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 β- and α-catenins from the AJ complex. As a consequence, MDCK cells did not develop the hypertonicity-induced differentiated epithelial cell phenotype.Fil: Favale, Nicolas Octavio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Santacreu, Bruno Jaime. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Pescio, Lucila Gisele. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Marquez, Maria Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de la Rioja. Departamento de Ciencias de la Salud y Educación; ArgentinaFil: Sterin, Norma Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentin

Implication of Sphingosine-1-Phosphate Receptor 2 (S1PR2) in Differentiation and Dedifferentiation of Epithelial Renal Cells

Epithelial cell differentiation is a process that involves the mesenchymal-epithelial transition (MET) and includes cell cycle arrest, cell-cell junction maturation in addition to changes in cell migration capacity. The epithelial-mesenchymal transition (EMT) is a dynamic process by which fully differentiated epithelial cells can acquire a mesenchymal phenotype. During EMT, cell adhesion and apical-basal polarity are lost, and the cytoskeleton is reorganized. Previous results from our laboratory showed that in Madin-Darby canine kidney cells (MDCK) under different culture conditions can achieve different stages of differentiation resembling MET. Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid, produced by the phosphorylation of sphingosine by sphingosine kinases (SKs), which is involved in different processes such as proliferation, cell growth, differentiation, and migration. S1P can act both intracellularly as a second messenger or extracellularly as a ligand of 5 different G protein-coupled receptors (S1PR1-5). In the present work, we evaluated the importance of S1P acting on S1PR2 in the modulation of MET and EMT. We found that there are differences in the action of S1PR2 in MDCK cells that depends on the differentiation stage. S1PR2 positively modulates the passage from polarized to differentiated cells through MET. Inhibition of S1PR2 blocks adherens junction establishment, as well as apical and basal polarity. On the other hand, once cells have acquired the differentiated phenotype, S1PR2 induces the dedifferentiation of epithelial cells through EMT. Inhibition of S1PR2 triggers changes in EMT markers, such as rearrangements of the actin cytoskeleton, expression of vimentin, and nuclear translocation of beta-catenin, as well as Slug. The expression levels of S1PR2 in the different stages of differentiation of MDCK cells did not show significant differences. Instead, immunofluorescence studies showed that during cell differentiation, S1PR2 was progressively enriched at the plasma membrane. These results suggest that the location of S1PR2 depends on the stage of cell differentiation, and this determines its role. These findings highlight the great versatility of S1P on the control of physiological and pathophysiological processes.Fil: Romero, Daniela Judith. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Santacreu, Bruno Jaime. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Tarallo, Estefania. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Favale, Nicolas Octavio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaLVI Annual Meeting Argentine Society for Biochemistry and Molecular Biology; XV Annual Meeting Argentinean Society for General MicrobiologyArgentinaSociedad Argentina de Investigación Bioquímica y Biología Molecula

Sphingosine kinase and sphingosine-1phosphate 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/β-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.Fil: Santacreu, Bruno Jaime. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Pescio, Lucila Gisele. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Romero, Daniela Judith. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Biología Celular y Molecular; ArgentinaFil: Corradi, Gerardo Raul. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Sterin, Norma Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Favale, Nicolas Octavio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentin

Bradykinin mediates the association of collecting duct cells to form migratory colonies, through B2 receptor activation

It is known that bradykinin (BK) B2 receptor (B2R) is expressed in the collecting duct (CD) cells of the newborn rat kidney, but little is known about its role during early postnatal life. Therefore, we hypothesize that BK could participate in the mechanisms that mediate CD formation during the postnatal renal development. Performing primary cultures, combined with biochemical, immunocytochemical, and time‐lapse analysis, we studied the role of BK in CD cell behavior isolated from renal papilla of neonatal rats. A reverse relationship was observed between B2R expression and the degree of CD epithelial cell sheet maturation. BK stimulation induced CD cell association upon B2R activation. The lack of B2R expression in cells showing mature adherens junctions suggested that BK is mostly involved in early adhesive events, thus favoring the initial formation of CD during development. Time‐lapse analysis revealed that BK induced a high protrusive activity of CD cells, denoted by ruffle formation and lamellipodia extension. PI3K was involved in the BK‐induced CD cell‐cell association and the acquisition of the migratory phenotype since, when inhibited, membrane ruffles, and filopodia between cells diminished. Results indicate that the actions of BK mediated by PI3K activation were due to the downstream Akt and Rac pathways. This study, performed with CD cells that were not genetically manipulated, provides new experimental evidence supporting a novel role of BK in rat renal CD organization. As B2R blockade results in abnormal tubular differentiation, our results contribute to better understanding the etiology of human congenital renal malformation and diseases.Fil: Guaytima, Edith del Valle. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Rioja. Departamento de Ciencias de la Salud y Educación. Instituto de Investigaciones en Ciencias de la Salud Humana; ArgentinaFil: Brandán, Yamila Romina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Rioja. Departamento de Ciencias de la Salud y Educación. Instituto de Investigaciones en Ciencias de la Salud Humana; ArgentinaFil: Favale, Nicolas Octavio. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Santacreu, Bruno Jaime. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas; ArgentinaFil: Sterin, Norma Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Marquez, Maria Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Rioja. Departamento de Ciencias de la Salud y Educación. Instituto de Investigaciones en Ciencias de la Salud Humana; Argentin

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.Fil: Canals, Daniel. Stony Brook University; State University of New York;Fil: Salamone, Silvia. Stony Brook University; State University of New York;Fil: Santacreu, Bruno Jaime. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Nemeth, Erika. Stony Brook University; State University of New York;Fil: Aguilar, Daniel. Biomedical Research Networking Center in Hepatic and Digestive Diseases; EspañaFil: Hernandez Corbacho, María José. Stony Brook University; State University of New York;Fil: Adada, Mohamad. Stony Brook University; State University of New York;Fil: Staquicini, Daniela I.. Rutgers Cancer Institute of New Jersey; Estados UnidosFil: Arap, Wadih. Rutgers Cancer Institute of New Jersey; Estados UnidosFil: Pasqualini, Renata. Rutgers Cancer Institute of New Jersey; Estados UnidosFil: Haley, John. Stony Brook University; State University of New York;Fil: Obeid, Lina M.. Stony Brook University; State University of New York;Fil: Hannun, Yusuf A.. Stony Brook University; State University of New York

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.Fil: Canals, Daniel. Stony Brook University; State University of New York;Fil: Salamone, Silvia. Stony Brook University; State University of New York;Fil: Santacreu, Bruno Jaime. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Nemeth, Erika. Stony Brook University; State University of New York;Fil: Aguilar, Daniel. Biomedical Research Networking Center in Hepatic and Digestive Diseases; EspañaFil: Hernandez Corbacho, María José. Stony Brook University; State University of New York;Fil: Adada, Mohamad. Stony Brook University; State University of New York;Fil: Staquicini, Daniela I.. Rutgers Cancer Institute of New Jersey; Estados UnidosFil: Arap, Wadih. Rutgers Cancer Institute of New Jersey; Estados UnidosFil: Pasqualini, Renata. Rutgers Cancer Institute of New Jersey; Estados UnidosFil: Haley, John. Stony Brook University; State University of New York;Fil: Obeid, Lina M.. Stony Brook University; State University of New York;Fil: Hannun, Yusuf A.. Stony Brook University; State University of New York

Participación de la esfingosina cinasa en la diferenciación celular y organización tisular

Sphingosine kinase (SphK) biosynthesizes sphingosine-1-phosphate (S1P). S1P is a bioactive sphingolipid and has been shown to exert a variety of biological responses intracellularly or through extracellular specific receptors. Renal epithelial cell differentiation is a process that involves the mesenchymal epithelium transition (MET). During the MET, mesenchymal cells become polarized and establish cell junctions. The goal of this thesis was to study the role of S1P in the MET process of renal epithelial cells, as well as in the homeostasis once the differentiated state has already been established. We found a synchronization between the SphK / S1P pathway during MET, generated by a decrease in the activity of the SphK while cell differentiation progresses. In the less differentiated stages this was necessary to produce an inhibition of sphingolipids synthesis, to avoid ceramide accumulation and to correctly deliver of the adherens junction proteins. Whereas, the low global level of S1P in differentiated cells mobilizes the S1P(2) receptor to the plasma membrane. Moreover, it allows locally increases of S1P by SphK2 to mediate apoptotic cell extrusion. Thus, this synchronization guarantees the integrity of the epithelial tissue.Fil: Santacreu, Bruno Jaime. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Buenos Aires, ArgentinaLa esfingosina cinasa (SphK) biosintetiza esfingosina-1-fosfato (S1P), un esfingolípido bioactivo con importantes acciones biológicas. La S1P puede actuar intracelularmente o a través de receptores. La diferenciación celular de las células epiteliales renales es un proceso que involucra la transición mesénquima epitelio (MET). Durante la MET, células mesenquima?ticas se tornan polarizadas y establecen uniones célula-célula maduras. El objetivo de la presente tesis fue estudiar la participación de la S1P en el proceso de MET de células epiteliales renales, así como también en el mecanismos de homeostasis epitelial, una vez que el estado diferenciado ya se ha establecido. Se encontró que existe una disminución de la actividad de la vía SphK/S1P conforme el avance de la diferenciación celular. En estadios menos diferenciados, la S1P actúa por un mecanismo intrácrino controlando la síntesis de novo de los esfingolípidos, para garantizar el correcto tráfico de las proteínas de la unión adherente. Mientras que, en células diferenciadas el bajo tono de síntesis global moviliza al receptor de S1P(2) a la membrana plasmática. Esto permite que las células apoptóticas aumenten localmente la S1P por medio de la SphK2 produciendo la extrusión de la célula señalizadora y garantizando así la integridad del tejido epitelial

Participación de la Esfingosina Cinasa en la Diferenciación Celular y Organización Tisular

La esfingosina cinasa (SphK) sintetiza esfingosina-1-fosfato (S1P), un esfingolípido bioactivo con importantes acciones biológicas. Los esfingolípidos son moléculas que no solo presentan una fun- cionalidad estructural, sino que, además, cada uno posee una actividad biológica característica, encontrándose acciones biológicas asociadas a un tipo molecular especifico. La S1P puede actuar intracelularmente o a través de alguno de los cinco receptores acoplados a proteína G, por lo que resulta un ligando con acciones autocrinas y paracrinas.La diferenciación celular de las células epiteliales renales es un proceso que involucra la transi- ción mesénquima-epitelio (MET). Durante la MET, células mesenquimales se tornan polarizadas, desarrollando un dominio apical y uno basolateral, al mismo tiempo que establecen sus uniones célula-célula y célula-matriz. Esta transición se desencadena por una intrincada señalización mo- lecular, en donde es necesaria una hipertonicidad extracelular, que además es requerida para el mantenimiento del estado diferenciado. En este sentido, la homeostasis celular depende de un correcto recambio celular que garantice la integridad del tejido, permitiendo la funcionalidad del mismo.El objetivo de la presente tesis consistió en estudiar la participación de la S1P en el proceso de MET de células epiteliales renales, así como también en mecanismos de homeostasis una vez que el estado diferenciado ya se ha establecido. Para ello, se utilizó la línea celular MDCK, derivada de células de túbulo colector renal, un modelo prototipo para estudiar polarización y diferenciación celular.Inicialmente, se abordó el estudio de la vía SphK1/S1P durante el desencadenamiento de la di- ferenciación celular. Los resultados mostraron que tanto la expresión ezimática, la síntesis y los niveles endógenos de S1P descienden progresivamente a medida que las células se diferencian. Para evaluar, si existía una coordinación entre dicho descenso y la diferenciación celular, se in- hibió farmacologicamente a la SphK al momento de desencadenar la diferenciación celular y se encontró que las células no establecieron uniones adherentes maduras, manteniendo una morfo- logía fibroblastoide. Además, se encontró que las uniones adherentes no se establecieron correc- tamente porque el complejo de proteínas que lo conforma se acumulaba en el aparato de Golgi. El impedimento en el avance de las proteínas a través de la vía secretoria resultó estar mediado por la modulación que lleva a cabo la S1P sobre la síntesis de esfingolípidos, en especial por la acumulación de ceramida.Posteriormente, se evaluó si la baja actividad de la vía SphK/S1P detectada en el estado de dife- renciación celular tenía significancia biológica para la homeostasis de dicho estado. Los resultados demostraron que la inhibición de la SphK no produjo alteración de la morfología ni de las uniones adherentes, pero produjo pérdida de la integridad de la monocapa de células. Se relacionó dicha alteración con una falla en el proceso de extrusión celular, mecanismo dependiente de S1P, por el cual se eliminan células apoptóticas del tejido epitelial hacia la luz del mismo. Además, encontramos que dicho proceso es dependiente de la SphK2 y que durante la MET existe una relocalización del receptor de S1P implicado en la extrusión celular.Los resultados sugieren que, en etapas tempranas de la diferenciación celular, la vía SphK/S1P se encuentra más activa, limitando la síntesis de esfingolípidos. Sin embargo, con el avance en la diferenciación celular, progresivamente disminuye la actividad de la misma, contribuyendo a la síntesis de esfingolípidos, principalmente SM y GluCer, necesarios para el establecimiento de la unión adherente y la polaridad celular. Por otro lado, el bajo tono de síntesis en células diferencia- das moviliza al receptor desde vesículas a la membrana plasmática, posibilitando el mecanismo de extrusión celular que garantiza la homeostasis tisular. De esta manera, en este trabajo de tesis doctoral se pone en evidencia la sincronización existente entre la vía SphK/S1P, la diferenciación celular y la organización tisular.Sphingosine kinase (SphK) biosynthesizes sphingosine-1-phosphate (S1P). S1P is a bioactive sphingolipid and has been shown to exert a variety of biological responses, intracellularly or through extracellular specific receptors. Sphingolipids are molecules that not only have a structural functionality, each one is related with a characteristic biological activity. Morover, specific molecular species are associated with specific biological actions. Renal epithelial cell differentiation is a process that involves the mesenchymal to epithelium transition (MET). During the MET, mesenchymal cells become polarized, developing an apical and a basolateral domain, at the same time establishing cell junctions. This transition is triggered by an intricate molecular signaling where extracellular hypertonicity is necessary, which is also required for maintenance of the differentiated state. Moreover, the tissue homeostasis depends on a correct cell turnover, which guarantees the integrity of the tissue and allows its functionality. The goal of this thesis was to study the role of S1P in the MET process of renal epithelial cells, as well as in the homeostasis once the differentiated state has already been established. As experimental model we used the MDCK cell line, which derived from renal collector duct cells. MDCK cells are a prototype model to study cell polarization and differentiation. Initially, we studied SphK1 / S1P pathway during the onset of cell differentiation. The results showed that the enzymatic expression, the synthesis and the endogenous levels of S1P progressively decrease as the cell becomes more differentiated. To evaluate if it was due to a coordination between the downregulation of the pathway and cell differentiation, SphK was inhibited pharmacologically at the time of triggering cell differentiation. We found that cells did not establish mature adherens junctions and maintained a fibroblastoid morphology. In addition, we found that the adherens junction proteins were accumulated in the Golgi apparatus. This protein accumulation was due to the modulation on the sphingolipid synthesis mediated by S1P. Subsequently, we evaluated whether the downregulation of the SphK / S1P pathway affected the differentiated cells. We found that the inhibition of SphK did not affect cell morphology or maduration of cell junctions, but produced loss of monolayer integrity. This alteration was related to a failure in cell extrusion, a procces in which apoptotic cells are removed from the epithelial tissue, by S1P -dependent mechanism. In addition, we found that this process was dependent on SphK2, and that during MET the S1PR2 involved in cell extrusion was relocated. The results suggested that, in early stages of cell differentiation, the pathway is more active, limiting the synthesis of sphingolipids, but progressively decrease its activity in synchrony with the progress of cell differentiation. This regulation contributes to the synthesis of sphingolipids, mainly sphingomyelin and glucosylceramide, essential for the establishment of adherens junctions and cell polarity. On the other hand, low activity of SphK in differentiated cells mobilizes the rec eptor from vesicles to the plasma membrane, activating the cell extrusion mechanism that guarantees tissue homeostasis. Finally, we propose that SphK / S1P pathway is synchronized during cellular differentiation.Fil: Santacreu, Bruno Jaime. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentin

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.Fil: Santacreu, Bruno Jaime. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Romero, Daniela Judith. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Pescio, Lucila Gisele. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Tarallo, Estefania. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Sterin, Norma Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Favale, Nicolas Octavio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentin

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, β-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.Fil: Romero, Daniela Judith. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Biología Celular y Molecular; ArgentinaFil: Pescio, Lucila Gisele. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Biología Celular y Molecular; ArgentinaFil: Santacreu, Bruno Jaime. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Biología Celular y Molecular; ArgentinaFil: Mosca, Jazmín María. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Biología Celular y Molecular; ArgentinaFil: Sterin, Norma Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Favale, Nicolas Octavio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Biología Celular y Molecular; Argentin