Ligand-dependent down-regulation of stably transfected human glucocorticoid receptors is associated with the loss of functional glucocorticoid responsiveness.
Ligand-dependent down-regulation of stably transfected human glucocorticoid receptors is associated with the loss of functional glucocorticoid responsiveness.
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DOI：
10.1210/mend.6.12.1491690
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发表时间：
1992-12
影响因子：
--
通讯作者：
D. Bellingham;M. Sar;J. Cidlowski
中科院分区：
文献类型：
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作者：
D. Bellingham;M. Sar;J. Cidlowski
糖皮质激素对稳定转染的人糖皮质激素受体的调节作用已经被检测。将含有稳定转染的人糖皮质激素受体基因和糖皮质激素响应性二氢叶酸还原酶基因的中国仓鼠卵巢衍生细胞系暴露于5 nM地塞米松导致糖皮质激素受体蛋白水平在类固醇治疗5 h内快速、时间依赖性降低至对照水平的50%。这种受体蛋白的减少是持续的，甚至在地塞米松治疗4周后观察到最大70%的减少。地塞米松对稳定转染的糖皮质激素受体的影响的免疫细胞化学分析显示，激素治疗1小时内受体有效易位到细胞核。然而，在较长时间暴露于地塞米松（5小时），免疫反应性糖皮质激素受体主要定位于细胞质。到24小时的治疗，糖皮质激素受体的细胞质和细胞核中不存在，这表明配体诱导的糖皮质激素受体的损失可能是一个细胞质事件。转染糖皮质激素受体蛋白的减少在很大程度上反映在人糖皮质激素受体mRNA稳态水平的类似变化上;然而，激素对受体蛋白水平的影响比对受体mRNA的影响更深刻。有一个初步的快速减少转染糖皮质激素受体mRNA的50%的控制水平内2小时的地塞米松治疗。激素处理12小时后，mRNA表达短暂升高。随着地塞米松暴露时间的延长（> 12 h），观察到人糖皮质激素受体mRNA的第二次、更缓慢的下降。这种糖皮质激素受体基因表达的双相模式在受体蛋白水平上没有反映出来，这表明转录和翻译控制机制可能参与配体依赖性受体调控。当细胞在长达48小时的处理后从地塞米松中取出时，糖皮质激素受体mRNA水平在12小时内完全恢复。在同一时期，受体蛋白仅部分恢复。地塞米松在稳定转染细胞中下调糖皮质激素受体蛋白和mRNA水平导致对两个糖皮质激素调节基因的激素敏感性相应降低：瞬时转染的氯霉素乙酰转移酶受体基因和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，糖皮质激素受体的自动调节似乎是一个高度保守的机制，减弱细胞对激素的反应。
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.