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B
Gene Transcription by PPAR
Institut National de la Santé et de la Recherche Médicale U.545 (P.D., J.-C.F., B.S.), Département d’Athérosclérose, 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.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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B (NF-B) signaling pathway. We recently demonstrated that PPAR
activators increase IB mRNA and protein levels in human aortic smooth muscle cells. Here, we studied the molecular mechanisms by which PPAR controls IB expression. Using transient transfection assays, it is demonstrated that PPAR potentiates p65-stimulated IB transcription in a ligand-dependent manner. Site-directed mutagenesis experiments revealed that PPAR activation of IB transcription requires the NF-B and Sp1 sites within IB promoter. Chromatin immunoprecipitation assays demonstrate that PPAR activation enhances the occupancy of the NF-B response element in IB promoter in vivo. Overexpression of the oncoprotein E1A failed to inhibit PPAR-mediated IB 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 IB 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 IB and demonstrate, for the first time, that PPAR may positively regulate gene transcription in the absence of functional PPAR response elements. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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, 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 IB 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 IBs (13). Inducers of NF-B, which include inflammatory cytokines, reactive oxygen species, and viral products, activate a dimeric IB kinase complex (14, 15, 16), which phosphorylates IB on Ser-32 and Ser-36, leading to subsequent ubiquitination and degradation of IB 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 IB 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 IB 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 IB mRNA and protein levels in a PPAR-dependent manner (10). Induction of IB 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 IB transcription remain elusive. The aim of this study was to dissect further these mechanisms. Here, we demonstrate that PPAR potentiates p65-stimulated IB gene transcription through its ligand-binding domain (LBD). Furthermore, it is shown that PPAR regulates IB 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 IB promoter transcription in a DRIP205-dependent manner.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Potentiates p65-Stimulated IB Transcription activators strongly induce IB gene expression in cooperation with inflammatory stimuli (10) and because IB promoter is driven by NF-B (23, 24), we hypothesized that PPAR overexpression may enhance p65-induced IB gene transcription. As expected, cotransfection of p65 led to a strong induction of IB 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 IB promoter activity.
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LBD Is Required for IB Promoter Activation are involved in this transcriptional activation, we next tested the influence of different PPAR constructs on p65-mediated IB 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 IB 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 IB 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 IB gene transcription.
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-Mediated IB Promoter Activation Requires the Presence of the B1 and Sp1 SitesB1 and Sp1 sites are necessary for IB promoter induction (18) and since no functional PPRE was found within this promoter (data not shown), we next studied whether PPAR regulates IB 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 IB promoter, which was further enhanced by PPAR cotransfection in the presence of its specific ligand (Fig. 3). The IB 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 IB 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 IB 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).
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Activator GW9578 Enhances the Occupancy of the IB-NF-B Response Element in Vivo-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 IB-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 IB (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 IB-NF-B response element in vivo, which may explain, at least in part, the enhanced promoter activity.
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-Mediated IB Promoter Activation by glutathione-S-transferase pull-down assays (data not shown), we hypothesized that PPAR may regulate IB 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 IB 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 IB promoter (Fig. 6). These results suggest that PPAR may regulate IB gene transcription in a CBP-independent manner.
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Regulates IB Transcription By Recruiting the DRIP Complex-induced IB 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 IB 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.
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-induced IB 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 IB gene transcription, but not for PPAR-mediated repression of p65-stimulated transcription.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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induces IB gene transcription via its LBD. Site-directed mutagenesis experiments demonstrate that PPAR increases IB 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 IB promoter activation. Finally, using a dominant-negative form of DRIP205, we show that the DRIP complex is necessary for PPAR-induced IB transcription, but not for PPAR-mediated repression of p65 transcriptional activity. Altogether, these data provide a molecular basis for the induction of IB 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 IB mRNA and protein levels in human aortic smooth muscle cells. This action of PPAR on IB expression likely contributes to the overall antiinflammatory activities of PPAR agonists. Interestingly, the IB promoter contains three NF-B binding sites (23, 24), ensuring an inducible autoregulatory loop (17). PPAR cotransfection failed to inhibit p65-stimulated IB 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 IB promoter activity by PPAR requires the presence of p65 in COS cells. However, we previously reported that fibrates, synthetic PPAR activators, induce IB gene expression in human aortic smooth muscle cells, and this induction is further enhanced by cytokine treatment (10). The absence of activation of IB 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 IB induction by inflammatory stimuli (18). Mutation of both sites within IB promoter confirmed this functional cooperation between Sp1 and B1 sites (Fig. 3) (18) and demonstrated the requirement of these two sites for IB 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 IB 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 IB 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 IB 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 IB 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 IB promoter. Instead, it reduces p65-induced promoter activation (42) despite its strong interaction with DRIP205. This observation emphasizes the specificity of PPAR-mediated IB 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 IB induction in mouse embryonic fibroblasts from TRAP220-deficient mice.
In conclusion, our results provide a molecular basis for PPAR-induced IB transcription and raise a possibility that PPAR may positively regulate a number of genes via such indirect mechanisms.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 PPARLBD-V5 were constructed by PCR-amplifying the hPPARABC or DEF domains, respectively. The resulting PCR products were cloned in pcDNA3-GS (Invitrogen, San Diego, CA). The wild-type human IB 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-CMV2–205Box 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 50–60% 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 0.3–1.5 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 IB 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).
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ACKNOWLEDGMENTS |
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B promoter constructs. |
FOOTNOTES |
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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.
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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negatively regulates the vascular inflammatory gene response by negative cross-talk with transcription factors NF-B and AP-1. J Biol Chem 274:32048–32054 is a negative regulator of macrophage activation. Nature 391:79–82[CrossRef][Medline]
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/ß)-dependent activation at GC-rich (Sp1) promoter elements. J Biol Chem 275:5379–5387-regulated gene expression. Proc Natl Acad Sci USA 96:1585–1590B-dependent mechanism. Mol Pharmacol 56:797–806 and retinoid X receptor in recruiting nuclear receptor coactivators. Mol Cell Biol 20:8008–8017B by interfering with serine-2 phosphorylation of the RNA polymerase II carboxy-terminal domain. Genes Dev 14:2314–2329This article has been cited by other articles:
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L. Gao, F. Wang, B. Wang, B. Gong, J. Zhang, X. Zhang, and J. Zhao Cilostazol Protects Diabetic Rats from Vascular Inflammation via Nuclear Factor-{kappa}B-Dependent Down-Regulation of Vascular Cell Adhesion Molecule-1 Expression J. Pharmacol. Exp. Ther., July 1, 2006; 318(1): 53 - 58. [Abstract] [Full Text] [PDF]
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 A. Majdalawieh, L. Zhang, I. V. Fuki, D. J. Rader, and H.-S. Ro Adipocyte enhancer-binding protein 1 is a potential novel atherogenic factor involved in macrophage cholesterol homeostasis and inflammation PNAS, February 14, 2006; 103(7): 2346 - 2351. [Abstract] [Full Text] [PDF]
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T. Deng, S. Shan, P.-P. Li, Z.-F. Shen, X.-P. Lu, J. Cheng, and Z.-Q. Ning Peroxisome Proliferator-Activated Receptor-{gamma} Transcriptionally Up-Regulates Hormone-Sensitive Lipase via the Involvement of Specificity Protein-1 Endocrinology, February 1, 2006; 147(2): 875 - 884. [Abstract] [Full Text] [PDF]
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 A. Planavila, R. Rodriguez-Calvo, M. Jove, L. Michalik, W. Wahli, J. C. Laguna, and M. Vazquez-Carrera Peroxisome proliferator-activated receptor {beta}/{delta} activation inhibits hypertrophy in neonatal rat cardiomyocytes Cardiovasc Res, March 1, 2005; 65(4): 832 - 841. [Abstract] [Full Text] [PDF]
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 T. Okaya and A. B. Lentsch Peroxisome proliferator-activated receptor-{alpha} regulates postischemic liver injury Am J Physiol Gastrointest Liver Physiol, April 1, 2004; 286(4): G606 - G612. [Abstract] [Full Text] [PDF]
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 K. De Bosscher, W. Vanden Berghe, and G. Haegeman The Interplay between the Glucocorticoid Receptor and Nuclear Factor-{kappa}B or Activator Protein-1: Molecular Mechanisms for Gene Repression Endocr. Rev., August 1, 2003; 24(4): 488 - 522. [Abstract] [Full Text] [PDF]
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