Ligand-dependent down-regulation of stably transfected human glucocorticoid receptors is associated with the loss of functional glucocorticoid responsiveness.
The effect of glucocorticoids on the regulation of stably transfected human glucocorticoid receptors has been examined. Exposure of a Chinese hamster ovary-derived cell line containing stably transfected human glucocorticoid receptor genes and glucocorticoid-responsive dihydrofolate reductase genes to 5 nM dexamethasone resulted in a rapid, time-dependent reduction in the level of glucocorticoid receptor protein to 50% of control levels within 5 h of steroid treatment. This decrease in receptor protein was persistent, with a maximal 70% reduction observed even after 4 weeks of dexamethasone treatment. Immunocytochemical analysis of the influence of dexamethasone on stably transfected glucocorticoid receptors revealed efficient translocation of receptors to the nucleus within 1 h of hormone treatment. However, upon longer exposure to dexamethasone (5 h), immunoreactive glucocorticoid receptors were localized primarily to the cytoplasm. By 24 h of treatment, glucocorticoid receptors were absent from the cytoplasm and the nucleus, suggesting that the ligand-induced loss of glucocorticoid receptors may be a cytoplasmic event. The decrease in transfected glucocorticoid receptor protein was largely reflected by similar changes in steady state levels of human glucocorticoid receptor mRNA; however, the effects of hormone on receptor protein levels were more profound than on receptor mRNA. There was an initial rapid reduction in transfected glucocorticoid receptor mRNA to 50% of control levels within 2 h of dexamethasone treatment. This reduction was followed by a transient rise in mRNA expression after 12 h of hormone treatment. With prolonged exposure to dexamethasone (> 12 h) a second, more gradual decline in human glucocorticoid receptor mRNA was observed. This biphasic pattern of glucocorticoid receptor gene expression was not reflected at the level of receptor protein, suggesting that both transcriptional and translational control mechanisms may be involved in ligand-dependent receptor regulation. When cells were removed from dexamethasone after up to 48 h of treatment, glucocorticoid receptor mRNA levels fully recovered within 12 h. Receptor protein recovered only partially during this same time period. Down-regulation of glucocorticoid receptor protein and mRNA levels by dexamethasone in stably transfected cells led to corresponding reductions in the hormone sensitivity to two glucocorticoid-regulated genes: a transiently transfected chloramphenicol acetyltransferase receptor gene and a stably integrated dihydrofolate reductase gene. These results demonstrate that stably transfected human glucocorticoid receptors are subject to ligand-induced down-regulation in a heterologous cell line. Moreover, glucocorticoid receptor autoregulation appears to be a highly conserved mechanism for attenuating cellular responsiveness to hormone.
Bellingham
D L
DL
Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill 27599.
Sar
M
M
Cidlowski
J A
JA
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003907:Dexamethasone / Q000494:pharmacology*; D015536:Down-Regulation; D004361:Drug Tolerance; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D008024:Ligands; D014176:Protein Biosynthesis / Q000187:drug effects; D012333:RNA, Messenger / Q000096:biosynthesis; D011965:Receptors, Glucocorticoid / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis / Q000235:genetics; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection
D008024:Ligands; D012333:RNA, Messenger; D011965:Receptors, Glucocorticoid; D011993:Recombinant Fusion Proteins; C028697:dexamethasone receptor; D003907:Dexamethasone; D013762:Tetrahydrofolate Dehydrogenase