Novel Endogenous Regulation of Lysosomal Stress Response in Lung Endothelium

 描述（由申请人提供）：该博士前奖学金培训提案旨在阐明在基线和压力（如香烟烟雾（CS）暴露诱导的压力）期间调节内皮肺细胞命运的基本机制。这项工作与理解和开发肺气肿的未来疗法有关，肺气肿是一种以吸烟相关的肺结构细胞损失为特征的疾病，目前是美国主要的死亡原因之一。在肺气肿中，内皮和上皮细胞损伤的主要机制是细胞凋亡导致的细胞死亡，细胞凋亡通常在细胞自噬之前，细胞自噬是通过其成分的再循环来实现细胞存活的一种尝试。肺气肿发病机制中一个基本但未回答的问题是自噬期间促存活信号传导转变为促凋亡信号传导的分子基础是什么。为了回答这个问题，我们专注于生物活性鞘脂的作用，鞘脂是细胞内压力生物传感器，通过复杂调节的代谢途径快速产生。CS暴露增加神经酰胺的内皮水平，神经酰胺是一种典型的促凋亡鞘脂，最近也参与自噬。申请方获得的初步数据表明，溶酶体酸性鞘磷脂酶（ASM）（一种从鞘磷脂产生神经酰胺的酶）的活化水平可能感知和控制自噬，并可能诱导细胞凋亡以响应CS暴露。尽管CS快速激活ASM以产生促凋亡神经酰胺，但在CS暴露期间抑制ASM不仅没有减少，而且实际上刺激了自噬。此外，即使基础抑制ASM活性显着增加了自噬溶酶体的形成在体外和体内。这些数据表明，以前未被怀疑的ASM参与调节内皮细胞自噬。鉴于ASM的溶酶体膜位置，我们建议ASM参与溶酶体控制自噬。我们假设ASM活性是自噬和细胞凋亡的变阻器，其中溶酶体ASM抑制的抑制通过溶酶体营养感测（LYNUS）机制触发自噬，而其通过氧化应激的激活触发细胞凋亡。我们的目的是研究基础溶酶体ASM活性是否稳定LYNUS复合物并提供自噬的内源性控制，并确定CS刺激ASM对自噬通量和肺内皮细胞命运的功能影响。

 DESCRIPTION (provided by applicant): This proposal for predoctoral fellowship training seeks to elucidate a fundamental mechanism that regulates endothelial lung cell fate at baseline and during stress such as that induced by cigarette smoke (CS) exposure. This work is relevant to understanding and developing future therapies for emphysema, a disease characterized by loss of structural cells in the lung associated with cigarette smoking, and currently one of the leading causes of death in the US. In emphysema, a main mechanism of endothelial and epithelial cellular injury is cell death by apoptosis, which is typically preceded by cell autophag, an attempt at cellular survival via recycling of its constituents. A fundamental but unanswered question in emphysema pathogenesis is what is the molecular basis for the switch from pro-survival signaling during autophagy to pro-apoptotic signaling. To answer this question, we focused on the role of bioactive sphingolipids, which are intracellular stress biosensors, rapidly generated by intricately regulated metabolic pathways. CS exposure increases endothelial levels of ceramide, a prototypical pro-apoptotic sphingolipid, recently also involved in autophagy. The applicant obtained preliminary data indicating that the levels of activation of the lysosomal acid sphingomyelinase (ASM), an enzyme which produces ceramide from sphingomyelin, could be sensing and controlling autophagy and could induce apoptosis in response to CS exposure. Whereas CS rapidly activates ASM to produce pro- apoptotic ceramides, inhibition of ASM during CS exposure not only did not reduce, but actually stimulated autophagy. Furthermore, even basal inhibition of ASM activity markedly increased the formation of autophagolysosomes in vitro and in vivo. These data suggest a previously unsuspected involvement of ASM in the regulation of endothelial cell autophagy. Given the lysosomal membrane location of the ASM, we propose that ASM is involved in the lysosomal control of autophagy. We hypothesize that ASM activity is a rheostat for autophagy and apoptosis, wherein the inhibition of lysosomal ASM inhibition triggers autophagy via the lysosomal nutrient sensing (LYNUS) machinery, while its activation by oxidative stress triggers apoptosis. We aim to investigate if basal lysosomal ASM activity stabilizes the LYNUS complex and provides endogenous control of autophagy and to determine the functional impact of ASM stimulation by CS on the autophagic flux and on the lung endothelial cell fate.