Constitutive transcription of the osteocalcin gene in osteosarcoma cells is reflected by altered protein-DNA interactions at promoter regulatory elements.
The bone-specific osteocalcin (OC) gene is transcribed only after completion of proliferation in normal diploid calvarial-derived osteoblasts during extracellular matrix mineralization. In contrast, the OC gene is expressed constitutively in both proliferating and nonproliferating ROS 17/2.8 osteosarcoma cells. To address molecular mechanisms associated with these tumor-related modifications in transcriptional control, we examined sequence-specific interactions of transactivation factors at key basal and hormone-responsive elements in the OC gene promoter. In ROS 17/2.8 cells compared to normal diploid osteoblasts, the absence of a stringent requirement for cessation of proliferation to support both induction of OC transcription and steroid hormone-mediated transcriptional modulation is reflected by modifications in transcription factor binding at (i) the two primary basal regulatory elements, the OC box (which contains a CCAAT motif as a central core) and the TATA/glucocorticoid-responsive element domain, and (ii) the vitamin D-responsive element. Particularly striking are two forms of the vitamin D receptor complex that are present in proliferating osteoblasts and osteosarcoma cells. Both forms of the complex are sensitive to vitamin D receptor antibody and retinoic X receptor antibody. After the down-regulation of proliferation, only the lower molecular weight complex is found in normal diploid osteoblasts. Both forms of the complex are present in nonproliferating ROS 17/2.8 cells with increased representation of the complex exhibiting reduced electrophoretic mobility that is phosphorylation-dependent.
Bortell
R
R
Department of Cell Biology, University of Massachusetts Medical Center, Worcester 01655.
Owen
T A
TA
Shalhoub
V
V
Heinrichs
A
A
Aronow
M A
MA
Rochette-Egly
C
C
Lutz
Y
Y
Stein
J L
JL
Lian
J B
JB
Stein
G S
GS
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002117:Calcitriol / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003907:Dexamethasone / Q000494:pharmacology; D005333:Fetus; D015870:Gene Expression / Q000187:drug effects; D006657:Histones / Q000096:biosynthesis / Q000235:genetics; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000378:metabolism; D009838:Oligodeoxyribonucleotides; D010006:Osteoblasts / Q000502:physiology*; D015675:Osteocalcin / Q000235:genetics*; D012516:Osteosarcoma; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D012045:Regulatory Sequences, Nucleic Acid; D016385:TATA Box; D014158:Transcription, Genetic / Q000187:drug effects; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D006657:Histones; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D015675:Osteocalcin; D003907:Dexamethasone; D002117:Calcitriol