Enhancement of VDR-Mediated Transcription by Phosphorylation: Correlation with Increased Interaction Between the VDR and DRIP205, a Subunit of the VDR-Interacting Protein Coactivator Complex

Frank Barletta, Leonard P. Freedman and Sylvia Christakos

Department of Biochemistry and Molecular Biology (F.B., S.C.), University of Medicine and Dentistry of New Jersey-New Jersey Medical School and Graduate School of Biomedical Sciences, Newark, New Jersey 07103; and Cell Biology and Genetics Program (L.P.F.), Memorial Sloan-Kettering Cancer Center, New York, New York 10021

Address all correspondence and requests for reprints to: Dr. Sylvia Christakos, Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, 185 South Orange Avenue, Newark, New Jersey 07103. E-mail: christak{at}umdnj.edu


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
When UMR-106 osteoblastic cells, LLCPK1 kidney cells, and VDR transfected COS-7 cells were transfected with the rat 24-hydroxylase [24(OH)ase] promoter (-1,367/+74) or the mouse osteopontin (OPN) promoter (-777/+79), we found that the response to 1,25dihydroxyvitamin D3 [1,25-(OH)2D3] could be significantly enhanced 2- to 5-fold by the protein phosphatase inhibitor, okadaic acid (OA). Enhancement of 1,25-(OH)2D3-induced transcription by OA was also observed using a synthetic reporter gene containing either the proximal 24(OH)ase vitamin D response element (VDRE) or the OPN VDRE, suggesting that the VDRE is sufficient to mediate this effect. OA also enhanced the 1,25-(OH)2D3-induced levels of 24(OH)ase and OPN mRNA in UMR osteoblastic cells. The effect of OA was not due to an up-regulation of VDR or to an increase in VDR-RXR interaction with the VDRE. To determine whether phosphorylation regulates VDR-mediated transcription by modulating interactions with protein partners, we examined the effect of phosphorylation on the protein-protein interaction between VDR and DRIP205, a subunit of the vitamin D receptor-interacting protein (DRIP) coactivator complex, using glutathione-S-transferase pull-down assays. Similar to the functional studies, OA treatment was consistently found to enhance the interaction of VDR with DRIP205 3- to 4-fold above the interaction observed in the presence of 1,25-(OH)2D3 alone. In addition, studies were done with the activation function-2 defective VDR mutant, L417S, which is unable to stimulate transcription in response to 1,25-(OH)2D3 or to interact with DRIP205. However, in the presence of OA, the mutant VDR was able to activate 24(OH)ase and OPN transcription and to recruit DRIP205, suggesting that OA treatment may result in a conformational change in the activation function-2 defective mutant that creates an active interaction surface with DRIP205. Taken together, these findings suggest that increased interaction between VDR and coactivators such as DRIP205 may be a major mechanism that couples extracellular signals to vitamin D action.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
STEROID HORMONE ACTION is mediated through intracellular receptors that function as transcription factors in target cells (1). These receptors are comprised of modular domains that possess ligand binding, DNA binding, dimerization, and transactivation activities that are essential to their function. The binding of ligand results in a conformational change of the steroid receptor into an active form (2, 3, 4). The ligand-receptor complex binds DNA sequences within target gene promoters and modulates transcription (5). In addition to ligand binding, a number of steroid receptors are activated by different signaling pathways: growth factors [epidermal growth factor and IGF (6, 7)], the neurotransmitter dopamine (8), activators of PKA (9), and the protein phosphatase inhibitor, okadaic acid (10, 11). Phosphorylation has been implicated in transcriptional activation as well as repression (12). It has been suggested that the phosphorylation of nuclear hormone receptors may play a role in nuclear localization, hormone binding, DNA binding, and cofactor recruitment (12).

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] is a key regulator of calcium homeostasis and has been reported to have additional functions, including effects on differentiation, immune function, and cell proliferation (13). The action of 1,25-(OH)2D3, the active form of vitamin D, is mediated through the VDR. VDR is a member of the superfamily of nuclear receptors, the activity of which is also affected by phosphorylation-dependent signaling pathways (14, 15, 16). VDR, like the PR (17), GR (18, 19), AR (20, 21), ER (22, 23), and the RARs (RAR{alpha}, -ß, and -{gamma}) (24, 25, 26), is a phosphoprotein. The majority of the phosphorylation of VDR is on serine residues (27). VDR becomes hyperphosphorylated upon 1,25-(OH)2D3 treatment (28, 29). It has been demonstrated that VDR can be phosphorylated by PKA, PKC, and casein kinase II (30, 31, 32). VDR, similar to other steroid receptors, exerts its transcriptional activity by interaction with proteins of the preinitiation complex and by interaction with coactivators that may bridge the interaction between VDR and the basal transcription machinery (5, 33). Steroid receptor coactivators (SRCs) include SRC-1/NcoA1, GR-interacting protein 1 (GRIP-1)/transcriptional intermediary factor 2, and activator of thyroid and RARs (ACTR)/p300-CBP cointegrator-associated protein (pCIP). These coactivators exhibit histone acetylase (HAT) activity and can associate with additional proteins that possess HAT activity, such as CREB binding protein (CBP)/p300. HAT activity is thought to destabilize the interaction between DNA and the histone core, liberating DNA for transcription (34, 35). In addition to the SRC family of coactivators, VDR-mediated transcription is also modulated by a coactivator complex, DRIP [VDR-interacting protein, also called TRAP and ARC, (33, 35)]. The DRIP complex does not possess HAT activity but rather functions, at least in part, through recruitment of RNA polymerase II (36). Due to the fact that steroid receptors are phosphorylated, and given the interplay between these steroid receptors and other proteins that may also be affected by phosphorylation (37), it was important to examine the role that phosphorylation plays in vitamin D-mediated gene expression. The results of this investigation show that okadaic acid (OA), an inhibitor of protein phosphatase 1 and 2A (PP-1 and PP-2A), enhances 1,25-(OH)2D3-dependent transcription and suggest that this enhancement may be due, at least in part, to an increased interaction between the VDR and the coactivator protein DRIP205.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Enhancement of 1,25-(OH)2D3-Induced 24-Hydroxylase [24(OH)ase] Transcription by Inhibition of PP-1 and PP-2A
To investigate the role of phosphorylation in 1,25-(OH)2D3-dependent transcriptional activation, OA (an inhibitor of PP-1 and PP-2A) was used to enhance overall cellular phosphorylation. Enhancement of cellular phosphorylation resulted in an increase in 1,25-(OH)2D3-induced 24(OH)ase transcription. LLCPK1-, UMR-106-, or human VDR (hVDR)-transfected COS-7 cells were transfected with the rat 24(OH)ase promoter construct (-1,367/+74) and treated for 24 h with vehicle (-D), 10-8 M 1,25-(OH)2D3 (+D), 50 nM OA (OA), or 1,25-(OH)2D3 and 50 nM OA (OA+D) (Fig. 1Go). Treatment with OA alone was unable to significantly increase transcription over the levels observed with vehicle treatment. However, the response to 1,25-(OH)2D3 was significantly enhanced 2- to 5-fold by cotreatment with OA in all three cell lines. Enhancement of 1,25-(OH)2D3-induced transcription was also observed using 25 or 30 nM OA, and maximal enhancement of chloramphenicol acetyltransferase (CAT) activity was observed at 50 nM (not shown).



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Figure 1. Enhancement of 1,25-(OH)2D3-Induced 24(OH)ase Transcription by Inhibition of PP-1 and PP-2A

LLCPK1, UMR-106, or VDR expression vector (pAVhVDR) transfected COS-7 cells were transfected with 3 µg of the CAT construct of the rat 24(OH)ase promoter -1,357/+74 (which contains both VDREs at -258/-244 and at -151/-137). Transfected cells were treated with vehicle (-D), 10-8 M 1,25-(OH)2D3 (+D), 50 nM OA (OA), or 1,25-(OH)2D3 plus 50 nM OA (OA + D) for 24 h. After treatments, cells were harvested and lysed, and CAT activity was measured as described in Materials and Methods. Shown are representative autoradiograms and the graphic representation of three or more separate experiments for each cell line. Longer radiographic exposure time was needed to visualize CAT activity under basal conditions. The results are expressed as the percent of maximal response ± SE after normalization for ß-galactosidase activity. Transfection of the -1,357/+74 rat 24(OH)ase promoter construct and treatment with 10-8 M 1,25-(OH)2D3 resulted in a 16.5 ± 2.5-fold increase in CAT activity in LLCPK1 cells and a 22.7 ± 4- and a 24.5 ± 2-fold increase in CAT activity in UMR and COS cells, respectively. In all three cell lines the 1,25-(OH)2D3 and OA cotreatment led to significant enhancement of CAT activity compared with treatment with 1,25-(OH)2D3 (P < 0.05).

 
Enhancement of 1,25-(OH)2D3-Induced Osteopontin (OPN) Transcription by Phosphorylation
To determine whether the enhancement of 1,25-(OH)2D3-induced transcription is specific for 24(OH)ase, we tested the ability of OA to enhance 1,25-(OH)2D3-induced OPN transcription. COS-7 cells were transfected with a mouse OPN promoter construct (-777/+79) and pAVhVDR expression vector. After transfection, the cells were incubated with vehicle (-D), 10-8 M 1,25-(OH)2D3 (+D), 50 nM OA (OA), or 1,25-(OH)2D3 plus 50 nM OA (OA + D) for 24 h followed by analysis of CAT expression (Fig. 2Go). Similar to the experiments using the 24(OH)ase reporter construct (Fig. 1Go), the cotreatment with OA resulted in a 2- to 3-fold enhancement of 1,25-(OH)2D3-induced OPN transcription. Unlike VDR-mediated 24(OH)ase transcription, OA, in the absence of ligand, was found to activate VDR-mediated OPN transcription (Fig. 2Go). The transcription of the pRSV-CAT reporter construct, which places CAT under the control of the Rous sarcoma virus (RSV) long terminal repeat enhancer/promoter, was not affected by OA (Fig. 3Go). The activity of a thymidine kinase (tk) CAT construct, containing the minimal promoter region of the herpes virus thymidine kinase gene and cotransfected with hVDR in COS-7 cells, was also not affected by 50 nM OA treatment (not shown). The inability of OA to enhance transcription of the minimal promoter constructs and experiments using the OPN promoter construct indicate that the enhancement of 1,25-(OH)2D3-induced 24(OH)ase transcription by inhibition of PP-1 and 2A is neither specific for 24(OH)ase nor due to a general increase in transcription.



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Figure 2. Enhancement of 1,25-(OH)2D3 Induced OPN Transcription by OA

COS-7 cells were cotransfected with 500 ng of the VDR expression vector pAVhVDR and 3 µg of a CAT reporter plasmid containing the -777/+79 region of the mouse OPN promoter (VDRE at -757/-743). Transfected cells were treated with vehicle (-D), 10-8 M 1,25-(OH)2D3 (+D), 50 nM OA (OA), or 1,25-(OH)2D3 plus 50 nM OA (OA + D) for 24 h. After treatment, cells were harvested and lysed, and CAT activity was measured as described in Materials and Methods. A representative autoradiogram is shown. After normalization for ß-galactosidase activity, the results are expressed as the percent of maximal response and represent the mean of three separate experiments ± SE. Treatment with 10-8 M 1,25-(OH)2D3 resulted in a 2.9 ± 0.4-fold increase in CAT activity over basal (-D) levels. Cotreatment with 1,25-(OH)2D3 plus OA results in a significant enhancement of 1,25-(OH)2D3 induced OPN transcription (P < 0.05).

 


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Figure 3. Enhancement of 1,25-(OH)2D3-Induced Transcription by Inhibition of PP-1 and -2A is Not Due to a General Increase in Transcription

UMR osteoblast-like cells, that contain endogenous VDR, were transfected with 4 µg of a control reporter plasmid pRSV-CAT, which places CAT under the control of Rous sarcoma virus long terminal repeat. Cells were treated with vehicle or 50 nM OA for 24 h, harvested, and lysed, and CAT activity was measured. Shown is a representative autoradiogram. Similar results were observed in three experiments. OA treatment did not affect the transcription of the pRSV-CAT reporter construct (P > 0.5; basal vs. 50 nM OA treatment).

 
Effect of OA on the Endogenous Expression of 24(OH)ase and OPN mRNAs in UMR Cells
Northern blot analysis of mRNA from UMR-106 cells was performed to assess the effect of enhanced phosphorylation on the endogenous expression of 24(OH)ase and OPN mRNAs (Fig. 4Go). UMR-106 cells were treated with vehicle (-D), 1,25-(OH)2D3 (+D), OA alone (OA), or 1,25-(OH)2D3 and OA (OA + D) for 24 h. 24(OH)ase and OPN mRNA levels were significantly increased upon 1,25-(OH)2D3 treatment, and cotreatment with the phosphatase inhibitor led to an enhancement over the 1,25-(OH)2D3-induced levels. Similar to the findings using the OPN and 24(OH)ase promoter constructs (Fig. 4Go), OA alone resulted in a significant increase in OPN mRNA but not in 24(OH)ase mRNA levels in UMR cells (Fig. 4Go).



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Figure 4. OA Treatment Results in an Enhancement of 1,25-(OH)2D3-Induced 24(OH)ase and OPN mRNA Levels

Northern analysis was performed using 8 µg poly (A+) RNA per lane from UMR cells that had been treated with vehicle (-D), 1,25-(OH)2D3 (10-7 M; +D), OA alone (50 nM), or 1,25-(OH)2D3 plus OA (50 nM) for 24 h. Right panel, Representative autoradiogram. The filter was hybridized with 32P-labeled rat 24(OH)ase cDNA and then stripped and rehybridized sequentially with 32P-labeled mouse OPN and ß-actin cDNAs. Left panel, Graphic representation of the data normalized on the basis of results obtained after rehybridization with ß-actin cDNA. The results of four separate experiments are expressed as percent maximal response ± SE. A significant induction of 24(OH)ase and OPN mRNA levels compared with 1,25-(OH)2D3-induced levels was observed with cotreatment with OA + 1,25-(OH)2D3 (P < 0.05). 24(OH)ase mRNA was not observed under basal conditions (-D) or after treatment with OA alone even after longer autoradiographic exposure time. OA alone significantly induced OPN mRNA levels above the levels observed under basal (-D) conditions (P < 0.05).

 
Mechanism of the Effect of Phosphorylation on 1,25-(OH)2D3-Induced Transcription
To determine a mechanism by which OA enhances 1,25-(OH)2D3-induced VDR-mediated transcription, we examined the effect of OA treatment on VDR expression. Northern (Fig. 5AGo) and Western blot (Fig. 5BGo) analyses were performed using mRNA and protein from UMR-106 cells. VDR mRNA and protein levels were induced upon treatment with 1,25-(OH)2D3. However, there was no increase in the expression of VDR mRNA or VDR protein after treatment with 1,25-(OH)2D3 plus OA as compared with levels observed after treatment with 1,25-(OH)2D3 alone (Fig. 5Go, A and B). There was also no change in VDR mRNA or protein levels when compared with basal (-D) levels after treatment with OA alone. These findings indicate that the OA-mediated enhancement of transcription is not due to an increase in VDR expression.



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Figure 5. Enhancement of 1,25-(OH)2D3-Induced Transcription by Inhibition of PP-1 and PP-2A is Not Due to an Increase in VDR Expression

A, Northern analysis was performed using 8 µg poly (A+) RNA per lane from UMR cells that had been treated with vehicle (-D), 1,25-(OH)2D3 (10-7 M; +D), OA (50 nM), or 1,25-(OH)2D3 plus OA (OA + D) for 24 h. A representative autoradiogram is shown. The filter was hybridized with 32P-labeled VDR cDNA. The blot was stripped and rehybridized with 32P-labeled ß-actin cDNA. Treatment with 1,25-(OH)2D3 plus OA did not result in any significant changes in VDR mRNA levels compared with treatment with 1,25-(OH)2D3 alone (+D, 1.6 ± 0.2, OA + D 1.5 ± 0.2-fold induction over basal; P > 0.5; results of three experiments). B, Western blot analysis was performed using 50 µg of nuclear protein prepared from UMR cells treated with vehicle (-D), 1,25-(OH)2D3 (+D), OA, or 1,25-(OH)2D3 plus OA (OA + D) as described in panel A for 24 h. Detection was by immunoblotting with a monoclonal anti-VDR antibody. An increase in VDR protein levels was not observed when comparing 1,25-(OH)2D3 and 1,25-(OH)2D3 plus OA-treated cells (80% of the VDR protein levels observed after treatment with 1,25-(OH)2D3 was observed after treatment with OA and 1,25-(OH)2D3).

 
Gel shift analysis was performed to determine whether OA may modify the binding of VDR/RXR to the vitamin D response element (VDRE) (Fig. 6Go). Binding reactions were done using nuclear extracts prepared from UMR cells and an oligonucleotide containing the proximal 24(OH)ase VDRE. Protein-DNA interaction was induced using nuclear extracts from 1,25-(OH)2D3-treated UMR cells. No further increase in DNA binding activity was observed using nuclear extracts prepared from 1,25-(OH)2D3 plus OA-treated cells (Fig. 6Go). When nuclear extracts were prepared from cells treated with OA alone, the protein-DNA interaction was similar to that obtained under basal (-D) conditions (Fig. 6Go; -D and OA). These results suggest that the effect of OA is not due to an increase in VDR/RXR binding to the VDRE.



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Figure 6. OA Treatment Does Not Enhance VDR-RXR Interactions with the VDRE

Gel mobility shift assay using 3 µg of nuclear protein prepared from UMR cells treated with vehicle (-D), 1,25-(OH)2D3 (+D), OA, or 1,25-(OH)2D3 plus OA (OA +D) for 24 h and 32P-labeled oligonucleotide containing the 24(OH)ase VDRE (-150/-136). Preincubation with cold VDRE or monoclonal VDR antibody (anti-VDR) depleted the binding of the VDR/RXR to the labeled probe. Data are representative of three experiments.

 
To test whether the enhancement by OA occurs through the VDRE and not via other sequences in the promoter, we examined the effect of OA using the rat proximal 24(OH)ase VDRE-tk CAT construct or the mouse OPN VDRE-tk CAT construct (Fig. 7Go). The cotreatment of 1,25-(OH)2D3 and OA enhanced the 1,25-(OH)2D3-induced transcription of the VDRE-tk CAT constructs, similar to the effect seen with the larger promoter constructs, indicating that the VDRE is sufficient to mediate the effect of phosphatase inhibition. Similar to the activity of the -1,367/+74 24(OH)ase promoter CAT construct (Fig. 1Go), 24(OH)ase VDRE-tk CAT activity was not significantly affected by treatment with OA alone when compared with basal (-D) levels (Fig. 7Go; OA, left panel). Although the response to OA alone using the -777/+79 OPN promoter construct was equivalent to the 1,25-(OH)2D3 response (Fig. 2Go), the response of the OPN VDRE-tk CAT construct to OA alone was 1.5 ± 0.3-fold over basal [P > 0.1; basal (-D) vs. OA treatment] and not equivalent to the 1,25-(OH)2D3 response (5.6 ± 0.9-fold induction). The inability of OA alone to increase the transcription of the OPN-tk CAT construct suggests that the previously observed activation by OA (Fig. 2Go) may be mediated through promoter elements other than the VDRE. These findings suggest that sequences in the OPN promoter contribute to the ligand-independent induction of OPN expression and transcription.



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Figure 7. Enhancement of 1,25-(OH)2D3-Induced VDRE-tk CAT Transcription by OA

COS-7 cells were cotransfected with 500 ng of the VDR expression vector pAVhVDR and 3 µg of a CAT reporter plasmid containing multiple copies of the rat 24(OH)ase VDRE (-150/-136) or the mouse OPN VDRE (-757/-743). Transfected cells were treated with vehicle (-D), 10-8 M 1,25-(OH)2D3 (+D), OA (50 nM), or 1,25-(OH)2D3 plus 50 nM OA (OA + D) for 24 h. After treatments cells were harvested and lysed, and CAT activity was measured as described in Materials and Methods. Representative autoradiograms are shown. The results of three separate experiments are expressed as the percent of maximal response ± SE after normalization for ß-galactosidase activity. Cells transfected with the 24(OH)ase VDRE-tk CAT construct or the OPN VDRE-tk CAT construct and treated with 10-8 M 1,25-(OH)2D3 exhibited a 9.4 ± 0.2-fold and a 5.6 ± 0.9-fold induction in CAT activity, respectively. Longer radiographic exposure time was needed to visualize CAT activity under basal conditions. Cotreatment with 1,25-(OH)2D3 plus OA results in an enhancement of 1,25-(OH)2D3-induced VDRE-tk CAT transcription (P < 0.05).

 
Previous studies have indicated that OA can induce phosphorylation of VDR (14, 43). To examine whether phosphorylation of VDR is a mechanism involved in the enhancement of 1,25-(OH)2D3-induced transcription by OA, we used VDR mutants that have mutations in two major phosphorylation sites, serine 208 [phosphorylated by casein kinase II (32)] and serine 51 [phosphorylated by PKC (31)]. Using mutant VDR expression vector constructs S51A and S208A (mutation of serine to alanine), the rat 24(OH)ase -1,367/+74 promoter construct and transfection in COS-7 cells, a 3.9 ± 0.5- and a 3.8 ± 0.2-fold enhancement, respectively, of 1,25-(OH)2D3-induced transcription was still observed in the presence of OA, suggesting that the enhancement is not due to phosphorylation of these specific sites.

To further investigate the mechanism involved in the phosphorylation-dependent enhancement of VDR-mediated transcription, we used glutathione-S-transferase (GST) pull-down assays to assess any effect that OA may have on the binding of VDR to cofactor proteins. The binding of VDR to two cofactors, DRIP205 and GRIP-1, was analyzed (Fig. 8Go). The interaction of VDR to each cofactor was increased when nuclear extracts were used from 1,25-(OH)2D3-treated VDR-transfected COS cells, consistent with previous results indicating that VDR cofactor interactions are 1,25-(OH)2D3 dependent (36, 38). The cotreatment of 1,25-(OH)2D3 and OA was unable to increase GRIP-1 and VDR interaction over that seen with 1,25-(OH)2D3 alone (Fig. 8AGo). However, the interaction between VDR and DRIP205 was significantly increased when nuclear extracts were used from 1,25-(OH)2D3- and OA-treated cells as compared with extracts treated with 1,25-(OH)2D3 alone (P < 0.05) (Fig. 8BGo). Treatment with OA alone resulted in a VDR-DRIP interaction that was 32.5% of the interaction observed with 1,25-(OH)2D3 and OA treatment. Additional GST pull-down experiments using VDR that was in vitro phosphorylated by purified casein kinase II did not exhibit increased interaction with in vitro translated DRIP205 (not shown), suggesting that phosphorylation of VDR on serine 208 is not involved in the OA-mediated enhancement of VDR-DRIP205 interaction.



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Figure 8. Phosphorylation-Mediated Enhancement of VDR-DRIP205 Interaction

A, GST pull-down assay was performed using 15 µg of GST-GRIP-1 fusion protein and equal amounts of VDR protein from nuclear extracts of VDR expression vector, pAVhVDR, transfected COS-7 cells treated as described in Fig. 7Go with vehicle (-D), 1,25-(OH)2D3 (+D), OA, or 1,25-(OH)2D3 plus OA (OA +D) for 24 h. Western blot analysis with a monoclonal anti-VDR antibody was performed to visualize VDR binding. B, GST pull-down assays were performed as in panel A except using GST-DRIP205 (527–970) fusion protein instead of GST-GRIP-1. Representative Western blots are shown (left panels, A and B). The results of three to six separate experiments are expressed as relative signal intensity (100 = maximal intensity) ± SE after normalization to VDR input (right panels, A and B).

 
Additionally, we evaluated the role of the activation function-2 (AF-2) domain of VDR in transcriptional activation by OA. The C-terminal helix 12, the core AF-2 domain, contains residues that function as an interaction surface for coactivators (33). We used the AF-2 defective mutant L417S (the leucine residue 417 at the C terminus of the predicted helix 12 in VDR was mutated to serine). When COS-7 cells were cotransfected with the L417S mutant VDR and the rat proximal 24(OH)ase VDRE-tk CAT construct or the mouse OPN VDRE-tk CAT construct, 1,25-(OH)2D3-dependent transcriptional activation was not observed [Fig. 9, A and B (-D, +D)]. The failure of the AF-2 defective VDR to induce transcription in response to 1,25-(OH)2D3 correlates with the inability of the L417S mutant VDR to interact with DRIP205 in the presence of 1,25-(OH)2D3 (Fig. 9CGo, -D, +D). In contrast, in the presence of OA, the AF-2 defective mutant was able to activate the VDRE-tk CAT constructs in a ligand-independent manner (3- to 4-fold induction compared with basal, P < 0.05). Similar findings were observed using the -1,367/+74 rat 24(OH)ase promoter construct or the -777/+79 mouse OPN promoter construct. The activity of a tk CAT construct cotransfected with L417S VDR was not affected by OA treatment (not shown). Thus, activation of VDR-mediated transcription by phosphorylation can be independent of the conserved leucine in the AF-2 domain. In addition, phosphorylation rescued the recruitment of DRIP205 to the AF-2 defective mutant (Fig. 9CGo). These results suggest, similar to the findings shown in Fig. 8Go, that stimulation of transcription by inhibition of phosphatase involves increased interaction between VDR and the DRIP coactivator complex.



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Figure 9. Activation of AF-2 Defective VDR Mutant by Phosphorylation

COS cells were cotransfected with AF-2 defective VDR (L417S VDR) and either the 24(OH)ase VDRE-tk CAT construct (A) or the OPN VDRE-tk CAT construct (B). Transfected cells were treated as previously described with vehicle (-D), 1,25-(OH)2D3 (+D), OA, or 1,25-(OH)2D3 plus OA (OA + D) for 24 h. After treatment, cells were harvested and lysed, and CAT activity was measured as described in Materials and Methods. Representative autoradiograms are shown. The results of three separate experiments are expressed as the percent of maximal response ± SE after normalization for ß-galactosidase activity. C, GST pull-down assay was performed using 15 µg of GST-DRIP205 fusion protein and equal amounts mutant VDR (L417S) protein from nuclear extracts of transfected COS-7 cells treated as previously described with vehicle (-D), 1,25-(OH)2D3 (+D), OA, or 1,25-(OH)2D3 plus OA (OA +D) for 24 h. Western blot analysis was performed with a monoclonal anti-VDR antibody to visualize VDR binding. Representative VDR Western blots are shown (left panel). The results of three or more separate experiments are expressed as relative signal intensity (100 = maximal intensity) ± SE after normalization to VDR input.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The results of this study suggest that inhibition of cellular phosphatases can enhance 1,25-(OH)2D3-induced transcription and mRNA expression of 24(OH)ase and OPN, at least in part, by enhancing cofactor recruitment. This is the first report of a mechanism involved in the enhancement of 1,25-(OH)2D3-induced transcription by inhibition of protein phosphatases. The binding of DRIP205 to VDR but not the VDR/GRIP-1 interaction was enhanced. The DRIP complex lacks HAT activity characteristic of GRIP-1 and other p160 coactivators, and the individual DRIP subunits, including DRIP205, are not homologous to other known coactivators (36). In addition, GRIP-1 is structurally unrelated to DRIP205 except for the presence of a LXXLL motif that is required for the interaction with the ligand-binding domain (LBD) of nuclear receptors (35), and the specific amino acids needed for VDR binding to the two coactivators may indeed be different (36). The 20-epi-analogs of 1,25-(OH)2D3 are more potent than 1,25-(OH)2D3 and enhance cellular responses, such as differentiation of myeloid cells and p21 expression (39). Interestingly, these analogs also result in enhanced recruitment of DRIP205 but not GRIP-1 to VDR (39). Taken together with our results, this indicates that the DRIP complex is an important and perhaps preferred coactivator complex not only for the enhanced response to 20-epi analogs but also for enhancement of VDR-mediated transcription by phosphorylation.

OA enhanced 1,25-(OH)2D3-induced VDR-mediated transcription without increasing VDR levels or the 1,25-(OH)2D3-dependent binding of VDR-RXR to the VDRE. Enhancement of hormone-activated receptor-mediated transcription by OA has also been reported using human and chick PR (10, 40), rat GR (41), human RAR{alpha} and RARß, human RXR{alpha}, and mouse RXRß and RXR{gamma} (15). Similar to our study, Beck et al. (40) using the human PR and Moyer et al. (41) using the rat GR reported that the enhancing effects of OA are not due to changes in receptor content. Beck et al. (40) also noted that receptor DNA binding was unaffected by OA treatment. In these studies OA was found not to alter receptor phosphorylation. It was suggested that OA could possibly modulate transcription by altering the interaction with coactivators that may function as intermediaries between the receptor and the basal transcription machinery (40).

The phosphorylation of human VDR involves serine residues in distinct functional domains (30, 31, 32, 42). The phosphorylation of VDR by casein kinase II at serine 208 in the ligand binding domain has been reported to account for 60% of the phosphorylation of the receptor and to play an important role in transcriptional activation (16). In previous studies, metabolic 32P labeling and measurement of total VDR phosphorylation using ROS17/2.8 cells or VDR-transfected CV1 cells indicated that OA can induce phosphorylation of VDR (14, 43). In this study we found that the OA enhancement of 1,25-(OH)2D3-induced VDR-mediated transcription was not abolished using VDR mutants with either serine 208 or serine 51 phosphorylation sites mutated to alanine. In addition, casein kinase II phosphorylation of VDR did not result in enhanced binding of VDR to DRIP205. Although OA-mediated transcriptional enhancement was observed using the mutant VDRs, it is possible that novel sites may be phosphorylated as a result of OA treatment, resulting in an increased interaction with DRIP205 binding. Hilliard et al. (42) found that mutations in serine 208 and 51 could not eliminate VDR phosphorylation and that these mutations cause subsequent phosphorylation on amino acids that are not normally modified. It is also possible that OA may be acting by enhancing the phosphorylation of another protein that may stabilize the formation of the VDR-DRIP205 complex, resulting in enhanced VDR-DRIP interaction observed in this study. Mapping of VDR phosphorylation sites in response to inhibition of the cellular phosphatases will be required to determine whether OA increases site-specific phosphorylation of VDR. Knowledge of the crystal structure of VDR (4) and computer modeling of the conformational changes upon ligand binding and hyperphosphorylation will facilitate the identification of the exact mechanism by which phosphorylation enhances receptor-coactivator interactions and transactivation.

In this study we found that although OA can enhance 1,25-(OH)2D3-induced VDR-mediated transcription of both 24(OH)ase and OPN, significant ligand-independent activation by OA was observed only for the OPN promoter construct -777/+79. OA alone was unable to stimulate VDR-mediated transcription using the OPN VDRE-tk CAT construct, the 24(OH)ase VDRE-tk CAT construct, or the 24(OH)ase promoter (-1,367/+74). These findings suggest, similar to what has been reported for the AR (44, 45), that ligand-independent activation depends on promoter context. However, the refractory effect is lost in the context of the AF-2 defective VDR mutant. In contrast to the action of 1,25-(OH)2D3, in the presence of OA, the AF-2 defective mutant was able to activate both VDRE-tk CAT constructs, and OA alone was able to induce an interaction between DRIP205 and the mutant VDR (Fig. 9Go). These results suggest a subtle conformational effect in the AF-2 defective mutant, not as yet understood, that is more permissive to ligand-independent activation by OA. Recent studies have indicated that the Ets-1 transcription factor, a target of the Ras-MAPK signaling pathway, can also result in a ligand- and AF-2-independent activation of nuclear receptors, including VDR (46). It was suggested that Ets acts by inducing a conformational change in the receptor that creates an active interaction surface with coactivators even in the AF-2 defective mutant (46). The results obtained using Ets-1 (46), as well as our findings using OA and the AF-2 defective VDR mutant, suggest novel mechanisms by which inactive VDR can be converted to an effective transcriptional activator.

In addition to factors that may induce a conformational change in the receptor, previous studies have shown that species specificity of the steroid receptor and cell type are also important in determining ligand-independent activation (10, 40, 44, 47). Ligand-independent activation by modulation of cellular phosphorylation has been reported for chick PR (10) but not for human PR (40). Also, Chinese hamster ovary cells stably transfected with human AR do not exhibit ligand-independent transcriptional activation (44). However, studies performed in CV1 cells and PC-3 cells indicated ligand-independent activation of hAR (47). Ligand-independent transcriptional activation of VDR has been noted using CV1 cells and either the promoter of the human osteocalcin (OC) gene (14) or multiple copies of the rat OC VDRE ligated to tk CAT (15). However, when the rat OC promoter or the rat OC VDRE-tk CAT was used to transfect ROS17/2.8 cells, ligand-independent activation by OA was not observed (43). The cell type and species specificity of ligand-independent activation may be due to the relative abundance of coactivators in different cell types and differences in the interaction of specific nuclear receptors with coactivators.

Our findings suggest that the enhancement of 1,25-(OH)2D3-induced transcription by extracellular signals involves phosphorylation-dependent modulation of cofactor recruitment. Phosphorylation-mediated cofactor interaction was first shown to be important for the recruitment of CBP by the cAMP response element binding protein (CREB) (48). More recent studies have shown that cofactor recruitment by PPAR{gamma} and TR is also affected by phosphorylation. EGF was found to enhance binding of PPAR{gamma} to the corepressor SMRT (silencing mediator of retinoid and thyroid hormone receptor) (49). However, phosphorylation of SMRT by MEKK1 inhibits the binding of SMRT to TR (50). Phosphorylation of ERß by MAPK leads to ligand-independent activation and recruitment of the coactivator SRC-1 (51). Phosphorylation of the orphan receptor SF-1 by MAPK has also been reported to enhance cofactor recruitment (52). It was suggested that enhanced cofactor recruitment could then modulate the coordinate regulation of multiple SF-1 target genes. Most recently, phosphorylation of SRC-1, induced by cAMP on two MAPK kinase sites (threonine 1,179 and serine 1,185), was found to be necessary for optimal transactivation by PR and for optimal interaction between SRC-1 and p300/CBP-associated factor (53). Thus, it is becoming increasingly evident that phosphorylation-dependent cofactor recruitment plays an important role in the cross-talk between steroid receptors and signal transduction pathways. It will be of interest in the future to further consider the functional significance of cofactor phosphorylation as well as nuclear receptor phosphorylation and to examine the levels of various cofactors present endogenously in specific cell types. Differences in cofactor levels may account for the differential regulation of steroid-responsive genes in multiple cell types in response to activation of signaling pathways.

Although a number of previous studies have shown that inhibition of phosphatases can enhance steroid hormone-induced transcription (10, 11, 14, 15, 40, 41), our study is the first to show that endogenous VDR-mediated responses can be affected by OA treatment. 1,25-(OH)2D3 induction of 24(OH)ase and OPN mRNAs was enhanced in UMR osteoblastic cells by the inhibition of phosphatases. 24(OH)ase hydroxylates 25(OH)D3 and 1,25-(OH)2D3 and is involved in the catabolism of 1,25-(OH)2D3 (54). Thus, by inducing the 24(OH)ase enzyme, 1,25-(OH)2D3 induces its own deactivation. The 24(OH)ase gene is the most transcriptionally responsive vitamin D-inducible gene identified to date (54). OPN, a phosphorylated protein abundant in the bone matrix, is also induced by 1,25-(OH)2D3 and has been reported to modulate both mineralization and bone resorption (55). Previous studies in endogenous systems, including osteoblastic cells, have indicated that both OPN and 24(OH)ase can be modulated by factors that affect signal transduction pathways (55, 56, 57, 58, 59). The physiological significance of the enhancement of 1,25-(OH)2D3 induction of 24(OH)ase in osteoblastic cells by activation of signaling cascades may be to contribute to the prevention of elevated intracellular 1,25-(OH)2D3 levels that can adversely affect mineralization (60). Enhancement of 1,25-(OH)2D3 induction of OPN expression by activation of signaling cascades can result in enhanced effects on mineralization or resorption. It will be important in future studies to identify the phosphorylation sites in the coactivator and/or the VDR as well as to identify the specific kinases that catalyze the phosphorylations involved in 24(OH)ase- and OPN-enhanced transcription.

In summary, our findings suggest that increased interaction between VDR and specific coactivators is needed for enhanced expression of 1,25-(OH)2D3 target genes in response to activation of signaling pathways and that differential cofactor recruitment mediated by phosphorylation may be a major mechanism involved in the integration of signal transduction pathways and vitamin D action.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
[14C]Chloramphenicol (50 mCi/mmol), [{alpha}-32P]-dCTP [3,000 Ci (111 TBq)/mmol], [{gamma}-32P]-ATP [3,000 Ci (111 TBq)/mmol], Easytag L-[35S]-methionine [>1,000 Ci (37.0 TBq)/mmol], nylon membranes, and enhanced chemiluminescent (ECL) detection system were purchased from NEN Life Science Products (Boston, MA). Casein kinase II and TNT Coupled Reticulocyte Lysate System were from Promega Corp. (Madison, WI). Acetyl coenzyme-A, formamide, and OA (sodium salt) were obtained from Sigma (St. Louis, MO). DMEM plus Ham’s F12 nutrient mixture (DMEM-F12), DMEM, Medium 199, Random Primers DNA Labeling kit, T4 polynucleotide kinase, and oligo (dt) cellulose were purchased from Life Technologies, Inc. (Gaithersburg, MD). RNAzol was obtained from Tel-Test (Friendswood, TX). Rat monoclonal anti-VDR antibody was purchased from Affinity BioReagents, Inc. (Neshanic Station, NJ). 1,25-(OH)2D3 was a generous gift from M. Uskokovic (Hoffmann-LaRoche Inc., Nutley, NJ).

Cell Culture
COS-7 African green monkey kidney cells, UMR-106 rat osteoblastic cells, and LLCPK1 porcine kidney cells were obtained from American Type Culture Collection (ATCC, Manassas, VA) and were cultured in DMEM supplemented with 10% heat-inactivated FBS from Gemini Biological Products (Calabasas, CA), DMEM-F12 supplemented with 5% FBS, and Medium 199 supplemented with 3% FBS, respectively. All cell lines were grown in a humidified atmosphere of 95% O2-5% CO2 at 37 C. Cells were grown to 60–70% confluence in 100-mm2 tissue culture dishes, and 24 h before the start of experiments medium was replaced with medium supplemented with 2% charcoal-stripped serum. Cells were treated with vehicle or the compounds noted at the concentrations and times indicated.

Transfection and CAT Assay
Promoter constructs containing the rat 24(OH)ase promoter (-1,367/+74) (61) or the mouse OPN promoter (-777/+79) (62) linked to the CAT reporter gene were used. A tk CAT reporter plasmid containing multiple copies of the rat 24(OH)ase VDRE (-150/-136) (63) was also used. The mouse OPN VDRE-tk CAT construct, containing multiple copies of the mouse OPN VDRE (-757/-743) was prepared in the laboratory of L. P. Freedman. All cells were cotransfected with the appropriate reporter plasmid and a ß-galactosidase expression vector (pCH110, Pharmacia Biotech, Piscataway, NJ) as an internal control for transfection efficiency, using the calcium phosphate DNA precipitation method (64). In addition to the reporter plasmid and a ß-galactosidase expression vector, COS-7 cells were also transfected with the hVDR expression vector pAVhVDR (from J. W. Pike, University of Cincinnati, Cincinnati, OH), mutated VDR expression vector constructs [S208A and S51A were also a gift from J. W. Pike (42)], or the AF-2 defective VDR mutant L417S expression vector construct (prepared in the laboratory of L. P. Freedman). The pRSV-CAT expression plasmid (American Type Culture Collection) and the pBL2CAT construct (tk CAT; from J. W. Pike) were used in certain transfections as negative controls. Sixteen hours after transfection cells were shocked for 1 min with PBS containing 10% dimethylsulfoxide, washed with PBS, and treated for 24 h with the indicated treatments [1,25-(OH)2D3 (10-8 M) or OA (50 nM)] in the appropriate medium supplemented with 2% of charcoal-dextran-treated FBS.

Twenty-four hours after treatment cells were harvested by trypsinization, pelleted, washed with PBS and resuspended in 0.25 M Tris-HCl, pH 8.0, and lysed by freezing and thawing five times. Cellular extract was collected and used for ß-galactosidase or protein analysis. CAT assay was performed by standard protocols on the cell extract normalized to ß- galactosidase activity or total protein (64, 65). Autoradiograms were analyzed by densitometric scanning using the Shimadzu CS9000U Dual-Wavelength Flying spot scanner (Shimadzu Scientific Instruments, Princeton, NJ). For some experiments several autoradiographic exposure times were needed for densitometric analysis. CAT activity was also quantitated by scanning TLC plates using the Packard Constant Imager System (Packard Instrument Co., Meriden, CT).

Northern Blot Analysis
Total RNA was prepared from UMR-106 cells with RNAzol RNA extraction solution following the manufacturer’s instructions. Polyadenylated [poly (A+)] mRNA was isolated from the total RNA using oligo (deoxythymidine)-cellulose column chromatography. Northern blot analysis was performed as previously described (66). Briefly, Northern analysis prehybridization and hybridization were performed in Ultrahyb solution from Ambion, Inc. (Austin, TX). 32P-labeled cDNA probes were prepared using Random Primers DNA Labeling Systems (Life Technologies, Inc.) according to the random prime method (64). The 1-kb mouse OPN cDNA, used for Northern analyses, was generated by HindIII digestion and was a gift from D. Denhardt (Rutgers University, Piscataway, NJ) (67). A 1.7-kb rat VDR cDNA was obtained by digestion of pIBI76 with EcoRI (68). The 3.2-kb rat 24(OH)ase cDNA was obtained by EcoRI digestion and was a gift from K. Okuda (Hiroshima University School of Dentistry, Hiroshima, Japan) (69). The ß-actin cDNA was purchased from CLONTECH Laboratories, Inc. (Palo Alto, CA). The blot was hybridized to the specific 32P-labeled cDNA probe for 16 h at 42 C, washed, and subjected to autoradiography. Autoradiograms were analyzed by densitometric scanning using the Shimadzu CS9000U Dual-Wavelength Flying spot scanner (Shimadzu Scientific Instruments). Results are expressed as the optical density ratio of test probe to ß-actin control probe.

VDR Western Blot Analysis
UMR-106 cells were treated with the indicated compounds for 24 h and harvested by trypsinization, and nuclear extracts were prepared following the method of Dignam et al. (70). After isolation of nuclear extracts, 50 µg of protein were separated by 10% SDS-PAGE. Proteins were transferred to a polyvinylidene difluoride membrane from Bio-Rad Laboratories, Inc. (Hercules, CA). The membrane was blocked with PBST (0.5% Tween 20 in PBS) containing 5% nonfat milk for at 4 C. After 16 h of blocking the blot was incubated with rat monoclonal anti-VDR antibody (Affinity BioReagents, Inc.) for 2 h at 27 C, washed with PBST, and incubated for an additional hour with a goat antirat IgG conjugated to horseradish peroxidase (Sigma). Again the membrane was washed with PBST and the enhanced chemiluminescence Western blotting detection system (NEN Life Science Products) was used to detect the antigen/antibody complex.

EMSA
Nuclear extracts were prepared by the method of Dignam et al. (70) from cells incubated with the indicated treatments for 24 h. A DNA fragment of the rat 24(OH)ase promoter (-165/-122) containing the proximal VDRE (-150/-136) (Ref. 61 ; prepared by the UMD Molecular Resource Facility, Newark, NJ) was used as a probe. Overlapping and reverse strands were heat denatured and annealed overnight. Fifty nanograms of duplex oligo were end labeled with [{gamma}-32P]-ATP using T4 polynucleotide kinase (Life Technologies, Inc.) and purified using a micro bio-spin p-30 column (Bio-Rad Laboratories, Inc.). The eluted probe was used for EMSA as described (64). Briefly, aliquots of the nuclear preparations (3–5 µg of protein) were incubated for 20 min at 27 C with 0.3–0.5 ng of the labeled oligonucleotide probe (~100,000 cpm) in binding buffer (4 mM Tris-HCl, pH 7.9, 1 mM EDTA, pH 8.0, 60 mM KCl, 12% glycerol, 12 mM HEPES, 1 mM dithiothreitol). The samples were separated by electrophoresis on a 6% nondenaturing polyacrylamide gel. Gels were dried and exposed to Kodak XAR-5 film (Eastman Kodak Co., Rochester, NY).

GST Fusion Protein Pull-Down Assay
Nuclear extracts were prepared from COS-7 cells transiently transfected with a wild-type or mutated (L417S) VDR expression vector and incubated with the indicated test compound for 24 h. VDR protein in each sample was assessed by Western blot analysis using monoclonal anti-VDR antibody (Affinity BioReagents, Inc.). The GST-DRIP205 (527–970) or GST-GRIP-1 fusion proteins (39), immobilized on Sepharose beads, were incubated with GST binding buffer (20 mM Tris-HCl, pH 7.9, 180 mM KCl, 0.2 mM EDTA, pH 8.0, 0.05% Nonidet P-40, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol) that contained 1 mg/ml BSA for 2 h. The GST binding buffer was removed and nuclear extract aliquots, containing equal amounts of VDR protein, were incubated with 15 µg of the appropriate fusion protein for 16 h at 4 C. Bound proteins were washed four times with GST binding buffer containing 0.1% Nonidet P-40 and then incubated with elution buffer [GST binding buffer containing 0.1% Nonidet P-40 and 0.15% Sarkosyl (Sigma)] at 4 C for 20 min. The eluted proteins were analyzed by SDS-PAGE followed by Western blotting with VDR antibody. Additional GST pull-down assays were performed using GST-VDR-LBD- (110–427) (39) that was phosphorylated in vitro with casein kinase II. Briefly, 15 µg of GST-VDR-LBD fusion protein were incubated in assay buffer (25 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 200 mM NaCl, and 0.1 mM ATP) with 1 U purified casein kinase II for 30 min. GST-VDR-LBD was washed three times with GST binding buffer. Phosphorylation of GST-VDR-LBD was judged by performing the phosphorylation reaction in the presence of 25 µCi of [{gamma}-32P]-ATP followed by SDS-PAGE and autoradiography. GST pull-down assays were performed in the presence or absence of 1,25-(OH)2D3 (10-8 M) with the phosphorylated or unphosphorylated GST-VDR-LBD and Easytag L-[35S]-methionine-labeled DRIP205 that was prepared using the TNT Coupled Reticulocyte Lysate System from Promega Corp. After washing, bound DRIP205 was analyzed by SDS-PAGE and autoradiography.

Statistical Analysis
Significance was determined by t test or Dunnett’s multiple comparison t statistic.


    ACKNOWLEDGMENTS
 
We gratefully acknowledge Dr. Wen Yang and Dr. Michael Huening for helpful suggestions.


    FOOTNOTES
 
This work was supported by NIH Grants DK-38961 (to S.C.) and DK-45460 (to L.P.F.).

Abbreviations: AF-2, Activation function-2; CAT, chloramphenicol acetyltransferase; CBP, CREB binding protein; CREB, cAMP response element binding protein; DRIP, VDR-interacting protein; GRIP, GR-interacting protein; GST, glutathione-S-transferase; HAT, histone acetylase; hVDR, human VDR; LBD, ligand-binding domain; OA, okadaic acid; OC, osteocalcin; 24(OH)ase, 24-hydroxylase; 1,25-(OH)2D3, 1,25-dihydroxyvitamin D3; OPN, osteopontin; PP-1 and PP-2A, protein phosphatase 1 and 2A; RSV, Rous sarcoma virus; tk, thymidine kinase; SMRT, silencing mediator of retinoid and thyroid hormone receptor; SRC, steroid receptor coactivator; VDRE, vitamin D response element.

Received for publication January 5, 2001. Accepted for publication October 1, 2001.


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