A comparative analysis of TonEBP conditional knockout mouse models reveals inter-dependency between compartments of the intervertebral disc.

Interactions between notochord and sclerotome are required for normal embryonic spine patterning, but whether the postnatal derivatives of these tissues also require interactions for postnatal intervertebral disc (IVD) growth and maintenance is less established. We report here the comparative analysis of four conditional knockout mice deficient for TonEBP, a transcription factor known to allow cells to adapt to changes in extracellular osmotic pressure, in specific compartments of the IVD. We show that TonEBP deletion in nucleus pulposus (NP) cells does not affect their survival or aggrecan expression, but promoted cell proliferation in the NP and in adjacent vertebral growth plates (GPs). In cartilage end plates/GPs, TonEBP deletion induced cell death, but also structural alterations in the adjacent NP cells and vertebral bodies. Embryonic or postnatal TonEBP loss generated similar IVD changes. In addition to demonstrating the requirement of TonEBP in the different compartments of the IVD, this comparative analysis uncovers the in vivo interdependency of the different IVD compartments during the growth of the postnatal IVD-vertebral units.

TonEBP inhibits ciliogenesis by controlling aurora kinase A and regulating centriolar satellite integrity.

BACKGROUND: Primary cilia on the surface of eukaryotic cells serve as sensory antennas for the reception and transmission in various cell signaling pathways. They are dynamic organelles that rapidly form during differentiation and cell cycle exit. Defects in these organelles cause a group of wide-ranging disorders called ciliopathies. Tonicity-responsive enhancer-binding protein (TonEBP) is a pleiotropic stress protein that mediates various physiological and pathological cellular responses. TonEBP is well-known for its role in adaptation to a hypertonic environment, to which primary cilia have been reported to contribute. Furthermore, TonEBP is involved in a wide variety of other signaling pathways, such as Sonic Hedgehog and WNT signaling, that promote primary ciliogenesis, suggesting a possible regulatory role. However, the functional relationship between TonEBP and primary ciliary formation remains unclear. METHODS: TonEBP siRNAs and TonEBP-mCherry plasmids were used to examine their effects on cell ciliation rates, assembly and disassembly processes, and regulators. Serum starvation was used as a condition to induce ciliogenesis. RESULTS: We identified a novel pericentriolar localization for TonEBP. The results showed that TonEBP depletion facilitates the formation of primary cilia, whereas its overexpression results in fewer ciliated cells. Moreover, TonEBP controlled the expression and activity of aurora kinase A, a major negative regulator of ciliogenesis. Additionally, TonEBP overexpression inhibited the loss of CP110 from the mother centrioles during the early stages of primary cilia assembly. Finally, TonEBP regulated the localization of PCM1 and AZI1, which are necessary for primary cilia formation. CONCLUSIONS: This study proposes a novel role for TonEBP as a pericentriolar protein that regulates the integrity of centriolar satellite components. This regulation has shown to have a negative effect on ciliogenesis. Investigations into cilium assembly and disassembly processes suggest that TonEBP acts upstream of the aurora kinase A - histone deacetylase 6 signaling pathway and affects basal body formation to control ciliogenesis. Taken together, our data proposes previously uncharacterized regulation of primary cilia assembly by TonEBP.

TonEBP: A Key Transcription Factor in Microglia Following Intracerebral Hemorrhage Induced-Neuroinflammation.

Transcription factors within microglia contribute to the inflammatory response following intracerebral hemorrhage (ICH). Therefore, we employed bioinformatics screening to identify the potential transcription factor tonicity-responsive enhancer-binding protein (TonEBP) within microglia. Inflammatory stimuli can provoke an elevated expression of TonEBP in microglia. Nevertheless, the expression and function of microglial TonEBP in ICH-induced neuroinflammation remain ambiguous. In our recent research, we discovered that ICH instigated an increased TonEBP in microglia in both human and mouse peri-hematoma brain tissues. Furthermore, our results indicated that TonEBP knockdown mitigates lipopolysaccharide (LPS)-induced inflammation and the activation of NF-κB signaling in microglia. In order to more deeply comprehend the underlying molecular mechanisms of how TonEBP modulates the inflammatory response, we sequenced the transcriptomes of TonEBP-deficient cells and sought potential downstream target genes of TonEBP, such as Pellino-1 (PELI1). PELI has been previously reported to mediate nuclear factor-κB (NF-κB) signaling. Through the utilization of CUT & RUN, a dual-luciferase reporter, and qPCR, we confirmed that TonEBP is the transcription factor of Peli1, binding to the Peli1 promoter. In summary, TonEBP may enhance the LPS-induced inflammation and activation of NF-κB signaling via PELI1.

Tissue Sodium Accumulation Induces Organ Inflammation and Injury in Chronic Kidney Disease.

High salt intake is a primary cause of over-hydration in chronic kidney disease (CKD) patients. Inflammatory markers are predictors of CKD mortality; however, the pathogenesis of inflammation remains unclear. Sodium storage in tissues has recently emerged as an issue of concern. The binding of sodium to tissue glycosaminoglycans and its subsequent release regulates local tonicity. Many cell types express tonicity-responsive enhancer-binding protein (TonEBP), which is activated in a tonicity-dependent or tonicity-independent manner. Macrophage infiltration was observed in the heart, peritoneal wall, and para-aortic tissues in salt-loading subtotal nephrectomized mice, whereas macrophages were not prominent in tap water-loaded subtotal nephrectomized mice. TonEBP was increased in the heart and peritoneal wall, leading to the upregulation of inflammatory mediators associated with cardiac fibrosis and peritoneal membrane dysfunction, respectively. Reducing salt loading by a diuretic treatment or changing to tap water attenuated macrophage infiltration, TonEBP expression, and inflammatory marker expression. The role of TonEBP may be crucial during the cardiac fibrosis and peritoneal deterioration processes induced by sodium overload. Anti-interleukin-6 therapy improved cardiac inflammation and fibrosis and peritoneal membrane dysfunction. Further studies are necessary to establish a strategy to regulate organ dysfunction induced by TonEBP activation in CKD patients.

NFAT5 contributes to the pathogenesis of experimental autoimmune encephalomyelitis (EAE) and decrease of T regulatory cells in female mice.

Multiple sclerosis disproportionally affects women. The present study was undertaken to determine whether NFAT5 contributed to the pathogenesis of experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis, and if it did, whether the impact was sex associated. NFAT5 haplodeficiency reduced the disease severity only in female mice. This effect was associated with significant increases in frequency of T regulatory (Treg) cells in the CNS (from 1.45 ± 0.39% to 3.73 ± 0.94%) and spleen from (0.31 ± 0.06% to 0.94 ± 0.29%) without significantly affecting the CNS CD4+ subsets frequency. NFAT5 haploinsufficiency also significantly reduced the frequency of CD11c+CD8α+ dendritic cells in the female CNS. However, increase of their frequency in the CNS via intraperitoneal Flt3L injection at peak EAE had no significant effect on the disease courses. We conclude that NFAT5 contributes to pathogenesis of EAE in female mice, possibly through decreasing tissue specific frequency of Treg cells.

Tonicity-responsive enhancer-binding protein promotes diabetic neuroinflammation and cognitive impairment via upregulation of lipocalin-2.

BACKGROUND: Diabetic individuals have increased circulating inflammatory mediators which are implicated as underlying causes of neuroinflammation and memory deficits. Tonicity-responsive enhancer-binding protein (TonEBP) promotes diabetic neuroinflammation. However, the precise role of TonEBP in the diabetic brain is not fully understood. METHODS: We employed a high-fat diet (HFD)-only fed mice or HFD/streptozotocin (STZ)-treated mice in our diabetic mouse models. Circulating TonEBP and lipocalin-2 (LCN2) levels were measured in type 2 diabetic subjects. TonEBP haploinsufficient mice were used to investigate the role of TonEBP in HFD/STZ-induced diabetic mice. In addition, RAW 264.7 macrophages were given a lipopolysaccharide (LPS)/high glucose (HG) treatment. Using a siRNA, we examined the effects of TonEBP knockdown on RAW264 cell' medium/HG-treated mouse hippocampal HT22 cells. RESULTS: Circulating TonEBP and LCN2 levels were higher in experimental diabetic mice or type 2 diabetic patients with cognitive impairment. TonEBP haploinsufficiency ameliorated the diabetic phenotypes including adipose tissue macrophage infiltrations, neuroinflammation, blood-brain barrier leakage, and memory deficits. Systemic and hippocampal LCN2 proteins were reduced in diabetic mice by TonEBP haploinsufficiency. TonEBP (+ / -) mice had a reduction of hippocampal heme oxygenase-1 (HO-1) expression compared to diabetic wild-type mice. In particular, we found that TonEBP bound to the LCN2 promoter in the diabetic hippocampus, and this binding was abolished by TonEBP haploinsufficiency. Furthermore, TonEBP knockdown attenuated LCN2 expression in lipopolysaccharide/high glucose-treated mouse hippocampal HT22 cells. CONCLUSIONS: These findings indicate that TonEBP may promote neuroinflammation and cognitive impairment via upregulation of LCN2 in diabetic mice.

NFAT5-Mediated Signalling Pathways in Viral Infection and Cardiovascular Dysfunction.

The nuclear factor of activated T cells 5 (NFAT5) is well known for its sensitivity to cellular osmolarity changes, such as in the kidney medulla. Accumulated evidence indicates that NFAT5 is also a sensitive factor to stress signals caused by non-hypertonic stimuli such as heat shock, biomechanical stretch stress, ischaemia, infection, etc. These osmolality-related and -unrelated stimuli can induce NFAT5 upregulation, activation and nuclear accumulation, leading to its protective role against various detrimental effects. However, dysregulation of NFAT5 expression may cause pathological conditions in different tissues, leading to a variety of diseases. These protective or pathogenic effects of NFAT5 are dictated by the regulation of its target gene expression and activation of its signalling pathways. Recent studies have found a number of kinases that participate in the phosphorylation/activation of NFAT5 and related signal proteins. Thus, this review will focus on the NFAT5-mediated signal transduction pathways. As for the stimuli that upregulate NFAT5, in addition to the stresses caused by hyperosmotic and non-hyperosmotic environments, other factors such as miRNA, long non-coding RNA, epigenetic modification and viral infection also play an important role in regulating NFAT5 expression; thus, the discussion in this regard is another focus of this review. As the heart, unlike the kidneys, is not normally exposed to hypertonic environments, studies on NFAT5-mediated cardiovascular diseases are just emerging and rapidly progressing. Therefore, we have also added a review on the progress made in this field of research.

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.

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.

COX-2 expression mediated by calcium-TonEBP signaling axis under hyperosmotic conditions serves osmoprotective function in nucleus pulposus cells.

The nucleus pulposus (NP) of intervertebral discs experiences dynamic changes in tissue osmolarity because of diurnal loading of the spine. TonEBP/NFAT5 is a transcription factor that is critical in osmoregulation as well as survival of NP cells in the hyperosmotic milieu. The goal of this study was to investigate whether cyclooxygenase-2 (COX-2) expression is osmoresponsive and dependent on TonEBP, and whether it serves an osmoprotective role. NP cells up-regulated COX-2 expression in hyperosmotic media. The induction of COX-2 depended on elevation of intracellular calcium levels and p38 MAPK pathway, but independent of calcineurin signaling as well as MEK/ERK and JNK pathways. Under hyperosmotic conditions, both COX-2 mRNA stability and its proximal promoter activity were increased. The proximal COX-2 promoter (-1840/+123 bp) contained predicted binding sites for TonEBP, AP-1, NF-κB, and C/EBP-ß. While COX-2 promoter activity was positively regulated by both AP-1 and NF-κB, AP-1 had no effect and NF-κB negatively regulated COX-2 protein levels under hyperosmotic conditions. On the other hand, TonEBP was necessary for both COX-2 promoter activity and protein up-regulation in response to hyperosmotic stimuli. Ex vivo disc organ culture studies using hypomorphic TonEBP+/- mice confirmed that TonEBP is required for hyperosmotic induction of COX-2. Importantly, the inhibition of COX-2 activity under hyperosmotic conditions resulted in decreased cell viability, suggesting that COX-2 plays a cytoprotective and homeostatic role in NP cells for their adaptation to dynamically loaded hyperosmotic niches.

Inverse Regulation of Lipocalin-2/24p3 Receptor/SLC22A17 and Lipocalin-2 Expression by Tonicity, NFAT5/TonEBP and Arginine Vasopressin in Mouse Cortical Collecting Duct Cells mCCD(cl.1): Implications for Osmotolerance.

The rodent collecting duct (CD) expresses a 24p3/NGAL/lipocalin-2 (LCN2) receptor (SLC22A17) apically, possibly to mediate high-affinity reabsorption of filtered proteins by endocytosis, although its functions remain uncertain. Recently, we showed that hyperosmolarity/-tonicity upregulates SLC22A17 in cultured mouse inner-medullary CD cells, whereas activation of toll-like receptor 4 (TLR4), via bacterial lipopolysaccharides (LPS), downregulates SLC22A17. This is similar to the upregulation of Aqp2 by hyperosmolarity/-tonicity and arginine vasopressin (AVP), and downregulation by TLR4 signaling, which occur via the transcription factors NFAT5 (TonEBP or OREBP), cAMP-responsive element binding protein (CREB), and nuclear factor-kappa B, respectively. The aim of the study was to determine the effects of osmolarity/tonicity and AVP, and their associated signaling pathways, on the expression of SLC22A17 and its ligand, LCN2, in the mouse (m) cortical collecting duct cell line mCCD(cl.1). Normosmolarity/-tonicity corresponded to 300 mosmol/L, whereas the addition of 50-100 mmol/L NaCl for up to 72 h induced hyperosmolarity/-tonicity (400-500 mosmol/L). RT-PCR, qPCR, immunoblotting and immunofluorescence microscopy detected Slc22a17/SLC22A17 and Lcn2/LCN2 expression. RNAi silenced Nfat5, and the pharmacological agent 666-15 blocked CREB. Activation of TLR4 was induced with LPS. Similar to Aqp2, hyperosmotic/-tonic media and AVP upregulated Slc22a17/SLC22A17, via activation of NFAT5 and CREB, respectively, and LPS/TLR4 signaling downregulated Slc22a17/SLC22A17. Conversely, though NFAT5 mediated the hyperosmolarity/-tonicity induced downregulation of Lcn2/LCN2 expression, AVP reduced Lcn2/LCN2 expression and predominantly apical LCN2 secretion, evoked by LPS, through a posttranslational mode of action that was independent of CREB signaling. In conclusion, the hyperosmotic/-tonic upregulation of SLC22A17 in mCCD(cl.1) cells, via NFAT5, and by AVP, via CREB, suggests that SLC22A17 contributes to adaptive osmotolerance, whereas LCN2 downregulation could counteract increased proliferation and permanent damage of osmotically stressed cells.

TNF-α promotes nuclear enrichment of the transcription factor TonEBP/NFAT5 to selectively control inflammatory but not osmoregulatory responses in nucleus pulposus cells.

Intervertebral disc degeneration (IDD) causes chronic back pain and is linked to production of proinflammatory molecules by nucleus pulposus (NP) and other disc cells. Activation of tonicity-responsive enhancer-binding protein (TonEBP)/NFAT5 by non-osmotic stimuli, including proinflammatory molecules, occurs in cells involved in immune response. However, whether inflammatory stimuli activate TonEBP in NP cells and whether TonEBP controls inflammation during IDD is unknown. We show that TNF-α, but not IL-1ß or LPS, promoted nuclear enrichment of TonEBP protein. However, TNF-α-mediated activation of TonEBP did not cause induction of osmoregulatory genes. RNA sequencing showed that 8.5% of TNF-α transcriptional responses were TonEBP-dependent and identified genes regulated by both TNF-α and TonEBP. These genes were over-enriched in pathways and diseases related to inflammatory response and inhibition of matrix metalloproteases. Based on RNA-sequencing results, we further investigated regulation of novel TonEBP targets CXCL1, CXCL2, and CXCL3 TonEBP acted synergistically with TNF-α and LPS to induce CXCL1-proximal promoter activity. Interestingly, this regulation required a highly conserved NF-κB-binding site but not a predicted TonE, suggesting cross-talk between these two members of the Rel family. Finally, analysis of human NP tissue showed that TonEBP expression correlated with canonical osmoregulatory targets TauT/SLC6A6, SMIT/SLC5A3, and AR/AKR1B1, supporting in vitro findings that the inflammatory milieu during IDD does not interfere with TonEBP osmoregulation. In summary, whereas TonEBP participates in the proinflammatory response to TNF-α, therapeutic strategies targeting this transcription factor for treatment of disc disease must spare osmoprotective, prosurvival, and matrix homeostatic activities.

VEGF-C-mediated cardiac lymphangiogenesis in high salt intake accelerated progression of left ventricular remodeling in spontaneously hypertensive rats.

High salt (HS) diet can accelerate the progress of hypertensive left ventricular (LV) remodeling. But the detailed mechanism remains poorly understood. We hypothesized HS intake could impact cardiac lymphangiogenesis through tonicity-responsive enhancer binding protein (TonEBP)/vascular endothelial growth factor-C (VEGF-C) signaling pathway which might play an important role in HS intake accelerated LV remodeling. Eight-week-old male spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) were randomized to 0.5% NaCl (Low salt, LS) and 8% NaCl (high salt, HS) diets for 12 weeks. LV remodeling was determined by echocardiography. LV invasive hemodynamic analysis and morphologic staining (cardiomyocyte hypertrophy, collagen deposition, TonEBP expression, macrophage infiltration and lymphatic density) were performed at the time of sacrifice. The blood pressure of SHR-HS group was significantly increased compared to SHR-LS and WKY groups. Meanwhile, The LV chamber size was markedly enlargement, LV function apparently compromised accompanied with a severe macrophage infiltration, and fibrosis in the perivascular and interstitium of LV compared with SHR-LS group. Furthermore, the expression levels of VEGF-C, TonEBP, and lymphatic markers in SHR-HS group were significantly increased parallel with apparent lymphangiogenesis compared with SHR-LS group. Our work indicates that TonEBP/VEGF-C signaling pathway was up-regulated in HS intake accelerated hypertensive LV remodeling process that may be valuable for further investigation.

TonEBP suppresses adipocyte differentiation via modulation of early signaling in 3T3-L1 cells.

TonEBP belongs to the Rel family of transcription factors and plays important roles in inflammation as well as kidney homeostasis. Recent studies suggest that TonEBP expression is also involved in differentiation of several cell types such as myocytes, chondrocytes, and osteocytes. In this study, we investigated the roles of TonEBP during adipocyte differentiation in 3T3-L1 cells. TonEBP mRNA and protein expression was dramatically reduced during adipocyte differentiation. Sustained expression of TonEBP using an adenovirus suppressed the formation of lipid droplets as well as the expression of FABP4, a marker of differentiated adipocytes. TonEBP also inhibited the expression of PPARγ, a known master regulator of adipocytes. RNAi-mediated knock down of TonEBP promoted adipocyte differentiation. However, overexpression of TonEBP did not affect adipogenesis after the initiation of differentiation. Furthermore, TonEBP expression suppressed mitotic clonal expansion and insulin signaling, which are required early for adipocyte differentiation of 3T3-L1 cells. These results suggest that TonEBP may be an important regulatory factor in the early phase of adipocyte differentiation.

Sfmbt2 10th intron-hosted miR-466(a/e)-3p are important epigenetic regulators of Nfat5 signaling, osmoregulation and urine concentration in mice.

Sfmbt2-hosted miR-466a-3p and its close relatives are often among the most significantly up-regulated or down-regulated miRNAs in responses to numerous deleterious environmental stimuli. The exact roles of these miRNAs in cellular stress responses, however, are not clear. Here we showed that many Sfmbt2-hosted miRNAs were highly hypertonic stress responsive in vitro and in vivo. In renal medulla, water deprivation induced alterations in the expression of miR-466(a/b/c/e/p)-3p in a pattern similar to that of miR-200b-3p, a known regulator of osmoresponsive transcription factor Nfat5. Remarkably, exposure of mIMCD3 cells to an arginine vasopressin analog time-dependently down-regulated the expression of miR-466(a/b/c/e/p)-3p and miR-200b-3p, which provides a novel regulatory mechanism for these osmoresponsive miRNAs. In cultured mIMCD3 cells we further demonstrated that miR-466a-3p and miR-466g were capable of targeting Nfat5 by interacting with its 3'UTR. In transgenic mice overexpressing miR-466a-3p, significant down-regulation of Nfat5 and many other osmoregulation-related genes was observed in both the renal cortex and medulla. Moreover, sustained transgenic over-expression of miR-466a-3p was found to be associated with polydipsia, polyuria and disturbed ion homeostasis and kidney morphology. Since the mature sequence of miR-466a-3p is completely equivalent to that of miR-466e-3p and that the seed sequence of miR-466a-3p is completely equivalent to that of miR-297(a/b/c)-3p, miR-466d-3p, miR-467g and miR-669d-3p, and that miR-466a-3p differs from miR-466(b/c/p)-3p only in a 5' nucleotide, we propose that miR-466a-3p and many of its close relatives are important epigenetic regulators of renal Nfat5 signaling, osmoregulation and urine concentration in mice.

Tonicity-responsive enhancer binding protein regulates the expression of aldose reductase and protein kinase C δ in a mouse model of diabetic retinopathy.

Recent studies revealed that Tonicity-responsive enhancer binding protein (TonEBP) directly regulates the transcription of aldose reductase (AR), which catalyzes the first step of the polyol pathway of glucose metabolism. Activation of protein kinase C δ (PKCδ) is dependent on AR and it has been linked to diabetic complications. However, whether TonEBP affects expressions of AR and PKCδ in diabetic retinopathy was not clearly shown. In this study, we used TonEBP heterozygote mice to study the role of TonEBP in streptozotocin (STZ)-induced diabetic retinopathy. We performed immunofluorescence staining and found that retinal expressions of AR and PKCδ were significantly reduced in the heterozygotes compared to wild type littermates, particularly in ganglion cell layer. To examine further the effect of TonEBP reduction in retinal tissues, we performed intravitreal injection of TonEBP siRNA and confirmed the decrease in AR and PKCδ levels. In addition, we found that a proapoptotic factor, Bax level was reduced and a survival factor, Bcl2 level was increased after injection of TonEBP siRNA, indicating that TonEBP mediates apoptotic cell death. In parallel, TonEBP siRNA was applied to the in vitro human retinal pigment epithelial (ARPE-19) cells cultured in high glucose media. We have consistently found the decrease in AR and PKCδ levels and changes in apoptotic factors for survival. Together, these results clearly demonstrated that hyperglycemia-induced TonEBP plays a crucial role in increasing AR and PKCδ levels and leading to apoptotic death. Our findings suggest that TonEBP reduction is an effective therapeutic strategy for diabetic retinopathy.

Macrophage-induced Expression of TonEBP/NFAT5 Is Associated With Gefitinib Resistance and Migration in PC-9 Cells.

BACKGROUND/AIM: Macrophages prevail in the microenvironment of several tumors, including non-small-cell lung cancer (NSCLC), where they secrete pro-tumorigenic factors that contribute to cancer progression. This study investigated the role of macrophages on the resistance of epidermal growth factor receptor (EGFR)-mutated NSCLC cells to tyrosine kinase inhibitors (TKIs). MATERIALS AND METHODS: EGFR-mutated cell lines PC-9 and HCC827 were cocultured with macrophages and treated with TKIs (erlotinib and gefitinib). The effects of the macrophage-conditioned medium (macrophage CM) on gefitinib resistance and cell migration were also evaluated. RESULTS: Co-culture with macrophages significantly enhanced the resistance to erlotinib and gefitinib in PC-9 and HCC827 cells compared to that in cells cultured independently. Macrophage CM markedly induced gefitinib resistance in PC-9 cells, with maximum resistance observed at 50% CM concentration. This resistance persisted for up to 48 h post-CM removal. Macrophage CM inhibited gefitinib-induced apoptosis, as evidenced by the decreased expression of cleaved caspase-3, PARP, and BIM. Additionally, macrophage CM increased the migration ability of PC-9 cells, as shown by the wound healing and transwell migration assays. Studies have shown that TonEBP is crucial in cancer metastasis and drug resistance; we found that inhibition of TonEBP/NFAT5 expression reduced gefitinib resistance and migration in macrophage CM-induced PC-9 cells, indicating its role as mediator of these effects. CONCLUSION: Macrophages contribute to TKI resistance and enhance the migration of EGFR-mutated NSCLC cells through mechanisms involving TonEBP/NFAT5. Therefore, targeting TonEBP/NFAT5 represents a potential therapeutic strategy for overcoming macrophage-induced TKI resistance in NSCLC cells.

TonEBP/NFAT5 expression is associated with cisplatin resistance and migration in macrophage-induced A549 cells.

BACKGROUND: Macrophages promote angiogenesis, metastasis, and drug resistance in several cancers. Similarly, TonEBP/NFAT5 induces metastasis in renal carcinoma and colon cancer cells. However, the role of this transcription factor and that of macrophages in lung cancer cells remains unclear. Therefore, this study investigated the effects of macrophages and TonEBP/NFAT5 expression on cisplatin resistance and migration in A549 lung adenocarcinoma cells. RESULTS: A549 cells were cultured alone or indirectly co-cultured with THP-1-derived macrophages using a transwell culture chamber. Cisplatin-induced cell death was markedly decreased and migration increased in co-cultured A549 cells. Macrophage-conditioned media (CM) showed a similar effect on drug resistance and migration. Cisplatin-induced apoptosis, DNA fragmentation, and cleaved apoptotic proteins PARP and caspase-3 were markedly reduced in macrophage CM-induced A549 cells. Here, ERK, p38, JNK, and NF-κB activities were increased by macrophage CM. Furthermore, the proteins involved in cisplatin resistance and cancer cell migration were identified using specific inhibitors of each protein. ERK and NF-κB inhibition considerably reduced cisplatin resistance. The increase in macrophage CM-induced migration was partially reduced by treatment with ERK, JNK, and NF-κB inhibitors. TonEBP/NFAT5 expression was increased by macrophages, resulting in increased cisplatin resistance, cell migration, and invasion. Moreover, RNAi-mediated knockdown of TonEBP/NFAT5 reduced cisplatin resistance, migration, and invasion in macrophage CM-induced A549 cells. CONCLUSIONS: These findings demonstrate that paracrine factors secreted from macrophages can change A549 cells, resulting in the induction of drug resistance against cisplatin and migration. In addition, the TonEBP/NFAT5 ratio, increased by macrophages, is an important regulator of the malignant transformation of cells.

Water and Electrolyte Content in Salt-Dependent HYpertension in the SKIn (WHYSKI): Effect of Surgical Cure of Primary Aldosteronism.

 INTRODUCTION: This study will test the hypothesis that primary aldosteronism (PA) involves alterations in Na+, K+, and water content in the skin that are corrected by adrenalectomy. AIM AND METHODS: In skin biopsies, we will measure the content of Na+, K+, water, by physical-chemical methods and the osmotic-stress-responsive transcription factor Tonicity-responsive Enhancer Binding Protein (TonEBP, NFAT5) mRNA copy number by droplet digital PCR, in sex-balanced cohorts of 18 -75-year-old consecutive consenting patients with unilateral and bilateral PA, primary (essential) hypertension, and normotension. Before surgery, the patients with unilateral PA will receive the mineralocorticoid receptor antagonist (MRA) canrenone at doses that correct hypokalemia and high blood pressure values. They will be reassessed in an identical way one month after surgical cure, while off MRA. PA patients not selected for adrenalectomy will similarly be assessed at diagnosis and follow-up while on stable MRA treatment. Since a pilot study showed a direct correlation of dry weight (DW) with skin electrolytes and water content and significant differences of biopsy DW between surgery and follow-up, meaningful comparison of the skin cations and water content and TonEBP mRNA copy number, between specimen obtained at different time points, will require DW- and total mRNA-adjustment, respectively. CONCLUSION: This study will provide novel information on the skin Na+, K+ and water content in PA, the paradigm of salt-dependent hypertension, and novel knowledge on the effect of surgical cure of hyperaldosteronism. The TonEBP-mediated regulation of Na+, K+ and water content in the skin will also be unveiled. TRAIL REGISTRY: Trial Registration number: NCT06090617. Date of Registration: 2023-10-19.

The roles of TonEBP in the DNA damage response: From DNA damage bypass to R-loop resolution.

Tonicity-responsive enhancer binding protein (TonEBP) is a stress-responsive protein that plays a critical role in the regulation of gene expression and cellular adaptation to stressful environments. Recent studies uncovered the novel role of TonEBP in the DNA damage response, which significantly impacts genomic stability. This review provides a comprehensive overview of the novel role of TonEBP in DNA damage repair, including its involvement in the DNA damage bypass pathway and the recognition and resolution of DNA damage-induced R-loop structures.