ROS诱导CaMKIIγ核转位在醛固酮合成中的作用及其机制

Dysregulation of aldosterone biosynthesis has been demonstrated to be involved not only in the pathogenesis of various cardiovascular diseases, but also in the occurrence of primary aldosteronism. Results of our previous studies documented that Nox2-derived reactive oxygen species (ROS) plays an important role in aldosterone biosynthesis. Further experiments proved that ROS promote aldosterone synthase (CYP11B2) gene expression and increase the phosphorylation of CaMKII, CaMKIV and ATF2. More importantly, we observed CaMKIIγ and CaMKIV expressions in the cytoplasm and nucleus of adrenal aldosterone-producing adenoma. Studies have reported that CaMKIIγ contains a nuclear localization signal within its DNA binding domain. And activating transcription factor 2 (ATF2) is the crucial factor for CYP11B2 transcription. Therefore, we propose that ROS may regulate aldosterone synthesis by CaMKIIγ/ATF2 pathway. The project intends to use mass spectrometry, point-mutation and laser confocal to clarify the mechanism of ROS-dependent aldosterone synthesis through CaMKIIγ nuclear translocation. This research may reveal novel mechanism of aldosterone biosynthesis, and provide scientific basis for prevention and cure of aldosterone-induced target organ damage.

醛固酮在心脑血管疾病的发生发展中起着重要的介导作用。醛固酮合成调控过程异常不仅与高血压发生发展密切相关，也与原发性醛固酮增多症的形成有关；而醛固酮分泌调控及其失常机制仍有诸多环节有待阐明。课题组前期研究发现NADPH氧化酶亚型Nox2来源的ROS是调节醛固酮合成的重要因素；进一步实验证实ROS促进醛固酮合成酶（CYP11B2）表达，伴有CaMKII、CaMKIV和ATF2磷酸化；且发现CaMKIIγ和CaMKIV在醛固酮瘤细胞胞浆和胞核均有表达。研究报道CaMKIIγ存在核定位信号，而ATF2是直接调控CYP11B2基因的重要转录调控因子。由此，我们提出ROS可能通过CaMKIIγ/ATF2通路调节醛固酮合成。本项目拟采用质谱分析、定点突变和激光共聚焦等方法，阐明ROS促进CaMKIIγ核转位调控醛固酮合成的机制。该工作的完成有望为遏制醛固酮介导的靶器官损害提供新的理论基础和防治依据。

醛固酮是肾上腺皮质球状带细胞合成和分泌的盐皮质激素。醛固酮在调节肾脏钠水重吸收、维持正常血压等方面起着重要的生理作用。课题组前期研究发现NADPH氧化酶亚型Nox2来源的ROS是调节醛固酮合成的重要因素。在此基础上，应用免疫组化的技术发现CaM、CaMKIIβ和CaMKIIγ在醛固酮瘤细胞胞浆和胞核均有表达。应用电镜检测正常肾上腺球状带细胞和醛固酮瘤细胞的细胞结构和细胞器的改变，结果显示醛固酮瘤细胞核核仁明显，线粒体丰富，提示重要细胞器改变在醛固酮合成中起重要作用。在细胞水平，阻断CaN可抑制外源性ROS和内源性ROS诱导CaMKIIγ表达和醛固酮合成。此外，应用siRNA干扰等技术证实Nox2来源的ROS上调CaMKIIβ和CaMKIIγ基因和蛋白表达；进一步干扰CaMKIIγ可降低ATF2磷酸化和CYP11B2表达，证实ROS通过CaMKIIγ触发细胞核ATF2磷酸化，启动醛固酮的合成。由此可见，ROS可能通过ROS-CaN-CaMKIIγ-ATF2信号通路调控醛固酮合成。本研究从细胞内氧化还原状态的角度认识生理性和病理性醛固酮合成的机制，为选择新的药物靶点提供科学依据，为高血压等心脑血管疾病的防治提供理论依据。