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

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

The 9,000 M_r calcium-binding protein calbindin-D_9k (CaBP_9k) 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 CaBP_9k gene (at -490/-472), the CaBP_9k promoter is weakly transactivated by 1,25-(OH)₂D₃. Previous studies indicated that when MCF-7 cells are transfected with the rat CaBP_9k 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 CaBP_9k natural promoter to 1,25-(OH)₂D₃ could be enhanced by T₃, CaBP_9k 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 CaBP_9k 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, and chick T3R and 24(OH)ase, osteocalcin, osteopontin, and CaBP_9k 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.

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