Bellingham D L, Sar M, Cidlowski J A
Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill 27599.
Mol Endocrinol. 1992 Dec;6(12):2090-102. doi: 10.1210/mend.6.12.1491690.
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.
已研究了糖皮质激素对稳定转染的人糖皮质激素受体调节的影响。将含有稳定转染的人糖皮质激素受体基因和糖皮质激素反应性二氢叶酸还原酶基因的中国仓鼠卵巢来源的细胞系暴露于5 nM地塞米松，导致在类固醇处理后5小时内，糖皮质激素受体蛋白水平迅速、随时间依赖性降低至对照水平的50%。受体蛋白的这种降低持续存在，即使在地塞米松处理4周后，仍观察到最大降低70%。免疫细胞化学分析地塞米松对稳定转染的糖皮质激素受体的影响，显示在激素处理1小时内受体有效地转运至细胞核。然而，在地塞米松更长时间暴露（5小时）后，免疫反应性糖皮质激素受体主要定位于细胞质。处理24小时后，细胞质和细胞核中均不存在糖皮质激素受体，提示配体诱导的糖皮质激素受体丧失可能是一个细胞质事件。转染的糖皮质激素受体蛋白的降低在很大程度上反映为人糖皮质激素受体mRNA稳态水平的类似变化；然而，激素对受体蛋白水平的影响比对受体mRNA的影响更显著。在地塞米松处理后2小时内，转染的糖皮质激素受体mRNA最初迅速降低至对照水平的50%。这种降低之后，在激素处理12小时后mRNA表达出现短暂升高。随着对地塞米松的长时间暴露（>12小时），观察到人类糖皮质激素受体mRNA第二次更逐渐的下降。糖皮质激素受体基因表达的这种双相模式在受体蛋白水平未得到体现，提示转录和翻译控制机制可能均参与配体依赖性受体调节。当细胞在处理长达48小时后从地塞米松中移除时，糖皮质激素受体mRNA水平在12小时内完全恢复。在此相同时间段内，受体蛋白仅部分恢复。地塞米松在稳定转染的细胞中对糖皮质激素受体蛋白和mRNA水平的下调导致对两个糖皮质激素调节基因的激素敏感性相应降低：一个瞬时转染的氯霉素乙酰转移酶受体基因和一个稳定整合的二氢叶酸还原酶基因。这些结果表明，稳定转染的人糖皮质激素受体在异源细胞系中受到配体诱导的下调。此外，糖皮质激素受体自身调节似乎是减弱细胞对激素反应性的一种高度保守机制。
