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Graduate Schools of Medicine (R.A., I.S., M.M.) and Frontier Biosciences (I.S., M.M.), Osaka University, Osaka 565-0871, Japan; Howard Hughes Medical Institute and Department of Pharmacology (A.I.S., D.J.M.), University of Texas Southwestern Medical Center, Dallas, Texas 75390-9050; Institute of Biomaterials and Bioengineering and School of Biomedical Science (K.Y., S.Y.), Tokyo Medical and Dental University, Tokyo 101-0062, Japan
Address all correspondence and requests for reprints to: Makoto Makishima, Graduate School of Frontier Biosciences, Osaka University, 2–2 Yamadaoka, H2, Suita, Osaka 565-0871, Japan. E-mail: maxima{at}fbs.osaka-u.ac.jp.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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,25-dihydroxyvitamin D3 [1,25(OH)2D3], regulates calcium metabolism, cellular proliferation and differentiation, immune responses, and other physiological processes. Recently, secondary bile acids such as lithocholic acid (LCA) were identified as endogenous VDR agonists. To identify structural determinants required for VDR activation by 1,25(OH)2D3 and LCA, we generated VDR mutants predicted to modulate ligand response based on sequence homology to pregnane X receptor, another bile acid-responsive nuclear receptor. In both vitamin D response element activation and mammalian two-hybrid assays, we found that VDR-S278V is activated by 1,25(OH)2D3 but not by LCA, whereas VDR-S237M can respond to LCA but not to 1,25(OH)2D3. Competitive ligand binding analysis reveals that LCA, but not 1,25(OH)2D3, effectively binds to VDR-S237M and both 1,25(OH)2D3 and LCA bind to VDR-S278V. We propose a docking model for LCA binding to VDR that is supported by mutagenesis data. Comparative analysis of the VDR-LCA and VDR-1,25(OH)2D3 structure-activity relationships should be useful in the development of bile acid-derived synthetic VDR ligands that selectively target VDR function in cancer and immune disorders without inducing adverse hypercalcemic effects. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The active form of vitamin D, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], regulates calcium metabolism, cellular differentiation, and immunity through VDR activation (3, 4). Vitamin D is a secosteroid, in which the B ring of steroid structure is ruptured (5). UV irradiation induces a photochemical reaction of 7-dehydrocholesterol, which is synthesized from acetyl-coenzyme A and is a precursor of cholesterol, to produce the secosteroid vitamin D3 in the skin. Vitamin D3 is hydroxylated at the 25 position by vitamin D3 25-hydroxylase (CYP27A1) in the liver to yield 25-hydroxyvitamin D3, the major form of vitamin D in the circulation. The 25-hydroxyvitamin D3 is further hydroxylated in the 1 position by CYP27B1. This reaction is tightly regulated and occurs exclusively in the kidney to yield the active metabolite, 1,25(OH)2D3. Three-dimensional modeling and the solution of a crystal structure have yielded valuable insight into the mode of binding of 1,25(OH)2D3 in the VDR ligand-binding pocket (LBP) (6, 7, 8).
Synthetic vitamin D analogs have been used successfully in the treatment of bone and skin disorders. However, their adverse effects, including hypercalcemia, bone resorption, and soft tissue calcification, limit the clinical application of VDR agonists in the management of malignant tumors and immune disorders (3). The need for VDR ligands with potent anticancer activity that lack adverse effects on calcium metabolism has led to a major synthetic chemistry effort. Several analogs exhibit efficient antiproliferation and prodifferentiation activities with fewer calcemic side effects than 1,25(OH)2D3, but the underlying molecular mechanism of this functional specificity is still not understood (3). Structure-function analysis of vitamin D analogs suggests that these secosteroids also act on a membrane receptor and that adverse effects are at least partly due to a poorly characterized nongenomic mechanism of action (9). To develop efficient VDR-targeting therapy, it is very important to elucidate the structure-function relationship of VDR and its ligands.
Recently, we discovered that VDR functions as a receptor for a number of secondary bile acids including lithocholic acid (LCA) (10). LCA is also a weak agonist for the nuclear receptors farnesoid X receptor (NR1H4) and pregnane X receptor [PXR, also called SXR (NR1I2)]. The primary bile acids, cholic acid and chenodeoxycholic acid, are synthesized from cholesterol in liver and secreted in bile as glycine or taurine conjugates (11). After assisting in the digestion and intestinal absorption of lipids and fat-soluble vitamins, including dietary vitamin D, the majority of bile acids are reabsorbed and returned to the liver through the enterohepatic circulation. Bile acids that escape reabsorption in the ileum are converted to the secondary bile acids deoxycholic acid and LCA by intestinal microflora. Whereas farnesoid X receptor serves as a sensor for both primary and secondary bile acids (12, 13, 14), PXR and VDR are selectively activated by secondary bile acids (10, 15, 16). PXR, which shares the most sequence identity with VDR, responds to steroid hormone metabolites and xenobiotics, but not to 1,25(OH)2D3 (17, 18). The crystal structures of the VDR and PXR ligand binding domains (LBDs) reveal strong structural conservation (8, 19). In this study, we compared the LBD structures of VDR and PXR to generate VDR point mutants that selectively respond to 1,25(OH)2D3 or LCA. Computational docking analysis was used to model the structural requirement for the mutated residues in ligand discrimination. Identification of critical residues for response to endocrine and bile acid ligands should aid the development of VDR agonists with improved pharmacological specificity.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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,25(OH)2D3, we hypothesized that substitution of VDR residues with PXR amino acids at critical LBP positions that hydrogen bond with the hydroxyl groups of 1,25(OH)2D3 might yield LCA-selective mutants. The VDR-1,25(OH)2D3 cocrystal shows that the 1-hydroxyl group of 1,25(OH)2D3 contacts S237 and R274, the 3ß-hydroxyl group is coordinated by S278 and Y143, and the 25-hydroxyl group is hydrogen bonded to H397 and H305 (8). These amino acids, except for Y143, are conserved among human, mouse, rat, and chicken VDRs (Fig. 1A
). Y143 is replaced with phenylalanine in chicken VDR, which is identical to the corresponding amino acid in PXRs. R274 in hVDR is conserved in VDRs and PXRs, and H397 of hVDR is identical among other VDRs and hPXR. The alignment indicates that S237, S278, and H305 are unique to VDRs and that PXR has other amino acids at these positions. Although the C backbones of the VDR and PXR structures are very similar, the presence of a flexible loop between the ß-sheet and H7 instead of the H6 found in VDR might be responsible for the ability of PXR to respond to diverse ligands (19) (Fig. 1, B and C). In addition, the crystal structures defined for the VDR-LBD (
165–215) and the PXR-LBD (8, 19) indicate that small differences in the amino acid identity of LBD residues cause significant change in the shape of the LBP (Fig. 1). These findings suggest that mutational analysis of VDR based on an amino acid alignment and structural comparison with PXR should be very useful in elucidating the structure-function relationship of VDR and its ligands.
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(20, 21). Alanine scanning mutagenesis was used to analyze the interaction of VDR with several vitamin D analogs and bile acids (22). To further analyze structure-activity relationships between VDR and two different natural ligands [1,25(OH)2D3 and LCA], we replaced ligand-coordinating residues with the corresponding PXR amino acids. We generated hVDR mutants of Y143 and S278, which make hydrogen bonds with the 3-hydroxyl group of 1,25(OH)2D3, and of S237, which interacts with the 1-hydroxyl group of 1,25(OH)2D3. These amino acids were changed to the corresponding amino acids of hPXR (Fig. 1A
) or alanine. Vitamin D-resistant rickets-associated mutants of VDR (R274L and H305Q) were also examined (4). Because hPXR F288 contributes to structural differences between VDR and PXR (19), the corresponding amino acid in VDR (S275) was replaced with either phenylalanine or alanine (VDR-S275F and VDR-S275A). The crystal structure of VDR-LBD was determined in a deletion mutant (165–215), because a long flexible loop between helices 1 and 3 prevents the preparation of stable crystals (8). This loop does not seem to contribute to ligand interaction, because VDR (165–215) can be transactivated by 1,25(OH)2D3, its synthetic analogs, and LCA as efficiently as wild-type VDR (22, 23) (data not shown).
We examined ligand-responsive transcriptional activation by the full-length VDR point mutants. Human embryonic kidney (HEK)293 cells were transfected with wild-type VDR or VDR mutants and a luciferase reporter containing a VDR-responsive everted repeat-6 element from the CYP3A4 promoter (10) (Fig. 2A). Because kidney-derived HEK293 cells express endogenous VDR (data not shown), the addition of 1,25(OH)2D3 or LCA basally induced luciferase activity. Transfection of wild-type VDR effectively increased induction by both ligands. Mutants of residues that coordinate the 3-hydoxyl group of 1,25(OH)2D3, Y143A, Y143F, S278A, and S278V, were activated by 1,25(OH)2D3 but not by LCA. 1,25(OH)2D3 weakly activated S237A and had no effect on S237M activity, whereas LCA activated both mutants. The rickets-causing mutants R274L and H305Q were unresponsive to both 1,25(OH)2D3 and LCA. S275A maintained responsiveness to 1,25(OH)2D3 and LCA, but the S275F mutation abolished ligand response. The basal luciferase activity induced by 1,25(OH)2D3 was repressed by transfection of S237M, R274L, and S275F. This may be due to dominant negative effects of these mutants on endogenous VDR activity through sequestration of retinoid X receptor or cofactors and competitive binding to the vitamin D response element. The data for S237A, S275A, and S278A mutants are consistent with previous data utilizing a reporter with a DR-3 element from the osteopontin promoter (22). In that study, Y143A did not respond to 10 nM 1,25(OH)2D3. The discrepancy between these results may be due to the difference in ligand concentration or the response element construct tested.
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,25(OH)2D3 strongly induced association of VDR, Y143F, S275A, and S278A, and moderate association of Y143A, S237A, S278V, and H305Q with SRC-1. LCA was able to induce association of VDR, Y143F, S237A, S237M, and S275A with SRC-1 (Fig. 2B). The mammalian two-hybrid assay using the GAL4-N-CoR chimeric corepressor shows 1,25(OH)2D3-dependent dissociation of N-CoR from VDR, Y143A, Y143F, S237A, S275A, S278A, S278V, and S305Q, as well as LCA-dependent dissociation from VDR, Y143F, S237A, S237M, S275A, S278A, and H305Q (Fig. 2C). Difference in sensitivity of these assays may account for differential responsiveness of VDR mutants to ligands. The data indicate that S237 is more critical for induction by 1,25(OH)2D3 and that Y143 and S278 are more important for LCA activation. To evaluate the expression of VDR mutants in the cells, we transfected VP16-VDR chimeric mutants together with the VDRE-responsive reporter. Because VP16 chimeric receptors exhibit ligand-independent activity, the luciferase activity gives an indirect measure of the protein expression levels of transfected receptors. All of the VDR mutants show luciferase activities similar to wild-type VDR (Fig. 2D), indicating similar expression of functional VDR mutants in the cells.
The data shown in Fig. 2 suggest that VDR-S278V and VDR-S237M respond selectively to 1,25(OH)2D3 and LCA, respectively. To further investigate the preference of these VDR mutants for the ligands, dose-response curves were obtained with the GAL4-VDR system, which eliminates the activity of endogenous VDR (Fig. 3). VDR S278A was activated by 1,25(OH)2D3 as effectively as wild-type VDR. The S278V mutation partially decreased 1,25(OH)2D3 response. S237M completely abolished 1,25(OH)2D3 response, whereas S237A had a more moderate effect (Fig. 3A). Importantly, LCA was able to activate S237M as effectively as S237A (Fig. 3B). LCA was unable to activate S278V and weakly activated S278A (Fig. 3B). The data indicate that the S278V mutant is unresponsive to LCA and the S237M mutant loses 1,25(OH)2D3 response. Therefore, S278V selectively responds to 1,25(OH)2D3 whereas S237M is specifically activated by LCA.
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,25(OH)2D3 was incubated with glutathione-S-transferase (GST)-VDR proteins in the presence or absence of excess unlabeled 1,25(OH)2D3, and specific binding of 1,25(OH)2D3 was calculated. 1,25(OH)2D3 effectively bound to wild-type VDR and S278V but only interacted weakly with S237M (Fig. 4A). 1,25(OH)2D3 did not bind to GST control protein. The binding of labeled 1,25(OH)2D3 to wild-type VDR and the S278V mutant was competed by addition of unlabeled 1,25(OH)2D3 (Fig. 4B). Addition of unlabeled LCA inhibited the interaction of 1,25(OH)2D3 with wild-type VDR, VDR-S237M, and VDR-S278V, indicating that LCA binds directly to these VDR proteins. For example, 30 µM LCA inhibited binding of 1 nM 1,25(OH)2D3 to wild-type VDR, VDR-S237M, and VDR-S278V to 74%, 70%, and 79%, respectively, of the control, and 100 µM LCA inhibited those to 59, 38, and 39%, respectively. These findings suggest that the inability of LCA to activate VDR-S278V is not due to loss of binding and is more likely to be caused by a reduction in the ligand-induced shift to an active conformation.
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,25(OH)2D3 model and the VDR crystal structure, S237 hydrogen bonds with the 1-hydroxyl group, Y143 and S278 interact with the 3-hydroxyl group, and H305 interacts with the 25-hydroxyl group (8) (Fig. 5A). The S237M mutation loses the ability to interact with 1,25(OH)2D3 (Figs. 2 and 3). The model shows that a methionine residue at position 237 is too close to the 1-hydroxyl group of 1,25(OH)2D3 and would be expected to destabilize binding (Fig. 5B). Y143F and S278V do not affect activation by 1,25(OH)2D3. This may be due to the relatively conservative substitutions in these mutants and an apparent weak contribution of contacts between VDR and the 3-hydroxyl group of 1,25(OH)2D3 to overall binding energy. S275F mutation causes a marked change in the LBP conformation such that it is unable to accommodate 1,25(OH)2D3 (Fig. 5B). The mutational analysis in this study confirms the importance of contacts with the 1-hydroxyl group of 1,25(OH)2D3 in ligand binding and receptor activation (6, 8).
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). The carboxyl group of the LCA side chain is positioned within hydrogen bond distance of H305. Residues Y143 and S278 weakly interact with the 3-hydroxyl group of LCA. Mutation of H305 to glutamine decreased LCA response (Fig. 2), indicating that contacts of Y143 and S278 with LCA are insufficient for VDR activation. The S278V mutation abolished LCA response, although S278A maintained LCA response (Fig. 3). A valine residue at position 278 would be expected to sterically interfere with the 3-hydroxyl group of LCA, an unfavorable interaction that would be absent in S278A (Fig. 5D). Although the Y143F mutant was responsive, Y143A was not activated by LCA (Fig. 2). The conservative tyrosine to phenylalanine mutation maintains a large aromatic amino acid, whereas the alanine mutation is expected to more severely alter the LBP conformation. S237M mutation abolished 1,25(OH)2D3 response but did not affect activation by LCA (Fig. 3). As predicted by the docking model, S237 does not interact with LCA, and the S237M mutation has little effect on LCA activity. S275F prevents both LCA and 1,25(OH)2D3 from docking in the LBP (Fig. 5D). Taken together, these data indicate that LCA and 1,25(OH)2D3 make nonoverlapping critical interactions with residues of the VDR LBP. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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(NR1B3) (8, 19, 26). CAR and PXR are functionally redundant xenobiotic sensors in that both can regulate common target genes such as CYP3A and CYP2B (27). VDR is also able to regulate CYP3A transcription by binding to the same response element as PXR and CAR (10, 27), suggesting that NR1I receptors have evolved from a common ancestor and have a shared role in mediating the detoxification response to xenobiotics. These findings suggest a potential role of VDR as a xenobiotic sensor and the possibility that VDR responds to natural or synthetic compounds other than vitamin D and bile acid.
VDR is distinct from other NR1I receptors in that it interacts with bile acids with low affinity (at micromolar levels) like PXR and CAR but also responds to an endocrine ligand, 1,25(OH)2D3, with high affinity, similar to steroid hormone receptors. In this study, we demonstrate that distinct amino acid residues in the LBP are important for interaction with 1,25(OH)2D3 and LCA. This finding leads to the possibility that 1,25(OH)2D3 and LCA induce different activated receptor conformations that might recruit distinct sets of cofactors (28). Further analysis of ligand structure-function relationships should be helpful in elucidating the dual functions of VDR as an endocrine receptor for 1,25(OH)2D3 and as a xenobiotic sensor for secondary bile acids produced by intestinal microflora.
We generated several VDR mutants at residues which hydrogen bond with hydroxyl groups of 1,25(OH)2D3 and compared their responses to 1,25(OH)2D3 and LCA in VDRE activation and mammalian two-hybrid assays (Fig. 2). There are some discrepancies in the behavior of VDR mutants in the three assays shown in Fig. 2, A–C. 1,25(OH)2D3 induced the activation of Y143A as effectively as that of wild-type VDR, but the recruitment of SRC-1 by Y143A was weak. The effect of LCA on Y143F in VDRE activation was very weak, although LCA induced strong interaction with SRC-1 in Y143F as in wild-type VDR. Thus, the VDRE activation by full-length VDR was not completely correlated with recruitment of SRC-1. This may be because NR activation is mediated by sequential interaction with several sets of cofactor complexes (2). LCA induced N-CoR dissociation from Y143F, S278A, and H305Q, but these mutants showed low or undetectable transactivation by LCA in the VDRE-based assay. The data indicate the ligand-inducible dissociation of corepressor is not sufficient to induce VDR transactivation. Transfection experiments using the VDRE activation and mammalian two-hybrid assays in combination are useful in the detection of VDR-ligand interactions and ligand-induced receptor activation. The significance of interaction with particular cofactors on overall VDR transactivation potential requires further investigation.
We found that VDR-S278V and VDR-S237M mutants respond selectively to 1,25(OH)2D3 and LCA, respectively. These mutants should be useful not only for analysis of structure-activity relationships but also for development of new synthetic VDR agonists. Selective screening using VDR-S278V and VDR-S237M may lead to discovery of a new synthetic ligand that lacks hypercalcemic activity. Recently, mice with humanized PXR or CAR were generated because human PXR and CAR have different ligand selectivity from their rodent homologs (29, 30). Substitution of endogenous VDR with VDR-S278V may produce mice with defects in bile acid response with normal calcium metabolism whereas VDR-S237M-expressing mice may show selective dysfunction in vitamin D response. Development of ligand-specific VDR-substituted mice should provide a valuable tool in clarifying VDR function in vivo.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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,25(OH)2D3 and LCA were obtained from Calbiochem (San Diego, CA) and Nacalai (Kyoto, Japan), respectively.
Graphical Manipulation and Docking
Graphical manipulations were performed using SYBYL 6.7 (Tripos, St. Louis, MO) (6, 31). The atomic coordinates of the crystal structures of hVDR-LBD (165–215) (1DB1) and hPXR-LBD (1ILH) were retrieved from the Protein Data Bank. LCA was docked into VDR and PXR using the docking software FlexX (version 1.11.0; Tripos). The active site (in the case of NRs, the LBP) was defined to include all amino acids within 6.5 Å of the cocrystallized ligand.
Plasmids
A fragment of hVDR (GenBank accession no. J03258) was inserted into pCMX-flag vector to make pCMX-VDR (10, 32). The LBD of hVDR was inserted into pCMX-GAL4 vector to make pCMX-GAL4-VDR, and full-length hVDR was inserted into pCMX-VP16 vector to make pCMX-VP16-VDR (10). Nuclear hormone receptor-interacting domains of SRC-1 (amino acids 595–771; GenBank accession no. U90661) and N-CoR (1990–2416; GenBank accession no. U35312) were inserted into pCMX-GAL4 vector for pCMX-GAL4-SRC-1 and pCMX-GAL4-N-CoR, respectively. Mutations were introduced into pCMX-VDR, pCMX-GAL4-VDR, and pCMX-VP16-VDR using QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). VDR-responsive hCYP3A4-ER-6x3-tk-LUC and GAL4-responsive MH100(UAS)x4-tk-LUC reporters were used (10, 32). All plasmids were sequenced before use to verify DNA sequence fidelity.
Cell Culture and Cotransfection Assay
Human embryonic kidney HEK293 cells were cultured in DMEM containing 5% fetal bovine serum and antibiotic-antimycotic (Nacalai) at 37 C in a humidified atmosphere of 5% CO2 in air. Transfections were performed by the calcium phosphate coprecipitation assay as previously described (33). Eight hours after transfection, ligands were added. Cells were harvested approximately 16–20 h after the treatment, and luciferase and ß-galactosidase activities were assayed using a luminometer and a microplate reader (Molecular Devices, Sunnyvale, CA). DNA cotransfection experiments included 50 ng reporter plasmid, 20 ng pCMX-ß-galactosidase, 15 ng of each receptor and/or cofactor expression plasmid, and pGEM carrier DNA for a total of 150 ng DNA per well in a 96-well plate. Luciferase data were normalized to an internal ß-galactosidase control and represent the mean (±SD) of triplicate assays.
Ligand-Binding Assay
LBDs of hVDR and its mutants were cloned into the GST-fusion vector pGEX-4T1 (Amersham Pharmacia Biotech, Piscataway, NJ). GST-VDR fusion proteins were expressed in BL21 DE3 cells (Promega Corp., Madison, WI) and purified with glutathione sepharose beads (Amersham Pharmacia Biotech). Competitive ligand-binding assay was performed by modification of previous reports (34, 35). Briefly, 500 ng GST fusion proteins were bound to glutathione sepharose and incubated with [26,27-methyl-3H]1,25(OH)2D3 (Amersham Pharmacia Biotech) in the presence or absence of nonradioactive ligand in a buffer (10 mM Tris-HCl, pH 7.6; 1 mM EDTA; 300 mM KCl; 1 mM dithiothreitol; 10% glycerol) for 3 h at 4 C. After washing twice, the protein and bound 1,25(OH)2D3 were resuspended in 200 µl of the binding buffer, and 150 µl were assessed by liquid scintillation counting.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: CAR, Constitutive androstane receptor; FXR, farnesoid X receptor; GST, glutathione-S-transferase; h, human; HEK, human embryonic kidney; LBD, ligand binding domain; LBP, ligand binding pocket; LCA, lithocholic acid; NR, nuclear receptor; 1,25(OH)2D3, 1,25-dihydroxyvitamin D3; PXR, pregnane X receptor; SRC, steroid receptor coactivator; VDR, vitamin D receptor; VDRE, vitamin D response element.
Received for publication June 24, 2003. Accepted for publication September 22, 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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,25-dihydroxy-vitamin D3. J Cell Biochem 74:323–333[CrossRef][Medline]
(PPAR) generates a PPAR
phenotype. Mol Endocrinol 14:733–740NURSA Molecule Pages Link:
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