ZIP12介导的锌信号在肺动脉高压发病机制中的作用

There are numerous key molecules playing pivotal roles in the development of pulmonary arterial hypertension (PAH), some of which may be of therapeutic significance. Our preliminary study showed a sustained activation of CREB pathway in the pathogenesis of PAH, which is associated with the activation of multiple protein kinases that positively regulate CREB and down-regulation of phosphatases that negatively regulate CREB. However, its upstream regulatory mechanism is still unclear. Recently, it was found that intracellular free zinc plays an important role in the regulation of CREB pathway and ZIP12 is a major transporter mediating the influx of extracellular zinc. Based on above available data, we hypothesize that the upregulated ZIP12 induced by hypoxia and monocrotaline (MCT) triggers the zinc signaling, which regulates the CREB pathway directly or indirectly by inhibiting phosphatases, resulting in the sustained activation of CREB, the proliferation and migration of pulmonary artery smooth muscle cell (PASMC) and pulmonary vascular remodeling. In this study, we use PAH rat induced by hypoxia and MCT, ZIP12 knockout rat model and PASMC, and many techniques such as qPCR, western blot, transcriptome sequencing and others to reveal the role of zinc signaling mediated by ZIP12 in the regulation of CREB pathway and in the pathogenesis of PAH, hoping that potential critical targets may be found for therapy of PAH.

肺动脉高压（PAH）发生中有许多起重要作用的关键分子，其中一些可能为潜在的治疗靶点。预实验发现CREB通路在PAH发病过程中持续性激活，与多种正调控CREB磷酸化的激酶激活和负调控CREB的磷酸酶下调有关，但其上游的调控机制仍然不清。近年来发现细胞内的游离锌调控CREB的活性，而ZIP12为介导细胞外锌流入的主要转运体。据此，我们假设：缺氧和野百合碱（MCT）诱导ZIP12的表达触发锌信号，锌信号通过抑制磷酸酶直接或间接调控CREB通路，引起CREB的持续性激活，促进肺动脉平滑肌细胞（PASMC）增生、迁移和肺血管重构。本项目拟以缺氧和MCT诱导的PAH大鼠、ZIP12敲除的大鼠和PASMC为对象，采用qPCR、蛋白印迹和转录组测序等手段，从整体、细胞和分子多个层面探讨ZIP12介导的锌信号在调控CREB通路、促进PAH发病中的作用，从而为PAH提供潜在的关键治疗靶点。

肺动脉高压（PH）发生中有许多起重要作用的关键分子，其中一些可能为潜在的治疗靶点。通过转录组学和生物信息学分析发现，细胞外空间被最显著的富集，结合动态变化的基因表达谱，分别鉴别出Lcn2、Adamts4和Slc26a4为潜在的早期诊断诊断PH的血液学标志物，并使用qPCR和ELISA实验证实了以上结果。转录因子CREB在 PH发病过程中持续性激活，与多种正调控CREB磷酸化的激酶激活和负调控CREB的磷酸酶下调有关，但其上游的调控机制仍然不清。我们通过生物信息学分析发现，在野百合碱（MCT）和缺氧诱导的PH中含CRE的差异表达基因大量存在。Western blot发现从MCT诱导的PH大鼠分离的肺动脉平滑肌细胞（PASMCs）中总CREB和磷酸化CREB表达持续升高。PASMCs中持续激活的CREB磷酸化与多种磷酸化CREB的激酶有关。此外聚类分析发现，在PH中主要的CREB磷酸酶表达水平下降，包括PP2A、Ppp2r2c和Ppp2r3a调节亚基。细胞增殖分析发现MCT诱导的PH-PASMCs增殖依赖于CREB介导的转录活性。进一步分析揭示依赖于ZIP12的细胞内锌离子浓度升高可能磷酸酶表达下降，CREB介导的转录活性升高及细胞增殖有关。此外我们研究还发现，ZIP2通过促进PASMCs的增殖和迁移，参与MCT诱导的肺血管重构，其机制可能与AKT/ERK通路部分激活相关。锌离子及其调控的信号通路参与PH的发生发展，并可能作为PH潜在治疗靶点。