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Hypertonicity activates the transcription factor TonEBP/OREBP, resulting in increased expression of osmoprotective genes, including those responsible for accumulation of organic osmolytes and heat-shock proteins. Phosphorylation of TonEBP/OREBP contributes to its activation. Several of the kinases that are involved were previously identified, but the phosphatases were not. In the present studies we screened a genomewide human phosphatase siRNA library in human embryonic kidney (HEK)293 cells for effects on TonEBP/OREBP transcriptional activity. We found that siRNAs against 57 phosphatases significantly alter TonEBP/OREBP transcriptional activity during normotonicity (290 mosmol/kg) or hypertonicity (500 mosmol/kg, NaCl added) or both. Most siRNAs increase TonEBP/OREBP activity, implying that the targeted phosphatases normally reduce that activity. We further studied in detail SHP-1, whose knockdown by its specific siRNA increases TonEBP/OREBP transcriptional activity at 500 mosmol/kg. We confirmed that SliP-1 is inhibitory by overexpressing it, which reduces TonEBP/OREBP transcriptional activity at 500 mosmol/kg. SHP-1 dephosphorylates TonEBP/OREBP at a known regulatory site, Y143, both in vivo and in vitro. It inhibits TonEBP/OREBP by both reducing TonEBP/OREBP nuclear localization, which is Y143 dependent, and by lowering high NaCl-induced TonEBP/OREBP transactivating activity. SHP-1 coimmunoprecipitates with TonEBP/OREBP and vice versa, suggesting that they are physically associated in the cell. High NaCl inhibits the effect of SHP-1 on TonEBP/OREBP by increasing phosphorylatlon of SHP-1 on Ser591, which reduces its phosphatase activity and localization to the nucleus. Thus, TonEBP/OREBP is extensively regulated by phosphatases, including SHP-1, whose inhibition by high NaCl increases phosphorylation of TonEBP/OREBP at Y143, contributing to the nuclear localization and activation of TonEBP/OREBP. hypertonicity | NFAT5 | nuclear localization | phosphorylation | siRNA library doi/ 10.1073/pnas.1002795107

High NaCl elevates activity of the osmoprotective transcription factor TonEBP/OREBP by increasing its phosphorylation, trans-activating activity, and localization to the nucleus. We investigated the possible role in this activation of phospholipase C-[gamma]1 (PLC-[gamma]1), which has a predicted binding site at TonEBP/OREB-Pphospho-Y143. We find the following. (i) Activation of TonEBP/ OREBP transcriptional activity by high NaCl is reduced in PLC-[gamma]1 null cells and in HEK293 cells in which PLC-[gamma]1 is knocked down by a specific siRNA. (ii) High NaCI increases phosphorylation of TonEBP/ OREBP at Y143. (iii) Wild-type PLC-[gamma]1 coimmunoprecipitates with wild-type TonEBP/OREBP but not TonEBP/OREBP-Y143A, and the coimmunoprecipitation is increased by high NaCl. (iv) PLC-[gamma]1 is part of the protein complex that associates with TonEBP/OREBP at its DNA binding site. (v) Knockdown of PLC-[gamma]1 or overexpression of a PLC-[gamma]1-SH3 deletion mutant reduces high NaCl-dependent TonEBP/OREBP transactivating activity. (vi) Nuclear localization of PLC-[gamma]1 is increased by high NaCl. (vii) High NaCl-induced nuclear localization of TonEBP/OREBP is reduced if cells lack PLC-[gamma]1, if PLC[gamma]1 mutated in its SH2C domain is overexpressed, or if Y143 in TonEBP/OREBP is mutated to alanine. (viii) Expression of recombinant PLC-[gamma]1 restores nuclear localization of wild-type TonEBP/ OREBP in PLC-[gamma]1 null cells but not of TonEBP/OREBP-Y143A. (ix) The PLC-[gamma]1 phospholipase inhibitor U72133 inhibits nuclear localization of TonEBP/OREBP but not the increase of its transactivating activity. We conclude that, when NaCl is elevated, TonEBP/OREBP becomes phosphorylated at Y143, resulting in binding of PLC-[gamma]1 to that site, which contributes to TonEBP/OREBP transcriptional activity, transactivating activity, and nuclear localization. hypertonicity | phosphorylation | proteomics www.pnas.org/cgi/doi/10.1073/pnas.0913415107

Glycerophosphocholine (GPC) is an abundant osmoprotective renal medullary organic osmolyte. We previously found that its synthesis from phosphatidylcholine is catalyzed by tonicity-regulated activity of the phospholipase B, neuropathy target esterase. We also found that its degradation is catalyzed by glycerophosphocholine phosphodiesterase (GPC-PDE) activity and that elevating osmolality from 300 to 500 mosmol/kg by adding NaCl or urea, inhibits GPC-PDE activity, which contributes to the resultant increase of GPC. In the present studies we identify GDPD5 (glycerophosphodiester phosphodiesterase domain containing 5) as a GPC-PDE that is rapidly inhibited by high NaCI or urea. Recombinant GDPD5 colocalizes with neuropathy target esterase in the perinuclear region of HEK293 cells, and immuno-precipitated recombinant GDPD5 degrades GPC in vitro. The in vitro activity is reduced when the cells from which the GDPD5 is immuno-precipitated have been exposed to high NaCI or urea. In addition, high NaCI decreases mRNA abundance of GDPD5 via an increase of its degradation rate, although high urea does not. At 300 mosmol/kg siRNA knockdown of GDPD5 increases GPC in mouse inner medullary collecting duct-3 cells, and over expression of recombinant GDPD5 increases cellular GPC-PDE activity, accompanied by decreased GPC. We conclude that GDPD5 is a GPC-PDE that contributes to osmotic regulation of cellular GPC. high urea | hypertonicity | RNA stability

High NaCI rapidly activates p38 MAPK by phosphorylating it, the phosphorylation presumably being regulated by a balance of kinases and phosphatases. Kinases are known, but the phosphatases are uncertain. Our initial purpose was to identify the phosphatases. We find that in HEK293 cells transient overexpression of MAPK phosphatase-1 (MKP-1), a dual-specificity phosphatase, inhibits high NaCI-induced phosphorylation of p38, and that overexpression of a dominant negative mutant of MKP-1 does the opposite. High NaCI lowers MKP-1 activity by increasing reactive oxygen species, which directly inhibit MKP-1, and by reducing binding of MKP-1 to p38. Because inhibition of p38 is reported to reduce hypertonicity-induced activation of the osmoprotective transcription factor, TonEBP/OREBP, we anticipated that MKP-1 expression might also. However, overexpression of MKP-1 has no significant effect on Ton EBP/OREBP activity. This paradox is explained by opposing effects of p38[alpha] and p38[delta], both of which are activated by high NaCI and inhibited by MKP-1. Thus, we find that overexpression of p38[alpha] increases high NaCI-induced TonEBP/ OREBP activity, but overexpression of p38[delta] reduces it. Also, siRNAmediated knockdown of p38[delta] enhances the activation of TonEBP/OREBP. We conclude that high NaCI inhibits MKP-1, which contributes to the activation of p38. However, opposing actions of p38[alpha] and p38[delta] negate any effect on TonEBP/OREBP activity. Thus, activation of p38 isoforms by hypertonicity does not contribute to activation of TonEBP/OREBP because of opposing effects of p38[alpha] and p38[delta], and effects of inhibitors of p38 depend on which isoform is affected, which can be misleading. hypertonicity | reactive oxygen species | transcription HEK293 cells | mlMCD3 cells

Glycerophosphocholine (GPC) is an osmoprotective compatible and counteracting organic osmolyte that accumulates in renal inner medullary cells in response to high NaCl and urea. We previously found that high NaCl increases GPC in renal [Madin-Darby canine kidney (MDCK)] cells. The GPC is derived from phosphatidylcholine, catalyzed by a phospholipase that was not identified at that time. Neuropathy target esterase (NTE) was recently shown to be a phospholipase B that catalyzes production of GPC from phosphatidylcholine. The purpose of the present study was to test whether NTE contributes to the high NaCl-induced increase of GPC synthesis in renal cells. We find that in mouse inner medullary collecting duct cells, high NaCl increases NTE mRNA within 8 h and NTE protein within 16 h. Diisopropyl fluorophosphate, which inhibits NTE esterase activity, reduces GPC accumulation, as does an siRNA that specifically reduces NTE protein abundance. The 20-h half-life of NTE mRNA is unaffected by high NaCl. TonEBP/OREBP is a transcription factor that is activated by high NaCl. Knockdown of TonEBP/OREBP by a specific siRNA inhibits the high NaCl-induced increase of NTE mRNA. Further, the lower renal inner medullary interstitial NaCl concentration that occurs chronically in [CICK1.sup.-/-] mice and acutely in normal mice given furosemide is associated with lower NTE mRNA and protein. We conclude that high NaCl increases transcription of NTE, likely mediated by TonEBP/OREBP, and that the resultant increase of NTE expression contributes to increased production and accumulation of GPC in mammalian renal cells in tissue culture and in vivo. diisopropyl fluorophosphate | phospholipase B

High NaCl 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 NaCl 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, p110[alpha], reduces high NaCl-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 NaCl. High NaCl-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 NaCl activates PI3K-IA, which, in turn, contributes to full activation of TonEBP/OREBP via ATM. DNA damage | osmotic stress | TonEBP/OREBP

Pax2 is a transcription factor that is crucial for kidney development, and it is also expressed in the normal adult kidney, where its physiological function is unknown. In the present study, we find by cDNA microarray analysis that Pax2 expression in second-passage mouse inner-medullary epithelial cells is increased by a high NaCl concentration, which is significant because NaCl levels are normally high in the inner medulla in vivo, and varies with urinary concentration. Furthermore, a high NaCl concentration increases Pax2 mRNA and protein expression in mouse inner medullary collecting duct (mlMCD3) cells, and its transcriptional activity. Pax2 mRNA and protein expression is high in normal adult mouse renal inner medulla but much lower in renal cortex. Pax2 protein is present in collecting duct cells in both renal medulla and cortex and in thin descending limbs of Henle's loop in inner medulla. Treating Brattleboro rats with desamino-Cys-1, D-Arg-8 vasopressin, which increases inner-medullary NaCl concentration, causes a 4-fold increase in inner-medullary Pax2 protein. Treatment with furosemide, which decreases inner-medullary NaCl, reduces inner-medullary Pax2 mRNA and protein. Pax2-specific short interfering RNA increases high NaCl concentration-induced activation of caspase-3 and apoptotic bodies in mlMCD3 cells. We thus conclude that (i) Pax2 is expressed in normal renal medulla, (ii) its expression is regulated there by the normally high and variable NaCl concentration, and (iii) it protects renal medullary cells from high NaCl concentration-induced apoptosis. apoptosis | osmotic stress | renal inner medulla

High NaCl activates the transcription factor tonicity-responsive enhancer/osmotic response element-binding protein (TonEBP/ OREBP), resulting in increased transcription of several protective genes, including the glycine betaine/[gama]-aminobutyric acid transporter (BGT1). High NaCl damages DNA, and DNA damage activates ataxia telangiectasia mutated (ATM) kinase through autophosphorylation on Ser-1981. TonEBP/OREBP contains ATM consensus phosphorylation sites at Ser-1197, Ser-1247, and Ser-1367. The present studies test whether ATM is involved in activation of TonEBP/OREBP by high NaCl. We find that raising osmolality from 300 to 500 mosmol/kg by adding NaCl activates ATM, as indicated by phosphorylation at Ser-1981. High urea and radiation also activate ATM, but they do not increase TonEBP/OREBP transcriptional activity like high NaCl does. Wortmannin, which inhibits ATM, reduces NaCl-induced TonEBP/OREBP transcriptional activation and BGT1 mRNA increase. Overexpression of wild-type TonEBP/OREBP increases ORE/TonE reporter activity much more than does overexpression of TonEBP/OREBP S1197A, $1247A, or $1367A. In AT cells (which express nonfunctional ATM), TonEBP/ OREBP transcriptional and transactivating activity are further increased by expression of wild-type ATM but not of S1981A ATM. TonEBP/OREBP reciprocally coimmunoprecipitates with ATM kinase, demonstrating physical association. Additionally, antibody to ATM kinase supershifts TonEBP/OREBP bound to its cognate ORE/TonE DNA element. In AT cells, wortmannin further decreases high NaCl-induced increase in transcriptional activity, consistent with participation of signaling kinase(s) in addition to ATM. In conclusion, signaling via ATM is necessary for full activation of TonEBP/OREBP by high NaCl, but it is not sufficient.

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.

Hypertonicity-induced binding of the transcription factor TonEBP/ OREBP to its cognate DNA element, ORE/TonE, is associated with increased transcription of several osmotically regulated genes. Previously, it was found that hypertonicity rapidly causes nuclear translocation and phosphorylation of TonEBP/OREBP and, more slowly, increases TonEBP/OREBP abundance. Also, the C terminus of TonEBP/OREBP was found to contain a transactivation domain (TAD). We have now tested for tonicity dependence of the TAD activity of the 983 C-terminal amino acids of TonEBP/OREBP. HepG2 cells were cotransfected with a reporter construct and one of several TAD expression vector constructs. The reporter construct contained GAL4 DNA binding elements, a minimal promoter, and the Photinus luciferase gene. TAD expression vectors generate chimeras comprised of the GAL4 DNA binding domain fused to (i) the 983 C-terminal amino acids of TonEBP/OREBP, (ii) 17 glutamine residues, (iii) the TAD of c-Jun, or (iv) no TAD. All TAD-containing chimeras were functional at normal extracellular osmolality (300 mosmol/kg), but the activity only of the chimera containing the 983 C-terminal amino acids of TonEBP/OREBP varied with extracellular NaCl concentration, decreasing by >80% at 200 mosmol/kg and increasing 8-fold at 500 mosmol/kg. The chimera containing the 983 C-terminal amino acids of TonEBP/OREBP was constitutively localized to the nucleus and showed tonicity-dependent posttranslational modification consistent with phosphorylation. The activity at 500 mosmol/kg was reduced by herbimycin, a tyrosine kinase inhibitor and by 5,6-dichloro-1-[beta]-D-ribofuranosyl-benzimidazole, a protein kinase CK2 inhibitor. Thus, the 983 C-terminal amino acids of TonEBP/OREBP contain a TAD that is regulated osmotically, apparently by tonicity-dependent phosphorylation.

Cloning and characterization of aldose reductase (AR) gene from rabbit to examine the modulation of the gene expression by hypersomalality reveal that the upstream region of rAR promoter contains osmotic response elements (OREs) in a DNA fragment that facilitates AR gene modulation during osmotic stress. Use of a 235-base pair fragment and the upstream sequences in luciferase reporter gene constructs enhances luciferase gene expression by 40 fold. Further analysis of OREs and their trans-acting factors help define the molecular processes that mediate the gene regulation during osmotic stress.

Nuclear Factor of Activated T cells 5 (NFAT5) is a transcription factor (TF) that mediates protection from adverse effects of hypertonicity by increasing transcription of genes, including those that lead to cellular accumulation of protective organic osmolytes. NFAT5 has three intrinsically ordered (ID) activation domains (ADs). Using the NFAT5 N-terminal domain (NTD), which contains AD1, as a model, we demonstrate by biophysical methods that the NTD senses osmolytes and hypertonicity, resulting in stabilization of its ID regions. In the presence of sufficient NaCl or osmolytes, trehalose and sorbitol, the NFAT5 NTD undergoes a disorder-to-order shift, adopting higher average secondary and tertiary structure. Thus, NFAT5 is activated by the stress that it protects against. In its salt and/or osmolyte-induced more ordered conformation, the NTD interacts with several proteins, including HMGI-C, which is known to protect against apoptosis. These findings raise the possibility that the increased intracellular ionic strength and elevated osmolytes caused by hypertonicity activate and stabilize NFAT5. [ABSTRACT FROM AUTHOR]

Glycerophosphocholine (GPC) is high in cells of the renal inner medulla where high interstitial NaCl and urea power concentration of the urine. GPC protects inner medullary cells against the perturbing effects of high NaCl and urea by stabilizing intracellular macromolecules. Degradation of GPC is catalyzed by the glycerophosphocholine phosphodiesterase activity of glycerophosphodiester phosphodiesterase domain containing 5 (GDPD5). We previously found that inhibitory posttranslational modification (PTM) of GDPD5 contributes to high NaCl- and urea-induced increase of GPC. The purpose of the present studies was to identify the PTM(s). We find at least three such PTMs in HEK293 cells: (i) Formation of a disulfide bond between C25 and C571. High NaCl and high urea increase reactive oxygen species (ROS). The ROS increase disulfide bonding between GDPD5-C25 and -C571, which inhibits GDPD5 activity, as supported by the findings that the antioxidant N-acetylcysteine prevents high NaCl- and urea-induced inhibition of GDPD5; GDPD5-C25S/C571S mutation or over expression of peroxiredoxin increases GDPD5 activity; H2O2 inhibits activity of wild type GDPD5, but not of GDPD5-C25S/C571S; and peroxiredoxin is relatively low in the renal inner medulla where GPC is high. (ii) Dephosphorylation of GDPD5-T587. GDPD5 threonine 587 is constitutively phosphorylated. High NaCl and high urea dephosphorylate GDPD5-T587. Mutation of GDPD5-T587 to alanine, which cannot be phosphorylated, decreases GPC-PDE activity of GDPD5. (iii) Alteration at an unknown site mediated by CDK1. Inhibition of CDK1 protein kinase reduces GDE-PDE activity of GDPD5 without altering phosphorylation at T587, and CDK1/5 inhibitor reduces activity of GDPD5- C25S/C571S-T587A. [ABSTRACT FROM AUTHOR]

Separate reports that hypertonicity activates p38 via a Rac1-OSM-MEKK3-MKK3-p38 pathway and that p38a contributes to activation of TonEBP/OREBP led us to the hypothesis that Rac1 might activate TonEBP/OREBP via p38. The present studies examine that possibility. High NaCl is hypertonic. We find that siRNA knockdown of Rac1 reduces high NaCl-induced increase of TonEBP/OREBP transcriptional activity (by reducing its transactivating activity but not its nuclear localization). Similarly, siRNA knockdown of osmosensing scaffold for MEKK3 (OSM) also reduces high NaCl-dependent TonEBP/OREBP transcriptional and transactivating activities. Simultaneous siRNA knockdown of Rac1 and OSM is not additive in reduction of TonEBP/OREBP transcriptional activity, indicating a common pathway. However, siRNA knockdown of MKK3 does not reduce TonEBP/OREBP transcriptional activity, although siRNA knockdown of MKK6 does. Nevertheless, the effect of Rac1 on TonEBP/OREBP is also independent of MKK6 because it occurs in MKK6-null cells. Furthermore, we find that siRNA knockdown of Rac1 or OSM actually increases activity (phosphorylation) of p38, rather than decreasing it, as previously reported. Thus, the effect of Rac1 on TonEBP/OREBP is independent of p38. We find instead that phospholipase C-γ1 (PLC-γ1) is involved. When transfected into PLC-γ1-null mouse embryonic fibroblast cells, catalytically active Rac1 does not increase TonEBP/OREBP transcriptional activity unless PLC-γ1 is reconstituted. Similarly, dominant-negative Rac1 also does not inhibit TonEBP/OREBP in PLC-γ1-null cells unless PLC-γ1 is reconstituted. We conclude that Rac1/OSM supports TonEBP/OREBP activity and that this activity is mediated via PLC-γ1, not p38. [ABSTRACT FROM AUTHOR]

Hypertonicity activates the transcription factor TonEBP/OREBP, resulting in increased expression of osmoprotective genes, including those responsible for accumulation of organic osmolytes and heat-shock proteins. Phosphorylation of TonEBP/OREBP contributes to its activation. Several of the kinases that are involved were previously identified, but the phosphatases were not. In the present studies we screened a genomewide human phosphatase siRNA library in human embryonic kidney (HEK)293 cells for effects on TonEBP/OREBP transcriptional activity. We found that siRNAs against 57 phosphatases significantly alter TonEBP/OREBP transcriptional activity during normotonicity (290 mosmol/kg) or hypertonicity (500 mosmol/kg, NaCl added) or both. Most siRNAs increase TonEBP/OREBP activity, implying that the targeted phosphatases normally reduce that activity. We further studied in detail SHP-1, whose knockdown by its specific siRNA increases TonEBP/OREBP transcriptional activity at 500 mosmol/kg. We confirmed that SHP-1 is inhibitory by overexpressing it, which reduces TonEBP/OREBP transcriptional activity at 500 mosmol/kg. SHP-1 dephosphorylates TonEBP/OREBP at a known regulatory site, Y143, both in vivo and in vitro. It inhibits TonEBP/OREBP by both reducing TonEBP/OREBP nuclear localization, which is Y143 dependent, and by lowering high NaCl-induced TonEBP/OREBP transactivating activity. SHP-1 coimmunoprecipitates with TonEBP/OREBP and vice versa, suggesting that they are physically associated in the cell. High NaCl inhibits the effect of SHP-1 on TonEBP/OREBP by increasing phosphorylation of SHP-1 on Ser591, which reduces its phosphatase activity and localization to the nucleus. Thus, TonEBP/OREBP is extensively regulated by phosphatases, including SHP-1, whose inhibition by high NaCl increases phosphorylation of TonEBP/OREBP at Y143, contributing to the nuclear localization and activation of TonEBP/OREBP. [ABSTRACT FROM AUTHOR]

High NaCI elevates activity of the osmoprotective transcription factor TonEBP/OREBP by increasing its phosphorylation, transactivating activity, and localization to the nucleus. We investigated the possible role in this activation of phospholipase C-γ1 (PLC-γ1). which has a predicted binding site at TonEBP/OREBPphospho-Y143. We find the following. (i) Activation of TonEBP/ OREBP transcriptional activity by high NaCI is reduced in PLC-γ1 null cells and in HEK293 cells in which PLC-γ1 is knocked down by a specific siRNA. (ii) High NaCI increases phosphorylation of TonEBP/ OREBP at Y143. (iii) Wild-type PLC-γ1 coimmunoprecipitates with wild-type TonEBP/OREBP but not TonEBP/OREBP-Y143A, and the coimmunoprecipitation is increased by high NaCI. (iv) PLC-γ1 is part of the protein complex that associates with TonEBP/OREBP at its DNA binding site. (v) Knockdown of PLC-γ1 or overexpression of a PLC-γ1-5H3 deletion mutant reduces high NaCI-dependent TonEBP/OREBP transactivating activity. (vi) Nuclear localization of PLC-yl is increased by high NaCI. (vii) High NaCI-induced nuclear localization of TonEBP/OREBP is reduced if cells lack PLC-γ1, if PLCγ1 mutated in its SH2C domain is overexpressed, or if Y143 in TonEBP/OREBP is mutated to alanine. (viii) Expression of recombinant PLC-γ1 restores nuclear localization of wild-type TonEBP/ OREBP in PLC-γ1 null cells but not of TonEBP/OREBP-Y143A. (ix) The PLC-γ1 phospholipase inhibitor U72133 inhibits nuclear localization of TonEBP/OREBP but not the increase of its transactivating activity. We conclude that, when NaCI is elevated, TonEBP/OREBP becomes phosphorylated at Y143, resulting in binding of PLC-γ1 to that site, which contributes to TonEBP/OREBP transcriptional activity-transactivating activity, and nuclear localization. [ABSTRACT FROM AUTHOR]

High NaCl rapidly activates p38 MAPK by phosphorylating it, the phosphorylation presumably being regulated by a balance of kinases and phosphatases. Kinases are known, but the phosphatases are uncertain. Our initial purpose was to identify the phosphatases. We find that in HEK293 cells transient overexpression of MAPK phosphatase-1 (MKP-1), a dual-specificity phosphatase, inhibits high NaCl-induced phosphorylation of p38, and that over-expression of a dominant negative mutant of MKP-1 does the opposite. High NaCl lowers MKP-1 activity by increasing reactive oxygen species, which directly inhibit MKP-1, and by reducing binding of MKP-1 to p38. Because inhibition of p38 is reported to reduce hypertonicity-induced activation of the osmoprotective transcription factor, TonEBP/OREBP, we anticipated that MKP-1 expression might also. However, overexpression of MKP-1 has no significant effect on Ton EBP/OREBP activity. This paradox is explained by opposing effects of p38α and p38δ, both of which are activated by high NaCl and inhibited by MKP-1. Thus, we find that overexpression of p38α increases high NaCI-induced TonEBP/OREBP activity, but overexpression of p38δ reduces it. Also, siRNA-mediated knockdown of p38δ enhances the activation of TonEBP/OREBP. We conclude that high NaCl inhibits MKP-1, which contributes to the activation of p38. However, opposing actions of p38α and p38δ negate any effect on TonEBP/OREBP activity. Thus, activation of p38 isoforms by hypertonicity does not contribute to activation of TonEBP/OREBP because of opposing effects of p38α and p38δ, and effects of inhibitors of p38 depend on which isoform is affected, which can be misleading. [ABSTRACT FROM AUTHOR]

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 tonicitydependent 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. [ABSTRACT FROM AUTHOR]