Role of IGSF3 in Lung Epithelial Barrier Disruption Induced by Cigarette Smoke Exposure

Abstract

RationaleCigarette smoke (CS) exposure disrupts lung epithelial and endothelial cell barrier function which is linked to lung injury and inflammation. We identified a tetraspanin‐interacting protein, IGSF3, with roles in cell proliferation, adhesion, and migration via unclear mechanisms. We have shown that lung IGSF3 is decreased by cigarette smoke (CS) exposure and that loss of IGSF3 can disrupt sphingolipid metabolites. Of these, sphingosine‐1 phosphate (S1P) protects endothelial barrier function but may disrupt epithelial barrier via distinct receptors. We investigated if IGSF3 and S1P are required for maintaining the lung epithelial barrier in response to CS.MethodsHuman bronchial lung epithelial cells (Beas2b) were stably transfected with lentivirus control or IGSF3 shRNA (shIGSF3). Trans‐epithelial resistance (TER) was measured using electric cell‐substrate impedance sensing. CS extract was prepared using research cigarettes (3R4F, University of KY). Phosphatidylinositol lipid strips (Echelon) were used to study IGSF3‐lipid binding.ResultsCompared to control shRNA, knockdown of IGSF3 did not alter epithelial barrier function as measured by TER. Exposure to CS (7.5%), as expected, decreased epithelial cell monolayers TER in control cells by 35%. CS‐induced effects on TER were attenuated by treatment with N‐acetylcysteine or with (R)‐FTY720‐phosphonate (1R), a sphingosine‐1 phosphate receptor 1 (S1PR1) agonist, and were worsened by sphingosine kinase‐1 inhibition using SKI2. The CS‐induced decrease in TER was significantly worsened (by 55%, p<0.01 vs. control cells exposed to CS) in shIGSF3 ‐ transfected cells which responded in turn to the barrier‐enhancing effect of N‐acetylcysteine treatment (by 30%, p<0.05). A putative Pleckstrin‐homology (PH) domain was identified in the C‐terminal cytoplasmic domain of IGSF3 which predicted binding to PI(3,4)P2, which was then experimentally confirmed. Further, site directed mutagenesis within PH domain disrupted binding of IGSF3 with tetraspanin CD81.ConclusionsIGSF3 is required for partial maintenance of the lung epithelial barrier function in response to CS injury. Loss of IGSF3 may worsen CS injury of the airways, contributing to inflammation, an effect that could be alleviated by antioxidants and S1P1‐mediated signaling. The mechanisms of IGSF3 barrier effects may include binding to and tethering PI(3,4)P2 and CD81 at the cell membrane via its PH domain.Support or Funding InformationFunding: Wollowick Chair of Pulmonary Research (IP), Department of Medicine Research Microgrant (KSS) from National Jewish Health.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.