Nuclear Factor Of Activated T Research Articles

The Structure Analysis and mRNA Expression of CaV2 Gene Responding to Hypoxia Stress in Anadara granosa

The blood clam (Anadara granosa) is an economic bivalve that is relatively tolerant to hypoxia, but its molecular mechanism of hypoxia tolerance is unclear. We found that a significant decrease in extracellular Ca2+ concentration and a marked increase in intracellular Ca2+ concentration was observed in the blood clam through the fluorescence probe method, under hypoxic conditions at 0.5 mg/L. Concomitantly, there was a downward trend in the expression level of CaV2 mRNA, whereas NFAT (nuclear factor of activated T cells) expression increased by qRT-PCR. These findings suggest that the elevated intracellular Ca2+ concentration may activate negative transcription factors of NFAT, which subsequently suppresses the transcription of CaV2, leading to its decreased expression. Then, the NFAT RNA interference experiments supported this hypothesis. Sequence analysis and 3D structure prediction revealed conserved and mutated residue sites in blood clam compared to other bivalves. Hypoxia-induced changes in intracellular and extracellular Ca2+ concentrations, activating transcription factor NFAT and suppressing CaV2 expression. This study highlights the key roles of CaV2 and NFAT in hypoxia adaptation, paving the way for further exploration of hypoxia tolerance mechanisms in mollusca.

The expression of NFAT family genes in subcutaneous adipose tissue before and after weight loss in obese individuals

Background and aimsAdipose tissue (AT) serves as a vital energy storage site and plays a pivotal role in metabolic regulation, exhibiting a high response to insulin. Impairment in this response may closely associate with obesity, and NFAT (nuclear factor of activated T cells) family genes may be involved in the process. However, human data linking NFAT and AT remains elusive. The aim of this study was to assess the expression of NFAT family genes and markers of adipogenesis in subcutaneous adipose tissue (SAT) among normal-weight and overweight/obese individuals before and after weight loss, in relation to insulin sensitivity. Methods and resultsThe study included 45 participants, 15 normal-weight (control group) and 30 overweight or obese, who underwent a 12-week dietary intervention (DI) program. Before and after the program hyperinsulinemic-euglycemic clamp and SAT biopsy were conducted. Before DI, a positive correlations was observed in the expression of NFATc1, NFATc4, and NFAT5 with insulin sensitivity. The expression of NFAT family genes and markers of adipogenesis in SAT was lower in individuals with overweight or obesity compared to normal-weight. Additionally, a positive correlation was noted between NFAT family genes and adipogenesis markers both before and after weight loss. Following the DI program, there was an increase in the expression of NFATc3, NFATc4, and NFAT5 in SAT. ConclusionDecreased SAT expression of NFAT genes in obesity is partly reversed in response to weight loss. NFAT genes in SAT are associated with insulin sensitivity and adipogenesis.Registration number for clinical trial: NCT01393210.

G protein subunit alpha i2's pivotal role in angiogenesis.

Here we explored the potential role of Gαi2 (G protein subunit alpha i2) in endothelial cell function and angiogenesis. Methods: Genetic methodologies such as shRNA, CRISPR/Cas9, dominant negative mutation, and overexpression were utilized to modify Gαi2 expression or regulate its function. Their effects on endothelial cell functions were assessed in vitro. In vivo, the endothelial-specific Gαi2 shRNA adeno-associated virus (AAV) was utilized to silence Gαi2 expression. The impact of this suppression on retinal angiogenesis in control mice and streptozotocin (STZ)-induced diabetic retinopathy (DR) mice was analyzed. Results: Analysis of single-cell RNA sequencing data revealed Gαi2 (GNAI2) was predominantly expressed in retinal endothelial cells and expression was increased in retinal endothelial cells following oxygen-induced retinopathy (OIR) in mice. Moreover, transcriptome analysis linking Gαi2 to angiogenesis-related processes/pathways, supported by increased Gαi2 expression in experimental OIR mouse retinas, highlighted its possible role in angiogenesis. In various endothelial cell types, shRNA-induced silencing and CRISPR/Cas9-mediated knockout (KO) of Gαi2 resulted in substantial reductions in cell proliferation, migration, invasion, and capillary tube formation. Conversely, Gαi2 over-expression in endothelial cells induced pro-angiogenic activities, enhancing cell proliferation, migration, invasion, and capillary tube formation. Furthermore, our investigation revealed a crucial role of Gαi2 in NFAT (nuclear factor of activated T cells) activation, as evidenced by the down-regulation of NFAT-luciferase reporter activity and pro-angiogenesis NFAT-targeted genes (Egr3, CXCR7, and RND1) in Gαi2-silenced or -KO HUVECs, which were up-regulated in Gαi2-overexpressing endothelial cells. Expression of a dominant negative Gαi2 mutation (S48C) also down-regulated NFAT-targeted genes, slowing proliferation, migration, invasion, and capillary tube formation in HUVECs. Importantly, in vivo experiments revealed that endothelial Gαi2 knockdown inhibited retinal angiogenesis in mice, with a concomitant down-regulation of NFAT-targeted genes in mouse retinal tissue. In contrast, Gαi2 over-expression in endothelial cells enhanced retinal angiogenesis in mice. Single-cell RNA sequencing data confirmed increased levels of Gαi2 specifically in retinal endothelial cells of mice with streptozotocin (STZ)-induced diabetic retinopathy (DR). Importantly, endothelial Gαi2 silencing ameliorated retinal pathological angiogenesis in DR mice. Conclusion: Our study highlights a critical role for Gαi2 in NFAT activation, endothelial cell activation and angiogenesis, offering valuable insights into potential therapeutic strategies for modulating these processes.

Sustained store-operated calcium entry utilizing activated chromatin state leads to instability in iTregs.

Thymus-originated tTregs and in vitro induced iTregs are subsets of regulatory T cells. While they share the capacity of immune suppression, their stabilities are different, with iTregs losing their phenotype upon stimulation or under inflammatory milieu. Epigenetic differences, particularly methylation state of Foxp3 CNS2 region, provide an explanation for this shift. Whether additional regulations, including cellular signaling, could directly lead phenotypical instability requires further analysis. Here, we show that upon TCR (T cell receptor) triggering, SOCE (store-operated calcium entry) and NFAT (nuclear factor of activated T cells) nuclear translocation are blunted in tTregs, yet fully operational in iTregs, similar to Tconvs. On the other hand, tTregs show minimal changes in their chromatin accessibility upon activation, in contrast to iTregs that demonstrate an activated chromatin state with highly accessible T cell activation and inflammation related genes. Assisted by several cofactors, NFAT driven by strong SOCE signaling in iTregs preferentially binds to primed-opened T helper (TH) genes, resulting in their activation normally observed only in Tconv activation, ultimately leads to instability. Conversely, suppression of SOCE in iTregs can partially rescue their phenotype. Thus, our study adds two new layers, cellular signaling and chromatin accessibility, of understanding in Treg stability, and may provide a path for better clinical applications of Treg cell therapy.

Crosstalk between NFAT and NFκB Signaling Regulates NK Cell Immunosurveillance

One of the major transcriptional regulators in lymphoid cells is NFAT (Nuclear Factor of Activated T Cells), controlling lymphocyte development and activity. The role of NFAT signaling is well defined in T cells, the cytotoxic lymphocytes of the adaptive immunity. However, surprisingly little is known regarding the relevance of this transcription factor family in NK cells as effector cells of the innate immunity. Available data indicate that NFAT activity is dispensable for development of NK cells, whereas effects of the immunosuppressive drugs cyclosporin A and tacrolimus, that inhibit calcineurin and consecutively NFAT, implicate an involvement of the NFAT family in NK cell function. We here employed different genetic mouse models and functional analyses to unravel the role of NFAT1 (NFATc2) and NFAT2 (NFATc1) in NK cell reactivity. In vitro knockout (KO) of NFAT1 (NFATc2) or NFAT2 (NFATc1) enhanced NK cell degranulation and resulted in increased production of granzyme B and perforin upon stimulation of activating receptors like NK1.1 or Nkp46 or upon co-culture with different leukemia and solid tumor cells. In line, cytotoxicity assays revealed increased lysis of YAC-1 and B16F10 tumor cells by both NFAT1- and NFAT2-deficient NK cells as compared to wildtype (WT) controls. The inhibitory effect of NFAT transcription factors on NK cell effector function could also be confirmed in vivo by employing WT and NFAT KO animals in syngeneic B16F10 melanoma, RMA-S lymphoma and LL/2-luc lung carcinoma models, which revealed a significantly reduced tumor burden in NFAT1 and NFAT2 KO mice. Furthermore, we detected higher numbers of tumor infiltrating NK cells in NFAT KO mice, which suggested improved NK cell migration and homing to the tumor side. Comparative analyses with single NFAT as well as NFAT1+NFAT2 double KO and WT animals further confirmed the inhibitory effect of NFAT1 and NFAT2 and pointed to additive effects of NFAT1 and NFAT2 in NK cell tumor immunosurveillance. Proteomics analysis of NK cells from the different NFAT KO strains revealed altered expression of molecules which are regulating chemokine receptor expression as well as a decreased expression of NFκB inhibitory molecules, like NFKBIB (NFKB inhibitor beta) Commd6 (COMM domain containing 6). NFκB signaling was shown to regulate granzyme B and perforin synthesis and expression. Thus, we suggest that NFAT1 and NFAT2 inhibit NFκB signaling in NK cells, and NFAT1 and NFAT2 KO reinforce NFκB induced granzyme B and perforin expression and thereby increase NK cell cytotoxicity. Taken together, our results identify NFAT as a negative regulator of NK cell function. In addition, we provide the first evidence for a direct functional involvement of NFAT1 and NFAT2 in NK cell antitumor reactivity by regulation tumor infiltration and cytotoxicity of NK cell.

Role of APR3 in cancer: apoptosis, autophagy, oxidative stress, and cancer therapy.

APR3 (Apoptosis-related protein 3) is a gene that has recently been identified to be associated with apoptosis. The gene is located on human chromosome 2p22.3 and contains both transmembrane and EGF (epidermal growth factor)-like domains. Additionally, it has structural sites, including AP1, SP1, and MEF2D, that indicate NFAT (nuclear factor of activated T cells) and NF-κB (nuclear factor kappa-B) may be transcription factors for this gene. Functionally, APR3 participates in apoptosis due to the induction of mitochondrial damage to release mitochondrial cytochrome C. Concurrently, APR3 affects the cell cycle by altering the expression of Cyclin D1, which, in turn, affects the incidence and growth of malignancies and promotes cell differentiation. Previous reports indicate that APR3 is located in lysosomal membranes, where it contributes to lysosomal activity and participates in autophagy. While further research is required to determine the precise role and molecular mechanisms of APR3, earlier studies have laid the groundwork for APR3 research. There is growing evidence supporting the significance of APR3 in oncology. Therefore, this review aims to examine the current state of knowledge on the role of the newly discovered APR3 in tumorigenesis and to generate fresh insights and suggestions for future research.

The activation of CaN/NFAT signaling pathway in macrophages aggravated Lactobacillus casei cell wall extract-induced Kawasaki disease vasculitis

ObjectivesBy using GWAS(genome-wide association studies) and linkage disequilibrium analysis to investigate the susceptibility genes of KD(Kawasaki disease), previous studies have identified that the CaN(calcineurin)-NFAT(the nuclear factor of activated T cell) signal pathway were significantly associated with susceptibility to KD. However, little is known about the molecular basis of the CaN/NFAT pathway involved in KD. Therefore, in our study we investigate the role of Ca2+/CaN/NFAT signaling pathway in macrophages in vitro and in vivo on coronary artery lesions induced by LCWE (Lactobacillus casei cell wall extract). Methods and resultsWe observed that LCWE could increase the expression of NFAT1 and NFAT2 in macrophages in vitro, and also enhance the transcriptional activity of NFAT by promoting the nucleus translocation. Similarly, in LCWE-induced mice model, the expression of NFAT1 and NFAT2 and associated proinflammatory factors were increased significantly. In addition, by knocking down or overexpressing NFAT1 or NFAT2 in macrophages, the results indicated that NFAT signaling pathway mediated LCWE-induced immune responses in macrophages and regulated the synthesis of IL(interleukin)-6, IL-1β and TNF(tumor necrosis factor)-α in LCWE-induced macrophage activation. As well, we found that this process could be suppressed by CaN inhibitor CsA(cyclosporinA). ConclusionsTherefore, the CaN/NFAT signaling pathway mediated LCWE-induced immune responses in macrophages, and also participated in the LCWE-induced CALs(coronary artery lesions). And also the inhibitory effect of CsA in LCWE-induced cell model towards a strategy to modulate the CaN/NFAT pathway during the acute course of KD might be helpful in alleviate KD-induced CALs.

The role of FOXO4/NFAT2 signaling pathway in dysfunction of human coronary endothelial cells and inflammatory infiltration of vasculitis in Kawasaki disease.

The Ca+/NFAT (Nuclear factor of activated T cells) signaling pathway activation is implicated in the pathogenesis of Kawasaki disease (KD); however, we lack detailed information regarding the regulatory network involved in the human coronary endothelial cell dysfunction and cardiovascular lesion development. Herein, we aimed to use mouse and endothelial cell models of KD vasculitis in vivo and in vitro to characterize the regulatory network of NFAT pathway in KD. Among the NFAT gene family, NFAT2 showed the strongest transcriptional activity in peripheral blood mononuclear cells (PBMCs) from patients with KD. Then, NFAT2 overexpression and knockdown experiments in Human coronary artery endothelial cells (HCAECs) indicated that NFAT2 overexpression disrupted endothelial cell homeostasis by regulation of adherens junctions, whereas its knockdown protected HCAECs from such dysfunction. Combined analysis using RNA-sequencing and transcription factor (TF) binding site analysis in the NFAT2 promoter region predicted regulation by Forkhead box O4 (FOXO4). Western blotting, chromatin immunoprecipitation, and luciferase assays validated that FOXO4 binds to the promoter and transcriptionally represses NFAT2. Moreover, Foxo4 knockout increased the extent of inflamed vascular tissues in a mouse model of KD vasculitis. Functional experiments showed that inhibition NFAT2 relieved Foxo4 knockout exaggerated vasculitis in vivo. Our findings revealed the FOXO4/NFAT2 axis as a vital pathway in the progression of KD that is associated with endothelial cell homeostasis and cardiovascular inflammation development.

Orai1 Inhibitors as Potential Treatments for Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is characterized by progressive distal pulmonary artery (PA) obstruction, leading to right ventricular hypertrophy and failure. Exacerbated intracellular calcium (Ca2+) signaling contributes to abnormalities in PA smooth muscle cells (PASMCs), including aberrant proliferation, apoptosis resistance, exacerbated migration, and arterial contractility. Store-operated Ca2+ entry is involved in Ca2+ homeostasis in PASMCs, but its properties in PAH are unclear. Using a combination of Ca2+ imaging, molecular biology, in vitro, ex vivo, and in vivo approaches, we investigated the roles of the Orai1 SOC channel in PA remodeling in PAH and determined the consequences of pharmacological Orai1 inhibition in vivo using experimental models of pulmonary hypertension (PH). Store-operated Ca2+ entry and Orai1 mRNA and protein were increased in human PASMCs (hPASMCs) from patients with PAH (PAH-hPASMCs). We found that MEK1/2 (mitogen-activated protein kinase kinase 1/2), NFAT (nuclear factor of activated T cells), and NFκB (nuclear factor-kappa B) contribute to the upregulation of Orai1 expression in PAH-hPASMCs. Using small interfering RNA (siRNA) and Orai1 inhibitors, we found that Orai1 inhibition reduced store-operated Ca2+ entry, mitochondrial Ca2+ uptake, aberrant proliferation, apoptosis resistance, migration, and excessive calcineurin activity in PAH-hPASMCs. Orai1 inhibitors reduced agonist-evoked constriction in human PAs. In experimental rat models of PH evoked by chronic hypoxia, monocrotaline, or Sugen/hypoxia, administration of Orai1 inhibitors (N-{4-[3,5-bis(Trifluoromethyl)-1H-pyrazol-1-yl]phenyl}-4-methyl-1,2,3-thiadiazole-5-carboxamide [BTP2], 4-(2,5-dimethoxyphenyl)-N-[(pyridin-4-yl)methyl]aniline [JPIII], or 5J4) protected against PH. In human PAH and experimental PH, Orai1 expression and activity are increased. Orai1 inhibition normalizes the PAH-hPASMCs phenotype and attenuates PH in rat models. These results suggest that Orai1 should be considered as a relevant therapeutic target for PAH.

Chronic Alcohol Exposure Promotes Cancer Stemness and Glycolysis in Oral/Oropharyngeal Squamous Cell Carcinoma Cell Lines by Activating NFAT Signaling.

Alcohol consumption is associated with an increased risk of several cancers, including oral/oropharyngeal squamous cell carcinoma (OSCC). Alcohol also enhances the progression and aggressiveness of existing cancers; however, its underlying molecular mechanism remains elusive. Especially, the local carcinogenic effects of alcohol on OSCC in closest contact with ingestion of alcohol are poorly understood. We demonstrated that chronic ethanol exposure to OSCC increased cancer stem cell (CSC) populations and their stemness features, including self-renewal capacity, expression of stem cell markers, ALDH activity, and migration ability. The ethanol exposure also led to a significant increase in aerobic glycolysis. Moreover, increased aerobic glycolytic activity was required to support the stemness phenotype of ethanol-exposed OSCC, suggesting a molecular coupling between cancer stemness and metabolic reprogramming. We further demonstrated that chronic ethanol exposure activated NFAT (nuclear factor of activated T cells) signaling in OSCC. Functional studies revealed that pharmacological and genetic inhibition of NFAT suppressed CSC phenotype and aerobic glycolysis in ethanol-exposed OSCC. Collectively, chronic ethanol exposure promotes cancer stemness and aerobic glycolysis via activation of NFAT signaling. Our study provides a novel insight into the roles of cancer stemness and metabolic reprogramming in the molecular mechanism of alcohol-mediated carcinogenesis.

Abstract 2104: NK cell immunosurveillance of tumors is regulated by NFAT1 and NFAT2

Abstract NK cells are lymphoid components of innate immunity and play an important role in tumor immunosurveillance. One of the major transcriptional regulators in lymphoid cells is NFAT (Nuclear Factor of Activated T cells), controlling lymphocyte development and activity. However, while the role of NFAT signaling is well defined in T cells, the cytotoxic lymphocytes of adaptive immunity, surprisingly little is known regarding the relevance of this transcription factor family in NK cells as effector cells of innate immunity. Available data indicate that NFAT activity is dispensable for development of NK cells. However, several lines of evidence including reports on the effects of the immunosuppressive drugs cyclosporin A and tacrolimus, which mediate their effects through inhibition of calcineurin and consecutively NFAT, implicate an involvement of the NFAT family in NK cell function. We here employed different genetic mouse models to unravel the role of NFAT1 (NFATc2) and NFAT2 (NFATc1) in NK cell reactivity. When NK cells with knockout (KO) of NFAT1 or NFAT2 compared to NK cells of control mice (WT) were employed in in vitro analyses, lack of either NFAT was found to surprisingly result in enhanced NK cell degranulation as well as increased production of granzyme B and perforin upon stimulation of activating receptors like NK1.1 or Nkp46 or upon co-culture with different leukemia and solid tumor tumor cells. In line, cytotoxicity assays revealed increased lysis of YAC-1 and B16F10 tumor cells by both NFAT1- and NFAT2-deficient NK cells as compared to WT controls. The inhibitory effect of NFAT transcription factors on NK cell effector function could also be confirmed in vivo by employing WT and NFAT KO animals in syngeneic B16F10 melanoma and RMA-S flank tumor models, which revealed a significantly reduced tumor burden in NFAT1 and NFAT2 KO mice. Comparative analyses with single NFAT as well as NFAT1+NFAT2 double KO and WT animals further confirmed the inhibitory effect of NFAT1 and NFAT2 and pointed to additive effects of NFAT1 and NFAT2 in NK cell tumor immunosurveillance. Taken together, our results provide the first evidence for the direct functional involvement of NFAT1 and NFAT2 in NK cell antitumor reactivity and, in contrast to T and B cells, identify NFAT as a negative regulator of NK cell function. Citation Format: Melanie Märklin, Samuel Holzmayer, Kübra Kaban, Martin R. Müller, Helmut R. Salih. NK cell immunosurveillance of tumors is regulated by NFAT1 and NFAT2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2104.

Delivery of the VIVIT Peptide to Human Glioma Cells to Interfere with Calcineurin-NFAT Signaling.

The activation of NFAT (nuclear factor of activated T cells) transcription factors by calcium-dependent phosphatase calcineurin is a key step in controlling T cell activation and plays a vital role during carcinogenesis. NFATs are overexpressed in many cancers, including the most common primary brain tumor, gliomas. In the present study, we demonstrate the expression of NFATs and NFAT-driven transcription in several human glioma cells. We used a VIVIT peptide for interference in calcineurin binding to NFAT via a conserved PxIxIT motif. VIVIT was expressed as a fusion protein with a green fluorescent protein (VIVIT-GFP) or conjugated to cell-penetrating peptides (CPP), Sim-2 or 11R. We analyzed the NFAT expression, phosphorylation, subcellular localization and their transcriptional activity in cells treated with peptides. Overexpression of VIVIT-GFP decreased the NFAT-driven activity and inhibited the transcription of endogenous NFAT-target genes. These effects were not reproduced with synthetic peptides: Sim2-VIVIT did not show any activity, and 11R-VIVIT did not inhibit NFAT signaling in glioma cells. The presence of two calcineurin docking sites in NFATc3 might require dual-specificity blocking peptides. The cell-penetrating peptides Sim-2 or 11R linked to VIVIT did not improve its action making it unsuitable for evaluating NFAT dependent events in glioma cells with high expression of NFATc3.

Functional analyses of STIM1 mutations reveal a common pathomechanism for tubular aggregate myopathy and Stormorken syndrome.

Tubular aggregate myopathy (TAM) is a progressive disorder characterized by muscle weakness, cramps, and myalgia. TAM clinically overlaps with Stormorken syndrome (STRMK), combining TAM with miosis, thrombocytopenia, hyposplenism, ichthyosis, short stature, and dyslexia. TAM and STRMK arise from gain-of-function mutations in STIM1 (stromal interaction molecule 1) or ORAI1, both encoding key regulators of Ca2+ homeostasis, and mutations in either gene result in excessive extracellular Ca2+ entry. The pathomechanistic similarities and differences between TAM and STRMK are only partially understood. Here we provide functional in vitro experiments demonstrating that STIM1 harboring the TAM D84G or the STRMK R304W mutation similarly cluster and exert a dominant effect on the wild-type protein. Both mutants recruit ORAI1 to the clusters, increase cytosolic Ca2+ levels, promote major nuclear import of the Ca2+ -dependent transcription factor NFAT (nuclear factor of activated T cells), and trigger the formation of circular membrane stacks. In conclusion, the analyzed TAM and STRMK mutations have a comparable impact on STIM1 protein function and downstream effects of excessive Ca2+ entry, highlighting that TAM and STRMK involve a common pathomechanism.

Aromadendrin Inhibits T Cell Activation via Regulation of Calcium Influx and NFAT Activity.

The objective of this study was to assess the inhibitory effect of the flavonoid aromadendrin on T cell activity to identify a non-cytotoxic immunosuppressive reagent. Conventional and qualitative PCR, MTT assays, flow cytometry and Western blotting were used to evaluate the effect of aromadendrin on the activity, cell viability and confluency, and proximal signal transduction of activated T cells. Aromadendrin effectively regulated IL-2 and IFNγ production in vitro from activated Jurkat T cells without cytotoxicity. Pre-treatment with aromadendrin also suppressed the expression levels of surface molecules CD69, CD25, and CD40L. Reduced calcium (Ca2+) influx in activated T cells pre-treated with aromadendrin was observed. Western blotting revealed that aromadendrin blocked the dephosphorylation of nuclear factor of activated T (NFAT) cells and its nuclear translocation. Involvement of the NFκB and MAPK pathways in the inhibitory effect of aromadendrin was also demonstrated. Results obtained demonstrated the suppressive effect of aromadendrin on T cell activation by Ca2+ influx regulation through NFAT activity suppression of the activated T cells.

Issue Cover (September 2020)

Journal of NeurochemistryVolume 154, Issue 5 Issue CoverFree Access Issue Cover (September 2020) First published: 20 August 2020 https://doi.org/10.1111/jnc.14759 Read the full article: ‘Combination of antioxidants and NFAT (nuclear factor of activated T cells) inhibitor protects auditory hair cells from ototoxic insult’ by M. Sekulic-Jablanovic, K. Voronkova, D. Bodmer, V. Petkovic, (J. Neurochem. 2020, vol. 154 (5), pp. 519–529) on doi:10.1111/jnc.14921 AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinked InRedditWechat Abstract Front cover: Auditory hair cell degeneration underlies hearing loss, and finding effective treatment has been a holy grail quest of hearing research. So far, a combination of antioxidants and other protective substances have proven successful to a limited extent. Our findings show that the combination of nuclear factor of activated T-cells (NFAT) inhibitor and N-acetylcysteine provides full protection from gentamicin damage in hair cells. The main advantage is that hair cell protection can be achieved using lower concentrations of each component, and thus decreasing their potential side effects. This approach could be beneficial as treatment not just in aminoglycoside-induced ototoxicity, but in all cases of ROS-induced cochlear damage, with noise-induced hearing loss being one of them. Image content: Representative image of green phalloidin-stained auditory hair cells of the cultured organ of Corti. Read the full article ‘Combination of antioxidants and NFAT (nuclear factor of activated T cells) inhibitor protects auditory hair cells from ototoxic insult’ by M. Sekulic-Jablanovic, K. Voronkova, D. Bodmer, V. Petkovic, (J. Neurochem. 2020, vol. 154 (5), pp. 519–529) on doi:10.1111/jnc.14921 Volume154, Issue5September 2020 RelatedInformation

RNA sequencing reveals an additional Crz1-binding motif in promoters of its target genes in the human fungal pathogen Candida albicans

BackgroundThe calcium/calcineurin signaling pathway is mediated by the transcription factors NFAT (nuclear factor of activated T cells) in mammals and Crz1 (calcineurin-responsive zinc finger 1) in yeasts and other lower eukaryotes. A previous microarray analysis identified a putative Crz1-binding motif in promoters of its target genes in Candida albicans, but it has not been experimentally demonstrated.MethodsAn inactivation mutant for CaCRZ1 was generated through CRISPR/Cas9 approach. Transcript profiling was carried out by RNA sequencing of the wild type and the inactivation mutant for CaCRZ1 in response to 0.2 M CaCl2. Gene promoters were scanned by the online MEME (Multiple Em for Motif Elicitation) software. Gel electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) analysis were used for in vitro and in vivo CaCrz1-binding experiments, respectively.ResultsRNA sequencing reveals that expression of 219 genes is positively, and expression of 59 genes is negatively, controlled by CaCrz1 in response to calcium stress. These genes function in metabolism, cell cycling, protein fate, cellular transport, signal transduction, transcription, and cell wall biogenesis. Forty of these positively regulated 219 genes have previously been identified by DNA microarray analysis. Promoter analysis of these common 40 genes reveals a consensus motif [5′-GGAGGC(G/A)C(T/A)G-3′], which is different from the putative CaCrz1-binding motif [5′-G(C/T)GGT-3′] identified in the previous study, but similar to Saccharomyces cerevisiae ScCrz1-binding motif [5′-GNGGC(G/T)CA-3′]. EMSA and ChIP assays indicate that CaCrz1 binds in vitro and in vivo to both motifs in the promoter of its target gene CaUTR2. Promoter mutagenesis demonstrates that these two CaCrz1-binding motifs play additive roles in the regulation of CaUTR2 expression. In addition, the CaCRZ1 gene is positively regulated by CaCrz1. CaCrz1 can bind in vitro and in vivo to its own promoter, suggesting an autoregulatory mechanism for CaCRZ1 expression.ConclusionsCaCrz1 differentially binds to promoters of its target genes to regulate their expression in response to calcium stress. CaCrz1 also regulates its own expression through the 5′-TGAGGGACTG-3′ site in its promoter.3QEHhwZ2kG3j3zrcs7CSeNVideo abstract

Combination of antioxidants and NFAT (nuclear factor of activated T cells) inhibitor protects auditory hair cells from ototoxic insult.

Hair cell (HC) degeneration causes hearing loss in millions of people worldwide. Aminoglycoside exposure is one major cause of sensory HC damage. Aminoglycosides generate free radicals within the inner ear, permanently damaging sensory cells, and thus causing hearing loss. Hearing protection requires strategies to overcome the apparently irreversible loss of HCs in mammals. The nuclear factor of activated T cells (NFAT) inhibitor 11R-VIVIT reportedly protects HCs from gentamicin toxicity. Here we investigated whether the combination of 11R-VIVIT with the antioxidant L-carnitine or N-acetylcysteine could protect mouse cochlear HCs from gentamicin damage. Compared to single-component treatment, combined treatment with 11R-VIVIT plus L-carnitine yielded significant protection from gentamicin, and 11R-VIVIT plus N-acetylcysteine provided almost complete protection of HCs from gentamicin. Caspase activity in organ of Corti was significantly reduced by combined treatment with 11R-VIVIT+N-acetylcysteine+gentamicin, compared to 11R-VIVIT+gentamicin or gentamicin alone. Analysis of relative gene expression by qPCR revealed down-regulation of the pro-apoptotic genes Fasl and Casp9, and up-regulation of the antioxidant genes Hmox1 and Nrf2 after treatment with 11R-VIVIT+N-acetylcysteine+gentamicin, compared to single-compound treatment or gentamicin alone in cultures. Selective NFAT inhibition by 11R-VIVIT may be a good strategy for preventing gentamicin-induced HC damage. L-carnitine and N-acetylcysteine, with their ROS-reducing properties, contribute to the synergistic effectiveness with 11R-VIVIT by decreasing ROS-induced NFAT translocation. Our data suggest that a combined approach of NFAT inhibition together with an antioxidant, like N-acetylcysteine, could be useful for hearing loss treatment and/or prevention. Cover Image for this issue: https://doi.org/10.1111/jnc.14759.

NR4A transcription factors limit CAR T cell function in solid tumours.

T cells expressing chimeric antigen receptors (CAR) targeting human CD19 (huCD19) have exhibited impressive clinical efficacy against B cell malignancies1,2. CAR-T cells have been less effective against solid tumors3–5, in part because they enter a hyporesponsive (“exhausted” or “dysfunctional”) state6–9 triggered by chronic antigen stimulation and characterized by upregulation of inhibitory receptors and loss of effector function. To investigate the function of CAR-T cells in solid tumors, we transferred huCD19-reactive CAR-T cells into huCD19+ tumor-bearing mice. CD8+ CAR+ tumor-infiltrating lymphocytes (TILs) and endogenous TILs expressing inhibitory receptors PD-1 and TIM3 exhibited similar profiles of gene expression and chromatin accessibility, associated with secondary activation of nuclear receptor transcription factors (TFs) Nr4a1 (Nur77), Nr4a2 (Nurr1) and Nr4a3 (Nor1) by the initiating TF NFAT (nuclear factor of activated T cells)10–12. CD8+ T cells from humans with cancer or chronic viral infections13,14,15 expressed high levels of Nr4a TFs and displayed enrichment of Nr4a binding motifs in accessible chromatin regions. CAR-T cells lacking all three Nr4a TFs (Nr4aTKO) promoted tumor regression and prolonged the survival of tumor-bearing mice. Nr4aTKO CAR-TILs displayed phenotypes and gene expression profiles characteristic of CD8+ effector T cells, and chromatin regions uniquely accessible in Nr4aTKO CAR-TILs compared to WT were enriched for binding motifs for NFκB and AP-1, TFs involved in T cell activation. Our data identify Nr4a TFs as major players in the cell-intrinsic program of T cell hyporesponsiveness and point to Nr4a inhibition as a promising strategy for cancer immunotherapy.

Functional Analysis of Hepatic TCF7L2 Gene Polymorphism in Glucose Metabolism by Allele-Specific CRISPR/Cas 9 Gene Editing

Background We previously demonstrated a close relevance between a single nucleotide polymorphism, rs290487 of TCF7L2 gene, in donor grafts and new-onset diabetes mellitus in liver transplant recipients. In this study, we aim to make a functional analysis of hepatic rs290487 variant in glucose metabolism homeostasis. Method We used CRISPR/Cas 9 technology to construct a variant type in rs290487 locus in PLC-PRF-5 cell line. The base Cytosine in rs290487 SNP site of TCF7L2 gene was replaced with Thymine in PLC-PRF-5 cell line. A transcriptome sequencing analysis was preformed to detect the differential expression profiles between C/C and T/T genotype PLC-PRF-5 cells. Result Compared to C/C genotype, T/T genotype PLC-PRF-5 cells showed increased glucose uptake and insulin sensitivity, and decreased glucose production (all P < 0.05). There was a significant decrease of TCF7L2 mRNA expression and protein content after C to T transition at rs290487 in PLC-PRF-5 cells (fold change = 0.61, adjusted P < 0.001). In alternative splicing analysis, C/C genotype lost expression of variant 5 and showed significantly decreased expression of variant 2 as compared to T/T genotype (both P < 0.05). mRNA-seq revealed 896 and 714 genes significantly up- and down-regulated, respectively, in T/T genotype compared with C/C according to a criteria that adjusted P value < 0.05 and fold change > 2. KEGG analysis demonstrated a very strong enrichment in metabolic pathways with 553 significantly differentially expressed genes involved. We focused on glucose metabolism and found significant up- (e.g., LEPR, GPD1, G6PC, and GYS2) and down-regulated key genes (e.g., RBP4, PDK4, HK2, and FOXA2) after C to T transition. Of note, 30 genes in drug metabolism-cytochrome P450 pathway and nuclear factor of activated T (NFAT) family (key calcineurin targets) were significantly down-regulated after C to T transition. This may affect the calcineurin inhibitors metabolism and be associated with new-onset diabetes mellitus in liver transplant recipients. Conclusion TCF7L2 gene rs290487 is a functional intronic variant and modulates hepatic glucose metabolism.

Hypoxia-Induced Mitogenic Factor Promotes Cardiac Hypertrophy via Calcium-Dependent and Hypoxia-Inducible Factor-1α Mechanisms.

HIMF (hypoxia-induced mitogenic factor/found in inflammatory zone 1/resistin like α) is a secretory and cytokine-like protein and serves as a critical stimulator of hypoxia-induced pulmonary hypertension. With a role for HIMF in heart disease unknown, we explored the possible roles for HIMF in cardiac hypertrophy by overexpressing and knocking down HIMF in cardiomyocytes and characterizing HIMF gene (himf) knockout mice. We found that HIMF mRNA and protein levels were upregulated in phenylephrine-stimulated cardiomyocyte hypertrophy and our mouse model of transverse aortic constriction-induced cardiac hypertrophy, as well as in human hearts with dilated cardiomyopathy. Furthermore, HIMF overexpression could induce cardiomyocyte hypertrophy, as characterized by elevated protein expression of hypertrophic biomarkers (ANP [atrial natriuretic peptide] and β-MHC [myosin heavy chain-β]) and increased cell-surface area compared with controls. Conversely, HIMF knockdown prevented phenylephrine-induced cardiomyocyte hypertrophy and himf ablation in knockout mice significantly attenuated transverse aortic constriction-induced hypertrophic remodeling and cardiac dysfunction. HIMF overexpression increased the cytosolic Ca2+ concentration and activated the CaN-NFAT (calcineurin-nuclear factor of activated T cell) and MAPK (mitogen-activated protein kinase) pathways; this effect could be prevented by reducing cytosolic Ca2+ concentration with L-type Ca2+ channel blocker nifedipine or inhibiting the CaSR (Ca2+ sensing receptor) with Calhex 231. Furthermore, HIMF overexpression increased HIF-1α (hypoxia-inducible factor) expression in neonatal rat ventricular myocytes, and HIMF knockout inhibited HIF-1α upregulation in transverse aortic constriction mice. Knockdown of HIF-1α attenuated HIMF-induced cardiomyocyte hypertrophy. In conclusion, HIMF has a critical role in the development of cardiac hypertrophy, and targeting HIMF may represent a potential therapeutic strategy.