Crosstalk Between Tetraspanin‐Interacting Protein Igsf3 and Sphingolipid Metabolism

Abstract

RationaleBuilding on our finding that the tetraspanin‐interacting protein Igsf3 controls glucosylceramide levels in lung endothelial and epithelial cells, we investigated the mechanism and functional impact of Igsf3 regulation of sphingolipid metabolism. We focused on tetraspanin‐controlled cell functions, such as cell migration and adhesion and effectors such as the tight junction proteins, including claudins.MethodsPrimary rat lung endothelial cells and human bronchial epithelial cells (Beas2b) were stably transfected with lentiviral IGSF3 shRNA (R‐shIGSF3 and B2B‐shIGSF3, respectively) or control shRNA or with pIGSF3‐V5His overexpression plasmid. Primary human lung microvascular cells (HMVECL) were treated with IGSF3 siRNA or non‐targeting siRNA. Gene expression profiles were investigated using TaqMan Low Density Arrays (TLDA) for genes involved in the sphingolipid metabolism in epithelial cells with IGSF3 knockdown and in lymphocytes lacking IGSF3. Cell migration and cell adhesion assays were performed using standard methods.ResultsLoss of IGSF3 in B2B‐shIGSF3 and in lymphocytes, in parallel with a dysregulation of glucosylceramide levels, was associated with significant increases in Dihydroceramide Desaturase 1 (DEGS1, p<0.05, p<0.0001), Neutral Sphingomyelinase (SMPD2, p=0.07, p<0.05), Acid Ceramidase (ASAH1, p<0.0001, p<0.01), and Glucosylceramide Synthase (UGCG, p<0.01; ns) mRNA. Functionally, all approaches to decrease IGSF3 caused a significant delay in cell migration, whereas cells overexpressing IGSF3 had increased migration compared to controls (p<0.001). The decrease in migration was attenuated with pharmacological inhibition of Ugcg (p<0.001). In addition, adherent or non‐adherent cells with decreased IGSF3 expression exhibited enhanced cell adhesion to fibronectin (p<0.0001) or to other cells (p<0.01), respectively, which was attenuated with pharmacological inhibition of Asah1 (p<0.05) and was associated with increased expression of claudin 6 (CLDN6, p<0.05, p<0.05, respectively), and decreased expression of claudin 7 (CLDN7, p<0.001, p<0.0001, respectively) mRNA.ConclusionsThe tetraspanin‐interacting protein IGSF3 promotes cell migration and inhibits cell adhesion through inhibition of glucosylceramide synthase and acid ceramidase, respectively. Conditions characterized by loss of IGSF3, through an upregulation of sphingolipid production towards glycosphingolipids and decreased cell migration, may impair functions that require proper cell migration, such as lung tissue repair.Support or Funding InformationFunding: Wollowick Chair of Pulmonary Research, Department of Medicine Research Microgrant, National Jewish HealthThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.