mTOR信号对内皮细胞骨架及屏障功能的调控

The activation of cell contraction machinery which is triggered by MLC phosphorylation represents an important mechanism of endothelial (EC) barrier dysfunction. The compromised barrier function of endothelial monolayer plays a critical role in the pathogenesis of many diseases, such as acute lung injury, sepsis. Mechanistic target of rapamycin (mTOR) is a highly conservative serine/threonine protein kinase. It interacts with several proteins to form two multi-protein complexes, i.e., complex 1(mTORC1) and complex 2（mTORC2）which participate in the regulation of many cellular events. Hence the inhibitor of mTOR is widely used in clinic for treatment of cancer or as immune repressor after organ transplantation. However, so far how mTOR signaling regulates EC barrier function remains unknown. Also unknown is the mechanism of mTOR inhibition-caused side effect in clinic such as lung inflammation and injury. In our preliminary studies, we found inhibition of mTOR with compound or siRNA significantly promoted reorganization of F-actin, MLC phosphorylation and EC contraction. Upon mTOR inhibition, MLC kinase (MLCK) expression was upregulated and the activity of MLC phosphotase (MLCP) decreased via increased phosphorylation of its MYPT1 subunit at Ser696. Knockdown of either mTORC1 or mTORC2-specific component, Raptor or Rictor to disrupt mTORC1 or mTORC2 increased MLC phophorylation, indicating that both mTORC1 and mTORC2 contribute to the regulation of MLC phosphorylation and EC barrier function. This project aims to 1) explore how mTOR signaling modulates MLCK and MLCP to control MLC phosphorylation; 2) distinguish the distinct contribution and mechanism of mTORC1 and mTORC2; 3) seek direct evidence of the mTORC1 and/or mTORC2 inhibition in promoting EC barrier dysfunction and inflammation by using EC-specific mTOR，Raptor and Rictor knockout mice and experimental model of LPS-induced acute lung injury. The findings obtained from this project may shed light on the molecular regulation of mTOR signaling on EC barrier function, and potentiate therapeutic effects and minimize side effect of mTOR-inhibitory drugs in clinic.

肌球蛋白轻链（MLC）磷酸化由相应的激酶（MLCK）与磷酸酶（MLCP）正负调控，其触发的细胞收缩是内皮（EC）屏障功能降低的关键机制，后者为炎症的重要病理基础。mTOR是一种高度保守的Ser/Thr蛋白激酶，通过mTOR复合物（mTORC）1和2调控细胞生长与增殖等，其抑制剂雷帕霉素被广泛应用于抗肿瘤等治疗。我们在预实验中发现抑制mTOR，或敲降mTORC1或2成员Raptor或Rictor，促进EC收缩与MLC磷酸化、上调MLCK并降低MLCP活性，提示抑制mTOR信号可能降低EC屏障功能，并与雷帕霉素所致肺炎副作用相关。本课题将1）深入研究mTORC1和/或mTORC2对EC屏障功能的分子调控；2）以组织特异mTOR、Raptor和Rictor基因敲除小鼠，验证抑制EC内mTOR信号促进炎症。结果将揭示mTOR信号调控EC屏障功能的新机制；并提出抗mTOR治疗导致肺炎副作用的新途径。

肺微血管内皮（EC）屏障功能障碍与肺炎发生发展密切相关。虽然mTOR抑制剂药物广泛应用于治疗肿瘤和抗器官移植后排斥反应，但显著增加非感染性肺炎发生的风险。迄今为止，mTOR抑制是否影响肺EC屏障功能及其分子机制不明。本课题运用EC特异的mTOR复合物成员基因（Mtor, Rictor 和 Rptor）敲除小鼠，以LPS诱导的肺损伤模型证实了mTOR信号对于维持正常EC屏障功能至关重要。以药物抑制和基因沉默的方法处理体外培养的肺EC，结果显示：抑制mTOR通过PKCd/p38/NF-kB信号上调MLC激酶的表达；通过PKCa/RhoA/MYPT1途径抑制MLC磷酸酶活性。这两者均促进MLC磷酸化，EC细胞骨架收缩和高通透性。进一步的研究显示：mTOR复合物需要其激酶活性抑制EC中PKCd/p38/NF-κB，而Raptor不依赖于mTOR复合物促进PKCa的激活。这些mTOR抑制如何导致EC功能障碍的机制研究将为减轻临床上mTOR抑制剂药物的炎症副作用提供方向。