Identification of pyrogallol from Awa-tea as an anti-allergic compound that suppresses nasal symptoms and IL-9 gene expression.

As the expression level of allergic disease sensitive genes are correlated with the severity of allergic symptoms, suppression of these gene expressions could be promising therapeutics. We demonstrated that protein kinase Cδ / heat shock protein 90-mediated H1R gene expression signaling and nuclear factor of activated T-cells (NFAT)-mediated IL-9 gene expression signaling are responsible for the pathogenesis of pollinosis. Treatment with Awa-tea combined with wild grape hot water extract suppressed these signaling and alleviated nasal symptoms in toluene-2,4-diisocyanate (TDI)-sensitized rats. However, the underlying mechanism of its anti-allergic activity is not elucidated yet. Here, we sought to identify an anti-allergic compound from Awa-tea and pyrogallol was identified as an active compound. Pyrogallol strongly suppressed ionomycin-induced up-regulation of IL-9 gene expression in RBL-2H3 cells. Treatment with pyrogallol in combination with epinastine alleviated nasal symptoms and suppressed up-regulation of IL-9 gene expression in TDI-sensitized rats. Pyrogallol itself did not inhibit calcineurin phosphatase activity. However, pyrogallol suppressed ionomycin-induced dephosphorylation and nuclear translocation of NFAT. These data suggest pyrogallol is an anti-allergic compound in Awa-tea and it suppressed NFAT-mediated IL-9 gene expression through the inhibition of dephosphorylation of NFAT. This might be the underlying mechanism of the therapeutic effects of combined therapy of pyrogallol with antihistamine. J. Med. Invest. 67 : 289-297, August, 2020.

Beneficial effect of 2'-acetylacteoside on ovariectomized mice via modulating the function of bone resorption.

2'-Acetylacteoside-(2'-AA), a bioactive constituent isolated from Cistanche deserticola, has been proven to possess a variety of important pharmacological effects, thus brought an increased amount of scientists' attention. As the extract of C. deserticola exhibited significant anti-osteoporotic bioactivity in our previous study, we proposed that 2'-AA maybe one of the responsibilities. As a result, 2'-AA (10, 20 and 40 mg/kg body weight/day) exhibited significant anti-osteoporotic effects on ovariectomized (OVX) mice after 12 weeks of oral administration, confirmed by the increased bone mineral density, enhanced bone strength and improved trabecular bone micro-architecture including bone mineral content, tissue mineral content, trabecular number, and trabecular separation of OVX mice. Moreover, the properties of bone resorption markers including cathepsin K, TRAP and deoxypyridinoline were significantly suppressed, whereas the activities of bone formation index like ALP and BGP as well as the weights of the body, uterus, and vagina were seemingly not influenced by 2'-AA intervention. Mechanistically, the above therapeutic effect of 2'-AA on bone resorption of OVX mice operated maybe mainly through RANKL/RANK/TRAF6-mediated NF-κB/NFATc1 pathway, which was confirmed by the down-regulated expressions of RANK, TRAF6, IκB kinase ß, NF-κB and NFATc1. Summarily, 2'-AA exhibited significant anti-osteoporotic activity and may be regarded as a promising anti-osteoporotic candidate for future clinical trial.

Blocking NFATc3 ameliorates azoxymethane/dextran sulfate sodium induced colitis-associated colorectal cancer in mice via the inhibition of inflammatory responses and epithelial-mesenchymal transition.

Ulcerative colitis-associated colorectal cancer (UC-CRC) is the most serious complication of ulcerative colitis (UC). Nuclear factor of activated T cells 3 (NFATc3) is participated in inflammation and cancer. In this study, we investigated the effects of NFATc3 on experimental UC-CRC in vivo and in vitro, and explored the underlying mechanisms. Administration of azoxymethane (AOM) and dextran sulfate sodium (DSS) induced UC-CRC model in C57BL/6 mice. Body weight was monitored weekly. Colon tissues were harvested at week 14. We examined changes in the histopathology, inflammatory cytokines, carcinogenesis factors, and epithelial-mesenchymal transition (EMT) markers in colon tissues. We found that NFATc3 expression was significantly up-regulated in AOM/DSS treated mice compared with control. Mice lacking NFATc3 showed decreased tumor number and size, decreased mucosal damage, and increased survival rate. Moreover, down-regulation of NFATc3 could inhibit the proliferation and EMT of UC-CRC, decrease the levels of pro-inflammatory cytokines, reduce the colonic infiltration by neutrophils and macrophages, and suppress the activation of P38 and JNK signal pathway in mice. In In vitro experiments, silencing NFATc3 suppressed the proliferation and EMT of CRC cells, and reduced the activation of P38 and JNK. In addition, miR-370-3p could bind to NFATc3. Down-regulation of miR-370-3p promoted proliferation and EMT of CRC cells, while silencing NFATc3 could reverse these effects. In conclusion, NFATc3 was involved in the pathogenesis of experimental UC-CRC and NFATc3 knockdown ameliorated experimental UC-CRC progression via the inhibition of inflammatory responses and EMT. NFATc3 mediated the inhibitory effects of miR-370-3p on CRC cells proliferation and EMT. Targeting NFATc3 may be effective in treating UC-CRC.

The evolving role of TonEBP as an immunometabolic stress protein.

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

Nfatc1 Promotes Interstitial Cell Formation During Cardiac Valve Development in Zebrafish.

RATIONALE: The transcription factor NFATC1 (nuclear factor of activated T-cell 1) has been implicated in cardiac valve formation in humans and mice, but we know little about the underlying mechanisms. To gain mechanistic understanding of cardiac valve formation at single-cell resolution and insights into the role of NFATC1 in this process, we used the zebrafish model as it offers unique attributes for live imaging and facile genetics. OBJECTIVE: To understand the role of Nfatc1 in cardiac valve formation. METHODS AND RESULTS: Using the zebrafish atrioventricular valve, we focus on the valve interstitial cells (VICs), which confer biomechanical strength to the cardiac valve leaflets. We find that initially atrioventricular endocardial cells migrate collectively into the cardiac jelly to form a bilayered structure; subsequently, the cells that led this migration invade the ECM (extracellular matrix) between the 2 endocardial cell monolayers, undergo endothelial-to-mesenchymal transition as marked by loss of intercellular adhesion, and differentiate into VICs. These cells proliferate and are joined by a few neural crest-derived cells. VIC expansion and a switch from a promigratory to an elastic ECM drive valve leaflet elongation. Functional analysis of Nfatc1 reveals its requirement during VIC development. Zebrafish nfatc1 mutants form significantly fewer VICs due to reduced proliferation and impaired recruitment of endocardial and neural crest cells during the early stages of VIC development. With high-speed microscopy and echocardiography, we show that reduced VIC formation correlates with valvular dysfunction and severe retrograde blood flow that persist into adulthood. Analysis of downstream effectors reveals that Nfatc1 promotes the expression of twist1b-a well-known regulator of epithelial-to-mesenchymal transition. CONCLUSIONS: Our study sheds light on the function of Nfatc1 in zebrafish cardiac valve development and reveals its role in VIC formation. It also further establishes the zebrafish as a powerful model to carry out longitudinal studies of valve formation and function.

Genetic loss of NFAT2 (NFATc1) impairs B cell development of B1 and B2 B cells.

NFAT2 activity was shown to be of critical importance in B cell receptor signaling, development and proliferation; however its role in B cell development in the periphery is still not completely understood. We confirmed that NFAT2 deletion leads to impaired B1 B cell development, supported by our finding of limited B1 progenitors in the bone marrow and spleen of NFAT2 deficient mice. Moreover, we show for the first time that loss of NFAT2 increases immature B cells in particular transitional T2 and T3 as well as mature follicular B cells while marginal zone B cells are decreased. We further demonstrate that NFAT2 regulates the expression of B220, CD23, CD38, IgM/IgD and ZAP70 in murine B cells. In vivo analyses revealed decreased proliferation and increased apoptosis of NFAT2 deficient B cells. In summary, this study provides an extensive analysis of the role of NFAT2 in peripheral B lymphocyte development.

Dehydrocostus lactone inhibits NFATc1 via regulation of IKK, JNK, and Nrf2, thereby attenuating osteoclastogenesis.

Excessive and hyperactive osteoclast activity causes bone diseases such as osteoporosis and periodontitis. Thus, the regulation of osteoclast differentiation has clinical implications. We recently reported that dehydrocostus lactone (DL) inhibits osteoclast differentiation by regulating a nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), but the underlying mechanism remains to be elucidated. Here we demonstrated that DL inhibits NFATc1 by regulating nuclear factor-κB (NF-κB), activator protein-1 (AP-1), and nuclear factor-erythroid 2- related factor 2 (Nrf2). DL attenuated IκBα phosphorylation and p65 nuclear translocation as well as decreased the expression of NF-κB target genes and c-Fos. It also inhibited c-Jun N-terminal kinase (JNK) but not p38 or extracellular signalregulated kinase. The reporter assay revealed that DL inhibits NF-κB and AP-1 activation. In addition, DL reduced reactive oxygen species either by scavenging them or by activating Nrf2. The DL inhibition of NFATc1 expression and osteoclast differentiation was less effective in Nrf2-deficient cells. Collectively, these results suggest that DL regulates NFATc1 by inhibiting NF-κB and AP-1 via down-regulation of IκB kinase and JNK as well as by activating Nrf2, and thereby attenuates osteoclast differentiation. [BMB Reports 2020; 53(4): 218-222].

Osmolarity and calcium regulate connective tissue growth factor (CTGF/CCN2) expression in nucleus pulposus cells.

OBJECTIVE: The objective of our study was to verify the hypothesis that the expression of connective tissue growth factor (CTGF/CCN2), a key molecule essential for the maintenance of nucleus pulposus (NP) matrix homeostasis, is regulated by osmolarity and intracellular calcium in NP cells. METHODS: Gene and protein expression levels of CCN2 were assessed using quantitative real-time PCR and western blot. Transfections and dual luciferase assays were performed to measure the effect of hyperosmolarity, tonicity enhancer binding protein (TonEBP) and Ca2+-calcineurin (Cn)-NFAT signaling on CCN2 promoter activity. RESULTS: Cultured in hyperosmotic media, there was a significant decrease in the levels of CCN2 promoter activity, gene and protein expression in NP cells. The JASPAR database was used to analyze the construction of human CCN2 promoter, we found conserved TonE and NFAT binding sites. We then investigated whether TonEBP controlled CCN2 expression. Forced expression of TonEBP in NP cells showed that TonEBP negatively regulated CCN2 promoter activity, while suppression of TonEBP induced CCN2 promoter activity and expression. We then examined if Ca2+-Cn-NFAT signaling participated in the regulation of CCN2 expression. Co-expression of CCN2 reporter with individual NFAT1-4 expression plasmids and/or calcineurin A/B constructs suggested this signaling pathway played a role in the regulation of CCN2expression in NP cells. CONCLUSIONS: Results of these studies illustrated that the expression of CCN2 in NP cells was regulated by the NFAT family through a signaling pathway network involving both activator (Ca2+-Cn-NFAT signaling) and suppressor (Hyperosmolarity-TonEBP) molecules.

Down-Regulation of Nfatc1 Suppresses Proliferation, Migration, Invasion, and Warburg Effect in Prostate Cancer Cells.

BACKGROUND Prostate cancer (PCa), accounting for 28% of all male cancer cases, is the second leading cause of cancer-related death among men. NFATc1, belonging to the NFAT family, is overexpressed in PCa and is correlated with the risk of recurrence after radical prostatectomy. MATERIAL AND METHODS In the present study, the expression of NFATc, c-myc, and PKM2 in PCa cells was regulated by lentiviruses and then detected by real-time PCR and Western blot analysis. Further, proliferation, invasion, and migration assays were performed. The glucose consumption and lactate production were assessed by biochemical detection. RESULTS We found that NFATc1 down-regulation significantly suppressed the proliferation and Warburg effect of PCa cells, concurrent with a decrease of c-myc and PKM2 expression. Likewise, the abilities of migration and invasion were also inhibited in NFATc1-silenced PCa cells. In addition, NFATc1 down-regulation-induced inhibition of cell proliferation, migration, invasion, and Warburg effect were counteracted by up-regulation of c-myc or PKM2. The expression of PKM2 was positively regulated by NFATc1 and c-myc expression. CONCLUSIONS These results indicate that NFATc1 down-regulation can suppress the proliferation, Warburg effect, and migration and invasion abilities of PCa cells, probably by regulating c-myc and PKM2 expression. NFATc1 may be a potential therapeutic target for PCa and could be used as a diagnosis or prognosis indicator of PCa.

Regulation of nuclear factor of activated T cells (NFAT) and downstream myogenic proteins during dehydration in the African clawed frog.

Xenopus laevis, otherwise known as the African clawed frog, undergoes natural dehydration of up to 30% of its total body water during the dry season in sub-Saharan Africa. To survive under these conditions, a variety of physiological and biochemical changes take place in X. laevis. We were interested in understanding the role that the calcineurin-NFAT pathway plays during dehydration stress response in the skeletal muscles of X. laevis. Immunoblotting was performed to characterize the protein levels of NFATc1-4, calcium signalling proteins, in addition to myogenic proteins (MyoD, MyoG, myomaker). In addition, DNA-protein interaction ELISAs were used to assess the binding of NFATs to their consensus binding sequence, and to identify the effect of urea on NFAT-binding. Our results showed that NFATc1 and c4 protein levels decreased during dehydration, and there were no changes in NFATc2, c3, and calcium signalling proteins. However, MyoG and myomaker both showed increases in protein levels during dehydration, thus indicating that the late myogenic program involving myoblast differentiation, but not satellite cell activation and myoblast proliferation, could be involved in preserving the skeletal muscle of X. laevis during dehydration. In addition, we observed that urea seems to reduce NFATc3-binding to DNA during control, but not during dehydration, possibly indicating that NFATc3 is protected from the denaturing effects of urea as it accumulates during dehydration. These findings expand upon our knowledge of adaptive responses to dehydration, and they identify specific protein targets that could be used to protect the skeletal muscle from damage during stress.

Angiotensin-(1-7) reduces cardiac effects of thyroid hormone by GSK3Β/NFATc3 signaling pathway.

Patients with hyperthyroidism exhibit increased risk of development and progression of cardiac diseases. The activation of the renin-angiotensin system (RAS) has been indirectly implicated in these cardiac effects observed in hyperthyroidism. Angiotensin-(1-7) (Ang-(1-7)) has previously been shown to counterbalance pathological effects of angiotensin II (Ang II). The aim of the present study was to investigate the effects of elevated circulating Ang-(1-7) levels on cardiac effects promoted by hyperthyroidism in a transgenic rat (TG) model that constitutively overexpresses an Ang-(1-7)-producing fusion protein [TGR(A1-7)3292]. TG and wild-type (WT) rats received daily injections (i.p.) of triiodothyronine (T3; 7 µg/100 g of body weight (BW)) or vehicle for 14 days. In contrast with WT rats, the TG rats did not develop cardiac hypertrophy after T3 treatment. Indeed, TG rats displayed reduced systolic blood pressure (SBP) and cardiac hyperdynamic condition induced by hyperthyroidism. Moreover, increased plasma levels of Ang II observed in hyperthyroid WT rats were prevented in TG rats. TG rats were protected from glycogen synthase kinase 3ß (GSK3ß) inactivation and nuclear factor of activated T cells (NFAT) nuclear accumulation induced by T3. In vitro studies evidenced that Ang-(1-7) prevented cardiomyocyte hypertrophy and GSK3ß inactivation induced by T3. Taken together, these data reveal an important cardioprotective action of Ang-(1-7) in experimental model of hyperthyroidism.

Ethyl Acetate Fraction from Dendropanax morbifera Leaves Increases T Cell Growth by Upregulating NF-AT-Mediated IL-2 Secretion.

Dendropanax morbifera Leveille (Araliaceae) is an endemic species that grows in Southwestern Korea and has been used as a folk medicine. Several studies reported that D. morbifera leaves have diverse therapeutic potentials. We found that the water extract of D. morbifera leaves increased the growth of EL-4 T cells. The water extract was divided into five fractions: [Formula: see text]-hexane, chloroform, ethyl acetate, [Formula: see text]-butanol, and water layers. The ethyl acetate (W-EA) fraction showed a more significant effect than the other fractions on the growth of EL-4 T cells, splenocytes, and isolated murine CD4[Formula: see text] T cells. We evaluated the W-EA fraction for its immunomodulatory effects focusing on T cell functions. First, we tested the effect of the W-EA fraction on the regulation of interleukin-2 (IL-2), a potent T cell growth factor. The W-EA fraction significantly increased IL-2 secretion in EL-4 T cells activated with phorbol 12-myristate 13-acetate (PMA) and ionomycin (Io). In addition, the W-EA fraction increased interferon-gamma (IFN-[Formula: see text] production in isolated murine splenocytes activated with Concanavalin A (ConA). Next, we examined the effect of the W-EA fraction on the regulation of transcriptional factors related to IL-2 production in T cells. The W-EA fraction significantly increased PMA/Io-induced promoter activity of a nuclear factor of activated T cells (NF-AT) in EL-4 T cells, but did not show any significant effects on the promoters of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-[Formula: see text]B). These results indicate that the W-EA fraction from water extract of D. morbifera leaves enhances IL-2 production at the transcriptional levels via the up-regulation of NF-AT in PMA/Io-activated EL-4 T cells.

Induction of Short NFATc1/αA Isoform Interferes with Peripheral B Cell Differentiation.

In lymphocytes, immune receptor signals induce the rapid nuclear translocation of preformed cytosolic NFAT proteins. Along with co-stimulatory signals, persistent immune receptor signals lead to high levels of NFATc1/αA, a short NFATc1 isoform, in effector lymphocytes. Whereas NFATc1 is not expressed in plasma cells, in germinal centers numerous centrocytic B cells express nuclear NFATc1/αA. When overexpressed in chicken DT40 B cells or murine WEHI 231 B cells, NFATc1/αA suppressed their cell death induced by B cell receptor signals and affected the expression of genes controlling the germinal center reaction and plasma cell formation. Among those is the Prdm1 gene encoding Blimp-1, a key factor of plasma cell formation. By binding to a regulatory DNA element within exon 1 of the Prdm1 gene, NFATc1/αA suppresses Blimp-1 expression. Since expression of a constitutive active version of NFATc1/αA interfered with Prdm1 RNA expression, LPS-mediated differentiation of splenic B cells to plasmablasts in vitro and reduced immunoglobulin production in vivo, one may conclude that NFATc1/αA plays an important role in controlling plasmablast/plasma cell formation.

Unique properties of TCR-activated p38 are necessary for NFAT-dependent T-cell activation.

Nuclear factor of activated T cells (NFAT) transcription factors are required for induction of T-cell cytokine production and effector function. Although it is known that activation via the T-cell antigen receptor (TCR) results in 2 critical steps, calcineurin-mediated NFAT1 dephosphorylation and NFAT2 up-regulation, the molecular mechanisms underlying each are poorly understood. Here we find that T cell p38, which is activated by an alternative pathway independent of the mitogen-activated protein (MAP) kinase cascade and with different substrate specificities, directly controls these events. First, alternatively (but not classically) activated p38 was required to induce the expression of the AP-1 component c-Fos, which was necessary for NFAT2 expression and cytokine production. Second, alternatively (but not classically) activated p38 phosphorylated NFAT1 on a heretofore unidentified site, S79, and in its absence NFAT1 was unable to interact with calcineurin or migrate to the nucleus. These results demonstrate that the acquisition of unique specificities by TCR-activated p38 orchestrates NFAT-dependent T-cell functions.

[CD137 induces vascular muscle cells phenotype transformation through activating nuclear factor of activated T-cells 1 signaling].

Objective: To investigate whether CD137 induces primary vascular muscle cells (VSMCs) phenotype transformation through activating nuclear factor of activated T-cells 1(NFATc1) signaling. Methods: VSMCs were obtained from aorta of C57BL/6J mice (8 weeks, male) through tissue-piece inoculating. Cells were divided into control group, CD137 agonist group (treated with CD137L recombinant protein) and anti-CD137 group (treated with anti-CD137 antibody). In si-RNA transfection assay, cells were divided into si-control group and si-NFATc1 group which were transfected with control or si-NFATc1 sequence respectively. The levels of NFATc1 and other phenotype related protein such as α-smooth muscle actin (α-SMA), smooth muscle myosin heavy chain (SM-MHC), vimentin were detected by Q-PCR and Western blot. Nuclear protein expression and activity of NFATc1 were detected by immunofluorescence and Western blot. Transwell assay was performed to measure the migration of VSMCs. Results: According to Western blot, the expression of NFATc1 and vimentin was significantly upregulated (5.07±0.36 vs. 1.00±0.00, P<0.05; 3.23±0.27 vs. 1.00±0.00, P<0.05) while α-SMA and SM-MHC expressions was significantly downregulated (0.73±0.15 vs. 1.00±0.00, P<0.05; 0.45±0.05 vs. 1.00±0.00, P<0.05) in CD137 agonist group compare to control group. Compared with CD137 agonist group, the expression of NFATc1 and vimentin was significantly downregulated (1.56±0.27 vs. 5.07±0.36, P<0.05; 1.21±0.17 vs. 3.23±0.27, P<0.05), but the levels of α-SMA and SM-MHC were significantly upregulated (2.01±0.43 vs. 0.73±0.15, P<0.05; 2.85 ±0.32 vs. 0.45±0.05, P<0.05) in anti-CD137 group. Compared with si-con group, the expression of SM-MHC and α-SMA was significantly upregulated while the expression of vimentin was significantly downregulated in si-NFATc1 group. Transwell assay results demonstrated that migration cell numbers was significantly higher in CD137L group compared with control group(3.85±0.31 vs. 1.00±0.00, P<0.05), this effect was significantly attenuated by inhibiting NFATc1. Conclusion: CD137 could induce VSMC phenotype transformation through activating NFATc1 signaling.

Calcium-NFAT transcriptional signalling in T cell activation and T cell exhaustion.

A cornerstone of the adaptive immune response is the T cell receptor (TcR)-calcium-calcineurin signalling pathway leading to T cell activation. The 'nuclear factor of activated T cells' proteins NFAT1, NFAT2, and NFAT4 are transcription factors that promote expression of a panel of genes required for activation. It has become apparent that these same NFAT transcription factors underlie an alternative transcriptional program in T cells that serves to limit the immune response. This duality in NFAT transcriptional functions raises the possibility that NFAT transcriptional complexes could be targeted therapeutically to alter the relative strength of the effector and alternative transcriptional programs, thereby modulating immune responses.

Leukotrienes provide an NFAT-dependent signal that synergizes with IL-33 to activate ILC2s.

Group 2 innate lymphoid cells (ILC2s) and type 2 helper T cells (Th2 cells) are the primary source of interleukin 5 (IL-5) and IL-13 during type 2 (allergic) inflammation in the lung. In Th2 cells, T cell receptor (TCR) signaling activates the transcription factors nuclear factor of activated T cells (NFAT), nuclear factor κB (NF-κB), and activator protein 1 (AP-1) to induce type 2 cytokines. ILC2s lack a TCR and respond instead to locally produced cytokines such as IL-33. Although IL-33 induces AP-1 and NF-κB, NFAT signaling has not been described in ILC2s. In this study, we report a nonredundant NFAT-dependent role for lipid-derived leukotrienes (LTs) in the activation of lung ILC2s. Using cytokine reporter and LT-deficient mice, we find that complete disruption of LT signaling markedly diminishes ILC2 activation and downstream responses during type 2 inflammation. Type 2 responses are equivalently attenuated in IL-33- and LT-deficient mice, and optimal ILC2 activation reflects potent synergy between these pathways. These findings expand our understanding of ILC2 regulation and may have important implications for the treatment of airways disease.

GSK-3ß/NFAT Signaling Is Involved in Testosterone-Induced Cardiac Myocyte Hypertrophy.

Testosterone induces cardiac hypertrophy through a mechanism that involves a concerted crosstalk between cytosolic and nuclear signaling pathways. Nuclear factor of activated T-cells (NFAT) is associated with the promotion of cardiac hypertrophy, glycogen synthase kinase-3ß (GSK-3ß) is considered to function as a negative regulator, mainly by modulating NFAT activity. However, the role played by calcineurin-NFAT and GSK-3ß signaling in testosterone-induced cardiac hypertrophy has remained unknown. Here, we determined that testosterone stimulates cardiac myocyte hypertrophy through NFAT activation and GSK-3ß inhibition. Testosterone increased the activity of NFAT-luciferase (NFAT-Luc) in a time- and dose-dependent manner, with the activity peaking after 24 h of stimulation with 100 nM testosterone. NFAT-Luc activity induced by testosterone was blocked by the calcineurin inhibitors FK506 and cyclosporine A and by 11R-VIVIT, a specific peptide inhibitor of NFAT. Conversely, testosterone inhibited GSK-3ß activity as determined by increased GSK-3ß phosphorylation at Ser9 and ß-catenin protein accumulation, and also by reduction in ß-catenin phosphorylation at residues Ser33, Ser37, and Thr41. GSK-3ß inhibition with 1-azakenpaullone or a GSK-3ß-targeting siRNA increased NFAT-Luc activity, whereas overexpression of a constitutively active GSK-3ß mutant (GSK-3ßS9A) inhibited NFAT-Luc activation mediated by testosterone. Testosterone-induced cardiac myocyte hypertrophy was established by increased cardiac myocyte size and [3H]-leucine incorporation (as a measurement of cellular protein synthesis). Calcineurin-NFAT inhibition abolished and GSK-3ß inhibition promoted the hypertrophy stimulated by testosterone. GSK-3ß activation by GSK-3ßS9A blocked the increase of hypertrophic markers induced by testosterone. Moreover, inhibition of intracellular androgen receptor prevented testosterone-induced NFAT-Luc activation. Collectively, these results suggest that cardiac myocyte hypertrophy induced by testosterone involves a cooperative mechanism that links androgen signaling with the recruitment of NFAT through calcineurin activation and GSK-3ß inhibition.

Therapeutic potential of targeting acinar cell reprogramming in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a common pancreatic cancer and the fourth leading cause of cancer death in the United States. Treating this life-threatening disease remains challenging due to the lack of effective prognosis, diagnosis and therapy. Apart from pancreatic duct cells, acinar cells may also be the origin of PDAC. During pancreatitis or combined with activating KRas(G12D) mutation, acinar cells lose their cellular identity and undergo a transdifferentiation process called acinar-to-ductal-metaplasia (ADM), forming duct cells which may then transform into pancreatic intraepithelial neoplasia (PanIN) and eventually PDAC. During ADM, the activation of mitogen-activated protein kinases, Wnt, Notch and phosphatidylinositide 3-kinases/Akt signaling inhibits the transcription of acinar-specific genes, including Mist and amylase, but promotes the expression of ductal genes, such as cytokeratin-19. Inhibition of this transdifferentiation process hinders the development of PanIN and PDAC. In addition, the transdifferentiated cells regain acinar identity, indicating ADM may be a reversible process. This provides a new therapeutic direction in treating PDAC through cancer reprogramming. Many studies have already demonstrated the success of switching PanIN/PDAC back to normal cells through the use of PD325901, the expression of E47, and the knockdown of Dickkopf-3. In this review, we discuss the signaling pathways involved in ADM and the therapeutic potential of targeting reprogramming in order to treat PDAC.

Local TNF causes NFATc1-dependent cholesterol-mediated podocyte injury.

High levels of circulating TNF and its receptors, TNFR1 and TNFR2, predict the progression of diabetic kidney disease (DKD), but their contribution to organ damage in DKD remains largely unknown. Here, we investigated the function of local and systemic TNF in podocyte injury. We cultured human podocytes with sera collected from DKD patients, who displayed elevated TNF levels, and focal segmental glomerulosclerosis (FSGS) patients, whose TNF levels resembled those of healthy patients. Exogenous TNF administration or local TNF expression was equally sufficient to cause free cholesterol-dependent apoptosis in podocytes by acting through a dual mechanism that required a reduction in ATP-binding cassette transporter A1-mediated (ABCA1-mediated) cholesterol efflux and reduced cholesterol esterification by sterol-O-acyltransferase 1 (SOAT1). TNF-induced albuminuria was aggravated in mice with podocyte-specific ABCA1 deficiency and was partially prevented by cholesterol depletion with cyclodextrin. TNF-stimulated free cholesterol-dependent apoptosis in podocytes was mediated by nuclear factor of activated T cells 1 (NFATc1). ABCA1 overexpression or cholesterol depletion was sufficient to reduce albuminuria in mice with podocyte-specific NFATc1 activation. Our data implicate an NFATc1/ABCA1-dependent mechanism in which local TNF is sufficient to cause free cholesterol-dependent podocyte injury irrespective of TNF, TNFR1, or TNFR2 serum levels.