Sphingosine 1 Phosphate Synthesis Controls the Preservation of Epithelial Barrier During MDCK Cell Differentiation

SANTACREU BJ; STERIN SPEZIALE, NB; FAVALE, NO

Boston

Congreso; Experimental Biology 2015; 2015

American Society of Biochemistry and Molecular Biology (ASBMB)

Sphingosine 1-Phophate (S1P) is a sphingolipid mediator of cellular fate. We have demonstrated that hypertonicity induces epithelial tissue organization by the establishment of mature adherent junctions. Organization and preservation of epithelial tissue requires an efficient cell extrusion followed by a closing of cellular gap to preserve tissue permeability. We study the involvement of sphingosine kinase (SK) activity in the transition from polarized to differentiated MDCK cells. Confluent MDCK cells were subjected to hypertonic medium with the concomitant knock down of SK or not (control), or pharmacological inhibition of SK. After 48 h, cell phenotype was visualized by fluorescence microscopy, evaluating actin cytoskeleton and Adherens Junction (AJ) formation. By performing a confocal z-plane reconstruction we visualized extruding cells and found that the inhibition of SK evoked an alteration of efficient cell extrusion. SK knock-down induced AJ disassembling, and actin cytoskeleton reorganization. SK inhibition produced an increase in de novo sphingolipid synthesis with Ceramide (Cer) accumulation. In order to evaluate whether AJ disassembly is due to Cer accumulation, Fumonicin B1 (FB1) was used, as inhibitor of Ceramide Synthase. FB1 treatment recovers MDCK phenotype, suggesting that the disassembly of AJ due to inhibition of SK activity is an indirect effect produced by Cer accumulation.