Molecular Mechanism of the Vitamin D Antagonistic Actions of (23S)-25-Dehydro-1
-Hydroxyvitamin D3-26,23-Lactone Depends on the Primary Structure of the Carboxyl-Terminal Region of the Vitamin D Receptor
Eiji Ochiai,
Daishiro Miura,
Hiroshi Eguchi,
Sachiko Ohara,
Kazuya Takenouchi,
Yoshiaki Azuma,
Takashi Kamimura,
Anthony W. Norman and
Seiichi Ishizuka
Teijin Institute for Bio-Medical Research (E.O., D.M., H.E., S.O., K.T., Y.A., T.K., S.I.), Hino, Tokyo 191-8512, Japan; and Department of Biochemistry (A.W.N.) and Division of Biomedical Sciences, University of California, Riverside, California 92521
Address all correspondence and requests for reprints to: Dr. Anthony W. Norman, Department of Biochemistry University of California Riverside, California 92521 E-mail: Anthony.Norman{at}ucr.edu.
 |
ABSTRACT
|
---|
We reported that (23S)-25-dehydro-1
-hydroxyvitamin D3-26,23-lactone (TEI-9647) antagonizes vitamin D receptor (VDR)-mediated genomic actions of 1
,25-dihydroxyvitamin D3 [1
,25(OH)2D3] in human cells but is agonistic in rodent cells. Human and rat VDR ligand-binding domains are similar, but differences in the C-terminal region are important for ligand binding and transactivation and might determine the agonistic/antagonistic effects of TEI-9647. We tested TEI-9647 on 1
,25(OH)2D3 transactivation using SaOS-2 cells (human osteosarcoma) or ROS 24/1 cells (rat osteosarcoma) cotransfected with human or rodent VDR and a reporter. In both cell lines, TEI-9647 was antagonistic with wild-type human (h)VDR, but agonistic with overexpressed wild-type rat (r)VDR. VDR chimeras substituting the hVDR C-terminal region (activation function 2 domain) with corresponding rVDR residues diminished antagonism and increased agonism of TEI-9647. However, substitution of 25 C-terminal rVDR residues with corresponding hVDR residues diminished agonism and increased antagonism of TEI-9647. hVDR mutants (C403S, C410N) demonstrated that Cys403 and/or 410 was necessary for TEI-9647 antagonism of 1
,25(OH)2D3 transactivation. These results suggest that species specificity of VDR, especially in the C-terminal region, determines the agonistic/antagonistic effects of TEI-9647 that determine, in part, VDR interactions with coactivators and emphasize the critical interaction between TEI-9647 and the two C-terminal hVDR Cys residues to mediate the antagonistic effect of TEI-9647.
 |
INTRODUCTION
|
---|
IT IS WIDELY ACCEPTED that the fundamental biological activities of the hormonal form of vitamin D3, 1
,25-dihydroxyvitamin D3 [1
,25(OH)2D3], are to stimulate intestinal calcium absorption and to increase bone calcium mobilization (1). Recently, however, many other biological functions have been reported (2); these include inhibition of cell proliferation and induction of cell differentiation (3), modulation of immunological responses (4, 5), stimulation of insulin secretion (6, 7), and neurobiological functions (8, 9, 10). 1
,25(OH)2D3 generates biological responses both by interaction with the vitamin D receptor (VDR) to regulate gene transcription (genomic responses) (11, 12, 13, 14) and/or with a membrane-associated receptor (VDRmem) (15, 16) to activate a variety of signal transduction pathways that generate rapid responses (17) including the opening of voltage-gated calcium and chloride channels (18), and activation of MAPK (19).
The VDR is a member of the nuclear hormone receptor superfamily (13) and consists of several functional domains including the DNA-binding and ligand-binding domains (LBDs) (14). The VDR and the retinoid X receptor (RXR) form a heterodimeric complex and bind cooperatively to vitamin D-responsive elements with high affinity to activate or repress the transcription of multiple genes (20, 21, 22). The extreme C-terminal portion of the VDR, which is designated as the activation function 2 (AF-2) core domain, is required for ligand-dependent transactivation (11, 23). The x-ray crystallography studies of the LBD (residues 118427;
165215) of the VDR bound to 1
,25(OH)2D3 have shown that the LBD consists of 12
-helices and three ß-sheets (24, 25). In the VDR LBD, helices 111 form the ligand-binding pocket, whereas helix 12 is believed to undergo a major reorientation from an open to a closed portal configuration as it functions as a gatekeeper to facilitate entrance and then retention of 1
,25(OH)2D3 into the LBD.
Ishizuka et al. (26, 27, 28) and Ishizuka and Norman (29) found that the (23S,25R)-1
,25-dihydroxyvitamin D3-26,23-lactone [1
,25(OH)2D3-lactone] is a major metabolite of 1
,25(OH)2D3 that occurs under both in vivo and in vitro conditions. We reported that the naturally occurring 1
,25(OH)2D3-lactone has unique biological activities in comparison with 1
,25(OH)2D3. Although the VDR binding affinity of the naturally occurring 1
,25(OH)2D3-lactone is very low [0.5% of 1
,25(OH)2D3] (27, 30), it can stimulate collagen synthesis in osteoblasts (31, 32) and inhibit formation of osteoclast-like multinucleated cells from bone marrow mononuclear cells and bone resorption induced by 1
,25(OH)2D3 (31, 33, 34). The 1
,25(OH)2D3-lactone can also stimulate proteoglycan synthesis and type II collagen synthesis in chondrocytes from rabbit costal growth cartilage (35).
To date, a large number of vitamin D3 analogs have been synthesized; however, almost all these compounds are agonists (2). 1ß,25(OH)2D3 blocks rapid nongenomic responses but is without effect on the classical nuclear VDR (36). Currently, only two types of 1
,25(OH)2D3 analog nuclear VDR antagonists have been described. These are the (23S,25R)-1
,25(OH)2D3-26,23-lactone derivatives [e.g. (23S)-25-dehydro-1
-hydroxyvitamin D3-26,23-lactone (TEI-9647); Fig. 1
] from Ishizuka and co-workers (37, 38) and the 25-carboxylic ester derivatives of 1
,25(OH)2D3 (e.g. ZK159222) from Herdick et al. (39). Recently, Ishizuka and colleagues (37) reported that TEI-9647, an analog of the naturally occurring metabolite (23S,25R)-1
,25(OH)2D3-26,23-lactone failed to induce human promyelocytic leukemia cell (HL-60 cell) differentiation even at a high concentration (106 M). Intriguingly, TEI-9647 was found to inhibit differentiation of HL-60 cells induced by 1
,25(OH)2D3. In contrast, TEI-9647 did not block the actions of retinoic acid and 12-O-tetradecanoylphorbol-13-acetate on HL-60 cell differentiation, suggesting that the inhibitory action might be 1
,25(OH)2D3/VDR specific (37). More recently, it has been reported that TEI-9647 inhibits the heterodimer formation between VDR and RXR and between VDR and steroid receptor coactivator 1 (SRC-1) or transcriptional intermediary factor 2, the coactivator proteins of the p160 family (38, 40, 41). Further, it is known that coactivators interact with the VDR-RXR heterodimer via the C-terminal region of the VDR, especially the AF-2 domain (42, 43).

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 1. Structures of 1 ,25(OH)2D3, Its Lactone Metabolite, and Related Analogs
The chemical names of the analogs are as follows: 1 ,25(OH)2D3, 1 ,25-dihydroxyvitamin D3; 1 ,25(OH)2D3-lactone, (23S,25R)-1 ,25-dihydroxyvitamin D3-26,23-lactone [a natural metabolite of 1 ,25(OH)2D3]; 1ß-OH-TEI-9647, (23S)-25-dehydro-1ß-hydroxyvitamin D3-26,23-lactone; TEI-9647, (23S)-25-dehydro-1 -hydroxyvitamin D3-26,23-lactone; ZK159222, 1 ,3ß,24(OH)3-22-ene-24-cyclopropyl-25-n-butyl-ester-vitamin D3.
|
|
In this report, the effects of TEI-9647 and ZK159222 on the alkaline phosphatase (ALP) activity induced by 1
,25(OH)2D3 in human and rat osteosarcoma cells were compared. We tested the effect of species specificity of the VDR on the actions of the two structurally different VDR antagonists using a reporter gene assay. The results demonstrate that the antagonistic effect of TEI-9647, but not ZK159222, depends on the species of origin of the VDR.
 |
RESULTS
|
---|
TEI-9647 Is Not an Antagonist in Rat-Derived Cells
Previous studies on human cells demonstrated that TEI-9647 antagonized the transactivation of the hVDR by 1
,25(OH)2D3 (37, 38, 40, 44, 45, 46). In the course of developing TEI-9647 as a therapeutic agent, we compared the agonist and antagonist properties of TEI-9647 in rat osteoblast-derived cells (ROS 17/2.8) and in the human osteoblast-derived MG-63 cell. As shown in Fig. 2
, A and B, each cell line in the ALP activity was induced, in a dose-dependent manner, by 1
,25(OH)2D3 (Fig. 2
, A and B). As shown in Fig. 2
, C and D, TEI-9647 had no agonistic activity in the human MG-63 cells but was an agonist only at the highest (106 M) concentration in the rat ROS 17/2.8 cells (Fig. 2D
). As expected, TEI-9647 was a potent antagonist of 1
,25(OH)2D3-induced ALP in the human MG-63 cells (Fig. 2C
) but did not inhibit in the rat ROS 17/2.8 cells. These results indicate that TEI-9647 is only a weak agonist and not an antagonist in rat-derived cells. In contrast, in human-derived cells, TEI-9647 is an antagonist and not an agonist.
The Species Specificity of VDR Affects the Antagonistic/Agonistic Activity of TEI-9647
The different effects in terms of the agonistic/antagonistic activities of TEI-9647, as shown in Fig. 2
, may be due to any of several components related to this phenomenon, e.g. VDR, RXR, coactivators, etc. We hypothesized that the VDR is the key determinant of the species specificity. To investigate whether a species- specific VDR affects the activity of TEI-9647, ROS24/1 cells, which do not express endogenous VDR (47), were cotransfected with a human vitamin D-responsive Cyp24 promoter-reporter plasmid and either the hVDR or rVDR expression plasmids by transient transfection and assayed for their transcriptional activity in response to 1
,25(OH)2D3 in the presence or absence of TEI-9647. As shown in Fig. 3
, when hVDR was expressed in ROS 24/1 cells, TEI-9647 displayed a strong antagonistic effect whereas in ROS 24/1 cells that express the rVDR, TEI-9647 functioned as an agonist and was not able to antagonize the transactivation by 1
,25(OH)2D3. These results indicate that the VDR is the key determinant of the species specificity of the activity of TEI-9647 between rat and human cells.

View larger version (11K):
[in this window]
[in a new window]
|
Fig. 3. Comparison of Human vs. Rat VDR on the Transcriptional Activities of 1 ,25(OH)2D3 and TEI-9647 in Rat Cells
ROS24/1 cells were cotransfected with the human vitamin D-responsive Cyp24 promoter-reporter plasmid and either human (A) or rat (B) VDR expression plasmid by transient transfection and assayed for the resulting transcriptional activity in response to 1 ,25(OH)2D3 in the presence or absence of TEI-9647. Values shown represent the mean ± SD from three separate transfections.
|
|
Comparison of hVDR vs. rVDR on the Transcriptional Activities of 1
,25(OH)2D3 and TEI-9647 in Human Osteosarcoma Cells
To study whether the species specificity of VDR is the cause of the alternative responses of TEI-9647 in human- and rat-derived cells, SaOS-2 cells were cotransfected with the human vitamin D-responsive Cyp24 promoter-reporter plasmid and either the hVDR or rVDR expression plasmid by transient transfection. Then the resulting transcriptional activity was assayed in response to the presence 1
,25(OH)2D3 and/or TEI-9647 (see Fig. 4
, A and B). Without cotransfection of the VDR, the SaOS-2 cells were not transactivated by 108 M 1
,25(OH)2D3 stimulation (data not shown), which demonstrates that the measured transactivation depends mainly on the overexpressed VDR in this system. As can be seen in Fig. 4A
, in hVDR-expressing cells, TEI-9647 exhibited a suppressive effect on the function of 108 M 1
,25(OH)2D3 and no agonistic effect when present alone. On the contrary, in rVDR-expressing cells (Fig. 4B
), TEI-9647 present alone was an agonist and was not able to suppress the induction by 108 M 1
,25(OH)2D3. Similar results were obtained in ROS24/1 cells (Fig. 3
). Thus, in these two systems, the agonistic/antagonistic profile of TEI-9647 depends on the species origin of the VDR and not on the other cellular components. Collectively, these results indicate that the species origin of VDR directly determines the activity profile of TEI-9647.

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 4. Comparison of Human vs. Rat VDR on the Transcriptional Activities of 1 ,25(OH)2D3 and TEI-9647 in Human Osteosarcoma Cells
SaOS-2 cells were cotransfected with the human vitamin D-responsive Cyp24 promoter-reporter plasmid and either human (A) or rat (B) VDR expression plasmid by transient transfection and assayed for the resulting transcriptional activity in response to 1 ,25(OH)2D3 in the presence or absence of TEI-9647. Values shown represent the mean ± SD from three separate transfections.
|
|
ZK159222 Is an Antagonist in Either hVDR- or rVDR-Expressing Cells
The 1
,25(OH)2D3 analog ZK159222 (Fig. 1
) has been shown by several laboratories to be an effective antagonist for 1
,25(OH)2D3 (39, 41, 50, 51). It was of interest to determine whether the VDR antagonist properties of ZK159222 were affected by the species of origin (human vs. rat) of the VDR. As shown in Fig. 5
, A and B, when ZK159222 was evaluated in human MG-63 cells or rat ROS 17/2.8 cells, in the presence and absence of 1
,25(OH)2D3, it was found to be an antagonist in both cells with either an hVDR or a rVDR. This result was confirmed in the SaOS-2 cells that were transfected with either the hVDR (Fig. 5C
) or the rVDR (Fig. 5D
). Collectively, these results show that the antagonistic properties of ZK159222 are not related to the amino acid sequence differences between the hVDR and the rVDR.
Amino Acid Residues C403 and/or C410 Are Crucial for the Antagonistic Actions of TEI-9647
The C-terminal amino acid sequences of the human and rat VDRs are highly conserved, but there are three specific residues that are changed in the rVDR from residues L378, C403, and C410 of the hVDR (Fig. 6A
) (48, 49). The C-terminal region of the LBD of the VDR is related to the transactivation by the ligands (11) and also is the region containing three amino acid changes in the rVDR as compared with other hVDRs. We focused on the C403 and C410 residues in the hVDR, but which are serine (S399) or asparagine (N406) in the rVDR (Fig. 6A
). Thus, two expression plasmids for chimera VDRs were constructed (see Fig. 6B
). One is hVDR, of which the C-terminal region of the LBD (403427) is mutated to corresponding residues of the rVDR (hrVDR); the other chimera is the rVDR, which has the human C-terminal region of LBD (hVDR403427) (rhVDR) (Fig. 6B
). These were used as VDR expression plasmids and their reporter activity was assayed (Fig. 7
, A and B). When the hVDR with the rat-derived C-terminal sequence (hrVDR) was used, TEI-9647 did not antagonize the transactivation by 1
,25(OH)2D3, but could function as an agonist in the absence of 1
,25(OH)2D3. On the contrary, using rVDR with the human-derived C-terminal sequence (rhVDR), the TEI-9647 acted as an antagonist of 1
,25(OH)2D3 but was not an agonist itself. These results clearly indicate that the C-terminal region of the LBD of VDR plays an important role for determining the antagonistic/agonistic profile of TEI-9647 and suggest that one or both of the two Cys residues at 403 and 410 in hVDR were the key residues for the antagonist activity of TEI-9647.

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 6. Schematic Representation of the VDR Expression Plasmids Used in This Study
A, Comparison of the amino acid sequence of the C-terminal regions of human and rat VDRs. The C-terminal amino acid sequence of hVDR (48 ) and rVDR (49 ) are shown. The C-terminal regions of the LBDs of the two receptors are aligned. The shaded areas indicate homologous amino acids between the two VDRs. The asterisks indicate the two locations of amino acid sequence difference: specifically, the L378, C403, and C410 of the hVDR; and Q374, S399, and N406 in the rVDR. B, The following VDR plasmids were used: wild-type human, wild-type rat, chimera, and mutant VDR. Solid and shaded lines represent hVDR and rVDR sequences, respectively. Chimera VDRs were generated by the transfer of the 25 C-terminal residues of human and rat VDRs. The residues at 403 and 410 residues in the hVDR and corresponding residues in the rVDR are indicated by the two vertical lines.
|
|
To clarify which or both of the Cys residues in the C-terminal region of LBD of hVDR determine the agonistic/antagonistic profile of TEI-9647, we prepared two hVDR constructs with point mutations in residues 403 and 410 (see Fig. 6B
). The C403 and C410 were altered to Ser (C403S) and Asn (C410N), respectively [the double mutant hVDR (C403S/C410N) is the hrVDR described above]. These mutated hVDR constructs were used as expression plasmid and analyzed for transactivation by 1
,25(OH)2D3 or/and TEI-9647 with SaOS-2 cells. As shown in Fig. 7
, C and D, in the cells where either the hVDR (C403S) or the hVDR (C410N) was expressed, 1
,25(OH)2D3 transactivation of the Cyp24 promoter was antagonized by the presence of TEI-9647. Thus, the presence of only one cysteine (either C403 or C410) is sufficient for an antagonistic effect of TEI-9647; however, when both cysteines are absent, as in the rVDR, TEI-9647 acts as agonist rather than antagonist.
To examine the interaction between various mutant VDRs and SRC-1, we used a mammalian two-hybrid system. When using hVDR, rhVDR, or hVDR (C403S/C410N), TEI-9647 antagonized the interaction of these VDRs and SRC-1 induced by 1,25-(OH)2D3 (Fig. 8
, A, DF). On the contrary, using rVDR and hrVDR, TEI-9647 acted as an agonist, but was not an antagonist itself (Fig. 8
, B and C). These results confirm the results seen in Figs. 4
and 7
. The agonist/antagonist activities of TEI-9647 are based, in part, on the difference of the interaction of VDRs and SRC-1.

View larger version (37K):
[in this window]
[in a new window]
|
Fig. 8. Effects of TEI-9647 on the Interaction between Various Mutant VDRs and Coactivator SRC-1 Induced by 1 ,25(OH)2D3 in SaOS-2 Cells
SaOS-2 cells were transiently cotransfected using FuGENE 6 (Roche Diagnostics Corp.) with pM-SRC-1, pVP16-based receptor expression plasmid for wild-type, chimeric VDR, and site-directed mutant VDRs (pVP16-VDR), pM-SRC-1, pGVP2-GAL4BS, and pRL-TK, and assayed for the luciferase activity in response to 1 ,25(OH)2D3 (108 M) and/or TEI-9647 (107 M). Panel A, hVDR; panel B, rVDR; panel C; hrVDR; panel D, rhVDR; panel E, hVDR (C403S); panel F, hVDR (C410N). Values shown represent the mean ± SD from three separate transfections. Similar results were obtained in three independent experiments.
|
|
 |
DISCUSSION
|
---|
TEI-9647 was the first 1
,25(OH)2D3 analog to be identified as being an antagonist of 1
,25(OH)2D3 gene transactivation; these antagonist properties occurred both in human- or monkey-derived cells (37, 38). In this report we confirmed that TEI-9647 is an antagonist when acting on the hVDR but report that it is an agonist when acting on the rVDR. We showed that in rVDR-negative ROS24/1 cells (52), the antagonist or agonist actions of TEI-9647 were determined by the transfection of either the hVDR or rVDR, respectively (Fig. 3
). Similar results were obtained in the human cell line SaOS-2 (Fig. 4
). This indicates that it is the VDR that is the determinant of the species-specific effects of TEI-9647. Further, we showed that the antagonistic effects of TEI-9647 depend on the presence of one or two Cys residues in the C-terminal region of the hVDR.
Since the identification of the antagonistic actions of TEI-9647, many reports have studied the mechanism(s) of action of the antagonistic action of this compound. It is known that the antagonistic activity of TEI-9647 is dependent upon binding to the VDR and is dependent upon certain side-chain structures (see Fig. 1
). The 1-hydroxy stereoisomer of TEI-9647 is (23S)-25-dehydro-1ß-hydroxy-vitamin D3-26,23-lactone (1ß-OH-TEI-9647). Its binding affinity is about 200 times weaker than that of TEI-9647 and, accordingly, does not inhibit differentiation of HL-60 cells mediated by 1
,25(OH)2D3 (44). (23S)-1
-OH-D3-26,23-lactone (TEI-9616) functions as an agonist, not as an antagonist, in cells with an hVDR. This suggests that the 25-dehydro functionality is essential for the antagonistic property of the TEI-9647 (45).
Recently, the crystal structure of the natural 1
,25(OH)2D3 ligand and VDRs LBD was obtained with a truncated version of the receptor VDR that lacks amino acids 165215 (24). The x-ray structure of this ligand-receptor complex revealed that the side chain of this ligand interacted with the C-terminal region of VDR LBD. In other reports, TEI-9647 was shown to inhibit the heterodimer formation between VDR and RXR
, and the VDR interaction with coactivator proteins of the SRC/p160 family (38, 39, 40, 41). The x-ray structure of the estrogen receptor bound to two antagonists (4-hydroxytamoxifen and raloxifene) illustrated the presence of a unique antagonist ligand-receptor complex in which the position of helix 12 was significantly altered from that of an agonist-ligand-receptor complex. In the antagonistic conformation, helix 12 of the estrogen receptor LBD seems to be positioned incorrectly, so that the AF-2 domain on this helix is unable to interact with the LXXLL (L, leucine; X, any amino acid) core nuclear interaction motifs of coactivator proteins (53, 54). If the antagonist-ligand-receptor complex for TEI-9647/VDR has a similar helix 12 structure, then this would suggest that TEI-9647 stabilizes the hVDR helix 12 in a conformation that blocks an interaction with coactivators because the side chain of the ligand interacts with the C-terminal region of the LBD.
Thus, we focused on the C-terminal region of the VDR LBD. The C-terminal regions (the last 67 residues) of the LBD of human and rat VDR are highly conserved but differ from each other only at the L378, C403, and C410 of hVDR and the corresponding residues of rVDR (Fig. 6A
). We demonstrated that the C403 and/or C410 of hVDR is critical for the antagonistic activity of TEI-9647 (Fig. 7
).
Unlike TEI-9647, ZK159222, another well-known VDR antagonist, exhibited its antagonistic effect regardless of the species of VDR (Fig. 5
). This suggests the molecular mechanisms of these compounds are different from each other, although both types of compounds have relatively bulky ring structures in their side chains compared with the natural ligand. The trypsin protease sensitivity of the hVDR when liganded with TEI-9647 or ZK159222 is slightly different (41, 55). This suggests that these two compounds stabilize the VDR in different antagonistic conformations. The extended side chain of ZK159222 prevents the interaction between residues H397 and F422 (56). The disturbance of the interaction with these residues makes helix 12 deviate from an optimized position for coactivator recruitment. As mentioned above, TEI-9647 has been described to stabilize a different antagonistic conformation than ZK159222. Also, the TEI-9647 side chain is not so bulky as that of ZK159222. Thus, TEI-9647 may disturb the correct positioning of helix 12 via other amino acid residues within the ligand-binding pocket. Our data also support and extend this report. The tissue-specific agonism of ZK159222 is also reported (57), but the exact mechanism of the phenomena is not yet clear. On the other hand, as we show in this study, the species specificity of ZK159222 has not been demonstrated.
In the hVDR, C403 is included in helix 11, and C410 is in the linker region between helix 11 and helix 12. How the interaction between TEI-9647 and these residues can disturb the optimal positioning of helix 12 is unclear. The agonistic activity of TEI-9647 is approximately 10 times weaker than the natural ligand, 1
,25(OH)2D3. In the rVDR, the binding affinity of TEI-9647 is also about 10 times less than the natural ligand (data not shown). TEI-9647 binds to VDR, and the side chain (especially the 25-exo-methylene) might interact with C403 and/or C410 of the LBD. Thus, TEI-9647 could disturb the correct positioning of helix 12, resulting in an antagonistic effect on the transactivation properties of hVDR. On the other hand, TEI-9647 is an agonist when bound to the rVDR where there cannot be an appropriate interaction with side-chain cysteines. As shown in Fig. 8
, the agonist/antagonist activities of TEI-9647 are correlated, in part, with the difference of the interaction of VDRs and the coactivator SRC-1. Thus, the presence of either C403 or C410 or both residues (as in hVDR) results in TEI-9647 being an antagonist. Also, the mechanisms of antagonistic action of this compound are clearly a species-dependent phenomenon in the human vs. rat VDR.
In summary, this is the first report that presents not only the identification of two critical amino acid residues of the VDR that mediate the TEI-9647 functioning as an antagonist, but also shows that the species specificity of VDR can be essential in determining the agonist/antagonist profile of this analog as a ligand for the VDR.
 |
MATERIALS AND METHODS
|
---|
Compounds
The natural hormone 1
,25(OH)2D3, TEI-9647, and another type vitamin D antagonist, ZK159222, were synthesized at the Teijin Institute for Bio-Medical Research (44); their structures are shown in Fig. 1
. Each compound was dissolved in absolute ethanol.
Cells and Cell Culture
Rat osteosarcoma ROS17/2.8 cells were obtained from the Riken Cell Bank (cell no. RCB462; RCB, Ibargi, Japan) and human osteosarcoma MG-63 cells were obtained from the Japanese Collection of Research Bioresources (JCRB Cell Bank; Tokyo, Japan). Rat osteosarcoma ROS24/1 cells were kindly provided by Dr. Janet L. Stein (Department of Molecular Biology, University of Massachusetts Medical School, Worcester, MA). Human osteosarcoma SaOS-2 cells were kindly provided by Dr. T. Michigami (Departments of Environmental Medicine, Osaka Medical Center and Research Institute for Maternal and Child Health, Osaka, Japan). Cells were passaged twice each week to maintain an exponential proliferating phase.
-MEM (Invitrogen Corp., Carlsbad, CA) containing 10% heat-inactivated fetal bovine serum (FBS) was used as culture medium and maintained at 37 C under a 5% CO2 atmosphere.
Assay of ALP Activity
For the analog treatment of MG-63 cells, the cell culture was done in medium containing 50 µg/ml L-ascorbic acid and 10 mM ß-glycerol phosphate (58). For the ROS17/2.8 cells, medium containing neither ascorbic acid nor ß-glycerol phosphate was used. Treatment duration was 3 d for ROS17/2.8 cells and 6 d for MG-63 cells, respectively. At the end of the incubation with the vitamin D3 analogs, the treatment was stopped by removing the medium, followed by washing with calcium/magnesium-free Hanks salt solution, and sonicating the cells in 50 mM Tris-HCl (pH 7.3) (59). The samples were kept frozen until the ALP assays. ALP activity and protein content in the samples were determined by a Hitachi 7070 autoanalyzer (Hitachi Science Systems, Ltd., Hitachinaka, Ibargi, Japan) with the Bessey-Lowry method using 4-nitrophenol phosphate as a substrate and the Pyrogallol-Red method, respectively. ALP activity was expressed as IU/ mg protein.
Reporter Plasmid Construct
The PCR corresponding to the region of the human 24-hydroxylase gene (Cyp24), which contains two vitamin D-responsive elements (60), was inserted into a luciferase reporter plasmid pGL3-basic (Promega Corp. Madison, WI).
Site-Directed Mutagenesis and Plasmid Construction
The full-length cDNA for hVDR, rVDR, chimeric VDRs, and mutant VDR fragments was subcloned into the CMV2 promoter-driven pTracer expression plasmid (Stratagene, Heidelberg, Germany).
Transfection and Luciferase Reporter Gene Assays
SaOS-2 or ROS24/1 cells were seeded into 24-well tissue culture plates (4 x 104 cells per well) and cultured overnight in
-MEM containing 10% FBS. Cells were transfected using FuGENE 6 (Roche Diagnostics Corp., Indianapolis, IN) with 0.025 µg of pTracer-based receptor expression plasmid for VDR, and 0.25 µg of the reporter plasmid and Renilla luciferase plasmid, pRL-TK (0.025 µg) (Promega Corp.), which serves as an internal control for transfection efficiency, were included in each transfection. After 24 h transfection, the cells were incubated in
-MEM containing 10% FBS with or without vitamin D3 analogs. The cells were washed 24 h after onset of stimulation and prepared for dual luciferase assays according to the manufacturers instruction (Dual-Luciferase Reporter Assay System; Promega Corp.). Luciferase activities were determined using a Fluoroskan Ascent FL (Thermo Electron Business, Vantaa, Finland).
Mammalian Two-Hybrid Assays
To investigate the interaction of SRC-1 and VDR, the PCR products for wild-type, chimera and site-directed VDRs were fused in-frame into pVP16 expression vector, which contains the activation domain of a herpes virus (CLONTECH Laboratories, Inc., Palo Alto, CA) (pVP16-VDR) as described previously (38). pVP, pM-SRC-1, and pGVP2-GAL4BS plasmids were a gift from Dr. Michigami (Osaka Medical Center and Research Institute for Maternal and Child Health, Osaka, Japan). SaOS-2 cells were seeded into 24-well tissue culture plates and cultured overnight in
-MEM containing 10% FBS. Cells were transfected using FuGENE 6 with 0.25 µg of pM-SRC-1, 0.25 µg of pVP16-based receptor expression plasmid for wild type, chimeric VDR, and site-directed mutant VDRs, and 0.25 µg of pGVP2-GAL4BS and Renilla luciferase plasmid, pRL-TK (0.025 µg) (Promega Corp.), which serves as an internal control for transfection efficiency, were included in each transfection. After 24 h transfection, the cells were incubated in
-MEM containing 10% FBS with or without vitamin D analogs. The cells were washed 24 h after onset of stimulation and prepared for dual luciferase assays by Dual-Luciferase Reporter Assay System according to the manufacturers instruction. Luciferase activities were determined using a Fluoroscan Ascent FL.
 |
FOOTNOTES
|
---|
This work was partially supported by National Institutes of Health Grant DK-09012037 (to A.W.N.).
First Published Online January 13, 2005
Abbreviations: AF-2, Activation function 2; ALP, alkaline phosphatase; FBS, fetal bovine serum; LBD, ligand-binding domain; 1
,25(OH)2D3, 1
,25-dihydroxyvitamin D3; (23S,25R)-1
,25(OH)2D3-lactone, (23S,25R)-1
,25-dihydroxyvitamin D3-26,23-lactone; RXR, retinoid X receptor; SRC-1, steroid receptor coactivator 1; TEI-9647, (23S)-25-dehydro-1
-hydroxyvitamin D3-26,23-lactone; VDR, vitamin D receptor.
Received for publication June 8, 2004.
Accepted for publication January 5, 2005.
 |
REFERENCES
|
---|
- Carmeliet G, Van Cromphaut S, Daci E, Maes C, Bouillon R 2003 Disorders of calcium homeostasis. Best Pract Res Clin Endocrinol Metab 17:529546[CrossRef][Medline]
- Bouillon R, Okamura WH, Norman AW 1995 Structure-function relationships in the vitamin D endocrine system. Endocr Rev 16:200257[CrossRef][Medline]
- Abe E, Miyaura C, Sakagami H, Takeda M, Konno K, Yamazaki T, Yoshiki S, Suda T 1981 Differentiation of mouse myeloid leukemia cells induced by 1
,25-dihydroxyvitamin D3. Proc Natl Acad Sci USA 78:49904994[Abstract]
- Mathieu C, Adorini L 2002 The coming of age of 1,25-dihydroxyvitamin D3 analogs as immunodulatory agents. Trends Mol Med 8:174179[CrossRef][Medline]
- van Etten E, Branisteanu DD, Verstuyf A, Waer M, Bouillon R, Mathieu C 2000 Analogs of 1,25-dihydroxyvitamin D3 as dose-reducing agents for classical immunosuppressants. Transplantation 69:19321942[CrossRef][Medline]
- Cade C, Norman AW 1987 Rapid normalization/stimulation by 1,25(OH)2-vitamin D3 of insulin secretion and glucose tolerance in the vitamin D-deficient rat. Endocrinology 120:14901497[Abstract]
- Kajikawa M, Ishida H, Fujimoto S, Mukai E, Nishimura M, Fujita J, Tsuura Y, Okamoto Y, Norman AW, Seino Y 1999 An insulinotropic effect of vitamin D analog with increasing intracellular Ca2+ concentration in pancreatic ß-cells through nongenomic signal transduction. Endocrinology 140:47064712[Abstract/Free Full Text]
- Neveu I, Naveilhan P, Menaa C, Wion D, Brachet P, Garabédian M 1994 Synthesis of 1,25-dihydroxyvitamin D3 by rat brain macrophages in vitro. J Neurosci Res 38:214220[CrossRef][Medline]
- McGrath J, Feron F, Eyles D 2001 Vitamin D: the neglected neurosteroid? Trends Neurosci 24:570572[CrossRef][Medline]
- Wion D, MacGrogan D, Neveu I, Jehan F, Houlgatte R, Brachet P 1991 1,25-Dihydroxyvitamin D3 is a potent inducer of nerve growth factor synthesis. J Neurosci Res 28:110114[CrossRef][Medline]
- Haussler MR, Whitfield GK, Haussler CA, Hsieh JC, Thompson PD, Selznick SH, Dominguez CE, Jurutka PW 1998 The nuclear vitamin D receptor: biological and molecular regulatory properties revealed. J Bone Miner Res 13:325349[Medline]
- Pike JW 1997 The vitamin D receptor and its gene. In: Feldman D, Glorieux FH, Pike JW, eds. Vitamin D. San Diego: Academic Press; 105125
- Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schütz G, Umesono K, Blumberg B, Kastner P, Mark M, Chambon P, Evans RM 1995 The nuclear receptor superfamily: the second decade. Cell 83:835839[CrossRef][Medline]
- Norman AW, Collins ED 2001 Vitamin D and gene expression. In: Moussa NN, Berdanier C, eds. Nutrient-gene interactions in health and disease. Orlando, FL: CRC Press; 348391
- Kim YS, MacDonald PN, Dedhar S, Hruska KA 1996 Association of 1
,25-dihydroxyvitamin D3-occupied vitamin D receptors with cellular membrane acceptance sites. Endocrinology 137:36493658[Abstract]
- Huhtakangas JA, Olivera CJ, Bishop JE, Zanello LP, Norman AW 2004 The vitamin D receptor is present in caveolae-enriched plasma membranes and binds 1,25(OH)2-vitamin D3 in vivo and in vitro. Mol Endocrinol 18:26602671[Abstract/Free Full Text]
- Norman AW 1998 Receptors for 1
,25(OH)2D3: past, present, and future. J Bone Miner Res 13:13601369[Medline]
- Zanello LP, Norman AW 1997 Stimulation by 1
,25(OH)2-vitamin D3 of whole cell chloride currents in osteoblastic ROS 17/2.8 cells: a structure-function study. J Biol Chem 272:2261722622[Abstract/Free Full Text]
- Song X, Bishop JE, Okamura WH, Norman AW 1998 Stimulation of phosphorylation of mitogen-activated protein kinase by 1
,25-dihydroxyvitamin D3 in promyelocytic NB4 leukemia cells: a structure-function study. Endocrinology 139:457465[Abstract/Free Full Text]
- Akutsu N, Lin R, Bastien Y, Bestawros A, Enepekides DJ, Black MJ, White JH 2001 Regulation of gene expression by 1
,25-dihydroxyvitamin D3 and its analog EB1089 under growth-inhibitory conditions in squamous carcinoma Cells. Mol Endocrinol 15:11271139[Abstract/Free Full Text]
- Hannah SS, Norman AW 1994 1
,25(OH)2-vitamin D3-regulated expression of the eukaryotic genome. Nutr Rev 52:376382[Medline]
- Carlberg C, Polly P 1998 Gene regulation by vitamin D3. Crit Rev Eukaryot Gene Expr 8:1942[Medline]
- Masuyama H, Brownfield CM, St-Arnaud R, MacDonald PN 1997 Evidence for ligand-dependent intramolecular folding of the AF-2 domain in vitamin D receptor-activated transcription and coactivator interaction. Mol Endocrinol 11:15071517[Abstract/Free Full Text]
- Rochel N, Wurtz JM, Mitschler A, Klaholz B, Moras D 2000 The crystal structure of the nuclear receptor for vitamin D bound to its natural ligand. Mol Cell 5:173179[CrossRef][Medline]
- Tocchini-Valentini G, Rochel N, Wurtz JM, Mitschler A, Moras D 2001 Crystal structures of the vitamin D receptor complexed to superagonist 20-epi ligands. Proc Natl Acad Sci USA 98:54915496[Abstract/Free Full Text]
- Ishizuka S, Yamaguchi H, Yamada S, Nakayama K, Takayama H 1981 Stereochemistry of 25-hydroxyvitamin D3-26,23-lactone and 1
,25-dihydroxyvitamin D3-26,23-lactone in rat serum. FEBS Lett 134:207211[CrossRef][Medline]
- Ishizuka S, Ishimoto S, Norman AW 1984 Biological activity assessment of 1
,25-dihydroxyvitamin D3-26,23-lactone in the rat. J Steroid Biochem 20:611615[CrossRef][Medline]
- Ishizuka S, Ishimoto S, Norman AW 1984 Isolation and identification of 1
,25-dihydroxy-24-oxo-vitamin D3, 1
,25-dihydroxyvitamin D3-26,23-lactone and 1
,24(S),25-trihydroxyvitamin D3: in vivo metabolites of 1
,25-dihydroxy-vitamin D3. Biochemistry 23:14731478[CrossRef][Medline]
- Ishizuka S, Norman AW 1987 Metabolic pathways of 1
,25-dihydroxyvitamin D3 to 1
,25-dihydroxyvitamin D3-26,23-lactone: stereo-retained and stereo-selective lactonization. J Biol Chem 262:71657170[Abstract/Free Full Text]
- Ishizuka S, Oshida J, Tsuruta H, Norman AW 1985 The stereochemical configuration of the natural 1
,25-dihydroxyvitamin D3-26,23-lactone. Arch Biochem Biophys 242:8289[CrossRef][Medline]
- Kiyoki M, Kurihara N, Ishizuka S, Ishii S, Hakeda Y, Kumegawa M, Norman AW 1985 The unique action for bone metabolism of 1
,25-(OH)2D3-26,23-lactone. Biochem Biophys Res Commun 127:693698[Medline]
- Ishizuka S, Kiyoki M, Kurihara N, Hakeda Y, Ikeda K, Kumegawa M, Norman AW 1988 Effects of diastereoisomers of 1
,25-dihydroxyvitamin D3-26,23-lactone on alkaline phosphatase and collagen synthesis in osteoblastic cells. Mol Cell Endocrinol 55:7786[CrossRef][Medline]
- Ishizuka S, Kurihara N, Hakeda S, Maeda N, Ikeda K, Kumegawa M, Norman AW 1988 1
,25-Dihydroxyvitamin D3 [1
,25-(OH)2D3]-26,23-lactone inhibits 1
,25-(OH)2D3-mediated fusion of mouse bone marrow mononuclear cells. Endocrinology 123:781786[Abstract]
- Ishizuka S, Sumitani K, Hiura K, Kawata T, Okawa M, Hakeda Y, Kumegawa M 1990 Biological activity assessment of 1
,25-dihydroxyvitamin D3-26,23-lactone and its intermediate metabolites in vivo and in vitro. Endocrinology 127:695701[Abstract]
- Ishizuka S, Mimura H, Hayashi T, Oshida J, Ishizeki K, Takigawa M, Norman AW 1997 23(S)25(R)-1
,25-dihydroxyvitamin D3-2623-lactone stimulates matrix synthesis in chondrocytes from rabbit costal growth cartilage. In: Norman AW, Bouillon R, Thomasset M, eds. Vitamin D: chemistry, biology and clinical applications of the steroid hormone. Riverside, CA: University of California; 683684
- Norman AW, Bouillon R, Farach-Carson MC, Bishop JE, Zhou L-X, Nemere I, Zhao J, Muralidharan KR, Okamura WH 1993 Demonstration that 1ß,25-dihydroxyvitamin D3 is an antagonist of the nongenomic but not genomic biological responses and biological profile of the three A-ring diastereomers of 1
,25-dihydroxyvitamin D3. J Biol Chem 268:2002220030[Abstract/Free Full Text]
- Miura D, Manabe K, Ozono K, Saito M, Gao Q, Norman AW, Ishizuka S 1999 Antagonistic action of novel 1
,25-dihydroxyvitamin D3-26,23-lactone analogs on differentiation of human leukemia cells (HL-60) induced by 1
,25-dihydroxyvitamin D3. J Biol Chem 274:1639216399[Abstract/Free Full Text]
- Ozono K, Saito M, Miura D, Michigami T, Nakajima S, Ishizuka S 1999 Analysis of the molecular mechanism for the antagonistic action of a novel 1
,25-dihydroxyvitamin D3 analogue toward vitamin D receptor function. J Biol Chem 274:3237632381[Abstract/Free Full Text]
- Herdick M, Steinmeyer A, Carlberg C 2000 Antagonistic action of a 25-carboxylic ester analogue of 1
,25-dihydroxyvitamin D3 is mediated by a lack of ligand-induced vitamin D receptor interaction with coactivators. J Biol Chem 275:1650616512[Abstract/Free Full Text]
- Ishizuka S, Miura D, Ozono K, Saito M, Eguchi H, Chokki M, Norman AW 2001 (23S)- and (23R)-25-dehydro-1
-hydroxyvitamin D3-26,23-lactone function as antagonists of vitamin D receptor-mediated genomic actions of 1
,25-dihydroxyvitamin D3. Steroids 66:227237[CrossRef][Medline]
- Toell A, Gonzalez MM, Ruf D, Steinmeyer A, Ishizuka S, Carlberg C 2001 Different molecular mechanisms of vitamin D3 receptor antagonists. Mol Pharmacol 59:14781485[Abstract/Free Full Text]
- Herdick M, Bury Y, Quack M, Uskokovic MR, Polly P, Carlberg C 2000 Response element and coactivator-mediated conformational change of the vitamin D3 receptor permits sensitive interaction with agonists. Mol Pharmacol 57:12061217[Abstract/Free Full Text]
- Malloy PJ, Xu R, Peng L, Clark PA, Feldman D 2002 A novel mutation in helix 12 of the vitamin D receptor impairs coactivator interaction and causes hereditary 1,25-dihydroxyvitamin D-resistant rickets without alopecia. Mol Endocrinol 16:25382546[Abstract/Free Full Text]
- Miura D, Manabe K, Gao Q, Norman AW, Ishizuka S 1999 1
,25-Dihydroxyvitamin D3-26,23-lactone analogs antagonize differentiation of human leukemia cells (HL60 cells) but not of human acute promyelocytic leukemia cells (NB4 cells). FEBS Lett 460:297302[CrossRef][Medline]
- Ishizuka S, Miura D, Eguchi H, Ozono K, Chokki M, Kamimura T, Norman AW 2000 Antagonistic action of novel 1
,25-dihydroxyvitamin D3-26,23-lactone analogs on 25-hydroxyvitamin-D3-24-hydroxylase gene expression induced by 1
,25-dihydroxy-vitamin D3 in human promyelocytic leukemia (HL-60) cells. Arch Biochem Biophys 380:92102[CrossRef][Medline]
- Ishizuka S, Miura D, Ozono K, Chokki M, Mimura H, Norman AW 2001 Antagonistic actions in vivo of (23S)-25-dehydro-1
-hydroxyvitamin D3-26,23-lactone on calcium metabolism induced by 1
,25-dihydroxyvitamin D3. Endocrinology 142:5967[Abstract/Free Full Text]
- Dokoh S, Donaldson CA, Haussler MR 1984 Influence of 1,25-dihydroxyvitamin D3 on cultured osteogenic sarcoma cells: correlation with the 1,25-dihydroxyvitamin D3 receptor. Cancer Res 44:21032109[Abstract]
- Baker AR, McDonnell DP, Hughes M, Crisp TM, Mangelsdorf DJ, Haussler MR, Pike JW, Shine J, OMalley BW 1988 Cloning and expression of full-length cDNA encoding human vitamin D receptor. Proc Natl Acad Sci USA 85:32943298[Abstract]
- Burmester JK, Maeda N, DeLuca HF 1988 Isolation and expression of rat 1,25-dihydroxyvitamin D3 receptor cDNA. Proc Natl Acad Sci USA 85:10051009[Abstract]
- Bury Y, Steinmeyer A, Carlberg C 2000 Structure activity relationship of carboxylic ester antagonists of the vitamin D3 receptor. Mol Pharmacol 58:10671074[Abstract/Free Full Text]
- Carlberg C 2003 Molecular basis of the selective activity of vitamin D analogues. J Cell Biochem 88:274281[CrossRef][Medline]
- Shen J, Montecino M, Lian JB, Stein GS, Van Wijnen AJ, Stein JL 2002 Histone acetylation in vivo at the osteocalcin locus is functionally linked to vitamin D-dependent, bone tissue-specific transcription. J Biol Chem 277:2028420292[Abstract/Free Full Text]
- Brzozowski AM, Pike ACW, Dauter Z, Hubbard RE, Bonn T, Engstrom O, Ohman L, Greene GL, Gustafsson J-A, Carlquist M 1997 Molecular basis of agonism and antagonism of the oestrogen receptor. Nature 389:753758[CrossRef][Medline]
- Shiau AK, Barstad D, Loria PM, Cheng L, Kushner PJ, Agard DA, Greene GL 1998 The structural basis of estrogen receptor/coactivator recognition and the antagonism of this interaction by tamoxifen. Cell 95:927937[CrossRef][Medline]
- Bula CM, Bishop JE, Ishizuka S, Norman AW 2000 25-Dehydro-1
-hydroxyvitamin D3-26,23S-lactone antagonizes the nuclear vitamin D receptor by mediating a unique noncovalent conformational change. Mol Endocrinol 14:17881796[Abstract/Free Full Text]
- Vaisanen S, Perakyla M, Karkkainen JI, Steinmeyer A, Carlberg C 2002 Critical role of helix 12 of the vitamin D3 receptor for the partial agonism of carboxylic ester antagonists. J Mol Biol 315:229238[CrossRef][Medline]
- Herdick M, Steinmeyer A, Carlberg C 2001 Carboxylic ester antagonists of 1
,25-dihydroxyvitamin D3 show cell-specific actions. Chem Biol 7:885894[CrossRef]
- Franceschi RT, Young J 1990 Regulation of alkaline phosphatase by 1,25-dihydroxyvitamin D3 and ascorbic acid in bone-derived cells. J Bone Miner Res 5:11571167[Medline]
- Rao LG, Wylie JN, Sutherland MS, Murray TM 1994 17ß-Estradiol and parathyroid hormone potentiate each others stimulatory effects on alkaline phosphatase activity in SaOS-2 cells in a differentiation-dependent manner. Endocrinology 134:614620[Abstract]
- Chen K-S, DeLuca HF 1995 Cloning of the human 1
,25-dihydroxyvitamin D3 24hydroxylase gene promoter and identification of two vitamin D-responsive elements. Biochim Biophys Acta 1263:19[Medline]