Implication of sphingolipid metabolism in MDCK cells transition from polarized to differentiated phenotype

NICOLÁS O. FAVALE; LUCILA G. PESCIO; MARÍA GABRIELA MÁRQUEZ; NORMA B. STERIN-SPEZIALE

Congreso; 52nd International Conference on the Bioscience of Lipids (ICBL); 2011

We have previously demonstrated that hypertonicity induced MDCK differentiation accompanied by increase in glycosphingolipids (GSLs) and sphingomyelin (SM) biosynthesis. While GSLs are involved apical domain maturation, SM is the major sphingolipid of basolateral membrane. We aim to study the importance of SM synthesis in the transition from polarized to differentiated MDCK cells. Confluent MDCK cells were submitted to hypertonicity and concomitantly treated or not (control) with 7.5; 10.0; or 20.0_molar of D609 an SM synthase1 inhibitor. Sphingolipid metabolism was determined by using [14C]Palmitic acid precursor. After 48 h, cell viability was determined by tripan blue and their phenotype visualized by confocal immunofluorescence of the cytoskeleton using phalloidin. Cell?cell adhesion was determined with anti alfa and beta catenin. As inhibitor concentration rise the cell?cell adhesions were impaired, and the characteristic polarized phenotype of the cells were lost. First cells appeared lengthened, and thereafter acquired a fibroblast like phenotype. Beta catenin signal decreased and appeared discontinued. Polymerized actin seemed not to be altered, cortically located and increased stress fibers were observed at the highest D609 concentration. From these results we suggest, instead to differentiate, polarized MDCK cells undergo mesenchymal transition when induced to differentiate in a condition of inhibition of SM synthesis.