Search

Biological information, even in highly specialized fields, is increasing at a volume that no single investigator can assimilate. The existence of this vast knowledge base creates the need for specialized computer databases to store and selectively sort the information. We have developed a manually curated database of the effects of hypertonicity on target proteins. Effects include changes in mRNA abundance and protein abundance, activity, phosphorylation state, binding, and cellular compartment. The biological information used in this database was derived from three research approaches: transcriptomic, proteomic, and reductionist (hypothesis-driven). The data are presented in the form of grammatical triplets consisting of subject, verb phrase, and object. The purpose of this format is to allow the data to be read from left to right as an English sentence. It is readable either by humans or by computers using natural language processing algorithms. An example of a data entry reads 'Hypertonicity increases activity of ABL1 in HEK293.' This database was created to provide access to a wealth of information on the effects of hypertonicity in a format that can be selectively sorted. [ABSTRACT FROM AUTHOR]

Having previously found that high NaCl causes rapid exit of 14-3-3 isoforms from the nucleus, we used siRNA-mediated knockdown to test whether 14-3-3s contribute to the high NaCl-induced increase in the activity of the osmoprotective transcription factor NFAT5. We find that, when NaCl is elevated, knockdown of 14-3-3- β and/or 14-3-3- ε decreases NFAT5 transcriptional activity, as assayed both by luciferase reporter and by the mRNA abundance of the NFAT5 target genes aldose reductase and the sodium- and chloride-dependent betaine transporter, BGT1. Knockdown of other 14-3-3 isoforms does not significantly affect NFAT5 activity. 14-3-3- β and/or 14-3-3- ε do not act by affecting the nuclear localization of NFAT5, but by at least two other mechanisms: (1) 14-3-3- β and 14-3-3- ε increase protein abundance of NFAT5 and (2) they increase NFAT5 transactivating activity. When NaCl is elevated, knockdown of 14-3-3- β and/or 14-3-3- ε reduces the protein abundance of NFAT5, as measured by Western blot, without affecting the level of NFAT5 mRNA, and the knockdown also decreases NFAT5 transactivating activity, as measured by luciferase reporter. The 14-3-3s increase NFAT5 protein, not by increasing its translation, but by decreasing the rate at which it is degraded, as measured by cycloheximide chase. It is not clear at this point whether the 14-3-3s affect NFAT5 directly or indirectly through their effects on other proteins that signal activation of NFAT5. [ABSTRACT FROM AUTHOR]

The transcription factor TonEBP/OREBP promotes cell survival during osmotic stress. High NaCl-induced phosphorylation of TonEBP/OREBP at tyrosine-143 was known to be an important factor in increasing its activity in cell culture. We now find that TonEBP/OREBP also is phosphorylated at tyrosine-143 in rat renal inner medulla, dependent on the interstitial osmolality. c-Abl seemed likely to be the kinase that phosphorylates TonEBP/OREBP because Y143 is in a consensus c-Abl phosphorylation site. We now confirm that, as follows. High NaCl increases c-Abl activity. Specific inhibition of c-Abl by imaunib, siRNA, or c-Abl kinase dead drastically reduces high NaCl-induced TonEBP/OREBP activity by reducing its nuclear location and transactivating activity. c-Abl associates with TonEBP/OREBP (coimmunopre-cipitation) and phosphorylates TonEBP/OREBP-Y143 both in cell and in vitro. High NaCl-induced activation of ataxia telangiectasia mutated, previously known to contribute to activation of TonEBP/OREBP, depends on c-Abl activity. Thus, c-Abl is the kinase responsible for high NaCl-induced phosphorylation of TonEBP/OREBP-Y143, which contributes to its increased activity. [ABSTRACT FROM AUTHOR]

Hypertonicity activates the transcription factor TonEBP, which protects cells by stimulating the transcription of transporters and enzymes involved in organic osmolyte accumulation, p38 kinase is activated by hypertonicity and contributes to the activation of TonEBP. Rac1 GTPase signals an activation of p38 kinase, associated with hypertonicity-induced cytoskeletal reorganization. An important part of the activation of TonEBP is its movement from cytosol to nucleus. The signaling pathways involved in this translocation are incompletely understood. Since Rac1 and p38 are activated by hypertonicity and p38 is involved in TonEBP activation, we hypothesized that Rac1 and p38 activity might both be necessary for TonEBP translocation. In the present experiments, to test for a role of Rac1 we transiently transfected Hek293 cells with expression plasmids containing either catalytically active (CA) Rac1 or dominant negative (DN) Rac1 or empty vector (EV). To test for a role of p38, we added the inhibitor SB203580. The cells were incubated for 2 hours in 200 (hypotonic), 300 (normotonic) or 500 (hypertonic) mosmol/kg medium (NaCl varied). We extracted cytosolic and nuclear proteins and measured TonEBP in both fractions by Western analysis, then calculated the total amount of TonEBP in each compartment and the nuclear to cytosolic ratio (N/C). We found that at 500 mosmol/kg N/C TonEBP is increased by expression of Rac1CA and is decreased by expression of Rac1DN or by addition of SB203580. We conclude that Rac1 and p38 contribute to high NaCl-induced movement of TonEBP from the cytoplasm to the nucleus. [ABSTRACT FROM AUTHOR]

High NaCl activates TonEBP/OREBP, resulting in increased transcription of osmoprotective genes. Kinases known to contribute to signaling activation of TonEBP/OREBP include p38, Fyn, PKA, and also the phosphatidylinositol kinase (PIK) family members, ATM and PI3K IA. The purpose of the present studies was to investigate the possible role of PLC-γ1, using MEF PLC-γ1-null and HEK293 cells. Suppression of PLC-γ1 inhibits both high NaCl-induced increase of TonEBP/OREBP transcriptional activity and the accompanying nuclear translocation. Further, mutation of Y143 in TonEBP/OREBP, identified as a likely site of interaction with the SH2 domain of PLC-γ1, also reduces high NaCl-induced nuclear translocation of TonEBP/OREBP. PLC-γ1 co-immunoprecipitates with TonEBP/OREBP. This interaction involves the n-terminal (1-547), but not the c-terminal (548-1531) part of TonEBP/OREBP, and is increased in the cytoplasm by high NaCl. Conclusions: 1) PLC-γ1 is a positive regulator of TonEBP/OREBP transcriptional activity and nuclear translocation. 2) This regulation involves interaction of the SH2 domain of PLC-γ1 with Y143 of TonEBP/OREBP. [ABSTRACT FROM AUTHOR]

GPC is an abundant renal medullary organic osmolyte. We previously found that its synthesis from phosphatidylcholine is catalyzed by tonicity-regulated activity of the phospholipase B, NTE (PNAS 103:15260, 2006). We also found that its degradation is catalyzed by a GPC-PDE activity that is inhibited by high NaCl or high urea, contributing to the resultant increase of GPC. We have now identified a specific GPC-PDE that is inhibited by high NaCl, resulting in increased GPC. In the present studies we found that high NaCl causes a 2.5-fold increase of GPC in mIMCD3 cells within 24hours. mRNA abundance of Gdpd5 decreases by 70% within 8 hours after adding NaCl. In normotonic medium a specific siRNA against Gdpd5 decreases its mRNA expression by 55% while increasing GPC by 60%. High urea also increases GPC in these cells, but does not affect Gdpd5 mRNA expression, leaving the mechanism unexplained for now. We conclude that Gdpd5 degrades cellular GPC and that inhibition of Gdpd5 is involved in tonicity-induced accumulation of GPC. [ABSTRACT FROM AUTHOR]

High NaCl increases the number of DNA breaks in renal cells in culture and in the kidney inner medulla in vivo (Dmitrieva, PNAS, 2004). Ku86 is a DNA end binding protein that normally aligns the ends of broken DNA while it is being repaired. High NaCl disrupts chromatin, which leads to chromosomal aberrations. These are particularly pronounced in Ku86-/- cells (Dmitrieva, PNAS, 2005). P16INK4 is a member of INK4-class of cell cycle inhibitors. It is upregulated in cells whose DNA is damaged, preventing them from growing aberrantly. It is also upregulated in senescent cells and in cells that have exhausted their regenerative ability in the course of aging. In the present studies we show by immunohistochemical staining that the P16INK4 level in kidneys increases with age, and is highest in the inner medulla. Thus, in wild type mice the level of P16INK4 is higher in inner medulla than in the cortex, and is much greater at 12 months than at 3-4 months. Further, restricting water intake for two days increases renal p16INK4 level in wild type mice, and the greatest increase is in the inner medulla. In Ku86-/- mice, renal medullary p16INK4 is already high at 4 months, and the mice have damaged glomeruli. We speculate that the p16INK4 level is elevated in renal medullas because of the normally high level of DNA breaks that occurs there and that deficiency of the DNA repair protein Ku86 exacerbates the response. The role and consequences of the high level of p16INK4 remains to be established. [ABSTRACT FROM AUTHOR]

Hypertonicity increases the activity of TonEBP, resulting in increased transcription of osmo-protective genes. Hypertonicity also increases phosphorylation of TonEBP and there is indirect evidence that phosphorylation of some specific amino acids may be involved in its activation. However, there has been no direct demonstration of phosphorylation of any specific amino acid. The goal of the present studies was to identify amino acids in TonEBP that are phosphorylated. We used HEK293 cells stably transfected with TonEBP-1-547-V5. We incubated them at 200 or 500 mosmol/kg (NaCl varied) for two hours, extracted nuclear and cytoplasmic proteins, immunoprecipitated TonEBP-V5, subjected the immunoprecipitate to in-solution digestion, enriched for phosphopeptides by immobilized metal affinity chromatography (IMAC), and analyzed peptides from both the eluate and flow-through by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In order to maximize coverage of possible phosphorylation sites, we used 3 different proteolytic enzymes (trypsin, endoproteinase Arg-C, and proteinase K). We find a high probability that several different serines, threonines and tyrosines are phosphorylated in at least one of the conditions. We are mutating the amino acids that we identified to determine their role in TonEBP activity. [ABSTRACT FROM AUTHOR]

High NaCl and urea increase reactive oxygen species (ROS) of mitochondrial origin and cause oxidative damage to proteins and DNA in cell culture. Also, kidney medullas of normal mice have oxidative stress as indicated by high protein carbonylation. To understand how the kidney cells adapt to oxidative stress, we measured expression of antioxidant proteins. Superoxide dismutase (SOD) lowers the ROS, superoxide, by converting it to H2O2. We found that in vivo protein expression of MnSOD (the mitochondrial SOD) increases progressively between cortex, outer medulla (OM) and inner medulla (IM). Further, water restriction increases it in all three regions. In cultures of mlMCD3 cells, high NaCl, but not high urea, increases MnSOD protein expression. To understand the relationship between oxidative stress and MnSOD in the kidney, we measured its enzymatic activity. The activity of MnSOD per unit of its protein, is lower in IM than in cortex and, following water restriction, it falls in all regions of the kidney. We suggest that high NaCl reduces MnSOD activity, and that MnSOD protein abundance increases in compensation. In contrast to MnSOD, protein expression of neither Cu/ZnSOD (the cytosolic SOD), or thioredoxin 1 (a co-enzyme predominantly located in cytosol) is higher in the medulla than in the cortex, and neither changes significantly with water restriction. The anti-apoptotic and anti-oxidant protein Bcl-2 often acts in concert with MnSOD to prevent oxidative injury to cells. Bcl-2 protein abundance is higher in the renal medulla than in the cortex, but water restriction does not affect its expression in any of these regions. In conclusion, expression of MnSOD and Bcl-2 proteins is elevated in the renal medulla, which may contribute to survival of its cells despite hyperosmolality-induced oxidative stress. [ABSTRACT FROM AUTHOR]

Focuses on signals arising from desiccation in mammalian cells and their effects on hypertonicity. Role of sodium chloride in mammalian renal cell response to hypertonicity; Effect of hypertonicity on cells; Result of experiments in which messenger RNA for tonicity-responsive enhancer/osmotic response element-binding protein target genes were measured.