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NFAT5 is an osmoregulated transcription factor that particularly increases expression of genes involved in protection against hypertonicity. Transcription factors often contain unstructured regions that bind co-regulatory proteins that are crucial for their function. The NH2-terminal region of NFAT5 contains regions predicted to be intrinsically disordered. We used peptide aptamer-based affinity chromatography coupled with mass spectrometry to identify protein preys pulled down by one or more overlapping 20 amino acid peptide baits within a predicted NH2-terminal unstructured region of NFAT5. We identify a total of 467 unique protein preys that associate with at least one NH2-terminal peptide bait from NFAT5 in either cytoplasmic or nuclear extracts from HEK293 cells treated with elevated, normal, or reduced NaCl concentrations. Different sets of proteins are pulled down from nuclear vs. cytoplasmic extracts. We used GeneCards to ascertain known functions of the protein preys. The protein preys include many that were previously known, but also many novel ones. Consideration of the novel ones suggests many aspects of NFAT5 regulation, interaction and function that were not previously appreciated, for example, hypertonicity inhibits NFAT5 by sumoylating it and the NFAT5 protein preys include components of the CHTOP complex that desumoylate proteins, an action that should contribute to activation of NFAT5.

Hypertonicity (e.g., high NaCl) activates the transcription factor tonicity-responsive enhancer/osmotic response element-binding protein (TonEBP/OREBP), increasing transcription of protective genes. In the present studies, by stably expressing amino acids 1-547 of TonEBP/OREBP in HEK 293 cells and immunoprecipitating it plus associated proteins from the nuclei of cells exposed to high NaCl, we identify 14 proteins that are physically associated with TonEBP/OREBP. The associated proteins fall into several classes: 1) DNA-dependent protein kinase, both its catalytic subunit and regulatory subunit, Ku86; 2) RNA helicases, namely RNA helicase A, nucleolar RNA helicase II/Gu, and DEAD-box RNA helicase p72; 3) small or heterogeneous nuclear ribonucleoproteins (snRNPs or hnRNPs), namely U5 snRNP-specific 116 kDa protein, U5 snRNP-specific 200 kDa protein, hnRNP U, hnRNP M, hnRNP K, and hnRNP F; 4) heat shock proteins, namely Hsp90beta and Hsc70; and 5) poly(ADP-ribose) polymerase-1 (PARP-1). We confirm identification of most of the proteins by Western analysis and also demonstrate by electrophoretic mobility-shift assay that they are present in the large complex that binds specifically along with TonEBP/OREBP to its cognate DNA element. In addition, we find that PARP-1 and Hsp90 modulate TonEBP/OREBP activity. PARP-1 expression reduces TonEBP/OREBP transcriptional activity and the activity of its transactivating domain. Hsp90 enhances those activities and sustains the increased abundance of TonEBP/OREBP protein in cells exposed to high NaCl.

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]

High NaCI causes DNA double-strand breaks and activates the transcription factor, TonEBP/OREBP, resulting in increased transcription of several protective genes, including those involved in accumulation of compatible organic osmolytes. Several kinases are known to contribute to signaling activation of TonEBP/OREBP, including ATM, which is a member of the phosphatidylinositol 3-kinase (PI3K)-like kinase family and is activated by DNA double- strand breaks. The purpose of the present studies was to investigate a possible role of PI3K Class IA (PI3K-IA). We found that high NaCI increases PI3K-IA lipid kinase activity. Inhibiting PI3K-IA either by expressing a dominant negative of its regulatory subunit, p85, or by small interfering RNA-mediated knockdown of its catalytic subunit, p110α, reduces high NaCI-induced increases in TonEBP/OREBP transcriptional activity and transactivation, but not nuclear translocation of TonEBP/OREBP, or increases in its abundance. Further, suppression of PI3K-IA inhibits the activation of ATM that is caused by either ionizing radiation or high NaCI. High NaCI-induced increase in TonEBP/OREBP activity is reduced equally by inhibition of ATM or PI3K-IA, and the effects are not additive. The conclusions are as follows: (i) PI3K-IA activity is necessary for both high NaCl- and ionizing radiation-induced activation of ATM and (ii) high NaCI activates PI3K-IA, which, in turn, contributes to full activation of TonEBP/OREBP via ATM. [ABSTRACT FROM AUTHOR]

Hypertonicity increases mRNA and protein abundance of the transcription factor tonicity-responsive enhancer/osmotic response element binding protein (TonEBP/OREBP), contributing to increased transcription of downstream osmoprotective genes. Previously, this was attributed to increased transcription of TonEBP/OREBP because no change was found in its mRNA stability. However, there is no direct evidence for increased transcription, and the 3′-untranslated region (UTR) of TonEBP/OREBP contains numerous adenylate/uridylate-rich elements, which can modulate RNA stability. Therefore, we have reinvestigated the effect of hypertonicity on TonEBP/OREBP mRNA stability. We find that, in mouse inner medullary collecting duct cells, raising osmolality from 300 to 500 mosmol/kgH2O by adding NaCl increases TonEBP/OREBP mRNA to a peak of 2.3-fold after 4 h, followed by a decline. TonEBP/OREBP protein increases to a sustained peak of 3.0-fold at 8 h. To determine the stability of TonEBP/OREBP mRNA, we measured the rate of its decrease after inhibiting transcription with actinomycin D, finding that it is stabilized for 6 h after addition of NaCl. This stabilization is sufficient to explain the increase in mRNA without any change in transcription. To investigate how hypertonicity stabilizes TonEBP/OREBP mRNA, we tested luciferase reporters containing parts of the TonEBP/OREBP mRNA UTR. Inclusion of both the 5′- and 3′-UTR increases reporter activity, consistent with mRNA stabilization. Surprisingly, however, it is the 5′-UTR that stabilizes; the 3′-UTR, by itself, decreases reporter activity. We concluded that 1) hypertonicity stabilizes TonEBP/OREBP mRNA, contributing to its increase, and 2) stabilization depends on the presence of the 5′-UTR. [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.

Tonicity-responsive enhancer/osmotic response element-binding protein (TonEBP/OREBP) is a Rel protein that activates transcription of osmoprotective genes at high extracellular NaCl. Other Rel proteins NFAT1-4 and NF-κB complex with activator protein-1 (AP-1) to transactivate target genes through interaction at composite NFAT/NF-κB·AP-1 sites. TonEBP/OREBP target genes commonly have one or more conserved AP-1 binding sites near TonEBP/OREBP cognate elements (OREs). Also, TonEBP/OREBP and the AP-1 proteins c-Fos and c-Jun are all activated by high NaCl. We now find, using an ORE·AP-1 reporter from the target aldose reductase gene or the same reporter with a mutated AP-1 site, that upon stimulation by high extracellular NaCl, 1) the presence of a wild type, but not a mutated, AP-1 site contributes to TonEBP/OREBP-dependent transcription and 2) AP-1 dominant negative constructs inhibit TonEBP/OREBP-dependent transcription provided the AP-1 site is not mutated. Using supershifts and an ORE·AP-1 probe, we find c-Fos and c-Jun present in combination with TonEBP/OREBP. Also, c-Fos and c-Jun coimmunoprecipitate with TonEBP/OREBP, indicating physical association. Small interfering RNA knockdown of either c-Fos or c-Jun inhibits high NaCl-induced increase of mRNA abundance of the TonEBP/ OREBP target genes AR and BGT1. Furthermore, a dominant negative AP-1 also reduces high NaCI-induced increase of TonEBP/ OREBP transactivating activity. Inhibition of p38, which is known to stimulate TonEBP/OREBP transcriptional activity, reduces high NaCl-dependent transcription of an ORE·AP-1 reporter only if the AP-1 site is intact. Thus, AP-1 is part of the TonEBP/OREBP enhanceosome, and its roe in high NaCl-induced activation of TonEBP/OREBP may require p38 activity. [ABSTRACT FROM AUTHOR]