Regions of the rat osteocalcin gene which mediate the effect of 1,25-dihydroxyvitamin D3 on gene transcription.
We have cloned the genomic DNA encoding rat osteocalcin and have isolated fragments in the 5' flanking region which mediate the effects of 1,25-(OH)2D3 (1,25-dihydroxyvitamin D3) on osteocalcin gene transcription. Approximately 3 kilobase pairs of the osteocalcin gene's 5' flanking region, including the promoter and transcription start site, were fused to the reporter gene chloramphenicol acetyltransferase. Transfection into ROS 17/2.8 rat osteosarcoma cells demonstrated low level basal expression of the chloramphenicol acetyltransferase gene. The expression increased markedly in the presence of 1,25-(OH)2D3; induction was observed at doses as low as 10(-11) M 1,25-(OH)2D3. Chloramphenicol acetyltransferase activity increased as early as 16 h after stimulation with 10(-9) M 1,25-(OH)2D3. Basal chloramphenicol acetyltransferase activity in ROS 24/1 and 25/1 cells was much lower than in ROS 17/2.8 cells. In these two cell lines, there was little induction of chloramphenicol acetyltransferase activity in the presence of 10(-9) M 1,25-(OH)2D3. Deletion studies of the 5' flanking region demonstrated two regions that contribute to the induction by 1,25-(OH)2D3. Deletion of a 650-base pair fragment ending 1.4 kilobase pairs upstream from the initiator ATG led to an 80% decrease in responsiveness. Removal of an additional 1.1 kilobase pairs, leaving a 300-base pair promoter containing fragment obliterated responsiveness to 1,25-(OH)2D3.
Endocrine Unit, Massachusetts General Hospital, Boston 02114.
M B
MB
Demay
D A
DA
Roth
H M
HM
Kronenberg
D000818:Animals; D001842:Bone and Bones / Q000378:metabolism; D002117:Calcitriol / Q000494:pharmacology*; D002135:Calcium-Binding Proteins / Q000235:genetics*; D002460:Cell Line; D005796:Genes; D005809:Genes, Regulator; D015675:Osteocalcin; D012516:Osteosarcoma; D010957:Plasmids; D051381:Rats; D014158:Transcription, Genetic / Q000187:drug effects*; D014162:Transfection
D002135:Calcium-Binding Proteins; D015675:Osteocalcin; D002117:Calcitriol