Nicolás O. Favale

Hypertonic induction of COX2 expression requires TonEBP/NFAT5 in renal epithelial cells.

TonEBP/NFAT5 transcription factor is a master regulator of genes involved in osmoprotection. Cyclooxygenase 2 (COX2) has been reported to be a cytoprotective molecule in the inner renal medulla, where cells are physiologically exposed to the highest osmolality of the body. Our aim was to study whether COX2 expression requires TonEBP/NFAT5. Incubation of MDCK cells in hypertonic NaCl medium (500 mOsm/kg H2O) caused fully translocation of TonEBP/NFAT5 from cytoplasm to nucleoplasm and significantly increased COX2 mRNA, protein and activity levels. TonEBP/NFAT5-siRNA prevented hypertonic induction of COX2 mRNA and protein, leading to a depressed-prostaglandin synthesis and to a decreased cell survival. By using COX2-siRNA and COX2 specific inhibitor NS398, we found that cell survival does not depend on endogenous COX2-induced prostaglandin synthesis, but that cytoprotection strongly correlates with COX2 protein levels. These results demonstrate a new function for TonEBP/NFAT5, i.e., to mediate hypertonic-induced COX2 expression, and suggest that osmoprotection strongly depends on COX2 protein levels.

The rate-limiting enzyme in phosphatidylcholine synthesis is associated with nuclear speckles under stress conditions

Phosphatidylcholine (PtdCho) is the most abundant phospholipid in eukaryotic membranes and its biosynthetic pathway is generally controlled by CTP:Phosphocholine Cytidylyltransferase (CCT), which is considered the rate-limiting enzyme. CCT is an amphitropic protein, whose enzymatic activity is commonly associated with endoplasmic reticulum (ER) translocation; however, most of the enzyme is intranuclearly located. Here we demonstrate that CCTα is concentrated in the nucleoplasm of MDCK cells. Confocal immunofluorescence revealed that extracellular hypertonicity shifted the diffuse intranuclear distribution of the enzyme to intranuclear domains in a foci pattern. One population of CCTα foci colocalised and interacted with lamin A/C speckles, which also contained the pre-mRNA processing factor SC-35, and was resistant to detergent and salt extraction. The lamin A/C silencing allowed us to visualise a second more labile population of CCTα foci that consisted of lamin A/C-independent foci non-resistant to extraction. We demonstrated that CCTα translocation is not restricted to its redistribution from the nucleus to the ER and that intranuclear redistribution must thus be considered. We suggest that the intranuclear organelle distribution of CCTα is a novel mechanism for the regulation of enzyme activity.

Glycosphingolipid synthesis is essential for MDCK cell differentiation

Glycosphingolipids (GSLs), which are highly concentrated at the apical membrane of polarized epithelial cells, are key components of cell membranes and are involved in a large number of processes. Here, we investigated the ability of hypertonicity (high salt medium) to induce Madin-Darby Canine Kidney (MDCK) cell differentiation and found an increase in GSL synthesis under hypertonic conditions. Then, we investigated the role of GSLs in MDCK cell differentiation induced by hypertonicity by using two approaches. First, cultured cells were depleted of GSLs by exposure to D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP). Second, cells were transfected with an siRNA specific to glucosylceramide synthase, the key enzyme in GSL synthesis. Exposure of cells to both treatments resulted in the impairment of the development of the apical membrane domain and the formation of the primary cilium. Enzymatic inhibitions of the de novo and the salvage pathway of GSL synthesis were used to determine the source of ceramide responsible of the GSL increase involved in the development of the apical membrane domain induced by hypertonicity. The results from this study show that extracellular hypertonicity induces the development of a differentiated apical membrane in MDCK cells by performing a sphingolipid metabolic program that includes the formation of a specific pool of GSLs. The results suggest as precursor a specific pool of ceramides formed by activation of a Fumonisin B1-resistant ceramide synthase as a component of the salvage pathway.

The Cytochrome b5 dependent C-5(6) sterol desaturase DES5A from the endoplasmic reticulum of Tetrahymena thermophila complements ergosterol biosynthesis mutants in Saccharomyces cerevisiae

Tetrahymena thermophila is a free-living ciliate with no exogenous sterol requirement. However, it can perform several modifications on externally added sterols including desaturation at C5(6), C7(8), and C22(23). Sterol desaturases in Tetrahymena are microsomal enzymes that require Cyt b(5), Cyt b(5) reductase, oxygen, and reduced NAD(P)H for their activity, and some of the genes encoding these functions have recently been identified. The DES5A gene encodes a C-5(6) sterol desaturase, as shown by gene knockout in Tetrahymena. To confirm and extend that result, and to develop new approaches to gene characterization in Tetrahymena, we have now, expressed DES5A in Saccharomyces cerevisiae. The DES5A gene was codon optimized and expressed in a yeast mutant, erg3Δ, which is disrupted for the gene encoding the S. cerevisiae C-5(6) sterol desaturase ERG3. The complemented strain was able to accumulate 74% of the wild type level of ergosterol, and also lost the hypersensitivity to cycloheximide associated with the lack of ERG3 function. C-5(6) sterol desaturases are expected to function at the endoplasmic reticulum. Consistent with this, a GFP-tagged copy of Des5Ap was localized to the endoplasmic reticulum in both Tetrahymena and yeast. This work shows for the first time that both function and localization are conserved for a microsomal enzyme between ciliates and fungi, notwithstanding the enormous evolutionary distance between these lineages. The results suggest that heterologous expression of ciliate genes in S. cerevisiae provides a useful tool for the characterization of genes in Tetrahymena, including genes encoding membrane protein complexes.

Membrane Lipid Composition Plays a Central Role in the Maintenance of Epithelial Cell Adhesion to the Extracellular Matrix

Focal contacts (FC) are membrane-associated multi-protein complexes that mediate cell-extracellular matrix (ECM) adhesion. FC complexes are inserted in detergent-resistant membrane microdomains enriched in phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2); however, the influence of membrane lipid composition in the preservation of FC structures has not been extensively addressed. In the present work, we studied the contribution of membrane lipids to the preservation of renal epithelial cell adhesion structures. We biochemically characterized the lipid composition of membrane-containing FC complexes. By using cholesterol and PtdIns(4,5)P2 affecting agents, we demonstrated that such agents did not affect any particular type of lipid but induced the formation of new FC-containing domains of completely different lipid composition. By using both biochemical approaches and fluorescence microscopy we demonstrated that phospholipid composition plays an essential role in the in vivo maintenance of FC structures involved in cell-ECM adhesion.

Sphingolipid Metabolism Is a Crucial Determinant of Cellular Fate in Nonstimulated Proliferating Madin-Darby Canine Kidney (MDCK) Cells

The present report was addressed to study the influence of sphingolipid metabolism in determining cellular fate. In nonstimulated proliferating Madin-Darby canine kidney (MDCK) cells, sphingolipid de novo synthesis is branched mainly to a production of sphingomyelin and ceramide, with a minor production of sphingosylphosphocholine, ceramide 1-phosphate, and sphingosine 1-phosphate. Experiments with 32P as a radioactive precursor showed that sphingosine 1-phosphate is produced mainly by a de novo independent pathway. Enzymatic inhibition of the de novo pathway and ceramide synthesis affected cell number and viability only slightly, without changing sphingosine 1-phosphate production. By contrast, inhibition of sphingosine kinase-1 activity provoked a significant reduction in both cell number and viability in a dose-dependent manner. When sphingolipid metabolism was studied, an increase in de novo formed ceramide was found, which correlated with the concentration of enzyme inhibitor and the reduction in cell number and viability. Knockdown of sphingosine kinase-1 expression also induced an accumulation of de novo synthesized ceramide, provoking a slight reduction in cell number and viability similar to that induced by a low concentration of the sphingosine kinase inhibitor. Taken together, our results indicate that the level of de novo formed ceramide is controlled by the synthesis of sphingosine 1-phosphate, which appears to occur through a de novo synthesis-independent pathway, most probably the salvage pathway, that is responsible for the MDCK cell fate, suggesting that under proliferating conditions, a dynamic interplay exists between the de novo synthesis and the salvage pathway.

Changes in membrane lipid composition cause alterations in epithelial cell-cell adhesion structures in renal papillary collecting duct cells.

In epithelial tissues, adherens junctions (AJ) mediate cell-cell adhesion by using proteins called E-cadherins, which span the plasma membrane, contact E-cadherin on other cells and connect with the actin cytoskeleton inside the cell. Although AJ protein complexes are inserted in detergent-resistant membrane microdomains, the influence of membrane lipid composition in the preservation of AJ structures has not been extensively addressed. In the present work, we studied the contribution of membrane lipids to the preservation of renal epithelial cell-cell adhesion structures. We biochemically characterized the lipid composition of membranes containing AJ complexes. By using lipid membrane-affecting agents, we found that such agents induced the formation of new AJ protein-containing domains of different lipid composition. By using both biochemical approaches and fluorescence microscopy we demonstrated that the membrane phospholipid composition plays an essential role in the in vivo maintenance of AJ structures involved in cell-cell adhesion structures in renal papillary collecting duct cells.

Physiologically induced restructuring of focal adhesions causes mobilization of vinculin by a vesicular endocytic recycling pathway.

In epithelial cells, vinculin is enriched in cell adhesion structures but is in equilibrium with a large cytosolic pool. It is accepted that when cells adhere to the extracellular matrix, a part of the soluble cytosolic pool of vinculin is recruited to specialized sites on the plasma membrane called focal adhesions (FAs) by binding to plasma membrane phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2). We have previously shown that bradykinin (BK) induces both a reversible dissipation of vinculin from FAs, by the phospholipase C (PLC)-mediated hydrolysis of PtdIns(4,5)P2, and the concomitant internalization of vinculin. Here, by using an immunomagnetic method, we isolated vinculin-containing vesicles induced by BK stimulation. By analyzing the presence of proteins involved in vesicle traffic, we suggest that vinculin can be delivered in the site of FA reassembly by a vesicular endocytic recycling pathway. We also observed the formation of vesicle-like structures containing vinculin in the cytosol of cells treated with lipid membrane-affecting agents, which caused dissipation of FAs due to their deleterious effect on membrane microdomains where FAs are inserted. However, these vesicles did not contain markers of the recycling endosomal compartment. Vinculin localization in vesicles has not been reported before, and this finding challenges the prevailing model of vinculin distribution in the cytosol. We conclude that the endocytic recycling pathway of vinculin could represent a physiological mechanism to reuse the internalized vinculin to reassembly new FAs, which occurs after long time of BK stimulation, but not after treatment with membrane-affecting agents.

Environmental hyperosmolality regulates phospholipid biosynthesis in the renal epithelial cell line MDCK.

Hyperosmolality is a key signal for renal physiology. On the one hand, it contributes to the differentiation of renal medullary structures and to the development of the urinary concentrating mechanism. On the other, it is a stress factor. In both cases, hyperosmolality activates processes that require an adequate extension of cellular membranes. In the present work, we examined whether hyperosmolality regulates phospholipid biosynthesis, which is needed for the membrane biogenesis in the renal epithelial cell line Madin-Darby canine kidney (MDCK). Because phospholipids are the structural determinants of all cell membranes, we evaluated their content, synthesis, and regulation in MDCK cultures subjected to different hyperosmotic concentrations of NaCl, urea, or both. Hyperosmolality increased phospholipid content in a concentration-dependent manner. Such an effect was exclusively due to changes in NaCl concentration and occurred at the initial stage of hyperosmolar treatment concomitantly with the expression of the osmoprotective protein COX-2. The hypertonic upregulation of phosphatidylcholine (PC) synthesis, the main constituent of all cell membranes, involved the transcriptional activation of two main regulatory enzymes, choline kinase (CK) and cytidylyltransferase α (CCTα) and required ERK1/2 activation. Considering that physiologically, renal medullary cells are constantly exposed to high and variable NaCl, these findings could contribute to explaining how renal cells could maintain cellular integrity even in a nonfavorable environment.

Bradykinin induces formation of vesicle-like structures containing vinculin and PtdIns(4,5)P2 in renal papillary collecting duct cells

Focal adhesions (FAs) are structures of cell attachment to the extracellular matrix. We previously demonstrated that the intrarenal hormone bradykinin (BK) induces the restructuring of FAs in papillary collecting duct cells by dissipation of vinculin, but not talin, from FAs through a mechanism that involves PLCbeta activation, and that it also induces actin cytoskeleton reorganization. In the present study we investigated the mechanism by which BK induces the dissipation of vinculin-stained FAs in collecting duct cells. We found that BK induces the internalization of vinculin by a noncaveolar and independent pinocytic pathway and that at least a fraction of this protein is delivered to the recycling endosomal compartment, where it colocalizes with the transferrin receptor. Regarding the reassembly of vinculin-stained FAs, we found that BK induces the formation of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]-enriched vinculin-containing vesicles, which, by following a polarized exocytic route, transport vinculin to the site of FA assembly, an action that depends on actin filaments. The present study, which was carried out with cells that were not genetically manipulated, shows for the first time that BK induces the formation of vesicle-like structures containing vinculin and PtdIns(4,5)P2, which transport vinculin to the site of FA assembly. Therefore, the modulation of the formation of these vesicle-like structures could be a physiological mechanism through which the cell can reuse the BK-induced internalized vinculin to be delivered for newly forming FAs in renal papillary collecting duct cells.