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Inhibition of peroxisome proliferator-activated receptor α signaling by vitamin D receptor
BBRC Biochemical and Biophysical Research Communications 312 (2003) 513–519 www.elsevier.com/locate/ybbrc
Inhibition of peroxisome proliferator-activated receptor a signaling by vitamin D receptorq Takahiro Sakuma, Takahide Miyamoto,* Wei Jiang, Tomoko Kakizawa, Shin-ich Nishio, Satoru Suzuki, Teiji Takeda, Ako Oiwa, and Kiyoshi Hashizume Department of Aging Medicine and Geriatrics, Shinshu University School of Medicine, Matsumoto 390-8621, Japan Received 6 October 2003
Abstract Peroxisome proliferator-activated receptors (PPARs) are nuclear fatty acid receptors that have been implicated to play an important role in lipid and glucose homeostasis. PPARa potentiates fatty acid catabolism in the liver and is activated by the lipidlowering fibrates, whereas PPARc is essential for adipocyte differentiation. Here we report that nuclear vitamin D3 receptor (VDR) represses the transcriptional activity of PPARa but not PPARc in a 1,25(OH)2 D3 -dependent manner. The analysis using chimeric receptors revealed that ligand binding domain of PPARa and VDR was involved in the molecular basis of this functional interaction and that the DNA binding domain of VDR was not required for the suppression, suggesting a novel mechanism that might involve protein–protein interactions rather than a direct DNA binding. Furthermore, the treatment of rat hepatoma H4IIE cells with 1,25(OH)2 D3 diminishes the induction of AOX mRNA by PPARa ligands, Wy14,643. VDR signaling might be considered as a factor regulating lipid metabolism via PPARa pathway. We report here the novel action of VDR in controlling gene expression through PPARa signaling. Ó 2003 Elsevier Inc. All rights reserved.
The peroxisome proliferator-activated receptors are members of the nuclear receptor superfamily of transcription factors that regulate gene expression in response to the binding of small molecular weight lipophilic ligands. The identification of fatty acids as endogenous ligands for peroxisome proliferator-activated receptors (PPARs) has provided a unique approach to study lipid homeostasis at the molecular level [1–5]. Like other nuclear receptors that include the thyroid hormone receptor (TR), retinoic acid receptor (RAR), and vitamin D3 receptor (VDR), PPARs regulate transcription by binding to PPAR response elements (PPRE) normally present in the promoter of their target genes. PPARs form heterodimers with 9-cis-retinoic acid receptor (RXR) to recognize the PPRE that is composed of direct repeats (DRs) of the consensus q
Abbreviations: PPAR, peroxisome proliferator-activated receptor; RXR, retinoid X receptor; VDR, vitamin D receptor; GR, glucocorticoid receptor; LBD, ligand binding domain. * Corresponding author. Fax: +81-263-37-2710. E-mail address: [email protected] (T. Miyamoto). 0006-291X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2003.10.131
motif AGGTCA separated by 1 nucleotide (DR1 motif). Furthermore, ligand-dependent transcriptional activation by PPARs involves co-activator proteins to the C-terminal receptor region [6,7]. Three subtypes, PPARa (NR1C1), PPARc (NR1C2), and PPARd (NR1C3), have been identified with distinct tissue distributions and biological activities. PPARa is expressed in liver, heart, muscle, and kidney where it regulates fatty acid catabolism [8,9]. PPARc is expressed in adipocyte and macrophage and is involved in adipocyte differentiation, lipid storage, and glucose homeostasis [10,11]. PPARd is expressed ubiquitously. It has been implicated in keratinocyte differentiation and wound healing and, more recently, in mediating VLDL signaling of the macrophage [12–15]. PPARa have been shown to induce peroxisomal and microsomal enzymes involved in lipid metabolism [16,17]. Liver is the key site of metabolic integration where fatty acids are mobilized and, depending on the body’s needs, either stored or used as an energy source. Earlier studies have demonstrated that in the liver, PPARa directly regulates genes involved in fatty acid uptake [fatty acid binding protein
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(FABP)], b-oxidation (acyl-CoA oxidase), and x-oxidation (cytochrome P450). PPAR response elements (PPRE) have been identified in the 50 flanking sequences of peroxisome proliferator-inducible genes such as the rat acyl-CoA oxidase (AOX) gene [18,19], the gene for cytochrome P450 CYP4A6 [20], and many other genes that were involved in fatty acid metabolism. Gene targeting studies confirmed that PPARa is essential for the up-regulation of these genes caused by fasting [21,22] or by pharmacological stimulation with synthetic ligands such as the fibrates [9,23,24]. In an attempt to investigate the cross-talk among the nuclear receptors, we have found that VDR, which mediates the effects of 1a,25-dihydroxyvitamin D3 , represses the transcriptional activity of PPARa in a 1,25(OH)2 D3 dependent manner. In addition to the best known actions of vitamin D3 on classic target organs related to mineral homeostasis (i.e., the intestine, kidney, and bone), vitamin D receptors, as first demonstrated by thaw-mount receptor autoradiography and later by immunohistochemistry and molecular biological techniques [25,26], are present in a variety of cell lines (including keratinocytes, lymphocytes or hematopoietic cells), as well as in numerous tissues (such as pituitary, pancreas, brain, thyroid, adrenal, prostate, thymus, testes, muscle, gastrointestinal tract, breast, and skin) suggesting other functions of the hormone beyond bone metabolism and mineral homeostasis [27,28]. Indeed, at the liver level, it has been shown that 1,25(OH)2 D3 induced DNA replication enzymes [29–31] stimulate the hepatic glycogen synthetic pathway and the synthesis of transferrin [32,33], as well as significantly promoted normal liver recovery after partial hepatectomy [34]. Recently, it has been reported that VDR also functions as a receptor for the secondary bile acid, lithocholic acid (LCA), which is hepatotoxic and a potential enteric carcinogen. We obtained results, which indicated that VDR negatively regulated the PPARa action on PPRE. In general, transcriptional regulation of genes is controlled by proteins that bind to specific DNA sequences. We report here the novel action of VDR in controlling gene expression through PPREs. Studies using chimeric receptors show that the ligand binding domain of VDR is responsible for the inhibition and that the DNA binding domain of the receptor is indispensable. VDR negatively regulates PPARa signaling by a novel mechanism that involves protein–protein interactions rather than a direct DNA binding. VDR signaling might be considered as a factor regulating lipid metabolism via PPARa pathway.
Materials and methods Plasmids constructions. Full length of rPPARa was inserted into BamHI site of pCMV expression plasmid using BamHI linker [35].
PPRE-TK-luciferase reporter plasmid harbors three copies of PPRE from AOX promoter in front of TK promoter [36]. VDR cDNA was obtained from Dr. O’Malley and expressed under the control of CMV promoter (pcDNA, Invitrogen). To construct the mammalian expression vector for Gal4 DBD fusion protein, PCR amplified ligand binding domain of PPARa was inserted in-frame into BamHI and XbaI cloning sites of the pM vector (Clonetech). Correct insertion was confirmed by di-deoxy nucleotide sequencing. Mammalian expression vectors for PPARc [37], retinoid X receptor a (RXRa) [38], retinoic acid receptor a (RARa) [39], glucocorticoid receptor (GR) [40], and vitamin D3 receptor (VDR) [41] were described previously. VDRE 2 E1b-luciferase reporter plasmid is the kind gift from Dr. Freedman [42]. In order to create a DNA binding defective mutant VDR, an artificial mutation at a base coding for a cysteine residue in the P box [43] of the first zinc finger of the DNA binding domain in VDR was substituted to phenylalanine (C66F) using QuikChange Multi Sitedirected Mutagenesis kit (Stratagene). RNA Northern analysis. Total RNA was isolated using RNeasy kit (Qiagen). Fifteen micrograms of total RNA was size fractionated in 1% denaturing agarose–formaldehyde gel, transferred onto a HybondNþ nylon membrane (Amersham–Pharmacia Biotech), and crosslinked with UV light (Stratalinker, Stratagene). Hybridizations were performed in Express Hybri solution (Clonetech) at 65 °C for 2 h with AOX cDNA labeled by [32 P]dCTP using random primer labeling kit (Amersham). After hybridization, membranes were washed in 0.1% SDS, 0.1 SSC buffer at 65 °C. The results were visualized using a Phosphor Imager (Fuji BAS 1500). Blots were stripped and reprobed with a cDNA for b-actin to control for RNA loading. Transcription assay. COS 1 cells were obtained from ATCC (CRL1777) as frozen stocks, then thawed and grown in 10 cm plates in DMEM supplemented by 10% fetal calf serum (Gibco), 100 U/ml penicillin, and 100 mg/ml streptomycin. Transfection was done in COS1 cells using the standard calcium phosphate procedure. Typically, 250 ng luciferase reporter was co-transfected with 100 ng of the indicated expression vectors. Cells were incubated for 12 h, and the medium on the cells was replaced with fresh medium and the indicated ligand was added. Cells were harvested after 24 h and b-galactosidase activity was measured by the method previously described using chlorophenol red-b-D -galactopyranoside (CPRG) as a substrate [44]. Luciferase assays were performed using the PicaGene Luciferase Assay System (Toyo Inki, Tokyo). Luciferase activity was determined using Lumat LB9501 (Berthold Japan K.K., Tokyo, Japan) and expressed as RLU (Relative Light Units) normalized to the b-galactosidase activity. Troglitazone was obtained from Sankyo Co., Ltd. 1,25(OH)2 D3 was from Chugai Co., Ltd.
Results VDR inhibits the PPARa-mediated transcription in a ligand-dependent manner We first examined the effect of VDR and 1,25(OH)2 D3 on a ligand-dependent transcriptional activity of PPARa and c in transient transfection assays using COS1 cells. As shown in Fig. 1, the PPRE 3 TK luciferase reporter was activated by PPARa or PPARc in the presence of their ligands, clofibric acid and troglitazone, respectively. Co-transfection of VDR significantly inhibited the PPARa activity in a 1,25(OH)2 D3 -dependent manner but did not affect the PPARc function. Addition of increasing amounts of 1,25(OH)2 D3 to the cells, which were co-transfected with VDR, revealed the dose-dependent
T. Sakuma et al. / Biochemical and Biophysical Research Communications 312 (2003) 513–519
Fig. 1. VDR inhibits the PPARa but not PPARc action on PPRE. COS1 cells were transfected with PPRE-TK-luciferase reporter plasmid (250 ng) along with the expression plasmid for PPARa or PPARc. As much as 12.5 ng of the parental pcDNA expression vector or the VDR expression vector was co-transfected. Cells were treated with either dimethyl sulfoxide as vehicle or 1 mM clofibric acid in the presence or absence of 1,25(OH)2 D3 as indicated. Cell extracts were assayed for luciferase activity. Each luciferase activity was corrected for transfection efficiency by measuring b-galactosidase activity transfected together. Each transfection was conducted in triplicate and data represent means SD of more than three individual experiments.
inhibition of the PPARa induced reporter activity (Fig. 2). Half-maximal inhibition was obtained at about 1–2 109 M, which was consistent with reported affinity constant and transcriptional activation of VDR,
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suggesting physiological role of VDR in controlling the gene expression through PPRE. In order to confirm that inhibition occurred at physiological state, it might be important to titrate the dose of VDR expression plasmid necessary for inhibition of PPARa. We compared the titration curve of VDR expression plasmid in the inhibition of PPARa on PPRE with that in the D3-dependent transactivation of VDRE. As shown in Fig. 3, experiments with co-transfection of an increasing amount of VDR expression vector showed that the inhibitory effect of VDR on PPARa occurred in a similar dose of VDR necessary for activation of VDRE, suggesting physiological role of VDR in controlling the gene expression through PPRE in vivo. Then we tested the specificity of the inhibitory effect among the nuclear receptor family. Expression plasmid for glucocorticoid receptor (GR) or retinoic acid receptor (RAR) was co-transfected with the expression vector for PPARa and PPRE-TK-luciferase reporter construct. As shown in Fig. 4, none of the nuclear receptors used but VDR showed the inhibition of PPRE-TK-luciferase reporter. DNA binding domain of VDR is not indispensable for the inhibitory effect on PPAR In order to define the molecular mechanism underlying the inhibitory effect of VDR, we examine the contribution of DNA binding of VDR to the inhibition. A mutation was introduced in the DNA binding domain of VDR and tested whether the mutant VDR still possessed the ability to inhibit the PPARa action.
Fig. 2. 1,25(OH)2 D3 dose-dependency in the inhibition of PPARa. COS1 cells were transiently transfected with 250 ng PPRE-TK-luciferase reporter plasmid and 12.5 ng pCMV-PPARa expression vector (left), or VDRE-luciferase vector (right) along with 12.5 ng VDR expression plasmid. Cells were treated with indicated concentration of 1,25(OH)2 D3 in the presence (left) or absence (right) of 1 mM clofibric acid as shown. Twelve hours later, cells were collected for analysis of reporter gene assays. Each luciferase activity was corrected for transfection efficiency by measuring b-galactosidase activity transfected together. Each transfection was conducted in triplicate and data represent means SD of more than three individual experiments.
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Fig. 3. Dose-dependency of VDR expression plasmid for PPARa inhibition and VDRE activation. COS1 cells were transfected with PPRE-TKluciferase reporter plasmid (250 ng) and 12.5 ng of pCMV-PPARa expression vector (left) and VDRE-luciferase reporter plasmid (250 ng) (right). Indicated amounts of VDR expression plasmid were co-transfected. Cells were treated with 1 mM clofibric acid and 108 M of 1,25(OH)2 D3 (left) or 108 M of 1,25(OH)2 D3 (right). Each transfection was conducted in triplicate and data represent means SD of more than three individual experiments.
PPAR-mediated activation not by competing for binding to DNA. To further clarify the molecular mechanism whereby VDR inhibits the PPARa transcription, chimeric receptor was created in which the ligand binding domain of PPARa was tethered to Gal4 DNA binding domain. Interestingly, this Gal4-PPARa (LBD) chimera is still repressed by VDR in a ligand-dependent manner, indicating that specific DNA binding of PPARa is not required for the transcriptional repression (Fig. 6). These findings strongly suggested that VDR negatively regulated the PPARa transcription through its ligand binding domain that might cross-talk with other transcription factors. VDR modulate the PPARa-dependent transcriptional activation in AOX gene Fig. 4. Effect of RAR and GR on PPAR activity. COS1 cells were transfected with PPRE-TK-luciferase reporter plasmid (250 ng) and 12.5 ng pCMV-PPARa. Indicated parental expression vector or receptor expression vector (12.5 ng each) was co-transfected. Cells were treated with either dimethyl sulfoxide as vehicle or 103 M clofibric acid in the presence or absence of 107 M dexamethasone for GR, 107 M all trans-RA for RAR or 108 M of 1,25(OH)2 D3 for VDR. Cell extracts were assayed for luciferase activity. Each luciferase activity was corrected for transfection efficiency by measuring b-galactosidase activity transfected together. Each transfection was conducted in triplicate and data represent means SD of more than three individual experiments.
In transient co-transfection assay, as shown in Fig. 5, the mutant VDR showed the similar inhibitory effect on PPARa. These results indicated that VDR inhibited the
We confirmed the evidence for the negative regulation by VDR in a native gene using an AOX gene promoter. AOX-luciferase reporter plasmid was activated by PPARa in the presence of clofibric acid. This activation was completely suppressed by co-transfection of VDR expression plasmid (Fig. 7). These results are consistent with those observed when the PPRE-TK luciferase was used. The effects of PPARa agonist on AOX mRNA expression in rat hepatoma cells, H4IIE cells, were studied. As shown in Fig. 8, treatment of H4IIE cells with 1 mM Wy14,643 increased the AOX mRNA expression, however, co-incubation with 108 M 1,25(OH)2 D3 significantly diminished the activation by Wy14,643.
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Fig. 5. Mutation in DNA binding domain of VDR did not affect the inhibition of PPARa. (A) COS1 cells were transfected with PPRE-TK-luciferase reporter plasmid (250 ng) and 12.5 ng pCMV-PPARa. Indicated parental expression vector, wild-type or mutant VDR expression vector (12.5 ng each) was co-transfected. Cells were treated with either dimethyl sulfoxide as vehicle or 103 M clofibric acid in the presence of 108 M of 1,25(OH)2 D3 . (B) COS1 cells were transfected with VDRE-E1b-luciferase reporter plasmid (250 ng) along with 12.5 ng wild-type or mutant VDR expression vector. Cells were treated with ethanol as vehicle or 108 M of 1,25(OH)2 D3 . Cell extracts were assayed for luciferase activity. Each luciferase activity was corrected for transfection efficiency by measuring b-galactosidase activity transfected together. Each transfection was conducted in triplicate and data represent means SD of more than three individual experiments.
Fig. 6. VDR represses the transcriptional activation by Gal4VDR(LBD). COS1 cells were transfected with UAS 4-TK-luciferase reporter plasmid (250 ng) and 25 ng pM-PPARa (LBD). Indicated parental expression vector or wild-type VDR expression vector (12.5 ng each) was co-transfected. Cells were treated with either dimethyl sulfoxide as vehicle or 103 M clofibric acid in the presence of 108 M of 1,25(OH)2 D3 . Cell extracts were assayed for luciferase activity. Each luciferase activity was corrected for transfection efficiency by measuring b-galactosidase activity transfected together. Each transfection was conducted in triplicate and data represent means SD of more than three individual experiments.
Fig. 7. VDR modulate the ligand-dependent transcriptional activation by PPARa in AOX gene. COS1 cells were transfected with 250 ng of the luciferase reporter plasmid containing the AOX promoter and 12.5 ng pCMV-PPARa expression plasmid. As much as 12.5 ng of either the parental pcDNA expression vector or VDR expression plasmid was co-transfected. Cells were treated with either dimethyl sulfoxide as vehicle or 103 M clofibric acid in the presence or absence of 1,25(OH)2 D3 . Cell extracts were assayed for luciferase activity. Each luciferase activity was corrected for transfection efficiency by measuring b-galactosidase activity transfected together.
Discussion
b-oxidation of fatty acids in rodent liver [45–49]. Regulation of the expression of genes involved in lipid metabolism by hypolipidemic drugs and hormones is of great physiological and clinical interest. In this paper we show that VDR negatively regulates PPARa-dependent
It is well documented that hypolipidemic drugs, such as clofibrate, induce peroxisome proliferation and increase the activity of enzymes of the peroxisomal
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Fig. 8. Effect of 1,25(OH)2 D3 on AOX mRNA expression. Effect of PPARa agonist treatment on AOX mRNA expression in rat hepatoma cell line, H4IIE. H4IIE cells were treated with Wy14,643 in the absence or presence of 108 M of 1,25(OH)2 D3 . Total RNA (15 lg/lane) was isolated from H4IIE cells with indicated treatment. Isolated RNA was separated by electrophoresis, blotted to nylon membrane, and hybridized with 32 P-labeled rat AOX cDNA. b-Actin levels are shown to demonstrate that equivalent amounts of RNA were loaded in each lane.
transcription. The inhibition was 1,25(OH)2 D3 -dependent and specific among the nuclear receptors. Moreover, the inhibition by VDR was PPARa specific; especially PPARc-dependent activation was not altered by VDR. This is a novel mechanism of VDR to regulate gene expression through DR1 motif (PPRE). To date, several enzymes, which are involved in peroxisomal boxidation, have been shown to be regulated by PPARa through PPRE. Peroxisomal fatty acid acyl-CoA oxidase [17–19], peroxisomal enoil-CoA hydratase/3hydroxyacyl-CoA dehydrogenase (bifunctional enzyme) [50,51], the liver fatty acid binding protein [52], and the rabbit P450 4A6 fatty acid x-hydroxylase [20] have been shown to be regulated by PPARa. Our results strongly suggest that these enzymes might be modulated by VDR through convergence of PPARa signaling pathway. We next tried to clarify the mechanism for negative regulation of PPARa-mediated transcription by VDR. In general, three different mechanisms are proposed for inhibition of transcription factors, namely competition for binding to response element, formation of inactive heterodimer, and squelching cofactor. Redundancy of DNA binding domain of VDR for the inhibition of PPARa was demonstrated using a DNA binding defective mutant of VDR. An artificial mutation at a base coding for a cysteine residue in the P box [43] of the first zinc finger of the DNA binding domain in VDR destroyed binding to DNA. This DNA binding defective mutant still possesses the inhibitory effect on PPARa action in transient transfection assays, while it lost the function to activate the classical VDRE reporter (Fig. 5). These results also indicate that it is quite unlikely for VDR to induce a factor, which mediates the specific inhibition of PPARa. We have now shown that DNA binding is not required for inhibition of PPARa activity. Moreover, we demonstrated that Gal4-PPARa was similarly inhibited by VDR on UAS-luciferase reporter, indicating that VDR targeted the LBD of PPARa independently of DNA
element where the PPARa bound. Second possible mechanism is formation of inactive VDR/PPARa heterodimer. We tried to detect the interaction between VDR and PPARa in several methods using pull-down assay, two-hybrid assay both in yeast and mammalian, however, either method failed to demonstrate the interaction (data not shown). Third possible mechanism is squelching. Co-expression of RXRa, steroid receptor coactivator-1 (SRC-1) [53], CREB-binding protein (CBP/p300) [54] or ACTR cannot reverse the inhibitory effect of VDR (data not shown), suggesting that the inhibition by VDR might not be mediated through the sequestration of limiting amounts of these common cofactors by VDR. Given that the inhibition is specific between PPARa and VDR, VDR probably targets the specific cofactor for PPARa that is essential for PPARa activity. Mechanism of direct or indirect specific cross-talk between PPARa and VDR appears to be important for PPARa inhibition by VDR. Further studies will be required to elucidate the precise molecular mechanism underlying the inhibitory effect of VDR on PPARa. The inhibitory effect of 1,25(OH)2 D3 on the gene expression of 25(OH)D3 1a-hydroxylase or parathyroid hormone is a well-documented phenomenon. It will be of interest to explore the possibility that VDR-dependent inhibition in this study might be involved in the molecular basis of the feedback repression of certain genes that are known to be negatively regulated by 1,25(OH)2 D3 . Since repression plays a role in many physiological and pathological processes, a better understanding of the specificity and determinants of repression is likely to provide novel approaches to human biology and disease. In conclusion, we suggest the remarkable potential of VDR to couple with PPAR signaling pathway regulating lipid metabolism, or cell growth and differentiation. Action of nuclear receptors appeared to have a great diversity with their promiscuous interaction. We presented the evidence for enormous possibilities of crosstalk among nuclear receptor signaling pathway.
Acknowledgments We thank Dr. Ronald M. Evans (The Salk Institute for Biological Studies, La Jolla, California) for providing PPRE-TK-luciferase, AOX-luciferase reporter plasmid, RXRa, and GR cDNA. We also thank Dr. Bert O’Malley for VDR and SRC-1 cDNA, Dr. Richard H Goodman for mouse CBP expression vector. PPARc expression plasmid is kind gift from Dr. Alex Elbrecht. We thank Sankyo for Troglitazone and Chugai for 1,25(OH)2 D3 .
References [1] G. Krey, O. Braissant, F. L’Horset, E. Kalkhoven, M. Perroud, M.G. Parker, W. Wahli, Mol. Endocrinol. 11 (1997) 779–791.
T. Sakuma et al. / Biochemical and Biophysical Research Communications 312 (2003) 513–519 [2] S.A. Kliewer, S.S. Sundseth, S.A. Jones, P.J. Brown, G.B. Wisely, C.S. Koble, P. Devchand, W. Wahli, T.M. Willson, J.M. Lenhard, J.M. Lehmann, Proc. Natl. Acad. Sci. USA 94 (1997) 4318–4323. [3] S.A. Kliewer, J.M. Lenhard, T.M. Willson, I. Patel, D.C. Morris, J.M. Lehmann, Cell 83 (1995) 813–819. [4] B.M. Forman, P. Tontonoz, J. Chen, R.P. Brun, B.M. Spiegelman, R.M. Evans, Cell 83 (1995) 803–812. [5] B.M. Forman, J. Chen, R.M. Evans, Proc. Natl. Acad. Sci. USA 94 (1997) 4312–4317. [6] S. Westin, R. Kurokawa, R.T. Nolte, G.B. Wisely, E.M. McInerney, D.W. Rose, M.V. Milburn, M.G. Rosenfeld, C.K. Glass, Nature 395 (1998) 199–202. [7] R.T. Nolte, G.B. Wisely, S. Westin, J.E. Cobb, M.H. Lambert, R. Kurokawa, M.G. Rosenfeld, T.M. Willson, C.K. Glass, M.V. Milburn, Nature 395 (1998) 137–143. [8] C. Dreyer, H. Keller, A. Mahfoudi, V. Laudet, G. Krey, W. Wahli, Biol. Cell 77 (1993) 67–76. [9] J.M. Peters, N. Hennuyer, B. Staels, J.C. Fruchart, C. Fievet, F.J. Gonzalez, J. Auwerx, J. Biol. Chem. 272 (1997) 27307–27312. [10] Y. Barak, M.C. Nelson, E.S. Ong, Y.Z. Jones, P. Ruiz-Lozano, K.R. Chien, A. Koder, R.M. Evans, Mol. Cell 4 (1999) 585– 595. [11] E.D. Rosen, P. Sarraf, A.E. Troy, G. Bradwin, K. Moore, D.S. Milstone, B.M. Spiegelman, R.M. Mortensen, Mol. Cell 4 (1999) 611–617. [12] L. Michalik, B. Desvergne, N.S. Tan, S. Basu-Modak, P. Escher, J. Rieusset, J.M. Peters, G. Kaya, F.J. Gonzalez, J. Zakany, D. Metzger, P. Chambon, D. Duboule, W. Wahli, J. Cell Biol. 154 (2001) 799–814. [13] N.S. Tan, L. Michalik, N. Noy, R. Yasmin, C. Pacot, M. Heim, B. Fluhmann, B. Desvergne, W. Wahli, Genes Dev. 15 (2001) 3263– 3277. [14] N. Di-Poi, N.S. Tan, L. Michalik, W. Wahli, B. Desvergne, Mol. Cell 10 (2002) 721–733. [15] A. Chawla, C.H. Lee, Y. Barak, W. He, J. Rosenfeld, D. Liao, J. Han, H. Kang, R.M. Evans, Proc. Natl. Acad. Sci. USA 100 (2003) 1268–1273. [16] I. Issemann, S. Green, Nature 347 (1990) 645–650. [17] C. Dreyer, G. Krey, H. Keller, F. Givel, G. Helftenbein, W. Wahli, Cell 68 (1992) 879–887. [18] T. Osumi, J.K. Wen, T. Hashimoto, Biochem. Biophys. Res. Commun. 175 (1991) 866–871. [19] J.D. Tugwood, I. Issemann, R.G. Anderson, K.R. Bundell, W.L. McPheat, S. Green, EMBO J. 11 (1992) 433–439. [20] A.S. Muerhoff, K.J. Griffin, E.F. Johnson, J. Biol. Chem. 267 (1992) 19051–19053. [21] T.C. Leone, C.J. Weinheimer, D.P. Kelly, Proc. Natl. Acad. Sci. USA 96 (1999) 7473–7478. [22] S. Kersten, J. Seydoux, J.M. Peters, F.J. Gonzalez, B. Desvergne, W. Wahli, J. Clin. Invest. 103 (1999) 1489–1498. [23] S.S. Lee, T. Pineau, J. Drago, E.J. Lee, J.W. Owens, D.L. Kroetz, P.M. Fernandez-Salguero, H. Westphal, F.J. Gonzalez, Mol. Cell. Biol. 15 (1995) 3012–3022. [24] T. Aoyama, J.M. Peters, N. Iritani, T. Nakajima, K. Furihata, T. Hashimoto, F.J. Gonzalez, J. Biol. Chem. 273 (1998) 5678– 5684. [25] M.R. Walters, Endocr. Rev. 13 (1992) 719–764. [26] W.E. Stumpf, Histochem. Cell Biol. 104 (1995) 417–427. [27] W.E. Stumpf, M. Sar, F.A. Reid, Y. Tanaka, H.F. DeLuca, Science 206 (1979) 1188–1190.
519
[28] A.W. Norman, I. Nemere, L.X. Zhou, J.E. Bishop, K.E. Lowe, A.C. Maiyar, E.D. Collins, T. Taoka, I. Sergeev, M.C. FarachCarson, J. Steroid Biochem. Mol. Biol. 41 (1992) 231–240. [29] T. Youdale, J.F. Whitfield, R.H. Rixon, Can. J. Biochem. Cell Biol. 63 (1985) 319–324. [30] R.H. Rixon, R.J. Isaacs, J.F. Whitfield, J. Cell. Physiol. 139 (1989) 354–360. [31] M. Sikorska, I. de Belle, J.F. Whitfield, P.R. Walker, Biochem. Cell Biol. 67 (1989) 345–351. [32] W.L. Davis, J.L. Matthews, D.B. Goodman, FASEB J. 3 (1989) 1651–1655. [33] K. Taniguchi, S. Morimoto, K. Itoh, R. Morita, K. Fukuo, S. Imanaka, T. Ogihara, Biochem. Int. 22 (1990) 37–44. [34] C. Ethier, R. Kestekian, C. Beaulieu, C. Dube, J. Havrankova, M. Gascon-Barre, Endocrinology 126 (1990) 2947–2959. [35] T. Miyamoto, A. Kaneko, T. Kakizawa, H. Yajima, K. Kamijo, R. Sekine, K. Hiramatsu, Y. Nishii, T. Hashimoto, K. Hashizume, J. Biol. Chem. 272 (1997) 7752–7758. [36] S.A. Kliewer, K. Umesono, D.J. Noonan, R.A. Heyman, R.M. Evans, Nature 358 (1992) 771–774. [37] A. Elbrecht, Y. Chen, C.A. Cullinan, N. Hayes, M. Leibowitz, D.E. Moller, J. Berger, Biochem. Biophys. Res. Commun. 224 (1996) 431–437. [38] D.J. Mangelsdorf, E.S. Ong, J.A. Dyck, R.M. Evans, Nature 345 (1990) 224–229. [39] V. Giguere, E.S. Ong, P. Segui, R.M. Evans, Nature 330 (1987) 624–629. [40] S.M. Hollenberg, C. Weinberger, E.S. Ong, G. Cerelli, A. Oro, R. Lebo, E.B. Thompson, M.G. Rosenfeld, R.M. Evans, Nature 318 (1985) 635–641. [41] A.R. Baker, D.P. McDonnell, M. Hughes, T.M. Crisp, D.J. Mangelsdorf, M.R. Haussler, J.W. Pike, J. Shine, B.W. O’Malley, Proc. Natl. Acad. Sci. USA 85 (1988) 3294–3298. [42] C. Rachez, Z. Suldan, J. Ward, C.P. Chang, D. Burakov, H. Erdjument-Bromage, P. Tempst, L.P. Freedman, Genes Dev. 12 (1998) 1787–1800. [43] K. Umesono, R.M. Evans, Cell 57 (1989) 1139–1146. [44] T. Miyamoto, T. Kakizawa, K. Hashizume, Mol. Cell. Biol. 19 (1999) 2644–2649. [45] M.R. Nemali, M.K. Reddy, N. Usuda, P.G. Reddy, L.D. Comeau, M.S. Rao, J.K. Reddy, Toxicol. Appl. Pharmacol. 97 (1989) 72–87. [46] J.K. Reddy, G.P. Mannaerts, Annu. Rev. Nutr. 14 (1994) 343– 370. [47] J. Vamecq, J.P. Draye, J. Biochem. (Tokyo) 106 (1989) 216–222. [48] E.A. Lock, A.M. Mitchell, C.R. Elcombe, Annu. Rev. Pharmacol. Toxicol. 29 (1989) 145–163. [49] R.K. Berge, L.H. Hosoy, A. Aarsland, O.M. Bakke, M. Farstad, Toxicol. Appl. Pharmacol. 73 (1984) 35–41. [50] B. Zhang, S.L. Marcus, K.S. Miyata, S. Subramani, J.P. Capone, R.A. Rachubinski, J. Biol. Chem. 268 (1993) 12939–12945. [51] O. Bardot, T.C. Aldridge, N. Latruffe, S. Green, Biochem. Biophys. Res. Commun. 192 (1993) 37–45. [52] I. Issemann, R. Prince, J. Tugwood, S. Green, Biochem. Soc. Trans. 20 (1992) 824–827. [53] S.A. Onate, S.Y. Tsai, M.J. Tsai, B.W. O’Malley, Science 270 (1995) 1354–1357. [54] Y. Kamei, L. Xu, T. Heinzel, J. Torchia, R. Kurokawa, B. Gloss, S.C. Lin, R.A. Heyman, D.W. Rose, C.K. Glass, M.G. Rosenfeld, Cell 85 (1996) 403–414.
Inhibition of peroxisome proliferator-activated receptor a signaling by vitamin D receptorq Takahiro Sakuma, Takahide Miyamoto,* Wei Jiang, Tomoko Kakizawa, Shin-ich Nishio, Satoru Suzuki, Teiji Takeda, Ako Oiwa, and Kiyoshi Hashizume Department of Aging Medicine and Geriatrics, Shinshu University School of Medicine, Matsumoto 390-8621, Japan Received 6 October 2003
Abstract Peroxisome proliferator-activated receptors (PPARs) are nuclear fatty acid receptors that have been implicated to play an important role in lipid and glucose homeostasis. PPARa potentiates fatty acid catabolism in the liver and is activated by the lipidlowering fibrates, whereas PPARc is essential for adipocyte differentiation. Here we report that nuclear vitamin D3 receptor (VDR) represses the transcriptional activity of PPARa but not PPARc in a 1,25(OH)2 D3 -dependent manner. The analysis using chimeric receptors revealed that ligand binding domain of PPARa and VDR was involved in the molecular basis of this functional interaction and that the DNA binding domain of VDR was not required for the suppression, suggesting a novel mechanism that might involve protein–protein interactions rather than a direct DNA binding. Furthermore, the treatment of rat hepatoma H4IIE cells with 1,25(OH)2 D3 diminishes the induction of AOX mRNA by PPARa ligands, Wy14,643. VDR signaling might be considered as a factor regulating lipid metabolism via PPARa pathway. We report here the novel action of VDR in controlling gene expression through PPARa signaling. Ó 2003 Elsevier Inc. All rights reserved.
The peroxisome proliferator-activated receptors are members of the nuclear receptor superfamily of transcription factors that regulate gene expression in response to the binding of small molecular weight lipophilic ligands. The identification of fatty acids as endogenous ligands for peroxisome proliferator-activated receptors (PPARs) has provided a unique approach to study lipid homeostasis at the molecular level [1–5]. Like other nuclear receptors that include the thyroid hormone receptor (TR), retinoic acid receptor (RAR), and vitamin D3 receptor (VDR), PPARs regulate transcription by binding to PPAR response elements (PPRE) normally present in the promoter of their target genes. PPARs form heterodimers with 9-cis-retinoic acid receptor (RXR) to recognize the PPRE that is composed of direct repeats (DRs) of the consensus q
Abbreviations: PPAR, peroxisome proliferator-activated receptor; RXR, retinoid X receptor; VDR, vitamin D receptor; GR, glucocorticoid receptor; LBD, ligand binding domain. * Corresponding author. Fax: +81-263-37-2710. E-mail address: [email protected] (T. Miyamoto). 0006-291X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2003.10.131
motif AGGTCA separated by 1 nucleotide (DR1 motif). Furthermore, ligand-dependent transcriptional activation by PPARs involves co-activator proteins to the C-terminal receptor region [6,7]. Three subtypes, PPARa (NR1C1), PPARc (NR1C2), and PPARd (NR1C3), have been identified with distinct tissue distributions and biological activities. PPARa is expressed in liver, heart, muscle, and kidney where it regulates fatty acid catabolism [8,9]. PPARc is expressed in adipocyte and macrophage and is involved in adipocyte differentiation, lipid storage, and glucose homeostasis [10,11]. PPARd is expressed ubiquitously. It has been implicated in keratinocyte differentiation and wound healing and, more recently, in mediating VLDL signaling of the macrophage [12–15]. PPARa have been shown to induce peroxisomal and microsomal enzymes involved in lipid metabolism [16,17]. Liver is the key site of metabolic integration where fatty acids are mobilized and, depending on the body’s needs, either stored or used as an energy source. Earlier studies have demonstrated that in the liver, PPARa directly regulates genes involved in fatty acid uptake [fatty acid binding protein
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(FABP)], b-oxidation (acyl-CoA oxidase), and x-oxidation (cytochrome P450). PPAR response elements (PPRE) have been identified in the 50 flanking sequences of peroxisome proliferator-inducible genes such as the rat acyl-CoA oxidase (AOX) gene [18,19], the gene for cytochrome P450 CYP4A6 [20], and many other genes that were involved in fatty acid metabolism. Gene targeting studies confirmed that PPARa is essential for the up-regulation of these genes caused by fasting [21,22] or by pharmacological stimulation with synthetic ligands such as the fibrates [9,23,24]. In an attempt to investigate the cross-talk among the nuclear receptors, we have found that VDR, which mediates the effects of 1a,25-dihydroxyvitamin D3 , represses the transcriptional activity of PPARa in a 1,25(OH)2 D3 dependent manner. In addition to the best known actions of vitamin D3 on classic target organs related to mineral homeostasis (i.e., the intestine, kidney, and bone), vitamin D receptors, as first demonstrated by thaw-mount receptor autoradiography and later by immunohistochemistry and molecular biological techniques [25,26], are present in a variety of cell lines (including keratinocytes, lymphocytes or hematopoietic cells), as well as in numerous tissues (such as pituitary, pancreas, brain, thyroid, adrenal, prostate, thymus, testes, muscle, gastrointestinal tract, breast, and skin) suggesting other functions of the hormone beyond bone metabolism and mineral homeostasis [27,28]. Indeed, at the liver level, it has been shown that 1,25(OH)2 D3 induced DNA replication enzymes [29–31] stimulate the hepatic glycogen synthetic pathway and the synthesis of transferrin [32,33], as well as significantly promoted normal liver recovery after partial hepatectomy [34]. Recently, it has been reported that VDR also functions as a receptor for the secondary bile acid, lithocholic acid (LCA), which is hepatotoxic and a potential enteric carcinogen. We obtained results, which indicated that VDR negatively regulated the PPARa action on PPRE. In general, transcriptional regulation of genes is controlled by proteins that bind to specific DNA sequences. We report here the novel action of VDR in controlling gene expression through PPREs. Studies using chimeric receptors show that the ligand binding domain of VDR is responsible for the inhibition and that the DNA binding domain of the receptor is indispensable. VDR negatively regulates PPARa signaling by a novel mechanism that involves protein–protein interactions rather than a direct DNA binding. VDR signaling might be considered as a factor regulating lipid metabolism via PPARa pathway.
Materials and methods Plasmids constructions. Full length of rPPARa was inserted into BamHI site of pCMV expression plasmid using BamHI linker [35].
PPRE-TK-luciferase reporter plasmid harbors three copies of PPRE from AOX promoter in front of TK promoter [36]. VDR cDNA was obtained from Dr. O’Malley and expressed under the control of CMV promoter (pcDNA, Invitrogen). To construct the mammalian expression vector for Gal4 DBD fusion protein, PCR amplified ligand binding domain of PPARa was inserted in-frame into BamHI and XbaI cloning sites of the pM vector (Clonetech). Correct insertion was confirmed by di-deoxy nucleotide sequencing. Mammalian expression vectors for PPARc [37], retinoid X receptor a (RXRa) [38], retinoic acid receptor a (RARa) [39], glucocorticoid receptor (GR) [40], and vitamin D3 receptor (VDR) [41] were described previously. VDRE 2 E1b-luciferase reporter plasmid is the kind gift from Dr. Freedman [42]. In order to create a DNA binding defective mutant VDR, an artificial mutation at a base coding for a cysteine residue in the P box [43] of the first zinc finger of the DNA binding domain in VDR was substituted to phenylalanine (C66F) using QuikChange Multi Sitedirected Mutagenesis kit (Stratagene). RNA Northern analysis. Total RNA was isolated using RNeasy kit (Qiagen). Fifteen micrograms of total RNA was size fractionated in 1% denaturing agarose–formaldehyde gel, transferred onto a HybondNþ nylon membrane (Amersham–Pharmacia Biotech), and crosslinked with UV light (Stratalinker, Stratagene). Hybridizations were performed in Express Hybri solution (Clonetech) at 65 °C for 2 h with AOX cDNA labeled by [32 P]dCTP using random primer labeling kit (Amersham). After hybridization, membranes were washed in 0.1% SDS, 0.1 SSC buffer at 65 °C. The results were visualized using a Phosphor Imager (Fuji BAS 1500). Blots were stripped and reprobed with a cDNA for b-actin to control for RNA loading. Transcription assay. COS 1 cells were obtained from ATCC (CRL1777) as frozen stocks, then thawed and grown in 10 cm plates in DMEM supplemented by 10% fetal calf serum (Gibco), 100 U/ml penicillin, and 100 mg/ml streptomycin. Transfection was done in COS1 cells using the standard calcium phosphate procedure. Typically, 250 ng luciferase reporter was co-transfected with 100 ng of the indicated expression vectors. Cells were incubated for 12 h, and the medium on the cells was replaced with fresh medium and the indicated ligand was added. Cells were harvested after 24 h and b-galactosidase activity was measured by the method previously described using chlorophenol red-b-D -galactopyranoside (CPRG) as a substrate [44]. Luciferase assays were performed using the PicaGene Luciferase Assay System (Toyo Inki, Tokyo). Luciferase activity was determined using Lumat LB9501 (Berthold Japan K.K., Tokyo, Japan) and expressed as RLU (Relative Light Units) normalized to the b-galactosidase activity. Troglitazone was obtained from Sankyo Co., Ltd. 1,25(OH)2 D3 was from Chugai Co., Ltd.
Results VDR inhibits the PPARa-mediated transcription in a ligand-dependent manner We first examined the effect of VDR and 1,25(OH)2 D3 on a ligand-dependent transcriptional activity of PPARa and c in transient transfection assays using COS1 cells. As shown in Fig. 1, the PPRE 3 TK luciferase reporter was activated by PPARa or PPARc in the presence of their ligands, clofibric acid and troglitazone, respectively. Co-transfection of VDR significantly inhibited the PPARa activity in a 1,25(OH)2 D3 -dependent manner but did not affect the PPARc function. Addition of increasing amounts of 1,25(OH)2 D3 to the cells, which were co-transfected with VDR, revealed the dose-dependent
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Fig. 1. VDR inhibits the PPARa but not PPARc action on PPRE. COS1 cells were transfected with PPRE-TK-luciferase reporter plasmid (250 ng) along with the expression plasmid for PPARa or PPARc. As much as 12.5 ng of the parental pcDNA expression vector or the VDR expression vector was co-transfected. Cells were treated with either dimethyl sulfoxide as vehicle or 1 mM clofibric acid in the presence or absence of 1,25(OH)2 D3 as indicated. Cell extracts were assayed for luciferase activity. Each luciferase activity was corrected for transfection efficiency by measuring b-galactosidase activity transfected together. Each transfection was conducted in triplicate and data represent means SD of more than three individual experiments.
inhibition of the PPARa induced reporter activity (Fig. 2). Half-maximal inhibition was obtained at about 1–2 109 M, which was consistent with reported affinity constant and transcriptional activation of VDR,
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suggesting physiological role of VDR in controlling the gene expression through PPRE. In order to confirm that inhibition occurred at physiological state, it might be important to titrate the dose of VDR expression plasmid necessary for inhibition of PPARa. We compared the titration curve of VDR expression plasmid in the inhibition of PPARa on PPRE with that in the D3-dependent transactivation of VDRE. As shown in Fig. 3, experiments with co-transfection of an increasing amount of VDR expression vector showed that the inhibitory effect of VDR on PPARa occurred in a similar dose of VDR necessary for activation of VDRE, suggesting physiological role of VDR in controlling the gene expression through PPRE in vivo. Then we tested the specificity of the inhibitory effect among the nuclear receptor family. Expression plasmid for glucocorticoid receptor (GR) or retinoic acid receptor (RAR) was co-transfected with the expression vector for PPARa and PPRE-TK-luciferase reporter construct. As shown in Fig. 4, none of the nuclear receptors used but VDR showed the inhibition of PPRE-TK-luciferase reporter. DNA binding domain of VDR is not indispensable for the inhibitory effect on PPAR In order to define the molecular mechanism underlying the inhibitory effect of VDR, we examine the contribution of DNA binding of VDR to the inhibition. A mutation was introduced in the DNA binding domain of VDR and tested whether the mutant VDR still possessed the ability to inhibit the PPARa action.
Fig. 2. 1,25(OH)2 D3 dose-dependency in the inhibition of PPARa. COS1 cells were transiently transfected with 250 ng PPRE-TK-luciferase reporter plasmid and 12.5 ng pCMV-PPARa expression vector (left), or VDRE-luciferase vector (right) along with 12.5 ng VDR expression plasmid. Cells were treated with indicated concentration of 1,25(OH)2 D3 in the presence (left) or absence (right) of 1 mM clofibric acid as shown. Twelve hours later, cells were collected for analysis of reporter gene assays. Each luciferase activity was corrected for transfection efficiency by measuring b-galactosidase activity transfected together. Each transfection was conducted in triplicate and data represent means SD of more than three individual experiments.
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Fig. 3. Dose-dependency of VDR expression plasmid for PPARa inhibition and VDRE activation. COS1 cells were transfected with PPRE-TKluciferase reporter plasmid (250 ng) and 12.5 ng of pCMV-PPARa expression vector (left) and VDRE-luciferase reporter plasmid (250 ng) (right). Indicated amounts of VDR expression plasmid were co-transfected. Cells were treated with 1 mM clofibric acid and 108 M of 1,25(OH)2 D3 (left) or 108 M of 1,25(OH)2 D3 (right). Each transfection was conducted in triplicate and data represent means SD of more than three individual experiments.
PPAR-mediated activation not by competing for binding to DNA. To further clarify the molecular mechanism whereby VDR inhibits the PPARa transcription, chimeric receptor was created in which the ligand binding domain of PPARa was tethered to Gal4 DNA binding domain. Interestingly, this Gal4-PPARa (LBD) chimera is still repressed by VDR in a ligand-dependent manner, indicating that specific DNA binding of PPARa is not required for the transcriptional repression (Fig. 6). These findings strongly suggested that VDR negatively regulated the PPARa transcription through its ligand binding domain that might cross-talk with other transcription factors. VDR modulate the PPARa-dependent transcriptional activation in AOX gene Fig. 4. Effect of RAR and GR on PPAR activity. COS1 cells were transfected with PPRE-TK-luciferase reporter plasmid (250 ng) and 12.5 ng pCMV-PPARa. Indicated parental expression vector or receptor expression vector (12.5 ng each) was co-transfected. Cells were treated with either dimethyl sulfoxide as vehicle or 103 M clofibric acid in the presence or absence of 107 M dexamethasone for GR, 107 M all trans-RA for RAR or 108 M of 1,25(OH)2 D3 for VDR. Cell extracts were assayed for luciferase activity. Each luciferase activity was corrected for transfection efficiency by measuring b-galactosidase activity transfected together. Each transfection was conducted in triplicate and data represent means SD of more than three individual experiments.
In transient co-transfection assay, as shown in Fig. 5, the mutant VDR showed the similar inhibitory effect on PPARa. These results indicated that VDR inhibited the
We confirmed the evidence for the negative regulation by VDR in a native gene using an AOX gene promoter. AOX-luciferase reporter plasmid was activated by PPARa in the presence of clofibric acid. This activation was completely suppressed by co-transfection of VDR expression plasmid (Fig. 7). These results are consistent with those observed when the PPRE-TK luciferase was used. The effects of PPARa agonist on AOX mRNA expression in rat hepatoma cells, H4IIE cells, were studied. As shown in Fig. 8, treatment of H4IIE cells with 1 mM Wy14,643 increased the AOX mRNA expression, however, co-incubation with 108 M 1,25(OH)2 D3 significantly diminished the activation by Wy14,643.
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Fig. 5. Mutation in DNA binding domain of VDR did not affect the inhibition of PPARa. (A) COS1 cells were transfected with PPRE-TK-luciferase reporter plasmid (250 ng) and 12.5 ng pCMV-PPARa. Indicated parental expression vector, wild-type or mutant VDR expression vector (12.5 ng each) was co-transfected. Cells were treated with either dimethyl sulfoxide as vehicle or 103 M clofibric acid in the presence of 108 M of 1,25(OH)2 D3 . (B) COS1 cells were transfected with VDRE-E1b-luciferase reporter plasmid (250 ng) along with 12.5 ng wild-type or mutant VDR expression vector. Cells were treated with ethanol as vehicle or 108 M of 1,25(OH)2 D3 . Cell extracts were assayed for luciferase activity. Each luciferase activity was corrected for transfection efficiency by measuring b-galactosidase activity transfected together. Each transfection was conducted in triplicate and data represent means SD of more than three individual experiments.
Fig. 6. VDR represses the transcriptional activation by Gal4VDR(LBD). COS1 cells were transfected with UAS 4-TK-luciferase reporter plasmid (250 ng) and 25 ng pM-PPARa (LBD). Indicated parental expression vector or wild-type VDR expression vector (12.5 ng each) was co-transfected. Cells were treated with either dimethyl sulfoxide as vehicle or 103 M clofibric acid in the presence of 108 M of 1,25(OH)2 D3 . Cell extracts were assayed for luciferase activity. Each luciferase activity was corrected for transfection efficiency by measuring b-galactosidase activity transfected together. Each transfection was conducted in triplicate and data represent means SD of more than three individual experiments.
Fig. 7. VDR modulate the ligand-dependent transcriptional activation by PPARa in AOX gene. COS1 cells were transfected with 250 ng of the luciferase reporter plasmid containing the AOX promoter and 12.5 ng pCMV-PPARa expression plasmid. As much as 12.5 ng of either the parental pcDNA expression vector or VDR expression plasmid was co-transfected. Cells were treated with either dimethyl sulfoxide as vehicle or 103 M clofibric acid in the presence or absence of 1,25(OH)2 D3 . Cell extracts were assayed for luciferase activity. Each luciferase activity was corrected for transfection efficiency by measuring b-galactosidase activity transfected together.
Discussion
b-oxidation of fatty acids in rodent liver [45–49]. Regulation of the expression of genes involved in lipid metabolism by hypolipidemic drugs and hormones is of great physiological and clinical interest. In this paper we show that VDR negatively regulates PPARa-dependent
It is well documented that hypolipidemic drugs, such as clofibrate, induce peroxisome proliferation and increase the activity of enzymes of the peroxisomal
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Fig. 8. Effect of 1,25(OH)2 D3 on AOX mRNA expression. Effect of PPARa agonist treatment on AOX mRNA expression in rat hepatoma cell line, H4IIE. H4IIE cells were treated with Wy14,643 in the absence or presence of 108 M of 1,25(OH)2 D3 . Total RNA (15 lg/lane) was isolated from H4IIE cells with indicated treatment. Isolated RNA was separated by electrophoresis, blotted to nylon membrane, and hybridized with 32 P-labeled rat AOX cDNA. b-Actin levels are shown to demonstrate that equivalent amounts of RNA were loaded in each lane.
transcription. The inhibition was 1,25(OH)2 D3 -dependent and specific among the nuclear receptors. Moreover, the inhibition by VDR was PPARa specific; especially PPARc-dependent activation was not altered by VDR. This is a novel mechanism of VDR to regulate gene expression through DR1 motif (PPRE). To date, several enzymes, which are involved in peroxisomal boxidation, have been shown to be regulated by PPARa through PPRE. Peroxisomal fatty acid acyl-CoA oxidase [17–19], peroxisomal enoil-CoA hydratase/3hydroxyacyl-CoA dehydrogenase (bifunctional enzyme) [50,51], the liver fatty acid binding protein [52], and the rabbit P450 4A6 fatty acid x-hydroxylase [20] have been shown to be regulated by PPARa. Our results strongly suggest that these enzymes might be modulated by VDR through convergence of PPARa signaling pathway. We next tried to clarify the mechanism for negative regulation of PPARa-mediated transcription by VDR. In general, three different mechanisms are proposed for inhibition of transcription factors, namely competition for binding to response element, formation of inactive heterodimer, and squelching cofactor. Redundancy of DNA binding domain of VDR for the inhibition of PPARa was demonstrated using a DNA binding defective mutant of VDR. An artificial mutation at a base coding for a cysteine residue in the P box [43] of the first zinc finger of the DNA binding domain in VDR destroyed binding to DNA. This DNA binding defective mutant still possesses the inhibitory effect on PPARa action in transient transfection assays, while it lost the function to activate the classical VDRE reporter (Fig. 5). These results also indicate that it is quite unlikely for VDR to induce a factor, which mediates the specific inhibition of PPARa. We have now shown that DNA binding is not required for inhibition of PPARa activity. Moreover, we demonstrated that Gal4-PPARa was similarly inhibited by VDR on UAS-luciferase reporter, indicating that VDR targeted the LBD of PPARa independently of DNA
element where the PPARa bound. Second possible mechanism is formation of inactive VDR/PPARa heterodimer. We tried to detect the interaction between VDR and PPARa in several methods using pull-down assay, two-hybrid assay both in yeast and mammalian, however, either method failed to demonstrate the interaction (data not shown). Third possible mechanism is squelching. Co-expression of RXRa, steroid receptor coactivator-1 (SRC-1) [53], CREB-binding protein (CBP/p300) [54] or ACTR cannot reverse the inhibitory effect of VDR (data not shown), suggesting that the inhibition by VDR might not be mediated through the sequestration of limiting amounts of these common cofactors by VDR. Given that the inhibition is specific between PPARa and VDR, VDR probably targets the specific cofactor for PPARa that is essential for PPARa activity. Mechanism of direct or indirect specific cross-talk between PPARa and VDR appears to be important for PPARa inhibition by VDR. Further studies will be required to elucidate the precise molecular mechanism underlying the inhibitory effect of VDR on PPARa. The inhibitory effect of 1,25(OH)2 D3 on the gene expression of 25(OH)D3 1a-hydroxylase or parathyroid hormone is a well-documented phenomenon. It will be of interest to explore the possibility that VDR-dependent inhibition in this study might be involved in the molecular basis of the feedback repression of certain genes that are known to be negatively regulated by 1,25(OH)2 D3 . Since repression plays a role in many physiological and pathological processes, a better understanding of the specificity and determinants of repression is likely to provide novel approaches to human biology and disease. In conclusion, we suggest the remarkable potential of VDR to couple with PPAR signaling pathway regulating lipid metabolism, or cell growth and differentiation. Action of nuclear receptors appeared to have a great diversity with their promiscuous interaction. We presented the evidence for enormous possibilities of crosstalk among nuclear receptor signaling pathway.
Acknowledgments We thank Dr. Ronald M. Evans (The Salk Institute for Biological Studies, La Jolla, California) for providing PPRE-TK-luciferase, AOX-luciferase reporter plasmid, RXRa, and GR cDNA. We also thank Dr. Bert O’Malley for VDR and SRC-1 cDNA, Dr. Richard H Goodman for mouse CBP expression vector. PPARc expression plasmid is kind gift from Dr. Alex Elbrecht. We thank Sankyo for Troglitazone and Chugai for 1,25(OH)2 D3 .
References [1] G. Krey, O. Braissant, F. L’Horset, E. Kalkhoven, M. Perroud, M.G. Parker, W. Wahli, Mol. Endocrinol. 11 (1997) 779–791.
T. Sakuma et al. / Biochemical and Biophysical Research Communications 312 (2003) 513–519 [2] S.A. Kliewer, S.S. Sundseth, S.A. Jones, P.J. Brown, G.B. Wisely, C.S. Koble, P. Devchand, W. Wahli, T.M. Willson, J.M. Lenhard, J.M. Lehmann, Proc. Natl. Acad. Sci. USA 94 (1997) 4318–4323. [3] S.A. Kliewer, J.M. Lenhard, T.M. Willson, I. Patel, D.C. Morris, J.M. Lehmann, Cell 83 (1995) 813–819. [4] B.M. Forman, P. Tontonoz, J. Chen, R.P. Brun, B.M. Spiegelman, R.M. Evans, Cell 83 (1995) 803–812. [5] B.M. Forman, J. Chen, R.M. Evans, Proc. Natl. Acad. Sci. USA 94 (1997) 4312–4317. [6] S. Westin, R. Kurokawa, R.T. Nolte, G.B. Wisely, E.M. McInerney, D.W. Rose, M.V. Milburn, M.G. Rosenfeld, C.K. Glass, Nature 395 (1998) 199–202. [7] R.T. Nolte, G.B. Wisely, S. Westin, J.E. Cobb, M.H. Lambert, R. Kurokawa, M.G. Rosenfeld, T.M. Willson, C.K. Glass, M.V. Milburn, Nature 395 (1998) 137–143. [8] C. Dreyer, H. Keller, A. Mahfoudi, V. Laudet, G. Krey, W. Wahli, Biol. Cell 77 (1993) 67–76. [9] J.M. Peters, N. Hennuyer, B. Staels, J.C. Fruchart, C. Fievet, F.J. Gonzalez, J. Auwerx, J. Biol. Chem. 272 (1997) 27307–27312. [10] Y. Barak, M.C. Nelson, E.S. Ong, Y.Z. Jones, P. Ruiz-Lozano, K.R. Chien, A. Koder, R.M. Evans, Mol. Cell 4 (1999) 585– 595. [11] E.D. Rosen, P. Sarraf, A.E. Troy, G. Bradwin, K. Moore, D.S. Milstone, B.M. Spiegelman, R.M. Mortensen, Mol. Cell 4 (1999) 611–617. [12] L. Michalik, B. Desvergne, N.S. Tan, S. Basu-Modak, P. Escher, J. Rieusset, J.M. Peters, G. Kaya, F.J. Gonzalez, J. Zakany, D. Metzger, P. Chambon, D. Duboule, W. Wahli, J. Cell Biol. 154 (2001) 799–814. [13] N.S. Tan, L. Michalik, N. Noy, R. Yasmin, C. Pacot, M. Heim, B. Fluhmann, B. Desvergne, W. Wahli, Genes Dev. 15 (2001) 3263– 3277. [14] N. Di-Poi, N.S. Tan, L. Michalik, W. Wahli, B. Desvergne, Mol. Cell 10 (2002) 721–733. [15] A. Chawla, C.H. Lee, Y. Barak, W. He, J. Rosenfeld, D. Liao, J. Han, H. Kang, R.M. Evans, Proc. Natl. Acad. Sci. USA 100 (2003) 1268–1273. [16] I. Issemann, S. Green, Nature 347 (1990) 645–650. [17] C. Dreyer, G. Krey, H. Keller, F. Givel, G. Helftenbein, W. Wahli, Cell 68 (1992) 879–887. [18] T. Osumi, J.K. Wen, T. Hashimoto, Biochem. Biophys. Res. Commun. 175 (1991) 866–871. [19] J.D. Tugwood, I. Issemann, R.G. Anderson, K.R. Bundell, W.L. McPheat, S. Green, EMBO J. 11 (1992) 433–439. [20] A.S. Muerhoff, K.J. Griffin, E.F. Johnson, J. Biol. Chem. 267 (1992) 19051–19053. [21] T.C. Leone, C.J. Weinheimer, D.P. Kelly, Proc. Natl. Acad. Sci. USA 96 (1999) 7473–7478. [22] S. Kersten, J. Seydoux, J.M. Peters, F.J. Gonzalez, B. Desvergne, W. Wahli, J. Clin. Invest. 103 (1999) 1489–1498. [23] S.S. Lee, T. Pineau, J. Drago, E.J. Lee, J.W. Owens, D.L. Kroetz, P.M. Fernandez-Salguero, H. Westphal, F.J. Gonzalez, Mol. Cell. Biol. 15 (1995) 3012–3022. [24] T. Aoyama, J.M. Peters, N. Iritani, T. Nakajima, K. Furihata, T. Hashimoto, F.J. Gonzalez, J. Biol. Chem. 273 (1998) 5678– 5684. [25] M.R. Walters, Endocr. Rev. 13 (1992) 719–764. [26] W.E. Stumpf, Histochem. Cell Biol. 104 (1995) 417–427. [27] W.E. Stumpf, M. Sar, F.A. Reid, Y. Tanaka, H.F. DeLuca, Science 206 (1979) 1188–1190.
519
[28] A.W. Norman, I. Nemere, L.X. Zhou, J.E. Bishop, K.E. Lowe, A.C. Maiyar, E.D. Collins, T. Taoka, I. Sergeev, M.C. FarachCarson, J. Steroid Biochem. Mol. Biol. 41 (1992) 231–240. [29] T. Youdale, J.F. Whitfield, R.H. Rixon, Can. J. Biochem. Cell Biol. 63 (1985) 319–324. [30] R.H. Rixon, R.J. Isaacs, J.F. Whitfield, J. Cell. Physiol. 139 (1989) 354–360. [31] M. Sikorska, I. de Belle, J.F. Whitfield, P.R. Walker, Biochem. Cell Biol. 67 (1989) 345–351. [32] W.L. Davis, J.L. Matthews, D.B. Goodman, FASEB J. 3 (1989) 1651–1655. [33] K. Taniguchi, S. Morimoto, K. Itoh, R. Morita, K. Fukuo, S. Imanaka, T. Ogihara, Biochem. Int. 22 (1990) 37–44. [34] C. Ethier, R. Kestekian, C. Beaulieu, C. Dube, J. Havrankova, M. Gascon-Barre, Endocrinology 126 (1990) 2947–2959. [35] T. Miyamoto, A. Kaneko, T. Kakizawa, H. Yajima, K. Kamijo, R. Sekine, K. Hiramatsu, Y. Nishii, T. Hashimoto, K. Hashizume, J. Biol. Chem. 272 (1997) 7752–7758. [36] S.A. Kliewer, K. Umesono, D.J. Noonan, R.A. Heyman, R.M. Evans, Nature 358 (1992) 771–774. [37] A. Elbrecht, Y. Chen, C.A. Cullinan, N. Hayes, M. Leibowitz, D.E. Moller, J. Berger, Biochem. Biophys. Res. Commun. 224 (1996) 431–437. [38] D.J. Mangelsdorf, E.S. Ong, J.A. Dyck, R.M. Evans, Nature 345 (1990) 224–229. [39] V. Giguere, E.S. Ong, P. Segui, R.M. Evans, Nature 330 (1987) 624–629. [40] S.M. Hollenberg, C. Weinberger, E.S. Ong, G. Cerelli, A. Oro, R. Lebo, E.B. Thompson, M.G. Rosenfeld, R.M. Evans, Nature 318 (1985) 635–641. [41] A.R. Baker, D.P. McDonnell, M. Hughes, T.M. Crisp, D.J. Mangelsdorf, M.R. Haussler, J.W. Pike, J. Shine, B.W. O’Malley, Proc. Natl. Acad. Sci. USA 85 (1988) 3294–3298. [42] C. Rachez, Z. Suldan, J. Ward, C.P. Chang, D. Burakov, H. Erdjument-Bromage, P. Tempst, L.P. Freedman, Genes Dev. 12 (1998) 1787–1800. [43] K. Umesono, R.M. Evans, Cell 57 (1989) 1139–1146. [44] T. Miyamoto, T. Kakizawa, K. Hashizume, Mol. Cell. Biol. 19 (1999) 2644–2649. [45] M.R. Nemali, M.K. Reddy, N. Usuda, P.G. Reddy, L.D. Comeau, M.S. Rao, J.K. Reddy, Toxicol. Appl. Pharmacol. 97 (1989) 72–87. [46] J.K. Reddy, G.P. Mannaerts, Annu. Rev. Nutr. 14 (1994) 343– 370. [47] J. Vamecq, J.P. Draye, J. Biochem. (Tokyo) 106 (1989) 216–222. [48] E.A. Lock, A.M. Mitchell, C.R. Elcombe, Annu. Rev. Pharmacol. Toxicol. 29 (1989) 145–163. [49] R.K. Berge, L.H. Hosoy, A. Aarsland, O.M. Bakke, M. Farstad, Toxicol. Appl. Pharmacol. 73 (1984) 35–41. [50] B. Zhang, S.L. Marcus, K.S. Miyata, S. Subramani, J.P. Capone, R.A. Rachubinski, J. Biol. Chem. 268 (1993) 12939–12945. [51] O. Bardot, T.C. Aldridge, N. Latruffe, S. Green, Biochem. Biophys. Res. Commun. 192 (1993) 37–45. [52] I. Issemann, R. Prince, J. Tugwood, S. Green, Biochem. Soc. Trans. 20 (1992) 824–827. [53] S.A. Onate, S.Y. Tsai, M.J. Tsai, B.W. O’Malley, Science 270 (1995) 1354–1357. [54] Y. Kamei, L. Xu, T. Heinzel, J. Torchia, R. Kurokawa, B. Gloss, S.C. Lin, R.A. Heyman, D.W. Rose, C.K. Glass, M.G. Rosenfeld, Cell 85 (1996) 403–414.