Influence of sphingomyelin metabolism during epithelial-mesenchymal transition associated with aging in the renal papilla.

The renal collecting ducts (CD) are formed by a fully differentiated epithelium, and their tissue organization and function require the presence of mature cell adhesion structures. In certain circumstances, the cells can undergo de-differentiation by a process called epithelial-mesenchymal transition (EMT), in which the cells lose their epithelial phenotype and acquire the characteristics of the mesenchymal cells, which includes loss of cell-cell adhesion. We have previously shown that in renal papillary CD cells, cell adhesion structures are located in sphingomyelin (SM)-enriched plasma membrane microdomains and the inhibition of SM synthase 1 activity induced CD cells to undergo an EMT process. In the present study, we evaluated the influence of SM metabolism during the EMT of the cells that form the CD of the renal papilla during aging. To this end, primary cultures of renal papillary CD cells from young, middle-, and aged-rats were performed. By combining biochemical and immunofluorescence studies, we found experimental evidence that CD cells undergo an increase in spontaneous and reversible EMT during aging and that at least one of the reasons for this phenomenon is the decrease in SM content due to the combination of decreased SM synthase activity and an increase in SM degradation mediated by neutral sphingomyelinase. Age is a risk factor for many diseases, among which renal fibrosis is included. Our findings highlight the importance of sphingolipids and particularly SM as a modulator of the fate of CD cells and probably contribute to the development of treatments to avoid or reverse renal fibrosis during aging.

The doxorubicin-induced cell motility network is under the control of the ceramide-activated protein phosphatase 1 alpha.

We have recently reported that a specific pool of ceramide, located in the plasma membrane, mediated the effects of sublethal doses of the chemotherapeutic compound doxorubicin on enhancing cancer cell migration. We identified neutral sphingomyelinase 2 (nSMase2) as the enzyme responsible to generate this bioactive pool of ceramide. In this work, we explored the role of members of the protein phosphatases 1 family (PP1), and we identified protein phosphatase 1 alpha isoform (PP1 alpha) as the specific PP1 isoform to mediate this phenotype. Using a bioinformatics approach, we build a functional interaction network based on phosphoproteomics data on plasma membrane ceramide. This led to the identification of several ceramide-PP1 alpha downstream substrates. Studies on phospho mutants of ezrin (T567) and Scrib (S1378/S1508) demonstrated that their dephosphorylation is sufficient to enhance cell migration. In summary, we identified a mechanism where reduced doses of doxorubicin result in the dysregulation of cytoskeletal proteins and enhanced cell migration. This mechanism could explain the reported effects of doxorubicin worsening cancer metastasis in animal models.

Ceramide launches an acute anti-adhesion pro-migration cell signaling program in response to chemotherapy.

Chemotherapy has been reported to upregulate sphingomylinases and increase cellular ceramide, often linked to the induction to cell death. In this work, we show that sublethal doses of doxorubicin and vorinostat still increased cellular ceramide, which was located predominantly at the plasma membrane. To interrogate possible functions of this specific pool of ceramide, we used recombinant enzymes to mimic physiological levels of ceramide at the plasma membrane upon chemotherapy treatment. Using mass spectrometry and network analysis, followed by experimental confirmation, the results revealed that this pool of ceramide acutely regulates cell adhesion and cell migration pathways with weak connections to commonly established ceramide functions (eg, cell death). Neutral sphingomyelinase 2 (nSMase2) was identified as responsible for the generation of plasma membrane ceramide upon chemotherapy treatment, and both ceramide at the plasma membrane and nSMase2 were necessary and sufficient to mediate these "side" effects of chemotherapy on cell adhesion and migration. This is the first time a specific pool of ceramide is interrogated for acute signaling functions, and the results define plasma membrane ceramide as an acute signaling effector necessary and sufficient for regulation of cell adhesion and cell migration under chemotherapeutical stress.

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

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.

Sphingomyelin metabolism is involved in the differentiation of MDCK cells induced by environmental hypertonicity.

Sphingolipids (SLs) are relevant lipid components of eukaryotic cells. Besides regulating various cellular processes, SLs provide the structural framework for plasma membrane organization. Particularly, SM is associated with detergent-resistant microdomains. We have previously shown that the adherens junction (AJ) complex, the relevant cell-cell adhesion structure involved in cell differentiation and tissue organization, is located in an SM-rich membrane lipid domain. We have also demonstrated that under hypertonic conditions, Madin-Darby canine kidney (MDCK) cells acquire a differentiated phenotype with changes in SL metabolism. For these reasons, we decided to evaluate whether SM metabolism is involved in the acquisition of the differentiated phenotype of MDCK cells. We found that SM synthesis mediated by SM synthase 1 is involved in hypertonicity-induced formation of mature AJs, necessary for correct epithelial cell differentiation. Inhibition of SM synthesis impaired the acquisition of mature AJs, evoking a disintegration-like process reflected by the dissipation of E-cadherin and ß- and α-catenins from the AJ complex. As a consequence, MDCK cells did not develop the hypertonicity-induced differentiated epithelial cell phenotype.

Sphingosine-1-phosphate receptor 2 plays a dual role depending on the stage of cell differentiation in renal epithelial cells.

Epithelial renal cells have the ability to adopt different cellular phenotypes through epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET). These processes are increasingly recognized as important repair factors following acute renal tubular injury. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid with impact on proliferation, growth, migration, and differentiation which has significant implication in various diseases including cancer and kidney fibrosis. Here we demonstrated that S1P can exert by activating S1P receptor 2 (S1PR2) different functions depending on the stage of cell differentiation. We observed that the differences in the migratory profile of Madin-Darby canine kidney (MDCK) cells depend both on their stage of cell differentiation and the activity of S1PR2, a receptor that can either promote or inhibit the migratory process. Meanwhile in non-differentiated cells S1PR2 activation avoids migration, it is essential on fully differentiated cells. This is the first time that an antagonist effect of S1PR2 was reported for the same cell type. Moreover, in fully differentiated cells, S1PR2 activation is crucial for the progression of EMT - characterized by adherent junctions disassembly, ß-catenin and SNAI2 nuclear translocation and vimentin expression- and depends on ERK 1/2 activation and nuclear translocation. These findings provide a new perspective about the different S1PR2 functions depending on the stage of cell differentiation that can be critical to the modulation of renal epithelial cell plasticity, potentially paving the way for innovative research with pathophysiologic relevance.

Apoptotic cell extrusion depends on single-cell synthesis of sphingosine-1-phosphate by sphingosine kinase 2.

Collecting duct cells are physiologically subject to the hypertonic environment of the kidney. This condition is necessary for kidney maturation and function but represents a stress condition that requires active strategies to ensure epithelial integrity. Madin-Darby Canine Kidney (MDCK) cells develop the differentiated phenotype of collecting duct cells when subject to hypertonicity, serving as a model to study epithelial preservation and homeostasis in this particular environment. The integrity of epithelia is essential to achieve the required functional barrier. One of the mechanisms that ensure integrity is cell extrusion, a process initiated by sphingosine-1-phosphate (S1P) to remove dying or surplus cells while maintaining the epithelium barrier. Both types start with the activation of S1P receptor type 2, located in neighboring cells. In this work, we studied the effect of cell differentiation induced by hypertonicity on cell extrusion in MDCK cells, and we provide new insights into the associated molecular mechanism. We found that the different stages of differentiation influence the rate of apoptotic cell extrusion. Besides, we used a novel methodology to demonstrate that S1P increase in extruding cells of differentiated monolayers. These results show for first time that cell extrusion is triggered by the single-cell synthesis of S1P by sphingosine kinase 2 (SphK2), but not SphK1, of the extruding cell itself. Moreover, the inhibition or knockdown of SphK2 prevents cell extrusion and cell-cell junction protein degradation, but not apoptotic nuclear fragmentation. Thus, we propose SphK2 as the biochemical key to ensure the preservation of the epithelial barrier under hypertonic stress.

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

Sphingolipids regulate several aspects of cell behavior and it has been demonstrated that cells adjust their sphingolipid metabolism in response to metabolic needs. Particularly, sphingosine-1-phosphate (S1P), a final product of sphingolipid metabolism, is a potent bioactive lipid involved in the regulation of various cellular processes, including cell proliferation, cell migration, actin cytoskeletal reorganization and cell adhesion. In previous work in rat renal papillae, we showed that sphingosine kinase (SK) expression and S1P levels are developmentally regulated and control de novo sphingolipid synthesis. The aim of the present study was to evaluate the participation of SK/S1P pathway in the triggering of cell differentiation by external hypertonicity. We found that hypertonicity evoked a sharp decrease in SK expression, thus activating the de novo sphingolipid synthesis pathway. Furthermore, the inhibition of SK activity evoked a relaxation of cell-cell adherens junction (AJ) with accumulation of the AJ complex (E-cadherin/ß-catenin/α-catenin) in the Golgi complex, preventing the acquisition of the differentiated cell phenotype. This phenotype alteration was a consequence of a sphingolipid misbalance with an increase in ceramide levels. Moreover, we found that SNAI1 and SNAI2 were located in the cell nucleus with impairment of cell differentiation induced by SK inhibition, a fact that is considered a biochemical marker of epithelial to mesenchymal transition. So, we suggest that the expression and activity of SK1, but not SK2, act as a control system, allowing epithelial cells to synchronize the various branches of sphingolipid metabolism for an adequate cell differentiation program.