Kurokawa R, Ota M
Department of Biochemistry, Iwate Medical University School of Dentistry, Japan.
Biochim Biophys Acta. 1988 Jul 29;970(3):292-304. doi: 10.1016/0167-4889(88)90129-2.
[3H]Triamcinolone acetonide glucocorticoid receptor complexes from human salivary gland adenocarcinoma cells (HSG cells) were shown to be activated with an accompanying decrease in molecular weight in intact cells, as analyzed by gel filtration, DEAE chromatography, the mini-column method and glycerol gradient centrifugation. Glucocorticoid receptor complexes consist of steroid-binding protein (or glucocorticoid receptor) and non-steroid-binding factors such as the heat-shock protein of molecular weight 90,000. To determine whether the steroid-binding protein decreases in molecular weight upon activation, affinity labeling of glucocorticoid receptor in intact cells by incubation with [3H]dexamethasone 21-mesylate, which forms a covalent complex with glucocorticoid receptor, was performed. Analysis by gel filtration and a mini-column method indicated that [3H]dexamethasone 21-mesylate-labeled receptor complexes can be activated under culture conditions at 37 degrees C. SDS-polyacrylamide gel electrophoresis of [3H]dexamethasone 21-mesylate-labeled steroid-binding protein resolved only one specific 92 kDa form. Furthermore, only one specific band at 92 kDa was detected in the nuclear fraction which was extracted from the cells incubated at 37 degrees C. These results suggest that there is no change in the molecular weight of steroid-binding protein of HSG cell glucocorticoid receptor complexes upon activation and that the molecular weight of nuclear-binding receptor does not change, although the molecular weight of activated glucocorticoid receptor complexes does decrease. Triamcinolone acetonide induced an inhibitory effect on DNA synthesis in HSG cells. Dexamethasone 21-mesylate exerted no such effect and blocked the action of triamcinolone acetonide on DNA synthesis. These results suggests that dexamethasone 21-mesylate acts as antagonist of glucocorticoid in HSG cells. The fact that dexamethasone 21-mesylate-labeled receptor complexes could be activated and could bind to DNA or nuclei as well as triamcinolone acetonide-labeled complexes suggests that dexamethasone 21-mesylate-labeled complexes can not induce specific gene expression after their binding to DNA.
通过凝胶过滤、DEAE 色谱法、微柱法和甘油梯度离心法分析表明，来自人涎腺腺癌细胞（HSG 细胞）的[3H]曲安奈德糖皮质激素受体复合物在完整细胞中被激活，同时分子量降低。糖皮质激素受体复合物由类固醇结合蛋白（或糖皮质激素受体）和非类固醇结合因子组成，如分子量为 90,000 的热休克蛋白。为了确定类固醇结合蛋白在激活后分子量是否降低，通过与[3H]地塞米松 21-甲磺酸盐孵育对完整细胞中的糖皮质激素受体进行亲和标记，[3H]地塞米松 21-甲磺酸盐与糖皮质激素受体形成共价复合物。凝胶过滤和微柱法分析表明，[3H]地塞米松 21-甲磺酸盐标记的受体复合物在 37℃培养条件下可被激活。[3H]地塞米松 21-甲磺酸盐标记的类固醇结合蛋白的 SDS-聚丙烯酰胺凝胶电泳仅显示一种特定的 92 kDa 形式。此外，在从 37℃孵育的细胞中提取的核部分中仅检测到一条 92 kDa 的特定条带。这些结果表明，HSG 细胞糖皮质激素受体复合物的类固醇结合蛋白在激活后分子量没有变化，核结合受体的分子量也没有变化，尽管激活的糖皮质激素受体复合物的分子量确实降低了。曲安奈德对 HSG 细胞中的 DNA 合成有抑制作用。地塞米松 21-甲磺酸盐没有这种作用，并阻断了曲安奈德对 DNA 合成的作用。这些结果表明，地塞米松 21-甲磺酸盐在 HSG 细胞中作为糖皮质激素的拮抗剂起作用。地塞米松 21-甲磺酸盐标记的受体复合物可以被激活，并且可以像曲安奈德标记的复合物一样与 DNA 或细胞核结合，这一事实表明，地塞米松 21-甲磺酸盐标记的复合物在与 DNA 结合后不能诱导特定基因表达。
