A Negative Vitamin D Response DNA Element in the Human Parathyroid Hormone-related Peptide Gene Binds to Vitamin D Receptor Along with Ku Antigen to Mediate Negative Gene Regulation by Vitamin D*

We found that the human parathyroid hormone-related peptide (hPTHrP) gene contained a DNA element (nVDREhPTHrP) homologous to a negative vitamin D response element in the human parathyroid hormone gene. It bound to vitamin D receptor (VDR) but not retinoic acid Xα receptor (RXRα) in the human T cell line MT2 cells. VDR binding to this element was confirmed by the Southwestern assay combined with immunodepletion using anti-VDR monoclonal antibody, and this binding activity was repressed by 1,25-dihydroxyvitamin D3. Such a repression was reversed by acid phosphatase treatment, suggesting that 1,25-dihydroxyvitamin D3 phosphorylates VDR to weaken its binding activity to nVDREhPTHrP. In electrophoretic mobility shift assay, we found anti-Ku antigen antibody specifically supershifted the MT2 nuclear proteinnVDREhPTHrP complex. The nVDREhPTHrP-bearing reporter plasmid produced vitamin D-dependent inhibition of the reporter activity in MT2 cells, which was markedly masked by the introduction of the Ku antigen expression vector in the antisense orientation. On the other hand, such a procedure did not perturb the vitamin D response element-mediated gene stimulation by vitamin D. These results indicate that nVDREhPTHrP interacts with Ku antigen in addition to VDR to mediate gene suppression by vitamin D.

The mechanism by which steroid/thyroid nuclear hormone receptors activate gene transcription has been extensively studied (1)(2)(3)(4)(5)(6)(7)(8). Among them there have been so many lines of solid evidence showing that vitamin D receptor (VDR), 1 thyroid hormone receptor, and retinoic acid receptor employ a common machinery to exert their ligand-dependent specific effects; all of them utilize a retinoic acid X receptor (RXR) in common as a partner of a heterodimer (2,4). However, this mechanism seems to be confined only to gene stimulation but not to gene repression. This situation led us to address one of the key points in hormonal biology, negative feedback mechanism. The levels of almost all the hormones synthesized at a specific organ are under rigid control to keep their levels within a very narrow range. One of the representative mechanisms is the so-called end product inhibition. In the cases of the nuclear hormone receptors, negative transcriptional regulation of several pituitary trophic peptide hormone genes by glucocorticoid or sex steroid hormones is a good example. Although individual nuclear hormone receptors might well be involved in such regulation, detailed unified molecular mechanisms such as the manner of dimerization are largely unknown, with a few exceptions (9 -14). Likewise, the mechanism of negative gene regulation by VDR and vitamin D is only partially understood (15)(16)(17)(18)(19)(20)(21). In this case, an active form of vitamin D, 1,25-dihydroxyvitamin D 3 , can be considered an end product of parathyroid hormone (PTH) action, and this metabolite, in turn, inhibits the synthesis of PTH mRNA to keep the blood calcium level constant. Demay et al. (16) first reported that a negative vitamin D response element (nVDRE) exists in the upstream region of the human PTH gene to mediate such gene repression. This element contains a homologous sequence to only one of the two hexameric DNA sequences that form the core sequence of the consensus DNA sequence (VDRE) for positive gene regulation by vitamin D (2,4,(22)(23)(24)(25). In this process, it was shown that VDR, but not RXR, was involved, and the presence of another unknown partner protein(s) of VDR was proposed (17). However, there have been no reports confirming that the nVDRE is conserved among the genes whose expression is negatively regulated by vitamin D. We have found that the human PTH-related peptide (PTHrP) gene, which is assumed to be derived from an ancestoral gene in common with the PTH gene (26), contains a DNA sequence very homologous to the nVDRE of hPTH. Inoue et al. (27) reported that expression of the hPTHrP gene was inhibited by 1,25-dihydroxyvitamin D 3 at the transcriptional level in human adult T cell lymphoma/ leukemia virus-infected T cells, MT2 cells. With these cells, the therapeutic potential of vitamin D to decrease the blood calcium level due to the inhibition of PTHrP synthesis was discussed. Unlike parathyroid cells, from which it is hard to establish cultured cell lines, MT2 cells are transfectable cultured cell lines. Here, we examined the molecular mechanism of negative regulation of the PTHrP gene by vitamin D in MT2 cells.

MATERIALS AND METHODS
Homology Search by Computer Analysis-The DNA sequences homologous to the nVDRE of human PTH (16) were searched in the EMBL gene bank, including the human PTHrP gene, and some of them are shown in Fig. 1. We also compared the nVDRE with other DNA elements reported to be responsible for negative as well as positive gene regulation by vitamin D (16 -24).
Transfection and CAT Assay-MT2 cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum unless otherwise mentioned. The CAT plasmids or the expression vector encoding p70 subunits of Ku antigen in the antisense orientation (29) were introduced into MT2 cells by the DEAE-dextran method (30). After transfection, cells were equally split into several dishes to avoid differences in transfectional efficiency among dishes. Final amounts of the transfected plasmids per dish were 5 g. Twelve h later, the medium was changed to RPMI 1640 containing 1% fetal bovine serum with several different concentrations of vitamin D metabolites (purchased from Dupher Co.) or vehicle (100% ethanol) alone. In each case, 40 h after transfection, cells were harvested, and CAT assay was performed. Where indicated, average CAT activity was calculated after three separate transfections using 14 C scintillation counting, and typical results are shown in the figures with a given CAT activity as 100 in each case. Details are shown in each figure.
Preparation of Nuclear Extracts and Gel Shift Assay-Nuclear extracts were prepared by the method of Schreiber et al. (31) from MT2 cells 40 h after maintaining the cells in the media as indicated. The synthetic oligonucleotides were end-labeled with [␥-32 P]ATP by T 4 polynucleotide kinase. 10 4 cpm of the probe (10 9 cpm/g) was incubated with 10 g of the nuclear protein along with 1 g of poly(dI-dC) in each reaction for 30 min at room temperature. Final KCl concentration was adjusted to 80 mM by the binding buffer containing no KCl (10,31). The total reaction volume was 25 l. DNA-protein complexes were resolved on 4% nondenaturing acrylamide gels, dried, and visualized by autoradiography as described (12)(13)(14). Where indicated, 0 -50-fold molar excesses of nonradiolabeled oligonucleotides were used as competitors. When anti-human VDR rat monoclonal antibody, 9A7 (BIOMOL Research Laboratory Inc.), or anti-human RXR␣ rabbit polyclonal antibody, D-20 (Santa Cruz Biotech. Inc.) was used, each of 1/20 and/or 1/4 dilution and the respective control IgGs (1 l) supplied by manufacturers were included in the reaction for 15 min before the addition of the radiolabeled probes. We also used 1 l of anti-Ku antigen human antiserum, which specifically recognizes the p70 subunit of Ku antigen (29) and control serum in the same way in electrophoretic mobility shift assay (EMSA). To explore the effects of vitamin D metabolites on the protein-DNA interaction, cells were treated in the same manner as described in transfection method. Where indicated, 0.1 unit of potato acid phosphatase (Sigma) was directly included in the reaction mixture at 37 C°for the indicated period. Protein concentrations were determined by the Bio-Rad assay kit.
Southwestern Analyses, Immunodepletion, and Immunoblotting-Thirty g of the nuclear proteins obtained as described above was mixed with an equal volume of 2ϫ denaturing buffer (5% N-lauroylsarcosine, 5 mM Tris-Cl (pH 6.8), 25% glycerol, 0.05% pyronin Y, and 200 mM dithiothreitol). After a 15-min incubation at room temperature, samples were loaded onto an 8% (or 10%) SDS-polyacrylamide gel. Further details were described elsewhere (28), except that we used a radiolabeled oligo(nVDRE hPTHrP or VDRE mop ) in this study. Where indicated, we used MT2 nuclear proteins in which VDR was immunodepleted with the 9A7 shown above, according to the method reported previously (28). Immunoblotting with 1 g/ml of the 9A7 or 1:1000 diluted anti-Ku antigen human antiserum also shown above was performed by the enhanced chemiluminescence method as reported (28).

Putative nVDRE Sequences in PTH and PTHrP Genes from
Several Species-In the upstream region of the human PTHrP gene, we found a DNA sequence homologous to a nVDRE in the hPTH gene (16). In the anti-stranded sequence of this DNA in the hPTHrP gene, 11 out of 13 bases or 10 out of 11 bases were identical to the nVDRE of hPTH (Fig. 1). Both contained one copy of the DNA motif AG(G/A)TTCA. This motif mimics a core sequence (AG(G/A)TCA) for the binding of the specific set of the nuclear hormone receptor including VDR (2). Interestingly, one VDRE found in the mouse osteopontin gene (VDRE mop ; Ref. 22) consists of a direct repeat of the same motif with two (or three) spacers. Furthermore, 4 bases, CTAT, flanking this motif were common between the two. These findings suggest that both might mediate negative gene regulation by 1,25-dihydroxyvitamin D 3 . Falzon (18) recently reported that the rat PTHrP gene possessed two distinct DNA elements (nVDREs), both of which were responsible for negative gene regulation by vitamin D. We compared our nVDRE of hPTHrP with Falzon's nVDREs in the rat PTHrP gene (18) and speculated that one of them might be equivalent to our nVDRE because of the similar location and overall sequence similarity (see "Discussion"). Furthermore, we also found a DNA sequence in the chicken  (16), nVDRE rPTHrP -1 and nVDRE rPTHrP -2 (18), nVDRE rPTHrP -3 (19), nVDRE cPTH (21), and the nVDRE-like sequence of chicken TGF␤2 (20). One of the positive VDREs, VDRE mop (22), was also included.
TGF␤2 gene very homologous to nVDREs of both hPTH and hPTHrP. Intriguingly, expression of the chicken TGF␤2 gene was shown to be inhibited by vitamin D in mesangial cells (20). On the other hand, as shown in Fig. 1, the sequences of other recently proposed nVDREs such as those in the chicken PTH (21) gene and the rat PTHrP gene (19) or positive VDRE in the human osteocalcin gene (23) are considerably different from these three nVDREs.
Interaction between Nuclear Protein(s) in MT2 Cells and nVDRE hPTHrP -We synthesized oligonucleotides corresponding to nVDRE hPTHrP and VDRE mop and examined the interactions between each of these oligonucleotides and nuclear protein(s) from MT2 cells by EMSA. As shown in Fig. 2, both of the oligonucleotides formed two protein-DNA complexes. Since each of the lower bands did not consistently appear, we focused on the upper bands in this study. Although the migrating positions of both complexes seemed similar in EMSA, the MT2 protein-nVDRE hPTHrP complex was competed out only by a 10 -50-fold molar excess of the nonradiolabeled nVDRE hPTHrP . The same amount of VDRE mop was ineffective as a competitor. Conversely, only a 10 -50-fold molar excess of VDRE mop , but not that of nVDRE hPTHrP , was able to abolish the formation of MT2 protein-VDRE mop complex. Nevertheless, a 200-fold molar excess of the reciprocal competitors in each EMSA similarly abolished the binding between MT2 nuclear proteins and each probe (not shown and see "Discussion"). These results indicate that the composition of the nuclear proteins is different, at least in part, between the two protein-DNA complexes.
We next explored the possibility that VDR and RXR␣, both of which were shown to be crucially involved in VDRE mop -mediated gene regulation by vitamin D in a wide variety of cells (2 and 4) other than MT2 cells, were present in the two MT2 nuclear protein-DNA complexes shown here. As shown in Fig.  3, both antibodies supershifted or attenuated the binding between VDRE mop and MT2 nuclear proteins. On the other hand, the formation of the complex between nVDRE hPTHrP and MT2 nuclear proteins was hardly affected by the addition of anti-RXR␣ antibody. Although the 1/4-diluted anti-VDR antibody weakened this complex, it did not supershift the complex (lane 3) nor did its 1/20 dilution affected the formation of the complex (lane 2), which was in contrast to the case of VDRE mop and MT2 nuclear proteins (lanes 4 -6). Together, these results suggest that nVDRE hPTHrP -MT2 protein complex contained VDR but not RXR␣, and VDR might be included in this complex in a manner different from the authentic VDR-RXR␣ heterodimer formation. Indeed, MT2 cells are known to contain large amounts of VDR (27). nVDRE hPTHrP Is Necessary for Negative Gene Regulation by Vitamin D in MT2 Cells-To examine whether nVDRE hPTHrP is necessary for negative gene regulation by vitamin D in MT2 cells (Fig. 4), we transfected the cells either with the parental TkCAT (28) or with the same CAT plasmid bearing nVDRE hPTHrP in the sense orientation. As shown in Fig. 4, 10 Ϫ8 M 1,25-dihydroxyvitamin D 3 selectively inhibited CAT activity driven by nVDRE hPTHrP -TkCAT but not TkCAT. Furthermore, less active vitamin D metabolites were ineffective.
Vitamin D Attenuates the Binding between nVDRE hPTHrP and VDR-Our next question was what is the effect of vitamin D on the binding between nVDRE hPTHrP and MT2 proteins. As shown in EMSA (Fig. 5A), 1,25-dihydroxyvitamin D 3 weakened the binding between nVDRE hPTHrP and MT2 nuclear proteins, although it strengthened the binding between VDRE mop and these proteins in a dose-dependent manner. In the Southwestern assay, using a radiolabeled nVDRE hPTHrP as a probe (Fig.  5B), similar dose-dependent effects of 1,25-dihydroxyvitamin D 3 were observed on the band corresponding to about 50 kDa in size but not on the other bands. We then employed MT2 nuclear proteins in which VDR was immunodepleted with the 9A7 shown above in the Southwestern assay. As shown in Fig. 5C, immunodepletion treatment clearly and selectively abolished the binding of this 50-kDa band but not higher molecular mass bands, strongly suggesting this 50-kDa band corresponded to the VDR itself. On the other hand, the radiolabeled VDRE mop yielded only one band around 96 kDa in size, which was unaffected by the immunodepletion. We assume these higher molecular weight proteins are nonspecific DNA binding proteins. Immunoblotting with 9A7 revealed that treatments with 1,25dihydroxyvitamin D 3 did not affect the amounts of VDR protein (Fig. 5D). Together, these results suggest that 1,25-dihydroxyvitamin D 3 treatment weakens the binding between nVDRE hPTHrP and VDR in MT2 cells, thereby exerting its inhibitory effect on the expression of nVDRE hPTHrP -bearing gene(s) in these cells.
Ku Antigen, Along with VDR, Mediates Negative Regulation of the Gene(s) Containing nVDRE hPTHrP by Vitamin D-In order to find a clue for a probable partner of VDR in the nVDRE hPTHrP -MT2 protein complex, we reexamined the sequence of nVDRE hPTHrP and noticed that it contained an octamer ATTTGCAT-like sequence following the core motif. This sequence, ATTTGGTT, is reminiscent of one of the negative calcium-responsive elements, oligo(A). We previously showed that the footprint in the oligo(A) HeLa nuclear protein was localized exactly in this position (15) and demonstrated that the nuclear protein specifically binding to oligo(A) contained Ku antigen (29). Various roles of Ku antigen such as double strand break repair or DNA recombination by virtue of its ability to bind DNA ends nonspecifically have been reported (32)(33)(34). However, a recent report demonstrating that Ku antigen recognizes a specific internal DNA element in the mouse mammary tumor virus long terminal repeat to inhibit glucocorticoid receptor-mediated transcriptional stimulation (35) prompted us to examine whether Ku antigen is involved in nVDRE hPTHrP -mediated gene inhibition by vitamin D. As shown in Fig. 6, inclusion of anti-Ku antigen antibody (29) in EMSA significantly reduced the nVDRE hPTHrP -MT2 protein complex but not the VDRE mop -MT2 protein complex. Of note, this treatment seemed to strengthen the binding activity of the faster migrating band when we used nVDRE hPTHrP as a probe. However, as noted earlier, because the migrating position of the faster migrating band overlaps that of the occasionally observed lower band (Fig. 2), we did not pursue this issue in this manuscript.
To show the effect of Ku antigen on nVDRE hPTHrP -mediated gene repression by vitamin D in vivo, we transiently introduced an expression vector encoding the p70 subunit of Ku antigen in its antisense orientation (29) along with the TkCAT-based reporter plasmids bearing either nVDRE hPTHrP or VDRE mop . Immunoblotting with anti-Ku antigen antiserum, which specifi-cally recognizes the p70 subunit of Ku antigen (Fig. 7A), revealed that this treatment inhibited the synthesis of the p70 subunit of Ku antigen by about 50%. As shown in Fig. 7B, although 1,25-dihydroxyvitamin D 3 had similar repressive effects on nVDRE hPTHrP -CAT activity in MT2 cells transfected with an empty vector, it lost the inhibitory action in MT2 cells when the p70 subunit of Ku antigen was expressed in the antisense orientation. On the other hand, 1,25-dihydroxyvitamin D 3 exerted similar stimulatory effects on the CAT activity by VDRE mop -TkCAT with or without expression of Ku antigen.
Phosphatase Treatment of VDR Restores Its Binding to nVDRE hPTHrP Once Attenuated by Vitamin D-Recently, Ku antigen was shown to have an inhibitory effect on the glucocorticoid receptor-mediated transcriptional stimulation of the mouse mouse mammary tumor virus long terminal repeat gene (35). It was subsequently shown that the catalytic subunit of Ku antigen (DNA-dependent protein kinase), along with both regulatory subunits of Ku antigen (p70 and p86), phosphorylates glucocorticoid receptor after binding to its specific binding DNA element called negative response element (36). Although the authors did not refer to the direct role of this type of phosphorylation on gene repression (36), their report prompted us to speculate that presumable phosphorylation of VDR by the Ku antigen, which would have been triggered by the treatment with 1,25-dihydroxyvitamin D 3 , might weaken its activity to bind to nVDRE hPTHrP , leading to vitamin D-mediated gene repression. To examine this possibility, we treated nuclear proteins obtained from vehicle-or 1,25-dihydroxyvitamin D 3administered MT2 cells with potato acid phosphatase for different times and examined their activity to bind nVDRE hPTHrP by the Southwestern assay as shown in Fig. 8. As expected, the binding of the VDR to nVDRE hPTHrP , once attenuated by the treatment with 10 Ϫ7 M 1,25-dihydroxyvitamin D 3 (see Fig. 5), but not with vehicle alone, was up-regulated by the phosphatase treatment. On the other hand, the binding of the nonspecific bands above the VDR was not altered by the treatment. These results suggest, albeit indirectly, that phosphorylation of VDR by a certain kinase (e.g. DNA-dependent protein kinase) might decrease its activity to bind to nVDRE hPTHrP .

DISCUSSION
We have shown that the human PTHrP gene contains a DNA element (nVDRE hPTHrP ) very homologous to the negative vitamin D response element found in the human PTH gene (16). Although it is in reverse orientation compared with the nVDRE hPTH , we demonstrated that nVDRE hPTHrP functioned similarly to nVDRE hPTH in terms of transcriptional repression by vitamin D. Sequence comparison between nVDRE hPTHrP and nVDRE hPTH revealed 11 identical bases out of 13 nucleotides. Furthermore, it contains a heptameric AG(G/A)TTCA, mimicking a hexameric AG(G/A)TCA motif proposed to be a core element (2) for the binding of the nuclear receptors for thyroid hormone, vitamin D (VDR), or retinoic acid (retinoic acid receptor and RXR). However, unlike VDRE, consisting of two such motifs separated by a three-base spacer (Refs. 2 and 22; see VDRE mop ), nVDRE hPTHrP and nVDRE hPTH possess only one motif. This observation led us to speculate that the content of nuclear proteins binding to the nVDREs was different from the usual VDR-RXR heterodimer, as is found in the authentic VDRE (see below). On the other hand, by computer search, we found the nVDRE motif in the Ϫ1100-bp upstream region of the mouse TGF␤2 gene (Fig. 1). 12 out of 13 bases were identical between the two. The only difference lay in the heptameric region. In the chick TGF␤2 gene, AGATTCA in the hPTHrP gene was changed to AGGTTCA, which was, in turn, identical to the heptamer in the hPTH gene. Very interestingly, expression of this gene in the mesangial cells was reported to be inhibited by 1,25-dihydroxyvitamin D 3 (20). This finding further supports that nVDRE might play a general role in vitamin D-mediated gene inhibition.
A couple of recent reports demonstrated that the rat PTHrP gene contains several DNA sequences responsible for negative gene regulation by vitamin D. Falzon (18) reported two nV-DREs in the upstream region of the rat PTHrP gene. He showed that both of them were necessary for vitamin D-mediated gene inhibition. One of them located at the Ϫ726-bp upstream region possesses one heptameric sequence, AGGTTCT.
This sequence is oriented similar to nVDRE hPTHrP and in reverse when compared with nVDRE hPTH . The similarity between nVDRE hPTHrP and nVDRE hPTH was found in the sequences adjacent to the heptamer. The five nucleotides, FIG. 6. Ku antigen exists in the nVDRE hPTHrP -MT2 nuclear protein complex but not in the VDRE mop -MT2 protein complex. EMSA using MT2 nuclear proteins and nVDRE hPTHrP (lanes 1-2) or VDRE mop (lanes 3-4) is shown. One microliter of preimmune human serum (lanes 1 and 3) or anti-Ku antigen antibody (lanes 2 and 4, see Ref. 29) was added in each lane. The solid arrows indicate the specific DNA protein complexes (Fig. 2). The upper dashed arrow in lane 2 indicates the supershifted band.  1 and 5) is shown. M, molarity. In B, a Southwestern assay using the same set of MT2 protein and radiolabeled nVDRE hPTHrP is shown. The arrow indicates a presumptive VDR monomer, which is strongly suggested in C. In C, VDR was immunodepleted with the 9A7 (each, right) or rat IgG2 (each, left) from nuclear proteins obtained from untreated MT2 cells and Southwestern assay with the radiolabeled nVDRE hPTHrP (left panel) or VDRE mop (right panel) was performed. In D, immunoblotting assay with the 9A7 and MT2 nuclear proteins treated similarly to A and B are shown. Immunoblotting of proteins corresponding to those in lane 1 with rat IgG2 alone is shown in lane 5. The arrow indicates the position of human VDR. The higher molecular weight proteins might be nonspecific proteins. In B, C, and D, molecular weight markers are shown on the left side.
GTGCT, lying Ϫ7 to Ϫ3 bases preceding the core heptamer were identical between the two nVDREs found in the human and rat PTHrP genes; such identity was not observed in nV-DRE hPTH . Interestingly, this AGGTTCA was repeated with a two-base spacer in the VDRE mop (22). Like our nVDRE hPTHrP (Figs. 3 and 5C), the nVDRE in the human PTH gene was reported to bind to VDR as well as another nuclear protein(s) other than RXR (17). On the other hand, Falzon reported another nVDRE at the Ϫ805-bp region of the rat PTHrP gene (18). This element resembles a prototype DR3 VDREs, and similar nVDREs mimicking DR3 VDRE were reported to lie at the Ϫ1100-bp region of the rat PTHrP gene (19) or at the Ϫ80-bp region of the chicken PTH gene (21). VDR and probably RXR could bind to all of them, although it is still unknown why this DR3-type structure was able to mediate negative gene regulation by vitamin D.
Our competition EMSA experiments (Fig. 2) suggested that nVDRE hPTHrP and VDRE mop bound to different sets of MT2 nuclear proteins, although each DNA-MT2 nuclear protein complex migrated similarly on a gel. Nonetheless, the use of a 200-fold molar excess of the reciprocal competitors in each EMSA similarly abolished the binding between MT2 nuclear proteins and each probe (not shown), suggesting that each complex might share a common protein(s). The obvious candidate of such a protein is VDR. Our EMSA employing the anti-VDR and anti-RXR␣ antibodies revealed that, unlike the classical interaction between VDR and RXR␣ found in the binding to the authentic VDRE, such as VDRE mop shown here (Fig. 4), nVDRE hPTHrP recognized only VDR, and the supershift pattern in EMSA (Fig. 4) predicted that VDR might exhibit a different conformation from that in the binding to RXR␣, suggesting that another unknown partner (16,17) might be involved. Although we cannot rule out the possibility that RXRs other than RXR␣ are involved here, the usual partnership composed of VDR and RXR␣, which is found in the binding to the positive VDRE mop as shown in Fig. 3, is not predominant in the binding between nVDRE hPTHrP and MT2 nuclear proteins. This notion was also supported by the Southwestern assay, in which MT2 VDR was immunodepleted by the anti-VDR antibody (Fig. 5C).
In this assay, we demonstrated that nVDRE hPTHrP could not bind to 50-kDa nuclear proteins after immunodepletion, whereas VDRE mop did not bind to this band irrespective of immunodepletion. This result suggests that VDRE mop, unlike nVDRE hPTHrP , can bind to MT2 VDR only when the latter is associated with RXR␣.
0n the other hand, our experiments using anti-Ku antigen antibody, which recognizes one of its subunits, p70, demonstrated that it supershifted the complex between MT2 nuclear proteins and nVDRE hPTHrP but not VDRE mop (Fig. 3). Furthermore, our experiments using the antisense-oriented Ku antigen expression vector (Fig. 6) demonstrated that negative regulation by vitamin D via the nVDRE hPTHrP was abrogated by the introduction of such a vector. Because such an abrogation was not observed in the case of the VDRE mop -TkCAT, we concluded that Ku antigen was crucially involved in negative gene regulation by vitamin D in MT2 cells. Although the mode of action of Ku antigen has been currently under extensive study (32)(33)(34), only recently has its role as a transcription factor been established (29,33). Ku antigen consists of two regulatory subunits, p70 and p86, as well as the catalytic unit of another large protein of DNA-dependent protein kinase, DNA-dependent protein kinase (32). Its ability to bind DNA ends nonspecifically could explain its function such as double strand break repair or DNA recombination. We (29) recently demonstrated that p70 and p86 subunits of Ku antigen could interact in association with another nuclear protein, redox factor 1 (1), with one of the negative calcium-responsive elements in a sequence-specific manner. We showed that such an interaction led to extracellular calcium-mediated transcriptional inhibition of the genes bearing negative calcium-responsive elements. Furthermore, of particular interest, the DNA sequence were treated with 0.1 unit of potato acid phosphatase (PAP) for 0 min (second and fourth lanes) or 30 min (first and third lanes) individually at 37°C followed by SDS-polyacrylamide gel electrophoresis and Southwestern assay. The radiolabeled nVDRE hPTHrP was used as a probe. An arrow indicated the position of VDR (Fig. 5C). Unlike VDR, whose binding activity was up-regulated by phosphatase treatment, several upper nonspecific bands were not affected by the treatment (compare the third and fourth lanes. On the other hand, the intensity of VDR in the first and second lanes did not differ. Molecular mass markers are shown on the left side. following the heptamer in the nVDRE hPTHrP , ATTTGGTT, was similar to the partial sequence of one of the negative calciumresponsive elements, oligo(A) (15,28), ATTTGTGT. The latter sequence was protected from DNase digestion in HeLa cells (15), and we had proposed that Ku antigen along with ref1 protein (29) bound to oligo(A) in a sequence-specific manner. Therefore, we hypothesized that two different portions within the nVDRE hPTHrP bound to two different nuclear proteins, VDR and Ku antigen, and that protein-protein interaction between VDR and Ku antigen might not occur. Such an independent binding of Ku antigen and another protein(s) to one DNA fragment was reported in mouse mammary tumor virus long terminal repeat where Ku antigen recognized one specific internal DNA sequence 20 -30 bases away from the glucocorticoid receptor binding site (35). However, since the examination of the exact DNA binding manner of Ku antigen requires very careful preparation of DNA samples due to the the Ku antigen nature of binding DNA ends nonspecifically, proving this hypothesis has so far been unsuccessful.
The underlying mechanism by which vitamin D weakens the binding between nVDRE hPTHrP and MT2 VDR as shown in Fig.  5 is currently unclear. Although there have been several reports describing up-regulatory effects of vitamin D on the amount of VDR protein or on its binding activity to VDRE (37), the mechanism by which vitamin D inhibits the binding of VDR to nVDRE has not been addressed. One attractive hypothesis is that Ku antigen might play some role in such negative regulation as shown in this manuscript. Particularly, the recent report suggesting that protein kinase activity of DNA-dependent protein kinase, the catalytic subunit of Ku antigen, would modulate glucocorticoid receptor activity after the specific binding to DNA (35,36) prompted us to carry out the Southwestern assay in which the VDR from 1,25-dihydroxyvitamin D 3 -administered MT2 cells was dephosphorylated by potato acid phosphatase (Fig. 8). Although dephosphorylation of VDR did not affect its binding to nVDRE hPTHrP when VDR was obtained from MT2 cells treated with vehicle alone, it reversed VDR binding to nVDRE hPTHrP pre-attenuated by the treatment with 1,25-dihydroxyvitamin D 3 . Therefore, we raise the possibility that phosphorylation of VDR by the Ku antigen, which would have been triggered by the treatment with 1,25dihydroxyvitamin D 3 , might weaken its activity to bind to nVDRE hPTHrP followed by vitamin D-mediated gene repression, although direct evidence to support this contention is still lacking.
If vitamin D mediates conditional inhibition of PTHrP gene as suggested here, what is the significance of the constitutively active binding between nVDRE hPTHrP and MT2 nuclear protein(s) in the unliganded state? We speculate that such a binding might be somewhat related to constitutively active production of PTHrP in such cell lines as MT2 cells, leading to humoral hypercalcemia of malignancy, the most common cause of human hypercalcemia (26). We suggest here that a therapeutic approach to targeting to the transcriptional apparatus would be of potential benefit.
In this paper, we focused on the difference between positive and negative VDREs as described above. Future investigation is directed to the detailed analyses of the interaction between nVDRE hPTHrP and VDR including mutagenesis of nVDRE-hPTHrP as well as characterization of VDR domain involved here, all of which are now in progress in our laboratory.