Role of NFAT5 in Inflammatory Disorders Associated with Osmotic Stress

Nuclear factor of activated T cells 5 (NFAT5) is the most recently described member of the Rel family of transcription factors, including NF-κB and NFAT1-4, which play central roles in inducible gene expression during the immune response. NFAT5 was initially described to drive osmoprotective gene expression in renal medullary cells, which are routinely faced by high extracellular osmolalities. Recent data however indicate profound biological importance of the mammalian osmotic stress response in view of NFAT5 dependent gene regulation in non-renal tissues. In mononuclear cells and epithelial cells, NFAT5 stimulates the expression of various pro-inflammatory cytokines during elevated ambient tonicity. Accordingly, compared to plasma, the interstitial tonicity of lymphoid organs like spleen and thymus and that of liver is substantially hypertonic under physiological conditions. In addition, anisotonic disorders (hypernatremia, diabetes mellitus, dehydration) entail systemic hyperosmolality, and, in inflammatory disorders, the skin, intestine, and cornea are sites of local hyperosmolality. This article summarizes the current knowledge regarding systemic and local osmotic stress in anisotonic and inflammatory disorders in view of NFAT5 activation and regulation, and NFAT5 dependent cytokine production

Heat shock proteins and the cellular response to osmotic stress

In antidiuresis, the intrarenal distribution of HSP25/27, alpha beta -crystallin, HSP72, OSP94 and HSP110 corresponds to the osmotic gradient between cortex and papilla: low amounts in the cortex and high values in the inner medulla and papilla. In addition, medullary HSP72 levels change appropriately with the diuretic state. Studies on MUCK cells suggest that, in the renal medulla in vivo, stressors, such as NaCl and low pH, may act in concert to induce HSP72 expression. Urea, added to the medium at high concentrations (600 mM), causes the majority of MUCK cells to die. Prior exposure of these cells to hypertonic media (NaCl addition), a maneuver that induces HSP72, protects the cells against the deleterious effects of high urea concentrations. Inhibition of HSP72 expression by stable antisense transfection or SB203580 treatment abolishes the beneficial effects of prior hypertonic stress. Conversely, overexpression of HSP72 under isotonic conditions by a dexamethasone-driven vector confers substantial resistance against subsequent exposure to high urea concentrations. Taken together these results suggest that also in the renal inner medulla, NaCl-induced enhancement of HSP72 expression may help counteract the detrimental effects of high urea concentrations. Copyright (C) 2000 S. Karger AG Basel

NFAT5 Contributes to Osmolality-Induced MCP-1 Expression in Mesothelial Cells

Increased expression of the C-C chemokine monocyte chemoattractant protein-1 (MCP-1) in mesothelial cells in response to high glucose concentrations and/or high osmolality plays a crucial role in the development of peritoneal fibrosis during continuous ambulatory peritoneal dialysis (CAPD). Recent studies suggest that in kidney cells osmolality-induced MCP-1 upregulation is mediated by the osmosensitive transcription factor, nuclear factor of activated T cells 5 (NFAT5). The present study addressed the question of whether activation of NFAT5 by hyperosmolality, as present in PD fluids, contributes to MCP-1 expression in the mesothelial cell line Met5A. Hyperosmolality, induced by addition of glucose, NaCl, or mannitol to the growth medium, increased NFAT5 activity and stimulated MCP-1 expression in Met5A cells. siRNA-mediated knockdown of NFAT5 attenuated osmolality-induced MCP-1 upregulation substantially. Hyperosmolality also induced activation of nuclear factor-κB (NF-κB). Accordingly, pharmacological inhibition of NF-κB significantly decreased osmolality-induced MCP-1 expression. Taken together, these results indicate that high osmolalities activate the transcription factor NFAT5 in mesothelial cells. NFAT5 in turn upregulates MCP-1, likely in combination with NF-κB, and thus may participate in the development of peritoneal fibrosis during CAPD

Dual effect of lithium on NFAT5 activity in kidney cells

Lithium salts are used widely for treatment of bipolar and other mental disorders. Lithium therapy is accompanied frequently by renal side effects, such as nephrogenic diabetes insipidus or chronic kidney disease (CKD),but the molecular mechanisms underlying these effects are still poorly understood. In the present study we examined the effect of lithium on the activity of the osmosensitive transcriptional activator nuclear factor of activated T cells 5 (NFAT5, also known as TonEBP),which plays a key role in renal cellular osmoprotection and urinary concentrating ability. Interestingly, we found different effects of lithium on NFAT5 activity, depending on medium osmolality and incubation time. When cells were exposed to lithium for a relative short period (24 h),NFAT5 activity was significantly increased, especially under isosmotic conditions, resulting in an enhanced expression of the NFAT5 target gene heat shock protein 70 (HSP70). Further analysis revealed that the increase of NFAT5 activity depended primarily on an enhanced activity of the c-terminal transactivation domain (TAD),while NFAT5 protein abundance was largely unaffected. Enhanced activity of the TAD is probably mediated by lithium-induced inhibitory phosphorylation of glycogen synthase kinase 3 beta (GSK-3 beta),which is in accordance with previous studies. When cells were exposed to lithium for a longer period (96 h),cellular NFAT5 activity and subsequently expression of HSP70 significantly decreased under hyperosmotic conditions, due to diminished NFAT5 protein abundance, also resulting from GSK-3 beta inhibition. Taken together, our results provide evidence that lithium has opposing effects on NFAT5 activity, depending on environmental osmolality and exposure duration. The potential impacts of these observations on the diverse effects of lithium on kidney function are discussed

NFAT5-mediated expression of S100A4 contributes to proliferation and migration of renal carcinoma cells

The osmosensitive transcription factor nuclear factor of activated T-cells (NEAT) 5, also known as tonicity enhancer binding protein (TonEBP),has been associated with the development of a variety of tumor entities, among them breast cancer, colon carcinoma, and melanoma. The aim of the present study was to determine whether NFAT5 is also involved in the development of renal cell carcinoma (RCC). The most common type of RCC, the clear cell RCC, originates from the proximal convoluted tubule. We tested our hypothesis in the clear cell RCC cell line CaKi-1 and the non-cancerous proximal tubule cell line HK-2, as control. Basal expression of NFAT5 and NFAT5 activity in CaKi-1 cells was several times higher than in HK-2 cells. Osmotic stress induced an increased NFAT5 activity in both CaKi-1 and HK-2 cells, again with significantly higher activities in CaKi-1 cells. Analysis of NFAT5-regulating signaling pathways in CaKi-1 cells revealed that inhibition of the MAP kinases p38, c-Jun-terminal kinase (JNK) and extracellular regulated kinase (ERK) and of the focal adhesion kinase (FAK) partially blunted NFAT5 activity. FAK and ERK were both constitutively active, even under isotonic conditions, which may contribute to the high basal expression and activity of NFAT5 in CaKi-1 cells. In contrast, the MAP kinases p38 and JNK were inactive under isotonic conditions and became activated under osmotic stress conditions, indicating that p38 and JNK mediate upregulation of NFAT5 activity under these conditions. siRNA-mediated knockdown of NFAT5 in CaKi-1 cells reduced the expression of S100A4, a member of the S100 family of proteins, which promotes metastasis. Knockdown of NFAT5 was accompanied by a significant decrease in proliferation and migration activity. Taken together, our results indicate that NFAT5 induces S100A4 expression in CaKi-1 cells, thereby playing an important role in RCC proliferation and migration

Focal adhesion kinase regulates the activity of the osmosensitive transcription factor TonEBP/NFAT5 under hypertonic conditions

Ton EBP/NFAT5 is a major regulator of the urinary concentrating process and is essential for the osmoadaptation of renal medullary cells. Focal adhesion kinase (FAK) is a mechanosensitive non-receptor protein tyrosine kinase expressed abundantly in the renal medulla. Since osmotic stress causes cell shrinkage, the present study investigated the contribution of FAK on TonEBP/NFAT5 activation. Osmotic stress induced time-dependent activation of FAK as evidenced by phosphorylation at Tyr-397, and furosemide reduces FAK Tyr-397 phosphorylation in the rat renal medulla. Both pharmacological inhibition of FAK and siRNA-mediated knockdown of FAK drastically reduced TonEBP/NFAT5 transcriptional activity and target gene expression in HEK293 cells. This effect was not mediated by impaired nuclear translocation or by reduced transactivating activity of TonEBP/NFAT5. However, TonEBP/NFAT5 abundance under hypertonic conditions was diminished by 50% by FAK inhibition or siRNA knockdown of FAK. FAK inhibition only marginally reduced transcription of the TonEBP/NFAT5 gene. Rather, TonEBP/NFAT5 mRNA stability was diminished significantly by FAK inhibition, which correlated with reduced reporter activity of the TonEBP/NFAT5 mRNA 3' untranslated region (3'-UTR). In conclusion, FAK is a major regulator of TonEBP/NFAT5 activity by increasing its abundance via stabilization of the mRNA. This in turn, depends on the presence of the TonEBP/NFAT5 3'-UTR

Impact of salt and the osmoprotective transcription factor NFAT-5 on macrophages during mechanical strain

Myeloid cells regulate bone density in response to increased salt (NaCl) intake via the osmoprotective transcription factor, nuclear factor of activated T cells-5 (NFAT-5). Because orthodontic tooth movement (OTM) is a pseudoinflammatory immunological process, we investigated the influence of NaCl and NFAT-5 on the expression pattern of macrophages in a model of simulated OTM. RAW264.7 macrophages were exposed for 4 h to 2 ⁻²g cm compressive or 16% tensile or no mechanical strain (control), with or without the addition of 40 mM NaCl. We analyzed the expression of inflammatory genes and proteins [tumor necrosis factor (TNF), interleukin (IL)-6 and prostaglandin endoperoxide synthase-2 (Ptgs-2)/prostaglandin E2 (PG-E2)] by real-time-quantitative PCR and ELISA. To investigate the role of NFAT-5 in these responses, NFAT-5 was both constitutively expressed and silenced. Salt and compressive strain, but not tensile strain increased the expression of NFAT-5 and most tested inflammatory factors in macrophages. NaCl induced the expression of Ptgs-2/PG-E2 and TNF, whereas secretion of IL-6 was inhibited. Similarly, a constitutive expression of NFAT-5 reduced IL-6 expression, while increasing Ptgs-2/PG-E2 and TNF expression. Silencing of NFAT-5 upregulated IL-6 and reduced Ptgs-2/PG-E2 and TNF expression. Salt had an impact on the expression profile of macrophages as a reaction to compressive and tensile strain that occur during OTM. This was mediated via NFAT-5, which surprisingly also seems to play a regulatory role in mechanotransduction of compressive strain. Sodium accumulation in the periodontal ligament caused by dietary salt consumption might propagate local osteoclastogenesis via increased local inflammation and thus OTM velocity, but possibly also entail side effects such as dental root resorptions or periodontal bone loss

Renal Sodium Gradient Orchestrates a Dynamic Antibacterial Defense Zone.

Lower urinary tract infections are among the most common human bacterial infections, but extension to the kidneys is rare. This has been attributed to mechanical forces, such as urine flow, that prevent the ascent of bladder microbes. Here, we show that the regional hypersalinity, required for the kidney's urine-concentrating function, instructs epithelial cells to produce chemokines that localize monocyte-derived mononuclear phagocytes (MNPs) to the medulla. This hypersaline environment also increases the intrinsic bactericidal and neutrophil chemotactic activities of MNPs to generate a zone of defense. Because MNP positioning and function are dynamically regulated by the renal salt gradient, we find that patients with urinary concentrating defects are susceptible to kidney infection. Our work reveals a critical accessory role for the homeostatic function of a vital organ in optimizing tissue defense

Probing Real Sensory Worlds of Receivers with Unsupervised Clustering

The task of an organism to extract information about the external environment from sensory signals is based entirely on the analysis of ongoing afferent spike activity provided by the sense organs. We investigate the processing of auditory stimuli by an acoustic interneuron of insects. In contrast to most previous work we do this by using stimuli and neurophysiological recordings directly in the nocturnal tropical rainforest, where the insect communicates. Different from typical recordings in sound proof laboratories, strong environmental noise from multiple sound sources interferes with the perception of acoustic signals in these realistic scenarios. We apply a recently developed unsupervised machine learning algorithm based on probabilistic inference to find frequently occurring firing patterns in the response of the acoustic interneuron. We can thus ask how much information the central nervous system of the receiver can extract from bursts without ever being told which type and which variants of bursts are characteristic for particular stimuli. Our results show that the reliability of burst coding in the time domain is so high that identical stimuli lead to extremely similar spike pattern responses, even for different preparations on different dates, and even if one of the preparations is recorded outdoors and the other one in the sound proof lab. Simultaneous recordings in two preparations exposed to the same acoustic environment reveal that characteristics of burst patterns are largely preserved among individuals of the same species. Our study shows that burst coding can provide a reliable mechanism for acoustic insects to classify and discriminate signals under very noisy real-world conditions. This gives new insights into the neural mechanisms potentially used by bushcrickets to discriminate conspecific songs from sounds of predators in similar carrier frequency bands