transcription factor
binding Moreover, PPARc:RXR complex binding to the latter probe was efficiently competed by an excess
of the corresponding nonlabelled ds PPARc⁄ RXR oligonucleotide (Fig 8B, lane 5) but was not competed
by an excess of ds oligonucleotides in which the PPARc(b) site, the RXR IV site or the PPARc(b) plus
A
B
Fig 7 Identification of the site of action 15d-PGJ2site of action within Prm3 by site-directed and deletion mutagenesis (A) Recombinant pGL3Basic plasmids encoding Prm3ab ( )320 to +1), Prm3abc ()192 to +1), Prm3abe ()186 to +1), Prm3abf ()175 to +1), Prm3abd ()170
to +1), Prm3aa ( )154 to +1) or the site-directed variants Prm3ab PPARc(a)
* or Prm3abRXRIV*, where the PPARc(a) and RXR IV half sites within Prm3ab were mutated, were transiently cotransfected along with pRL-TK into HEL 92.1.7 Thirty-six hours post-transfection, cells were incu-bated with either 15d-PGJ2(10 l M ) or the vehicle [0.1% (v ⁄ v) dimethylsulfoxide] for 16 h Mean firefly relative to renilla luciferase activity is expressed in arbitrary relative luciferase units (RLU ± SEM; n ¼ 5) The asterisks (*) indicate that luciferase expression in HEL cells was sig-nificantly reduced in 15d-PGJ 2 treated cells relative to vehicle treated cells; **P £ 0.02 (B) EMSAs were carried out using a c 32
P-radio-labelled ds DNA probe (Kin281 and its complement corresponding to nucleotides )168 to )141 of Prm3)and nuclear extract (4 lg) prepared from vehicle- (lanes 2–5) or 15d-PGJ 2 – (10 l M ; lanes 6–9) preincubated HEL 92.1.7 cells as described in the Experimental procedures sec-tion The c32P-radiolabelled probe was incubated: without nuclear extract (lane 1); with nuclear extract (lanes 2 and 6); with nuclear extract
in the presence of a 50-fold excess of: nonlabelled ds specific competitor oligonucleotide (Kin281 and its complement, lanes 3 and 7); non-labelled ds oligonucleotide containing a consensus acyl coA oxidase PPARc response element (Kin342 and its complement, lanes 4 and 8); nonlabelled ds noncompetitor oligonucleotide (Kin289 and its complement, corresponding to nucleotides )168 to )141 of Prm3 in which both the PPRE PPARc(b) * and RXR IV half-site half-sites were disrupted by site-directed mutagenesis (lanes 5 and 9) DNA ⁄ nuclear factor com-plexes were subject to polyacrylamide gel electroporesis followed by autoradiography, as outlined in Experimental procedures section.