THYROID-HORMONE RECEPTOR DOES NOT HETERODIMERIZE WITH THE VITAMIN-D-RECEPTOR BUT REPRESSES VITAMIN-D RECEPTOR-MEDIATED TRANSACTIVATION

The 9,000 M-r calcium-binding protein calbindin-D-9k (CaBP9k) is marke dly induced by 1,25-dihydroxyvitamin D-3 [1,25-(OH)(2)D-3] in mammalia n 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),D,. 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)(2)D-3 and T-3 there is an enhancement of the response o bserved with 1,25-(OH),D, 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 CaBP 9k natural promoter to 1,25-(OH)(2)D-3 could be enhanced by T-3, CaBP9 k promoter/reporter chloramphenicol acetyltransferase constructs were transfected in MCF-7 cells, and the cells were treated with the two ho rmones alone or in combination. No induction with T-3 alone and no enh ancement of reporter activity in the presence of both hormones was obs erved. To determine whether a lack of effect by T-3 was specific for t he CaBP9k promoter and to further examine the possibility of cross-tal k between the TR- and VDR-signaling pathways, the 1,25-(OH)(2)D-3-resp onsive rat 24 hydroxylase [24(OH)ase] promoter and the rat osteocalcin VDRE (-457/-430), both fused to reporter genes were similarly examine d in MCF-7 cells. Again, no enhancement of the response to 1,25-(OH),D , was observed in the presence of T-3. In addition, a similar lack of response to T-3 but responsiveness to 1,25-(OH)(2)D-3 was observed whe n UMR106-01 osteosarcoma cells [which, like MCF-7 cells, express VDR, TR, and the retinoid X receptor (RXR) endogenously] were transfected w ith a 1,25-(OH)(2)D-3 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, oste opontin, and CaBP9k VDRE oligonucleotide probes. No VDR-TR heterodimer binding on any of these VDREs was observed, although, as expected, th ere was binding by the VDR-RXR complex and strong TR-RXR binding to a consensus thyroid hormone response element. Simultaneous gel retardati on assays using similar and lower concentrations of TR with RXR showed strong binding of TR-RXR on a P-32-labeled thyroid response element. Studies using the yeast two-hybrid system also did not provide evidenc e for the formation of a VDR-TR protein-protein interaction. In additi on, in vivo data showed that transfection of TR, in fact, repressed VD R-mediated transcription and that the repression could be reversed by the addition of RXR. Thus, in vitro and in vivo experiments do not sup port ligand-sensitive transactivation mediated by VDR-TR heterodimer f ormation but rather suggest that TR expression can repress 1,25-(OH)(2 )D-3-induced transcription predominantly by sequestering RXR.