Abstract
Ceramides (Cers) and complex sphingolipids with defined acyl chain lengths play important roles in numerous cell processes. Six Cer synthase (CerS) isoenzymes (CerS1-6) are the key enzymes responsible for the production of the diversity of molecular species. In this study, we investigated the changes in sphingolipid metabolism during the differentiation of Madin-Darby canine kidney (MDCK) cells. By MALDI TOF TOF MS, we analyzed the molecular species of Cer, glucosylceramide (GlcCer), lactosylceramide (LacCer), and SM in nondifferentiated and differentiated cells (cultured under hypertonicity). The molecular species detected were the same, but cells subjected to hypertonicity 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 no differences during cell differentiation. We next evaluated the different synthesis pathways with sphingolipid inhibitors and found that cells subjected to hypertonicity in the presence of amitriptyline, an inhibitor of acid sphingomyelinase, showed decreased radiolabeled incorporation in LacCer and cells did not develop a mature apical membrane. These results suggest that hypertonicity induces the endolysosomal degradation of SM, generating the Cer used as substrate for the synthesis of specific molecular species of glycosphingolipids that are essential for MDCK cell differentiation.
Highlights
Ceramides (Cers) and complex sphingolipids with defined acyl chain lengths play important roles in numerous cell processes
We have previously demonstrated that hypertonicity induces a specific program of sphingolipid metabolism that includes an increase in GSL synthesis that is essential for Madin-Darby canine kidney (MDCK) cell differentiation [14]
We analyzed by MALDI TOF TOF MS the molecular species of GlcCer in cells subjected to hypertonicity treated or not treated with fumonisin B1 (FB1).The analysis showed that the presence of FB1 did not evoke changes in the GlcCer profile in cells subjected to hypertonicity (Fig. 5E), demonstrating that FB1 did not exert any selective modification in the molecular species of GlcCer during MDCK cell differentiation
Summary
Ceramides (Cers) and complex sphingolipids with defined acyl chain lengths play important roles in numerous cell processes. We evaluated the different synthesis pathways with sphingolipid inhibitors and found that cells subjected to hypertonicity in the presence of amitriptyline, an inhibitor of acid sphingomyelinase, showed decreased radiolabeled incorporation in LacCer and cells did not develop a mature apical membrane These results suggest that hypertonicity induces the endolysosomal degradation of SM, generating the Cer used as substrate for the synthesis of specific molecular species of glycosphingolipids that are essential for MDCK cell differentiation.—Pescio, L. We have previously demonstrated that during the differentiation of Madin-Darby canine kidney (MDCK) cells induced by hypertonicity, cells develop a program of sphingolipid metabolism that includes an increase in GSL and SM synthesis [14, 15] This increased production of GSLs is essential to acquire the MDCK cell-differentiated phenotype, reflected by the development of the mature apical membrane domain and the formation of the primary cilium, considered the last steps in the final epithelial cell differentiation. The aim of the present work was to investigate the changes in the sphingolipid profile that are essential for MDCK cell differentiation and the pathways displayed by cells to achieve this optimal combination of molecular species of sphingolipids
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