Identification of a DNA sequence responsible for binding of the 1,25-dihydroxyvitamin D3 receptor and 1,25-dihydroxyvitamin D3 enhancement of mouse secreted phosphoprotein 1 (SPP-1 or osteopontin) gene expression.
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.
Noda
M
M
Department of Bone Biology and Osteoporosis Research, Merck Sharp and Dohme Research Laboratories, West Point, PA 19486.
Vogel
R L
RL
Craig
A M
AM
Prahl
J
J
DeLuca
H F
HF
Denhardt
D T
DT
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002117:Calcitriol / Q000378:metabolism / Q000494:pharmacology*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D051379:Mice; D008969:Molecular Sequence Data; D053495:Osteopontin; D010750:Phosphoproteins / Q000235:genetics; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000187:drug effects*; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid / Q000378:metabolism*; D012689:Sequence Homology, Nucleic Acid; D012795:Sialoglycoproteins / Q000235:genetics*; D014162:Transfection
D010750:Phosphoproteins; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid; D012795:Sialoglycoproteins; C506896:Spp1 protein, mouse; D053495:Osteopontin; D004247:DNA; D002117:Calcitriol