In agreement with this conclusion, the overexpression of ectopic Cox-2 and PGI2 syn-thase PGIS resulted in massive PGI2 synthesis but did not activate the transcriptional activity of PPA
Trang 1by Raf signaling: failure of PGI2 to activate PPARb
Tanja Fauti1, Sabine Mu¨ller-Bru¨sselbach1, Mihaela Kreutzer1, Markus Rieck1, Wolfgang Meissner1, Ulf Rapp2, Horst Schweer3, Martin Ko¨mhoff3and Rolf Mu¨ller1
1 Institute of Molecular Biology and Tumor Research (IMT), Philipps-University, Marburg, Germany
2 MSZ, University of Wu¨rzburg, Germany
3 Department of Pediatrics, Philipps-University, Marburg, Germany
All prostaglandins [PGD2, PGE2, PGF2, PGI2
(prosta-cyclin), 15-deoxy-D12,14-PGJ2] and thromboxane A2are
synthesized from the common precursor PGH2, which
is generated by cyclooxygenase (Cox)-1 and Cox-2
from arachidonic acid (AA) (see [1] and references
therein) Cyclooxygenase-2 is regulated by
transcrip-tional and post-translatranscrip-tional mechanisms in response
to a plethora of stimuli, while Cox-1 expression is
con-stitutive Prostaglandin D2, PGE2, PGF2and PGI2can
trigger signaling cascades by interacting with G-protein
coupled membrane receptors Prostaglandin I2has also
been proposed as an agonist of the ‘peroxisome
prolif-erator activated receptor-b’ (PPARb; also known as
PPARoad) [2–5] Prostanoids play essential roles in many physiological processes, such as inflammation, pain, fever and platelet aggregation, but some compo-nents of the prostanoid signaling network also figure in tumorigenesis, including PGE2 and the PPARs While the former plays a predominant role in promoting tumor angiogenesis through upregulation of proangio-genic growth factors [6,7], PGI2and PPARb have been suggested to play a role in cell proliferation, differenti-ation and apoptosis [5,8–11]
A role for PPARb in tumorigenesis has been pro-posed for human colon cancer cells where the APC tumor suppressor gene product inhibits PPARb
Correspondence
R Mu¨ller, Institute of Molecular Biology and
Tumor Research (IMT), Philipps-University,
Emil-Mannkopff-Strasse 2, 35033 Marburg,
Germany
E-mail: rmueller@imt.uni-marburg.de
(Received 25 August 2005, revised 24
Octo-ber 2005, accepted 8 NovemOcto-ber 2005)
doi:10.1111/j.1742-4658.2005.05055.x
A role for the nuclear receptor peroxisome proliferator-activated recep-tor-b (PPARb) in oncogenesis has been suggested by a number of obser-vations but its precise role remains elusive Prostaglandin I2 (PGI2, prostacyclin), a major arachidonic acid (AA) derived cyclooxygenase (Cox) product, has been proposed as a PPARb agonist Here, we show that the 4-hydroxytamoxifen (4-OHT) mediated activation of a C-Raf-estrogen receptor fusion protein leads to the induction of both the PPARb and Cox-2genes, concomitant with a dramatic increase in PGI2 synthesis Sur-prisingly, however, 4-OHT failed to activate PPARb transcriptional activ-ity, indicating that PGI2is insufficient for PPARb activation In agreement with this conclusion, the overexpression of ectopic Cox-2 and PGI2 syn-thase (PGIS) resulted in massive PGI2 synthesis but did not activate the transcriptional activity of PPARb Conversely, inhibition of PGIS blocked PGI2 synthesis but did not affect the AA mediated activation of PPARb Our data obtained with four different cell types and different experimental strategies do not support the prevailing opinion that PGI2 plays a signifi-cant role in the regulation of PPARb
Abbreviations
AA, arachidonic acid; ASA, acetylsalicylic acid; Cox, cyclooxygenase (EC 1.44.99.1); cPGI, carbaprostacyclin; cPLA2, cytosolic phospholipase
A 2 (EC 3.1.1.5); DBD, DNA-binding domain; EPA, eicosapentaenoic acid; ERK, extracellular signal-regulated kinase; 6-k-PGF 1a , 6-keto-prostaglandin F 1a ; LBD, ligand-binding domain; mPGES, microsomal prostaglandin E 2 synthase (EC 5.3.99.3); 4-OHT, 4-hydroxytamoxifen; PGE2, prostaglandin E2; PGI2, prostaglandin I2(prostacyclin); PGIS, prostaglandin I2synthase (prostacyclin synthase; EC, 5.3.99.4); PPAR, peroxisome proliferator activated receptor; qPCR, quantitative PCR (real-time PCR).
Trang 2In line with a oncogenic function of the
pro-posed PPARb agonist PGI2 is the observation that in
human colon carcinoma PGI2 released by stromal
fibroblasts promotes the survival of the tumor cells [5],
and that apoptosis in mesenchymal renal medullary
interstitial cells is reduced by overexpression of PPARb
and further decreased upon administration of cPGI
[16] In apparent contrast to these observations is the
finding that the ectopic expression of prostaglandin I2
synthase (prostacyclin synthase; EC 5.3.99.4) inhibits
mouse lung tumorigenesis [17] and promotes apoptosis
[3] The interpretation of these studies is, however,
complicated because there is no definitive proof that
natural PGI2 is a PPARb agonist and other potential
PPARb ligands may exist [18] Moreover, other recent
studies support the hypothesis that PPARb inhibits cell
proliferation and promotes differentiation [11,19–22]
The Ras-Raf-ERK signaling pathway controls the
activity of numerous transcription factors that are
essential for the regulation of cell cycle progression
and cell survival [23,24] Different Ras-triggered
path-ways have also been implicated in the regulation of
genes involved in prostanoid synthesis and signaling,
such as group IVA cytosolic, calcium-dependent
phos-pholipase A2 (cPLA2), Cox-2 and PPARb, all of which
have been implicated in tumorigenesis (see [1] for
review) In the present study, we use a
4-hydroxy-tamoxifen (4-OHT) inducible system (N-BxB-ER cells)
[25] to show that multiple components of the
prosta-noid signaling network are targets of C-Raf signaling
pathways Triggering of C-Raf signaling resulted in a
dramatic Cox-2 and ERK-dependent increase in the
synthesis and release of PGE2 and PGI2 which was
mainly due to a strong transcriptional activation of the
Cox-2 gene (and to a lesser extent of PGIS and
mPGES-1) Under the same experimental conditions
expression of the PPARb gene was also augmented by
C-Raf signaling suggesting the presence of an
auto-crine or intraauto-crine PGI2–PPARb signaling mechanism
Surprisingly, however, the observed massive induction
of PGI2 synthesis did not lead to the transcriptional
activation of PPARb In agreement with this finding,
PPARb transcriptional activity was affected neither by
signaling
To investigate the effect of Raf signaling on prostanoid synthesis we made use of the 3T3-derived N-BxB-ER cells that express a 4-OHT inducible N-terminally truncated oncogenic Raf protein fused to the estrogen receptor [25] Cells were treated with 4-OHT for differ-ent times in the absence and presence of AA and the concentrations of prostanoids was measured in the cell culture supernatants by GC-MS Figure 1A shows a dramatic induction of both PGE2 and the stable PGI2 metabolite 6-k-PGF1a Induction of both prostanoids was detectable within 2 h of 4-OHT treatment and after 24 h reached values > 100-fold of the uninduced basal levels In the presence of AA (Fig 1A, bottom panel), synthesis of both prostanoids was greatly accel-erated and reached higher maximum levels, indicating that the level of endogenous AA generated by phos-pholipase A2 is rate-limiting even in the presence of activated Raf The induction of both prostanoids was almost completely blocked by the Cox-1⁄ 2 inhibitor acetylsalicylic acid (ASA) and the Cox-2 inhibitor SC-58125 (Fig 1B), pointing to a key role for Cox-2
in the induction of prostanoid synthesis by Raf In contrast to PGE2 and 6-k-PGF1a, no significant increase upon 4-OHT treatment was seen for throm-boxane B2 (TxB2), PGD2and PGF2a(Fig 1A)
Effects of c-Raf signaling on genes encoding prostanoid-synthesizing enzymes
We next analyzed by quantitative real-time PCR (qPCR) the effect of Raf activation on the expression
of genes that are relevant for the synthesis of PGE2 and PGI2 Figure 2A shows a strong induction of Cox-2 mRNA expression peaking at 240 min after 4-OHT addition, whereas no induction was seen for cPLA2 This finding was confirmed by northern blot-ting which showed a 10-fold induction of Cox-2 mRNA after 8 h (Fig 2B and C) Induction was
speci-fic for Cox-2, since no signispeci-ficant change in expression was seen with Cox-1 (Fig 2B) These observations explain the effects of exogenous AA and the Cox-2
Trang 3inhibitor on prostanoid synthesis in Fig 1 We also
observed a 4-OHT triggered increase in the levels of
PGIS mRNA (Fig 2A), but this was weak (1.3-fold)
and is therefore unlikely to contribute significantly
to the 4-OHT induced PGI2 synthesis Induction
mPGES-1 occurred relatively late after 4-OHT
treat-ment (4.7-fold 12 h post-treattreat-ment; Fig 2B) suggestive
of a secondary event Taken together, these results
indicate that Cox-2 is the key enzyme mediating the
dramatic induction of PGE2 and PGI2 synthesis after
Raf activation The strong induction of Cox-2
expres-sion was virtually abolished by both the ERK inhibitor
UO126 and the RNA polymerase inhibitor
actinomy-cin D (Fig 2C) indicating the Raf-triggered increase in
Cox-2 mRNA expression is due to an ERK-mediated
induction of Cox-2 transcription
Effects of C-Raf activation on PPARb expression
Activation of Raf not only led to a dramatic induction
of PGI2 synthesis as described above, but in the same
experimental setting also induced the expression of
the PPARb gene, which encodes the proposed nuclear
receptor for PGI2 As illustrated in Fig 3A, an
approximately threefold increase in the level of PPARb mRNA was seen within 8 h of 4-OHT treatment Induction was completely abolished by UO126 and actinomycin D (Fig 3B), suggesting an absolute requirement for ERK function and unimpaired tran-scription as already seen with Cox-2 above
Effect of Raf activation on the transcriptional activity of PPARb
The simultaneous upregulation of PGI2 synthesis and PPARb expression suggested the induction of an auto-crine⁄ intracrine signaling loop upon activation of Raf
We therefore investigated whether 4-OHT treatment of N-BxB-ER cells would lead to an activation of the transcriptional activity of PPARb To address this question we constructed a luciferase reporter construct consisting of seven LexA binding sites upstream of a TATA-Initiator (TATA-Inr) module without any addi-tional promoter elements This reporter plasmid on its own shows negligible luciferase activity and therefore allows for a highly sensitive detection of the transcrip-tional activity of a cotransfected transcriptranscrip-tional activa-tor harboring a LexA DNA binding domain (DBD)
A
B
Fig 1 Raf induces PGE 2 and PGI 2 synthesis (A) Prostanoid levels in the culture medium of RafER3T3 cells after treatment with 4-OHT for the indicated times in the absence ( )AA; upper panel) or presence of 20 l M arachidonic acid (+AA; bottom panel) 6-kPGF 1a is a stable metabolite
of the unstable PGI2that is used as a direct measure of PGI2synthesis (B) PGE2and 6-kPGF1alevels in the culture medium of RafER3T3 cells after treatment with 4-OHT in the presence of 100 l M ASA or 0.1 l M SC-58125 All data points represent the average of two measurements.
Trang 4In this system, the synthetic PPARb agonist
GW501516 gave a 30-fold induction with a fusion
protein consisting of the PPARb ligand binding
domain LBD and the LexA DBD (Fig 4) In contrast,
no induction was seen after treatment with 4-OHT in
spite of the massive synthesis of the presumptive PPARb agonist PGI2
We also analyzed the effect of 4-OHT on a PPRE-HSV-tk-pomoter-driven luciferase reporter construct [26] in N-BxB-ER cells, but again were unable to
Fig 2 Raf induces genes encoding enzymes with key functions in prostaglandin synthesis expression (A) RafER3T3 cells were treated with 4-OHT and mRNA levels of PLA2, Cox-2, mPGES-1 and PGIS were determined by qPCR Values represent the average of triplicates; error bars show the standard deviation Significant differences from untreated cells are indicated by an asterisk (paired t-test: P < 0.05) (B) Analy-sis of Cox-1 and Cox-2 expression in 4-OHT treated RafER3T3 cells by northern blotting Quantitative evaluation by PhosphoImaging showed that Cox-1 and PGIS mRNA levels did not fluctuate significantly during the time-course of the experiment For a quantification of Cox-2 expression see (C) PGES mRNA was induced 4.7-fold at 16 h (C) Analysis of Cox-2 induction in the presence of UO126 or actinomycin D Shown is the quantitative evaluation of a northern blot (PhosphorImager).
Trang 5detect any induction of transcriptional activity, both
in the presence and absence of a cotransfected
PPARb expression vector (data not shown) Likewise,
transcriptional activity was not increased by 4-OHT
in cells transfected with a RXRa expression vector
[26] and treated with the RXR agonist 9-cis retinoic
acid (data not shown) These findings strongly suggest
that the lack of PPARoad activation by Raf-induced
PGI2in the Lex system described above is not a
pecu-liarity of the experimental setup and is not due to a
rate-limiting level of the obligatory PPAR
heterodime-rization partner RXR These observations are
surpri-sing and indicate that, at least in the experimental
systems used, PGI2 may not act as agonist for
PPARb We therefore addressed this issue in further
detail below
Effect of PGI2synthesis on PPARb
Certain polyunsaturated fatty acids, such as AA and
eicosapentaenoic acid (EPA) have been described to
exert some agonistic effect on PPARb This effect was also observed in the LexA-DBD based luciferase assay
in the present study An approximately 3-fold stimula-tion of the transcripstimula-tional activity o PPARb was seen with 10 lm AA, whereas EPA had a modest effect only at a higher concentration of 30 lm (Fig 5A) Although the effect of AA was much weaker than that
of the synthetic PPARb agonists carbaprostacyclin (cPGI) and GW501516, it was consistently and repro-ducibly seen Treatment with AA resulted in an approximately sixfold increase in 6-k-PGF1ain the cul-ture medium, and this increase could be completely blocked by the PGIS inhibitor U51605 [27] (Fig 5B) U51605 also further reduced the low level of PGI2 synthesis in the absence of AA by about threefold (Fig 5B) Thus, the extent of PGI2 synthesis varied over an overall range of nearly 15-fold, but no correlation with PPARb transcriptional activity was observed (Fig 5B) Very similar results were obtained with the Cox inhibitors ASA and SC-58125 (data not shown)
Next, we overexpressed Cox-2 and⁄ or PGIS in HEK293 cells and monitored the effect on PGI2 synthesis and PPARb transcriptional activity As depicted in Fig 6, transfection of Cox-2 or PGIS expression vectors alone only had a marginal effect
on 6-k-PGF1a levels in the culture medium, but cotransfection of both vectors resulted in an almost 100-fold increased PGI2 synthesis, both in the
A
B
Fig 3 Raf induces PPARb gene expression (A) RafER3T3 cells
were treated with 4-OHT and PPARb mRNA levels were
deter-mined by qPCR Values represent the average of triplicates; error
bars show the standard deviation Significant differences from
untreated cells are indicated by an asterisk (paired t-test:
P < 0.005) (B) Analysis by northern blotting of PPARb induction in
the presence of UO126 or actinomycin D Shown is a quantitative
evaluation of a northern blot by PhosphorImaging.
Fig 4 Induction of PPARb transcriptional activity by AA is not dependent on PGI 2 synthesis (A) Stimulation of PPARb-LBD medi-ated transcriptional activity in NIH3T3 cells by polyunsaturmedi-ated fatty acids and the synthetic agonists carbaprostacyclin (cPGI) and GW01516 For experimental details see legend to Fig 6 Values represent the average of triplicates; error bars show the standard deviation Significant differences from untreated cells are indicated
by an asterisk (paired t-test: P ¼ 0.01) (B) Effect of the PGIS inhib-itor U51605 on 6-kPGF 1a accumulation in the cell culture superna-tant as a measure of PGI2synthesis (bar graph) and on PPARb-LBD mediated transcriptional activity (bottom row) PPAR activities are shown as the average of triplicates and standard deviation.
Trang 6absence and presence of exogenous AA But again,
this dramatic increase in PGI2 synthesis has no
inducing effect on the transcriptional activity of
PPARb
Discussion
In the present study, we used a 4-OHT inducible
sys-tem (N-BxB-ER cells) [25] to investigate which
com-ponents of the prostanoid signaling network are
targets of Raf signaling Our data show that C-Raf
activation leads to a dramatic ERK-dependent
induc-tion of Cox-2 transcripinduc-tion and to a modest increase
in mPGES-1 and PGIS mRNA expression (Fig 2)
Induction of Cox-2 by Ras-dependent signaling,
inclu-lack of cPLA2 induction, could be substantially enhanced by adding AA to the growth medium (Fig 1A) These data suggest that Raf oncogenes can contribute to tumorigenesis by augmenting the secre-tion of tumor growth promoting prostaglandins, such
as PGE2
In the same experimental system, we also observed a clear induction of PPARb transcription upon Raf acti-vation (Fig 3) PPARb has been shown to play a role
in diverse biological and biochemical processes, inclu-ding lipid metabolism, wound healing, placenta development and inflammation, but there is also con-siderable evidence suggesting a function for PPARb in oncogenesis [1,30] This assumption is mainly based on observations made with PPARb null mice where an altered growth behavior of intestinal polyps was observed [13–15] In spite of this central biological role for PPARb, the ligands that regulate its transcriptional activity in vivo remain largely obscure [31] Polyunsatu-rated fatty acids, such as EPA, undoubtedly have an agonistic effect, but this is weak and not isoform
speci-fic [32] PGI2, an AA derivative formed by the succes-sive action of Cox and PGIS, has been suggested as a PPARb specific agonist [2,33,34] Since 4-OHT induces both PGI2 synthesis and PPARb expression in
N-BxB-ER cells, we utilized this system to test whether Raf activation establishes an autocrine⁄ intracrine signaling loop consistent with the notion of PGI2 acting as PPARb agonist
Surprisingly, however, Raf activation did not lead
to any detectable increase in PPARb transcriptional activity This was seen with both a PPRE-tk reporter construct measuring total PPAR activity (data not shown) and with the b-isoform specific LexA-based system established in this study (Fig 4) The same observation was made when an expression vector for RxRa was cotransfected (data not shown), indicating that the lack of activation by PGI2 was not due to rate-limiting levels of the obligatory PPAR hetero-dimerization partner These results clearly suggested that PGI2 is not a PPARb agonist in this experimen-tal system (3T3 fibroblasts) We therefore performed several additional experiments that all confirm the
B
Fig 5 Overexpression of Cox-2 and PGIS does not induce PPARb
transcriptional activity HEK293 cells were transiently transfected
with expression vectors for Cox-2, PGIS or both Forty-eight hours
later, PPARb-LBD mediated transcriptional activity and 6-kPGF 1a
accumulation in the cell culture supernatant were determined For
experimental details see legend to Fig 6 Values represent the
average of triplicates; error bars show the standard deviation
Signi-ficant differences from untreated cells are indicated by an asterisk
(paired t-test: P ¼ 0.01).
Trang 7conclusion that PGI2 lacks agonistic activity for
PPARb in vivo
The ectopic expression of Cox-2 and PGIS in
HEK293 cells resulted in a dramatic induction of
PGI2 synthesis, but no increase in PPARb
transcrip-tional activity was observed (Fig 6) This is in
con-trast to a previously published observation made
with the human osteosarcoma cell line U2OS [2]
The reason for this discrepancy is not clear since we
were unable to reproduce the published results using
in the identical experimental set-up (U2OS cells and
Gal4-based reporter system; Tanja Fauti, unpublished
data) Prostacyclin-mediated regulation of PPARb
has also been claimed in another study using
HEK293 cells [3] In this study, a
PPRE-SV40-pro-moter-luciferase construct was used as the reporter,
raising the possibility that the observed
transcrip-tional activation was mediated by a different PPAR
or even by a PPAR-unrelated event, e.g through sti-mulation of the SV40 promoter and⁄ or via the PGI2 membrane receptor IP Unless supplemented by appropriate controls, these data therefore do not unequivocally show that PGI2 can invoke a direct transcriptional activation of PPARb
The addition of pure PGI2 (10 lm) to the culture medium of Chinese hamster ovary cells did not alter the transcriptional activity of PPARb to any significant extent (unpublished data) This is in agreement with two other previous studies First, U2OS cells trans-fected with a PPARb reporter did not show any response to the addition of PGI2 [35] In a second study, the same result was obtained with CV1 cells [36] Even though these results are in perfect agree-ment, they have to be considered with some caution since it is unclear how the biological instability of PGI2might affect these kinds of experiments
A weak agonistic effect was seen in 3T3 cells with exogenously supplied AA, but this increase in PPARb transcriptional activity was not influenced when PGI2 synthesis was blocked by inhibitors of PGIS or Cox (Fig 5) Taken together, our observations made with three different cell types and different experimental approaches provide no evidence that PGI2 acts as a PPARb agonist
Interestingly, in spite of the failure of PGI2 to acti-vate PPARb, the PGI2 analog cPGI showed strong agonistic properties in all four cell lines analyzed (Fig 5A; data not shown) It is possible that the subtle differences in the chemical structures of PGI2 and cPGI have an unexpected effect on the ability to inter-act with PPARb Alternatively, the half-life of PGI2 may be too short to allow for a sufficient concentra-tion of intact molecules in transcripconcentra-tion complexes in the nucleus While a very short interaction with the PGI2 membrane receptor (IP) may be sufficient for triggering a signal, a much greater stability may be required as a ligand of a nuclear receptor, where the presence of ligand may be necessary for an extended period of time
As expected, AA was able to activate PPARb activity, albeit at high concentrations (Fig 5A) Even though high local concentrations of specific lipids can be achieved in vivo, so that there may be no need for a high affinity ligand, it is unclear whether
AA itself can act as a PPARb agonist in vivo, or whether AA is converted to PPARb stimulatory metabolites by Cox-independent pathways Further-more, the existence of totally unrelated high affinity PPARb agonists cannot be excluded at present Fur-ther studies systematically addressing this are neces-sary to clarify this issue
Fig 6 Raf induction does not activate PPARb transcriptional
activ-ity PPARb-LBD mediated transcriptional activity was determined in
untreated and 4-OHT-treated RafER3T3 cells in the presence of
20 m M arachidonic acid Cells were transiently transfected with an
expression vector encoding the LexA-PPARb fusion protein
(Lex-PPARb-LBD) or the empty vector (pcDNA3.1) together with a
lexA-luciferase reporter plasmid (7 L-TATAi) Luciferase activity was
determined 48 h after transfection; 4-OHT treatment was for 24 h.
As a positive control, cells were also treated with 1 m M GW501516
for 24 h Values represent the average of triplicates; error bars
show the standard deviation Significant differences from untreated
cells are indicated by an asterisk (paired t-test: P < 0.003).
Trang 8land), SC-58125 (Calbiochem⁄ Merck Biosciences), U51605
(Cayman Chemical Company), UO126 (Promega,
Man-nheim, Germany)
Cell culture
NIH3T3, N-BxB-ER, HEK293 and CHO cells were
cul-tured in DMEM supplemented with 10% fetal bovine
serum, 100 UÆmL)1 penicillin and 100 lgÆmL)1
streptomy-cin Cells were maintained in culture at 37C with 5% CO2
in a humidified incubator
Plasmids
PGIS-pcDNA3.1 and COX2-pcDNA3.1 were obtained by
cloning the full-length human PGIS and Cox-2 cDNAs
into the expression vector pcDNA3.1(+) (Invitrogen,
Kahlsruhe, Germany) PPREx3-tk-pGL3 was constructed
by inserting the PPRE3-TK-fragment from PPRE3
-TK-LUC [36] (obtained from R.M Evans, La Jolla, CA,
USA) into the pGL3 basic luciferase vector (Promega)
7 L-TATAi has been described previously [37]
pcDNA3.1-LexA-PPARb-LBD was constructed as follows: the
PPARb-LBD fragment flanked by a 5¢-AseI- and a-3¢
BamHI-site was synthesized by PCR using
pCMX-mPPARb [36] as the template The LexA-DBD fragment,
including a Kozak and a nuclear localization sequence,
was amplified from vWFnLexA by RT–PCR The
remain-ing LexA-fragment was flanked with a 5¢ HindIII- and
a-3¢-NdeI-site The fragments were cut with NdeI and
AseI, ligated with T4 DNA ligase (Roche diagnostics),
treated with Taq DNA polymerase to add 3¢ oligo(A)
overhangs and cloned into pCRIITOPO (Invitrogen)
Finally the LexA-PPARb-LBD fragment was cut with
BamHI and HindIII and subcloned into pcDNA3.1zeo
(Invitrogen)
RNA isolation
RNA was isolated using the RNeasyTM kit from Qiagen
(Hilden, Germany) following the manufacturer’s protocol
Briefly 30 lg of tissue were homogenized in 600 lL RLT
buffer and 6 lL b-mercaptoethanol with a warring blender
(Ultra-Thurrax; IKA, Staufen, Germany) Qia shredders
Germany) with 10· NaCl ⁄ Cit and crosslinking under UV light (Stratalinker 2400, 254 nm, 1200 J m)2; Stratagene,
La Jolla, CA, USA) Hybridization to P32-labeled probes was performed as described [25] Signal intensites on mem-branes were quantitated by PhosphorImager (Fuji, Du¨ssel-dorf, Germany)
Reverse transcriptase PCR cDNA was synthesized using 1 lg of RNA, oligo dT primers and reverse transcriptase according to the manufacturer’s protocol (Roche Diagnostics, Mannheim, Germany) PCR was performed for 25 cycles at an annealing temperature of
55C (PPARb) respective 58 C (Cox-2) with Platinum Taq polymerase (Invitrogen) using primers obtained from MWG Biotech (Ebersberg, Germany) with the following sequences: Cox-2 forward, 5¢—CCTTCTCCAACCTCTCCTAC—3¢; Cox-2 reverse, 5¢—AGGGGGTGCCAGTGATAGAG—3¢; PPARb forward, 5¢—AAGAGGAGAAAGAGGAAG TGG—3¢; PPARb reverse, 5¢—ATTGAGGAAGAGGCTG CTGA—3¢; actin forward, 5¢—GATGATGATATCGCCGC GCTCGTCGTC—3¢; actin reverse, 5¢—GTGCCTCAGGG CAGCGGACCGCTCA—3¢
Quantitative PCR Quantitative PCR was performed in a Mx3000P Real-Time PCR system (Stratagene) for 45 cycles at an annealing tem-perature of 57C PCR reactions were carried out using the Absolute QPCR SYBR Green Mix (Abgene, Hamburg, Germany) and a primer concentration of 0.2 lm following the manufacturer’s instructions The following primers MWG Biotech were used: actin forward, 5¢—AGAGGGA AATCGTGCGTGAC—3¢; actin reverse, 5¢—CAATAGTG ATGACCTGGCCGT—3¢; PPARb forward, 5¢—GTCGCA CAACGCTATCC—3¢; PPARb reverse, 5¢—CTCCGGGCC TTCTTTTTGGTCA—3¢; cPLA2 forward, 5¢—CATAAGT TTACTGTTGTGGTTCTA—3¢; cPLA2 reverse, 5¢—AGT GTCTCGTTCGCTTCC—3¢; COX-2 forward, 5¢—CCATG GGTGTGAAGGGAAATAA—3¢; COX-2 reverse, 5¢—TTG AAAAACTGATGGGTGAAG—3¢; mPGES-1 forward, 5¢—GGTGGCCCAGGAAGGAGACAGC—3¢; reverse 5¢—TGGCCTTCATGGGTGGGTAATA—3¢
Trang 9Transient tansfections and luciferase assays
Transfections were performed with polyethylenimine (PEI,
average MW 25 000; Sigma-Aldrich) For each assay, 105
cells were transfected in DMEM plus 2% FCS with 5 lg of
plasmid DNA and 5 lL of a 1⁄ 1000 PEI dilution (adjusted
to pH 7.0) preincubated for 15 min in 100 lL NaCl⁄ Pifor
complex formation Four hours after transfection, the
med-ium was changed and cells were incubated in normal growth
medium for 24 h Luciferase assays were performed as
des-cribed [38] Values from three independent experiments were
combined to calculate averages and standard deviations
Sample preparation for prostanoids by
GC⁄ MS ⁄ MS-analysis
Samples were prepared as described [39] with minor
modifi-cations Briefly, cell culture supernatants were spiked with
10 ng of deuterated internal standards, and solvent was
removed The methoxime was obtained through reaction
with an O-methylhydroxylamine hydrochloride-acetate
buffer After acidification to pH 3.5, prostanoid derivatives
were extracted, and the pentafluorobenzylesters were
formed Samples were purified by TLC and two broad
zones with Rv 0.03–0.39 and 0.4–0.8 were eluted After
withdrawal of the organic layers, trimethylsilyl ethers were
prepared by reaction with
bis(trimethylsilyl)-trifluoroaceta-mide and thereafter subjected to GC⁄ MS ⁄ MS analysis
GC⁄ MS ⁄ MS analysis
A Finnigan (Thermo Electron Corp., Dreieich, Germany)
MAT TSQ700 GC⁄ MS ⁄ MS equipped with a Varian (Palo
Alto, CA, USA) 3400 gas chromatograph and a CTC
A200S autosampler was used [39]
Acknowledgements
We are grateful to Margitta Alt and Bernhard Watzer
for excellent technical assistance This work was
sup-ported by the Wihelm-Sander-Stiftung, the Dr Mildred
Scheel Stiftung and the Deutsche
Forschungsgemeinsc-haft (SFB-TR17)
References
1 Mu¨ller R (2004) Crosstalk of oncogenic and prostanoid
signaling pathways J Cancer Res Clin Oncol 130, 429–
444
2 Gupta RA, Tan J, Krause WF, Geraci MW, Willson
TM, Dey SK & DuBois RN (2000)
Prostacyclin-mediated activation of peroxisome proliferator-activated
receptor delta in colorectal cancer Proc Natl Acad Sci
USA 97, 13275–13280
3 Hatae T, Wada M, Yokoyama C, Shimonishi M & Tanabe T (2001) Prostacyclin-dependent apoptosis mediated by PPAR delta J Biol Chem 276, 46260–46267
4 Lim H & Dey SK (2002) A novel pathway of prostacy-clin signaling-hanging out with nuclear receptors Endo-crinology 143, 3207–3210
5 Cutler NS, Graves-Deal R, LaFleur BJ, Gao Z, Boman
BM, Whitehead RH, Terry E, Morrow JD & Coffey RJ (2003) Stromal production of prostacyclin confers an antiapoptotic effect to colonic epithelial cells Cancer Res 63, 1748–1751
6 Amano H, Hayashi I, Endo H, Kitasato H, Yamashina
S, Maruyama T, Kobayashi M, Satoh K, Narita M, Sugimoto Y, Murata T, Yoshimura H, Narumiya S & Majima M (2003) Host prostaglandin E(2)-EP3 signal-ing regulates tumor-associated angiogenesis and tumor growth J Exp Med 197, 221–232
7 Sonoshita M, Takaku K, Sasaki N, Sugimoto Y, Ush-ikubi F, Narumiya S, Oshima M & Taketo MM (2001) Acceleration of intestinal polyposis through prostaglan-din receptor EP2 in Apc (Delta 716) knockout mice Nat Med 7, 1048–1051
8 He TC, Chan TA, Vogelstein B & Kinzler KW (1999) PPARdelta is an APC-regulated target of nonsteroidal anti-inflammatory drugs Cell 99, 335–345
9 Di-Poi N, Tan NS, Michalik L, Wahli W & Desvergne
B (2002) Antiapoptotic role of PPARbeta in keratino-cytes via transcriptional control of the Akt1 signaling pathway Mol Cell 10, 721–733
10 Mao-Qiang M, Fowler AJ, Schmuth M, Lau P, Chang S, Brown BE, Moser AH, Michalik L, Des-vergne B, Wahli W, Li M, Metzger D, Chambon PH, Elias PM & Feingold KR (2004) Peroxisome-prolifera-tor-activated receptor (PPAR)-gamma activation sti-mulates keratinocyte differentiation J Invest Dermatol
123, 305–312
11 Kim DJ, Bility MT, Billin AN, Willson TM, Gonzalez
FJ & Peters JM (2005) PPARbeta/delta selectively induces differentiation and inhibits cell proliferation Cell Death Differdoi:10.1038/sj.cdd.4401713
12 Park BH, Vogelstein B & Kinzler KW (2001) Genetic disruption of PPARdelta decreases the tumorigenicity of human colon cancer cells Proc Natl Acad Sci USA 98, 2598–2603
13 Barak Y, Liao D, He W, Ong ES, Nelson MC, Olefsky
JM, Boland R & Evans RM (2002) Effects of peroxi-some proliferator-activated receptor delta on placenta-tion, adiposity, and colorectal cancer Proc Natl Acad Sci USA 99, 303–308
14 Harman FS, Nicol CJ, Marin HE, Ward JM, Gonzalez
FJ & Peters JM (2004) Peroxisome proliferator-acti-vated receptor-delta attenuates colon carcinogenesis Nat Med 10, 481–483
15 Gupta RA, Wang D, Katkuri S, Wang H, Dey SK & DuBois RN (2004) Activation of nuclear hormone
Trang 10murine lung cancer Cancer Res 62, 734–740.
18 Shaw N, Elholm M & Noy N (2003) Retinoic acid is
a high affinity selective ligand for PPAR-beta⁄ delta
J Biol Chem 278, 41589–41592
19 Westergaard M, Henningsen J, Svendsen ML,
Johan-sen C, JenJohan-sen UB, Schroder HD, Kratchmarova I,
Berge RK, Iversen L, Bolund L, Kragballe K &
Kristiansen K (2001) Modulation of keratinocyte gene
expression and differentiation by PPAR-selective
ligands and tetradecylthioacetic acid J Invest Dermatol
116, 702–712
20 Tan NS, Michalik L, Noy N, Yasmin R, Pacot C, Heim
M, Fluhmann B, Desvergne B & Wahli W (2001)
Criti-cal roles of PPAR beta⁄ delta in keratinocyte response
to inflammation Genes Dev 15, 3263–3277
21 Schmuth M, Haqq CM, Cairns WJ, Holder JC, Dorsam
S, Chang S, Lau P, Fowler AJ, Chuang G, Moser AH,
Brown BE, Mao-Qiang M, Uchida Y, Schoonjans K,
Auwerx J, Chambon P, Willson TM, Elias PM &
Fein-gold KR (2004) Peroxisome proliferator-activated
recep-tor (PPAR)-beta⁄ delta stimulates differentiation and
lipid accumulation in keratinocytes J Invest Dermatol
122, 971–983
22 Kim DJ, Murray IA, Burns AM, Gonzalez FJ, Perdew
GH & Peters JM (2005) Peroxisome
proliferator-acti-vated receptor-beta⁄ delta inhibits epidermal cell
prolifer-ation by down-regulprolifer-ation of kinase activity J Biol
Chem 280, 9519–9527
23 Kerkhoff E & Rapp UR (1998) Cell cycle targets of
Ras⁄ Raf signalling Oncogene 17, 1457–1462
24 Chang F, Steelman LS, Shelton JG, Lee JT, Navolanic
PM, Blalock WL, Franklin R & McCubrey JA (2003)
Regulation of cell cycle progression and apoptosis by
the Ras⁄ Raf ⁄ MEK ⁄ ERK pathway (Review) Int J
Oncol 22, 469–480
25 Kerkhoff E, Houben R, Loffler S, Troppmair J, Lee JE
& Rapp UR (1998) Regulation of c-myc expression by
Ras⁄ Raf signalling Oncogene 16, 211–216
26 Shi Y, Hon M & Evans RM (2002) The peroxisome
proliferator-activated receptor delta, an integrator of
transcriptional repression and nuclear receptor
signal-ing Proc Natl Acad Sci USA 99, 2613–2618
J Biol Chem 276, 42737–42743
30 Michalik L, Desvergne B & Wahli W (2004) Peroxi-some-proliferator-activated receptors and cancers: com-plex stories Nat Rev Cancer 4, 61–70
31 Bishop-Bailey D & Wray J (2003) Peroxisome prolifera-tor-activated receptors: a critical review on endogenous pathways for ligand generation Prostaglandins Other Lipid Mediat 71, 1–22
32 Forman BM, Tontonoz P, Chen J, Brun RP, Spiegel-man BM & Evans RM (1995) 15-Deoxy-delta
12, 14-prostaglandin J2 is a ligand for the adipocyte determination factor PPAR gamma Cell 83, 803–812
33 Lim H, Gupta RA, Ma WG, Paria BC, Moller DE, Morrow JD, DuBois RN, Trzaskos JM & Dey SK (1999) Cyclo-oxygenase-2-derived prostacyclin mediates embryo implantation in the mouse via PPARdelta Genes Dev 13, 1561–1574
34 Shao J, Sheng H & DuBois RN (2002) Peroxisome pro-liferator-activated receptors modulate K-Ras-mediated transformation of intestinal epithelial cells Cancer Res
62, 3282–3288
35 YuK, Bayona W, Kallen CB, Harding HP, Ravera CP, McMahon G, Brown M & Lazar MA (1995) Differential activation of peroxisome proliferator-activated receptors
by eicosanoids J Biol Chem 270, 23975–23983
36 Forman BM, Chen J & Evans RM (1997) Hypolipi-demic drugs, polyunsaturated fatty acids, and eicosa-noids are ligands for peroxisome proliferator-activated receptors alpha and delta Proc Natl Acad Sci USA 94, 4312–4317
37 Nettelbeck DM, Jerome V & Muller R (1999) A dual specificity promoter system combining cell cycle-regu-lated and tissue-specific transcriptional control Gene Ther 6, 1276–1281
38 Gehrke S, Jerome V & Muller R (2003) Chimeric tran-scriptional control units for improved liver-specific transgene expression Gene 322, 137–143
39 Schweer H, Watzer B & Seyberth HW (1994) Determi-nation of seven prostanoids in 1 ml of urine by gas chromatography-negative ion chemical ionization triple stage quadrupole mass spectrometry J Chromatogr 652, 221–227