Metabolic Profiling and Quantification of Sphingolipids by Liquid Chromatography-Tandem Mass Spectrometry

Abstract

Epithelial-to-mesenchymal transition is a differentiation switch from polarized epithelial cells to mesenchymaltype cells. The cellular event is triggered by a number of exogenously added cytokines, such as TGF-ß, and is often accompanied by an increase in migratory and invasive capacity as evidenced by altered expression of adhesion molecules and matrix degrading enzymes. The present study focuses on how the transition process modulates the expression of heparanase. Heparanase is a heparan sulfate-specific endo-ß-glucuronidase substantially involved in the degradation of heparan sulfate proteoglycan, a matrix component in the basement membrane, and thereby potentiates cell invasion. The murine mammary epithelial cell lines NMuMG and EpH4 expressing c-Ha-Ras gene when cultured with TGF-ß3 underwent epithelial-to-mesenchymal transition as confirmed by downmodulation of E-cadherin and upregulation of N-cadherin.In TGF-ß3-treated cells, heparanase expression was downmodulated at the mRNA and protein level. Heparan sulfate degradation activity of cell lysates was examined using radiolabeled basement membrane deposited by bovine corneal endothelial cells as a substrate. Cell lysate made from untreated epithelial cells but not from TGF-ß3- treated cells released downsized heparan sulfate fragments into the supernatant. Causal involvement of heparanase in the transition process was examined. Addition of the heparanase inhibitor Heparastatin (SF4) and overexpression of the heparanase transgene did not influence the transition process, clearly indicating that heparanase expression is dispensable for the process of epithelial-to-mesenchymal transition.