Vitamin D receptor binding to the negative human parathyroid hormone vitamin D response element does not require the...

An important physiological control of PTH gene expression is its transcriptional repression by 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)]. The mechanism of this 1,25-(OH)(2)D(3)-mediated transcriptional repression is poorly understood. Previous investigations have identified a DNA sequence in the 5'-regulatory region of the human PTH (hPTH) gene that binds the vitamin D receptor (VDR) and mediates transcription repression in response to 1,25(OH)(2)D(3) in GH4CI cells. The hPTH gene sequence does not mediate transcriptional repression in ROS 17/2.8 cells, even though up-regulatory vitamin D response elements (VDREs) are active in these cells. The hPTH DNA sequence differs from the upregulatory VDREs in that it contains a single copy of a hexameric motif (AGGUC) homologous to those repeated in the up-regulatory VDREs. The protein-DNA interactions of this sequence were examined using nuclear extracts from bovine parathyroid, GH4CI, and ROS 17/2.8 cells. In bovine parathyroid nuclear extracts, the VDR binds the down-regulatory hPTH DNA sequence independently of the retinoid X receptor (RXR). In GH4C1 nuclear extracts, two VDR-containing complexes are observed: one lacking RXR and one containing RXR. In ROS 17/2.8 nuclear extracts, a single VDRdependent complex containing RXR is observed. When the up-regulatory rat osteocalcin VDRE is used as a probe, only VDR-RXR-containing complexes are generated using nuclear extracts from all three cell types. These results demonstrate that the sequence that mediates transcriptional repression in response to 1 ,25-(OH)(2)D(3) differs from the up-regulatory response elements both in sequence composition and in its ability to bind VDR independently of RXR.