25-Dehydro-1{alpha}-Hydroxyvitamin D3- 26,23S-Lactone Antagonizes the Nuclear Vitamin D Receptor by Mediating a Unique Noncovalent Conformational Change

C. M. Bula, J. E. Bishop, S. Ishizuka and A. W. Norman

Department of Biochemistry (C.M.B., J.E.B., A.W.N.) University of California-Riverside Riverside, California 92521
Department of Bone and Calcium Metabolism (S.I.) Teijin Institute for Biomedical Research Tokyo 191-8512, Japan


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
(23S)-25-dehydro-1{alpha}-Dihydroxyvitamin D3-26,23-lactone (TEI-9647; MK) has been reported to antagonize the 1{alpha},25-dihydroxyvitamin D3 nuclear receptor (VDR)- mediated increase in transcriptional activity. Using a transient transfection system incorporating the osteocalcin VDRE (vitamin D response element) in Cos-1 cells, we found that 20 nM MK antagonizes VDR-mediated transcription by 50% when driven by 1 nM 1{alpha},25(OH)2D3. Four analogs of 1{alpha},25(OH)2D3, also at 1 nM, were antagonized 25 to 39% by 20 nM MK. However, analogs with 16-ene/23-yne or 20-epi modifications, which have a significantly lower agonist ED50 for the VDR than 1{alpha},25(OH)2D3, were antagonized by 20 nM MK only at 100 pM or 10 pM, respectively. One possible mechanism for antagonism is that the 25-dehydro alkene of MK might covalently bind the ligand-binding site of the VDR rendering it inactive. Utilization of a ligand exchange assay, however, demonstrated that MK bound to VDR is freely exchanged with 1{alpha},25(OH)2D3 in vitro. These data support the apparent correlation between VDR transcriptional activation by agonists and the effective range of MK antagonism by competition. Furthermore, protease sensitivity analysis of MK bound to VDR indicates the presence of a unique conformational change in the VDR ligand-binding domain, showing a novel doublet of VDR fragments centered at 34 kDa, whereas 1{alpha},25(OH)2D3 as a ligand produces only a single 34-kDa fragment. In comparison, the natural metabolite 1{alpha},25dihydroxyvitamin D3-26,23-lactone yields only the 30-kDa fragment that is produced by all ligands to varying degrees. Collectively, these results support that MK is a potent partial antagonist of the VDR for 1{alpha},25(OH)2D3 and its analogs when in appropriate excess of the agonist.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The seco-steroid hormone 1{alpha},25(OH)2-vitamin D3 [1{alpha},25(OH)2D3] is known to have numerous physiological functions (1). These functions may be manifested either by the classical genomic mechanism (2, 3) or by the initiation of a complex signal transduction cascade that has been termed a nongenomic rapid response (4). In the genomic response, 1{alpha},25(OH)2D3 binds to the ligand-binding domain of the nuclear vitamin D receptor protein (VDR) which heterodimerizes with retinoid X receptor {alpha} (RXR{alpha}) and recruits transcriptional coactivators such as SRC-1 (5) and the DRIP complex (6).

Among the many biological functions of 1{alpha},25(OH)2D3, the most studied are its role in calcium homeostasis and cellular differentiation. Both 1{alpha},25(OH)2D3 and many analogs have been shown to mediate inhibition of the growth of various cancers; however, the utility of the hormone as a chemotherapeutic agent is reduced by its calcemic action, which is harmful above physiological concentrations (7, 8). Currently, many researchers are striving to produce analogs of 1{alpha},25(OH)2D3 that are able to discriminate between actions for cellular differentiation and calcium absorption/mobilization (2).

In the search for ligands that discriminate between desirable and undesirable functionality, occasionally some have been found to be antagonists, such as raloxifene for the estrogen receptor (9) and RU-486 for the progesterone receptor (10). In the case of the estrogen system, some of these antagonists have been shown to confer only partial antagonism and show useful agonist properties in other cell types or metabolic pathways (raloxifene and tamoxifen for example).

Recently, an analog of the natural metabolite 1{alpha},25(OH)2D3-26,23S-lactone has been discovered to function as an antagonist to the VDR-mediated actions of 1{alpha},25(OH)2D3. This antagonistic analog, 25-dehydro-1{alpha}-OH-D3-26,23S-lactone (see Fig. 1Go) [TEI-9647 or MK], has been shown to block 1{alpha},25(OH)2D3-induced cellular differentiation in HL-60 but not in NB4 cells (11). In cells overexpressing a transiently transfected VDR, a 10-fold excess of analog MK in combination with 1{alpha},25(OH)2D3 results in a 50% reduction in transactivation of a reporter plasmid driven by the 24-hydroxylase promoter. The same concentration ratio also was shown to reduce 1{alpha},25(OH)2D3-induced p21WAF1,CIP1 expression (12) and modulate the protein-protein interaction between the VDR/RXR heterodimers and between VDR and SRC-1 without modifying the nuclear localization or DNA binding properties of the VDR (13).



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Figure 1. List of Chemical Structures and Comparative Values for 1{alpha},25(OH)2D3 and Its Analogs Used in This Paper

The RCI or relative competitive index is a measure of ability to compete with [3H]-1{alpha},25(OH)2D3 for binding to the VDR-LBD. The CDR or cell differentiation ratio is the ratio of ED50 of the analog vs. 1{alpha},25(OH)2D3 in HL-60 or U-937 cells in antiproliferation assays (2 12 13 17 ). Complete names of the analogs are as follows: BS, 23S-25R-1{alpha},25-(OH)2-D3-26,23-lactone (naturally occurring metabolite); MK, 25-dehydro-1{alpha},25-(OH)2-D3-26,23R-lactone; V, 1{alpha},25-(OH)2-16-ene-23-yne-D3; IE, 20-epi-1{alpha},25-(OH)2-D3; AW, 1-F-25-(OH)-16-ene-23-yne-D3; EV, 22-(m-(dimethylhydroxymethyl)phenyl)-23,24,25,26,27-pentanor-1{alpha}-OH-D3;Q, 1,23R,25-(OH)3-D3; HQ, 1,25-(OH)2-22,23-diene-D3.

 
Presently, the molecular basis of antagonistic action of the 25-dehydro-1{alpha}-OH-D3-26,23S-lactone is not known. One possibility is that the reactive C-25/C-26 primary alkene may bind covalently within the hydrophobic pocket of the ligand-binding domain of the VDR to block the transcriptional action of the receptor. In this communication we extend the action of the antagonistic analog to the osteocalcin vitamin D response element (VDRE) and indicate that it functions not by covalent modification of the VDR but rather by displacing the far more agonistic 1{alpha},25(OH)2D3 in a concentration-dependent manner. The antagonistic action occurs only over a limited concentration range, however, that is dependent on the activity of the agonist. Furthermore, evidence for a unique conformational change in the ligand-binding domain of the VDR upon antagonist binding is also shown. Together, these results support a mechanism by which MK, when in appropriate excess, acts as a potent partial antagonist for 1{alpha},25(OH)2D3 and its analogs.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Antagonistic Action of MK on Transactivation by 1{alpha},25(OH)2D3
The antagonist 25-dehydro-1{alpha}-OH-D3-26,23S-lactone (MK TEI-9647; see Fig. 1Go), has previously been shown to reduce transcriptional activation of 1{alpha},25(OH)2D3 for the 25(OH)D-24-hydroxylase gene by 50% in transiently expressed Cos-7 cells (12, 13). Figure 2Go shows the reporter activity as fold-activation relative to the vehicle control over a range of 0.1 to 100 nM 1{alpha},25(OH)2D3 with and without analog MK in 10-fold excess. Here we show that a 10-fold excess of MK over 10 nM 1{alpha},25(OH)2D3 results in a 50% reduction of transcriptional activity in transiently transfected Cos-1 cells using a promoter containing the osteocalcin VDRE linked to the human GH reporter. Unlike the earlier report (14), we found that treatment with MK alone results in a significant 2.5-fold agonist activity over the vehicle control for the osteocalcin reporter. For the purpose of approximating physiological concentrations and assuring that the transactivation achieved by 1{alpha},25(OH)2D3 is not attenuated by nearing the maximal activation of the system, we selected 1 nM 1{alpha},25(OH)2D3 to be used for further experiments. Thus, in this experiment a 10-fold excess of MK over 1{alpha},25(OH)2D3 is a significant inhibitor of the transactivation of the osteocalcin promoter.



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Figure 2. Antagonistic Action of a 10-fold Molar Excess of MK on the Transcriptional Activation of 1{alpha},25(OH)2D3 at Various Concentrations

Cos-1 cells treated with DEAE-dextran were transiently transfected to overexpress the VDR with an osteocalcin VDRE-driven reporter gene and exposed 30 h later to either vehicle alone, 1{alpha},25(OH)2D3 at the indicated concentration, and analog MK at a 10-fold excess, either alone or in combination with 1{alpha},25(OH)2D3. An osteocalcin VDRE-induced reporter was assayed at 30 h following addition of analogs. Error bars indicate SEM of triplicate samples. Symbols **, *, and # above a pair (1{alpha}, 25(OH)2D3 ± MK) indicate a significant difference for P < 0.99, 0.95 or 0.90, respectively.

 
To determine the effect of varying the concentration of MK around a constant concentration of 1{alpha},25(OH)2D3, the transfected cells were treated with 1 nM 1{alpha},25(OH)2D3 and MK ranging from 0.1 nM and 1000 nM (Fig. 3Go). In this experiment, transactivation of the osteocalcin reporter by 1 nM 1{alpha},25(OH)2D3 is significantly antagonized by 30% at 1 nM MK and 36% at 10 nM MK (Fig. 3Go). Although, the maximal antagonism of 52% occurs at a 100-fold excess (100 nM) of MK, only an 80% confidence level can be attributed to the combination being different from MK alone. It is evident (Fig. 3Go) that antagonism by MK increases with the concentration of MK although the agonist activity attributed to MK rises also. The agonist activity of MK and the antagonistic action on 1{alpha},25(OH)2D3 transactivation are equivalent at 1000 nM, but the maximum antagonism occurs at 100 nM. The inability of MK to antagonize 1{alpha},25(OH)2D3 at a level greater than 60% is the result of MK being both an antagonist and a weak agonist. Therefore, the antagonistic resultant of the MK-1{alpha},25(OH)2D3 interaction is a combination of MK agonism and antagonism for 1{alpha},25(OH)2D3. To focus on the antagonistic effect in the absence of a significant agonist effect, we chose an MK concentration of 20 nM. At this level, MK is only mildly agonistic (0.2–0.8 fold activation over background; data not shown) and produces a 40% antagonism of transactivation as compared with the same level of 1{alpha},25(OH)2D3 alone (Fig. 3Go).



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Figure 3. Effect of Varying the Concentration of Analog MK Relative to a Constant 1 nM Concentration of 1{alpha},25(OH)2D3

Cos-1 cells were transiently transfected to overexpress the VDR with a osteocalcin VDRE driven reporter gene and exposed to the indicated concentrations of analog MK with or without 1 nM 1{alpha},25(OH)2D3. Osteocalcin-VDRE-induced reporter activity was assayed after 30 h. Error bars indicate ± SEM of triplicate samples. The symbol immediately above a pair of bars indicates a significant difference between that pair (1{alpha}, 25(OH)2D3 treated relative to MK alone). Symbols * and # represent P < 0.95 or 0.90, respectively. The bar at the top of the graph refers to categories with a significant antagonistic difference with respect to 1{alpha},25(OH)2D3 (left bar) alone having P < 0.95.

 
Consideration of Covalent Binding as a Possible Mechanism for the Antagonistic Action of MK
Two hypotheses have been used to describe the mechanism of MK antagonism toward the VDR. One mechanism suggests that the antagonist acts to compete for the same receptor binding site as 1{alpha},25(OH)2D3 where it acts to generate a receptor conformation that is not competent with regard to transactivation. A second hypothesis is that the C-25, C-26 primary alkene adjacent to a carbonyl group of the lactone serves as a nucleophile that may form a 1,4-nucleophilic addition to either histidine 305 or histidine 397 of the VDR; these residues have been shown to hydrogen bond with the 25-hydroxyl of 1{alpha},25(OH)2D3 (14). In this case, the VDR would be covalently bound at the ligand-binding site by a relatively inactive ligand. A hypothetical mechanism for the covalent addition is shown in Fig. 4AGo. The neutral nitrogen of a histidine could donate its lone pair of electrons to form a bond with C-27 of MK. This bond would simultaneously shift the electron density to the C-25/C-26 bond to form a more highly substituted alkene and forming a oxygen anion of the carbonyl group that could be stabilized by the second local histidine residue, if it was positively charged. Decay of the oxygen anion would regenerate the carbonyl causing electron density in the adjacent alkene to be transferred to a bond between C-25 and hydrogen from the charged nitrogen. The hydrogen-nitrogen bond energy would be returned to regenerate the lone pair on nitrogen.



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Figure 4. After Exposure to MK the VDR Remains Fully Exchangeable with 1{alpha},25(OH)2D3

A, A hypothetical mechanism for the 1,4-nucleophilic addition of MK to the VDR is illustrated as described in the results. B, After preincubation with either vehicle control, partially saturating or fully saturating concentrations of 1{alpha},25(OH)2D3 or MK for 4 h at 0 C, the remaining unoccupied VDR-LBD was then irreversibly bound to TPCK. Ligand-occupied VDR was then exchanged with excess [3H]-1{alpha},25(OH)2D3 and the unbound ligand was washed away (see Materials and Methods). DPM from the exchanged radioactive ligand are reported here as percent reoccupancy of the VDR-LBD. Values are the average of triplicate samples minus a duplicate background. The data from two independent experiments are shown. Error bars indicate SEM.

 
To test this second hypothesis, we preincubated analog MK or 1{alpha},25(OH)2D3 as a control with VDR present in vitamin D-deficient chick duodenal mucosal chromatin. After a 4-h incubation at 0 C, the analog was washed away and TPCK (L-1-tosylamido-2phenylethyl-chloromethyl-ketone) was added to covalently bind in the ligand binding site of any unoccupied VDR (15). Once the unreacted TPCK was washed away, the VDR is incubated at 37 C in the presence of excess [3H]1{alpha},25(OH)2D3. The radioactive ligand can then exchange with the sites previously protected by a noncovalently bound ligand. Figure 4BGo shows exchanged radioactive ligand bound as percent reoccupancy. As expected, all of the unoccupied ligand binding domains (LBDs) are irreversibly occupied by TPCK in the vehicle control and, as a result, none of the VDR is available for reoccupancy. At the low concentration of both 1{alpha},25(OH)2D3 and MK, the VDR ligand binding site is only partially occupied, allowing TPCK to covalently bind only to a portion of the receptors resulting in partial reoccupancy. The high concentration of 1{alpha},25(OH)2D3 is able to protect nearly all of the receptors (better than 96%), which would indicate that the VDR is nearly saturated. For the high concentration of analog MK, 89% of the VDR was occupied and was later fully exchanged for the tritium-labeled ligand. This would indicate that analog MK does not bind irreversibly to the VDR. Therefore, the theory that antagonism of MK functions by displacing the more agonistic 1{alpha},25(OH)2D3 is our working hypothesis.

Antagonistic Action of MK on 1{alpha},25(OH)2D3 Analogs
If antagonism results from the ability of MK to interfere with the interaction of stronger agonists for the VDR, then MK should be able to antagonize other VDR agonists in a manner that is proportional to their agonistic activities. To test this theory, four ligands were chosen on the basis of their relative competitive index (RCI) and cell differentiation ratio values (2, 16, 17). The chemical structure, the RCI, and the ratio of ED50 for cell differentiation in HL-60 or U-937 cells vs. 1{alpha},25(OH)2D3 (CDR) of these analogs is shown in the bottom row of Fig. 1Go. Each of these analogs was added both in the presence and absence of 20 nM analog MK to transiently transfected Cos-1 cells overexpressing the VDR and a plasmid vector containing the osteocalcin VDRE reporter construct (18). The transcriptional activity of each of these analogs as compared with 1{alpha},25(OH)2D3 is shown in Fig. 5Go. It is evident that the transcriptional activity is not directly related to either RCI or the cell differentiation ratio values. For instance, both the RCI and cell differentiation ratio of AW [1-F-25-(OH)-16-ene-23-yne-D3 ]are much lower than EV [22-(m-(dimethylhydroxymethyl)phenyl)-23,24,25,26,27-pentanor-1{alpha}-OH-D3], yet AW induces transactivation by 34% more. However, antagonism by MK is directly proportional to the transactivational activity for each of these analogs including 1{alpha},25(OH)2D3. Antagonism ranges from 44% for analog AW to 30% for both analogs Q and HQ. Another apparent trend is that as the agonist activity weakens (transcriptional activity is reduced), the antagonist action of analog MK diminishes. This trend can also be explained by the stronger agonist displacement hypothesis. When weaker agonists are used, the transcriptional activation of the agonist is less distinguishable from the poorly agonistic analog MK.



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Figure 5. 1{alpha},25(OH)2D3 and Comparable Analogs Are Antagonized by Analog MK in Proportion to Their Transcriptional Activity

The indicated analogs, chosen for having a similar RCI and cell differentiation ratio (CDI) to 1{alpha},25(OH)2D3 (chemical structures and activity values shown in Fig. 1Go), were used to treat transfected Cos-1 cells at 1 nM concentration with and without 20 nM analog MK. Reporter activity was measured 30 h after dosing and is reported here as fold activation relative to control 0.1% ethanol. Data are the average of two separate triplicate experiments. Error bars indicate ± SEM. Symbols **, *, and # indicate an antagonistic difference for P < 0.99, 0.95, or 0.90, respectively.

 
A corollary to the stronger agonist displacement theory is that an analog with a significantly higher agonistic effect than 1{alpha},25(OH)2D3 will require an increased molar excess of the antagonist to induce antagonism. The 20-epi analog IE (19, 20) and the 16-ene,23-yne analog V (7, 21, 22), which have increased biological effectiveness when present as ligands for the VDR relative to 1{alpha},25(OH)2D3, were used to test whether this were true. To test this hypothesis, dose-response curves of both analogs IE and V from 0.001 to 10 nM were used in the transfected Cos-1 cell system with and without the addition of 20 nM MK. Reporter activity was assayed at 30 h after dosing, and the results are reported as fold activation over vehicle control (Fig. 6Go). Analog IE was antagonized by MK only in the 0.01–0.1 nM range. At 0.001 nM, IE does not show any activation of transcription. At 0.01 nM, IE shows a 2-fold activation that is antagonized 70% by the addition of 20 nM MK. This difference is significant at better than 99% confidence interval. MK antagonism toward analog IE retreats rapidly at higher IE concentrations as indicated by the 18% antagonism of 0.1 nM IE. Analog V at 0.1 nM (Fig. 6BGo) is antagonized 42% in the presence of 20 nM MK with a difference of better than a 95% confidence. Analog V at 0.01 nM shows only a small amount of activity (<1.5-fold of basal expression), which is less than the agonist response of MK in the control (1.6-fold).



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Figure 6. Dose Response of Ligands of Higher Affinity Than 1{alpha},25(OH)2D3 and Concentration Range Affected by Antagonism of Analog MK

Transiently transfected Cos-1 cells were dosed with the indicated concentration of 20-epi analog IE (A) or 16-ene/23-yne analog V (B) both in the presence and absence of 20 nM MK (see Fig. 1Go for chemical structure and activity values). Bars labeled "control" are 1 nM 1{alpha},25(OH)2D3 instead of IE or V as labeled. After 30 h, reporter was assayed and results are reported here as the average of triplicate ± SEM. Each experiment is a representative of three separate experiments. Symbols ** and * indicate a pair of significant difference at P <0.99 or P <0.95, respectively.

 
Detection of an Altered VDR Conformation Resulting from MK Binding
For MK to function as an antagonist when bound to the VDR ligand-binding domain, we assume that the resulting conformational change would be different than that shown to occur as a result of binding an agonist such as 1{alpha},25(OH)2D3. To address this hypothesis, the VDR was subjected to limited proteolysis by trypsin in the presence of a saturating concentration of ligand. Alterations in the conformation of the protein, as reflected by different trypsin-mediated cleavage sites, can then be detected on SDS-PAGE as the fragmentation pattern changes. This occurs by exposing or shielding lysine and arginine residues that are differentially protected from proteolysis by steric hindrance. In the case of the VDR, the binding of 1{alpha},25(OH)2D3 produces protease-resistant fragments at 34 kDa and 28 kDa (18, 19). The 34-kDa and 28-kDa fragments have been shown by N-terminal sequencing to be cleaved at the C terminus of arginine 173; thus, they contain the C-terminal ligand-binding domain (23). Using this method we have observed the formation of a unique conformation that occurs upon binding of the MK antagonist to the VDR. This fragment is characterized by a doublet of bands at 34 kDa (specifically 34.6 kDa and 33.3 kDa mass apparent; see Fig. 7Go) and a fragment at 30 kDa. This 30-kDa band is also present in a very small amount in the 1{alpha},25(OH)2D3 fragmentation pattern and is seen as the predominant band for the natural metabolite lactone BS, 1{alpha},25R(OH)2-D3-26,23S-lactone. The 30-kDa fragment, as is reported here, is thought to be the same fragment as has been previously referred to as the 28-kDa fragment (23).



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Figure 7. MK induces a Unique Conformational Change in the VDR LBD

Limited trypsin proteolysis of [35S]methionine VDR occupied with the indicated ligand is shown by autoradiography after SDS-PAGE. 1{alpha},25(OH)2D3, the antagonistic lactone analog MK, and the natural lactone BS were added at 10 µM concentration prior exposure to trypsin for fragmentation (see Materials and Methods).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Previous studies have demonstrated (11, 24) that 25-dehydro-1{alpha}-OH-D3-26,23S-lactone (MK) can function as an antagonist of the VDR. In this report we have studied the molecular basis of MK’s antagonistic properties with respect to its possible covalent attachment to the VDR-LBD and as to whether the presence of MK in the VDR-LBD alters the conformation of the protein as assessed by protease sensitivity analysis.

A model to describe the possible covalent addition of MK to the VDR is presented in Fig. 4AGo. However, our exchange assay has indicated that no significant covalent binding of MK to the VDR takes place within 4 h at 0 C (Fig. 4BGo). Thus, we conclude that the MK interaction with VDR is freely reversible.

It is obvious that there is a small but significant agonist activity that can be attributed to MK. At 1000 nM, MK alone shows 39% of the activity induced by 1 nM 1{alpha},25(OH)2D3 alone on the osteocalcin VDRE (Fig. 3Go). Agonist activity induced by a combination of both 1000 nM MK and 1 nM 1{alpha},25(OH)2D3 falls within the standard error of the agonist activity of 1{alpha},25(OH)2D3. As a partial antagonist, the antagonism of the MK and 1{alpha},25(OH)2D3 is actually the product of two components, the subtractive antagonistic effect of MK on the 1{alpha},25(OH)2D3 agonist and the additive effect of MK and 1{alpha},25(OH)2D3 agonism.

When the ligand being antagonized is comparatively much more active than MK, as is the case of V (21, 22) and IE (19, 20) (Fig. 6Go), the equilibrium of interaction with the VDR is shifted away from MK because of its relatively poorer affinity so that the antagonistic activity of MK is again reduced. These analogs require a 200- to 2,000-fold excess of MK, respectively, as compared with only a 20-fold excess shown for 1{alpha},25(OH)2D3. This would imply that when using this system, the effective range of MK antagonism is only useful for agonists with a level of transactivation in the range of 1{alpha},25(OH)2D3.

We have shown that as the VDR interacts with progressively weaker agonists, the antagonism by MK is correspondingly weaker (Fig. 5Go). When the ligand being antagonized by MK is comparatively less active, the agonist activity of MK is more significant because the subtractive effect is reduced. Therefore, the antagonism of MK results from the ability to displace ligands that have more agonist activity in a concentration-dependent manner.

The ability of a ligand to induce transactivation of the VDR can be described as a combination of affinity, kinetics, and effectiveness at producing an optimal protein conformation. Using the protease sensitivity assay, we could observe changes in the protein conformation of the ligand-binding domain. With 1{alpha},25(OH)2D3 as the ligand, a single 34-kDa trypsin fragment is produced. However, ligands that transactivate poorly or not at all, such as 1{alpha}-OH-D3 or 25-OH-D3, predominantly produce only a 30-kDa fragment (data not shown). 20-Epi-1{alpha},25(OH)2D3, which is 500–2,000 times more potent than 1{alpha},25(OH)2D3 in transactivation of the VDR (19), generates not only the 34-kDa fragment of 1{alpha},25(OH)2D3 and a minor amount of the 30-kDa fragment but also a novel 32 kDa fragment (18, 23). Antagonist MK, however, consistently produces a unique pattern of three fragments with nearly equal intensity at 34.6, 33.3, and 30 kDa (Fig. 7Go). The 30-kDa fragment is exactly the same size as that seen very faintly when 1{alpha},25(OH)2D3 is the ligand, and predominantly when the natural metabolite lactone BS is the ligand. However, both the 34.6- and 33.3-kDa fragments are somewhat different than the fragmentation pattern of any previously tested analog.

Previous researchers determined by N-terminal sequencing that both the 34-kDa and 30-kDa (previously cited as 28 kDa) bands are C-terminal fragments from cleavage at arginine-173 (23). The 34-kDa band contains a 19 residue portion of the hinge region and the entire LBD. Having the same N terminus as the 34-kDa band, the 30-kDa band must result from further trypsinization near the C terminus. An increase in the intensity of this fragment could be explained as the failure of the ligand to coordinate the active closed conformation (26, 27, 28) of helices 10–12, leaving them more susceptible to proteolytic cleavage. We speculate that the increase in the 30-kDa band protease sensitivity is indicative of more time spent in a transcriptionally inactive state.

The MK-induced doublet at 34 kDa, however, is less easily explained. It could possibly be formed as a consequence of altered VDR phosphorylation in the transcription/translation reticulocyte system used to produce the [35S]-VDR employed for the protease sensitivity assay. More likely, however, VDR binding of MK may result from the exposure of alternate trypsin sites at either end of the 34-kDa fragment produced by cleavage at Arg174 (23), which is characteristic of 1{alpha},25(OH)2D3 and other agonistic ligands. A candidate cleavage point located two amino acids before the beginning of helix 12, Lys413, is 1.5 kDa smaller than the Arg174 cleavage product as determined from the primary sequence (29). This value is almost exactly the difference in molecular mass between the two fragments reported here. The example of an altered positioning of helix 12 resulting from antagonist binding into the estrogen receptor has been previously reported (9, 30). This further supports the idea that the doublet of fragments centered at 34 kDa upon MK binding results from proteolysis of a differentially exposed trypsin site near helix 12 of the VDR.

This publication is the first report of MK antagonism toward the osteocalcin VDRE and in Cos-1 cells. For this reason, it is difficult to ascertain whether the differences between results seen for this system and those reported are the result of using a different promoter, the cell line, or a yet-unidentified factor. Transcriptional antagonism of the reporter plasmid containing the SV40 promoter having two human 25-OH-D3 24-hydroxlase gene VDREs has been previously shown in three different cell lines using 10 nM 1{alpha},25(OH)2D3 and 100 nM MK concentrations. In Cos-7 cells, MK shows approximately 60% antagonism compared with 1{alpha},25(OH)2D3 induction alone. MK agonist activity in these cells is only about 3% of the activation by 1{alpha},25(OH)2D3 (24). In HeLa cells, MK has a similar antagonism of approximately 45% but twice the MK agonist activity (~ 8% of 10 nM 1{alpha}, 25(OH)2D3 alone). Saos-2 cells show a much larger antagonistic response of around 85% with less than 3% MK agonist activity (13). In the present study, the Cos-1 cells display a 54% antagonism but have a larger MK agonist activity that is 11% of the 1{alpha},25(OH)2D3-induced activity at these same concentrations.

It has been suggested that the larger antagonistic response to MK in Saos-2 cells may be due to a possible 1{alpha},25(OH)2D3-specific functionality resulting from the bone-derived osteosarcoma cells, whereas, kidney (Cos-1 and Cos-7)- or cervical tissue-derived (HeLa) cells might not be as responsive due to an absence of such functionality (13). A reason for the larger agonist response of MK in the Cos-1/osteocalcin system, which is nearly 3 times that of the morphologically similar Cos-7 cells, may be due to the use of the osteocalcin reporter, but no data are presented to support such a hypothesis.

Previous to MK, no antagonists of the genomic action of 1{alpha},25(OH)2D3 on the VDR had been reported, although, 1ß,25(OH)2D3 has been shown to antagonize the nongenomic or rapid actions of 1{alpha},25(OH)2D3 to stimulate transcaltachia or to open chloride channels in ROS 17/2.8 cells (31, 32). However, antagonists or partial antagonists of other steroid hormones already have been shown to be useful pharmaceuticals. The estrogen system has a number of clinically relevant antagonists such as raloxifene, tamoxifen, and ICI182,780 (9). Another prominent pharmaceutical antagonist of both progesterone and glucocorticoid is mifepristone (RU486) (10).

In conclusion, we have shown that the antagonism of MK is not due to a covalent binding to the VDR but, rather, the freely reversible interaction with a ligand that is partial antagonist and agonist. For the osteocalcin VDRE in Cos-1 cells, this antagonism has been shown to occur against all ligands tested, but the effective range of antagonism is dependent on the transactivation activity of the agonist relative to MK. Also, we have shown that when the VDR-LBD is occupied by MK there results a unique conformational change in the protein which mediates the antagonist properties of the analog.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Culture and Transient Transfection Assays
Cos-1 monkey kidney cells were seeded at 8 x 105 cells per 150-mm culture dishes (Corning, Inc. Corning, NY) in DMEM Nutrient Mixture-F12 Ham (Sigma, St. Louis, MO) with 10% Rehatuin FBS (Intergen, Purchase, NY). These cells were passed near confluency at 4 x 106 cells per 150-mm plate using Cellgro 0.25% trypsin, 0.1% EDTA solution (Mediatech Inc., Herndon, VA). For transfection, Cos-1 cells were seeded at 3 x 105 cells per well on Costar six-well plates (Corning, Inc. Corning, NY). After 24 h incubation, PBS-washed cells at approximately 50% confluence were transfected using a 9-min pretreatment with 1 mg/ml diethylaminoethyl (DEAE)-dextran (Sigma) in PBS. After being washed twice, pretreated cells were incubated for 24 min with 0.5 µg/well pGEM-4 VDR plasmid and 1.5 µg/well pTKGH (18) plasmid, containing the VDRE from the osteocalcin gene (ocVDRE) with the tyrosine kinase promoter and human GH reporter gene (18). Transfected cells were incubated in 80 µM chloroquine in DMEM Nutrient Mixture-F12 Ham with 4.5% charcoal-stripped FBS for 3.5 h followed by the same culture medium without chloroquine for 27 h. Thirty hours after transfection, the cell medium was replaced with the same medium containing 1{alpha},25(OH)2D3, its analogs, or a combination with a final ethanol concentration of 0.1%. At 30 h after analog treatment, the cell medium was harvested to measure GH reporter by RIA (Nichols Institute Diagnostics, San Juan Capistrano, CA). The data is presented as fold activation relative to ethanol control. All experiments were carried out on triplicate samples and the data are expressed as the mean ± SEM.

Exchange Assay
Duodenal mucosa from 3-week-old vitamin D-deficient White Leghorn cockerels (Hyline International, Lakeview CA), fed from hatch on a standard rachitogenic diet (33), was homogenized in 10 mM Tris, 1.5 mM EDTA, 1 mM dithiothreitol, pH 7.4 (TED) to make a 10% homogenate. The crude chromatin fraction was prepared by our standard procedure (34) involving three washes of the nuclear fraction in TED with 0.5% Triton X-100 (Sigma), wash and resuspension in TED. The chromatin fraction was divided into five aliquots and treated with either ethanol (vehicle control, 5 µl/ml) or 1{alpha},25(OH)2D3 at 0.5 or 2 nM (final concentration) or MK at 5 or 200 nM. Ligands were incubated with the chromatin prep for 4 h on ice, followed by a wash and resuspension in TED. For the measurement of unoccupied receptor, a standard binding assay was set up using receptor from each treatment group binding to [3H]-1{alpha},25(OH)2D3 (100 Ci/mmol, Amersham Pharmacia Biotech, Arlington Heights, IL) in the presence or absence of a 200-fold excess of 1{alpha},25(OH)2D3. The proportion of occupied receptor in each sample was measured using our exchange assay (35). Briefly, aliquots of the chromatin receptor preparation were incubated with TPCK (Sigma) for 30 min on ice to covalently block unoccupied receptor binding sites. Next, aliquots of the TPCK-treated chromatin receptor were incubated with [3H]-1{alpha},25(OH)2D3 in the presence or absence of 1{alpha},25(OH)2D3 at 37 C for 30 min, followed by hydroxyapatite (Bio-Rad Laboratories, Inc. Hercules CA) separation of bound and free ligand (on ice) and liquid scintillation counting. Data are presented as percent reoccupancy, which is the percent normalized average of the disintegrations per minute (DPM) from triplicate samples divided by duplicate background samples containing a large excess of unlabeled 1{alpha},25(OH)2D3. The data from two separate experiments are shown as the mean ± SEM.

Protease Sensitivity Assay
The sensitivity of ligand-bound receptor to limited proteolysis was determined using our protease sensitivity assay (18). [35S]-labeled VDR was prepared using the TNT coupled transcription and translation system (Promega Corp., Madison, WI) from the pGEM-4 plasmid containing an insert for wild-type VDR cDNA and [35S]methionine (1,000 Ci/mmol, Amersham Pharmacia Biotech). Aliquots (5 µl) of the [35S]-VDR were incubated for 20 min at room temperature with ligand (10 µM), followed by treatment with 15 µg trypsin/ml (Sigma) for 20 min at room temperature. Samples were run on 12% SDS-PAGE. The radioactive bands showing the [35S]-VDR proteolytic fragments were visualized by autoradiography.


    FOOTNOTES
 
Address requests for reprints to: Dr. Anthony W. Norman, Department of Biochemistry, University of California, Riverside, Riverside, California 92521. E-mail: norman{at}ucrac1.ucr.edu

Received for publication May 9, 2000. Revision received August 3, 2000. Accepted for publication August 7, 2000.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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