Mechanism of glucocorticoid regulation of alkaline phosphatase gene expression in osteoblast-like cells.
In the rat osteosarcoma cell line ROS 17/2.8, glucocorticoids increase the activity of the plasma membrane enzyme, alkaline phosphatase. To determine the mechanisms responsible for this effect, we have studied the actions of dexamethasone on alkaline phosphatase activity, immunoreactive protein, and steady-state mRNA levels. Dexamethasone treatment increased both specific activity of alkaline phosphatase and the cell surface expression of immunoreactive protein in a dose-dependent manner, with a half-maximal increase at 2 nM. Steady-state alkaline phosphatase mRNA levels were also increased in a dose-dependent manner. The time course of dexamethasone induction occurred relatively slowly, with a lag period of 12 h before any discernable effect on alkaline phosphatase mRNA levels. The rise in alkaline phosphatase mRNA levels was attributable entirely to changes in gene transcription, with no effect on message stability. Treatment of ROS 17/2.8 cells with actinomycin D completely abolished the dexamethasone-induced rise in alkaline phosphatase mRNA levels. Measurement of alkaline phosphatase mRNA degradation, by incubation of cells with the transcriptional inhibitor 5,6-dichloro-ribofuranosylbenzimidazole, indicated an apparent half-life of 24 h in both untreated and dexamethasone-stimulated cells. The protein synthesis inhibitors cycloheximide and puromycin blocked the dexamethasone induction of alkaline phosphatase mRNA. These data suggest that the dexamethasone-induced rise in alkaline phosphatase gene transcription requires the synthesis of an unknown mediator protein.
Green
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Department of Medicine, University of Birmingham, Queen Elizabeth Hospital, Edgbaston, Birmingham, England.
Todd
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Heath
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D000469:Alkaline Phosphatase / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D015152:Blotting, Northern; D002460:Cell Line / Q000187:drug effects; D003513:Cycloheximide / Q000494:pharmacology; D003609:Dactinomycin / Q000494:pharmacology; D003907:Dexamethasone / Q000494:pharmacology*; D004004:Dichlororibofuranosylbenzimidazole / Q000494:pharmacology; D004347:Drug Interactions; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D015335:Molecular Probes; D010006:Osteoblasts / Q000187:drug effects / Q000201:enzymology*; D012516:Osteosarcoma / Q000201:enzymology; D011691:Puromycin / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats; D014158:Transcription, Genetic / Q000187:drug effects
D000911:Antibodies, Monoclonal; D015335:Molecular Probes; D012333:RNA, Messenger; D003609:Dactinomycin; D011691:Puromycin; D004004:Dichlororibofuranosylbenzimidazole; D003907:Dexamethasone; D003513:Cycloheximide; D000469:Alkaline Phosphatase