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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 Hsp9013 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. osmolarity; immunoprecipitation; mass spectrometry

The response of renal inner medullary (IM) collecting duct cells (mIMCD3) to high NaCl involves increased expression of Gadd45 and p53, both of which have important effects on growth and survival of the cells. However, mIMCD3 cells, being immortalized by SV40, proliferate rapidly, which is known to sensitize cells to high NaCl, whereas IM cells in situ proliferate very slowly and survive much higher levels of NaCl. In the present studies, we have examined the importance of Gadd45 and p53 for survival of normal IM cells in their usual high-NaCl environment by using more slowly proliferating second-passage mouse inner medullary epithelial (p2mIME) cells and comparing cells from wild-type and gene knockout mice. Acutely elevating NaCl (and/or urea) reduces Gadd45a, but increases Gadd45b and Gadd45g mRNA, depending on the mix of NaCl and urea and the rate of increase of osmolality. Nevertheless, p2mIME cells from Gadd45[b.sup.-/-], Gadd45[g.sup.-/-], and Gadd45[bg.sup.-/-] mice survive elevation of NaCl (or urea) essentially the same as do wild-type cells. P[53.sup.-/-] Cells do not tolerate as high a concentration of NaCl (or urea) as p[53.sup.+/+] cells, but urinary concentrating ability of p[53.sup.-/-] mice is normal, as is the histology of inner medullas from p[53.sup.-/-] and Gadd45[abg.sup.-/-] mice. Thus although Gadd45 and p53 may play roles in osmotically stressed mIMCD3 cells, we do not find that their expression makes an important difference, either for Gadd45 in slower proliferating p2mIME cells or for Gadd45 or p53 in normal inner medullary epithelial cells in situ. high NaCl; high urea; cell survival doi:10.1152/ajprenal.00518.2005

Hypertonicity activates the transcription factor tonicity-responsive enhancer/ osmotic response element binding protein (TonEBP/OREBP), resulting in increased expression of genes involved in osmoprotective accumulation of organic osmolytes, including glycine betaine, and in increased expression of osmoprotective heat shock proteins. Our previous studies showed that high NaCI increases reactive oxygen species (ROS), which contribute to activation of TonEBP/OREBP. Mitochondria are a major source of ROS. The purpose of the present study was to examine whether mitochondria produce the ROS that contribute to activation of TonEBP/OREBP. We inhibited mitochondrial ROS production in HEK293 cells with rotenone and myxothiazol, which inhibit mitochondrial complexes I and III, respectively. Rotenone (250 nM) and myxothiazol (12 nM) reduce high NaCl induced ROS over 40%, whereas apocynin (100 [micro]M), an inhibitor of NADPH oxidase, and allopurinol (100 [micro]M), an inhibitor of xanthine oxidase, have no significant effect. Rotenone and myxothiazol reduce high NaCl-induced increases in TonEBP/OREBP transcriptional activity (ORE/TonE reporter assay) and BGTI (betaine transporter) mRNA abundance ranging from 53 to 69%. They inhibit high NaCl induced TonEBP/OREBP transactivating activity, but not its nuclear translocation. Release of ATP into the medium on hypertonic stress has been proposed to be a signal that triggers cellular osmotic responses. However, we do not detect release of ATP into the medium or inhibition of high NaCl-induced ORE/TonE reporter activity by an ATPase, apyrase (20 U/ml), indicating that high NaCl-induced activation of TonEBP/OREBP is not mediated by release of ATP. We conclude that high NaCl increases mitochondrial ROS production, which contributes to the activation of TonEBP/OREBP by increasing its transactivating activity. superoxides; rotenone: myxothiazol: transactivation; nuclear translocation; ATP release

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/kg[H.sub.2]O 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. mRNA stability; hypertonicity

High NaCl activates the transcription factor tonicity-responsive enhancer/ osmotic response element binding protein (TonEBP/OREBP) by increasing its abundance and transactivation, the latter signaled by a variety of protein kinases. In addition, high NaCl causes TonEBP/ OREBP to translocate into the nucleus, but little is known about the signals directing this translocation. The result is increased transcription of protective genes, including those involved in accumulation of organic osmolytes. High NaCl also damages DNA, and DNA damage activates ataxia telangiectasia-mutated (ATM) kinase through autophosphorylation on serine 1981. We previously found that ATM is involved in the high NaCl-induced increase in TonEBP/OREBP transactivation. The purpose of the present studies was to test whether ATM is also involved in high NaCl-induced TonEBP/OREBP nuclear translocation. We quantified TonEBP/OREBP in nuclear and cytoplasmic extracts from cultured cells by Western blot analysis. In COS-7 cells, wortmannin, an inhibitor of ATM, reduces high NaCl-induced nuclear translocation of TonEBP/OREBP. We used AT cells (in which ATM is inactive) to test the specificity of this effect. Nuclear translocation of native TonEBP/OREBP and of its recombinant N[H.sub.2]-terminal rel homology domain, which contains the nuclear localization signal, is reduced in AT cells and is restored when the cells are reconstituted with functional ATM. In conclusion, activation of ATM contributes to high NaCl-induced nuclear translocation of TonEBP/OREBP. ataxia telangiectasia-mutated kinase; tonicity-responsive enhancer/ osmotic response element binding protein; wortmannin; AT cells

The signaling pathways leading to high NaCl-induced activation of the transcription factor tonicity-responsive enhancer binding protein/osmotic response element binding protein (TonEBP/OREBP) remain incompletely understood. High NaCl has been reported to produce oxidative stress. Reactive oxygen species (ROS), which are a component of oxidative stress, contribute to regulation of transcription factors. The present study was undertaken to test whether the high NaCl-induced increase in ROS contributes to tonicity-dependent activation of TonEBP/OREBP. Human embryonic kidney 293 cells were used as a model. We find that raising NaCl increases ROS, including superoxide. N-acetylcysteine (NAC), an antioxidant, and MnTBAP, an inhibitor of superoxide, reduce high NaCl-induced superoxide activity and suppress both high NaCl-induced increase in TonEBP/OREBP transcriptional activity and high NaCl-induced increase in expression of BGT1mRNA, a transcriptional target of TonEBP/OREBP. Catalase, which decomposes hydrogen peroxide, does not have these effects, whether applied exogenously or overexpressed within the cells. Furthermore, NAC and MnTBAP, but not catalase, blunt high NaCl-induced increase in TonEBP/ OREBP transactivation. [N.sup.G]-monomethyl-L-arginine, a general inhibitor of nitric oxide synthase, has no significant effect on either high NaCl-induced increase in superoxide or TonEBP/OREBP transcriptional activity, suggesting that the effects of ROS do not involve nitric oxide. Ouabain, an inhibitor of Na-K-ATPase, attenuates high NaCl-induced superoxide activity and inhibits TonEBP/OREBP transcriptional activity. We conclude that the high NaCl-induced increase in ROS, including superoxide, contributes to activation of TonEBP/OREBP by increasing its transactivation. superoxides; nitric oxide; ouabain; transactivation; nuclear translocation

Renal medullary cells normally are exposed to extraordinarily high interstitial NaCl concentration as part of the urinary concentrating mechanism, yet they survive and function. Acute elevation of NaCl to a moderate level causes transient cell cycle arrest in culture. Higher levels of NaCl, within the range found in the inner medulla, cause apoptosis. Recently, it was surprising to discover that even moderately high levels of NaCl cause DNA double-strand breaks. The DNA breaks persist in cultured cells that are proliferating rapidly after adaptation to high NaCl, and DNA breaks normally are present in the renal inner medulla in vivo. High NaCl inhibits repair of broken DNA both in culture and in vivo, but the DNA is rapidly repaired if the level of NaCl is reduced. The inhibition of DNA repair is associated with suppressed activity of some DNA damage-response proteins like Mre11, Chk1, and H2AX but not that of others, like GADD45, p53, ataxia telangiectasia-mutated kinase (ATM), and Ku86. In this review, we consider possible mechanisms by which the renal ceils escape the known dangerous consequences of persistent DNA damage. Furthermore, we consider that the persistent DNA damage may be a sensor of hypertonicity that activates ATM kinase to provide a signal that contributes to protective osmotic regulation. hypertonicity; renal medulla; apoptosis; cell cycle delay

In tests of osmotic tolerance of renal inner medullary cells in tissue culture, osmolality has usually been increased in a single step, whereas in vivo the increase occurs gradually over several hours. We previously found that more passage 2 mouse inner medullary epithelial (p2mIME) cells survive a linear increase in NaCl and urea from 640 to 1,640 mosmol/kg[H.sub.2]O over 20 h (which is similar to the change that may occur in vivo) than they do a step increase. The present studies examine accompanying differences in gene expression. Among mRNAs of genes known to be protective, tonicity-responsive enhancer binding protein and aldose reductase increase with a linear but decrease with a step increase; betaine transporter BGTI decreases with a step but not a linear increase; heat shock protein 70.1 (HSP70.1) and HSP70.3 increase more with a linear than a step increase; and osmotic stress protein 94 and heme oxygenase-1 increase with a linear but decrease with a step increase, mRNAs for known urea-responsive proteins, GADD153 and Egr-1, increase with both a step and linear increase. A step increase in urea alone reduces mRNAs, similar to the combination of NaCl and urea, but a step increase in NaCl alone does not. HSP70 protein increases substantially with a linear rise in osmolality but does not change significantly with a step rise. We speculate that poorer survival of p2mIME cells with a step than with linear increase in NaCl and urea is accounted for, at least in part, by urea-induced suppression of protective genes, particularly HSP70. osmotic stress; organic osmolyte; heat shock protein; mRNA abundance

TonEBP is a transcription factor that, when activated by hypertonicity, increases transcription of genes, including those involved in organic osmolyte accumulation. Surprisingly, it is expressed in virtually all tissues, including many never normally exposed to hypertonicity. We measured TonEBP mRNA (real-time PCR) and protein (Western blot analysis) in tissues of control (plasma osmolality 294 [+ or -] 1 mosmol/kg[H.sub.2]O) and hyposmotic (dDAVP infusion plus water loading for 3 days, 241 [+ or -] 2 mosmol/kg[H.sub.2.]O) rats to test whether the ubiquitous expression of TonEBP mRNA is osmotically regulated around the normal plasma osmolality. TonEBP protein is reduced by hyposmolality in thymus and liver, but not in brain, and is not detected in heart and skeletal muscle. TonEBP mRNA decreases in brain and liver but is unchanged in other tissues. There are no general changes in mRNA of TonEBP-mediated genes: aldose reductase (AR) does not change in any tissue, betaine transporter (BGT1) decreases only in liver, taurine transporter (TauT) only in brain and thymus, and inositol transporter (SMIT) only in skeletal muscle and liver. Heat shock protein (Hsp)70-1 and Hsp70-2 mRNA increase greatly in most tissues, which cannot be attributed to decreased TonEBP activity. The conclusions are as follows: 1) TonEBP protein or mRNA expression is reduced by hyposmolality in thymus, liver, and brain. 2) TonEBP protein and mRNA expression are differentially regulated in some tissues. 3) Although AR, SMIT, BGT1, and Taut are regulated by TonEBP in renal medullary cells, other sources of regulation may predominate in other tissues. 4) TonEBP abundance and activity are regulated by factors other than tonicity in some tissues. aldose reductase; transcription factor; TonEBP

Hypertonicity-induced increase in activity of the transcription factor tonicity-responsive enhancer/osmotic response element-binding protein (TonEBP/OREBP) protects renal cells by increasing transcription of genes, including those involved in increased accumulation of organic osmolytes. We previously showed that hypertonicity increases transactivating activity of TonEBP/OREBP. Assay with a binary GAL4 transactivation system showed that the 984 C-terminal amino acids of TonEBP/OREBP (amino acids 548-1531) contain a tonicity-dependent transactivation domain (TAD). Also, amino acids 548-1531 undergo tonicity-dependent phosphorylation, and some inhibitors of protein kinases reduce the tonicity-dependent transactivation. In the present studies we examined the role of protein kinase A (PKA). Results: (i) An inhibitor of PKA (H89) reduces tonicity-dependent increases in transactivation, ORE/TonE reporter activity, and induction of aldose reductase and betaine transporter mRNAs. (ii) Overexpression of the catalytic subunit of PKA (PKAc) increases transactivation activity of amino acids 548-1531 and activity of an ORE/TonE reporter. The increases are much greater under isotonic than under hypertonic conditions. (iii) A dominant-negative PKAc reduces activity of an ORE/TonE reporter. (iv) PKAc activity increases with tonicity but cAMP does not. (v) TonEBP/OREBP and PKAc coimmunoprecipitate. (vi) amino acids 872-1271, including N- and C-terminal polyglutamine stretches, demonstrate tonicity-dependent transactivation, albeit less than amino acids 548-1531, and a similar role for PKA. Conclusions: (i) PKA plays an important role in TonEBP/ OREBP activation of tonicity-dependent gene expression; (ii) PKA activation of TonEBP/OREBP appears to be cAMP-independent; and (iii) amino acids 872-1271 are sufficient for tonicity-dependent transactivation of TonEBP/OREBP.

Mammalian renal inner medullary (IM) cells routinely face and resist hypertonic stress. Such stress causes DNA damage to which IM cells respond with cell cycle arrest. We report that three growth arrest and DNA damage-inducible 45 (GADD45) isoforms (GADD45[alpha], GADDD45[beta], and GADD45[gamma]) are induced by acute hypertonicity in murine IM cells. Maximum induction occurs 16-18 h after the onset of hypertonicity. GADD45[gamma] is induced more strongly (7-fold) than GADD45[beta] (3-fold) and GADD45[alpha] (2-fold). GADD45[alpha] and GADD45[beta] protein induction is more pronounced and stable compared with the corresponding transcripts. Hypertonicity of various forms (NaCl, KCl, sorbitol, or mannitol) always induces GADD45 transcripts, whereas nonhypertonic hyperosmolality (urea) has no effect. Actinomycin D does not prevent hypertonic GADD45 induction, indicating that mRNA stabilization is the mechanism that mediates this induction. GADD45 induction patterns in IM cells exposed to 10 different stresses suggest isoform specificity, but similar functions, of individual isoforms during hypertonicity, heat shock, and heavy metal stress, when GADD45[gamma] induction is strongest (17-fold). These data associate all known GADD45 isoforms with the hypertonicity phenotype of renal IM cells. cell cycle; hypertonicity; nephrotoxins; kidney inner medulla

Each base of a minimal osmotic response element (ORE) having the sequence CGGAAAATCAC(C) was replaced with every other possible nucleotide and the resulting sequences were tested to study their binding activity. The hyperosmotic response was also categorized using electrophoretic mobility shift assays of a 17-bp sequence. Results showed that a functional consensus for the mammalian ORE is NGGAAAWDHMC(N).

The effect of osmolyte accumulation on betaine transporter mRNA abundance in rabbit renal papillary cells, PAP-HT25, was investigated using cDNA cloned from PAP-HT25 cells. The presence of the osmolytes betaine, sorbitol and inositol increased betaine transporter mRNA abundance but their accumulation caused a subsequent reduction in betaine transporter mRNA. Similar results were observed for aldose reductase. The results suggest that betaine transporter mRNA accumulation is regulated by betaine transporter mRNA gene expression.

Extracellular hypertonicity induces the formation of CD9 antigen mRNA in the renal epithelial cell lines Madin-Darby canine kidney and PAP-HT25. C9 is involved in the hyperosmotic stress response as hypertonicity increases the transcription of proteins, participating in metabolism and osmolyte transportation. The CD9 mRNA induction decreases with accumulation of organic osmolytes. Induction of C9 is specific rather than as a part of a generalized response system. Differential mRNA display polymerase chain reaction technique is used to detect the cDNA product of CD9 mRNA.

Compatible organic osmolytes decrease the increased expression of heat shock protein (HSP)-70 mRNA where the rise in expression is due to higher temperature and higher tonicity of the medium. Increase in temperature and hypertonicity alters the cellular proteins while osmolytes stabilize these cellular proteins. Two interrelated mechanisms explain the stabilization of intracellular proteins.

The perturbations caused by the hyperosmotic stresses in cells of the renal inner medulla due to high levels of NaCl and urea are discussed. The mechanisms of the adaptive osmoprotective responses of the cells are analyzed.