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The osmotic response element-binding protein (OREBP), also known as tonicity enhancer-binding protein (TonEBP) or NFAT5, is the only known osmo-sensitive transcription factor that mediates cellular adaptations to extracellular hypertonic stress. Although it is well documented that the subcellular localization and transactivation activity of OREBP/TonEBP are tightly regulated by extracellular tonicity, the molecular mechanisms involved remain elusive. Here we show that nucleocytoplasmic trafficking of OREBP/TonEBP is regulated by the dual phosphorylation of Ser-155 and Ser-158. Alanine scanning mutagenesis revealed that Ser-155 is an essential residue that regulates OREBP/TonEBP nucleocytoplasmic trafficking. Tandem mass spectrometry revealed that Ser-155 and Ser-158 of OREBP/TonEBP are both phosphorylated in living cells under hypotonic conditions. In vitro phosphorylation assays further suggest that phosphorylation of the two serine residues proceeds in a hierarchical manner with phosphorylation of Ser-155 priming the phosphorylation of Ser-158 and that these phosphorylations are essential for nucleocytoplasmic trafficking of the transcription factor. Finally, we have shown that the pharmacological inhibition of casein kinase 1 (CK1) abolishes the phosphorylation of Ser-158 and impedes OREBP/TonEBP nuclear export and that recombinant CK1 phosphorylates Ser-158. Knockdown of CK1alpha1L, a novel isoform of CK1, inhibits hypotonicity-induced OREBP/TonEBP nuclear export. Together these data highlight the importance of Ser-155 and Ser-158 in the nucleocytoplasmic trafficking of OREBP/TonEBP and indicate that CK1 plays a major role in regulating this process.

The osmotic response element-binding protein (OREBP), also known as tonicity enhancer-binding protein (TonEBP) or NFAT5, regulates the hypertonicity-induced expression of a battery of genes crucial for the adaptation of mammalian cells to extracellular hypertonic stress. The activity of OREBP/TonEBP is regulated at multiple levels, including nucleocytoplasmic trafficking. OREBP/TonEBP protein can be detected in both the cytoplasm and nucleus under isotonic conditions, although it accumulates exclusively in the nucleus or cytoplasm when subjected to hypertonic or hypotonic challenges, respectively. Using immunocytochemistry and green fluorescent protein fusions, the protein domains that determine its subcellular localization were identified and characterized. We found that OREBP/TonEBP nuclear import is regulated by a nuclear localization signal. However, under isotonic conditions, nuclear export of OREBP/TonEBP is mediated by a CRM1-dependent, leucine-rich canonical nuclear export sequence (NES) located in the N terminus. Disruption of NES by site-directed mutagenesis yielded a mutant OREBP/TonEBP protein that accumulated in the nucleus under isotonic conditions but remained a target for hypotonicity-induced nuclear export. More importantly, a putative auxiliary export domain distal to the NES was identified. Disruption of the auxiliary export domain alone is sufficient to abolish the nuclear export of OREBP/TonEBP induced by hypotonicity. By using bimolecular fluorescence complementation assay, we showed that CRM1 interacts with OREBP/TonEBP, but not with a mutant protein deficient in NES. Our findings provide insight into how nucleocytoplasmic trafficking of OREBP/TonEBP is regulated by changes in extracellular tonicity.

Osmotic-response element-binding protein (OREBP), also known as TonEBP or NFAT5, is thought to be responsible for the induction of osmolyte-accumulating genes when cells are under hypertonic stress. Recent studies suggest that OREBP also plays a role in water reabsorption in the kidney, T-cell proliferation, and embryonic development. We developed transgenic mice that express the dominant-negative OREBP (OREBPdn) specifically in the lens because our earlier studies showed that it is particularly sensitive to osmotic stress. The transgenic mice developed nuclear cataract soon after birth, suggesting defects in lens development. The developing transgenic lenses showed incomplete elongation of fiber cells and formation of vacuoles. This is accompanied by evidence of DNA strand breaks, activation of p53, and induction of checkpoint kinase, suggesting that the developing fiber cells lacking OREBP are in a similar physiological state as cells experiencing hypertonic stress. These results indicate that OREBP-mediated accumulation of osmolytes is essential during elongation of the lens fiber cells.

OREBP (osmotic response element-binding protein), also called TonEBP or NFAT5, is thought to induce the expression of genes that increase the accumulation of organic osmolytes to protect cells against a hypertonic environment. To investigate the consequences of lacking OREBP activity, transgenic (Tg) mice that overexpress OREBPdn (dominant negative form of OREBP) specifically in the epithelial cells of the renal collecting tubules were generated. These mice showed impairment in their urine concentrating mechanism, most likely due to reduced expression of the aquaporin AQP2 and the urea transporter UT-A1 and UT-A2 mRNAs. When deprived of water or after the administration of a vasopressin analogue, urine osmolality of the Tg mice was significantly increased but not to the same extent as that of the wild type mice. The expression of AQP2 and UT-A1, but not UT-A2 mRNAs, was increased to the same level as that of the wild type mice in the water deprivation state, indicating that the vasopressin regulatory mechanism was not affected by OREBPdn. These data indicate that in addition to vasopressin, OREBP is another essential regulator of the urine concentrating mechanism. Furthermore, the OREBPdn Tg mice developed progressive hydronephrosis soon after weaning, confirming the osmoprotective function of OREBP implicated by the in vitro experiments.

When cells are challenged by hyperosmotic stress, one of the crucial adaptive responses is the expression of osmoprotective genes that are responsible for raising the intracellular level of compatible osmolytes such as sorbitol, betaine, and myo-inositol. This is achieved by the activation of the transcription factor called OREBP (also known as TonEBP or NFAT5) that specifically binds to the osmotic response element (ORE) or tonicity-responsive enhancer that enhances the transcription of these genes. Here we show that p38, a subgroup of the mitogen-activated kinases activated by hypertonic stress, and Fyn, a shrinkage-activated tyrosine kinase, are both involved in the hypertonic activation of OREBP/TonEBP. Inhibition of p38 by SB203580 or by the dominant negative p38 mutant partially blocked the hypertonic induction of ORE reporter (reporter gene regulated by ORE). Similarly, hypertonic activation of ORE reporter was partially blocked by pharmacological inhibition of Fyn or by a dominant negative Fyn and was attenuated in Fyn-deficient cells. Importantly, inhibiting p38 in Fyn-deficient cells almost completely abolished the hypertonic induction of ORE reporter activity, indicating that p38 and Fyn are the major signaling pathways for the hypertonic activation of OREBP/TonEBP. Further we show that the transactivation domain of OREBP/TonEBP is the target of p38- and Fyn-mediated hypertonic activation. These results indicate a dual control in regulating the expression of the osmoprotective genes in mammalian cells.

Background: Osmotic Response Element-Binding Protein (OREBP), also known as TonEBP or NFAT5, is a unique transcription factor. It is hitherto the only known mammalian transcription factor that regulates hypertonic stress-induced gene transcription. In addition, unlike other monomeric members of the NFAT family, OREBP exists as a homodimer and it is the only transcription factor known to bind naked DNA targets by complete encirclement in vitro. Nevertheless, how OREBP interacts with target DNA, also known as ORE/TonE, and how it elicits gene transcription in vivo, remains unknown. Methodology: Using hypertonic induction of the aldose reductase (AR) gene activation as a model, we showed that OREs contained dynamic nucleosomes. Hypertonic stress induced a rapid and reversible loss of nucleosome(s) around the OREs. The loss of nucleosome(s) was found to be initiated by an OREBP-independent mechanism, but was significantly potentiated in the presence of OREBP. Furthermore, hypertonic induction of AR gene was associated with an OREBP-dependent hyperacetylation of histones that spanned the 59 upstream sequences and at least some exons of the gene. Nevertheless, nucleosome loss was not regulated by the acetylation status of histone. Significance: Our findings offer novel insights into the mechanism of OREBP-dependent transcriptional regulation and provide a basis for understanding how histone eviction and transcription factor recruitment are coupled. [ABSTRACT FROM AUTHOR]

The Osmotic Response Element-Binding Protein (OREBP), also known as TonEBP or NFAT5, is a key tonicity-regulated transcription factor that regulates the expression of a battery of genes crucial for the adaptation of mammalian cells to extracellular hypertonic stress. In this study, we investigated the role of nucleosomes in hypertonicity-induced and OREBP-dependent gene transcription by using chromatin immunoprecipitation and real-time quantitative PCR analysis. Here, we demonstrated that histones within 2 kb upstream of the aldose reductase (AR) promoter are rapidly acetylated and phosphorylated upon hypertonic stress, suggesting a link between histone modifications and tonicity-regulated AR gene transcription. Furthermore, we determined that histone modification is associated with the loss of nucleosome integrity specifically at ORE site, which couples the recruitment of OREBP to the site. However, OREBP is not required for the loss of nucleosome integrity under hypertonic stress, since nucleosome loss was also observed in mouse embryonic fibroblasts derived from OREBP-deficient mice, although the level of nucleosome loss was less than in the cells derived from the wild-type mice. Our present data suggest that hypertonicity modifies the chromatin environment of the AR promoter to facilitate OREBP-dependent AR gene transcriptions. [ABSTRACT FROM AUTHOR]