Decreased glucocorticoid receptor activity following glucocorticoid receptor antisense RNA gene fragment transfection.
Depression is often characterized by increased cortisol secretion caused by hyperactivity of the hypothalamic-pituitary-adrenal axis and by nonsuppression of cortisol secretion following dexamethasone administration. This hyperactivity of the hypothalamic-pituitary-adrenal axis could result from a reduced glucocorticoid receptor (GR) activity in neurons involved in its control. To investigate the effect of reduced neuronal GR levels, we have blocked cellular GR mRNA processing and/or translation by introduction of a complementary GR antisense RNA strand. Two cell lines were transfected with a reporter plasmid carrying the chloramphenicol acetyltransferase (CAT) gene under control of the mouse mammary tumor virus long terminal repeat (a glucocorticoid-inducible promoter). This gene construction permitted assay of the sensitivity of the cells to glucocorticoid hormones. Cells were also cotransfected with a plasmid containing 1,815 bp of GR cDNA inserted in the reverse orientation downstream from either a neurofilament gene promoter element or the Rous sarcoma virus promoter element. Northern (RNA) blot analysis demonstrated formation of GR antisense RNA strands. Measurement of the sensitivity of CAT activity to exogeneous dexamethasone showed that although dexamethasone increased CAT activity by as much as 13-fold in control incubations, expression of GR antisense RNA caused a 2- to 4-fold decrease in the CAT response to dexamethasone. Stable transfectants bearing the GR antisense gene fragment construction demonstrated a 50 to 70% decrease of functional GR levels compared with normal cells, as evidenced by a ligand-binding assay with the type II glucocorticoid receptor-specific ligand [3H]RU 28362. These results validate the use of antisense RNA to GR to decrease cellular response to glucocorticoids.
Pepin
M C
MC
Laboratory of Molecular Psychogenetics, CHUL Research Centre, Sainte Foy, Québec, Canada.
Barden
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N
D000818:Animals; D015152:Blotting, Northern; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003907:Dexamethasone / Q000494:pharmacology; D015870:Gene Expression; D051379:Mice; D011401:Promoter Regions, Genetic; D016372:RNA, Antisense; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000235:genetics*
D016372:RNA, Antisense; D012333:RNA, Messenger; D011965:Receptors, Glucocorticoid; D003907:Dexamethasone