Abstract
Dexamethasone increases glutamine synthetase activity and mRNA abundance in L6 muscle cells in culture, apparently by a glucocorticoid receptor-mediated process. The data in this report reveal that increased glutamine synthetase mRNA abundance is attributable at least in part to an enhanced rate of transcription of the glutamine synthetase gene. "Nuclear runoff" assays of glutamine synthetase gene expression were performed with purified myonuclei from dexamethasone-treated or untreated L6 skeletal muscle cells. These assays showed glutamine synthetase transcription to be increased approximately 2-fold as early as 1 h after incubation of cells with dexamethasone (10(-7) M); there was no increase in the rate of transcription of the beta-tubulin gene, which served as a control. The increase in glutamine synthetase gene transcription correlates with increased glutamine synthetase enzymatic activity after dexamethasone treatment. Studies with actinomycin D indicated that the half-life of glutamine synthetase mRNA (7-8 h) is not altered by dexamethasone. Therefore, the degradation of glutamine synthetase mRNA is not affected by dexamethasone, and the increased glutamine synthetase mRNA level is attributable to increased transcription. The dexamethasone-mediated increase in glutamine synthetase mRNA abundance is glucocorticoid receptor-mediated; RU38486 (a glucocorticoid receptor blocker) completely blocked the effect of dexamethasone. The dexamethasone-mediated increase in glutamine synthetase gene transcription and steady-state mRNA level was not blocked by cycloheximide, indicating a direct effect.
Highlights
Dexamethasone increases glutamine synthetase activity and mRNA abundance in L6 muscle cells in culture, apparently by a glucocorticoid receptor-mediated process
The degradation of glutamine synthetase mRNA is not affected by dexamethasone, and the increased glutamine synthetase mRNA level is attributable to increased transcription
We showed that the dexamethasoneinduced increase in glutamine synthetase activity was blocked by RU38486, a glucocorticoid antagonist (Max et al, 1987), supporting the notion that glutamine synthetase is responsive to glucocorticoid receptor activation
Summary
Total cellular RNA was isolated from L6 muscle cells using the guanidine isothiocynate procedure (Chirgwin et al, 1979). The RNA was transferred to nitrocellulose for 2 h and hybridized filters; the filters were baked in uucuo at 80 “C with a radioactively labeled glutamine synthetase cDNA probe (Thomas, 1980). The blots were hybridized to cDNA probe as an internal control gene (Lad et al, 1986). Both glutamine synthetase and cyclophilin probes were labeled to high specific activity through the “random primed” labeling method of Feinberg and Vogelstein Isolation of Nuclei from Lb Cells-The medium was aspirated and the cells washed twice with cold phosphate-buffered saline
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