DNA Binding-Independent Induction of I
B
Gene Transcription by PPAR
Philippe Delerive1,
Karolien De Bosscher,
Wim Vanden Berghe,
Jean-Charles Fruchart,
Guy Haegeman and
Bart Staels
Institut National de la Santé et de la Recherche Médicale U.545 (P.D., J.-C.F., B.S.), Département dAthérosclérose, Institut Pasteur de Lille, 59019 Lille, and Faculté de Pharmacie, Université de Lille II, 59000 Lille, France; Laboratory of Molecular Biology (K.D.B., W.V.B., G.H.), University of Gent and Vlaams Interuniversitair Instituut voor Biotechnologie, B-9000 Gent, Belgium
Address all correspondence and requests for reprints to: Dr. Philippe Delerive, Gene Regulation, Bone and Inflammation Research, Lilly Corporate Center, Building 98C-3 Drop Code 0434, Indianapolis, Indiana 46285. E-mail: delerive_philippe{at}lilly.com or Dr. Bart Staels, Institut National de la Santé et de la Recherche Médicale U.545, Institut Pasteur de Lille, 1 rue Calmette, BP 245, 59019 Lille, France. E-mail: Bart.Staels{at}pasteur-lille.fr.
 |
ABSTRACT
|
---|
PPARs are ligand-activated transcription factors that regulate energy homeostasis. In addition, PPARs furthermore control the inflammatory response by antagonizing the nuclear factor-
B (NF-
B) signaling pathway. We recently demonstrated that PPAR
activators increase I
B
mRNA and protein levels in human aortic smooth muscle cells. Here, we studied the molecular mechanisms by which PPAR
controls I
B
expression. Using transient transfection assays, it is demonstrated that PPAR
potentiates p65-stimulated I
B
transcription in a ligand-dependent manner. Site-directed mutagenesis experiments revealed that PPAR
activation of I
B
transcription requires the NF-
B and Sp1 sites within I
B
promoter. Chromatin immunoprecipitation assays demonstrate that PPAR
activation enhances the occupancy of the NF-
B response element in I
B
promoter in vivo. Overexpression of the oncoprotein E1A failed to inhibit PPAR
-mediated I
B
promoter induction, suggesting that cAMP response element binding protein-binding protein/p300 is not involved in this mechanism. By contrast, a dominant-negative form of VDR-interacting protein 205 (DRIP205) comprising its two LXXLL motifs completely abolished PPAR
ligand-mediated activation. Furthermore, cotransfection of increasing amounts of DRIP205 relieved this inhibition, suggesting that PPAR
requires DRIP205 to regulate I
B
promoter activity. By contrast, DRIP205 is not involved in PPAR
-mediated NF-
B transcriptional repression. Taken together, these data provide a molecular basis for PPAR
-mediated induction of I
B
and demonstrate, for the first time, that PPAR
may positively regulate gene transcription in the absence of functional PPAR response elements.
 |
INTRODUCTION
|
---|
PPARs ARE TRANSCRIPTION factors belonging to the nuclear receptor superfamily, which have been reported to regulate gene expression upon ligand binding (1). To date, three different PPAR subtypes have been identified: PPAR
, PPARß/
, and PPAR
. PPARs regulate gene expression by binding with RXR as heterodimeric partner to specific DNA sequences termed PPAR response elements (PPREs) (2). In addition to regulating gene transcription via PPREs, PPARs modulate gene transcription also by negatively interfering with other transcription factor pathways in a DNA binding-independent manner (3, 4). Among the three different PPARs, PPAR
activation has been reported to be involved in a number of cellular processes including lipid and lipoprotein metabolism (5), apoptosis (6), and the inflammatory response (3, 7, 8). Activated PPAR
represses cytokine-induced activation of a number of inflammatory genes such as vascular cell adhesion molecule-1, cyclooxygenase-2, and IL-6 by negatively interfering with nuclear factor-
B (NF-
B) transcriptional activity (8, 9). In addition, we recently demonstrated that PPAR
activators induce I
B
expression in human aortic smooth muscle cells and in human hepatocytes (10).
The NF-
B family of transcription factors plays a major role in the regulation of the expression of a number of genes implicated in cell growth, inflammation, and apoptosis (11, 12). This NF-
B/Rel family consists of five members, namely c-Rel, p65, Rel B, p50, and p52, which form heterodimeric complexes, the most frequently occurring being composed of p50 and p65. In most unactivated cells, NF-
B remains in a cytoplasmic inactive complex through its association with the inhibitory proteins I
Bs (13). Inducers of NF-
B, which include inflammatory cytokines, reactive oxygen species, and viral products, activate a dimeric I
B kinase complex (14, 15, 16), which phosphorylates I
B
on Ser-32 and Ser-36, leading to subsequent ubiquitination and degradation of I
B
and release of NF-
B proteins (11, 12). Free NF-
B dimers translocate to the nucleus where they regulate target gene transcription. Interestingly, NF-
B dimers also regulate I
B
gene expression, thus ensuring a feedback regulation leading to the inflammatory response arrest (17). Using in vivo genomic footprinting, it has been demonstrated that the adjacent NF-
B and Sp1 sites are necessary for I
B
transcriptional induction (18). In addition, NF-
B and Sp1 transcription factors have been reported to recruit coactivator complexes, namely ARC (activator-recruited cofactor) and CRSP (cofactor required for Sp1 activation), respectively (19, 20). The ARC coactivator complex was found to strongly stimulate NF-
B- and Sp1-directed activation of transcription using chromatin-assembled templates (19). Interestingly, the ARC complex is identical with the DRIP (VDR-interacting protein)/TRAP(TR-associated protein) complex, which is recruited in a ligand-dependent manner to the AF-2 domain of nuclear receptors via a single subunit DRIP205/TRAP220 (21, 22).
We recently reported that PPAR
activators induce I
B
mRNA and protein levels in a PPAR
-dependent manner (10). Induction of I
B
is associated with a decrease in p65-mediated gene activation. This mechanism may contribute to the antiinflammatory activities of PPAR
agonists. However, the molecular mechanisms by which PPAR
regulates I
B
transcription remain elusive. The aim of this study was to dissect further these mechanisms. Here, we demonstrate that PPAR
potentiates p65-stimulated I
B
gene transcription through its ligand-binding domain (LBD). Furthermore, it is shown that PPAR
regulates I
B
promoter activity in the absence of PPRE. This PPAR
-mediated promoter activation requires the presence of the NF-
B and the Sp1 sites within this promoter. Finally, using transient transfection experiments, we demonstrate that PPAR
activates I
B
promoter transcription in a DRIP205-dependent manner.
 |
RESULTS
|
---|
PPAR
Potentiates p65-Stimulated I
B
Transcription
Because we previously demonstrated that PPAR
activators strongly induce I
B
gene expression in cooperation with inflammatory stimuli (10) and because I
B
promoter is driven by NF-
B (23, 24), we hypothesized that PPAR
overexpression may enhance p65-induced I
B
gene transcription. As expected, cotransfection of p65 led to a strong induction of I
B
promoter activity in COS cells (Fig. 1A
). Incubation of COS cells with a synthetic PPAR
activator (GW9578 500 nM) did not affect this induction, consistent with the absence of endogenous PPAR
in this cell line (3). Surprisingly, cotransfection of PPAR
strongly potentiated p65-mediated promoter activation in a ligand-dependent manner in COS cells (Fig. 1A
). As a control, PPAR
significantly induced a PPRE-driven reporter activity (3.5-fold) in COS cells in a ligand-dependent manner (Fig. 1B
). Similar results were obtained in HeLa cells (data not shown). Taken together, these data demonstrate that PPAR
potentiates p65-stimulated I
B
promoter activity.
The PPAR
LBD Is Required for I
B
Promoter Activation
To determine which domains of PPAR
are involved in this transcriptional activation, we next tested the influence of different PPAR
constructs on p65-mediated I
B
gene regulation in COS cells. A PPAR
construct lacking the LBD and an LBD-containing construct were cotransfected either in the presence or absence of p65. p65 strongly enhanced I
B
promoter activity (Fig. 2A
). This induction was potentiated by the PPAR
LBD-containing construct in the presence of its ligand (Fig. 2A
), whereas the truncated PPAR
lacking the LBD failed to stimulate I
B
promoter activity (Fig. 2A
). Western blot analysis revealed that both PPAR
constructs were expressed at similar levels (Fig. 2B
). Together, these results indicate that the PPAR
LBD is required for PPAR
-induced I
B
gene transcription.
PPAR
-Mediated I
B
Promoter Activation Requires the Presence of the
B1 and Sp1 Sites
Because it has been demonstrated that both NF-
B1 and Sp1 sites are necessary for I
B
promoter induction (18) and since no functional PPRE was found within this promoter (data not shown), we next studied whether PPAR
regulates I
B
promoter via the NF-
B1 and Sp1 sites. To study a potential involvement of these sites, various mutated constructs were used as previously described (18). p65 transfection led to a strong activation of the wild-type I
B
promoter, which was further enhanced by PPAR
cotransfection in the presence of its specific ligand (Fig. 3
). The I
B
promoter mutated in the NF-
B1 site was also induced by p65 transfection but to a lesser extent compared with the wild-type promoter. This induction is likely due to the presence of other NF-
B sites within the promoter (23, 24). Interestingly, PPAR
cotransfection weakly increased p65-mediated promoter activation in the presence of the GW9578 compound (Fig. 3
). The I
B
promoter mutated in the Sp1 site was p65-responsive but also to a lesser extent compared with the wild-type promoter, suggesting functional cooperations between the
B1 and Sp1 sites as previously described (18). PPAR
cotransfection failed to activate the Sp1-mutated promoter construct activated by p65 transfection, suggesting that this site is crucial for PPAR
-mediated promoter activation. Finally, the promoter construct mutated in both
B1 and Sp1 sites was weakly activated by p65 and completely unresponsive to PPAR
activators. Taken together, these results indicate that both
B1 and Sp1 sites are required for maximal transcriptional activation of the I
B
promoter by PPAR
and confirm the existence of a functional cooperation between
B1 and Sp1 sites. In addition, PPAR
, in cooperation with p65, was found to activate the
B1-Sp1 enhancer in a heterologous promoter system (data not shown).
The Synthetic PPAR
Activator GW9578 Enhances the Occupancy of the I
B
-NF-
B Response Element in Vivo
To get further insight into the molecular mechanisms of PPAR
-mediated transactivation of the
B1-Sp1 enhancer, we performed chromatin immunoprecipitation assays (ChIP) using a p65 rabbit polyclonal antibody. Because COS cells do not contain endogenous PPAR
, PPAR
-transfected COS cells were used. PMA was used as an inflammatory stimulus because of the unresponsiveness of COS cells to inflammatory cytokines such as IL-1 and TNF
. Immunoprecipitates of GW9578 cotreated cells were significantly enriched in sequences containing the I
B
-NF-
B response element compared with PMA-only stimulated cells (Fig. 4A
). Amplification of the HSP70 promoter as a negative control shows that this enrichment was specific for I
B
(Fig. 4A
). Western blot analysis indicates that this increase in promoter occupancy upon GW9578 treatment was not due to an increase of p65 or PPAR
protein levels in nuclear extracts (Fig. 4B
). These results demonstrate that the synthetic PPAR
activator GW9578 enhances the occupancy of the I
B
-NF-
B response element in vivo, which may explain, at least in part, the enhanced promoter activity.
cAMP Response Element-Binding Protein-Binding Protein (CBP)/p300 and the p160 Coactivators Are Not Involved in PPAR
-Mediated I
B
Promoter Activation
Because we failed to detect a ternary complex between p65, Sp1, and PPAR
by glutathione-S-transferase pull-down assays (data not shown), we hypothesized that PPAR
may regulate I
B
gene transcription by recruiting and/or stabilizing additional factors on the
B1-Sp1 enhancer. It has recently been shown that CBP may act as an integrator of both NF-
B and nuclear receptor signaling (25, 26). To determine whether PPAR
regulates I
B
promoter activation by such a mechanism, we next tested the influence of the oncoprotein E1A, which has been reported to inhibit CBP-mediated gene activation (27). As expected, E1A overexpression dose dependently inhibited p65 and PPAR
transcriptional activities on a promoter construct driven by three copies of their respective response-elements (Fig. 5
). By contrast, high amounts of E1A expression plasmid failed to repress the ligand-dependent synergistic transcriptional activation between p65 and PPAR
on I
B
promoter (Fig. 6
). These results suggest that PPAR
may regulate I
B
gene transcription in a CBP-independent manner.
PPAR
Regulates I
B
Transcription By Recruiting the DRIP Complex
It has recently been shown that nuclear receptors regulate gene transcription by recruiting a multisubunit complex called DRIP or TRAP (21, 28). This complex is recruited in response to ligand binding to the LBD AF-2 core through a single subunit (DRIP205/TRAP220/PBP), similarly as reported for the p160 coactivators. Interestingly, the DRIP complex is identical with the ARC coactivator complex, which has been shown to potentiate p65- and Sp1-mediated activation of chromatin templates in vitro (19). Furthermore, CRSP, a transcriptional cofactor complex required for Sp1 activity, shares many subunits with the ARC/DRIP complex (20). To test the functional involvement of the DRIP complex in PPAR
-induced I
B
induction, we next tested the influence of DRIP205 overexpression on PPAR
-stimulated promoter activity. DRIP205 cotransfection did not potentiate p65 or PPAR
transcriptional activities on a promoter construct driven by three copies of their respective response-elements (Fig. 7
, A and B) as previously reported for the VDR (29). By contrast, a 190-amino acid fragment of DRIP205 that contains the two LXXLL motifs inhibited p65- and PPAR
-mediated promoter activation in a dose-dependent manner (Fig. 7
, C and D), as previously found for the VDR (29). Interestingly, overexpression of the DRIP205 fragment containing the two NR boxes abolished the ligand-dependent PPAR
activation of I
B
promoter, whereas mutation of the two NR boxes resulted in a loss of this dominant negative activity (Fig. 8
). Furthermore, cotransfection of increasing amounts of full-length DRIP205 relieved the dominant negative activity on PPAR
-stimulated promoter activity (Fig. 9
), demonstrating the specificity of this effect.

View larger version (27K):
[in this window]
[in a new window]
|
Figure 7. A Dominant Negative Form of DRIP205 Inhibits p65 and PPAR Transactivation
COS-1 cells were transfected with (NF- B)3-TK-Luc (panels A and C), or (PPRE)3-TK-Luc (panels B and D) in the presence of p65 (10 ng), PPAR (30 ng), or empty expression plasmids pRSV and pSG5, respectively, and increasing amounts (50, 100, 200 ng for p65; 25, 50, 100 ng for PPAR ) of DRIP205 (panels A and B) or 205-Box (panels C and D). Twenty-four hours post transfection, cells were refed with DMEM supplemented with 0.2% FCS in the presence of GW9578 (500 nM) or vehicle (DMSO 0.1%).
|
|
Having shown that DRIP205 is required for PPAR
-induced I
B
gene transcription, we next tested the influence of DRIP205 overexpression on PPAR
-mediated NF-
B transcriptional repression. As expected, cotransfection of p65 strongly induced a minimal promoter driven by multiple NF-
B response elements. PPAR
cotransfection in the presence of GW9578 resulted in a significant reduction of p65 reporter transactivation (Fig. 10
). DRIP205 overexpression did not affect p65-induced promoter activity (Fig. 7A
), in agreement with our previous results, nor abolish PPAR
-mediated NF-
B transcriptional repression (Fig. 10
). Taken together, these results suggest that the DRIP205, through its recruitment to PPAR
AF-2, is required for PPAR
-induced I
B
gene transcription, but not for PPAR
-mediated repression of p65-stimulated transcription.
 |
DISCUSSION
|
---|
In this manuscript, we demonstrate that PPAR
induces I
B
gene transcription via its LBD. Site-directed mutagenesis experiments demonstrate that PPAR
increases I
B
transcription via the NF-
B 1 and Sp1 sites and that this mechanism functions in a PPRE-independent manner. Cotransfections of E1A suggest that CBP/p300 is not required for PPAR
-induced I
B
promoter activation. Finally, using a dominant-negative form of DRIP205, we show that the DRIP complex is necessary for PPAR
-induced I
B
transcription, but not for PPAR
-mediated repression of p65 transcriptional activity. Altogether, these data provide a molecular basis for the induction of I
B
by PPAR
activators.
It has been demonstrated that PPAR
activators negatively regulate NF-
B-driven gene transcription in different cellular models (3, 8, 9, 30). We recently demonstrated that PPAR
activators increase I
B
mRNA and protein levels in human aortic smooth muscle cells. This action of PPAR
on I
B
expression likely contributes to the overall antiinflammatory activities of PPAR
agonists. Interestingly, the I
B
promoter contains three NF-
B binding sites (23, 24), ensuring an inducible autoregulatory loop (17). PPAR
cotransfection failed to inhibit p65-stimulated I
B
promoter activity, but, surprisingly, potentiated p65 activation in a ligand-dependent manner (Fig. 1A
). Similar results were obtained when we used the human immunodeficiency virus-1 promoter, which presents approximately the same promoter architecture, i.e. with multiple NF-
B binding sites and Sp1 sites (data not shown). A significant induction of I
B
promoter activity by PPAR
requires the presence of p65 in COS cells. However, we previously reported that fibrates, synthetic PPAR
activators, induce I
B
gene expression in human aortic smooth muscle cells, and this induction is further enhanced by cytokine treatment (10). The absence of activation of I
B
promoter in COS cells can be explained by the fact that primary vascular smooth muscle cells display nuclear p65 activity under basal conditions (31), whereas COS cells do not.
The
B1 and Sp1 sites have been reported to be crucial for I
B
induction by inflammatory stimuli (18). Mutation of both sites within I
B
promoter confirmed this functional cooperation between Sp1 and
B1 sites (Fig. 3
) (18) and demonstrated the requirement of these two sites for I
B
induction by PPAR
activators. Moreover, this
B1-Sp1 enhancer can function on a heterologous promoter (data not shown). To our knowledge, this is the first demonstration of PPAR
-mediated promoter activation in the absence of a PPRE. A functional interaction between PPAR
and Sp1 has been previously reported in the transcriptional control of the acyl-coenzyme-A oxidase promoter (32). However, in this case, PPAR
and Sp1 binding to their respective binding sites was required for promoter activation. These results indicate that PPAR
activators do not inhibit all NF-
B-driven target genes. Further studies will be necessary to determine whether additional regulatory sequences, adjacent to NF-
B sites and modulating PPAR
activity, exist.
In contrast to other steroid receptors such as ER
, ERß (33), and PR (34), immunoprecipitations and glutathione-S-transferase pull-down assays did not reveal any physical interactions between PPAR
and Sp1 (data not shown). Therefore, we hypothesized that PPAR
may recruit platform proteins leading to the formation of a large complex on the
B1-Sp1 enhancer. Owen et al. (34) demonstrated that liganded PR regulates p21 promoter activity through Sp1 proteins in a complex that also includes CBP/p300. In their report, overexpression of E1A [a powerful inhibitor of CBP-mediated gene activation (27)] completely abolished progesterone activation of the p21 promoter. To determine whether PPAR
regulates I
B
by such a mechanism, E1A overexpression experiments were performed. Our results indicate that CBP/p300 is not necessary for PPAR-mediated promoter activation (Fig. 6
). Because it has been demonstrated that E1A, through its association with the nuclear receptor coactivator domain of CBP, also inhibits p160-mediated nuclear receptor function (27), p160 coactivators such as transcriptional intermediary factor-2 or steroid receptor coactivator 1 are probably not involved in I
B
promoter stimulation by PPAR
. In line with this, PPAR
function is not affected in steroid receptor coactivator 1-deficient mice (35), and several observations suggest that steroid receptor coactivators are not involved in the PPAR
/NF-
B cross-talk (3). However, we cannot rule out a potential involvement of general coactivators such as TR-binding protein, which has been reported to potentiate NF-
B and nuclear receptor transcriptional activities (36).
It has been reported recently that nuclear receptors may regulate transcription by utilizing a general mechanism implicating the ligand-dependent recruitment of the DRIP/TRAP complex (37). Interestingly, the TRAP/DRIP complex is similar to the ARC coactivator complex and the CRSP complex, which are targeted by different classes of activators such as p65 and Sp1, respectively (19, 20). DRIP complex involvement in I
B
promoter induction by PPAR
was therefore tested. Overexpression of DRIP205 did not affect p65 and PPAR
function (Fig. 7
). In another study, DRIP205 overexpression was also shown not to affect GR transactivation (38). By contrast, a dominant-negative form of DRIP205 inhibited p65 and PPAR
function in a dose-dependent manner (Fig. 7
). Similar results were obtained by different groups on VDR and ER transactivation (29, 39). Cotransfection of this DRIP205 fragment comprising the two LXXLL motifs abolished PPAR
-induced I
B
promoter activity (Fig. 8
), whereas the same fragment mutated in the two NR boxes did not. These results demonstrate the functional requirement of DRIP205 LXXLL motifs in PPAR
-mediated promoter activation. Furthermore, increasing amounts of cotransfected DRIP205 relieved this dominant negative activity (Fig. 9
). Interestingly, the ARC complex, which is similar to the DRIP/TRAP complex, has been shown to greatly stimulate NF-
B and Sp-1-directed activation of human immunodeficiency virus-1 transcription with chromatin-assembled template (19). Nevertheless, these authors failed to observe a direct association between Sp1 and the ARC complex. Furthermore, Sp1 activity has been shown to be dependent on CRSP, a cofactor complex that shares several subunits with DRIP and ARC, but also contains specific subunits (20). However, the p200 subunit of CRSP is identical with TRAP220/DRIP205. Hence, DRIP205 is a protein common in the CRSP, the ARC, and the DRIP/TRAP coactivator complexes. DRIP205/PBP strongly interacts with PPAR
in a ligand-dependent manner (40). Furthermore, PPAR
displays a preference for binding to the second LXXLL motif of DRIP205 (41). In a recent paper, GR was reported to physically interact with both DRIP150 and DRIP205 through its AF-1 and AF-2 domains, respectively (38). Interestingly, the liganded GR is unable to potentiate p65-mediated activation of I
B
promoter. Instead, it reduces p65-induced promoter activation (42) despite its strong interaction with DRIP205. This observation emphasizes the specificity of PPAR
-mediated I
B
promoter induction. Based on our results, we propose a model in which NF-
B and Sp1 transcription factors recruit the ARC/CRSP coactivator complexes, which could be stabilized by the ligand-dependent interaction between PPAR
and DRIP205. Further studies will be required to determine the precise molecular interactions between these different partners. Two independent groups have recently generated mice lacking TRAP220/DRIP205 (43, 44). The absence of TRAP220 results in embryonic lethality and in an impaired TR-driven transcription, whereas p53- and RAR/RXR-driven transcription was not affected (43), suggesting gene-selective functions for TRAP220. Transient transfection assays in TRAP220-deficient mouse embryonic fibroblasts showed a modest reduction of PPAR
transcriptional activity on a PPRE-driven promoter (44). Interestingly, in a recent paper, Yang et al. (45) reported that PPAR
recruits DRIP205 through the first NR box, whereas PPAR
displays a preference for binding to the second LXXLL motif of DRIP205 (41), which suggests that PPAR
and PPAR
may distinctly recruit coactivator complexes on DNA. Thus, it would be interesting to test I
B
induction in mouse embryonic fibroblasts from TRAP220-deficient mice.
In conclusion, our results provide a molecular basis for PPAR
-induced I
B
transcription and raise a possibility that PPAR
may positively regulate a number of genes via such indirect mechanisms.
 |
MATERIALS AND METHODS
|
---|
Cell Culture and Chemical Reagents
COS-1 cells (ATCC, Manassas, VA) were grown in DMEM supplemented with 2 mM glutamine and 10% (vol/vol) FCS in a 5% CO2 humidified atmosphere at 37 C. The PPAR
activator GW9578 (46) was kindly provided by Dr. Peter Brown (GlaxoWellcome).
Plasmids
The pRSV-p65, p(NF-
B)3-TK-luc, pSG5-hPPAR
, and PPRE-containing reporter plasmids [(PPRE)3-TK-Luc] were previously described (3). The plasmids pcDNA3-PPAR
LBD-V5 and PPAR
LBD-V5 were constructed by PCR-amplifying the hPPAR
ABC or DEF domains, respectively. The resulting PCR products were cloned in pcDNA3-GS (Invitrogen, San Diego, CA). The wild-type human I
B
promoter construct and the corresponding mutants of the NF-
B or Sp1 sites, as well as the plasmid with both the
B1 and Sp1 sites mutated, have been described previously (18) and were generously provided by J. Hiscott (McGill University, Montréal, Québec, Canada). The pcDNA3-DRIP205, pFLAG-CMV2205Box wt, and pFLAG-CMV2-205Box mutant 1+2 were generously provided by L. P. Freedman (Memorial Sloan-Kettering Cancer Center, New York, NY) and have been previously described (29). The E1A12S expression plasmid was provided by E. Moran (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).
Transient Transfection Assays
COS-1 cells, grown in 24-well plates to 5060% confluence in DMEM supplemented with 10% FCS, were transiently transfected using a lipid cationic technique (RPR120535B, Aventis, Vitry, France) with reporter and expression plasmids as stated in the figure legends. The phosphoglycerate kinase-ß-galactosidase expression plasmid was cotransfected as a control for transfection efficiency. Twenty-four hours post transfection, cells were refed with DMEM supplemented with 0.2% FCS and GW9578 (500 nM) or vehicle [0.1% dimethylsulfoxide (DMSO)]. Forty-eight hours later, the COS-1 cells were collected and also subjected to luciferase and ß-galactosidase assays. All experiments were repeated at least three times.
ChIP Assay
ChIP assays were performed essentially as previously described (47). Briefly, 50 x 106 of PPAR
-transfected COS cells were treated with various compounds as stated in the figure legends and subsequently cross-linked for 30 min at 4 C by adding a 11% formaldehyde-containing solution. Cross-linking was stopped by adding glycine to a final concentration of 125 mM for 5 min. Then, cells were rinsed with PBS, harvested, and centrifuged at 600 x g for 5 min at 4 C. Pellets were resuspended in lysis buffer [50 mM HEPES-KOH at pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 140 mM NaCl, 10% glycerol, 0.5% NP-40, 0.25% Triton, 1 mM phenylmethylsulfonyl fluoride (PMSF), and leupeptin/pepstatin A/aprotinin, 5 µg/ml each] and rotated for 10 min at 4 C. The nuclei were collected by centrifugation and resuspended in 10 ml wash buffer (10 mM Tris-HCl at pH 8, 1 mM EDTA, 0.5 mM EGTA, 200 mM NaCl, 1 mM PMSF, and leupeptin/pepstatin A/aprotinin 5 µg/ml each) and rotated again. Washed nuclei were centrifuged and resuspended in 1x RIPA buffer (10 mM Tris-HCl at pH 8, 1 mM EDTA, 0.5 mM EGTA, 140 mM NaCl, 1% Triton, 0.1% SDS, 0.1% Na-deoxycholate, 1 mM PMSF, and leupeptin/pepstatin A/aprotinin, 5 µg/ml each) and subsequently sonicated, leading to the generation of DNA fragment sizes of 0.31.5 kb. Samples were cleared by centrifugation at 16,000 x g for 10 min at 4 C and immunoprecipitated using a rabbit p65 polyclonal antibody (sc-109, Santa Cruz Biotechnology, Inc.), as previously described (47). After extensive washings, proteins were digested by adding proteinase K (100 µg/ml) and placed at 55 C for 3 h followed by 6 h at 65 C to reverse cross-links. DNA was extracted by a standard procedure and pellets were resuspendend in Tris-EDTA buffer. The human I
B
and HSP70 promoter regions were PCR amplified using standard procedures as previously described (47). PCRs were analyzed by electrophoresis in a nondenaturing 5% polyacrylamide gel in 0.5x Tris-borate-EDTA. The gels were then dried and exposed at -80 C for autoradiography.
Western Blot Analysis
Protein extracts were fractionated on a 10% polyacrylamide gel under reducing conditions [sample buffer containing 10 mM dithiothreitol, transferred onto nitrocellulose membranes and probed with rabbit polyclonal p65 (sc-109, Santa Cruz Biotechnology, Inc.), PPAR
(Affinity BioReagents, Inc., Golden, CO), or V5 (Invitrogen) antibody. After incubation with a secondary peroxidase-conjugated antibody, signals were visualized by chemiluminescence (Amersham Pharmacia Biotech, Buckinghamshire, UK).
 |
ACKNOWLEDGMENTS
|
---|
The authors would like to thank L. P. Freedman and C. Rachez for providing the DRIP205 expression plasmids and J. Hiscott for the different I
B
promoter constructs.
 |
FOOTNOTES
|
---|
This work was supported by grants of the Institut Pasteur de Lille, Institut National de la Santé et de la Recherche Médicale, the Région Nord-Pas-de-Calais/Fonds Européen de Dévelopment Regional, and Interuniversitaire Attractie Polen (Belgium). W.V.B. is a postdoctoral fellow with the Fonds voor Wetenschappelijk Onderzoek (FWO)-Vlaanderen, and G.H. is a research director with the FWO-Vlaanderen.
1 Current address: Gene Regulation, Bone & Inflammation Research, Eli Lilly \|[amp ]\| Co. Research Laboratories, Lilly Corporate Centre, Indianapolis, Indiana 46285. 
Abbreviations: ARC, Activator-recruited cofactor; CBP, cAMP response element-binding protein-binding protein; ChIP, chromatin immunoprecipitation; CRSP, cofactor required to Sp1 activation; DMSO, dimethylsulfoxide; DRIP, VDR-interacting protein; LBD, ligand-binding domain; NF-
B, nuclear factor-
B; PMA, phorbol 12-myristate 13-acetate; PMSF, phenylmethylsulfonyl fluoride; PPRE, PPAR response element; TRAP, TR-associated protein.
Received for publication June 19, 2001.
Accepted for publication January 2, 2002.
 |
REFERENCES
|
---|
-
Desvergne B, Wahli W 1999 Peroxisome proliferator-activated receptors: nuclear control of metabolism. Endocr Rev 20:649688[Abstract/Free Full Text]
-
Tugwood JD, Issemann I, Anderson RG, Bundell KR, McPheat WL, Green S 1992 The mouse peroxisome proliferator-activated receptor recognizes a response element in the 5' flanking sequence of the rat acyl coA oxidase gene. EMBO J 11:433439[Abstract]
-
Delerive P, De Bosscher K, Besnard S, Vanden Berghe W, Peters JM, Gonzalez FJ, Fruchart JC, Tedgui A, Haegeman G, Staels B 1999 PPAR
negatively regulates the vascular inflammatory gene response by negative cross-talk with transcription factors NF-
B and AP-1. J Biol Chem 274:3204832054[Abstract/Free Full Text]
-
Ricote M, Li AC, Willson TM, Kelly CJ, Glass CK 1998 The peroxisome proliferator-activated receptor-
is a negative regulator of macrophage activation. Nature 391:7982[CrossRef][Medline]
-
Staels B, Dallongeville J, Auwerx J, Schoonjans K, Leitersdorf E, Fruchart JC 1998 Mechanism of action of fibrates on lipid and lipoprotein metabolism. Circulation 98:20882093[Abstract/Free Full Text]
-
Chinetti G, Griglio S, Antonucci M, Pineda Torra I, Delerive P, Majd Z, Fruchart JC, Chapman J, Najib J, Staels B 1998 Activation of peroxisome proliferator activated receptors
and
induces apoptosis of human monocyte-derived macrophages. J Biol Chem 273:2557325580[Abstract/Free Full Text]
-
Devchand PR, Keller H, Peters JM, Vasquez M, Gonzalez FJ, Wahli W 1996 The PPAR
-leukotriene B pathway to inflammation control. Nature 384:3943[CrossRef][Medline]
-
Staels B, Koenig W, Habib A, Merval R, Lebret M, Pineda-Torra I, Delerive P, Fadel A, Chinetti G, Fruchart JC, Najib J, Maclouf J, Tedgui A 1998 Activation of human aortic smooth-muscle cells is inhibited by PPAR
but not by PPAR
activators. Nature 393:790793[CrossRef][Medline]
-
Marx N, Sukhova GK, Collins T, Libby P, Plutzky J 1999 PPAR
activators inhibit cytokine-induced vascular cell adhesion molecule-1 expression in human endothelial cells. Circulation 99:31253131[Abstract/Free Full Text]
-
Delerive P, Gervois P, Fruchart J-C, Staels B 2000 Induction of I
B
expression as a mechanism contributing to the anti-inflammatory activities of PPAR
activators. J Biol Chem 275:3670336707[Abstract/Free Full Text]
-
Baeuerle PA, Baltimore D 1996 NF-
B: ten years after. Cell 87:1320[Medline]
-
Verma, IM, Stevenson JK, Schwartz EM, Van Antwerp D, Miyamoto S 1995 Rel/NF-
B/I
B family: intimate tales of association and dissociation. Genes Dev 9:27232735[CrossRef][Medline]
-
Baeuerle PA, Baltimore D 1988 I
B: a specific inhibitor of the NF-
B transcription factor. Science 242:540546[Medline]
-
DiDonato JA, Hayakawa M, Rothwarf DM, Zandi E, Karin M 1997 A cytokine-responsive I
B kinase that activates the transcription factor NF-
B. Nature 388:548554[CrossRef][Medline]
-
Mercurio F, Zhu H, Murray BW, Shevchenko A, Bennett BL, Li JW, Young DB, Barbosa M, Mann M, Manning A, Rao A 1997 IKK-1 and IKK-2: cytokine-activated I
B kinases essential for NF-
B activation. Science 278:860866[Abstract/Free Full Text]
-
Zandi E, Rothwarf DM, Delhase M, Hayakawa M, Karin M 1997 The I
B kinase complex (IKK) contains two kinase subunits, IKK
and IKKß, necessary for I
B phosphorylation and NF-
B activation. Cell 91:243252[Medline]
-
Sun SC, Ganchi PA, Ballard DW, Greene WC 1993 NF-
B controls expression of inhibitor I
B
: evidence for an inducible autoregulatory pathway. Science 259:19121915[Medline]
-
Algarté M, Kwon H, Génin P, Hiscott J 1999 Identification by in vivo footprinting of a transcriptional switch containing NF-
B and Sp1 that regulates the I
B
promoter. Mol Cell Biol 19:61406153[Abstract/Free Full Text]
-
Näär AM, Beaurang PA, Zhou S, Abraham S, Solomon W, Tjian R 1999 Composite co-activator ARC mediates chromatin-directed transcriptional activation. Nature 398:828832[CrossRef][Medline]
-
Ryu S, Zhou S, Ladurner AG, Tjian R 1999 The transcriptional cofactor complex CRSP is required for activity of the enhancer-binding protein Sp-1. Nature 397:446450[CrossRef][Medline]
-
Rachez C, Lemon BD, Suldan Z, Bromleigh V, Gamble M, Näär AM, Erdjument-Bromage H, Tempst P, Freedman LP 1999 Ligand-dependent transcription activation by nuclear receptors requires the DRIP complex. Nature 398:824828[CrossRef][Medline]
-
Yuan C-X, Ito M, Fondell JD, Fu Z-Y, Roeder RG 1998 The TRAP220 component of a thyroid hormone receptor-associated protein (TRAP) coactivator complex interacts directly with nuclear receptors in a ligand-dependent fashion. Proc Natl Acad Sci USA 95:79397944[Abstract/Free Full Text]
-
Ito CY, Kazantsev AG, Baldwin AS 1994 Three Nf-
B sites in the I
B-
promoter are required for induction of gene expression by TNF
. Nucleic Acids Res 22:37873792[Abstract]
-
Le Bail O, Schmidt-Ulrich R, Israël A 1993 Promoter analysis of the gene encoding the I
B
/MAD3 inhibitor of NF-
B: positive regulation by members of the rel/NF-
B family. EMBO J 12:50435049[Abstract]
-
De Bosscher K, Vanden Berghe W, Vermeulen L, Plaisance S, Boone E, Haegeman G 2000 Glucocorticoids repress NF-
B driven genes by disturbing the interaction of p65 with the basal transcription machinery, irrespective to coactivator levels in the cells. Proc Natl Acad Sci USA 97:39193924[Abstract/Free Full Text]
-
McKay LI, Cidlowski JA 2000 CBP (CREB binding protein) integrates NF-
B (nuclear factor-
B) and glucocorticoid receptor physical interactions and antagonism. Mol Endocrinol 14:12221234[Abstract/Free Full Text]
-
Kurokawa R, Kalafus D, Ogliastro M-H, Kioussi C, Xu L, Torchia J, Rosenfeld MG, Glass CK 1998 Differential use of CREB binding protein-coactivator complexes. Science 279:700703[Abstract/Free Full Text]
-
Fondell JD, Ge H, Roeder RG 1996 Ligand induction of a transcriptionally active thyroid hormone receptor coactivator complex. Proc Natl Acad Sci USA 93:83298333[Abstract/Free Full Text]
-
Rachez C, Gamble M, Chang C-PB, Atkins GB, Lazar MA, Freedman LP 2000 The DRIP complex and SRC-1/p160 coactivators share similar nuclear receptor binding determinants but constitute distinct complexes. Mol Cell Biol 20:27182726[Abstract/Free Full Text]
-
Poynter ME, Daynes RA 1998 Peroxisome proliferator-activated receptor
activation modulates cellular redox status, represses nuclear factor-
B signaling, and reduces inflammatory cytokine production in aging. J Biol Chem 273:3283332841[Abstract/Free Full Text]
-
Bellas RE, Lee JS, Sonenshein GA 1995 Expression of a constitutive NF-
B-like activity is essential for proliferation of cultured bovine vascular smooth muscle cells. J Clin Invest 96:25212527[Medline]
-
Krey G, Mahfoudi A, Wahli W 1995 Functional interactions of peroxisome proliferator-activated receptor, retinoid-X receptor, and Sp1 in the transcriptional regulation of the acyl-coenzyme-A oxidase promoter. Mol Endocrinol 9:219231[Abstract]
-
Saville B, Wormke M, Wang F, Nguyen T, Enmark E, Kuiper G, Gustafsson J-A, Safe S 2000 Ligand-, cell-, and estrogen receptor subtype (
/ß)-dependent activation at GC-rich (Sp1) promoter elements. J Biol Chem 275:53795387[Abstract/Free Full Text]
-
Owen GI, Richer JK, Takimoto G, Horwitz KB 1998 Progesterone regulates transcription of the p21waf1cyclin-dependent kinase inhibitor gene through Sp1 and CBP/p300. J Biol Chem 273:1069610701[Abstract/Free Full Text]
-
Qi C, Zhu Y, Pan J, Yeldandi AV, Sambasiva Rao M, Maeda N, Subbarao V, Pulikuri S, Hashimoto T, Reddy JK 1999 Mouse steroid receptor coactivator-1 is not essential for peroxisome proliferator-activated receptor
-regulated gene expression. Proc Natl Acad Sci USA 96:15851590[Abstract/Free Full Text]
-
Ko L, Cardona GR, Chin WW 2000 Thyroid hormone receptor-binding protein, an LXXLL motif containing protein, functions as a general coactivator. Proc Natl Acad Sci USA 97:62126217[Abstract/Free Full Text]
-
Freedman LP 1999 Increasing the complexity of coactivation in receptor signaling. Cell 97:58[Medline]
-
Hittelman AB, Burakov D, Iniguez-Lluhi JA, Freedman LP, Garabedian MJ 1999 Differential regulation of glucocorticoid receptor transcriptional activation via AF-1-associated proteins. EMBO J 18:53805388[Abstract/Free Full Text]
-
Burakov D, Wong CW, Rachez C, Cheskis BJ, Freedman LP 2000 Functional interactions between the estrogen receptor and DRIP205, a subunit of the heterodimeric DRIP coactivator complex. J Biol Chem 275:2092820934[Abstract/Free Full Text]
-
Zhu Y, Qi C, Jain S, Sambasiva Rao M, Reddy JK 1997 Isolation and Characterization of PBP, a protein that interacts with peroxisome proliferator-activated receptor. J Biol Chem 272:2550025506[Abstract/Free Full Text]
-
Ren Y, Behre E, Ren Z, Zhang J, Wang Q, Fondell JD 2000 Specific structural motifs determine TRAP220 interactions with nuclear hormone receptors. Mol Cell Biol 20:54335446[Abstract/Free Full Text]
-
Vanden Berghe W, Francesconi E, De Bosscher K, Resche-Rigon M, Haegeman G 1999 Dissociated glucocorticoids with anti-inflammatory potential repress interleulin-6 gene expression by a nuclear factor-
B-dependent mechanism. Mol Pharmacol 56:797806[Abstract/Free Full Text]
-
Ito M, C-X Yuan, Okano HK, Darnell RB, Roeder RG 2000 Involvement of the TRAP220 component of the TRAP/SMCC coactivator complex in embryonic development and thyroid hormone action. Mol Cell 5:683693[Medline]
-
Zhu Y, Qi C, Jia Y, Nye JS, Rao MS, Reddy JK 2000 Deletion of PBP/PPARBP, the gene for nuclear receptor coactivator peroxisome proliferator-activated receptor-binding protein, results in embryonic lethality. J Biol Chem 275:1477914782[Abstract/Free Full Text]
-
Yang W, Rachez C, Freedman LP 2000 Discrete roles for peroxisome proliferator-activated receptor
and retinoid X receptor in recruiting nuclear receptor coactivators. Mol Cell Biol 20:80088017[Abstract/Free Full Text]
-
Willson TM, Brown PJ, Sternbach DD, Henke BR 2000 The PPARs: from orphan receptors to drug discovery. J Med Chem 43:527550[CrossRef][Medline]
-
Nissen RM, Yamamoto KR 2000 The glucocorticoid receptor inhibits NF
B by interfering with serine-2 phosphorylation of the RNA polymerase II carboxy-terminal domain. Genes Dev 14:23142329[Abstract/Free Full Text]