ROS-dependent STIM1 activation and Ca2+ entry in lung inflammation

描述(申请人提供)：在人类或动物模型的脓毒症期间，内毒素激活天然免疫细胞，通过过度产生促炎细胞因子而导致全身炎症，从而导致急性肺损伤(ALI)。在这一过程中，内皮细胞经历了氧化应激、细胞内钙超载、细胞器功能障碍和内皮细胞死亡等多种途径。尽管有大量关于导致内毒素诱导ALI的潜在分子机制的信息，但该途径中的几个元件仍有待确定。确定ALI的分子组成对于了解ALI的发病机制和确定潜在的治疗靶点至关重要。已知旁分泌和自分泌衍生的氧化剂都可以调节内皮细胞的氧化还原生物学。我们最近的研究表明，免疫细胞和自分泌的氧化剂都激活了肺内皮细胞的钙信号转导，而阻断氧化剂的产生则抑制了钙离子信号转导的促炎反应。此外，我们最近还证明了内质网钙离子驻留传感器STIM1也是ROS的靶标。因此，STIM1的氧化促进质膜Ca~(2+)通道Orai1的激活，从而提高胞内Ca~(2+)水平和基因转录调控。STIM操作的通道能够提高细胞内钙离子水平并诱导转录活性，这促使我们研究它在内毒素引起的炎症中的作用。我们在这项工作中提出的假设是，血管炎症是由于内毒素诱导的ROS过度产生，导致钙信号紊乱，线粒体功能和能量代谢中断，血管张力和血管完整性丧失所致。该项目的具体目标是：1)表征ROS诱导的钙信号在内皮细胞激活和炎症中的作用；2)研究靶向CRAC通道组件是否控制血管炎症反应。我们的目标是描述新的机制，涉及ROS，Ca~(2+)和相关的细胞内事件，这些事件在体外或体内暴露于内毒素后调节内皮细胞的基因转录，这可能有助于脓毒症的发生。这些研究将为调节内皮功能和促进ALI发病的基本机制提供新的见解。

DESCRIPTION (provided by applicant): During sepsis in either humans or in animal models, activation of innate immune cells by endotoxin leads to systemic inflammation via overproduction of proinflammatory cytokines resulting in acute lung injury (ALI). During this process, several pathways are operative in endothelial cells including, oxidative stress, intracellular Ca2+ overload and organelle dysfunction and endothelial cell death. Despite substantial information regarding the underlying molecular mechanisms that lead to endotoxin-induced ALI, several elements in the pathway remain to be identified. Identification of the molecular components in ALI is of critical importance in order to understand the progression of ALI pathogenesis and to identify potential targets for treatment of the disease. Both paracrine and autocrine-derived oxidants are known to modulate the redox biology of the endothelial cells. Our recent studies demonstrate that both immune cell- and autocrine-derived oxidants activate pulmonary endothelial Ca2+ signaling, while blockade of oxidant production inhibits Ca2+ signaling induced proinflammatory responses. Further, we have recently demonstrated that the ER resident Ca2+ sensor STIM1 is also a ROS target. Hence, oxidation of STIM1 facilitates activation of the plasma membrane Ca2+ channel Orai1, thereby elevating cytosolic Ca2+ levels and regulation of gene transcription. The ability of STIM-operated channels to elevate intracellular Ca2+ levels and induce transcriptional activity prompted us to investigate its role in inflammation caused by endotoxin. Our hypothesis for work proposed in this application is that vascular inflammation results from LPS-induced ROS overproduction which leads to perturbation of Ca2+ signaling, disruption of mitochondrial function and energy metabolism, loss of vascular tone and vascular integrity. The specific aims of this project are: 1) Characterize the role of ROS-induced Ca2+ signaling in endothelial cell activation and inflammation; and 2) To examine whether targeting the CRAC channel components control vascular inflammatory responses. Our goal is to characterize novel new mechanisms involving ROS, Ca2+ and related intracellular events that modulate gene transcription in endothelial cells after either in vitro or in vivo exposure to endotoxin which could contribute to development of sepsis. These investigations will provide new insights into the fundamental mechanisms that modulate endothelial function and contribute to the ALI pathogenesis.