Sphingosine 1 phosphate controls Sphingolipids synthesis during MDCK cell differentiation

BRUNO JAIME SANTACREU; NORMA BEATRIZ STERIN-SPEZIALE; NICOLAS OCTAVIO FAVALE

Rosario

Congreso; L Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology Research (SAIB); 2014

Sphingosine 1-Phophate (S1P) is an important sphingolipid mediator in cell fate, synthetized by Sphingosine Kinase (SK). We have demonstrated that hypertonic media induce the establishment of differentiated phenotype of MDCK cells. In this situation the sphingolipid metabolism change and increase in the salvatage pathway. We study the involvement of SK activity in this process. For this end, confluent MDCK cells were subjected to hypertonic medium with the concomitant knock down of SK or not (control). Additionally we use D,L-threo-dihydrosphingosine (DHS) as an SK inhibitor. After 48 h of incubation, the cell phenotype was visualized by fluorescence microscopy, evaluating actin cytoskeleton and Adherens Junction (AJ) formation. SK knock down induces Adherens Junction protein redistribution to intracellular localization resulting in disassembling of AJ, nuclear accumulation of β-catenin and actin cytoskeleton reorganization. SK inhibition also induces 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, an 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.