Changes in ceramide metabolism are essential in Madin-Darby Canine Kidney cell differentiation

PESCIO, LUCILA GISELE; SANTACREU, BRUNO JAIME; LOPEZ, VANINA GISELA; PAVÁN, CARLOS HUMBERTO; ROMERO, DANIELA JUDITH; FAVALE, NICOLÁS OCTAVIO; STERIN-SPEZIALE, NORMA BEATRIZ

JLR PAPERS IN PRESS

AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

Año: 2017

0022-2275

Ceramides and complex sphingolipids with defined acyl-chain lengths play important roles in numerouscell processes. Six ceramide synthase isoenzymes (CerS1-6) are the key enzymes responsible for theproduction of the diversity of molecular species. In this study, we investigated the changes in sphingolipidmetabolism during the differentiation of Madin-Darby Canine Kidney (MDCK) cells. By MALDI TOFTOF MS, we analyzed the molecular species of ceramide (Cer), glucosylceramide (GlcCer),lactosylceramide (LacCer) and sphingomyelin (SM) in non-differentiated and differentiated cells(cultured under hypertonicity). The molecular species detected were the same, but cells subjected tohypertonicity presented higher levels of C24:1 Cer, C24:1 GlcCer, C24:1 SM and C16:0 LacCer.Consistently with the molecular species, MDCK cells expressed CerS2, CerS4 and CerS6, but with nodifferences during cell differentiation. We next evaluated the different synthesis pathways withsphingolipid inhibitors and found that cells subjected to hypertonicity in the presence of amitriptyline, aninhibitor of acid sphingomyelinase, showed decreased radiolabeled incorporation in LacCer and cells didnot develop a mature apical membrane. These results suggest that hypertonicity induces theendolysosomal degradation of SM, generating the Cer used as substrate for the synthesis of specificmolecular species of glycosphingolipids that are essential for MDCK cell differentiation.