HIF-1α调控GR在库欣病糖皮质激素抵抗中的机制研究

The mortality of cushing disease is rather high. Glucocorticoid resistance due to decreased expression of GR is an important pathogenesis. But nowadays there are no effective treatments for the disease. In previous study, we have found HIF-1α upregulated mRNA and protein expression levels of GR gene in AtT-20 cell lines in a hypoxia-dependent manner. However, GR expression was downregulated after treated with DEX in hypoxia states. We employed Co-IP and found a HIF-1α-GR complex in the cells treated with CoCl2 and DEX with an unkonwn mechanism. Furthermore, cells transfected with HIF-1α-siRNA were then treated with CoCl2 and dexamethasone, showing an increased GR expression levels. According to these findings, we suppose that in the context of cushing disease, HIF-1α’s regulation of GR may be in a direct protein-protein manner. With this in mind, we will construct different splicts deleted mutant of HIF-1α and GR gene, employ plasmids transfection and GST tags to clarify the specific fragments responsible for HIF-1α-GR interaction. Different methods are utilized to confirm that downregulation of GR by HIF-1α depends on the formation of HIF-1α-GR complex. Ubiquitinoylation will be detected to clarify the degradion pathway of GR by the complex. Cushing disease animal model will be constructed to verify the above mechanism and to test the functional role of HIF-1α-inhibitor in cushing disease, thus shedding light on a novel therapeutic target for cusing disease.

库欣病具有极高的死亡率，糖皮质激素受体（GR）表达下调所引起的糖皮质激素抵抗是重要的发病机制之一，但目前尚无有效的治疗方法。我们前期实验发现，低氧促进AtT-20细胞GR的表达，同时用地塞米松（DEX）处理细胞后，GR的表达会下调，下调程度与缺氧程度呈正相关；通过CO-IP实验，我们发现了HIF-1α-GR复合体的形成。相反，沉默HIF-1α后GR的表达即上调。据此，我们推测在低氧和DEX共存的微环境中，HIF-1α对GR的调控方式可能为蛋白-蛋白的直接作用。本项目拟通过构建HIF-1α和GR不同片段缺失的突变体、基因转染、GST标签等手段确认两者结合的特异片段，验证GR的表达下调依赖HIF-1α-GR复合体的形成；明确HIF-1α-GR复合体对GR泛素化的影响；并在裸鼠库欣病模型上验证该机制；同时检测HIF-1α抑制剂对糖皮质激素抵抗的改善作用，从而为库欣病的药物治疗提供新靶点。

库欣病具有极高的死亡率，糖皮质激素受体（GR）表达下调所引起的糖皮质激素抵抗是重要的发病机制之一，但其机制不明，也无有效的治疗方法。我们前期实验发现，低氧促进AtT-20细胞GR的表达，同时用地塞米松（DEX）处理细胞后，GR的表达会下调，下调程度与缺氧程度呈正相关；通过CO-IP实验，我们发现了HIF-1α-GR复合体的形成。相反，沉默HIF-1α后GR的表达即上调。据此，我们推测在低氧和DEX共存的微环境中，HIF-1α对GR的调控方式可能为蛋白-蛋白的直接作用。为了证实该假说，我们在本项目中按计划研究了以下内容：1、低氧环境下，验证HIF-1α对GR 转录活性的影响；2、验证低氧和地塞米松共存的微环境中，HIF-1α-GR 复合体的形成及其引起GR 降解的具体机制；3、建立皮下成瘤制备裸鼠库欣病模型以验证HIF-1α抑制剂LXY6006对糖皮质激素抵抗的改善作用。我们取得了以下研究成果：1、在库欣病中，HIF-1α和GR之间存在转录水平和翻译后水平的相互作用。2、缺氧和DEX共存时HIF1a通过泛素化蛋白酶体途径导致GR下降。3、在裸鼠模型中验证了HIF1a抑制剂对于库欣病的治疗效果。