Distinct Conformational Changes Induced by 20-epi Analogues of 1,25-Dihydroxyvitamin D Are Associated with Enhanced Activation of the Vitamin D Receptor

1,25-dihydroxyvitamin D3 [1,25(OH)2D3], plays an important role in the regulation of mineral ion homeostasis. As well as being the major steroid hormone that regulates calcium metabolism, 1,25(OH)2D3 suppresses transcription of the gene encoding parathyroid hormone, a peptide that plays a dominant role in regulating extracellular calcium levels. To identify DNA sequences that may mediate this transcriptional repression, nuclear extracts containing the 1,25(OH)2D3 receptor were examined for binding to sequences in the 5'-flanking region of the human parathyroid hormone gene. A 25-base-pair (bp) oligonucleotide containing the sequences from -125 to -101 from the start of exon I binds nuclear proteins recognized by monoclonal antibodies against the 1,25(OH)2D3 receptor. The sequences in this region contain a single copy of a motif (AGGTTCA) homologous to the motifs repeated in the up-regulatory 1,25(OH)2D3-response elements. When placed upstream to a heterologous viral promoter, the sequences contained in this 25-bp oligonucleotide mediate transcriptional repression in response to 1,25(OH)2D3 in GH4C1 cells but not in ROS 17/2.8 cells. This down-regulatory element, therefore, differs from the up-regulatory 1,25(OH)2D3-response elements both in sequence composition and in the requirement for particular cellular factors other than the 1,25(OH)2D3 receptor for repressing transcription.

Secreted phosphoprotein 1 (Spp-1; osteopontin) is one of the abundant noncollagenous proteins in bone matrix and is produced by osteoblasts. We examined the promoter region of the mouse Spp-1 gene and identified a sequence responsible for 1,25-dihydroxyvitamin D3 enhancement of the Spp-1 gene expression. This 24-base-pair (bp) sequence (vitamin D response element) is located 761 bp upstream of the transcription start site and consists of two direct repeats of a unique 9-bp motif, AGGTTCACG. The vitamin D response element confers responsiveness of a heterologous promoter to 1,25-dihydroxyvitamin D3 in a position- and orientation-independent and copy-number-dependent manner. The basal level of expression of the reporter constructs containing this sequence and its response to 1,25-dihydroxyvitamin D3 were not affected by cotreatment with transforming growth factor beta or the tumor promoter phorbol 12-myristate 13-acetate or by cotransfection with a JUN expression vector. The vitamin D response element forms DNA-protein complexes, as indicated by gel-retardation assays. The addition of a monoclonal antibody raised against the vitamin D receptor further retarded the mobility of the DNA-protein complex. Another antibody that recognizes the DNA binding region of the vitamin D receptor attenuated its binding to the sequence. These results indicate that this 24-bp sequence containing two 9-bp motifs binds to the vitamin D receptor and mediates the vitamin D3 enhancement of murine Spp-1 gene expression.