Heufelder A E, Wenzel B E, Bahn R S
Department of Internal Medicine, Mayo Clinic/Foundation, Rochester, MN 55905.
Acta Endocrinol (Copenh). 1993 Jan;128(1):41-50. doi: 10.1530/acta.0.1280041.
Glucocorticoids modulate numerous proliferative, metabolic and immunological functions in human fibroblasts, some of which appear to be mediated via glucocorticoid receptors. We studied the influence of glucocorticosteroids on the synthesis and expression of a 72 kDa heat shock protein that is thought to play a role in thyroid autoimmunity. Experiments were performed using orbital fibroblasts derived from patients with Graves' ophthalmopathy and normal individuals. Cell monolayers were exposed to various concentrations of dexamethasone, the specific glucocorticoid agonist RU 28362, the glucocorticoid antagonist RU 38486, or combinations thereof, prior to heat stress or exposure to hydrogen peroxide. Heat shock protein 72 expression was assessed using sodium dodecylsulfate polyacrylamide-gel electrophoresis of cellular extracts, followed by autoradiography or immunoblotting with a mouse monoclonal antibody against the 72 kDa heat shock protein and quantitative scanning densitometry. In addition, cellular distribution of the immunoreactivity for the 72 kDa heat shock protein was studied using indirect immunofluorescence on parallel cultures. In other experiments, aimed at studying heat shock protein synthesis, cell cultures were pulse-labeled with [35S]-methionine prior to harvesting. Treatment with dexamethasone or RU 28362 markedly attenuated the heat stress-enhanced synthesis and expression of the 72 kDa heat shock protein and several other heat shock proteins both in normal and in Graves' retroocular fibroblasts (p < 0.001). In addition, either treatment reduced baseline expression of the 72 kDa heat shock protein in Graves' retroocular fibroblasts (p < 0.01). These effects were dose-dependent and appeared to be mediated via the glucocorticoid receptor, because combined exposure to dexamethasone or RU 28362 plus RU 38486 completely restored synthesis and expression of the 72 kDa heat shock protein. Baseline or stress-enhanced expression of the 72 kDa heat shock protein was not altered by treatment of monolayers with RU 38486 alone. As demonstrated by immunofluorescence, the characteristic intracellular shifting of the 72 kDa heat shock protein in response to cellular stress was partially inhibited by glucocorticoid agonists and restored by simultaneous exposure to glucocorticoid agonists and RU 38486. These results demonstrate that dexamethasone and the specific glucocorticoid agonist RU 28362 can modulate baseline- and stress-induced synthesis and expression of the 72 kDa heat shock protein, as well as its subcellular distribution, in cultured retroocular fibroblasts. Our studies suggest that these compounds exert these effects via the glucocorticoid receptor.
糖皮质激素可调节人成纤维细胞的多种增殖、代谢和免疫功能，其中一些功能似乎是通过糖皮质激素受体介导的。我们研究了糖皮质激素对一种72 kDa热休克蛋白合成和表达的影响，该蛋白被认为在甲状腺自身免疫中起作用。实验使用了来自格雷夫斯眼病患者和正常人的眼眶成纤维细胞。在热应激或暴露于过氧化氢之前，将细胞单层暴露于不同浓度的地塞米松、特异性糖皮质激素激动剂RU 28362、糖皮质激素拮抗剂RU 38486或它们的组合中。使用细胞提取物的十二烷基硫酸钠聚丙烯酰胺凝胶电泳评估热休克蛋白72的表达，随后进行放射自显影或用针对72 kDa热休克蛋白的小鼠单克隆抗体进行免疫印迹以及定量扫描密度测定。此外，使用平行培养物上进行间接免疫荧光研究了72 kDa热休克蛋白免疫反应性的细胞分布。在其他旨在研究热休克蛋白合成的实验中，收获前用[35S] - 甲硫氨酸对细胞培养物进行脉冲标记。用地塞米松或RU 28362处理显著减弱了正常和格雷夫斯病眼后成纤维细胞中热应激增强的72 kDa热休克蛋白及其他几种热休克蛋白的合成和表达（p < 0.001）。此外，两种处理均降低了格雷夫斯病眼后成纤维细胞中72 kDa热休克蛋白的基础表达（p < 0.01）。这些效应呈剂量依赖性，似乎是通过糖皮质激素受体介导的，因为同时暴露于地塞米松或RU 28362加RU 38486可完全恢复72 kDa热休克蛋白的合成和表达。单独用RU 38486处理单层细胞不会改变72 kDa热休克蛋白的基础或应激增强表达。如免疫荧光所示，糖皮质激素激动剂部分抑制了72 kDa热休克蛋白在细胞应激时的特征性细胞内移位，而同时暴露于糖皮质激素激动剂和RU 38486可使其恢复。这些结果表明，地塞米松和特异性糖皮质激素激动剂RU 28362可调节培养的眼后成纤维细胞中72 kDa热休克蛋白的基础和应激诱导的合成与表达及其亚细胞分布。我们的研究表明，这些化合物通过糖皮质激素受体发挥这些作用。
