Department of Laboratory Medicine, the First Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong, China.
Int Immunopharmacol. 2012 Nov;14(3):334-40. doi: 10.1016/j.intimp.2012.08.012. Epub 2012 Aug 24.
Resistance to glucocorticoid (GC) is a challenge for the treatment of patients with idiopathic nephrotic syndrome (INS). Most of the effects of GC are mediated by the GC receptor (GR). Heat shock protein 90 (HSP90) is an important molecular chaperone for the GR and is supposed to be the key factor in regulating GC effects. In a previous study, we found that both the expression and nuclear distribution of HSP90 were increased in GC resistant INS patients. The aim of this study is to explore how these phenomena contribute to GC resistance in INS patients. Healthy subjects and INS patients with different GC responses were recruited. The total HSP90 expression was determined by reverse transcription-PCR and flow cytometric analysis. Western blot analysis was used to evaluate the expression of nuclear HSP90. Co-immunoprecipitation and electrophoretic mobility gel shift assays were performed to explore the interaction between HSP90 and the GR in the nucleus as well as the DNA-binding activity of GR. We induced the upregulation of the expression of total HSP90 in PBMCs by treatment with interleukin-6 in vitro and found that the nuclear HSP90 level, the DNA-binding activity of the GR and the cell apoptotic responsiveness to GC remained unchanged. Furthermore, an increased nuclear HSP90 was demonstrated mainly by binding to GR in the nucleus, while the DNA-binding activity of the GR dramatically decreased in GC resistant INS patients. The present results suggest that the accumulation of HSP90 in the nucleus potentially hinders DNA-binding activity and transactivation, which may contribute to GC resistance in patients with INS.
糖皮质激素（GC）耐药是特发性肾病综合征（INS）患者治疗的一大挑战。GC 的大多数作用是通过糖皮质激素受体（GR）介导的。热休克蛋白 90（HSP90）是 GR 的重要分子伴侣，被认为是调节 GC 作用的关键因素。在之前的研究中，我们发现 GC 耐药 INS 患者的 HSP90 表达和核分布均增加。本研究旨在探讨这些现象如何导致 INS 患者 GC 耐药。招募了不同 GC 反应的健康受试者和 INS 患者。通过逆转录-PCR 和流式细胞术分析测定总 HSP90 表达。Western blot 分析用于评估核 HSP90 的表达。通过共免疫沉淀和电泳迁移率凝胶阻滞试验研究 HSP90 与核内 GR 之间的相互作用以及 GR 的 DNA 结合活性。我们通过体外用白细胞介素-6 处理诱导 PBMCs 中总 HSP90 表达上调，发现核 HSP90 水平、GR 的 DNA 结合活性和细胞对 GC 的凋亡反应性保持不变。此外，在 GC 耐药 INS 患者中，核内 HSP90 的增加主要通过与 GR 结合来实现，而 GR 的 DNA 结合活性则显著降低。这些结果表明，HSP90 在核内的积累可能会阻碍 DNA 结合活性和反式激活，这可能导致 INS 患者的 GC 耐药。
