DNA sequences in the rat parathyroid hormone-related peptide gene responsible for 1,25-dihydroxyvitamin D₃-mediated transcriptional repression

Expression of the gene encoding PTH-related peptide (PTHrP), a protein that plays a primary role in the development of humoral hypercalcemia of malignancy, is down-regulated at the transcriptional level by 1,25- dihydroxyvitamin D₃ [1,25-(OH)₂D₃]. Deletions of the 5'-flanking region of the rat PTHrP gene, when fused to the chloramphenicol acetyltransferase gene and transfected into ROS 17/2.8 (rat osteosarcoma) cells, showed that the 1,25(OH)₂D₃ responsive region is located between -1.05 and -0.71 kb upstream of the transcription start site. Further mapping of this region revealed that a 123-bp fragment is able to confer 1,25(OH)₂D₃ responsiveness to a heterologous (SV40) promoter. This region contains two potential vitamin D response elements (VDREs). One of these motifs resembles the negative VDRE (nVDRE) from the PTH gene, which is also down-regulated by vitamin D₃. The other element resembles the canonical VDRE (two hexanucleotide motifs separated by three nucleotides), which has been characterized in a number of genes whose expression is modulated by vitamin D₃. Electrophoretic mobility shift assays using nuclear extracts from ROS 17/2.8 cells and from vitamin D receptor (VDR)-enriched COS I cells revealed that both elements interact with the VDR. This protein-DNA interaction is disrupted by an anti-VDR antibody. Therefore, modulation of PTHrP gene transcription by 1,25(OH)₂D₃ is mediated by the VDR interacting with one or both of the identified motifs in the 5'-flanking sequence of the gene.