Influence of dexamethasone on the vitamin D-mediated regulation of osteocalcin gene expression.
The influence of dexamethasone on expression of the osteocalcin gene which encodes the most abundant non-collagenous and only reported bone-specific protein was examined in ROS 17/2.8 osteosarcoma cells which express a broad spectrum of genes related to bone formation. Consistent with previous reports, quantitation of cellular osteocalcin mRNA levels by Northern blot analysis, osteocalcin gene transcription by activity of the osteocalcin gene promoter fused to a chloramphenicol acetyl-transferase (CAT) mRNA coding sequence following transfection into ROS 17/2.8 cells, and osteocalcin biosynthesis by radioimmunoassay indicate that dexamethasone in a concentration range of 10(-6) to 10(-9) M only modestly modifies basal levels of osteocalcin gene expression. However, dexamethasone significantly inhibits these parameters of the vitamin D-induced upregulation of osteocalcin gene expression in both proliferating and in confluent ROS 17/2.8 cells. In this study, we observed that the extent to which abrogation of the vitamin D response occurs is dependent on basal levels of osteocalcin gene expression as reflected by a complete inhibition of the vitamin D-induced upregulation in a ROS 17/2.8K subline with low basal expression and only a partial reduction of the vitamin D stimulation in a ROS 17/2.8C subline with eightfold higher levels of basal expression. This effect of glucocorticoid appears to be at the transcriptional and post-transcriptional levels as demonstrated by a parallel decline in the cellular representation of osteocalcin mRNA, osteocalcin gene promoter activity, and osteocalcin biosynthesis. The complexity of the glucocorticoid effect on vitamin D-mediated transcriptional properties of the osteocalcin gene is indicated by persistence of sequence-specific protein-DNA interactions at two principal osteocalcin gene promoter regulatory elements, the osteocalcin (CCAAT) box which modulates basal level of transcription, and the vitamin D responsive element, where vitamin D-mediated enhancement of osteocalcin gene transcription is controlled.
Schepmoes
G
G
University of Massachusetts Medical Center, Worcester 01655.
Breen
E
E
Owen
T A
TA
Aronow
M A
MA
Stein
G S
GS
Lian
J B
JB
D015152:Blotting, Northern; D002454:Cell Differentiation / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000378:metabolism; D003907:Dexamethasone / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D005938:Glucocorticoids / Q000494:pharmacology; D006801:Humans; D015675:Osteocalcin / Q000096:biosynthesis / Q000235:genetics*; D012516:Osteosarcoma / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis; D012334:RNA, Neoplasm / Q000235:genetics; D011863:Radioimmunoassay; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured; D015854:Up-Regulation; D014807:Vitamin D / Q000502:physiology*
D004268:DNA-Binding Proteins; D005938:Glucocorticoids; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D015675:Osteocalcin; D014807:Vitamin D; D003907:Dexamethasone