Endothelial Knockdown of the Tumor Suppressor, WWOX, Increases Inflammation in Ventilator-Induced Lung Injury

Abstract

BackgroundChronic cigarette smoke exposure downregulates lung expression of WWOX, an ARDS relevant tumor suppressor. Prior work has revealed a barrier protective function of WWOX during infectious models of ARDS. Proteomic analysis of WWOX-silenced lung endothelial cells suggest involvement of WWOX in protection against mechanical stretch-induced inflammation. MethodsProtein lysates from WWOX-silenced endothelial cells (ECs) were analyzed using tandem mass tag mass spectrometry (TMT-MS) to determine the differential expression status of the proteome compared to wild type ECs. WWOX-silenced ECs as well as those isolated from endothelial Wwox knockout (EC Wwox KO) mice were subjected to cyclic stretch (18% elongation, 0.5 Hz, 4 hours). Cellular lysates and media supernatant were harvested for assays of cellular signaling, protein expression, and cytokine release. Dual silencing of WWOX and zyxin was achieved to determine the role of zyxin upregulation in IL-8 production following mechanical stretch and during WWOX knockdown. Control and EC Wwox KO mice were subjected to high tidal volume ventilation (VILI, 40ml/kg, 65 breath/min, 4hours). Bronchoalveolar lavage fluid and mouse lung tissue were harvested for cellular signaling, cytokine secretion, and histologic assays. ResultsTMT-MS revealed upregulation of zyxin expression during WWOX knockdown which predicted a heightened inflammatory response to mechanical stretch. WWOX-silenced ECs and ECs isolated from EC Wwox mice displayed significantly increased cyclic stretch-mediated secretion of various cytokines (IL-6, KC/IL-8, IL-1{beta}, and MCP-1) relative to controls. This was associated with increased ERK and JNK phosphorylation but decreased p38 MAPK phosphorylation. EC Wwox KO mice subjected to VILI sustained a greater degree of injury than corresponding controls. Silencing of zyxin during WWOX knockdown abrogated stretch-induced increases in IL-8 secretion. ConclusionLoss of WWOX function in ECs is associated with a heightened inflammatory response during mechanical stretch that is associated with increased MAPK phosphorylation and appears to be dependent on upregulation of zyxin.