Extracellular sodium regulates fibroblast growth factor 23 (FGF23) formation.

The bone-derived hormone fibroblast growth factor-23 (FGF23) has recently received much attention due to its association with chronic kidney disease and cardiovascular disease progression. Extracellular sodium concentration ([Na+]) plays a significant role in bone metabolism. Hyponatremia (lower serum [Na+]) has recently been shown to be independently associated with FGF23 levels in patients with chronic systolic heart failure. However, nothing is known about the direct impact of [Na+] on FGF23 production. Here, we show that an elevated [Na+] (+20 mM) suppressed FGF23 formation, whereas low [Na+] (-20 mM) increased FGF23 synthesis in the osteoblast-like cell lines UMR-106 and MC3T3-E1. Similar bidirectional changes in FGF23 abundance were observed when osmolality was altered by mannitol but not by urea, suggesting a role of tonicity in FGF23 formation. Moreover, these changes in FGF23 were inversely proportional to the expression of NFAT5 (nuclear factor of activated T cells-5), a transcription factor responsible for tonicity-mediated cellular adaptations. Furthermore, arginine vasopressin, which is often responsible for hyponatremia, did not affect FGF23 production. Next, we performed a comprehensive and unbiased RNA-seq analysis of UMR-106 cells exposed to low versus high [Na+], which revealed several novel genes involved in cellular adaptation to altered tonicity. Additional analysis of cells with Crisp-Cas9-mediated NFAT5 deletion indicated that NFAT5 controls numerous genes associated with FGF23 synthesis, thereby confirming its role in [Na+]-mediated FGF23 regulation. In line with these in vitro observations, we found that hyponatremia patients have higher FGF23 levels. Our results suggest that [Na+] is a critical regulator of FGF23 synthesis.

Loss of adipose TET proteins enhances ß-adrenergic responses and protects against obesity by epigenetic regulation of ß3-AR expression.

ß-adrenergic receptor (ß-AR) signaling plays predominant roles in modulating energy expenditure by triggering lipolysis and thermogenesis in adipose tissue, thereby conferring obesity resistance. Obesity is associated with diminished ß3-adrenergic receptor (ß3-AR) expression and decreased ß-adrenergic responses, but the molecular mechanism coupling nutrient overload to catecholamine resistance remains poorly defined. Ten-eleven translocation (TET) proteins are dioxygenases that alter the methylation status of DNA by oxidizing 5-methylcytosine to 5-hydroxymethylcytosine and further oxidized derivatives. Here, we show that TET proteins are pivotal epigenetic suppressors of ß3-AR expression in adipocytes, thereby attenuating the responsiveness to ß-adrenergic stimulation. Deletion of all three Tet genes in adipocytes led to increased ß3-AR expression and thereby enhanced the downstream ß-adrenergic responses, including lipolysis, thermogenic gene induction, oxidative metabolism, and fat browning in vitro and in vivo. In mouse adipose tissues, Tet expression was elevated after mice ate a high-fat diet. Mice with adipose-specific ablation of all TET proteins maintained higher levels of ß3-AR in both white and brown adipose tissues and remained sensitive to ß-AR stimuli under high-fat diet challenge, leading to augmented energy expenditure and decreased fat accumulation. Consequently, they exhibited improved cold tolerance and were substantially protected from diet-induced obesity, inflammation, and metabolic complications, including insulin resistance and hyperlipidemia. Mechanistically, TET proteins directly repressed ß3-AR transcription, mainly in an enzymatic activity-independent manner, and involved the recruitment of histone deacetylases to increase deacetylation of its promoter. Thus, the TET-histone deacetylase-ß3-AR axis could be targeted to treat obesity and related metabolic diseases.

TFEB activation triggers pexophagy for functional adaptation during oxidative stress under calcium deficient-conditions.

BACKGROUND: Calcium is a ubiquitous intracellular messenger that regulates the expression of various genes involved in cell proliferation, differentiation, and motility. The involvement of calcium in diverse metabolic pathways has been suggested. However, the effect of calcium in peroxisomes, which are involved in fatty acid oxidation and scavenges the result reactive oxygen species (ROS), remains elusive. In addition, impaired peroxisomal ROS inhibit the mammalian target of rapamycin complex 1 (mTORC1) and promote autophagy. Under stress, autophagy serves as a protective mechanism to avoid cell death. In response to oxidative stress, lysosomal calcium mediates transcription factor EB (TFEB) activation. However, the impact of calcium on peroxisome function and the mechanisms governing cellular homeostasis to prevent diseases caused by calcium deficiency are currently unknown. METHODS: To investigate the significance of calcium in peroxisomes and their roles in preserving cellular homeostasis, we established an in-vitro scenario of calcium depletion. RESULTS: This study demonstrated that calcium deficiency reduces catalase activity, resulting in increased ROS accumulation in peroxisomes. This, in turn, inhibits mTORC1 and induces pexophagy through TFEB activation. However, treatment with the antioxidant N-acetyl-l-cysteine (NAC) and the autophagy inhibitor chloroquine impeded the nuclear translocation of TFEB and attenuated peroxisome degradation. CONCLUSIONS: Collectively, our study revealed that ROS-mediated TFEB activation triggers pexophagy during calcium deficiency, primarily because of attenuated catalase activity. We posit that calcium plays a significant role in the proper functioning of peroxisomes, critical for fatty-acid oxidation and ROS scavenging in maintaining cellular homeostasis. These findings have important implications for signaling mechanisms in various pathologies, including Zellweger's syndrome and ageing.

Macrophage transcription factor TonEBP promotes systemic lupus erythematosus and kidney injury via damage-induced signaling pathways.

Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by autoreactive B cells and dysregulation of many other types of immune cells including myeloid cells. Lupus nephritis (LN) is a common target organ manifestations of SLE. Tonicity-responsive enhancer-binding protein (TonEBP, also known as nuclear factor of activated T-cells 5 (NFAT5)), was initially identified as a central regulator of cellular responses to hypertonic stress and is a pleiotropic stress protein involved in a variety of immunometabolic diseases. To explore the role of TonEBP, we examined kidney biopsy samples from patients with LN. Kidney TonEBP expression was found to be elevated in these patients compared to control patients - in both kidney cells and infiltrating immune cells. Kidney TonEBP mRNA was elevated in LN and correlated with mRNAs encoding inflammatory cytokines and the degree of proteinuria. In a pristane-induced SLE model in mice, myeloid TonEBP deficiency blocked the development of SLE and LN. In macrophages, engagement of various toll-like receptors (TLRs) that respond to damage-associated molecular patterns induced TonEBP expression via stimulation of its promoter. Intracellular signaling downstream of the TLRs was dependent on TonEBP. Therefore, TonEBP can act as a transcriptional cofactor for NF-κB, and activated mTOR-IRF3/7 via protein-protein interactions. Additionally, TonEBP-deficient macrophages displayed elevated efferocytosis and animals with myeloid deficiency of TonEBP showed reduced Th1 and Th17 differentiation, consistent with macrophages defective in TLR signaling. Thus, our data show that myeloid TonEBP may be an attractive therapeutic target for SLE and LN.

TonEBP recognizes R-loops and initiates m6A RNA methylation for R-loop resolution.

R-loops are three-stranded, RNA-DNA hybrid, nucleic acid structures produced due to inappropriate processing of newly transcribed RNA or transcription-replication collision (TRC). Although R-loops are important for many cellular processes, their accumulation causes genomic instability and malignant diseases, so these structures are tightly regulated. It was recently reported that R-loop accumulation is resolved by methyltransferase-like 3 (METTL3)-mediated m6A RNA methylation under physiological conditions. However, it remains unclear how R-loops in the genome are recognized and induce resolution signals. Here, we demonstrate that tonicity-responsive enhancer binding protein (TonEBP) recognizes R-loops generated by DNA damaging agents such as ultraviolet (UV) or camptothecin (CPT). Single-molecule imaging and biochemical assays reveal that TonEBP preferentially binds a R-loop via both 3D collision and 1D diffusion along DNA in vitro. In addition, we find that TonEBP recruits METTL3 to R-loops through the Rel homology domain (RHD) for m6A RNA methylation. We also show that TonEBP recruits RNaseH1 to R-loops through a METTL3 interaction. Consistent with this, TonEBP or METTL3 depletion increases R-loops and reduces cell survival in the presence of UV or CPT. Collectively, our results reveal an R-loop resolution pathway by TonEBP and m6A RNA methylation by METTL3 and provide new insights into R-loop resolution processes.

Intracellular cholesterol transport inhibition Impairs autophagy flux by decreasing autophagosome-lysosome fusion.

BACKGROUND: Autophagy is an intracellular degradation process crucial for homeostasis. During autophagy, a double-membrane autophagosome fuses with lysosome through SNARE machinery STX17 to form autolysosome for degradation of damaged organelle. Whereas defective autophagy enhances cholesterol accumulation in the lysosome and impaired autophagic flux that results Niemann-Pick type C1 (NPC1) disease. However, exact interconnection between NPC1 and autophagic flux remain obscure due to the existence of controversial reports. RESULTS: This study aimed at a comparison of the effects of three autophagic inhibitor drugs, including chloroquine, U18666A, and bafilomycin A1, on the intracellular cholesterol transport and autophagy flux. Chloroquine, an autophagic flux inhibitor; U1866A, a NPC1 inhibitor, and bafilomycin A, a lysosomotropic agent are well known to inhibit autophagy by different mechanism. Here we showed that treatment with U1866A and bafilomycin A induces lysosomal cholesterol accumulation that prevented autophagic flux by decreasing autophagosome-lysosome fusion. We also demonstrated that accumulation of cholesterol within the lysosome did not affect lysosomal pH. Although the clearance of accumulated cholesterol by cyclodextrin restored the defective autophagosome-lysosome fusion, the autophagy flux restoration was possible only when lysosomal acidification was not altered. In addition, a failure of STX17 trafficking to autophagosomes plays a key role in prevention of autophagy flux caused by intracellular cholesterol transport inhibitors. CONCLUSIONS: Our data provide a new insight that the impaired autophagy flux does not necessarily result in lysosomal cholesterol accumulation even though it prevents autophagosome-lysosome fusion. Video abstract.

Catalase-deficient mice induce aging faster through lysosomal dysfunction.

BACKGROUND: Lysosomes are a central hub for cellular metabolism and are involved in the regulation of cell homeostasis through the degradation or recycling of unwanted or dysfunctional organelles through the autophagy pathway. Catalase, a peroxisomal enzyme, plays an important role in cellular antioxidant defense by decomposing hydrogen peroxide into water and oxygen. In accordance with pleiotropic significance, both impaired lysosomes and catalase have been linked to many age-related pathologies with a decline in lifespan. Aging is characterized by progressive accumulation of macromolecular damage and the production of high levels of reactive oxygen species. Although lysosomes degrade the most long-lived proteins and organelles via the autophagic pathway, the role of lysosomes and their effect on catalase during aging is not known. The present study investigated the role of catalase and lysosomal function in catalase-knockout (KO) mice. METHODS: We performed experiments on WT and catalase KO younger (9 weeks) and mature adult (53 weeks) male mice and Mouse embryonic fibroblasts isolated from WT and KO mice from E13.5 embryos as in vivo and in ex-vivo respectively. Mouse phenotyping studies were performed with controls, and a minimum of two independent experiments were performed with more than five mice in each group. RESULTS: We found that at the age of 53 weeks (mature adult), catalase-KO mice exhibited an aging phenotype faster than wild-type (WT) mice. We also found that mature adult catalase-KO mice induced leaky lysosome by progressive accumulation of lysosomal content, such as cathespin D, into the cytosol. Leaky lysosomes inhibited autophagosome formation and triggered impaired autophagy. The dysregulation of autophagy triggered mTORC1 (mechanistic target of rapamycin complex 1) activation. However, the antioxidant N-acetyl-L-cysteine and mTORC1 inhibitor rapamycin rescued leaky lysosomes and aging phenotypes in catalase-deficient mature adult mice. CONCLUSIONS: This study unveils the new role of catalase and its role in lysosomal function during aging. Video abstract.

Microglial TonEBP mediates LPS-induced inflammation and memory loss as transcriptional cofactor for NF-κB and AP-1.

BACKGROUND: Microglia are brain-resident myeloid cells involved in the innate immune response and a variety of neurodegenerative diseases. In macrophages, TonEBP is a transcriptional cofactor of NF-κB which stimulates the transcription of pro-inflammatory genes in response to LPS. Here, we examined the role of microglial TonEBP. METHODS: We used microglial cell line, BV2 cells. TonEBP was knocked down using lentiviral transduction of shRNA. In animals, TonEBP was deleted from myeloid cells using a line of mouse with floxed TonEBP. Cerulenin was used to block the NF-κB cofactor function of TonEBP. RESULTS: TonEBP deficiency blocked the LPS-induced expression of pro-inflammatory cytokines and enzymes in association with decreased activity of NF-κB in BV2 cells. We found that there was also a decreased activity of AP-1 and that TonEBP was a transcriptional cofactor of AP-1 as well as NF-κB. Interestingly, we found that myeloid-specific TonEBP deletion blocked the LPS-induced microglia activation and subsequent neuronal cell death and memory loss. Cerulenin disrupted the assembly of the TonEBP/NF-κB/AP-1/p300 complex and suppressed the LPS-induced microglial activation and the neuronal damages in animals. CONCLUSIONS: TonEBP is a key mediator of microglial activation and neuroinflammation relevant to neuronal damage. Cerulenin is an effective blocker of the TonEBP actions.

COL6A3-derived endotrophin links reciprocal interactions among hepatic cells in the pathology of chronic liver disease.

Extracellular matrix dysregulation is associated with chronic liver disease. CollagenVI-alpha3 chain (COL6A3) is a biomarker for hepatic fibrosis and poor prognosis of hepatocellular carcinoma (HCC), but its function in liver pathology remains unknown. High levels of COL6A3 and its cleaved product, endotrophin (ETP) in tumor-neighboring regions are strongly associated with poor prognosis in HCC patients. Here, we report that the high levels of ETP in injured hepatocytes induce JNK-dependent hepatocyte apoptosis and activate nonparenchymal cells to lead further activation of hepatic inflammation, fibrosis, and apoptosis. Nevertheless ETP per se showed limited phenotypic changes in normal liver tissues. Furthermore, inhibition of ETP activity by utilizing neutralizing antibodies efficiently suppressed the pathological consequences in chronic liver diseases. Our results implicate ETP mechanistically as a crucial mediator in reciprocal interactions among various hepatic cell populations in the pathogenesis of chronic liver disease, and it could be a promising therapeutic target particularly in individuals with high local levels of COL6A3. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Tonicity-responsive enhancer-binding protein promotes hepatocellular carcinogenesis, recurrence and metastasis.

OBJECTIVES: Hepatocellular carcinoma (HCC) is a common cancer with high rate of recurrence and mortality. Diverse aetiological agents and wide heterogeneity in individual tumours impede effective and personalised treatment. Tonicity-responsive enhancer-binding protein (TonEBP) is a transcriptional cofactor for the expression of proinflammatory genes. Although inflammation is intimately associated with the pathogenesis of HCC, the role of TonEBP is unknown. We aimed to identify function of TonEBP in HCC. DESIGN: Tumours with surrounding hepatic tissues were obtained from 296 patients with HCC who received completion resection. TonEBP expression was analysed by quantitative reverse transcription-quantitative real-time PCR (RT-PCR) and immunohfistochemical analyses of tissue microarrays. Mice with TonEBP haplodeficiency, and hepatocyte-specific and myeloid-specific TonEBP deletion were used along with HCC and hepatocyte cell lines. RESULTS: TonEBP expression is higher in tumours than in adjacent non-tumour tissues in 92.6% of patients with HCC regardless of aetiology associated. The TonEBP expression in tumours and adjacent non-tumour tissues predicts recurrence, metastasis and death in multivariate analyses. TonEBP drives the expression of cyclo-oxygenase-2 (COX-2) by stimulating the promoter. In mouse models of HCC, three common sites of TonEBP action in response to diverse aetiological agents leading to tumourigenesis and tumour growth were found: cell injury and inflammation, induction by oxidative stress and stimulation of the COX-2 promoter. CONCLUSIONS: TonEBP is a key component of the common pathway in tumourigenesis and tumour progression of HCC in response to diverse aetiological insults. TonEBP is involved in multiple steps along the pathway, rendering it an attractive therapeutic target as well as a prognostic biomarker.

Tonicity-Responsive Enhancer-Binding Protein Mediates Hyperglycemia-Induced Inflammation and Vascular and Renal Injury.

Diabetic nephropathy (DN) has become the single leading cause of ESRD in developed nations. Bearing in mind the paucity of effective treatment for DN and progressive CKD, novel targets for treatment are sorely needed. We previously reported that increased activity of tonicity-responsive enhancer-binding protein (TonEBP) in monocytes was associated with early DN in humans. We now extend these findings by testing the hypotheses that TonEBP in macrophages promotes hyperglycemia-mediated proinflammatory activation and chronic renal inflammation leading to DN and CKD, and TonEBP genetic variability in humans is associated with inflammatory, renal, and vascular function-related phenotypes. In a mouse model of DN, compared with the wild-type phenotype, TonEBP haplodeficiency associated with reduced activation of macrophages by hyperglycemia, fewer macrophages in the kidney, lower renal expression of proinflammatory genes, and attenuated DN. Furthermore, in a cohort of healthy humans, genetic variants within TonEBP associated with renal function, BP, and systemic inflammation. One of the genetic variants associated with renal function was replicated in a large population-based cohort. These findings suggest that TonEBP is a promising target for minimizing diabetes- and stress-induced inflammation and renovascular injury.

Roles of fluid shear stress and retinoic acid in the differentiation of primary cultured human podocytes.

Due to the distinct features that distinguish immortalized podocyte cell lines from their in vivo counterparts, primary cultured human podocytes might be a superior cell model for glomerular disease studies. However, the podocyte de-differentiation that occurs in culture remains an unresolved problem. Here, we present a method to differentiate primary cultured podocytes using retinoic acid (RA) and fluid shear stress (FSS), which mimic the in vivo environment of the glomerulus. RA treatment induced changes in the cell shape of podocytes from a cobblestone-like morphology to an arborized configuration with enhanced mobility. Moreover, the expression of synaptopodin and zonula occludens (ZO)-1 in RA-treated podocytes increased along with Krüppel-like factor 15 (KLF15) expression. Confocal microscopy revealed that RA increased the expression of cytoplasmic synaptopodin, which adopted a filamentous arrangement, and junctional ZO-1 expression, which showed a zipper-like pattern. To elucidate the effect of FSS in addition to RA, the podocytes were cultured in microfluidic devices and assigned to the static, static+RA, FSS, and FSS+RA groups. The FSS+RA group showed increased synaptopodin and ZO-1 expression with prominent spikes on the cell-cell interface. Furthermore, interdigitating processes were only observed in the FSS+RA group. Consistent with these data, the mRNA expression levels of synaptopodin, podocin, WT-1 and ZO-1 were synergistically increased by FSS and RA treatment. Additionally, the heights of the cells were greater in the FSS and FSS+RA groups than in the static groups, suggesting a restoration of the 3D cellular shape. Meanwhile, the expression of KLF15 increased in the RA-treated cells regardless of fluidic condition. Taken together, FSS and RA may contribute through different but additive mechanisms to the differentiation of podocytes. These cells may serve as a useful tool for mechanistic studies and the application of regenerative medicine to the treatment of kidney diseases.

One-hit kill: On the inactivation of RNA viruses by ultraviolet (UV)-C-induced genomic damage.

Large scale outbreaks of infectious respiratory disease have repeatedly plagued the globe over the last 100 years. The scope and strength of the outbreaks are getting worse as pathogenic RNA viruses are rapidly evolving and highly evasive to vaccines and anti-viral drugs. Germicidal UV-C is considered as a robust agent to disinfect RNA viruses regardless of their evolution. While genomic damage by UV-C has been known to be associated with viral inactivation, the precise relationship between the damage and inactivation remains unsettled as genomic damage has been analyzed in small areas, typically under 0.5 kb. In this study, we assessed genomic damage by the reduced efficiency of reverse transcription of regions of up to 7.2 kb. Our data seem to indicate that genomic damage was directly proportional to the size of the genome, and a single hit of damage was sufficient for inactivation of RNA viruses. The high efficacy of UV-C is already effectively adopted to inactivate airborne RNA viruses.

Multiple cell death pathways are independently activated by lethal hypertonicity in renal epithelial cells.

When hypertonicity is imposed with sufficient intensity and acuteness, cells die. Here we investigated the cellular pathways involved in death using a cell line derived from renal epithelium. We found that hypertonicity rapidly induced activation of an intrinsic cell death pathway-release of cytochrome c and activation of caspase-3 and caspase-9-and an extrinsic pathway-activation of caspase-8. Likewise, a lysosomal pathway of cell death characterized by partial lysosomal rupture and release of cathepsin B from lysosomes to the cytosol was also activated. Relationships among the pathways were examined using specific inhibitors. Caspase inhibitors did not affect cathepsin B release into the cytosol by hypertonicity. In addition, cathepsin B inhibitors and caspase inhibitors did not affect hypertonicity-induced cytochrome c release, suggesting that the three pathways were independently activated. Combined inhibition of caspases and cathepsin B conferred significantly more protection from hypertonicity-induced cell death than inhibition of caspase or cathepsin B alone, indicating that all the three pathways contributed to the hypertonicity-induced cell death. Similar pattern of sensitivity to the inhibitors was observed in two other cell lines derived from renal epithelia. We conclude that multiple cell death pathways are independently activated early in response to lethal hypertonic stress in renal epithelial cells.

TonEBP in Myeloid Cells Promotes Obesity-Induced Insulin Resistance and Inflammation Through Adipose Tissue Remodeling.

The phenotypic and functional plasticity of adipose tissue macrophages (ATMs) during obesity plays a crucial role in orchestration of adipose and systemic inflammation. Tonicity-responsive enhancer binding protein (TonEBP) (also called NFAT5) is a stress protein that mediates cellular responses to a range of metabolic insults. Here, we show that myeloid cell-specific TonEBP depletion reduced inflammation and insulin resistance in mice with high-fat diet-induced obesity but did not affect adiposity. This phenotype was associated with a reduced accumulation and a reduced proinflammatory phenotype of metabolically activated macrophages, decreased expression of inflammatory factors related to insulin resistance, and enhanced insulin sensitivity. TonEBP expression was elevated in the ATMs of obese mice, and Sp1 was identified as a central regulator of TonEBP induction. TonEBP depletion in macrophages decreased induction of insulin resistance-related genes and promoted induction of insulin sensitivity-related genes under obesity-mimicking conditions and thereby improved insulin signaling and glucose uptake in adipocytes. mRNA expression of TonEBP in peripheral blood mononuclear cells was positively correlated with blood glucose levels in mice and humans. These findings suggest that TonEBP in macrophages promotes obesity-associated systemic insulin resistance and inflammation, and downregulation of TonEBP may induce a healthy metabolic state during obesity.

PARP1-mediated PARylation of TonEBP prevents R-loop-associated DNA damage.

Lack of coordination between the DNA replication and transcription machineries can increase the frequency of transcription-replication conflicts, leading ultimately to DNA damage and genomic instability. A major source of these conflicts is the formation of R-loops, which consist of a transcriptionally generated RNA-DNA hybrid and the displaced single-stranded DNA. R-loops play important physiological roles and have been implicated in human diseases. Although these structures have been extensively studied, many aspects of R-loop biology and R-loop-mediated genome instability remain unclear. We found that in cancer cells, tonicity-responsive enhancer-binding protein (TonEBP, also called NFAT5) interacted with PARP1 and localized to R-loops in response to DNA-damaging agent camptothecin (CPT), which is associated with R-loop formation. PARP1-mediated PARylation was required for recruitment of TonEBP to the sites of R-loop-associated DNA damage. Loss of TonEBP increased levels of R-loop accumulation and DNA damage, and promoted cell death in response to CPT. These findings suggest that TonEBP mediates resistance to CPT-induced cell death by preventing R-loop accumulation in cancer cells.

Thrap3 promotes R-loop resolution via interaction with methylated DDX5.

Transcription-replication conflicts lead to DNA damage and genomic instability, which are closely related to human diseases. A major source of these conflicts is the formation of R-loops, which consist of an RNA-DNA hybrid and a displaced single-stranded DNA. Although these structures have been studied, many aspects of R-loop biology and R-loop-mediated genome instability remain unclear. Here, we demonstrate that thyroid hormone receptor-associated protein 3 (Thrap3) plays a critical role in regulating R-loop resolution. In cancer cells, Thrap3 interacts with DEAD-box helicase 5 (DDX5) and localizes to R-loops. Arginine-mediated methylation of DDX5 is required for its interaction with Thrap3, and the Thrap3-DDX5 axis induces the recruitment of 5'-3' exoribonuclease 2 (XRN2) into R-loops. Loss of Thrap3 increases R-loop accumulation and DNA damage. These findings suggest that Thrap3 mediates resistance to cell death by preventing R-loop accumulation in cancer cells.

The evolving role of TonEBP as an immunometabolic stress protein.

Tonicity-responsive enhancer-binding protein (TonEBP), which is also known as nuclear factor of activated T cells 5 (NFAT5), was discovered 20 years ago as a transcriptional regulator of the cellular response to hypertonic (hyperosmotic salinity) stress in the renal medulla. Numerous studies since then have revealed that TonEBP is a pleiotropic stress protein that is involved in a range of immunometabolic diseases. Some of the single-nucleotide polymorphisms (SNPs) in TONEBP introns are cis-expression quantitative trait loci that affect TONEBP transcription. These SNPs are associated with increased risk of type 2 diabetes mellitus, diabetic nephropathy, inflammation, high blood pressure and abnormal plasma osmolality, indicating that variation in TONEBP expression might contribute to these phenotypes. In addition, functional studies have shown that TonEBP is involved in the pathogenesis of rheumatoid arthritis, atherosclerosis, diabetic nephropathy, acute kidney injury, hyperlipidaemia and insulin resistance, autoimmune diseases (including type 1 diabetes mellitus and multiple sclerosis), salt-sensitive hypertension and hepatocellular carcinoma. These pathological activities of TonEBP are in contrast to the protective actions of TonEBP in response to hypertonicity, bacterial infection and DNA damage induced by genotoxins. An emerging theme is that TonEBP is a stress protein that mediates the cellular response to a range of pathological insults, including excess caloric intake, inflammation and oxidative stress.

TonEBP Promotes ß-Cell Survival under ER Stress by Enhancing Autophagy.

The endoplasmic reticulum (ER) stress response and autophagy are important cellular responses that determine cell fate and whose dysregulation is implicated in the perturbation of homeostasis and diseases. Tonicity-responsive enhancer-binding protein (TonEBP, also called NFAT5) is a pleiotropic stress protein that mediates both protective and pathological cellular responses. Here, we examined the role of TonEBP in ß-cell survival under ER stress. We found that TonEBP increases ß-cell survival under ER stress by enhancing autophagy. The level of TonEBP protein increased under ER stress due to a reduction in its degradation via the ubiquitin-proteasome pathway. In response to ER stress, TonEBP increased autophagosome formations and suppressed the accumulation of protein aggregates and ß-cell death. The Rel-homology domain of TonEBP interacted with FIP200, which is essential for the initiation of autophagy, and was required for autophagy and cell survival upon exposure to ER stress. Mice in which TonEBP was specifically deleted in pancreatic endocrine progenitor cells exhibited defective glucose homeostasis and a loss of islet mass. Taken together, these findings demonstrate that TonEBP protects against ER stress-induced ß-cell death by enhancing autophagy.

TonEBP in dendritic cells mediates pro-inflammatory maturation and Th1/Th17 responses.

Dendritic cells (DCs) are potent antigen-presenting cells that link the innate and adaptive immune responses; as such they play pivotal roles in initiation and progression of rheumatoid arthritis (RA). Here, we report that the tonicity-responsive enhancer-binding protein (TonEBP or NFAT5), a Rel family protein involved in the pathogenesis of autoimmune disease and inflammation, is required for maturation and function of DCs. Myeloid cell-specific TonEBP deletion reduces disease severity in a murine model of collagen-induced arthritis; it also inhibits maturation of DCs and differentiation of pathogenic Th1 and Th17 cells in vivo. Upon stimulation by TLR4, TonEBP promotes surface expression of major histocompatibility complex class II and co-stimulatory molecules via p38 mitogen-activated protein kinase. This is followed by DC-mediated differentiation of pro-inflammatory Th1 and Th17 cells. Taken together, these findings provide mechanistic basis for the pathogenic role of TonEBP in RA and possibly other autoimmune diseases.