Novel Endogenous Regulation of Lysosomal Stress Response in Lung Endothelium

National Jewish Health, Denver CO

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Abstract

? 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.