Tonicity-responsive Enhancer Binding Protein Research Articles

Hippocampal betaine/GABA transporter mRNA expression is not regulated by inflammation or dehydration post‐status epilepticus

Seizure activity can alter GABA transporter and osmoprotective gene expression, which may be involved in the pathogenesis of epilepsy. However, the response of the betaine/GABA transporter (BGT1) is unknown. The goal of the present study was to compare the expression of BGT1 mRNA to that of other osmoprotective genes and GABA transporters following status epilepticus (SE). The possible contributory role of dehydration and inflammation was also investigated because both have been shown to be involved in the regulation of GABA transporter and/or osmoprotective gene expression. BGT1 mRNA was increased 24 h post-SE, as were osmoprotective genes. BGT1 was decreased 72 h and 4 weeks post-SE, as were the GABA transporter mRNAs. The mRNA values for osmoprotective genes following 24-h water withdrawal were significantly lower than the values obtained 24 h post-SE despite similarities in their plasma osmolality values. BGT1 mRNA was not altered by lipopolysaccharide-induced inflammation while the transcription factor tonicity-responsive enhancer binding protein and the GABA transporters 1 and 3 were. These results suggest that neither plasma osmolality nor inflammation fully account for the changes seen in BGT1 mRNA expression post-SE. However, it is evident that BGT1 mRNA expression is altered by SE and displays a temporal pattern with similarities to both GABA and osmolyte transporters. Further investigation of BGT1 regulation in the brain is warranted.

TonEBP stimulates multiple cellular pathways for adaptation to hypertonic stress: organic osmolyte-dependent and -independent pathways

TonEBP (tonicity-responsive enhancer binding protein) is a transcription factor that promotes cellular accumulation of organic osmolytes in the hypertonic renal medulla by stimulating expression of its target genes. Genetically modified animals with deficient TonEBP activity in the kidney suffer from severe medullary atrophy in association with cell death, demonstrating that TonEBP is essential for the survival of the renal medullary cells. Using both TonEBP knockout cells and RNA interference of TonEBP, we found that TonEBP promoted cellular adaptation to hypertonic stress. Microarray analyses revealed that the genetic response to hypertonicity was dominated by TonEBP in that expression of totally different sets of genes was increased by hypertonicity in those cells with TonEBP vs. those without TonEBP activity. Of over 100 potentially new TonEBP-regulated genes, we selected seven for further analyses and found that their expressions were all dependent on TonEBP. RNA interference experiments showed that some of these genes, asporin, insulin-like growth factor-binding protein-5 and -7, and an extracellular lysophospholipase D, plus heat shock protein 70, a known TonEBP target gene, contributed to the adaptation to hypertonicity without promoting organic osmolyte accumulation. We conclude that TonEBP stimulates multiple cellular pathways for adaptation to hypertonic stress in addition to organic osmolyte accumulation.

Distinct cellular pathways for resistance to urea stress and hypertonic stress

During antidiuresis with elevated vasopressin, urea accumulates in the renal medulla to very high concentrations, imposing considerable cellular stress. How local cells cope with urea stress is relevant to the whole kidney because the renal medulla is the major site of residence for the renal stem cells. Previous studies showed that renal cells were incapable of preconditioning in moderate urea concentrations to enhance resistance to urea stress. Instead, preconditioning in moderately high salinity (moderate hypertonicity) has been shown to promote resistance to urea stress due to the induction of the molecular chaperone heat shock protein 70 (Hsp70), which is mediated by the transcription factor tonicity-responsive enhancer binding protein (TonEBP). Here we report that cell lines derived from the kidney and fibroblasts display enhanced resistance to urea stress after pretreatment in moderate, nonstressful concentrations of urea. Using TonEBP knockdown and immunoblot analyses, we demonstrate that TonEBP and Hsp70 are dispensable for the increased resistance to urea stress. These data suggest that cells in the renal medulla are capable of overcoming urea stress by activating distinct cellular pathways.

Polyol pathway and diabetic nephropathy revisited: Early tubular cell changes and glomerulopathy in diabetic mice overexpressing human aldose reductase.

Aims/Introduction: The polyol pathway has long been involved in the pathogenesis of diabetic nephropathy. It remains still unclear, however, how the polyol pathway is implicated in this process. We explored the effects of the enhanced polyol pathway on renocortical tubular cells and glomeruli in experimentally-induced diabetes. Transgenic mice (Tg) overexpressing human aldose reductase were made diabetic by streptozotocin and followed for 8 weeks. Renocortical pathology, expressions of tonicity-responsive enhancer binding protein (TonEBP) and carboxymethyllysine of advanced glycation end-products, were examined. Wild-type non-transgenic mice (Wt) were also made diabetic and served as controls. Diabetic Tg showed augmented expression of TonEBP in renocortical tubular cells with vacuolated degenerative changes. These structural changes were associated with pronounced deposition of carboxymethyllysine. There was a significant increase in kidney weight, glomerular size, and mesangial area in diabetic animals and there was a trend for more severe changes in these measures in diabetic transgenic mice compared with those in control diabetic mice. Treatment with aldose reductase inhibitor significantly prevented polyol accumulation, mesangial expansion and expressions of TonEBP and carboxymethyllysine in diabetic Tg, but its effects on the renal structure were equivocal in control diabetic Wt. Our findings suggest that tubuloglomerular change might contribute to early diabetic nephropathy under the influence of the enhanced polyol pathway. (J Diabetes Invest, doi: 10.1111/j.2040-1124.2010.00071.x, 2010).

Upregulation of ANP and NPR-C mRNA in the kidney and heart of eNOS knockout mice

The aim of the present studywas to examine the question of whether the atrial natriuretic peptide (ANP) system is altered by endothelial nitric-oxide synthase (eNOS). Male eNOS-deficient mice (eNOS-/-) and wild type control mice (eNOS+/+, C57B1/6J) were used. Blood pressure was measured in anesthetized mice by tail cuff plethysmography and renal function was measured. Expression of ANP, natriuretic peptide receptor (NPR)-A, NPR-C, and tonicity-responsive enhancer binding protein (TonEBP) mRNA was determined by real-time PCR. Localization of (125)I-ANP binding sites was measured using in vitro autoradiography. In eNOS-/- mice, systolic blood pressure increased and left ventricular hypertrophy was observed. Urine volume and osmolarity did not change. Expression of ANP markedly increased in the heart and kidney of eNOS-/- mice. Expression of NPR-A and NPR-C increased in the heart and tended to increase in the kidney of eNOS-/- mice. In the renal medulla in particular, increased expression of NPR-C was more prominent. Expression of TonEBP mRNA was markedly decreased in the renal medulla, but not in the renal cortex. Maximum binding capacity (B(max)) of ANP and C-ANP increased in the renal medulla in eNOS-/- mice. These results suggest that the eNOS-NO system may be partly involved in regulation of ANP, NPR-A, -C, and TonEBP mRNA expression in the kidney.

Physiological tonicity improves human chondrogenic marker expression through nuclear factor of activated T-cells 5 in vitro

IntroductionChondrocytes experience a hypertonic environment compared with plasma (280 mOsm) due to the high fixed negative charge density of cartilage. Standard isolation of chondrocytes removes their hypertonic matrix, exposing them to nonphysiological conditions. During in vitro expansion, chondrocytes quickly lose their specialized phenotype, making them inappropriate for cell-based regenerative strategies. We aimed to elucidate the effects of tonicity during isolation and in vitro expansion on chondrocyte phenotype.MethodsHuman articular chondrocytes were isolated and subsequently expanded at control tonicity (280 mOsm) or at moderately elevated, physiological tonicity (380 mOsm). The effects of physiological tonicity on chondrocyte proliferation and chondrogenic marker expression were evaluated. The role of Tonicity-responsive Enhancer Binding Protein in response to physiological tonicity was investigated using nuclear factor of activated T-cells 5 (NFAT5) RNA interference.ResultsModerately elevated, physiological tonicity (380 mOsm) did not affect chondrocyte proliferation, while higher tonicities inhibited proliferation and diminished cell viability. Physiological tonicity improved expression of chondrogenic markers and NFAT5 and its target genes, while suppressing dedifferentiation marker collagen type I and improving type II/type I expression ratios >100-fold. Effects of physiological tonicity were similar in osteoarthritic and normal (nonosteoarthritic) chondrocytes, indicating a disease-independent mechanism. NFAT5 RNA interference abolished tonicity-mediated effects and revealed that NFAT5 positively regulates collagen type II expression, while suppressing type I.ConclusionsPhysiological tonicity provides a simple, yet effective, means to improve phenotypical characteristics during cytokine-free isolation and in vitro expansion of human articular chondrocytes. Our findings will lead to the development of improved cell-based repair strategies for chondral lesions and provides important insights into mechanisms underlying osteoarthritic progression.

New Approaches to Pathogenesis and Management of Hypertension

New Approaches to Pathogenesis and Management of Hypertension

Osmotic Stress Regulates Mineralocorticoid Receptor Expression in a Novel Aldosterone-Sensitive Cortical Collecting Duct Cell Line

Aldosterone effects are mediated by the mineralocorticoid receptor (MR), a transcription factor highly expressed in the distal nephron. Given that MR expression level constitutes a key element controlling hormone responsiveness, there is much interest in elucidating the molecular mechanisms governing MR expression. To investigate whether hyper- or hypotonicity could affect MR abundance, we established by targeted oncogenesis a novel immortalized cortical collecting duct (CCD) cell line and examined the impact of osmotic stress on MR expression. KC3AC1 cells form domes, exhibit a high transepithelial resistance, express 11beta-hydroxysteroid dehydrogenase 2 and functional endogenous MR, which mediates aldosterone-stimulated Na(+) reabsorption through the epithelial sodium channel activation. MR expression is tightly regulated by osmotic stress. Hypertonic conditions induce expression of tonicity-responsive enhancer binding protein, an osmoregulatory transcription factor capable of binding tonicity-responsive enhancer response elements located in MR regulatory sequences. Surprisingly, hypertonicity leads to a severe reduction in MR transcript and protein levels. This is accompanied by a concomitant tonicity-induced expression of Tis11b, a mRNA-destabilizing protein that, by binding to the AU-rich sequences of the 3'-untranslated region of MR mRNA, may favor hypertonicity-dependent degradation of labile MR transcripts. In sharp contrast, hypotonicity causes a strong increase in MR transcript and protein levels. Collectively, we demonstrate for the first time that optimal adaptation of CCD cells to changes in extracellular fluid composition is accompanied by drastic modification in MR abundance via transcriptional and posttranscriptional mechanisms. Osmotic stress-regulated MR expression may represent an important molecular determinant for cell-specific MR action, most notably in renal failure, hypertension, or mineralocorticoid resistance.

Macrophages regulate salt-dependent volume and blood pressure by a vascular endothelial growth factor-C–dependent buffering mechanism

In salt-sensitive hypertension, the accumulation of Na(+) in tissue has been presumed to be accompanied by a commensurate retention of water to maintain the isotonicity of body fluids. We show here that a high-salt diet (HSD) in rats leads to interstitial hypertonic Na(+) accumulation in skin, resulting in increased density and hyperplasia of the lymphcapillary network. The mechanisms underlying these effects on lymphatics involve activation of tonicity-responsive enhancer binding protein (TonEBP) in mononuclear phagocyte system (MPS) cells infiltrating the interstitium of the skin. TonEBP binds the promoter of the gene encoding vascular endothelial growth factor-C (VEGF-C, encoded by Vegfc) and causes VEGF-C secretion by macrophages. MPS cell depletion or VEGF-C trapping by soluble VEGF receptor-3 blocks VEGF-C signaling, augments interstitial hypertonic volume retention, decreases endothelial nitric oxide synthase expression and elevates blood pressure in response to HSD. Our data show that TonEBP-VEGF-C signaling in MPS cells is a major determinant of extracellular volume and blood pressure homeostasis and identify VEGFC as an osmosensitive, hypertonicity-driven gene intimately involved in salt-induced hypertension.

Interstitial tonicity controls TonEBP expression in the renal medulla

Cells in the hyperosmotic kidney medulla, express a transcriptional activator termed tonicity responsive enhancer binding protein (TonEBP). Genes targeted by TonEBP protect kidney cells from the deleterious effects of hyperosmolality by inducing the expression of organic osmolytes and molecular chaperones, and other genes that mediate urine concentration such as aquaporin-2 and urea transporters. We tested here the effect of hypertonicity and hyperosmotic salt in the renal medullary interstitium on the expression TonEBP. When massive water diuresis was induced in rats the medullary sodium concentrations did not change, neither did TonEBP expression. In these animals the medullary tonicity was unchanged despite the production of dilute urine. On the other hand, treatment with the loop diurectic furosemide resulted in a dose-dependent decrease in the medullary sodium concentration causing a reduction in interstitial tonicity. Here, TonEBP expression was blunted in the outer and inner medulla which was due, in part, to decreased mRNA abundance. As expected, the expression of TonEBP target genes in the renal medulla also decreased in response to furosemide. Hence TonEBP expression in the renal medulla is stimulated by interstitial hypertonicity.

Aquaporin-2 abundance in the renal collecting duct: new insights from cultured cell models

The renal cortico-papillary osmotic gradient is generated by sodium reabsorption in the thick ascending limb. The antidiuretic hormone arginine vasopressin (AVP) increases collecting duct water permeability by enhancing aquaporin-2 (AQP2) water channel insertion in the apical membrane of principal cells, allowing water to passively flow along the osmotic gradient from the tubule lumen to the interstitium. In addition to short-term AQP2 redistribution between intracellular compartments and the cell surface, AQP2 whole cell abundance is tightly regulated. AVP is a major transcriptional activator of the AQP2 gene, and stimulation of insulin- and calcium-sensing receptors respectively potentiate and reduce its action. Extracellular tonicity is another key factor that determines the levels of AQP2 abundance. Its effect is dependent on activation of the tonicity-responsive enhancer binding protein that reinforces AVP-induced AQP2 transcriptional activation. Conversely, activation of the NF-kappaB transcriptional factor by proinflammatory factors reduces AQP2 gene transcription. Aldosterone additionally regulates AQP2 whole cell abundance by simultaneously reducing AQP2 gene transcription and stimulating AQP2 mRNA translation. These examples illustrate how cross talk between various stimuli regulates AQP2 abundance in collecting duct principal cells and consequently contributes to maintenance of body water homeostasis.

Does the intracellular ionic concentration or the cell water content (cell volume) determine the activity of TonEBP in NIH3T3 cells?

The transcription factor, tonicity-responsive enhancer binding protein (TonEBP), is involved in the adaptive response against hypertonicity. TonEBP regulates the expression of genes that catalyze the accumulation of osmolytes, and its transcriptional activity is increased by hypertonicity. The goal of the present investigation was to investigate whether cell shrinkage or high intracellular ionic concentration induced the activation of TonEBP. We designed a model system for isotonically shrinking cells over a prolonged period of time. Cells swelled in hypotonic medium and performed a regulatory volume decrease. Upon return to the original isotonic medium, cells shrank initially, followed by a regulatory volume increase. To maintain cell shrinkage, the RVI process was inhibited as follows: ethyl-isopropyl-amiloride inhibited the Na(+)/H(+) antiport, bumetanide inhibited the Na(+)-K(+)-2Cl(-) cotransporter, and gadolinium inhibited shrinkage-activated Na(+) channels. Cells remained shrunken for at least 4 h (isotonically shrunken cells). The activity of TonEBP was investigated with a Luciferase assay after isotonic shrinkage and after shrinkage in a high-NaCl hypertonic medium. We found that TonEBP was strongly activated after 4 and 16 h in cells in high-NaCl hypertonic medium, but not after 4 or 16 h in isotonically shrunken cells. Cells treated with high-NaCl hypertonic medium for 4 h had significantly higher intracellular concentrations of both K(+) and Na(+) than isotonically shrunken cells. This strongly suggested that an increase in intracellular ionic concentration and not cell shrinkage is involved in TonEBP activation.

NF-κB Modulates Aquaporin-2 Transcription in Renal Collecting Duct Principal Cells

Renal tubulo-interstitial inflammation is frequently associated with polyuria and urine concentration defects. This led us to investigate the effects of the major pro-inflammatory nuclear factor kappaB (NF-kappaB) pathway on aquaporin 2 (AQP2) expression by the collecting duct. Using immortalized collecting duct principal cells (mpkCCDcl4), we found that, acting independently of vasopressin, activation of NF-kappaB by lipopolysaccharide (LPS) decreased AQP2 mRNA and protein levels in a time- and dose-dependent manner but did not decrease AQP2 mRNA stability. Consistently, constitutively active IkappaB kinase beta decreased AQP2 expression. The LPS-induced decrease in AQP2 mRNA levels was confirmed in rat kidney slices and was reproduced both under conditions of elevated cAMP concentration and V(2) receptor antagonism. Computer analysis of the AQP2 promoter revealed two putative kappaB elements. Mutation of either kappaB element abolished the LPS-induced decrease of luciferase activity in cells expressing AQP2 promoter-luciferase plasmid constructs. Chromatin immunoprecipitation revealed that LPS challenge decreased p65, increased p50 and p52, and had no effect on RelB and c-Rel binding to kappaB elements of the AQP2 promoter. RNA-mediated interference silencing of p65, p50, and p52 confirmed controlled AQP2 transcription by these NF-kappaB subunits. We additionally found that hypertonicity activated NF-kappaB in mpkCCDcl4 cells, an effect that may counteract the Tonicity-responsive enhancer binding protein (TonEBP)-dependent increase in AQP2 gene transcription. Taken together, these findings indicate that NF-kappaB is an important physiological regulator of AQP2 transcription.

Effects of hyperosmolarity on the Na+-myo-inositol cotransporter SMIT2 stably transfected in the Madin-Darby canine kidney cell line

Myo-inositol (MI) is a compatible osmolyte used by cells to compensate for changes in the osmolarity of their surrounding milieu. In kidney, the basolateral Na(+)-MI cotransporter (SMIT1) and apical SMIT2 proteins are homologous cotransporters responsible for cellular uptake of MI. It has been shown in the Madin-Darby canine kidney (MDCK) cell line that SMIT1 expression was under the control of the tonicity-sensitive transcription factor, tonicity-responsive enhancer binding protein (TonEBP). We used an MDCK cell line stably transfected with SMIT2 to determine whether variations in external osmolarity could also affect SMIT2 function. Hyperosmotic conditions (+200 mosM raffinose or NaCl but not urea) generated an increase in SMIT2-specific MI uptake by three- to ninefold in a process that required protein synthesis. Using quantitative RT-PCR, we have determined that hyperosmotic conditions augment both the endogenous SMIT1 and the transfected SMIT2 mRNAs. Transport activities for both SMIT1 and SMIT2 exhibited differences in their respective induction profiles for both their sensitivities to raffinose, as well as in their time course of induction. Application of MG-132, which inhibits nuclear translocation of TonEBP, showed that the effect of osmolarity on transfected SMIT2 was unrelated to TonEBP, unlike the effect observed with SMIT1. Inhibition studies involving the hyperosmolarity-related MAPK suggested that p38 and JNK play a role in the induction of SMIT2. Further studies have shown that hyperosmolarity also upregulates another transfected transporter (Na(+)-glucose), as well as several endogenously expressed transport systems. This study shows that hyperosmolarity can stimulate transport in a TonEBP-independent manner by increasing the amount of mRNA derived from an exogenous DNA segment.

Nitric oxide decreases expression of osmoprotective genes via direct inhibition of TonEBP transcriptional activity

During antidiuresis, renal medullary cells adapt to the hyperosmotic interstitial environment by increased expression of osmoprotective genes, which is driven by a common transcriptional activator, tonicity-responsive enhancer binding protein (TonEBP). Because nitric oxide (NO) is abundantly produced in the renal medulla, the present studies addressed the effect of NO on expression of osmoprotective genes and TonEBP activation in MDCK cells. Several structurally unrelated NO donors blunted tonicity-induced up-regulation of TonEBP target genes involved in intracellular accumulation of organic osmolytes. These effects were mediated by reduced transcriptional activity of TonEBP, as assessed by tonicity-responsive elements- and aldose reductase promoter-driven reporter constructs. Neither total TonEBP abundance nor nuclear translocation of TonEBP was affected by NO. Furthermore, 8-bromo-cGMP and peroxynitrite failed to reproduce the inhibitory effect of NO, indicating that NO acts directly on TonEBP rather than through classical NO signaling pathways. In support of this notion, electrophoretic mobility shift assays showed reduced binding of TonEBP to its target sequence in nuclear extracts prepared from MDCK cells treated with NO in vivo and in nuclear extracts exposed to NO in vitro. Furthermore, immunoprecipitation of S-nitrosylated proteins and the biotin-switch method identified TonEBP as a target for S-nitrosylation, which correlates with reduced DNA binding and transcriptional activity. These observations disclose a novel direct inhibitory effect of NO on TonEBP, a phenomenon that may be relevant for regulation of osmoprotective genes in the renal medulla.

Osmotically-induced genes are controlled by the transcription factor TonEBP in cultured cardiomyocytes

Changes in cardiac osmolarity occur in myocardial infarction. Osmoregulatory mechanisms may, therefore, play a crucial role in cardiomyocyte survival. Tonicity-responsive enhancer binding protein (TonEBP) is a key transcription factor participating in the adaptation of cells to increases in tonicity. However, it is unknown whether cardiac TonEBP is activated by tonicity. Hypertonicity activated transcriptional activity of TonEBP, increased the amounts of both TonEBP mRNA and protein, and induced both the mRNA and protein of TonEBP target genes (aldose reductase and heat shock protein-70). Hypotonicity decreased the amount of TonEBP protein indicating bidirectional osmoregulation of this transcription factor. Adenoviral expression of a dominant negative TonEBP suppressed the hypertonicity-dependent increase of aldose reductase protein. These results indicated that TonEBP controls osmoregulatory mechanisms in cardiomyocytes.

The effect of water deprivation on the tonicity responsive enhancer binding protein (TonEBP) and TonEBP-regulated genes in the kidney of the Spinifex hopping mouse,Notomys alexis

In desert rodents, the production of concentrated urine is essential for survival in xeric environments in order to conserve water. Reabsorption of water in the kidney is dependent on large osmotic gradients in the renal medulla. This causes the renal cells to be bathed in a hypertonic extracellular fluid that can compromise cellular function. In response to hypertonicity, kidney cells accumulate compatible, non-ionic osmolytes that lower the ionic strength within the cells to isotonic levels by replacing intracellular ionic electrolytes. The tonicity-responsive enhancer binding protein (TonEBP) is a transcription factor that regulates the expression of genes that encode proteins that catalyse the accumulation of compatible osmolytes. We investigated the expression of TonEBP mRNA and protein and compatible osmolyte genes in the Spinifex hopping mouse, Notomys alexis, an Australian desert rodent that produces a highly concentrated urine. TonEBP mRNA expression was unchanged after 3 days of water deprivation but was significantly increased after 7 and 14 days of water deprivation. Immunohistochemistry showed that during water deprivation TonEBP had translocated from the cytoplasm into the nucleus of cells in the renal medulla and papilla. In addition, 3, 7 and 14 days of water deprivation caused a significant increase in aldose reductase (AR), myo-inositol (SMIT), betaine/GABA (BGT-1) and taurine (TauT) transporter mRNA expression, which is indicative of an increase in TonEBP activity. In desert rodents, TonEBP regulation of gene transcription is probably an important mechanism to protect renal cells in the face of the large corticomedullary gradient that is required to concentrate urine and conserve water.

Gene expression profiling in brain following acute systemic hypertonicity: novel genes possibly involved in osmoadaptation

In brain osmoprotective genes known to be involved in cellular osmoadaptation to hypertonicity, as well as the related transcription factor tonicity-responsive enhancer binding protein (TonEBP) are only expressed in some cell subsets. In the search for other genes possibly involved in osmoadaptation of brain cells we have analyzed, through microarray, the transcriptional profile of forebrain from rats subjected to 45 min, 90 min, and 6 h systemic hypertonicity. Microarray data were validated by quantitative real-time PCR. Around 23 000 genes gave a reliable hybridization signal. The number of genes showing a higher expression increased from around 15 (45 min) up to nearly 200 (6 h). Among about 30 immediate early genes (IEGs) encoding transcription factors, only Atf3, Verge, and Klf4 showed a rapid increased expression. TonEBP-mRNA tissue level and TonEBP-mRNA labeling in neurons remained unchanged whereas TonEBP labeling was rapidly increased in neurons. Sodium-dependent neutral amino acid transporter-2 (SNAT2) encoded by gene Slc38a2 showed a delayed increased expression. The rapid tonicity-induced activation of Atf3, Verge, and Klf4 may regulate genes involved in osmoadaptation. Nfat5 encoding TonEBP is not an IEG and the early tonicity-induced expression of TonEBP in neurons may result from translational activation. Increased expression of sodium-dependent neutral amino-acid transporter 2 may lead to the cellular accumulation of amino acids for adaptation to hypertonicity.

Protein kinase Cµ plays an essential role in hypertonicity-induced heat shock protein 70 expression

Heat shock protein 70 (HSP70), which evidences important functions as a molecular chaperone and anti-apoptotic molecule, is substantially induced in cells exposed to a variety of stresses, including hypertonic stress, heavy metals, heat shock, and oxidative stress, and prevents cellular damage under these conditions. However, the molecular mechanism underlying the induction of HSP70 in response to hypertonicity has been characterized to a far lesser extent. In this study, we have investigated the cellular signaling pathway of HSP70 induction under hypertonic conditions. Initially, we applied a variety of kinase inhibitors to NIH3T3 cells that had been exposed to hypertonicity. The induction of HSP70 was suppressed specifically by treatment with protein kinase C (PKC) inhibitors (Gö6976 and GF109203X). As hypertonicity dramatically increased the phosphorylation of PKCmu, we then evaluated the role of PKCmu in hypertonicity-induced HSP70 expression and cell viability. The depletion of PKCmu with siRNA or the inhibition of PKCmu activity with inhibitors resulted in a reduction in HSP70 induction and cell viability. Tonicity-responsive enhancer binding protein (TonEBP), a transcription factor for hypertonicity-induced HSP70 expression, was translocated rapidly into the nucleus and was modified gradually in the nucleus under hypertonic conditions. When we administered treatment with PKC inhibitors, the mobility shift of TonEBP was affected in the nucleus. However, PKCmu evidenced no subcellular co-localization with TonEBP during hypertonic exposure. From our results, we have concluded that PKCmu performs a critical function in hypertonicity-induced HSP70 induction, and finally cellular protection, via the indirect regulation of TonEBP modification.

Discovery of Osmosensitive Transcriptional Regulation of Human Cytochrome P450 3As by the Tonicity-Responsive Enhancer Binding Protein (Nuclear Factor of Activated T Cells 5)

We report the discovery of an osmosensitive transcriptional control of human CYP3A4, CYP3A7, and CYP3A5. Ambient hypertonicity (350-450 mOsmol/kg) increased mRNA expressions of the CYP3A by approximately 10- to 20-fold in human-intestinal C(2)bbe1 cells, followed by an increase of CYP3A protein. Hypotonicity, on the other hand, suppressed CYP3A mRNA levels, indicating that physiological isotonic conditions may regulate the basal expression of CYP3A. Similar responses to ambient tonicity were observed in other human-derived cell lines (intestinal LS180 and hepatic HepG2) and human primary colonic cells. The 11-base pair tonicity-responsive enhancer (TonE) is an osmosensitive regulator that is activated by the transcription factor, the nuclear factor of activated T-cells 5 (NFAT5). Luciferase-based reporter assays of 13 consensus TonE motifs within +/-10 kilobases (kb) from the transcription start sites of CYP3A showed that only the CYP3A7 intron 2 region ( approximately 5 kb downstream from the transcription start site), which contains two TonE motifs (+5076/+5086 and + 5417/+5427), was responsive to hypertonicity stimuli. This observation was confirmed upon cotransfection with an NFAT5 expression vector, small interfering RNA, or dominant-negative NFAT5. Deletion and mutation analyses suggested that the TonE (+5417/+5427) is indispensable for the enhancer activity. NFAT5 binding to the CYP3A7 intron 2 TonE motif was demonstrated with electrophoretic mobility shift assay and in a native cell context by chromatin immunoprecipitation. We conclude that transcription of human CYP3A is influenced by ambient tonicity. The physiological significance of the tonic regulation of CYP3A enzymes remains to be determined.