Changes in ceramide metabolism are essential in Madin-Darby canine kidney cell differentiation

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.

Sphingosine kinase and sphingosine-1-phosphate regulate epithelial cell architecture by the modulation of de novo sphingolipid synthesis

“…The quantitative analysis shows that cells in progress to differentiation, in response to treatment with SK inhibitors increased the incorporation of [ 14 C] palmitic acid associated with total sphingolipids ( Fig 7A ). The analysis of the individual sphingolipids, whose identity was determined by mass spectrometry [ 16 ], showed that the treatment of cells with t-DHS evoked an increase in the radioactivity associated with Cer (4.9-fold). A small but significant increase was observed in both GluCer (1.3-fold) and SM (2.3-fold).…”

“…We have previously shown that, by modulating the expression and activity of sphingolipid metabolic enzymes, MDCK cells can direct the formation of the final metabolites that define cellular fate. In fact, we have reported that SK is highly expressed in proliferative state, thus maintaining cell proliferation and survival, while in differentiated cells, increased expression and activity of SM synthase and GluCer synthase ensure the establishment and preservation of cell-cell adhesion and mature apical membrane domain, typical of differentiated renal collecting duct cells [ 14 – 16 ].…”

“…For Cer and GluCer analysis, the TLC was developed in two different solvents. First, the TLC was developed to two-thirds of the plate in the same solvent system as before and then, the plate was cut at Rf = 0.8 (just above the sphingosine standard), and the remaining piece of the plate was developed in chloroform:methanol (50:1.5) [ 15 , 16 ]. Radioactive resolved sphingolipids were scanned (Storm 840, GE Healthcare).…”

“…We have also previously reported that, under external hypertonicity, MDCK cells acquire the fully differentiated morphological epithelial cell phenotype, which is driven by changes in sphingolipid metabolism. In fact, cell differentiation is accompanied by an increase in SM synthesis and an increase in C24:0 and C24:1 glucosylceramide (GluCer) generated from the Cer recycling pathway [ 14 – 16 ]. While the increase in SM synthesis is involved in the assembly of a mature adherens junction (AJ) [ 15 ], the development of a differentiated apical membrane domain depends on GluCer synthesis [ 14 ].…”

Bioavailable wine pomace attenuates oxalate-induced type II epithelial mesenchymal transition and preserve the differentiated phenotype of renal MDCK cells

“…Previous studies (Favale et al, 2007;Pescio et al 2012Pescio et al , 2017Casali et al, 2013) have demonstrated that MDCK cultures subjected to high NaCl-hyperosmolar medium accelerate their polarization-differentiation process. Thus, after 48-72 h of hyperosmolar treatment cells undergo morphological changes from a fibroblast-like phenotype into a polarized epithelial phenotype that includes primary cilium formation on apical membranes almost reminiscent of medullary collecting duct cells.…”