Increased reactive oxygen species contribute to high NaCl-induced activation of the osmoregulatory transcription factor TonEBP/OREBP

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Separate reports that hypertonicity activates p38 via a Rac1-OSM-MEKK3-MKK3-p38 pathway and that p38α 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. osmotic stress | MAPK H ypertonicity (e.g., high NaCl) activates the transcription factor TonEBP/OREBP (also known as NFAT5), resulting in increased expression of osmoprotective genes (1). Hypertonicity increases TonEBP/OREBP activity by increasing its abundance (1), transactivating activity (1), nuclear localization (1), and phosphorylation (2, 3). The regulatory increase of phosphorylation depends on both increased kinase activity and reduced phosphatase activity (4, 5). The stress-activated MAP kinase p38 has been extensively studied in this regard, but understanding its role has been complicated because hypertonicity activates two different p38s, namely p38α (MAPK14) and p38δ (MAPK13), which have opposite effects on TonEBP/OREBP activity: p38α increases TonEBP/OREBP activity and p38δ decreases it (6). Furthermore, the phosphospecific antibodies commonly used to measure p38 activity do not directly distinguish between p38α activity and p38δ activity, nor do some forms of inhibition that have been used (6). In this article, we will continue to refer to "p38" unless p38α and p38δ are distinguished. Hypertonicity activates p38α via the upstream kinase MKK3 (MAP2K3) or MKK6 (MAP2K6) (7), and p38α and MEKK3 (MAP3K3) contribute to hypertonicity-induced activation of TonEBP/OREBP (6, 8, 9). Thus, p38α contributes to To...

Section: Methodsmentioning

confidence: 99%

“…All transfections were done by adding cell suspensions to plated DNA or siRNA and transfection reagent. Luciferase activity was measured as described previously (38).…”

Section: Methodsmentioning

confidence: 99%

Rac1/osmosensing scaffold for MEKK3 contributes via phospholipase C-γ1 to activation of the osmoprotective transcription factor NFAT5

Zhou

Yokoyama

Burg

et al. 2011

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“…1A). Furthermore, the antioxidant N-acetylcysteine (NAC), which prevents high NaCl-and urea-induced elevation of ROS (14), prevents high NaCl-and high urea-induced inhibition of GPC-PDE activity (Fig. 1B).…”

Section: Resultsmentioning

confidence: 99%

High NaCl- and urea-induced posttranslational modifications that increase glycerophosphocholine by inhibiting GDPD5 phosphodiesterase

Topanurak

Ferraris

et al. 2013

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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 ureainduced inhibition of GDPD5; GDPD5-C25S/C571S mutation or over expression of peroxiredoxin increases GDPD5 activity; H 2 O 2 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.peroxiredoxin | Phos-tag

“…ROS inactivate MKPs by direct oxidation of the thiolate anion to sulfenic acid (20). High NaCl increases ROS (3,19), and the high NaCl-induced ROS directly inhibit MKP-1 ( Fig. 3 A and B).…”

Section: Mkp-1 Regulatesmentioning

confidence: 98%

“…High NaCl increases reactive oxygen species (ROS) (3,19), and ROS inhibit MKP-1 activity (20). To determine whether high NaCl inhibits MKP-1 enzymatic activity by increasing ROS, we established stable clones overexpressing Flag-MKP-1 in mIMCD3 cells and measured the phosphatase activity of MKP-1 immunoprecipitated from the cells, using 3-O-methylfluorescein phosphate as substrate.…”

Section: Effect Of Antioxidant Treatment On High Nacl-induced Inhibitmentioning

confidence: 99%

MKP-1 inhibits high NaCl-induced activation of p38 but does not inhibit the activation of TonEBP/OREBP: Opposite roles of p38α and p38δ

Zhou

Ferraris

Dmitrieva

et al. 2008

Self Cite

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 overexpression 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 p38␣ and p38␦, both of which are activated by high NaCl and inhibited by MKP-1. Thus, we find that overexpression of p38␣ increases high NaCl-induced TonEBP/ OREBP activity, but overexpression of p38␦ reduces it. Also, siRNAmediated knockdown of p38␦ 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 p38␣ and p38␦ 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␣ and p38␦, and effects of inhibitors of p38 depend on which isoform is affected, which can be misleading.hypertonicity ͉ reactive oxygen species ͉ transcription ͉ HEK293 cells ͉ mIMCD3 cells