Pharmacological characterization of the zebrafish Hrh2a histamine H2 receptor.

The zebrafish, Danio rerio, is a widely adopted in vivo model that conserves organs such as the liver, kidney, stomach, and brain, being, therefore, suitable for studying human diseases, drug discovery and toxicology. The brain aminergic systems are also conserved and the histamine H1, H2 and H3 receptors were previously cloned and identified in the zebrafish brain. Genome studies identified another putative H2 receptor (Hrh2) with ∼50% sequence identity with H2 receptor orthologs. In this study, we recombinantly expressed both zebrafish H2 receptor paralogs (hrh2a and hrh2b) and compared their pharmacology with the human H2 receptor ortholog. Our results showed that both zebrafish receptors conserve all the class A GPCR motifs. However, in contrast with the Hrh2a paralog, the Hrh2b does not possess all the amino acid residues shown to participate in histamine binding. The zebrafish Hrh2a receptor displays high affinity for [3H]-tiotidine with a binding profile for H2 receptor ligands similar to that of the human H2 receptor. The zebrafish Hrh2a receptor couples to GαS and Gαq/11 proteins, resulting in cAMP accumulation and activation of several reporter genes linked to the Gαq/11 pathway. Additionally, this receptor shows high constitutive activity, with histamine potency in the low nanomolar range for cAMP accumulation and the micromolar range for the activation of the NFAT response element. Moreover, dimaprit and amthamine seem to preferentially activate GαS over Gαq/11 proteins via the zebrafish Hrh2a receptor. These results can contribute to clarifying the functional roles of the H2 receptor in zebrafish.

SHP2 mediates the ROS/JNK/NFAT4 signaling pathway in gastric cancer cells prompting lncRNA SNHG18 to drive gastric cancer growth and metastasis via CAR-T cells.

OBJECTIVE: In gastric cancer cells, the influence of CAR T cells can be produced in the process of inhibiting the progression of gastric cancer, and the role of tyrosine phosphatase SHP2 can be explored in this study, along with its molecular mechanisms. METHODS: The research utilized subcutaneous tumor models in nude mice to assess gastric cancer progression. Protein expression was detected using Western blotting, while Q-PCR examined the expression levels of lncRNA SNHG18 and miR-211-5p in MGC-803 cells. The relationship between miR-211-5p and lncRNA SNHG18 can be analyzed by dual luciferase reporter genes. The migratory ability of MGC-803 cells was determined through wound healing and transwell experiments, and cell proliferation was evaluated using a CCK-8 assay. RESULTS: SHP2 was found to inhibit the cytotoxic effects of CAR-T cells on MGC-803 cells, and it suppressed the expression of proteins related to the ROS/JNK/NFAT4 signaling pathway in MGC-803 cells and the miR-211-5p/BRD4 axis in CAR-T cells. In addition, the proliferation, invasion and migration of MGC-803 cells were promoted, and the expression of miR-211-5p could be inhibited specifically by ncRNA SNHG18, as shown below:SHP2 in gastric cancer cells mediates the ROS/JNK/NFAT4 signaling pathway and induces lncRNA SNHG18, which, through the miR-211-5p/BRD4 axis in CAR-T cells, promotes gastric cancer growth and metastasis.

Trifolirhizin protects ovariectomy-induced bone loss in mice by inhibiting osteoclast differentiation and bone resorption.

Background: Osteoporosis is a debilitating condition characterized by reduced bone density and microstructure, leading to increased susceptibility to fractures and increased mortality, particularly among older individuals. Despite the availability of drugs for osteoporosis treatment, the need for targeted and innovative agents with fewer adverse effects persists. Trifolirhizin, a natural pterostalin derived from the root of Sophora flavescens, has been previously studied for its effects on certain anticancer and antiinflammatory. The impact of trifolirhizin on the formation and function of osteoclasts remain unclear. Purpose: Herein, the possible roles of trifolirhizin the formation and function of osteoclasts and the underlying mechanism were explored. Methods: Bone marrow-derived macrophages (BMMs) were employed to evaluate the roles of trifolirhizin on steoclastogenesis, bone absorption and the underlying mechanism in vitro. Bone loss model was established by ovariectomy(OVX) in mice in vivo. Results: Trifolirhizin repressed osteoclastogenesis, bone resorption induced by receptor activator of nuclear factor kappa B ligand (RANKL) in vitro. Mechanistically, trifolirhizin inhibits RANKL-induced MAPK signal transduction and NFATc1 expression. Moreover, trifolirhizin inhibited osteoclast marker gene expression, including NFATc1, CTSK, MMP9, DC-STAMP, ACP5, and V-ATPase-D2. Additionally, trifolirhizin was found to protect against ovariectomy(OVX)-induced bone loss in mice. Conclusion: Trifolirhizin can effectively inhibit osteoclast production and bone resorption activity. The results of our study provide evidence for trifolirhizin as a potential drug for the prevention and treatment of osteoporosis and other osteolytic diseases.

Prolonged extracellular low sodium concentrations and subsequent their rapid correction modulate nitric oxide production dependent on NFAT5 in microglia.

Hyponatremia is the most common clinical electrolyte disorder. Chronic hyponatremia has been recently reported to be associated with falls, fracture, osteoporosis, neurocognitive impairment, and mental manifestations. In the treatment of chronic hyponatremia, overly rapid correction of hyponatremia can cause osmotic demyelination syndrome (ODS), a central demyelinating disease that is also associated with neurological morbidity and mortality. Using a rat model, we have previously shown that microglia play a critical role in the pathogenesis of ODS. However, the direct effect of rapid correction of hyponatremia on microglia is unknown. Furthermore, the effect of chronic hyponatremia on microglia remains elusive. Using microglial cell lines BV-2 and 6-3, we show here that low extracellular sodium concentrations (36 mmol/L decrease; LS) suppress Nos2 mRNA expression and nitric oxide (NO) production of microglia. On rapid correction of low sodium concentrations, NO production was significantly increased in both cells, suggesting that acute correction of hyponatremia partly directly contributes to increased Nos2 mRNA expression and NO release in ODS pathophysiology. LS also suppressed expression and nuclear translocation of nuclear factor of activated T cells-5 (NFAT5), a transcription factor that regulates the expression of genes involved in osmotic stress. Furthermore, overexpression of NFAT5 significantly increased Nos2 mRNA expression and NO production in BV-2 cells. Expressions of Nos2 and Nfat5 mRNA were also modulated in microglia isolated from cerebral cortex in chronic hyponatremia model mice. These data indicate that LS modulates microglial NO production dependent on NFAT5 and suggest that microglia contribute to hyponatremia-induced neuronal dysfunctions.

Nuclear factor of activated T-cells 5 is indispensable for a balanced adaptive transcriptional response of lung endothelial cells to hypoxia.

AIMS: Chronic hypoxia causes detrimental structural alterations in the lung, which may cause pulmonary hypertension and are partially mediated by the endothelium. While its relevance for the development of hypoxia-associated lung diseases is well known, determinants controlling the initial adaptation of the lung endothelium to hypoxia remain largely unexplored. METHODS AND RESULTS: We revealed that hypoxia activates the transcription factor nuclear factor of activated T-cells 5 (NFAT5) and studied its regulatory function in murine lung endothelial cells (MLECs). EC-specific knockout of Nfat5 (Nfat5(EC)-/-) in mice exposed to normobaric hypoxia (10% O2) for 21 days promoted vascular fibrosis and aggravated the increase in pulmonary right ventricular systolic pressure as well as right ventricular dysfunction as compared with control mice. Microarray- and single-cell RNA-sequencing-based analyses revealed an impaired growth factor-, energy-, and protein-metabolism-associated gene expression in Nfat5-deficient MLEC after exposure to hypoxia for 7 days. Specifically, loss of NFAT5 boosted the expression and release of platelet-derived growth factor B (Pdgfb)-a hypoxia-inducible factor 1 alpha (HIF1α)-regulated driver of vascular smooth muscle cell (VSMC) growth-in capillary MLEC of hypoxia-exposed Nfat5(EC)-/- mice, which was accompanied by intensified VSMC coverage of distal pulmonary arteries. CONCLUSION: Collectively, our study shows that early and transient subpopulation-specific responses of MLEC to hypoxia may determine the degree of organ dysfunction in later stages. In this context, NFAT5 acts as a protective transcription factor required to rapidly adjust the endothelial transcriptome to cope with hypoxia. Specifically, NFAT5 restricts HIF1α-mediated Pdgfb expression and consequently limits muscularization and resistance of the pulmonary vasculature.

Shifting phenotype and differentiation of CD11b+Gr.1+ immature heterogeneous myeloid derived adjuster cells support inflammation and induce regulators of IL17A in imiquimod induced psoriasis.

OBJECTIVE AND DESIGN: The exact immunological mechanism of widespread chronic inflammatory skin disorder psoriasis has not been fully established. CD11b+Gr.1+ myeloid-derived cells are immature heterogeneous cells with T-cell suppressive property in neoplasia; however, influence of these cells on adaptive immunity is highly contextual; therefore, we dubbed these cells as myeloid-derived adjuster cells (MDAC). We studied imiquimod induced psoriasis in mouse model and evaluated for the first time the RORγt-NFAT1 axis in MDACs and the function, differentiation and interaction of these cells with T cells. MATERIALS AND METHODS: The status of T cells and MDACs; their functionality and differentiation properties, and the roles of RORγt and NFAT1 in MDACs were evaluated using flow cytometry, qRT-PCR and confocal imaging. RESULTS: We found gradual increase in T cells and MDACs and an increase in the number of IL17 -secreting MDACs and T cells in the skin of psoriatic animals. We also noted that MDAC differentiation is biased toward M1 macrophages and DCs which perpetuate inflammation. We found that psoriatic MDACs were unable to suppress T-cell proliferation or activation but seemingly helped these T cells produce more IL17. Inhibition of the RORγt/NFAT1 axis in MDACs increased the suppressive nature of MDACs, allowing these cells to suppress the activity of psoriatic T-cells. CONCLUSION: Our results indicate that altered MDAC properties in psoriatic condition sustains pathological inflammation and RORγt and NFAT1 as promising intervention target for psoriasis management.

NFAT3-FasL axis synchronously regulates apoptosis and necroptosis in murine cochlear outer hair cells after noise trauma.

Existing studies have indicated that noise induces apoptosis and necroptosis in cochlear outer hair cells (OHCs). However, the role of the extrinsic cell death pathway, initiated by death ligands in the cochlea, remains unknown. In this study, we hypothesized that noise could induce the NFAT3/FasL axis-mediated extrinsic death pathway in the cochlea. We found that NFAT3/FasL signaling was silent in normal OHCs. Noise exposure induced apoptosis and necroptosis in OHCs with specifically high FasL expression. Multiplex immunofluorescence staining revealed that NFAT3 nuclear translocation and FasL upregulation were colocalized in the apoptotic and necroptotic OHCs following noise trauma. Administration of FK506 or 11R-vivit (an specific NFAT inhibitor) blocked NFAT3 nuclear translocation, inhibited FasL expression, mitigated apoptosis and necroptosis, and protected against noise-induced hearing loss (NIHL). Finally, FasL knockdown by delivering siRNA intratympanically attenuated apoptosis and necroptosis in OHCs and alleviated NIHL, confirming the role of FasL in OHC death. Collectively, our study demonstrates that the NFAT3/FasL axis mediates noise-induced extrinsic death pathway in OHCs, leading to their apoptosis and necroptosis.

Transcriptional upregulation of the myo-inositol biosynthesis pathway is enhanced by NFAT5 in hyperosmotically stressed tilapia cells.

Euryhaline fish experience variable osmotic environments requiring physiological adjustments to tolerate elevated salinity. Mozambique tilapia (Oreochromis mossambicus) possess one of the highest salinity tolerance limits of any fish. In tilapia and other euryhaline fish species, the myo-inositol biosynthesis (MIB) pathway enzymes, myo-inositol phosphate synthase (MIPS) and inositol monophosphatase 1 (IMPA1.1), are among the most upregulated mRNAs and proteins indicating the high importance of this pathway for hyperosmotic (HO) stress tolerance. These abundance changes must be precluded by HO perception and signaling mechanism activation to regulate the expression of MIPS and IMPA1.1 genes. In previous work using a O. mossambicus cell line (OmB), a reoccurring osmosensitive enhancer element (OSRE1) in both MIPS and IMPA1.1 was shown to transcriptionally upregulate these enzymes in response to HO stress. The OSRE1 core consensus (5'-GGAAA-3') matches the core binding sequence of the predominant mammalian HO response transcription factor, nuclear factor of activated T-cells (NFAT5). HO-challenged OmB cells showed an increase in NFAT5 mRNA suggesting NFAT5 may contribute to MIB pathway regulation in euryhaline fish. Ectopic expression of wild-type NFAT5 induced an IMPA1.1 promoter-driven reporter by 5.1-fold (P < 0.01). Moreover, expression of dominant negative NFAT5 in HO media resulted in a 47% suppression of the reporter signal (P < 0.005). Furthermore, reductions of IMPA1.1 (37-49%) and MIPS (6-37%) mRNA abundance were observed in HO-challenged NFAT5 knockout cells relative to control cells. Collectively, these multiple lines of experimental evidence establish NFAT5 as a tilapia transcription factor contributing to HO-induced activation of the MIB pathway.NEW & NOTEWORTHY In our study, we use a multi-pronged synthetic biology approach to demonstrate that the fish homolog of the predominant mammalian osmotic stress transcription factor nuclear factor of activated T-cells (NFAT5) also contributes to the activation of hyperosmolality inducible genes in cells of extremely euryhaline fish. However, in addition to NFAT5 the presence of other strong osmotically inducible signaling mechanisms is required for full activation of osmoregulated tilapia genes.

Ephrin type-A receptor 2-antisense RNA1/2 promote proliferation and migration of MDA-MB-231 cells through EPHA2-dependent Ras signaling pathway mediated by MAPK8/JNK1, MAPK9/JNK2-NFATC2/NFAT1 and JUND.

Ephrin type-A receptor 2 (EPHA2) is a receptor tyrosine kinase that is overexpressed in a variety of cancers, including breast cancer. EPHA2 expression may be causally related to tumorigenesis; therefore, it is important to understand how EPHA2 expression is regulated. We previously reported that EPHA2 antisense RNA (EPHA2-AS), a natural antisense transcript, is an important modulator of EPHA2 mRNA levels and hence production of EPHA2 protein. EPHA2-AS encodes two splice variants, EPHA2-AS1 and EPHA2-AS2. The two variants are constitutively expressed in a concordant manner with EPHA2 mRNA in human breast adenocarcinoma cell lines and in patient samples, with the highest levels detected in the basal-like/triple-negative molecular subtype of breast cancer cells. In this study, we investigated the mechanism of EPHA2-AS1/2 in triple-negative breast cancer using MDA-MB-231 cells. We performed RNA-seq transcriptome analyses of MDA-MB-231 cells treated with AHCC®, which suppressed expression of EPHA2-AS1/2 and EPHA2 mRNA, and EPHA2-AS1/2-silenced MDA-MB-231 cells. Bioinformatics analyses identified 545 overlapping differentially expressed genes that were significantly up- or down-regulated by these treatments. Subsequent functional enrichment analyses of the overlapping genes in combination with in vitro assays indicated that EPHA2-AS1/2 may promote the proliferation and migration of MDA-MB-231 cells through the EPHA2-dependent Ras signaling pathways mediated by MAPK8/JNK1, MAPK9/JNK2-NFATC2/NFAT1 (proliferation and migration) and JUND (migration). These results thus suggest that EPHA2-AS1/2 may represent a potential molecular target for triple-negative breast cancer treatment.

Structure-activity relationship studies of pyrogallol as a calcineurin/NFAT signaling suppressor.

Previously, we have shown that pyrogallol alleviated nasal symptoms and suppressed IL-9 gene up-regulation in allergy model rats by inhibiting calcineurin/NFAT signaling. As pyrogallol has antioxidative activity, it may be responsible for inhibiting calcineurin/NFAT signaling-mediated IL-9 gene expression. However, the relationship between antioxidative activity and suppression of IL-9 gene expression has not been elucidated yet. Here, we conducted the structure-activity relationship studies of pyrogallol and its structurally related compounds to understand the mechanism of IL-9 gene suppression by pyrogallol. 2, 2-Diphenyl-1-picrylhydrazyl radical scavenging assay showed that the antioxidative activity of catechol, resorcinol, phloroglucinol, and gallic acid is 60.1%, 10.4%, 18.8%, and 113.5% of pyrogallol, respectively. Catechol, resorcinol, and phloroglucinol did not suppress NFAT dephosphorylation. Gallic acid suppressed dephosphorylation of NFAT. Gallic acid also suppressed ionomycin-induced up-regulation of IL-9 gene expression with the IC50 value of 82.6 µM. However, catechol, resorcinol and phloroglucinol showed no suppressive activity. In addition, using gallic acid-immobilized beads, we isolated and identified Poly(U)-binding-splicing factor 60 (PUF60) as a pyrogallol binding protein. These results suggest that the antioxidative activity of pyrogallol is not likely to be the mechanism of IL-9 gene suppression. Data also suggest that PUF60 is one of its target molecules responsible for the suppression of calcineurin/NFAT signaling by pyrogallol.

Molecular Pathways and Animal Models of Tricuspid Atresia and Univentricular Heart.

The process of valve formation is a complex process that involves intricate interplay between various pathways at precise times. Although we have not completely elucidated the molecular pathways that lead to normal valve formation, we have identified a few major players in this process. We are now able to implicate TGF-ß, BMP, and NOTCH as suspects in tricuspid atresia (TA), as well as their downstream targets: NKX2-5, TBX5, NFATC1, GATA4, and SOX9. We know that the TGF-ß and the BMP pathways converge on the SMAD4 molecule, and we believe that this molecule plays a very important role to tie both pathways to TA. Similarly, we look at the NOTCH pathway and identify the HEY2 as a potential link between this pathway and TA. Another transcription factor that has been implicated in TA is NFATC1. While several mouse models exist that include part of the TA abnormality as their phenotype, no true mouse model can be said to represent TA. Bridging this gap will surely shed light on this complex molecular pathway and allow for better understanding of the disease process.

Harnessing Metformin's Immunomodulatory Effects on Immune Cells to Combat Breast Cancer.

Metformin, a medication known for its anti-glycemic properties, also demonstrates potent immune system activation. In our study, using a 4T1 breast cancer model in BALB/C WT mice, we examined metformin's impact on the functional phenotype of multiple immune cells, with a specific emphasis on natural killer T (NKT) cells due to their understudied role in this context. Metformin administration delayed the appearance and growth of carcinoma. Furthermore, metformin increased the percentage of IFN-γ+ NKT cells, and enhanced CD107a expression, as measured by MFI, while decreasing PD-1+, FoxP3+, and IL-10+ NKT cells in spleens of metformin-treated mice. In primary tumors, metformin increased the percentage of NKp46+ NKT cells and increased FasL expression, while lowering the percentages of FoxP3+, PD-1+, and IL-10-producing NKT cells and KLRG1 expression. Activation markers increased, and immunosuppressive markers declined in T cells from both the spleen and tumors. Furthermore, metformin decreased IL-10+ and FoxP3+ Tregs, along with Gr-1+ myeloid-derived suppressor cells (MDSCs) in spleens, and in tumor tissue, it decreased IL-10+ and FoxP3+ Tregs, Gr-1+, NF-κB+, and iNOS+ MDSCs, and iNOS+ dendritic cells (DCs), while increasing the DCs quantity. Additionally, increased expression levels of MIP1a, STAT4, and NFAT in splenocytes were found. These comprehensive findings illustrate metformin's broad immunomodulatory impact across a variety of immune cells, including stimulating NKT cells and T cells, while inhibiting Tregs and MDSCs. This dynamic modulation may potentiate its use in cancer immunotherapy, highlighting its potential to modulate the tumor microenvironment across a spectrum of immune cell types.

Heart-specific NFAT5 knockout suppresses type I interferon signaling and aggravates coxsackievirus-induced myocarditis.

Nuclear factor of activated T cells 5 (NFAT5) is an osmosensitive transcription factor that is well-studied in renal but rarely explored in cardiac diseases. Although the association of Coxsackievirus B3 (CVB3) with viral myocarditis is well-established, the role of NFAT5 in this disease remains largely unexplored. Previous research has demonstrated that NFAT5 restricts CVB3 replication yet is susceptible to cleavage by CVB3 proteases. Using an inducible cardiac-specific Nfat5-knockout mouse model, we uncovered that NFAT5-deficiency exacerbates cardiac pathology, worsens cardiac function, elevates viral load, and reduces survival rates. RNA-seq analysis of CVB3-infected mouse hearts revealed the significant impact of NFAT5-deficiency on gene pathways associated with cytokine signaling and inflammation. Subsequent in vitro and in vivo investigation validated the disruption of the cytokine signaling pathway in response to CVB3 infection, evidenced by reduced expression of key cytokines such as interferon ß1 (IFNß1), C-X-C motif chemokine ligand 10 (CXCL10), interleukin 6 (IL6), among others. Furthermore, NFAT5-deficiency hindered the formation of stress granules, leading to a reduction of important stress granule components, including plakophilin-2, a pivotal protein within the intercalated disc, thereby impacting cardiomyocyte structure and function. These findings unveil a novel mechanism by which NFAT5 inhibits CVB3 replication and pathogenesis through the promotion of antiviral type I interferon signaling and the formation of cytoplasmic stress granules, collectively identifying NFAT5 as a new cardio protective protein.

IRF4 impedes human CD8 T cell function and promotes cell proliferation and PD-1 expression.

Human CD8 tumor-infiltrating lymphocytes (TILs) with impaired effector functions and PD-1 expression are categorized as exhausted. However, the exhaustion-like features reported in TILs might stem from their activation rather than the consequence of T cell exhaustion itself. Using CRISPR-Cas9 and lentiviral overexpression in CD8 T cells from non-cancerous donors, we show that the T cell receptor (TCR)-induced transcription factor interferon regulatory factor 4 (IRF4) promotes cell proliferation and PD-1 expression and hampers effector functions and expression of nuclear factor κB (NF-κB)-regulated genes. While CD8 TILs with impaired interferon γ (IFNγ) production exhibit activation markers IRF4 and CD137 and exhaustion markers thymocyte selection associated high mobility group box (TOX) and PD-1, activated T cells in patients with COVID-19 do not demonstrate elevated levels of TOX and PD-1. These results confirm that IRF4+ TILs are exhausted rather than solely activated. Our study indicates, however, that PD-1 expression, low IFNγ production, and active cycling in TILs are all influenced by IRF4 upregulation after T cell activation.

Chromatin accessibility and epigenetic deoxyribose nucleic acid (DNA) modifications in chronic kidney disease (CKD) osteoblasts: a study of bone and osteoblasts from pediatric patients with CKD.

Maturation defects are intrinsic features of osteoblast lineage cells in CKD patients. These defects persist ex vivo, suggesting that CKD induces epigenetic changes in bone cells. To gain insights into which signaling pathways contribute to CKD-mediated, epigenetically driven, impairments in osteoblast maturation, we characterized RNA expression and DNA methylation patterns by RNA-Seq and MethylationEpic in primary osteoblasts from nine adolescent and young adult dialysis patients with end-stage kidney disease and three healthy references. ATAC-Seq was also performed on a subset of osteoblasts. Bone matrix protein expression was extracted from the iliac crest and evaluated by proteomics. Gene set enrichment analysis was used to establish signaling pathways consistently altered in chromatin accessibility, DNA methylation, and RNA expression patterns. Single genes were suppressed in primary osteoblasts using shRNA and mineralization characterized in vitro. The effect of nuclear factor of activated T cells (NFAT) signaling suppression was also assessed using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) incorporation. We found that signaling pathways critical for osteoblast differentiation were strongly downregulated in CKD osteoblasts. Gene set enrichment analysis identified highly significant methylation changes, differential chromatin accessibility, and altered RNA expression in NFAT signaling targets. NFAT inhibition reduced osteoblast proliferation. Combined analysis of osteoblast RNA expression and whole bone matrix composition identified 13 potential ligand-receptor pairs. In summary, epigenetic changes in CKD osteoblasts associate with altered expression of multiple osteoblast genes and signaling pathways. An increase in NFAT signaling may play a role in impaired CKD osteoblast maturation. Epigenetic changes also associate with an altered bone matrix, which may contribute to bone fragility. Further studies are necessary to elucidate the pathways affected by these genetic alterations since elucidating these pathways will be vital to correcting the underlying biology of bone disease in the CKD population.

LncRNA AP000842.3 Triggers the Malignant Progression of Prostate Cancer by Regulating Cuproptosis Related Gene NFAT5.

OBJECTIVES: Prostate cancer (PRAD) is a highly malignant disease with poor prognosis, and its development is regulated by a complex network of genes and signaling pathways. LncRNAs and miRNAs have significant regulatory roles in PRAD through the ceRNA network. Cuproptosis is a unique form of programmed cell death that is involved in various signaling pathways and biological processes related to tumor development. Nuclear factor of activated T cells 5 (NFAT5), a transcription factor that activates T cells, has been implicated in cuproptosis. However, the regulatory mechanism by which NFAT5 is involved in the ceRNA network in PRAD remains unclear. METHODS: Through bioinformatics analysis, we found the ceRNA axis that regulates cuproptosis. By performing ROS assay and copper ion concentration assay, we demonstrated that inhibiting NFAT5 can increase the sensitivity of PRAD to cuproptosis inducers. By using luciferase assay, we discovered that AP000842.3 acts as the ceRNA of miR-206 to regulate the expression of NFAT5. RESULTS: In this study, we found that lncRNA AP000842.3, as a ceRNA of miR-206, was involved in the regulation of levels of the transcription factor NFAT5 associated with cuproptosis in PRAD. First, knocking down NFAT5 can increase the sensitivity of PRAD to cuproptosis inducers. Meanwhile, changes in the expression of AP000842.3 and miR-206 could affect the proliferation of PRAD by regulating NFAT5. Mechanistically, AP000842.3 acts as the ceRNA of miR-206 to regulate the expression of NFAT5. In addition, the effects of lncRNA AP000842.3 on malignant progression of PRAD and NFAT5 were partially dependent on miR-206. CONCLUSION: Taken together, our study reveals a key ceRNA regulatory network in PRAD and can be regarded as a new potential target for PRAD diagnosis and treatment.

Dysregulated Wnt and NFAT signaling in a Parkinson's disease LRRK2 G2019S knock-in model.

Parkinson's disease (PD) is a progressive late-onset neurodegenerative disease leading to physical and cognitive decline. Mutations of leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of PD. LRRK2 is a complex scaffolding protein with known regulatory roles in multiple molecular pathways. Two prominent examples of LRRK2-modulated pathways are Wingless/Int (Wnt) and nuclear factor of activated T-cells (NFAT) signaling. Both are well described key regulators of immune and nervous system development as well as maturation. The aim of this study was to establish the physiological and pathogenic role of LRRK2 in Wnt and NFAT signaling in the brain, as well as the potential contribution of the non-canonical Wnt/Calcium pathway. In vivo cerebral Wnt and NFATc1 signaling activity was quantified in LRRK2 G2019S mutant knock-in (KI) and LRRK2 knockout (KO) male and female mice with repeated measures over 28 weeks, employing lentiviral luciferase biosensors, and analyzed using a mixed-effect model. To establish spatial resolution, we investigated tissues, and primary neuronal cell cultures from different brain regions combining luciferase signaling activity, immunohistochemistry, qPCR and western blot assays. Results were analyzed by unpaired t-test with Welch's correction or 2-way ANOVA with post hoc corrections. In vivo Wnt signaling activity in LRRK2 KO and LRRK2 G2019S KI mice was increased significantly ~ threefold, with a more pronounced effect in males (~ fourfold) than females (~ twofold). NFATc1 signaling was reduced ~ 0.5-fold in LRRK2 G2019S KI mice. Brain tissue analysis showed region-specific expression changes in Wnt and NFAT signaling components. These effects were predominantly observed at the protein level in the striatum and cerebral cortex of LRRK2 KI mice. Primary neuronal cell culture analysis showed significant genotype-dependent alterations in Wnt and NFATc1 signaling under basal and stimulated conditions. Wnt and NFATc1 signaling was primarily dysregulated in cortical and hippocampal neurons respectively. Our study further built on knowledge of LRRK2 as a Wnt and NFAT signaling protein. We identified complex changes in neuronal models of LRRK2 PD, suggesting a role for mutant LRRK2 in the dysregulation of NFAT, and canonical and non-canonical Wnt signaling.

Differentiation and immunosuppressive function of CD19+CD24hiCD27+ regulatory B cells are regulated through the miR-29a-3p/NFAT5 pathway.

BACKGROUND: Regulatory B cells (Bregs) is an indispensable element in inducing immune tolerance after liver transplantation. As one of the microRNAs (miRNAs), miR-29a-3p also inhibits translation by degrading the target mRNA, and yet the relationship between Bregs and miR-29a-3p has not yet been fully explored. This study aimed to investigate the impact of miR-29a-3p on the regulation of differentiation and immunosuppressive functions of memory Bregs (mBregs) and ultimately provide potentially effective therapies in inducing immune tolerance after liver transplantation. METHODS: Flow cytometry was employed to determine the levels of Bregs in peripheral blood mononuclear cells. TaqMan low-density array miRNA assays were used to identify the expression of different miRNAs, electroporation transfection was used to induce miR-29a-3p overexpression and knockdown, and dual luciferase reporter assay was used to verify the target gene of miR-29a-3p. RESULTS: In patients experiencing acute rejection after liver transplantation, the proportions and immunosuppressive function of mBregs in the circulating blood were significantly impaired. miR-29a-3p was found to be a regulator of mBregs differentiation. Inhibition of miR-29a-3p, which targeted nuclear factor of activated T cells 5 (NFAT5), resulted in a conspicuous boost in the differentiation and immunosuppressive function of mBregs. The inhibition of miR-29a-3p in CD19+ B cells was capable of raising the expression levels of NFAT5, thereby promoting B cells to differentiate into mBregs. In addition, the observed enhancement of differentiation and immunosuppressive function of mBregs upon miR-29a-3p inhibition was abolished by the knockdown of NFAT5 in B cells. CONCLUSIONS: miR-29a-3p was found to be a crucial regulator for mBregs differentiation and immunosuppressive function. Silencing miR-29a-3p could be a potentially effective therapeutic strategy for inducing immune tolerance after liver transplantation.

Calcineurin-NFAT signaling controls neutrophils´ ability of chemoattraction upon fungal infection.

Calcineurin - nuclear factor of activated T-cell (CN-NFAT) inhibitors are widely clinically used drugs for immunosuppression but besides their required T-cell response inhibition, they also undesirably affect innate immune cells. Disruption of innate immune cell function can explain the observed susceptibility of CN-NFAT inhibitors-treated patients to opportunistic fungal infections. Neutrophils play an essential role in innate immunity as a defense against pathogens, however, the effect of CN-NFAT inhibitors on neutrophil function was poorly described. Thus, we tested the response of human neutrophils to opportunistic fungal pathogens, namely Candida albicans and Aspergillus fumigatus in the presence of CN-NFAT inhibitors. Here, we report that the NFAT pathway members were expressed in neutrophils and mediated part of the neutrophil response to pathogens. Upon pathogen exposure, neutrophils underwent profound transcriptomic changes with subsequent production of effector molecules. Importantly, genes and proteins involved in the regulation of the immune response and chemotaxis, including the chemokines CCL2, CCL3, and CCL4 were significantly upregulated. The presence of CN-NFAT inhibitors attenuated the expression of these chemokines and impaired the ability of neutrophils to chemoattract other immune cells. Our results amend knowledge about the impact of CN-NFAT inhibition in human neutrophils.

Fluid shear force and hydrostatic pressure jointly promote osteogenic differentiation of BMSCs by activating YAP1 and NFAT2.

Natural bone tissue features a complex mechanical environment, with cells responding to diverse mechanical stimuli, including fluid shear stress (FSS) and hydrostatic pressure (HP). However, current in vitro experiments commonly employ a singular mechanical stimulus to simulate the mechanical environment in vivo. The understanding of the combined effects and mechanisms of multiple mechanical stimuli remains limited. Hence, this study constructed a mechanical stimulation device capable of simultaneously applying FSS and HP to cells. This study investigated the impact of FSS and HP on the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and examined the distinctions and interactions between the two mechanisms. The results demonstrated that both FSS and HP individually enhanced the osteogenic differentiation of BMSCs, with a more pronounced effect observed through their combined application. BMSCs responded to external FSS and HP stimulation through the integrin-cytoskeleton and Piezo1 ion channel respectively. This led to the activation of downstream biochemical signals, resulting in the dephosphorylation and nuclear translocation of the intracellular transcription factors Yes Associated Protein 1 (YAP1) and nuclear factor of activated T cells 2 (NFAT2). Activated YAP1 could bind to NFAT2 to enhance transcriptional activity, thereby promoting osteogenic differentiation of BMSCs more effectively. This study highlights the significance of composite mechanical stimulation in BMSCs' osteogenic differentiation, offering guidance for establishing a complex mechanical environment for in vitro functional bone tissue construction.