Retinoid X Receptor (RXR) Ligands Activate the Human 25-Hydroxyvitamin D3-24-hydroxylase Promoter via RXR Heterodimer Binding to Two Vitamin D-responsive Elements and Elicit Additive Effects with 1,25-Dihydroxyvitamin D3*

(Received for publication, December 3, 1996, and in revised form, April 21, 1997)

Aihua Zou , Marc G. Elgort and Elizabeth A. Allegretto Dagger

From the Department of Retinoid Research, Ligand Pharmaceuticals, Inc., San Diego, California 92121

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

We have previously shown that RNA levels of kidney 25-hydroxyvitamin D3-24-hydroxylase (24(OH)ase), a key metabolic enzyme for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), is up-regulated by retinoids in mice within hours. Deletion analysis of ~5500 base pairs of the human 24(OH)ase promoter showed that the sequence between -316 and -142 contained the information necessary and sufficient for retinoid-induced activation of the promoter. This region contains two previously defined vitamin D-responsive elements (VDREs) at -294 to -274 and -174 to -151. Mutation of either VDRE diminished responsiveness of the -316 to -22 promoter sequence to retinoids or 1,25(OH)2D3, while mutation of both VDREs essentially abolished the activity of the ligands via the promoter. Heterologous promoter vectors driven by the VDREs were responsive to a retinoid X receptor (RXR)-selective ligand (LG100268), a retinoic acid receptor (RAR)-selective ligand (TTNPB), or 1,25(OH)2D3, while combinations of LG100268 with either TTNPB or 1,25(OH)2D3 resulted in additive increases in activity. Band shift analyses showed that vitamin D receptor, RAR, or RXR alone did not bind to the VDREs; however, the combination of either vitamin D receptor or RAR with RXR led to retardation of each of the labeled probes. Treatment of nontransfected CV-1 cells with retinoids or 1,25(OH)2D3 resulted in induction of 24(OH)ase RNA, and ligand combinations led to increased RNA levels. These data imply that either or both of the heterodimer partners can be occupied with ligand to induce this enzyme, with dual receptor occupation leading to increased activation.


INTRODUCTION

The retinoic acid receptors (RARs)1 and retinoid X receptors (RXRs) are nuclear transcription factors that are activated by their retinoid ligands, all-trans-retinoic acid (tRA) and 9-cis-retinoic acid (9cRA) (1). While RARs bind to both tRA and 9cRA with high affinity, RXRs bind to 9cRA but not to tRA (2-5). Both receptor subfamilies are thought to mediate the biological actions of retinoids in processes such as cellular growth and differentiation and development by altering the production of certain proteins in various cells at the level of gene transcription (1, 6). The ligand-occupied receptors generally act by binding to retinoid receptor-responsive elements in the promoter regions of target genes. Ligand-bound RAR cooperates with RXR to form a heterodimer that is an efficient and high affinity binder of retinoic acid response elements, thereby activating transcription of certain tRA-responsive genes such as RARbeta (7-9) and cellular retinoic acid binding protein II (10). RXR is postulated to function by at least two modes of action. RXR has been shown to act as a silent (nonliganded) partner with a number of other intracellular receptors, including RAR, vitamin D receptor (VDR), and thyroid hormone receptor, in response to their respective ligands (11-16). RXR has also been shown to form homodimers in a complex with DNA upon binding to 9cRA (17). However, evidence for RXR homodimers as functional units in the transcription of biologically relevant target genes has yet to be demonstrated.

For example, while RXR binds to, and stimulates transcription from, an element within the cellular retinol binding protein II gene promoter in cotransactivation assays in mammalian cells (18) and in yeast cells (which do not contain RAR; Ref. 2), this gene has not been shown to be regulated by retinoids in nontransfected cells or in the animal. However, evidence is emerging that RXR may not function solely as a silent partner in hormone signaling pathways. Recently, 9cRA has been shown to activate RXR in heterodimeric interactions with two orphan receptors, LXR (19) and NGFI-B (20), to stimulate transcription from synthetic response elements in cotransfections assays. Additionally, RXR ligands have been shown to increase the effects of RAR ligands to induce certain RNA species (21). In these experiments, however, while an RXR ligand modulated the effects of an RAR ligand to activate certain genes, the RXR ligand alone did not induce RNA levels.

We show here that an RXR-selective ligand alone is able to activate transcription of the human 25-hydroxyvitamin D3-24-hydroxylase (24(OH)ase) promoter by binding to the RXR partner of RXR·VDR or RXR·RAR heterodimers and acting through previously defined VDRE sequences (22, 23) within the promoter. Either the RXR ligand alone or the VDR ligand alone leads to stimulation of the promoter, while the presence of both ligands leads to additive or more than additive induction of luciferase activity. These data indicate that ligand occupation of either or both heterodimeric receptor partners leads to a productive transcriptional event at this promoter, with maximal induction observed upon occupation of both receptors. Additionally, ligand-occupied RAR also activates this promoter through these sequences by binding with RXR, either with or with its ligand. Therefore, these two previously defined VDREs within the human 24(OH)ase promoter can also serve as retinoic acid response elements, since they are able to confer responsiveness of the promoter to retinoids.


EXPERIMENTAL PROCEDURES

Ligands

LG100268 (24), TTNPB (25, 26), and 9cRA (25) were synthesized and purified at Ligand Pharmaceuticals, Inc. (San Diego, CA). 1,25(OH)2D3 was from Solvay Duphar (The Netherlands).

Receptor and Reporter Vectors

Receptor expression vectors (pRShRXRalpha , pRSmRXRgamma , pRShRARgamma , and pRShVDR) were as described previously (2, 27). 24(OH)ase promoter-driven reporter constructs were derived from an ~6-kb human genomic clone (23) that included sequence 3' to the start site of transcription. The 6-kb promoter fragment (~-5500 to +455) was cloned into a promoterless luciferase expression vector, pLUCpl (2), at SalI and PstI sites upstream of the translation start site of the luciferase coding region of the plasmid. Deletion and mutant promoter constructs were generated from the 6-kb sequence with common 3' ends by digestion with NsiI at position -22 in the promoter sequence (~-5500 to -22)-LUC. (-1177 to -22)-LUC was generated by digesting (~-5500 to +455)-LUC with Van91I (blunted) and NsiI, and the resultant fragment was subcloned into pLUCpl digested with XhoI (blunted) and PstI. (-316 to -22)-LUC, (-294 to -22)-LUC, (-261 to -22)-LUC, and (-143 to -22)-LUC were generated by PCR utilizing 5' primers, some with an overhanging SalI site immediately upstream of the 5'-most base of the deletion construct (others without an enzyme site) and a common 3' primer encompassing the NsiI site. PCR products were digested with SalI (or used blunt-ended) and NsiI and subcloned into pLUCpl digested with XhoI (blunted or not) at the 5' end and PstI at the 3' end. (-261Delta 1)-LUC (-168CCC mutated to GTT) was created by site-specific oligonucleotide-directed mutagenesis of the (-261 to -22) PCR product in M13, digested with SalI and NsiI, and subcloned into pLUCpl cut with XhoI and PstI. (-316Delta 1)-LUC (-168CCC mutated to GTT) was made by digestion of (-316 to -22)-LUC with HindIII/BstEII, digestion of (-261Delta 1)-LUC with BstEII/BamHI, and ligation of the two fragments into pLUCpl digested with HindIII/BamHI. (-316Delta 2)-LUC (-289CACC to AAAA) was created utilizing the MORPHTM site-specific plasmid DNA mutagenesis kit (5 Prime right-arrow 3 Prime, Inc., Boulder, CO). (-316Delta 1Delta 2)-LUC (-168CCC to GTT and -289CACC to AAAA) was generated by digestion of (-316Delta 2)-LUC with HindIII/BstEII and (-261Delta 1)-LUC with BstEII/BamHI and combining the two fragments in pLUCpl digested with HindIII/BamHI. (-316Delta 3)-LUC (-308GGGAGGCGCGTTCG mutated to AGAAGGCGCAAATT) was generated by using PCR; the 5' primer contained the individual base changes as well as an XhoI site immediately upstream of position -316, and the 3' primer contained the NsiI site at -22. The resultant PCR product was digested with XhoI and NsiI and subcloned into pLUCpl digested with XhoI/PstI. Oligonucleotides corresponding to the individual VDREs (-294 to -274 and -174 to -151) were synthesized with HindIII ends, annealed, and subcloned into the Delta MTV-LUC vector. The identities of all constructs were confirmed by sequencing.

Cotransfection/Cotransactivation Assays

Receptor and reporter vectors were transfected along with carrier DNA and beta -galactosidase internal control plasmid in COS-1 cells by the use of calcium phosphate and N,N-bis(2-hydroxyethyl)-2-aminoethane-sulfonic acid-buffered saline. Briefly, transfections were performed in triplicate in 12-well plates with a total of 20 µg/ml DNA (0-0.5 µg of receptor expression vector, 5-10 µg of reporter plasmid, 5 µg of beta -galactosidase plasmid, and pGEM carrier plasmid to a total of 20 µg). Amounts of plasmids transfected in each experiment are listed in individual figure legends. Fifteen hours later, ligands were added, followed by an additional 30-h incubation. Cells were lysed, and measurements of luciferase and beta -galactosidase activities were as described previously (4). Luciferase values were normalized with beta -galactosidase values to control for variable transfection efficiencies. -Fold induction was calculated by dividing the maximal response by the response elicited with vehicle. Statistical analyses were performed using analysis of variance and Fisher's protected least squares determination.

Electrophoretic Mobility Shift Assays

Oligonucleotides (-294 to -274 with BamHI and HindIII sites at each end and -174 to -151 with HindIII ends) and a PCR fragment (-314 to -121) spanning the retinoid and 1,25(OH)2D3-responsive region of the human 24(OH)ase promoter were synthesized. Oligonucleotides containing a consensus DR3 sequence (GGGAGGTCATTTAGGTCAGGG) or a DR1 motif (AGGTCAGAGGTCA) were synthesized with HindIII, or SalI overhanging ends, respectively. The oligonucleotides and PCR fragment were end-labeled with 32P-labeled nucleotide triphosphates. Protein extracts were prepared from the yeast strain BJ2168 transformed without or with VDR, RXRalpha , or RARalpha expression vectors (2). In reactions including antibodies, anti-VDR 9A7gamma , anti-RXRalpha , anti-RARalpha , or anti-estrogen receptor antibodies (28, 29) were preincubated with crude protein extracts (~1 µl) for 1 h on ice prior to the addition of DNA probe. Reactions performed with ligand present included 9cRA (1 µM, 0.1% ethanol) or ethanol vehicle and RXR-containing extracts; incubation was at 4 °C for 1 h prior to the DNA addition. Protein-DNA binding reactions were carried out in buffer containing 50-100 mM KCl, 20 mM Hepes, pH 7.4, 20% glycerol, 12.5 mM MgCl2. Oligonucleotide probe (~20,000 dpm) was added to protein mixtures along with 1 µg of poly(dI-dC) competitor DNA/reaction and incubated on ice for 20 min, followed by 2 min at room temperature. Electrophoresis of protein-DNA complexes was performed on 6% acrylamide, 0.5 × TBE gels (Novex, San Diego) in 0.5 × TBE running buffer at 4 °C (~15 mA). Gels were dried and exposed to autoradiographic film for 1-2 h at -80 °C.

CV-1 Cell Studies

CV-1 cells were maintained in Dulbecco's modified Eagle's medium supplemented with L-glutamine and 10% fetal bovine serum. Cells at ~40% confluency were incubated in media containing 10% charcoal-stripped fetal bovine serum for 48 h prior to treatment with ligands. Cells were ~80% confluent upon the addition of ligands in 0.1% ethanol vehicle. 1,25(OH)2D3, LG100268, TTNPB, 9cRA, or combinations thereof were added in fresh media containing charcoal-stripped fetal bovine serum and incubated with cells for 6 h prior to harvest. Concentrations of ligands are indicated in the figure legends. Total cellular RNA was extracted, and Northern analysis was performed as per standard methodology. Probes included a 900-base pair EcoRI/XbaI DNA fragment of the rat 24(OH)ase cDNA (30) and a 1.4-kb human glyceraldehyde-3-phosphate dehydrogenase fragment (Clonetech). Hybridization was performed at 65 °C in Quik-Hyb solution (Amersham Corp.) for 2 h followed by washing (0.1 × SSC, 0.1% SDS at 65 °C for 1 h). Quantitation was by PhosphorImager analysis (Molecular Dynamics).


RESULTS

Two regions of the human 24(OH)ase promoter are responsive to retinoids and 1,25(OH)2D3. The cloning of the rat (31, 32) and human (22, 23) 24(OH)ase promoters has been reported previously. Two VDREs have been defined within both the rat and the human promoters that confer responsiveness of the promoters to 1,25(OH)2D3 (22, 32). We have previously reported that retinoids induce kidney 24(OH)ase activity in mice (27), and cursory experiments indicated that the human 24(OH)ase promoter was stimulated by retinoids in CV-1 cell cotransactivation assays (27). The in vivo effects were observed with a synthetic RXR-selective ligand, LG100268 (24), a synthetic RAR-selective compound, TTNPB, and the endogenous retinoid ligands, 9cRA and tRA. To determine the mechanism of retinoid activation of the 24(OH)ase promoter, an in depth promoter study was undertaken. Various deletions of the previously cloned ~6-kb human 24(OH)ase promoter (23) were constructed into a promoterless luciferase reporter vector (2) and tested for the ability to respond to various ligands in kidney cell lines cotransfected with retinoid receptors and/or VDR. The promoter fragment containing ~5.5 kb upstream of the start site of transcription ((-5500 to -22)-LUC) yielded 3-fold induction of luciferase activity by the RXR-selective ligand, LG100268, in either of two kidney cell lines, CV-1 (data not shown) or COS-1 (Fig. 1A). COS-1 cells were used for the remainder of the study, since they gave identical results to CV-1 cells and were more stable in the transfection assays than CV-1 cells. The magnitude of the response (~3-fold) to LG100268 with (-5500 to -22)-LUC was identical to that previously observed by us in the mouse (27) and was also identical to that observed with (-1177 to -22)-LUC and (-316 to -22)-LUC. (-261 to -22)-LUC yielded a less efficacious response (~1.8-fold activation; p < 0.05) with LG100268, and (-143 to -22)-LUC and pLUCpl did not respond to LG100268 (Fig. 1A). Therefore, two regions within the promoter conferred responsiveness to the RXR-selective ligand LG100268, one between -316 and -261 and the other between -261 and -143 (see Fig. 2). Interestingly, these two regions are also responsive to 1,25(OH)2D3 (Fig. 1B) and contain previously defined VDREs (see Fig. 2; Refs. 21 and 22). Sequence upstream of -1177 may contain an additional VDRE (Fig. 1B). The two LG100268-responsive regions also confer responsiveness of the promoter to TTNPB (Fig. 1C; p < 0.05) and to 9cRA treatment (Fig. 1D). COS-1 cells contain endogenous VDR, RAR, and RXR proteins (data not shown), and ligand-induced luciferase activity driven by the 24(OH)ase promoter sequences was also observed without transfected receptor expression vectors. The -fold induction varied from 20 to 80% of that in the presence of transfected receptors, depending on the ligand (Fig. 3). The endogenous retinoid receptor pan-agonist, 9cRA, elicited greater induction of luciferase activity through each of the promoter constructs both in the presence (~6-fold; Fig. 1D) and in the absence (~3-fold; Fig. 3) of transfected receptors, than either TTNPB or LG100268 alone, implying that occupancy of both RAR and RXR leads to greater activation of the promoter than either individual liganded retinoid receptor.


Fig. 1. Transactivational analysis of 5' deletion mutant human 24(OH)ase promoter sequences. COS-1 cells were transiently transfected with RXRgamma (A), VDR (B), RARgamma (C), or RARgamma and RXRgamma (D) expression vectors (0.5 µg) along with reporter vectors (10 µg) driven by human 24(OH)ase promoter sequences ranging from -5500 to -143 base pair at the 5' terminus to -22 at the 3' end (see "Experimental Procedures"). Cells were treated with vehicle (open bars) or increasing concentrations (10-10 M, 10-8 M, 10-6 M (A) or 10-9 M, 10-7 M, 10-5 M (B, C, and D) of the following ligands delivered in ethanol vehicle: LG100268 (A), 1,25(OH)2D3 (B), TTNPB (C), and 9cRA (D).
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Fig. 2. Human 24(OH)ase promoter sequence. The human 24(OH)ase promoter has been previously cloned (22, 23). A portion of the promoter sequence (-320 to +10) that includes the regions that are responsive to retinoids and 1,25(OH)2D3 is illustrated. VDRE sequences are boxed; dots over bases in the VDREs denote residues that were changed in mutation constructs (see "Experimental Procedures"). The region between -316 and -291 contains sequences that are homologous with various direct repeat motifs. Potential response elements are underlined and overlined; dots over bases denote residues that were changed in mutation constructs (see "Experimental Procedures"). The TATA box is underlined.
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Fig. 3. Transactivation from 24(OH)ase promoter sequences without transfected receptor plasmids. COS-1 cells were transiently transfected with (-316 to -22)-LUC reporter (5 µg) and treated with three doses of ligands or vehicle as indicated in the figure. All inductions were statistically significant (p < 0.05).
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RXR Binds as a Heterodimer with VDR or RAR, but Not as a Homodimer, to Retinoid-responsive Sequences of the Human 24(OH)ase Promoter

Upon identification of the retinoid-responsive regions of the human 24(OH)ase promoter (see Fig. 2), the ability of those sequences to bind directly to RXR and RAR was tested. It was previously demonstrated that RXR·VDR heterodimers could bind to these regions (22, 23). Oligonucleotides were synthesized spanning the regions from -294 to -274 and from -174 to -151. Wild type nontransformed yeast extracts did not display DNA binding activity via either of these sequences (Fig. 4, A-C). Extracts from yeast transformed with a human RXRalpha expression vector (2) were able to bind to each of these sequences in the presence of extracts from yeast expressing human VDR or human RARalpha (Fig. 4, A-C) or human RARgamma (data not shown). However, RXR did not bind alone to these sequences (Fig. 4, A-C) or to the entire responsive region between -314 and -121 (Fig. 4D) in the absence or presence of 9cRA (Fig. 4D and data not shown). Conversely, RXR was able to bind to an oligonucleotide containing a consensus direct repeat separated by 1 base pair (DR1) (Fig. 4D), as previously shown (17, 18). Therefore, we conclude that RXR is unable to bind as a homodimer to the retinoid-responsive elements of the 24(OH)ase promoter but that it does form heterodimers with either VDR or RAR. Also, VDR alone or RAR alone or the combination of the two receptors did not bind to any of the DNA probes (Fig. 4, A-C, and data not shown). Binding of RXR·RAR heterodimers to the human 24(OH)ase VDRE sequences was surprising in that the DR3 motifs that they contain are thought to bind preferentially to RXR·VDR heterodimers, while RXR·RAR heterodimers have been shown to prefer DR2 and DR5 type motifs (33). However, the 24(OH)ase promoter VDREs are not perfect consensus DR3 elements (see Fig. 2). Therefore, we tested the ability of RAR to heterodimerize with RXR on a consensus DR3 element and compared the binding with that via the 24(OH)ase VDREs. Fig. 4C shows that RXR-RAR interactions do occur on a consensus DR3-containing oligonucleotide probe (lane 4); however, this interaction is much weaker than the RXR-VDR interaction via the DR3 (lane 5). In contrast, the RXR-VDR and RXR-RAR interactions on the 24(OH)ase VDREs were both of similar high affinity (Fig. 4, A-C).


Fig. 4. RXR forms heterodimers with VDR or RAR, but not homodimers, on ligand-responsive human 24(OH)ase promoter sequences. Oligonucleotides corresponding to the 3' VDRE (VDRE1; -174 to -151; A and C), 5' VDRE (VDRE2; -294 to -274; B), or sequence from -314 to -121 (D) of the human 24(OH)ase promoter were radiolabeled and incubated with protein extracts (~1 µl) from nontransformed yeast (WT) or from yeast transformed with VDR, RXRalpha , RARalpha , or combinations thereof as denoted in the figure. Receptor amounts in each extract aliquot added to the binding reaction were approximately equal as determined by ligand binding assay. Additional oligonucleotide probes included DNA sequences containing a DR3 (C) or a DR1 motif (D) (see "Experimental Procedures"). alpha VDR, alpha RXR, alpha RAR, and alpha ER denote antibodies against VDR, human RXRalpha , human RARalpha , and human estrogen receptor, respectively. 9cRA (1 µM) or vehicle was preincubated with RXRalpha in some reactions (D).
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Mutations within the VDREs Abrogate Responsiveness of the Human 24(OH)ase Promoter to Retinoids

Two approaches were utilized to further delineate the cis-elements involved in the response of the promoter to retinoids: mutational analysis of the wild type promoter and the use of heterologous promoter constructs driven by the retinoid-responsive sequences. Mutations were made within the two previously defined VDREs and within DR-like elements between -316 and -291 in the context of the wild type human 24(OH)ase promoter-driven reporter vector. Point mutations within the 5' half-site of either the upstream VDRE (-316Delta 2-LUC) or the downstream VDRE (-316Delta 1-LUC) led to diminished -fold induction of luciferase activity by LG100268 or 1,25(OH)2D3 (Fig. 5, A and B). Mutation of both VDREs (-316Delta 1Delta 2-LUC) essentially abolished the response to each of the retinoids as well as to 1,25(OH)2D3 (Fig. 5, A-C). The mutations within the sequence between -316 and -294 (-316Delta 3-LUC) had no effect on retinoid or 1,25(OH)2D3 responsiveness (Fig. 5, A-C). Therefore, the two VDREs confer responsiveness of the promoter to retinoids as well as to 1,25(OH)2D3. The noninvolvement of the sequence between -316 and -294 was confirmed by a deletion construct containing promoter sequence from -294 to -22, which exhibited the same efficacy of response as the -316 to -22 construct to either retinoid or 1,25(OH)2D3 (data not shown).


Fig. 5. Mutational analysis of the human 24(OH)ase promoter. COS-1 cells were transiently cotransfected with RARgamma , RXRalpha , and VDR expression vectors (0.5 µg) along with various human 24(OH)ase promoter sequence-containing reporter constructs (10 µg). Cells were treated with vehicle (open bars) or three concentrations (10-9 M, 10-7 M, and 10-5 M) of LG100268 (A), 1,25(OH)2D3 (B), or TTNPB (C); luciferase activity was measured and normalized as described under "Experimental Procedures." Reporter constructs were as follows: -316 to -22 wild type sequence in pLUCpl (-316 to -22)-LUC or point mutants of VDRE 1 (-316Delta 1-LUC; -168CCC mutated to GTT), VDRE2 (-316Delta 2-LUC; -289CACC to AAAA), both VDREs (-316Delta 1Delta 2-LUC; -168CCC to GTT and -289CACC to AAAA), or site 3 (-316Delta 3-LUC; -308GGGAGGCGCGTTCG mutated to AGAAGGCGCAAATT) in the context of the -316 to -22 sequence; -261 to -22 wild type sequence in pLUCpl (-261 to -22)-LUC or a mutant of VDRE1 (-261Delta 1-LUC; -168CCC mutated to GTT) (see "Experimental Procedures" and Fig. 3 for details).
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Either VDRE Confers Retinoid Responsiveness to a Heterologous Promoter: LG100268 and 1,25(OH)2D3 Yield Additive Induction via Each VDRE

To confirm that the VDREs were able to function as cis-acting elements to confer responsiveness of the promoter to retinoids, oligonucleotides spanning the regions of activity were cloned into a Delta MTV-LUC (MTV-LUC with the glucocorticoid response element deleted) reporter vector. Fig. 6 shows that either VDRE alone as a single copy ((-294 to -274)-LUC or (-174 to -151)-LUC) was able to drive increased luciferase activity in response to 1,25(OH)2D3 (panels A and C) or LG100268 (panels A-C). 1,25(OH)2D3 treatment resulted in 12- and 11.3-fold induction of luciferase activity from the 3' and 5' VDREs, respectively (Fig. 6A). LG100268 alone yielded 4- and 3.5-fold induction from the 3' and 5' VDREs, respectively, while the combination of 1,25(OH)2D3 and LG100268 resulted in 17.4- and 16-fold responses from the 3' and 5' VDREs, respectively, which represent additive increases in luciferase activity (Fig. 6A). TTNPB also acted through these elements on its own and additively increased the activation elicited by LG100268 alone (Fig. 6B). In these experiments (Fig. 6, A and B), receptor plasmids were used at 0.1 µg/ml, and reporter constructs were at 5 µg/ml, typical concentrations used in our cotransfection experiments. To ensure that the additive effects observed were not due to monomeric receptor activation of individual reporter templates instead of heterodimer action on common templates, the amount of reporter used was decreased to 0.1 µg/ml. Fig. 6C shows that, using the VDRE1-Delta MTV-LUC reporter construct at this concentration, the overall luciferase values fall substantially, as expected (Fig. 6, compare panels A and B with panel C). However, the additive effect of 1,25(OH)2D3 with LG100268 or 9cRA is still observed, implying that the activity is on common templates and most likely through heterodimers. Therefore, these data show that each of the human 24(OH)ase VDREs is able to confer retinoid and 1,25(OH)2D3 responsiveness to a heterologous promoter and that saturating concentrations of each ligand in combination elicit greater reporter activity than either compound alone, implying that both receptors can be occupied with ligand to yield greater activation of the promoter.


Fig. 6. Analysis of human 24(OH)ase promoter sequences using heterologous promoter constructs. Delta MTV promoter luciferase reporter vectors (5 µg (A and B); 0.1 µg (C)) driven by oligonucleotides from the human 24(OH)ase promoter (VDRE1, -174 to -151; VDRE2, -294 to -274) were cotransfected with RARgamma (B), RXRalpha (A-C), or VDR (A and C) expression vectors (0.1 µg) into COS-1 cells. Cells were treated with vehicle (open bars) or three concentrations (10-10 M, 10-8 M, and 10-6 M (A) or 10-9 M, 10-7 M, and 10-5 M (B and C) of LG100268, 9cRA, TTNPB, 1,25(OH)2D3, or combinations thereof; luciferase activity was measured and normalized as described under "Experimental Procedures."
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24(OH)ase mRNA Is Induced by LG100268, TTNPB, 9cRA, or 1,25(OH)2D3 in Nontransfected CV-1 Cells: Combinations of Ligands Elicit Additive Effects

To determine if the additive effects of retinoids and 1,25(OH)2D3 in stimulation of the 24(OH)ase promoter observed in cotransfection assays were borne out in an endogenous setting, nontransfected CV-1 cells (which contain VDR, RARs, and RXRs; our data not shown) were treated with various concentrations of LG100268, TTNPB, 9cRA, or 1,25(OH)2D3, alone and in combination, and 24(OH)ase mRNA levels were assessed. Following ligand treatment for 6 h, cells were harvested, and total RNA was isolated and analyzed via Northern blotting with a rat 24(OH)ase cDNA probe (30). Fig. 7 illustrates that 1,25(OH)2D3 at 10-8 M (lane 3) or 10-7 M (lane 4) was an effective inducer of 24(OH)ase message levels (9.1- and 12.4-fold, respectively), while vehicle-treated cells expressed very low levels of 24(OH)ase RNA (lanes 1 and 2). LG100268 (lanes 5 and 6) and TTNPB (lanes 11 and 12) each at 10-7 and 10-6 M, also up-regulated RNA levels (2.1-3.3-fold and 4.1-5-fold, respectively). Combinations of 1,25(OH)2D3 and LG100268 yielded increased amounts (additive or superadditive) of 24(OH)ase RNA produced as compared with that resulting from cell treatment with identical concentrations of either ligand alone (compare lanes 3, 5, and 7; lanes 3, 6, and 8; lanes 4, 5, and 9; and lanes 4, 6, and 10). Similar effects were also observed with the combination of TTNPB and LG100268 versus either ligand alone (compare lanes 5, 11, and 13; lanes 6, 11, and 14; lanes 5, 12, and 15; and lanes 6, 12, and 16). The naturally occurring bifunctional retinoid, 9cRA, was also an efficacious inducer (9-10-fold) of 24(OH)ase RNA (lanes 17 and 18), an effect that was greater than that observed with either the RXR-selective (lanes 5 and 6) or the RAR-selective (lanes 11 and 12) ligand alone. 9cRA at 10 nM was as efficacious as 10 nM 1,25(OH)2D3 in induction of 24(OH)ase RNA levels in nontransfected CV-1 cells (compare lanes 3 and 17). Additionally, 9cRA led to increased levels of 24(OH)ase RNA in combination with 1,25(OH)2D3 (lanes 19 and 20). These data corroborate the information elucidated from the cotransfection/cotransactivation assays, since they indicate that endogenous 24(OH)ase production in kidney cells can be effected either by a retinoid alone or by 1,25(OH)2D3 alone at nanomolar concentrations. Furthermore, the data also show that combination treatment with retinoids and 1,25(OH)2D3 or two selective retinoids leads to increased levels of 24(OH)ase RNA, as was also demonstrated in the cotransfection assays. This information taken together with the DNA-binding data lead to the conclusion that ligand occupation of either or both receptor partners results in activation of 24(OH)ase, with increased levels of stimulation observed upon liganding of both receptors.


Fig. 7. Induction of 24(OH)ase RNA levels in CV-1 cells by treatment with retinoids and 1,25(OH)2D3. CV-1 cells were grown and treated with ligands for 6 h as described under "Experimental Procedures." Total RNA (15 µg) was analyzed via Northern blotting with a rat 24(OH)ase cDNA probe (B; Ref. 30). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Clonetech) was used to control for loading equivalency. Cell treatments were as follows: ethanol vehicle (V) (lanes 1 and 2); 10-8 M 1,25(OH)2D3 (lane 3); 10-7 M 1,25(OH)2D3 (lane 4); 10-7 M LG100268 (lane 5); 10-6 M LG100268 (lane 6); 10-8 M 1,25(OH)2D3 plus 10-7 M LG100268 (lane 7); 10-8 M 1,25(OH)2D3 plus 10-6 M LG100268 (lane 8); 10-7 M 1,25(OH)2D3 plus 10-7 M LG100268 (lane 9); 10-7 M 1,25(OH)2D3 plus 10-6 M LG100268 (lane 10); 10-7 M TTNPB (lane 11); 10-6 M TTNPB (lane 12); 10-7 M TTNPB plus 10-7 M LG100268 (lane 13); 10-7 M TTNPB plus 10-6 M LG100268 (lane 14); 10-6 M TTNPB plus 10-7 M LG100268 (lane 15); 10-6 M TTNPB plus 10-6 M LG100268 (lane 16); 10-8 M 9cRA (lane 17); 10-7 M 9cRA (lane 18); 10-7 M 9cRA plus 10-8 M 1,25(OH)2D3 (lane 19); 10-7 M 9cRA plus 10-7 M 1,25(OH)2D3 (lane 20). -Fold induction values versus vehicle average are indicated above each lane and depicted graphically in A.
[View Larger Version of this Image (37K GIF file)]


DISCUSSION

The experiments described here indicate that RXR-selective ligands are able to activate the human 24(OH)ase promoter in cotransactivation assays in COS-1 cells through binding to RXR, which has the ability to form heterodimers with either VDR or RAR. These heterodimers form on previously defined VDREs (22, 23), which as demonstrated here, also act as retinoic acid response elements. RXR is not observed to homodimerize on the retinoid-responsive sequences of the promoter as determined by electrophoretic mobility shift assays. Activation via the VDREs is achieved by specific ligands for either receptor, and the presence of both ligands leads to increased stimulation. Pan-agonists such as 9cRA lead to greater activation than either retinoid receptor-specific ligand alone. While we describe ligand-bound RXR interacting with VDR or RAR to activate this promoter, we cannot rule out the possibility of the occurrence of another partner for RXR in vivo, such as an orphan receptor (19, 20). However, the experiments in nontransfected CV-1 cells (Fig. 7) showing that a combination of saturating amounts of 1,25(OH)2D3 and an RXR ligand yields increased levels of 24(OH)ase RNA is difficult to reconcile with the involvement of another partner.

The formation of RXR·VDR and RXR·RAR heterodimers occurs with approximately equal affinity on each of the VDREs within the human 24(OH)ase promoter as determined by electrophoretic mobility shift assays. This was somewhat surprising, since the VDREs contain DR3 motifs that have been shown to be preferential binders of RXR·VDR heterodimers rather than other receptor combinations (33). However, Umesono et al. (33) used consensus DR sequences for their experiments. Upon comparison of consensus DR3-containing oligonucleotides and the nonconsensus 24(OH)ase VDREs, it was apparent that while the perfect DR3 sequence did have a higher affinity for RXR·VDR heterodimers than for RXR·RAR heterodimers, the human 24(OH)ase VDREs bound both heterodimer pairs with approximately equal affinity (Fig. 4). These VDRE sequences also confer responsiveness of the promoter to retinoids and 1,25(OH)2D3 in cotransactivation assays. Therefore, the VDREs within the human 24(OH)ase promoter also function as retinoic acid response elements.

The stimulation of the human 24(OH)ase promoter in COS-1 cell cotransfection/cotransactivation assays extends our previous work, which showed that retinoids, including an RXR-specific ligand, induced kidney 24(OH)ase RNA in mice within hours (27). This effect was observed in normally fed or vitamin D-deficient mice, implying that 1,25(OH)2D3 was not required for the activation by retinoids. We performed cursory promoter experiments in that report, which indicated that the activation by retinoids was not dependent on the 3' VDRE. Our present data show that while stimulation of the human 24(OH)ase (-316 to -22) promoter sequence by retinoids is retained with mutations in the 3' VDRE, it is diminished, and mutations in both VDREs essentially destroy the ability of retinoids to initiate transcription from the promoter. This work on the dissection of the human 24(OH)ase promoter in cotransfection/cotransactivation assays corroborates the effects previously observed in vitamin D-deficient and normally fed mice (27), i.e. an RXR-selective ligand is able to activate 24(OH)ase in the absence or presence of 1,25(OH)2D3.

The doses of 1,25(OH)2D3 that were administered to the mice maximized the induction of 24(OH)ase RNA, and the addition of LG100268 with 1,25(OH)2D3 had no effect on RNA levels at 32 h postdose (27). To test the effects of combinations of retinoids and 1,25(OH)2D3 at shorter times post dosing, we used CV-1 kidney cells as a model. CV-1 cells were found to produce 24(OH)ase RNA, and the levels of the RNA were modulated by 1,25(OH)2D3 and retinoids. 1,25(OH)2D3 induced 24(OH)ase RNA in CV-1 cells in a dose-dependent manner (Fig. 7 and data not shown). Each of the retinoids (LG100268, TTNPB, and 9cRA) tested also induced 24(OH)ase RNA in CV-1 cells. Combinations of 1,25(OH)2D3 with either LG100268 or 9cRA at saturating doses gave additive or superadditive increases in the amount of 24(OH)ase RNA that was produced. Additionally, 9cRA yielded a greater induction of RNA than either receptor-selective retinoid alone at the same concentrations. The combination of the two selective retinoids (LG100268 and TTNPB) also elicited an additive effect. Therefore, from these experiments it was demonstrated that while either a retinoid or 1,25(OH)2D3 alone induced 24(OH)ase RNA, the combination of both ligands gave an increased effect. Additionally, 1,25(OH)2D3 and 9cRA were approximately equipotent activators of 24(OH)ase in nontransfected CV-1 cells, since each ligand at 10-8 M gave a similar induction of RNA levels (9.1- and 9.4-fold, respectively).

We have concluded from the data described herein that 1,25(OH)2D3 and retinoids exert additive effects through RXR heterodimers at the VDREs within the 24(OH)ase promoter. To rule out the possibility that retinoids induce VDR or that vitamin D up-regulates retinoid receptors in these cells, CV-1 cells were treated for 6 h with 10 and 100 nM 9cRA or 1,25(OH)2D3 and 100 nM and 1 µM LG100268 (concentrations that gave effects in both analyses), and receptor levels were quantitated by ligand binding assays. Neither retinoid increased VDR levels, as assayed by specific binding of CV-1 cell extracts to tritiated 1,25(OH)2D3 (data not shown). Additionally, 1,25(OH)2D3 treatment of the cells did not increase RARs or RXRs as assayed by specific binding of the extracts to tritiated 9cRA (data not shown). Therefore, the additive effects of retinoids and 1,25(OH)2D3 via this promoter are not due to ligand-induced up-regulation of the receptor proteins.

Two other vitamin D target genes have also been shown to be regulated by retinoids: osteopontin (34, 35) and osteocalcin (36-38). Osteopontin RNA was induced in rats after a 4-h treatment with tRA regardless of vitamin A or D status and cooperated with 1,25(OH)2D3 to induce increased levels of osteopontin (34). Others have used a heterologous promoter containing two copies of the osteopontin VDRE to show differential effects of retinoids in cotransfection assays (35). Osteocalcin production has been shown to be stimulated by retinoids in primary human osteoblasts, and synergistic induction was observed with tRA and 1,25(OH)2D3 (37), although others have reported down-regulation of osteocalcin by 9cRA in cultured ROS17/2.8 osteosarcoma cells (36). Additionally, the human osteocalcin promoter has been shown to be stimulated by retinoids in cotransactivation assays in osteosarcoma cells through a sequence containing the VDRE (38). RXR ligands may have the ability to regulate a number of vitamin D (and thyroid hormone) target genes through perturbation of the structure of the heterodimer, which may lead to activation or repression. The potential for dual hormone regulation may depend on a number of factors including hormonal status of the organism, cellular receptor complement, promoter context, and the presence of specific receptor-interacting cofactors.

Interestingly, it has been shown that combinations of retinoids and vitamin D compounds have additive or synergistic effects in promoting apoptosis or growth inhibition in breast cancer cells (39), prostate cancer cells (40), and leukemia cells (41) and in growth inhibition and differentiation of leukemia cells (42-44). Therefore, lower concentrations of two ligands together may achieve efficacies that would require increased amounts of either compound alone. Clinically, combination therapy of retinoid and vitamin D analogues may potentially provide a drug treatment regimen that would exhibit a greater therapeutic index than either agent could achieve alone in diseases such as cancer and leukemia.


FOOTNOTES

*   The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed: Dept. of Retinoid Research, Ligand Pharmaceuticals, Inc., 10255 Science Center Dr., San Diego, CA 92121. Tel.: 619-550-7817; Fax: 619-550-7805; E-mail: ballegretto{at}ligand.com.
1   The abbreviations used are: RAR, retinoic acid receptor; RXR, retinoid X receptor; tRA, all-trans-retinoic acid; 9cRA, 9-cis-retinoic acid; VDR, vitamin D receptor; VDRE, vitamin D-responsive element; 24(OH)ase, 25-hydroxyvitamin D3-24-hydroxylase; 1,25(OH)2D3, 1,25-dihydroxyvitamin D3; kb, kilobase pair(s); PCR, polymerase chain reaction; DR, direct repeat.

ACKNOWLEDGEMENTS

We thank M. Boehm and L. Zhang for LG100268 and TTNPB, S. White for 9cRA, W. Lamph for pLUCpl, Y. Ohyama for rat 24(OH)ase cDNA, and J. W. Pike for anti-VDR 9A7gamma antibody.


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