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Endocrinology Vol. 150, No. 2 862-870
Copyright © 2009 by The Endocrine Society

A Selective Peroxisome Proliferator-Activated Receptor-{gamma} Modulator, Telmisartan, Binds to the Receptor in a Different Fashion from Thiazolidinediones

Tetsuya Tagami, Hiroyuki Yamamoto, Kenji Moriyama, Kuniko Sawai, Takeshi Usui, Akira Shimatsu and Mitsuhide Naruse

Clinical Research Institute, Kyoto Medical Center, National Hospital Organization, Kyoto 612-8555, Japan

Address all correspondence and requests for reprints to: Tetsuya Tagami, M.D., Ph.D., Clinical Research Institute, Kyoto Medical Center, National Hospital Organization, 1-1 Mukaihata-cho, Fukakusa, Fushimi-ku, Kyoto, 612-8555 Japan. E-mail: ttagami{at}kyotolan.hosp.go.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Angiotensin type 1 receptor blockers are widely used for the treatment of hypertension, and one angiotensin type 1 receptor blocker, telmisartan, specifically activates the peroxisome proliferator-activated receptor (PPAR)-{gamma}. We studied the impact of PPAR{gamma} mutants on transcriptional control and interaction with cofactors to elucidate differences in the molecular mechanism between telmisartan and other PPAR{gamma} agonists, thiazolidinediones (TZDs). We created several amino acid substitutions in the ligand binding domain of PPAR{gamma} that, based on molecular modeling, may affect the binding of these agents. In transient expression experiments, wild-type PPAR{gamma}-mediated transcription stimulated by telmisartan was more than one third of that stimulated by TZDs. The activation stimulated by TZDs was impaired, whereas activation stimulated by telmisartan was retained, in the H323Y, S342A, and H449A mutants. In the Y473A mutant, the TZD-induced activation was further impaired and lower than that of telmisartan-induced activation. Coexpression of coactivators enhanced the activation by both telmisartan and TZDs, but activation by telmisartan always exceeded that of TZDs in the Y473A mutant. Based on a mammalian two-hybrid assay, the interaction with corepressors was retained in Y473A. Telmisartan and TZDs, but not 9cis retinoic acid, dissociated corepressors from the wild-type PPAR{gamma}. Telmisartan most effectively dissociated corepressors from Y473A. The interaction with coactivators was enhanced by TZD activation of wild-type PPAR{gamma} and both telmisartan and TZD activation of Y473A. Thus, the Y473A mutant is selectively stimulated by telmisartan but not TZDs, suggesting that telmisartan and TZDs have differential effects on the transcriptional control. In conclusion, these PPAR{gamma} mutants could be powerful tools for developing novel therapeutic agents that retain the metabolic efficacy of PPAR{gamma} activation with fewer adverse effects, such as the increase in body weight associated with TZDs.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Angiotensin-II receptor blockers (ARBs) provide complete blockade of the renin-angiotensin system (RAS) by specifically inhibiting the actions of angiotensin II at the level of the angiotensin-II type 1 receptor (1). Because another class of antihypertensive agents targeting the RAS, angiotensin-converting enzyme inhibitors, may cause a dry cough, ARBs are increasingly used for the treatment of hypertension. Some ARBs are reported to lower the risk for type 2 diabetes (2, 3, 4) and improve insulin sensitivity in rodents (5) and humans (6). Telmisartan is an ARB that is highly selective for the angiotensin-II type 1 receptor (7). Recently telmisartan was reported to activate the peroxisome proliferator-activated receptor (PPAR)-{gamma} in transactivation assays (8). The effect of ARBs on PPAR{gamma} activation was strongest for telmisartan, followed by irbesartan and losartan. The effects of valsartan, candesartan, olmesartan, and eprosartan on PPAR{gamma} activation were minimal (8, 9). Another group of PPAR{gamma} ligands, thiazolidinediones (TZDs), is a new class of antidiabetic agents that act as insulin sensitizers, and the different effects of these drugs suggest that TZDs and telmisartan differentially modulate the expression of PPAR{gamma} target genes (9).

A subgroup of the nuclear receptor (NR) superfamily that includes the receptors for thyroid hormone, retinoic acid, vitamin D, and PPARs form heterodimers with retinoid X receptors (RXRs) to modulate the transcription of genes that contain hormone response elements (10, 11). These receptors act as ligand-dependent transcription factors that suppress or stimulate the expression of target genes (11). Transcriptional stimulation in response to ligand binding of these receptors is mediated by interactions with coactivator proteins (CoAs). Based on the {alpha}-helical LXXLL motif within CoAs, which interacts with the carboxyl terminus of the NRs, CoAs are classified into three subgroups, including members of the steroid receptor coactivator (SRC)-1 (12, 13), PPAR-binding protein (PBP)/thyroid hormone receptor-associated protein 220 (14, 15, 16), and PPAR{gamma} coactivator (PGC)-1 (17) families (18). cAMP response element-binding protein (CREB) binding protein (CBP)/p300 and p300/CBP-associated factor (pCAF), which possess intrinsic histone acetyl transferase activity (19, 20, 21, 22), also activate NR-mediated gene transcription. NRs can also function as potent repressors in the absence of hormone or in the presence of antagonist. Unliganded or antagonist-bound NRs suppress the basal activity of positively regulated promoters by binding to hormone response elements (23, 24, 25, 26). Two classes of nuclear corepressors (CoRs), termed nuclear receptor CoR (NCoR) (27, 28, 29) and silencing mediator for retinoid and thyroid hormone receptors (SMRT) (30, 31), have been identified, and are shown to mediate ligand-independent repression. These CoRs assemble a repression complex that includes Sin3 and histone deacetylases among other proteins (32, 33, 34). Therefore, transcriptional regulation involves chromatin remodeling caused by histone (de)acetylation by swapping these receptor-assembled histone acetyl transferase and histone deacetylase complexes in response to ligand binding to NR.

Crystal structures of the PPAR ligand binding domains (LBDs) revealed a large binding pocket, which may explain the diversity of PPAR{gamma} ligands (35). As a result, PPAR{gamma} may be activated by a different set of ligands and may exert distinct effects in vivo. The efficacy of activation may be due to the recruitment of a different set of cofactors on the receptor. Here, based on molecular modeling information, we picked four amino acids and created a substitution for each amino acid in the LBD of PPAR{gamma}, which may affect TZD binding (9, 35, 36). Superimposition of telmisartan bound to PPAR{gamma} on the cocrystal structure of rosiglitazone revealed that H323, H449, and Y473 are rosiglitazone-bound amino acids and S342 is a telmisartan-bound amino acid (9). To elucidate the molecular mechanism of the selective PPAR-modulating effects by telmisartan, the transactivation functions and interactions with cofactors were compared between TZDs and telmisartan using these PPAR{gamma} mutants.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
The chemical structures of troglitazone, pioglitazone, and telmisartan are shown in Fig. 1AGo. Troglitazone, pioglitazone, 9-cis retinoic acid, WY14643, and L165041 were purchased from Sigma Chemical Co. (St. Louis, MO). Telmisartan was provided by Boehringer Ingelheim Co. (Ingelheim, Germany). Antihuman PPAR{gamma} common mouse monoclonal antibodies were purchased from Perseus Proteomics Inc. (Tokyo, Japan).


Figure 1
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  		FIG. 1. The structure of TZDs and telmisartan, introduction of point mutations in the LBD of PPAR{gamma}, and Western blotting of mutant receptors. A, The chemical structures of troglitazone, pioglitazone, and telmisartan. B, The full-length mPPAR{gamma} is depicted at the top of the figure. The central DBD and the carboxyl-terminal LBD are shaded. In the middle, the LBD of hPPAR{gamma} was fused to the DBD of Gal4. At the bottom, the LBD of mPPAR{gamma} was fused to the activation domain of VP16. Amino acid substitutions are denoted by white dots. C, Ten micrograms of nuclear extracts (N) or cytosol fractions (C) from TSA-201 cells transfected with indicated PPAR{gamma} mutants were analyzed by SDS-PAGE with 10% acrylamide gel. The proteins were electroblotted onto nitrocellulose membranes, followed by reaction with anti-PPAR{gamma} monoclonal antibodies. *, Nonspecific band or endogenous human PPAR{gamma}.

 
Plasmid construction
The pCMX-mPPAR{gamma}1, -{alpha}, -{delta}, and pGal4-hPPAR{gamma} expression vectors were provided by R. M. Evans (Salk Institute, La Jolla, CA). The LBD of mPPAR{gamma} (BamHI-NheI fragment) was fused downstream of the VP16 activation domain in frame to create VP16-mPPAR{gamma} in pCMX. The mutant PPAR{gamma} cDNAs for each construct as shown in Fig. 1BGo were prepared by oligonucleotide-directed mutagenesis and verified by DNA sequencing as described previously (37). The numbering of the amino acid residues of PPAR{gamma} is based on the human PPAR{gamma}1 sequence (9). The expression vectors for F-SRC1, GRIP1, and ACTR were described previously (38). The pCX-PCAF was provided by Y. Nakatani (National Institutes of Health, Bethesda, MD), and pCMX-PBP and -PBP-T were provided by Y. Zhu (Northwestern University, Chicago, IL). The expression vectors for PGC1{alpha} and -β and Gal4-PGC1{alpha} and -β were provided by J. Szwaya (Dana Farber Cancer Institute, Boston, MA). Gal4-NCoR (residues 1552-2453), Gal4-SMRT (residues 876-1495), Gal4-SRC1 (residues 213-1061), Gal4-RXR (LBD), VP16-NCoR, VP16-SMRT, and VP16-RXR were described previously (39, 40). The reporter plasmid PPRE-tk-Luc was provided by J. Szwaya. UAS-tk-Luc was described previously (41, 42).

Transient expression assays
TSA-201 cells, a clone of human embryonic kidney 293 cells (38), were grown in phenol red-free DMEM (Nikken Biomedical Laboratory, Kyoto, Japan) with 10% charcoal-stripped fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 µg/ml) and were transfected by the calcium phosphate method (38). The total amount of expression plasmid DNA was kept constant in the different experimental groups by adding corresponding amounts of the same plasmids without receptor. After exposure to the calcium phosphate-DNA precipitate for 8 h, phenol red-free DMEM with 10% charcoal-stripped fetal bovine serum was added. Cells were harvested after 40 h for the measurement of luciferase activity, according to the manufacturer’s instructions (dual-luciferase reporter assay system; Promega, Madison, WI). The transfection efficiencies were corrected with the internal control.

Western blotting
Nuclear extracts or cytosol fractions (10 µg) from transfected TSA-201 cells prepared using nuclear extract kit (Active Motif, Carlsbad, CA) were analyzed by SDS-PAGE with 10% acrylamide gel. The proteins were electroblotted onto nitrocellulose membranes, followed by reaction with monoclonal antibodies against the amino-terminal domain (amino acids 3-108) of PPAR{gamma}1, as described previously (43).

Statistical analysis
Results are the mean ± SD from at least three transfections performed in triplicate. Data were analyzed by ANOVA with post hoc Dunnett’s tests to compare with the control.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Transcriptional activities of PPAR{gamma} mutants
Based on the molecular modeling (9, 35, 36), we introduced four types of mutations, i.e. H323A/Y, S342A, H449A, and Y473A, into mPPAR{gamma}1 cDNAs. The positions of the mutations were: H323 on helix 5, S342 on strand 3, H449 on helix 11, and Y473 on helix 12 [activation function (AF)-2]. Transient expression experiments were performed using TSA201 cells, which are derivatives of 293 cells. To test the protein expression of the PPAR mutants in the nucleus, we performed Western blotting using anti-PPAR{gamma} antibody. The protein expression level of each mutant in the nuclear and cytosolic fractions of transfected cells was confirmed except for H323A (Fig. 1CGo). Because the protein expressions of H323A both in the nucleus and cytosol were poor for unknown reasons, we used the H323Y mutant in subsequent studies. The transcriptional activities of these mutants were examined using a reporter gene, PPRE-tk-Luc, to characterize the functional properties in the presence of various ligands (Fig. 2AGo). First, all PPAR{gamma} ligands significantly increased luciferase activity in all constructs. Telmisartan (10 µmol) stimulated the wild-type PPAR{gamma}-mediated transcription by more than 35% that of the TZDs, such as troglitazone and pioglitazone. The activation induced by troglitazone was significantly impaired in the H323Y and S342A mutants and that by pioglitazone was impaired in the H449A PPAR{gamma} mutant. The effects of telmisartan were retained in all three of these mutants. In the Y473A mutant, TZD-induced activation was further impaired, such that the activation by telmisartan exceeded that of the TZDs. RXR is the heterodimeric partner of PPAR{gamma}. The 9-cis retinoic acid, which is an RXR ligand, stimulated the wild-type and mutant PPAR{gamma}s, probably mediated by RXR. Dose response studies were performed (Fig. 2BGo). The smaller dose (1 µM) of troglitazone, pioglitazone, or telmisartan slightly, but significantly, stimulated wild-type PPAR{gamma}-mediated transcription. The effects at the higher dose (100 µM) of troglitazone and pioglitazone were similar to those at 10 µM, respectively. Remarkably, telmisartan at 100 µM instead decreased the transcriptional activity compared with the smaller doses. In the H323Y mutant, stimulation by troglitazone or pioglitazone at the lower doses was less than that in the wild-type receptor, whereas stimulation by telmisartan at all doses was comparable with the wild type. In the Y473A mutant, the effects were the smallest at all doses of troglitazone or pioglitazone, but those of telmisartan were retained.


Figure 2
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  		FIG. 2. Function of mutant PPAR{gamma}s with respect to a PPRE-regulated reporter gene. A, The PPAR{gamma} expression plasmids for the indicated mutants or pCMX-hRXR{alpha} (10 ng) were transfected into TSA-201 cells together with PPRE-tk-Luc (50 ng) in the absence or presence of various ligands. B, Increasing amounts (1, 10, and 100 µM) of ligand were added. Results are the mean ± SD from at least three transfections performed in triplicate. *, P < 0.05, **, P < 0.01 (vs. corresponding ligand of WT); {dagger}, P < 0.05, {ddagger}, P < 0.01 (vs. respective control: no ligand).

 
The effects of coexpression of CoAs (18), such as class I (GRIP1/SRC2), class II (PBP/TRAP 220), and class III (PGC1) were examined. GRIP1 enhanced the wild-type PPAR{gamma}-mediated transcription by telmisartan and TZDs (Fig. 3AGo). GRIP1 also enhanced the Y473A-mediated transcription by these ligands, which resulted in greater activity induced by telmisartan than by TZD. Similar results were obtained by the coexpression of PBP (Fig. 3BGo) or PGC1s (Fig. 3CGo), both on the wild-type PPAR{gamma} and Y473A. Using the hPPAR{gamma}-LBD fused to the DNA-binding domain (DBD) of the Gal4 yeast transcription factor, Gal4-PPAR{gamma}, and a Gal4 reporter gene, UAS-tk-Luc, the effects of coexpression of CoAs were also examined. FSRC1 (SRC1), GRIP1 (SRC2), ACTR (SRC3), and a histone acetyltransferase, pCAF, proportionally enhanced the activities of Gal4-PPAR{gamma}WT and Gal4-Y473A induced, respectively, by telmisartan and TZDs (Fig. 4AGo). Similarly, PBP enhanced the activities of Gal4-PPAR{gamma}WT induced by telmisartan or TZD, but that of Gal4-Y473A was induced only by telmisartan (Fig. 4BGo).


Figure 3
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  		FIG. 3. Effects of CoAs on the function of the mutant PPAR{gamma} Y473A. A, The GRIP1 expression plasmids (50 ng) were cotransfected into TSA-201 cells together with pCMX-mPPAR{gamma} constructs (10 ng) and PPRE-tk-Luc (100 ng) in the absence or presence of various ligands. B, The PBP expression plasmids (50 ng) were cotransfected into TSA-201 cells together with pCMX-mPPAR{gamma} constructs (10 ng) and PPRE-tk-Luc (100 ng) in the absence or presence of various ligands. C, The PGC1 expression plasmids (50 ng) were cotransfected into TSA-201 cells together with pCMX-mPPAR{gamma} constructs (10 ng) and PPRE-tk-Luc (100 ng) in the absence or presence of various ligands. Results are the mean ± SD from at least three transfections performed in triplicate. *, P < 0.05; **, P < 0.01 (vs. corresponding ligand of WT); {dagger}, P < 0.05; {ddagger}, P < 0.01 (vs. corresponding ligand of control or PBP-T).

 

Figure 4
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  		FIG. 4. Effects of CoAs on the function of Gal4-PPAR{gamma} mutants. A and B, The various CoA expression plasmids (50 ng) were cotransfected into TSA-201 cells together with Gal4-hPPAR{gamma} (10 ng) constructs and the Gal4-responsive reporter gene, UAS-tk-Luc (100 ng), in the absence or presence of various ligands. Results are the mean ± SD from at least three transfections performed in triplicate. {dagger}, P < 0.05, {ddagger}, P < 0.01 (vs. corresponding ligand of respective control or PBP-T).

 
Cofactor binding of PPAR{gamma} mutants
The interaction with RXR{alpha} was studied using a mammalian two-hybrid assay (Fig. 5Go). PPAR{gamma}-RXR{alpha} binding was retained among mutants. The interactions with CoRs, such as SMRT and NCoR, were similarly examined. Both PPAR{gamma}-SMRT and -NCoR binding was relatively impaired in H449A but was retained in S342A and Y473A in the absence of ligand (Fig. 6AGo). To evaluate the effects of ligands on the PPAR{gamma}-CoR interaction, we constructed an mPPAR{gamma}-LBD fused to the transcriptional activation domain of VP16, VP16-PPAR{gamma}. The interaction between Gal4-CoR and VP16-PPAR{gamma}WT was disrupted by telmisartan and TZDs but not 9-cis retinoic acid (Fig. 6BGo). The dissociation of CoRs from the VP16-Y473A PPAR{gamma} mutant was mainly induced by telmisartan. A higher dose of telmisartan (100 µM) did not increase the dissociation of CoRs, consistent with the results of the transcriptional activity (Fig. 2BGo). The interaction with CoAs was also examined. The interaction of the Gal4-SRC1 with VP16-PPAR{gamma}WT was mainly enhanced by TZDs, but that with Y473A was enhanced by both telmisartan and TZDs (Fig. 7Go). In contrast, the interaction of the Gal4-PGCs with the VP16-Y473A PPAR{gamma} mutant was preserved and not influenced by any ligands, similar to the VP16-PPAR{gamma}WT (data not shown).


Figure 5
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  		FIG. 5. Interactions of RXR with mutant PPARs in a mammalian two-hybrid assay. The scheme of the mammalian two-hybrid experiment is shown at the top. VP16-RXR expression plasmids (50 ng) were cotransfected into TSA-201 cells together with UAS-tk-Luc (100 ng) and 10 ng of indicated Gal4-hPPAR{gamma} mutants in the absence of ligand. Results are the mean ± SD from at least three transfections performed in triplicate.

 

Figure 6
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  		FIG. 6. Interactions of CoRs with mutant PPARs in mammalian two-hybrid assays. A, The combination of chimeric proteins is shown at the top. VP16-CoR expression plasmids (50 ng) were cotransfected into TSA-201 cells together with UAS-tk-Luc (100 ng) and 10 ng of indicated Gal4-hPPAR{gamma} mutants in the absence of ligand. *, P < 0.05 (vs. WT). B, The combination of chimeric proteins is shown at the top. VP16-mPPAR{gamma} expression plasmids (50 ng) were cotransfected into TSA-201 cells together with UAS-tk-Luc (100 ng) and 10 ng of Gal4-CoR in the absence or presence of various ligands. Results are the mean ± SD from at least three transfections performed in triplicate. {dagger}, P < 0.05; {ddagger}, P < 0.01 (vs. respective control: no ligand).

 

Figure 7
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  		FIG. 7. Interactions of CoAs with mutant PPARs in mammalian two-hybrid assays. The combination of chimeric proteins is shown at the top. VP16-mPPAR{gamma} expression plasmids (50 ng) were cotransfected into TSA-201 cells together with UAS-tk-Luc (100 ng) and 10 ng of Gal4-SRC1 in the absence or presence of various ligands. Results are the mean ± SD from at least three transfections performed in triplicate. *, P < 0.05 (vs. corresponding control: no ligand).

 
Homologous mutants of PPAR{alpha} and {delta}
The PPAR{gamma}, -{alpha}, and -{delta} are well conserved from Xenopus to human. The corresponding amino acids in the context of mPPAR{alpha} and -{delta} with H323 mPPAR{gamma} are Y314 and H286, respectively. Those with Y473 mPPAR{gamma} are Y464 and Y436, respectively (Fig. 8AGo). Therefore, we next created mPPAR{alpha} mutant Y314H and Y464A and mPPAR{delta} mutants H286Y and Y436A. Although the effects of 9-cis retinoic acid were well preserved in the PPAR{alpha} and -{delta} mutant series, telmisartan did not stimulate any of the PPAR{alpha}, -{delta}, and their mutants (Fig. 8BGo).


Figure 8
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  		FIG. 8. Function of mutant PPAR isoforms with respect to a PPRE-regulated reporter gene. A, The full-length mPPAR{gamma}, -{alpha}, and -{delta} are illustrated. The amino acids of mPPAR{alpha} and -{delta} corresponding with H323 or Y473 in the context of mPPAR{gamma} were mutated as denoted by white dots. The central DBD and the carboxyl-terminal LBD are shaded. B, The PPAR expression plasmids for the indicated mutants (10 ng) were transfected into TSA-201 cells together with PPRE-tk-Luc (50 ng) in the absence or presence of various ligands. Results are the mean ± SD from at least three transfections performed in triplicate.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
RAS inhibition may prevent type 2 diabetes (44). Randomized clinical trials of ARB use such as Losartan Intervention for Endpoint Reduction in Hypertension, VALUE (Valsartan Antihypertensive Long-Term Use Evaluation), and CHARM (Candesartan in Heart Failure—Assessment of Mortality and Morbidity) indicate that these ARBs may reduce new-onset type 2 diabetes. Very recently telmisartan was reported to have effects on the incidence of diabetes that are comparable with ramipril (45), which potently reduces new-onset type 2 diabetes (46). The mechanism underlying this effect is unknown. Some ARBs and their metabolites might have PPAR{gamma}-activating properties (8, 9). TZDs, synthetic insulin-sensitizing drugs that include troglitazone, pioglitazone, and rosiglitazone, are highly effective in the treatment of type 2 diabetes, and their antidiabetic effect is believed to be mediated by activation of PPAR{gamma} (47). Thus, the structures of telmisartan and TZDs may have some similarities (Fig. 1AGo).

The carboxyl terminal activation domain (AF-2) in helix 12 of the NR-LBD is proposed to undergo induced conformational changes after binding to the ligand (26, 35, 36, 48, 49, 50, 51). Although the AF-2 of apo-PPAR{gamma} LBD is folded back toward the predicted ligand-binding pocket similar to other NR-LBDs, the pocket is larger and more accessible to the surface, in contrast to the other NR-LBDs (36). The hydrophobic groove within the ligand-bound NR-LBDs is postulated to interact with the conserved hydrophobic LXXLL motif that is found in several different CoAs (52, 53, 54, 55, 56). In the PPAR{gamma}, K301 in helix 3 and E471 in the AF-2 domain residues that are highly conserved in LBDs of NRs form a charge clamp that contacts the backbone atoms of the SRC-1 LXXLL motif (35). The critical region for CoR binding was identified more recently. Initially it was reported that mutations within the hinge region, or CoR box, disrupt CoR binding (27, 30, 57). Nagy et al. (58) showed that V284, K288, F293, Q301, and L305 in helices 3–6 in the thyroid hormone receptor are critical for SMRT binding, and those residues are well conserved among NR families. Among the PPARs, the binding strength with corepressor is stronger in PPAR{delta} than in PPAR{gamma} and PPAR{alpha}, whereas the precise binding sites for CoRs in the LBD of PPARs are not yet determined. PPAR binds to PPREs as heterodimers with RXR. The heterodimer interface is composed of conserved motifs in PPAR{gamma} and RXR{alpha} that form a coiled coil along helix 10 with additional charge interactions from helices 7 and 9 (59). The interaction between PPAR{delta} and NCoR is unaffected by coexpression of RXR{alpha} (60).

We created several amino acid substitutions based on the study of superimposition of telmisartan bound to PPAR{gamma} on the cocrystal structure of rosiglitazone (9). H323, H449, and Y473 were considered rosiglitazone-bound proteins, and S342 was considered a telmisartan-bound protein. Unexpectedly, however, telmisartan-induced activities were retained in H449A and Y473A as well as S342A; and TZD-induced activities were moderately impaired in H449A as well as S342A. H323A was an inactive mutant but H323Y retained some activities in which tyrosine is used in the context of wild-type PPAR{alpha}. Interestingly, the Y473A mutant was activated by telmisartan but not by TZDs. This specificity was partially explained by the relatively potent dissociation of CoRs and recruitment of CoAs by telmisartan on this mutant.

The region of the LBD occupied by telmisartan might be comparable with that occupied by other partial agonists of PPAR{gamma}, including GW0072 (61) and nTZDpa (62). Using the mammalian two-hybrid assay, GW0072 dissociated NCoR similarly to rosiglitazone but only partially recruited SRC-1 and CBP (61). In contrast, telmisartan or irbesartan partially dissociated NCoR and minimally recruited transcriptional intermediary factor-2 (SRC-2) using the fluorescence resonance energy transfer assay (63). Using the mammalian two-hybrid assay in this study, telmisartan dissociated NCoR and SMRT similarly to troglitazone but minimally recruited SRC-1 from the wild-type PPAR{gamma}. In addition, telmisartan dissociated CoRs and recruited SRC-1 in the Y473A mutant (Figs. 6BGo and 7Go), indicating that the mutation emphasizes the effects of ligand binding on conformational changes of the receptor.

Together these results clearly demonstrated that the Y473A mutant is selectively stimulated by telmisartan but not TZDs. Y473A interacts with RXR and its association with CoAs and dissociation from CoRs paralleled the activities of telmisartan and TZDs. The design of PPAR agonists or antagonists is of major medical interest. One agonist for PPAR{alpha} is the fibrate class of drugs that improve dyslipidemia. PPAR{gamma} agonists, such as the glitazone class of drugs, are used to treat diabetes. ARBs, which are widely used for the treatment of hypertension, also stimulate PPAR{gamma} and may have different effects. In conclusion, these PPAR{gamma} mutants may be powerful tools for screening and developing novel therapeutic agents that retain metabolic efficacy of PPAR{gamma} activation with fewer adverse effects than TZDs.


   Acknowledgments
 
The authors are grateful to R. M. Evans, Y. Nakatani, Y. Zhu, and J. Szwaya for providing plasmids. We also thank Ms. K. Matsuda, Ms. K. Kushii, and Ms. Y. Sakaguchi for their excellent secretarial assistance.


   Footnotes
 
This work was supported in part by Grant 17590973 from the Japanese Ministry of Education and Science (to T.T.) and grants from the Yamaguchi Endocrine Research Foundation.

First Published Online January 15, 2009.

Abbreviations: AF, Activation function; ARB, angiotensin-II receptor blocker; CoA, coactivator protein; CBP, cAMP response element-binding protein-binding protein; CoR, corepressor; DBD, DNA-binding domain; LBD, ligand binding domain; NCoR, nuclear receptor CoR; NR, nuclear receptor; PBP, PPAR-binding protein; PGC, PPAR{gamma} coactivator; PPAR, peroxisome proliferator-activated receptor; RAS, renin-angiotensin system; RXR, retinoid X receptor; SMRT, silencing mediator for retinoid and thyroid hormone receptors; SRC, steroid receptor coactivator; TZD, thiazolidinedione.

Received April 9, 2008.

Accepted for publication October 1, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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