Thyroid Hormone Receptor Does Not Heterodimerize with the Vitamin D Receptor but Represses Vitamin D Receptor-Mediated Transactivation

Mihali Raval-Pandya, Leonard P. Freedman, Hui Li and Sylvia Christakos

Department of Biochemistry and Molecular Biology (S.C., M.R.-P., H.L.) UMDNJ-New Jersey Medical School Newark, New Jersey 07103-2714
Cell Biology and Genetics Program (L.P.F.) Memorial Sloan-Kettering Cancer Center New York, New York 10021


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The 9,000 Mr calcium-binding protein calbindin-D9k (CaBP9k) is markedly induced by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] in mammalian intestine. However, although a vitamin D response element (VDRE) has been reported in the promoter of the rat CaBP9k gene (at -490/-472), the CaBP9k promoter is weakly transactivated by 1,25-(OH)2D3. Previous studies indicated that when MCF-7 cells are transfected with the rat CaBP9k VDRE ligated to the thymidine kinase promoter and treated with both 1,25-(OH)2D3 and T3 there is an enhancement of the response observed with 1,25-(OH)2D3 alone, suggesting direct cross-talk between thyroid hormone and the vitamin D endocrine system and activation via the formation of vitamin D receptor (VDR)-thyroid hormone receptor (TR) heterodimers. To determine whether the weak response of the rat CaBP9k natural promoter to 1,25-(OH)2D3 could be enhanced by T3, CaBP9k promoter/reporter chloramphenicol acetyltransferase constructs were transfected in MCF-7 cells, and the cells were treated with the two hormones alone or in combination. No induction with T3 alone and no enhancement of reporter activity in the presence of both hormones was observed. To determine whether a lack of effect by T3 was specific for the CaBP9k promoter and to further examine the possibility of cross-talk between the TR- and VDR-signaling pathways, the 1,25-(OH)2D3-responsive rat 24 hydroxylase [24(OH)ase] promoter and the rat osteocalcin VDRE (-457/-430), both fused to reporter genes were similarly examined in MCF-7 cells. Again, no enhancement of the response to 1,25-(OH)2D3 was observed in the presence of T3. In addition, a similar lack of response to T3 but responsiveness to 1,25-(OH)2D3 was observed when UMR106–01 osteosarcoma cells [which, like MCF-7 cells, express VDR, TR, and the retinoid X receptor (RXR) endogenously] were transfected with a 1,25-(OH)2D3 responsive mouse osteopontin promoter reporter. In vitro DNA binding assays were carried out using purified human VDR, human RXR{alpha}, and chick T3R{alpha} and 24(OH)ase, osteocalcin, osteopontin, and CaBP9k VDRE oligonucleotide probes. No VDR-TR heterodimer binding on any of these VDREs was observed, although, as expected, there was binding by the VDR-RXR complex and strong TR-RXR binding to a consensus thyroid hormone response element. Simultaneous gel retardation assays using similar and lower concentrations of TR with RXR showed strong binding of TR-RXR on a 32P-labeled thyroid response element. Studies using the yeast two-hybrid system also did not provide evidence for the formation of a VDR-TR protein-protein interaction. In addition, in vivo data showed that transfection of TR, in fact, repressed VDR-mediated transcription and that the repression could be reversed by the addition of RXR. Thus, in vitro and in vivo experiments do not support ligand-sensitive transactivation mediated by VDR-TR heterodimer formation but rather suggest that TR expression can repress 1,25-(OH)2D3-induced transcription predominantly by sequestering RXR.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Vitamin D is important for intestinal calcium absorption and for the development and maintenance of bone. In addition, vitamin D is involved in a number of diverse cellular processes including effects on cell proliferation and differentiation and on hormone secretion (1, 2). The genomic mechanism of action of the secosteroid hormone, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], the biologically active metabolite of vitamin D, is mediated by the vitamin D receptor (VDR) (3, 4), a member of the steroid/nuclear receptor super family. VDR, similar to the thyroid hormone receptor (TR) and the retinoic acid receptors (RARs), forms a heterodimeric complex with the retinoid X receptor (RXR) (5, 6, 7). This heterodimer binds to specific DNA sequences with high affinity and activates or represses the transcription of specific target genes (5).

Recent studies have begun to address the mechanisms involved in mediating transcription by these nuclear hormone receptors. It has been reported that VDR, similar to other nuclear receptors, can interact with the general transcription factors TFIIB and TFIIA, which may result in the recruitment of the basal components into the preinitiation complex (8, 9, 10, 11). In addition, TAFII135 has been reported to potentiate VDR as well as TR- and RAR-mediated transcription (12). Recent evidence has indicated that, in addition to the general transcription factors, another class of factors, known as coactivators, are needed for nuclear hormone receptor-mediated transcription. SRC-1 and its homologs have been reported to bind directly to VDR as well as to many other nuclear receptors (13, 14, 15) and a related protein, GRIP1, can potentiate VDR transactivation (16). Thus, heterodimerization of RXR with VDR, TR, and RAR, the interaction of the components of the preinitiation complex and coactivators with a number of nuclear hormone receptors, as well as some overlap in DNA recognition (17, 18, 19), suggest cross-talk of nuclear hormone receptor-signaling pathways. We are only now beginning to understand the numerous possibilities for interaction among different hormone systems and therefore for hormonal coregulation of genes. However, the physiological significance of many of the findings related to the convergence of the hormone-signaling pathways remains to be determined.

Although numerous genes have been reported to be regulated by 1,25-(OH)2D3, at this time only a small number have been reported to contain vitamin D responsive elements (VDREs) (2). The gene for 24(OH)ase, the enzyme thought to be involved in the catabolism of 1,25-(OH)2D3, is strongly transcriptionally responsive to 1,25-(OH)2D3 (20, 21). It is the first vitamin D-stimulated gene to be controlled by two independent VDREs [at -151/-137 and -259/-245 for the rat 24(OH)ase gene (20)]. VDREs have also been reported in the promoters for the genes encoding the cell cycle inhibitor p21, the bone proteins, osteocalcin (OC) and osteopontin (OP), and the calcium-binding proteins, calbindin-D28k (CaBP28k) and CaBP9k (2). CaBP28k and CaBP9k are vitamin D-dependent calcium-binding proteins that are thought to facilitate transcellular calcium transport primarily in mammalian kidney and intestine, respectively. Although a VDRE has been identified in the promoter of the CaBP9k gene (at -490/-472) (22), this promoter, as well as the CaBP28k promoter (23), is weakly transactivated by 1,25-(OH)2D3. Recent studies by Schräder et al. (24) indicated that when MCF7 cells were transfected with VDREtkCAT constructs and treated with T3 in combination with 1,25-(OH)2D3, a significant enhancement of the response observed with 1,25-(OH)2D3 alone was obtained, suggesting cross-talk between the thyroid hormone and the vitamin D endocrine system and activation by VDR-TR heterodimers. Understanding the cross-talk between VDR and TR would have physiological significance since it would suggest modulation by T3 of VDR-mediated transcription of target genes in tissues that express both receptors, such as intestine, bone, and pituitary (25, 26, 27). To further understand the potential interaction between TR and VDR, we examined whether the weak response of the CaBP9k promoter to 1,25-(OH)2D3 could be enhanced by T3. In addition, to determine whether cross-talk between T3 and the vitamin D endocrine system may be target gene specific, natural promoter/chloramphenicol acetyltransferase (CAT) constructs for rat 24(OH)ase and mouse OP as well as the rat OC VDRE (-457/-430) thymidine kinase (tk)CAT construct were similarly examined. Using both the natural promoters and the VDRE CAT construct, we consistently found no enhancement of the 1,25-(OH)2D3 response in the presence of T3. Studies using gel retardation assays as well as the yeast two-hybrid system also did not provide evidence for the formation of VDR-TR heterodimers. However, transfection of TR was found to repress VDR- mediated transactivation, and this inhibition was reversed by the addition of RXR. These findings suggest that cross-talk between the VDR- and TR-signaling pathways can occur indirectly due to sequestration by TR of RXR rather than by direct heterodimerization of VDR and TR, and in this sense TR may have an important role in modulating VDR-mediated transactivation.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The Rat CaBP9k Promoter Is Weakly Transactivated by 1,25-(OH)2D3
In mammalian intestine one of the most pronounced effects of 1,25-(OH)2D3 is increased synthesis of CaBP9k (2). However, when the CaBP9k promoter CAT constructs -1009/+61 and -580/+61 (which contain the previously identified VDRE at -490/-472) were cotransfected with human VDR (hVDR) in CV1 cells and treated with 1,25-(OH)2D3, weak transactivation was observed (Fig. 1AGo). Under the same conditions, a 21-fold and a 15-fold induction in CAT expression was detected using the rat 24(OH)ase promoter construct (-1367/+74, containing VDREs at -259/-245 and -151/-139) and the OC VDRE (-457/-430) tkCAT construct, respectively (Fig. 1AGo). To test the possibility that cell-specific factors may be important for 1,25-(OH)2D3-induced transactivation of the CaBP9k gene, CaBP9k promoter CAT constructs were transfected in MDBK bovine kidney cells and in Caco-2 human intestinal cells that contain endogenous VDR (28, 29). Weak responsiveness of the rat CaBP9k promoter to 1,25-(OH)2D3 was again observed (Fig. 1BGo). Similar results were observed when VDR levels were increased above endogenous levels by transfection of the VDR expression plasmid pAVhVDR in MDBK or Caco-2 cells (not shown). Although the CaBP9k promoter was weakly responsive to 1,25-(OH)2D3, when the CaBP9k promoter CAT construct -1009/+106, containing the reported functional estrogen- responsive element at +50/+65 (30), was cotransfected with the hER expression vector (pSG5-HEO) in T47-D breast cancer cells, an induction in CAT activity (up to 6-fold) was observed after estradiol treatment (10-9-10-7 M) (not shown).



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Figure 1. Weak Responsiveness of the CaBP9k Promoter to 1,25-(OH)2D3

A, CV1 cells were cotransfected with 6 µg of the VDR expression vector pAVhVDR and 4–6 µg CAT reporter plasmid containing the CaBP9k gene constructs (-590/+61 or -1009/+61), the rat 24(OH)ase promoter construct (-1367/+74) or multiple copies of the rat OC VDRE (-457/-430). Transfected cells, incubated in hormone-stripped medium, were either vehicle treated (Basal) or treated with 10-7 M 1,25-(OH)2D3 (cells transfected with CaBP promoter constructs) or with 10-8 M 1,25-(OH)2D3 (cells transfected with the rat 24(OH)ase promoter construct or with the OC VDRE tkCAT construct) for 24 h. After treatment, cells were harvested and lysed and CAT activity, measured as described in Materials and Methods, was normalized to ß-galactosidase activity. B, MDBK bovine kidney cells and Caco-2 human intestinal cells were transfected with CaBP9k promoter CAT constructs (-590/+61 or -1009/+61). Transfected cells were either vehicle treated (Basal) or treated with 10-7 M 1,25-(OH)2D3 (+D) for 24 h. For panels A and B the mean CAT activity ± SE (n = 4–6 observations per group) was quantitated by comparison to basal levels. In CV1 cells (A) the 24(OH)ase promoter and the OC VDRE were significantly responsive to 1,25-(OH)2D3 (P < 0.01). The CaBP9k promoter construct (-1009/+61) was induced 1.6-fold by 1,25-(OH)2D3 in CV1 cells (A) and in Caco-2 intestinal cells (B) (P < 0.05).

 
1,25(OH)2D3-Mediated Transactivation Is Not Enhanced by T3
Previous studies by Schräder et al. (24, 31) indicated when MCF-7 cells (which contain endogenous VDR and TR) or the Drosophila cell line SL-3 transfected with VDR and TR, was transfected with different VDREs, including the CaBP9k VDRE, an enhancement of the response observed with 1,25-(OH)2D3 alone was observed in the presence of T3 + 1,25-(OH)2D3, suggesting activation via VDR-TR heterodimers and cross-talk between thyroid hormone and the vitamin D endocrine system. To determine whether the weak response of the CaBP9k promoter to 1,25-(OH)2D3 could be enhanced by T3, the CaBP9k promoter CAT construct -1009/+61 was transfected in MCF-7 cells, and the cells were treated with 1,25-(OH)2D3 (10-7 M), T3 (10-7 M) or 1,25-(OH)2D3 + T3 for 24 h. No induction with T3 alone and no enhancement of CAT activity in the presence of both hormones was observed (Fig. 2Go). Similar results were obtained using the CaBP9k promoter CAT construct -590/+61 (data not shown). To determine whether a lack of effect of T3 was specific for the CaBP9k promoter, the rat OC VDRE (-457/-430) tkCAT construct and the rat 24(OH)ase promoter construct -1367/+74, which are strongly responsive to 1,25-(OH)2D3 (Fig. 2Go), as well as the CaBP28k promoter construct -390/+34 (VDRE at -200/-169) (not shown), were similarly examined in MCF-7 cells. Using these constructs, under these conditions, neither an activation with T3 nor an enhanced response in the presence of both ligands was observed (Fig. 2Go). In addition, a similar lack of response to T3 but responsiveness to 1,25-(OH)2D3 was also observed when a mouse OP promoter CAT construct (-777/+79) containing the mouse OP VDRE (-757/-743) was transfected in UMR106–01 osteosarcoma cells, which, like MCF-7 cells, express VDR, TR, and RXR endogenously (not shown). As a positive control the thyroid hormone response element (TRE) inverted repeat AGGTCATGACCT CAT construct was responsive to T3 in these cells (Fig. 2Go).



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Figure 2. Neither Transcriptional Activation with T3 Alone nor a T3-Mediated Enhancement of 1,25-(OH)2D3-Induced Transcription Is Observed

MCF-7 cells were transfected with reporter CAT constructs containing the indicated vitamin D-responsive promoter OC VDRE tkCAT or a TRE-inverted repeat CAT construct (MTV-TREpCAT; TRE). Transfected cells were either vehicle treated (Basal) or treated with 10-8 M 1,25-(OH)2D3 (+D), 10-7 M T3, or a combination of 1,25-(OH)2D3 and T3 for 24 h. Cells transfected with MTV-TREpCAT were treated only with T3 as a positive control. Results of six to eight independent experiments showed similar results. CAT activity in cells transfected with reporter CAT constructs CaBP9k -1009/+61, rat 24(OH)ase 1367/+74, or OC VDRE tkCAT and treated with T3 alone was not significantly different than basal (P > 0.1). In the presence of T3 + D, CAT activity was not significantly different than results obtained in the presence of D alone (P > 0.1).

 
Gel Retardation Analysis Does Not Provide Evidence for VDR-TR Heterodimeric Binding on VDREs
To determine whether VDR-TR heterodimer formation on DNA occurs in vitro, gel retardation analyses were performed using purified receptors. As can be seen in Fig. 3Go, there was strong binding of the TRE inverted repeat by the TR-RXR complex. However, VDR-TR heterodimer binding on the rat 24(OH)ase VDRE could not be demonstrated, although a prominent shifted complex was observed in the presence of VDR and RXR. Similar observations were made when the hOC VDRE, the mouse OP VDRE, and the rat CaBP9k VDRE were used for gel retardation assays (Fig. 3Go). Note in Fig. 3Go, as previously reported (32, 33), purified VDR can bind to the OP VDRE as a homodimer in the absence of RXR. Thus, for the OP VDRE, the band of retarded mobility observed in the presence of VDR/TR comigrates with the band observed in the presence of VDR alone, suggesting that it is due to VDR homodimerization. The binding observed in the presence of VDR/RXR to the CaBP9k VDRE was weak compared with VDR/RXR binding to the 24(OH)ase, OC, and OP VDREs. Results of these gel retardation assays, which do not provide evidence of binding of VDR-TR heterodimer to VDREs, are consistent with the lack of functional activity (Fig. 2Go).



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Figure 3. Gel Retardation Analysis Does Not Indicate VDR-TR Heterodimer Binding on VDREs

Gel shift experiments were carried out with purified hVDR, cTR{alpha}, and FLAG-hRXR{alpha} and 32P-labeled VDREs as described in Materials and Methods. First panel (TRE): Labeled probe was incubated with 10, 20, or 30 ng cTR{alpha} and 6.5 ng FLAG-hRXR{alpha}. Second panel (24(OH)ase): VDRE-Labeled probe was incubated with 20 ng VDR alone or 10 and 20 ng VDR in combination with 6.5 ng FLAG-hRXR{alpha} or 20 ng VDR in combination with 30 ng cTR{alpha}. Third panel (OC VDRE): Labeled probe was incubated with 10 ng VDR alone or 10 and 20 ng VDR in combination with 6.5 ng FLAG-hRXR{alpha} or 20 ng VDR in combination with 20 and 30 ng cTR{alpha}. In the last two lanes experiments were done in the presence of 20 ng VDR and 6.5 ng FLAG-hRXR{alpha}, labeled probe, and 100 fold molar excess cold unlabeled OC VDRE or TRE. The complex formed in the presence of VDR and RXR was competed with OC VDRE but not with TRE. Although the complex formed in the presence of cold TRE appears less intense than the complex formed in the absence of cold TRE, there was no significant difference in intensity when results of three separate gel shift experiments were compared. Fourth panel (OP VDRE): Labeled probe was incubated with the same amount of purified receptor protein used for gel shift analysis of binding to OC VDRE (third panel). In the last two lanes experiments were done in the presence of 20 ng VDR and 6.5 ng FLAG-hRXR{alpha}, labeled probe, and 100 fold molar excess cold unlabeled OP VDRE or TREp. Note as previously reported (32 33 ) purified VDR can bind to the OP VDRE in the absence of RXR, and the band of retarded mobility observed in the presence of VDR/TR comigrates with the band observed with VDR alone. Also note, the complex formed in the presence of VDR and RXR was competed with OP VDRE but not with TRE. Fifth panel (CaBP9k VDRE): Labeled probe was incubated with 10 and 20 ng VDR alone or 10 ng VDR in combination with 6.5 ng FLAG-hRXR{alpha} or 20, 50, and 100 ng VDR in combination with 13 ng FLAG h-RXR{alpha} or 100 ng VDR in combination with 30 ng cTR{alpha}. The complex formed in the presence of 100 ng VDR and 13 ng RXR{alpha} could be competed with 100-fold excess cold CaBP9k VDRE (not shown). Similar findings were observed under the same conditions in two additional gel shift experiments.

 
VDR Does Not Interact with TR in a Yeast Two-Hybrid Assay
A yeast two-hybrid assay was established in our laboratory to identify proteins that interact with VDR in vivo. One hybrid construct contained the GAL4 DNA-binding domain in pGBT9 fused to the ligand-binding domain of VDR (93–427; GBT9VDR). As a control interacting protein, a second hybrid construct contained the GAL4 activation domain in pGAD424 fused to the ligand-binding domain of hRXR{alpha} (223–462; GADRXR{alpha}). TR fusion was generated by ligating chick TR{alpha} (cTR{alpha}) (119–438) to the GAL4 DNA-binding domain as well as to the GAL4 activation domain (GBT9TR and GADTR, respectively). Interaction was characterized by monitoring ß-galactosidase expression and growth on selective media lacking tryptophan (Trp) and leucine (Leu). To characterize the specificity of the two-hybrid assay, as negative controls, the yeast strain SFY56 was transformed with GBT9VDR and the empty vector pGAD424 or with pGBT9 empty vector and GADRXR{alpha}. No ß-galactosidase staining was observed (Fig. 4AGo, VDR-pGAD and pGBT9-RXR). As positive controls, GBT9VDR and GADRXR{alpha}, as well as GBT9TR and GADRXR{alpha}, were transformed (see Fig. 4AGo, VDR-RXR and TR-RXR, note positive interaction). The interaction between VDR and TR was tested by simultaneous transformation of GBT9VDR and GADTR. Although the VDR-RXR and TR-RXR interactions were positive for ß-galactosidase, no interaction between VDR and TR was observed in either orientation (Fig. 4AGo; VDR-TR and TR-VDR). The transformation was repeated in yeast strain Y190 where growth on plates lacking Leu, Trp, and histidine (His) is dependent on protein interactions. When lysates were assayed for ß-galactosidase activity, similar results, indicating an interaction of VDR and TR with RXR but not an interaction between VDR and TR, were obtained (Fig. 4BGo).



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Figure 4. VDR Does Not Interact with TR in a Yeast Two-Hybrid System

A, Various two-hybrid combinations were examined in the yeast strain SFY526, and interaction in the presence of ligand was characterized by ß-galactosidase expression and growth on Trp and Leu-deficient media containing 10-7 M T3 and 10-8 M 1,25-(OH)2D3. Specific interaction is observed between TR and RXR and between VDR and RXR but not between empty vector (pGBT9) and RXR or between empty vector (pGAD) and VDR (negative controls). An interaction was also not observed between VDR and TR. B, Transformation was repeated in yeast strain Y190. Relative growth on Trp-, Leu-, and His-deficient plates containing 10-7 M T3, 10-8 M 1,25-(OH)2D3, and 30 mM 3-aminotriazole was assessed after 4 days, and ß-galactosidase expression was quantitated in liquid cultures as described in Materials and Methods. Similar results were observed for VDR and TR in the reverse orientation (not shown). Results are presented as mean (±SE) of triplicate cultures from two separate experiments.

 
TRs Repress VDR-Mediated Transcriptional Activation
Although in vivo and in vitro studies did not provide evidence for the formation of VDR-TR heterodimers, we further investigated the possibility of cross-talk between the TR and VDR hormone-signaling pathways by examining the effect of overexpression of TR{alpha} on VDR-mediated transactivation. A constant amount of VDR expression vector, which conferred close to maximal induction of CAT activity, was cotransfected in CV1 cells with 1 µg cTR{alpha} expression vector and either the 24(OH)ase -1369/+74 promoter CAT construct (Fig. 5AGo) or the OC VDRE tkCAT construct (Fig. 5BGo). In the presence of 1 µg cTR{alpha} expression vector, 1,25-(OH)2D3-induced transactivation was completely inhibited (Fig. 5Go). However, at this concentration of pEX-cTR{alpha}, 1,25-(OH)2D3- induced transactivation was not completely inhibited in the presence of T3. In the presence of 1,25-(OH)2D3 (10-8 M) and T3 (10-7 M), a 1.6- to 2.5-fold increase in CAT activity above basal levels was observed in CV1 cells transfected with pAVhVDR, 1 µg pEX-cTR{alpha}, and the rat 24(OH)ase CAT construct or OC VDREtkCAT (not shown). Experiments done with both the 24(OH)ase and the OC VDRE CAT reporter plasmids using higher concentrations of cTR{alpha} (3, 6, and 9 µg) also resulted in complete inhibition of VDR-mediated transactivation.



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Figure 5. TR Represses VDR-Mediated Transcriptional Activation

CV1 cells were cotransfected with CAT reporter plasmid [6 µg rat 24(OH)ase promoter construct -1367/+74 (A) or 4 µg rat OC VDRE tkCAT (B)] and 6 µg pAVhVDR (first two lanes; VDR). Cells transfected with reporter plasmid and pAVhVDR were also transfected with 1 µg pEX-cTR{alpha} (last two lanes; VDR+TR). The cells were incubated for 24 h in hormone-stripped medium without or with 1,25-(OH)2D3 (10-8 M). CAT activity was assayed as described in Materials and Methods using TLC. Similar results were observed in a second experiment using 1 µg cTR{alpha} expression vector.

 
Coexpression of RXR Alleviates TR-Dependent Repression
Dose-dependent inhibition of VDR-mediated transcription using lower concentrations of cTR{alpha} expression vector (0.1–0.5 µg) is shown in Fig. 6Go (left panel). pEXPRESS vector alone (0.5 µg) had no effect on 1,25-(OH)2D3- induced transactivation (not shown). Since it had previously been suggested that TR isoforms can repress transcription by the formation of heterodimers (34), we investigated the possibility that the repression by TR is mediated by heterodimer formation by cotransfection experiments in the presence of RXR{alpha}. In the presence of 2 µg hRXR{alpha} expression vector, the TR-negative effect on 1,25-(OH)2D3 mediated transcription could be reversed (Fig. 6Go). After cotransfection of a cTR{alpha} mutant that lacks the DNA-binding domain (1–120), inhibition of 1,25-(OH)2D3-dependent CAT activity was still observed (Fig. 6Go, left panel), suggesting that inhibition by TR does not primarily involve contact with DNA. Reversal of repression by 0.5 µg pEX-cTR{alpha} of 1,25-(OH)2D3- induced 24(OH)ase transcription was also observed using lower concentrations of hRXR{alpha} (0.25, 0.5, and 1 µg; not shown). At higher concentrations of pEX-cTR{alpha} (1 µg), 2 µg hRXR{alpha} expression vector also resulted in an 80% reversal of the repression of 1,25-(OH)2D3-induced 24(OH)ase transcription. However, at 1 µg pEX-cTR{alpha}, cells transfected with the OC VDREtkCAT construct were less sensitive to reversal of repression by RXR{alpha} (not shown). Repression of 1,25-(OH)2D3-induced transcription was also observed when cTR{alpha} expression vector and the OP promoter CAT construct (-777/+79) were cotransfected in UMR osteoblastic cells that contain endogenous VDR [relative CAT activity (fold induction compared with basal) in the presence of 1,25-(OH)2D3 (10-8 M) and in the absence of pEX-cTR{alpha} = 5.6 ± 0.7. In the presence of 0.25 µg pEX-cTR{alpha}, 1,25-(OH)2D3 induction of OP transcription was reduced to 1.6 ± 0.1 fold (P < 0.01). In the presence of 0.25 µg cTR{alpha} and 2 µg hRXR{alpha}, 1,25-(OH)2D3-induced transcription of the OP gene 4.2 ± 0.6 fold (n = 4–5)]. These findings, obtained using the 24(OH)ase, OP, and OC CAT reporter plasmids suggest that TR exerts its effect predominantly by sequestering RXR and reducing the available RXR needed to heterodimerize with VDR, thereby resulting in repression of 1,25-(OH)2D3-induced transactivation.



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Figure 6. Dose-Dependent Repression of VDR-Mediated Transcription by TR and Reversal of TR-Dependent Repression by RXR

Left panel, CV1 cells were cotransfected with CAT reporter plasmid [6 µg rat 24(OH)ase promoter construct -1367/+74 (top) or 4 µg rat OC VDRE thCAT (bottom)], 6 µg VDR expression vector pAVhVDR, and increasing amounts of pEX-cTR{alpha} (0.1 ng-0.5 µg). In the presence of 0.5 µg cTR{alpha} or 0.25 µg cTR{alpha} expression vector, cells transfected with the 24(OH)ase promoter CAT construct or OC VDREtkCAT, respectively, were also transfected with 2 µg hRXR{alpha} expression vector (RXR{alpha}). Cells transfected with CAT reporter plasmids and pAVhVDR were also transfected with 0.5 mg pEX-DBD-cTR{alpha} (mutant pEX-cTR{alpha}), which directs the expression of cTR{alpha} with the DNA-binding domain deleted. The cells were incubated in the presence or absence of 1,25-(OH)2D3 (10-8 M), and CAT activity was normalized to ß-galactosidase activity. Results, reported as fold induction (mean CAT activity ± SE; three to four observations per group) were quantitated by comparison to untreated transfected cells. Right panel, Representative autoradiograms: The rat 24(OH)ase promoter CAT construct (top) was cotransfected in CV1 cells with the following expression plasmids: 6 µg VDR and 0.5 µg cTR{alpha} (VDR+TR) or 6 µg VDR, 0.5 µg cTR{alpha}, and 2 µg hRXR{alpha} (VDR+TR+RXR). The cells were incubated in the presence or absence of 1,25-(OH)2D3 (10-8 M). The OC VDRE CAT construct (bottom) was cotransfected in CV1 cells with the following expression plasmids: 6 µg VDR and 0.25 µg cTR{alpha} (VDR+TR) or 6 µg VDR, 0.25 µg cTR{alpha}, and 2 µg RXR (VDR+TR+RXR). The cells were incubated in the presence or absence of 1,25-(OH)2D3 (10-8 M).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Findings from our in vitro and in vivo experiments do not support ligand-sensitive transactivation mediated by VDR-TR heterodimer formation. Similar to our findings, Yen et al. (35) also indicated that gel retardation assays did not provide evidence for the formation of VDR/TR heterodimers on different hormone response elements (direct repeats with three, four, or five nucleotide gaps or the F2 TRE) as well as on the CaBP9k and CaBP28k VDREs. Our observations differ from the findings reported by Schräder et al. (24, 31), who proposed TR-VDR heterodimerization and showed, using VDRE tkCAT constructs, that in the presence of T3 there is a further enhancement of the response observed with 1,25-(OH)2D3 alone. Differences in the results we observed and those reported by Schräder et al. (24, 31) may be due to the use of natural promoters in most of our functional studies as well as the performance of gel retardation assays in our laboratory using purified receptors rather than in vitro translated proteins. Experiments by Schräder et al. (24, 31) were carried out in MCF-7 cells as well as in the Drosophila cell line SL-3 transfected with VDR and TR. Although differences in cell type, constructs, and protocols may account for some of the differences observed, our data nevertheless invariantly and clearly indicate that RXR, and not TR, forms heterodimers with VDR.

Although our data do not support direct heterodimerization of VDR and TR, our findings do demonstrate that cross-talk between the vitamin D and T3 signaling pathways can intersect indirectly through competition by VDR and TR for a common limiting partner, namely RXR. This results in an attenuation of 1,25-(OH)2D3-induced transactivation when TR is overexpressed. In addition to repressing VDR-mediated transcription, TR has also been reported to block transcription mediated by other nuclear receptors (36, 37, 38, 39, 40, 41, 42, 43, 44, 45). Different mechanisms have been proposed to explain TR’s function as a repressor. One mechanism involves competitive DNA binding (36, 41). TR can also reduce hormone-dependent gene activation by receptors that interact with RXR by titrating out the common partner RXR, resulting in modulation and diversity of hormone responses (38, 39, 40). A third mechanism proposed for TR repression involves the direct interaction of unliganded TR with a nuclear receptor corepressor (43, 44) or with general initiation factors (45, 46). Since RXR was found to reverse the TR-negative effect on 1,25-(OH)2D3-mediated transcription and TR lacking its DNA-binding domain was also found to be an effective inhibitor (Fig. 6Go), our findings suggest that the mechanism of TR repression of VDR-mediated transcription is predominantly due to sequestration by TR of RXR, similar to the mechanism proposed for TR inhibition of RAR- and RXR-dependent activation of an RARE or an RXRE, respectively (38, 40). Modulation of VDR-mediated transcription by TR depends on the concentration of VDR, TR, and RXR and the dimerization status of RXR. In addition to sequestration of RXR by TR, it is possible that part of the TR inhibition of the 1,25-(OH)2D3 dependent transcriptional response may also involve other mechanisms. For example, since both VDR and TR interact with transcription factor TFIIB (8, 9, 10), unliganded TR may act as a transcriptional silencer in part by binding to TFIIB, preventing the formation of the preinitiation complex and precluding TFIIB from associating with liganded VDR. Further studies are needed to determine additional mechanisms that may be involved in TR silencing of VDR-mediated transcription.

VDR-TR cross-talk was also suggested in recent studies by Yen et al. (35), which indicated that in the presence or absence of 1,25-(OH)2D3, VDR can repress T3-mediated transcription. The mechanism proposed for VDR-mediated repression was competition by transcriptionally inactive VDR/RXR heterodimers with TR/RXR for TRE binding as well as titration of a common associated protein or coactivator(s) important for T3-mediated transcription. A VDR DNA binding mutant had some dominant negative activity, which was less than the dominant negative activity of wild-type VDR. Unlike our findings, the common associated protein did not appear to be RXR since cotransfection of RXR did not reverse VDR dominant negative activity. Thus, VDR-TR cross-talk can occur by VDR inhibition of T3 transactivation as well as by TR repression of VDR- mediated transcription.

In this study TR-mediated repression of VDR-mediated transcription was observed by examining the transcription of the 24(OH)ase, OP, and OC genes. We were unable to similarly examine VDR-TR cross-talk in studies related to CaBP9k gene transcription due to the weak responsiveness of the CaBP9k promoter to 1,25-(OH)2D3. Although one of the most pronounced effects of 1,25-(OH)2D3 in mammalian intestine is increased synthesis of CaBP9k, we found that the CaBP9k promoter is consistently weakly transactivated by 1,25-(OH)2D3. The weak transcriptional response reflects in vivo studies indicating that 1,25-(OH)2D3 induces the expression of the CaBP gene by a small rapid transcriptional stimulation followed by a large accumulation of calbindin mRNA long after 1,25-(OH)2D3 treatment (47, 48). Thus, the large induction of CaBP9k mRNA may be due primarily to posttranscriptional mechanisms. Our findings do not indicate that the presence of thyroid receptors and T3 can enhance this weak transcriptional responsiveness. Recent studies using transgenic mice have shown that responsiveness to 1,25-(OH)2D3 in the duodenum is contained in a 4.4-kb CaBP9k promoter construct and that a proximal element (from -117 to +400) as well as a distal element (from -3741 to -2894), together but not separately, can confer a 1,25-(OH)2D3-induced response (49, 50). Thus, the control of the transcription of the CaBP9k gene by 1,25-(OH)2D3 appears to be complex and may involve nonclassical pathways as well as specific intestinal transacting factors. Unlike studies related to the regulation of CaBP, studies concerning the regulation by 1,25-(OH)2D3 of OC and OP have resulted in the most information concerning transcriptional activation by 1,25-(OH)2D3 (2), and the 24(OH)ase gene is the gene that is most transcriptionally responsive to 1,25-(OH)2D3 (20). It is likely that further studies using the OC, OP, and 24(OH)ase genes, in which VDREs that contribute to 1,25-(OH)2D3 responsiveness in their natural promoters have been identified, will be important to better understand not only how T3, but also how other hormones and signaling pathways, are involved in the modulation of transcriptional regulation by 1,25-(OH)2D3.

1,25-(OH)2D3 has been reported to be involved in a number of diverse cellular processes (1, 2), and receptors for 1,25-(OH)2D3 have been identified not only in tissues involved in maintaining mineral homeostasis but also in many other tissues, including pancreas, thyroid, brain, and pituitary (51). Thus, cross-talk between VDR and TR may have significance in a number of different physiological systems. Previous reports have suggested cross-talk between vitamin D and thyroid hormone in studies in pituitary cells (GH4C1) as well as in bone. Receptors for 1,25-(OH)2D3 have been reported in pituitary (51, 52) where high levels of thyroid receptors are expressed (53). 1,25-(OH)2D3 was found to cause a dose-dependent down-regulation of TRs in GH4C1 pituitary cells and to block GH induction by T3 in these cells (27). It is possible that to increase GH synthesis and secretion, high levels of TR found endogenously in the pituitary would inhibit the action of 1,25-(OH)2D3 perhaps, as suggested by our studies, by sequestering RXR. The presence of T3 would then result in enhanced TR-mediated transcription of the GH gene. In bone, both T3 and 1,25-(OH)2D3 have been reported to be important for normal skeletal development and to affect bone turnover (2, 54, 55). Vitamin D deficiency is associated with rickets (56), and syndromes of resistance to thyroid hormone are associated with delayed skeletal maturation as well as growth retardation (57). Although osteoblasts are established target cells for 1,25-(OH)2D3 action and 1,25-(OH)2D3 responsive genes, including OP and OC, have been identified in osteoblasts (2), the molecular basis for T3 action in bone is less clearly defined, and genes responsive directly to T3 in bone have not been definitively identified. Previous studies indicated the presence of thyroid receptors in bone (58, 59). Recent studies by Williams et al. (26) reported the existence of different expression levels of TR{alpha}1 and TRß1 in three osteosarcoma cell lines (ROS25/1, UMR106, and ROS17/2.8) that express fibroblast-like, preosteoblast, and mature osteoblast phenotypes, respectively. The authors also studied the effect of hormones (1,25-(OH)2D3, T3, 9-cis-RA, and RA) on the induction of osteoblast phenotypic genes in these cells (60). Similar to our findings, under conditions in which 1,25-(OH)2D3 induced the expression of a target gene such as OC or OP, T3 was not found to have a significant additive effect over the effect observed with 1,25-(OH)2D3 alone, also suggesting lack of evidence for transactivation via a VDR/TR heterodimer. In addition to studies with cell lines, it will be of interest in future studies to determine the relative levels of VDR, TR, and RXR in bone at different stages of skeletal development to determine whether the concentration of the different receptors provides a molecular basis for the different action of the respective hormones at the various stages of development. Perhaps at different developmental stages when TR is overexpressed compared with VDR, not only in bone but also in other tissues, TR may have a repressor function preventing activation of 1,25-(OH)2D3 target genes under physiological conditions that do not require them to be expressed. In summary, although the physiological significance of VDR-TR cross-talk is speculative at this time, our findings nevertheless suggest an indirect interaction between TR- and VDR-signaling pathways that is dependent in part on the concentration of the receptors and modulation of the regulation of the expression of 1,25-(OH)2D3 target genes by competition for a common dimerization target.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
32P-radiolabeled and [14C]chloramphenicol (50 mCi/mmol) were from DuPont/New England Nuclear Corp (Boston, MA). DNA restriction and modifying enzymes as well as media for cell culture were obtained from GIBCO/BRL-Life Technologies (Gaithersburg, MD). Sera for cell culture were purchased from Gemini (Calabasas, CA). Oligonucleotides were synthesized by the UMD Molecular Resource Facility (Newark, NJ). The plasmids and yeast strains comprising the two-hybrid system as well as the yeast YPD medium (2% peptone, 1% yeast extract, 2% glucose) and synthetic minimal medium were from CLONTECH (Palo Alto, CA). 1,25-(OH)2D3 was a generous gift from Dr. M. Uskokovic (Hoffmann-La Roche. Co., Nutley, NJ). Acetyl-coenzyme A and T3 as well as general regents of molecular biology grade were purchased from Sigma Chemical Co. (St. Louis, MO).

Plasmids Used for Mammalian Cell Transfection
CaBP9k Promoter CAT Constructs
A rat genomic {lambda}gt11 library (CLONTECH) was screened using a 170-base CaBP9k cDNA (61) as well as a synthetic 42-base cDNA probe starting from the initiation site of the rat calbindin-D9k gene (62). EcoRI-PstI fragments of phage DNA from positive plaques were subcloned into the SmaI site of pBluescript SK (+) vector and sequenced by the dideoxynucleotide chain termination method (63). EcoRI-AvrII fragments in pBluescript SK(+) coordinated ~1.1 kb (-1009/+106) of the 5'-flanking region of the rat calbindin-D9k gene. This region is identical to the rat calbindin-D9k sequence -1009/+106 previously reported (22, 62). The 3'-end deletion mutant (+61 from the cap site) was obtained using the PCR. For the 3'-end deletion two 20-bp primers corresponding to region -1009/-989 of the rat CaBP9k promoter and to +42/+61 of its complementary strand were synthesized and used for amplification by PCR using the Gene Amp PCR Reagent kit (Perkin-Elmer Corp., Norwalk, CT). The 5'-deletion mutant -590/+61 was generated using the restriction endonuclease SspI. The CaBP9k gene construct -1009/+106 and deletion mutants (-1009/+61 and -590/+61), both containing the reported VDRE (-489/-445) (22), were fused at the SmaI site of pHCAT (which is derived from pSV2 CAT by deleting the simian virus 40 promoter; pHCAT was a gift from M. Tocci, Merck, Sharpe and Dohme). Insertion and orientation of inserts were confirmed by DNA sequencing.

Other Plasmids
The plasmid OC VDREtkCAT, previously described (64), contains multiple copies of the VDRE (-457/-430) found in the rat OC gene. The mouse OPN promoter CAT construct spans nucleotides -777/+79 (65) and was generously provided by Dr. D. Denhardt (UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ). The rat 24(OH)ase promoter CAT construct -1367/+74 phCAT was sent to us by Dr. John Omdahl (University of New Mexico, Albuquerque, NM) (20). The pAVhVDR directs expression of the full- length human VDR (66) and was a gift from J. W. Pike (University of Cincinnati, Cincinnati, OH). pCMX-hRXR{alpha} was provided by Drs. Ronald Evans (Salk Institute of Biological Sciences, San Diego, CA) and David Mangelsdorf (University of Texas Southwestern Medical Center, Dallas, TX). The hER expression vector (pSG5-HEO) was a gift from Dr. P. Chambon (Institut de Genetique et de Biologie Moleculaire et Cellulaire, Strasbourg, France). MTV-IR-CAT contains one copy of the inverted repeat AGGTCA TGACCT with no nucleotide gap cloned upstream of a mouse mammary tumor viral long-terminal repeat-CAT vector [referred to as IR or TREp (67)]. This construct as well as pEXPRESS-cTR{alpha} (or pEX-cTR{alpha}), which efficiently directs the expression of full-length chick TR (1–408) (68), and pEX-DBD-cTR{alpha} (120–408), which directs the expression of cTR{alpha} with DNA-binding domain deleted, were provided by Dr. Herbert Samuels (New York University Medical Center, New York, NY). The pCH110 plasmid (Pharmacia, Piscataway, NJ) contains the LacZ gene, which encodes ß-galactosidase. This expression vector was used to normalize for variation in transfection efficiency.

Mammalian Cell Culture Cell Transfection and CAT Assays
The African green monkey kidney cell line CV1, as well as Caco-2 cells (human colon carcinomas), MDBK cells (Madin Darby bovine kidney cells), and MCF-7 cells (human breast cancer cells) were obtained from ATCC (Rockville, MD) and maintained in DMEM supplemented with 10% FBS. Caco-2 cells are also supplemented with bovine insulin (10 ng/ml). For experiments examining CaBP9k transcription, MDBK cells and Caco-2 cells were used since bovine kidney contains both CaBP9k and CaBP28k (Ref. 28 and C. Fullmer, personal communication), and CaBP9k mRNA has been reported to be present in Caco-2 intestinal cells (69). Studies in MCF-7 cells were done in the presence or absence of 5 ng transfected cTR{alpha} expression vector that was used to supplement endogenous TR levels. Similar results for vitamin D-responsive CAT constructs were obtained in the presence or absence of 5 ng cTR{alpha} expression vector. UMR106 osteosarcoma cells, also from ATCC, were maintained in DMEM F-12 medium supplemented with 5% FBS. All cells were cultured at 37 C and 5% C02/95% air. Twenty four hours before transfection, cells were plated at 60–70% confluence in 100-mm tissue culture dishes. Transfection was carried out by the calcium phosphate DNA precipitation method (70) using the specified reporter plasmid, receptor expression vector, and the ß-galactosidase expression vector pCH110 as an internal control. Eighteen hours after transfection, cells were shocked for 1 min with 10% dimethylsulfoxide-PBS, washed with PBS, and then cultured in medium supplemented with 2% charcoal-stripped serum. Cells were harvested 24 h after treatment with vehicle (ethanol in 10 µl), T3, 1,25-(OH)2D3, or both hormones by trypsinization. Cells were pelleted, washed with PBS, resuspended in 50 µl 0.25 M Tris-HCl (pH 8.0), and lysed by pulse sonication three times at 10-sec intervals. Cell debris was spun down, and supernatants were saved for CAT assays (71), which were carried out as previously described (64). CAT assays were normalized by ß-galactosidase activity for each sample. CAT activity was quantitated by densitometric scanning of TLC autoradiograms (Shimadzu CS900 U densitomer, dual wavelength flying spot scanner, Shimadzu Scientific Instruments, Inc. Princeton, NJ). For some experiments several autoradiographic exposure times were needed for densitometric analysis to estimate changes in CAT activity. CAT activity was also quantitated by scanning TLC plates with the PACKARD instant imager system (Packard Instrument Co., Meriden, CT). Multiple experiments were performed and the results are reported as the mean ± SE. Significance was determined by Students’ t test or Dunnets’ multiple comparison t statistic.

Electrophoretic Mobility Shift Assays
The top strand of the oligonucleotide containing the rat 24(OH)ase proximal VDRE (at -150/-136, half-site recognition elements underlined) used as a probe is as follows: 5'-CTAGAGGATGGAGTCAGCGAGGTGAGTGAGGGCGCCGGGGCCTC-3'. The TREp probe (5'-AGCTTAGGTCATGACCTA-3'; TREp underlined) was obtained from Dr. H. Samuels (67). Overlapping forward and reverse strands of these response element probes were heat denatured and allowed to anneal overnight. Fifty nanograms of the annealed probes were end filled in the presence of [{alpha}-32P]dCTP and the Klenow fragment of DNA polymerase I (GIBCO/BRL). The other probes used for gel shift experiments containing sequences of the rat calbindin-D9k gene (-489/-445), the human OC gene (-511/-483), and the mouse OPN gene (-761/-738), which include the reported VDREs (2), were the same as previously described (22, 72, 73). Complementary strands of these oligonucleotide probes were annealed, and 50 ng of duplex oligo were end-labeled with {gamma}-[32P] ATP using T4 polynucleotide kinase. Overexpression and purification of hVDR were performed as described previously (74, 75). FLAG-hRXR{alpha} (epitope-tagged hRXR{alpha} expressed from the polyhedrin promoter) was overexpressed by baculovirus infection and purified from insect cells as described previously (11). cTR{alpha} was purified by diethylaminoethyl-Sephadex chromatography, heparin-agarose chromatography, and size exclusion chromatography as previously reported (68) and was a gift from Dr. H. Samuels. Purified receptor was incubated with ~0.5 ng of labeled probe at room temperature for 20 min in a reaction system containing 0.5 ng poly (deoxyinosinic-deoxycytidylic)acid and a dilution of binding buffer that resulted in a final concentration of 20 mM Tris-HCl (pH 7.9), 1 mM EDTA, 50 mM NaCl, 10% glycerol, 0.1% NP40, and 1 mM dithiothreitol. Where appropriate, double-stranded cold competitor oligonucleotide was added for 20 min at room temperature before addition of labeled probe. Complexes were resolved by electrophoresis on 8% low-ionic strength native polyacrylamide gels run at 26 V/cm at 4 C. Gels were dried and exposed to Kodak XAR-5 film (Eastman Kodak, Rochester, NY) at -80 C.

The Yeast Two-Hybrid System
For the yeast two-hybrid system the hinge region and the ligand-binding domain of the hVDRcDNA (93–427) were amplified as previously described (76) using VDR gene-specific primers containing EcoRI and BamHI linkers (5'tcctgaa TTCATTCTGACAGATGAGGA-3' and 5'-acttggaTCCTAGTCAGGAGATCTCAT-3', respectively). The amplified product was digested with EcoRI and BamHI and inserted into the pGBT9 plasmid (pGBT9 is used to generate a fusion between the GAL4 DNA-binding domain and the hinge region and ligand-binding domain of hVDR). As a control interacting protein, the HincII and EcoRI fragment of hRXR{alpha} cDNA (223–462; hRXR{alpha} cDNA was a gift of Dr. David Mangelsdorf and Dr. Ronald Evans) was excised and ligated to SmaI-digested pGAD424. pGAD424 was used to generate a fusion between the Gal4 activation domain and hRXR{alpha} (223–462). To test the interaction between TR and RXR (positive control) as well as a possible interaction between TR and VDR, TR fusion was generated between cTR{alpha} cDNA (119–438) and the GAL4 DNA-binding domain as well as the GAL4 activation domain by ligating the ~880-bp fragment of a NcoI/BamHI digest to SmaI/BamHI-digested pGBT9 and pGAD424. The product was end filled with Klenow and religated. A fusion between the Gal4 DNA-binding domain and the HincII/EcoRI digest of hRXR{alpha} cDNA was also generated by ligation of the hRXR{alpha} insert to the SmaI digest of pGBT9. The fusions were checked for the presence of insert by hybridization. The orientation and reading frame were confirmed by sequencing. The yeast strain SFY526 (MATta,ura3–52, his3–200, ade2–101, lys2–801, trp 1–901, leu2–3, 112, canr, gal4–542, gal80–538, URA3::GALUAS-GAL1TATA-lacZ) was used. SFY526, which contains the lacZ reporter gene under the control of the Gal4-responsive promoter, made competent with lithium acetate (77), was grown in YPD medium and then transformed with plasmid DNA using the Yeastmaker Yeast Transformation System (CLONTECH). Transformants were plated on media (SD synthetic medium; CLONTECH) without Trp and Leu and were grown for 4 days at 30 C. SFY526 is auxotrophic for Trp and Leu; thus, plating the transformed yeast culture on medium without Trp and Leu selects for colonies containing both types of plasmids (pGBT9 and pGAD424), which carry the wild-type genes for Trp and Leu, respectively. Interaction of two proteins in this two-hybrid system results in Gal4-dependent transcription of the lacZ reporter sequences integrated into the yeast genome and expression of ß-galactosidase. Colonies were assayed for ß-galactosidase expression using a colony lift filter assay (78) modified as described in the Matchmaker Two-Hybrid System (CLONTECH). To verify the results observed using SFY526 yeast in another yeast strain, experiments were also done using Y190 yeast (MATa, ura3–52, his3–200, ade2–101, lys2–801, tryp1–901, leu2–3, 112, gal4{Delta}, gal80{Delta}, cyhr2, LYS2::GAL1UAS-GAL1TATA-lacZ), and transformants were assayed for ß-galactosidase activity on liquid cultures. The indicated plasmid pairs encoding the hybrid constructs were transformed into Y190 yeast, plated on media lacking Leu, Trp, and histidine (His) containing 30 mM aminotriazole (inhibitor of His synthesis), and incubated for 4 days at 30 C. Triplicate colonies were grown overnight at 30 C in Leu-Trp-His- media. The culture was diluted 1:4 into fresh medium and further grown 3–5 h to midlog phase (A600 = 0.5–1.0). Cell pellets from 1.5-ml cultures were permeabilized by three cycles of freeze/thaw and assayed for ß-galactosidase activity using o-nitrophenyl ß-D-galactopyranoside (ONPG) as a substrate as described (79), modified as indicated in the Yeast Matchmaker Two-Hybrid System (CLONTECH). For calculation of ß-galactosidase units, 1 unit of ß-galactosidase is defined as the amount that hydrolyzes 1 µmol ONPG to o-nitrophenol and D-galactose per h at 30 C.


    ACKNOWLEDGMENTS
 
We gratefully acknowledge Dr. Paul MacDonald for experimental suggestions related to studies done using the yeast two-hybrid system. We also thank Terri Towers for advice concerning gel shift experiments. In addition, we acknowledge helpful discussions with Dr. Herbert Samuels and Bruce Raaka as well as the secretarial assistance of Mrs. Valerie Brooks and Ms. Connie Sheffield.


    FOOTNOTES
 
Address requests for reprints to: Dr. Sylvia Christakos, Department of Biochemistry and Molecular Biology, UMDNJ-New Jersey Medical School, 185 South Orange Avenue, Newark, New Jersey 07103-2714. E-mail: christak{at}umdnj.edu

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

Received for publication February 17, 1998. Revision received May 1, 1998. Accepted for publication May 20, 1998.


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 DISCUSSION
 MATERIALS AND METHODS
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