Nuclear Factor Of Activated T Cells C1 Expression Research Articles

The STING inhibitor C-176 attenuates osteoclast-related osteolytic diseases by inhibiting osteoclast differentiation.

Inflammatory osteolysis occurs primarily in the context of osteoarthritis, aseptic inflammation, prosthesis loosening, and other conditions. An excessive immune inflammatory response causes excessive activation of osteoclasts, leading to bone loss and bone destruction. The signaling protein stimulator of interferon gene (STING) can regulate the immune response of osteoclasts. C-176 is a furan derivative that can inhibit activation of the STING pathway and exert anti-inflammatory effects. The effect of C-176 on osteoclast differentiation is not yet clear. In this study, we found that C-176 could inhibit STING activation in osteoclast precursor cells and inhibit osteoclast activation induced by nuclear factor κB ligand receptor activator in a dose-dependent manner. After treatment with C-176, the expression of the osteoclast differentiation marker genes nuclear factor of activated T-cells c1(NFATc1), cathepsin K, calcitonin receptor, and V-ATPase a3 decreased. In addition, C-176 reduced actin loop formation and bone resorption capacity. The WB results showed that C-176 downregulated the expression of the osteoclast marker protein NFATc1 and inhibited activation of the STING-mediated NF-κB pathway. We also found that C-176 could inhibit the phosphorylation of mitogen-activated protein kinase signaling pathway factors induced by RANKL. Moreover, we verified that C-176 could reduce LPS-induced bone absorption in mice, reduce joint destruction in knee arthritis induced by meniscal instability, and protect against cartilage matrix loss in ankle arthritis induced by collagen immunity. In summary, our findings demonstrated that C-176 could inhibit the formation and activation of osteoclasts and could be used as a potential therapeutic agent for inflammatory osteolytic diseases.

α-Linolenic Acid Inhibits RANKL-Induced Osteoclastogenesis In Vitro and Prevents Inflammation In Vivo.

Inflammation is an important risk factor for bone-destroying diseases. Our preliminary research found that Zanthoxylum bungeanum seed oil (ZBSO) is abundant in unsaturated fatty acids and could inhibit osteoclastogenesis in receptor activator of nuclear factor κB ligand (RANKL)-induced RAW264.7 cells. However, the key constituents in ZBSO in the prevention of osteoclastogenesis and its possible mechanism related to inflammation are still unclear. Therefore, in this study, oleic acid (OA), linoleic acid (LA), palmitoleic acid (PLA), and alpha-linolenic acid (ALA) in ZBSO, havingthe strongest effect on RANKL-induced osteoclastogenesis, were selected by a tartrate-resistant acid phosphatase (TRAP) staining method. Furthermore, the effects of the selected fatty acids on anti-inflammation and anti-osteoclastogenesis in vitro and in vivo were assessed using RT-qPCR. Among the four major unsaturated fatty acids we tested, ALA displayed the strongest inhibitory effect on osteoclastogenesis. The increased expression of free fatty acid receptor 4 (FFAR4) and β-arrestin2 (βarr2), as well as the decreased expression of nuclear factor-kappa B (NF-κB), tumor necrosis factor-α (TNF-α), nuclear factor of activated T-cells c1 (NFATc1), and tartrate-resistant acid phosphatase (TRAP) in RAW264.7 cells after ALA treatment were observed. Moreover, in ovariectomized osteoporotic rats with ALA preventive intervention, we found that the expression of TNF-α, interleukin-6 (IL-6), interleukin-1β (IL-1β), NFATc1, and TRAP were decreased, while with the ALA therapeutic intervention, downregulated expression of NF-κB, NFATc1, TRAP, and transforming growth factor beta-activated kinase 1 (TAK1) were noticed. These results indicate that ALA, as the major unsaturated fatty acid in ZBSO, could inhibit RANKL-induced osteoclastogenesis via the FFAR4/βarr2 signaling pathway and could prevent inflammation, suggesting that ZBSO may be a promising potential natural product of unsaturated fatty acids and a dietary supplement for the prevention of osteoclastogenesis and inflammatory diseases.

Proliferation, apoptosis and invasion of human lung cancer cells are associated with NFATc1.

The expression of nuclear factor of activated T cells c1 (NFATc1) is closely associated with the progression of numerous types of cancer. When NFATc1 expression becomes dysregulated in some types of cancer, this alteration can promote malignant transformation and thereby progression of cancer. NFATc1 expression has been demonstrated to be upregulated in lung cancer cells. This suggests that knockdown of NFATc1 in lung cancer cells may be a therapeutic marker for the treatment of cancer. In the present study, the effects of NFATc1 on the proliferation, apoptosis, invasion and migration of NCI-H1299 and A549 lung cancer cell lines were explored. Lentivirus infection was used to establish a cell model of NFATc1 knockdown in A549 and NCI-H1299 lung cancer cells. Reverse transcription-quantitative PCR was subsequently performed to detect NFATc1 expression in these human lung cancer cells. MTT, wound healing, colony formation and Transwell invasion assays, and flow cytometry were then performed to measure the proliferation, invasion, apoptosis and cell cycle of the cells. Finally, western blot analysis was performed to investigate the mechanism underlying the involvement of NFATc1 in these processes. NFATc1 knockdown was found to significantly inhibit the proliferation, clone formation, migration and invasion of the cells. Furthermore, the cell cycle was arrested at the G1 phase and the expression levels of the target proteins located downstream in the signaling pathway, namely CDK4, c-Myc, ERK, p38 and N-cadherin, were decreased. Following NFATc1 knockdown, the percentages of apoptotic cells were increased, and the expression levels of Bax, cleaved caspase-3 and E-cadherin were also increased. Taken together, the results of the present study suggested that NFATc1 serves an oncogenic role in lung cancer. In terms of the underlying mechanism, NFATc1 promoted the proliferation of lung cancer cells by inhibiting the MAPK and epithelial-to-mesenchymal transition signaling pathways, suggesting that NFATc1 may be a novel target for therapeutic intervention for the treatment of lung cancer.

SOX2 is a positive regulator of osteoclast differentiation

Elucidating the mechanism underlying osteoclast differentiation is important to improve our understanding of the pathophysiologies related to skeletal diseases and osteolytic metastasis in cancer. Sex-determining region Y-box containing gene 2 (SOX2), a stemness marker, is known to affect osteoblast differentiation and cancer metastasis. However, its role in osteoclastogenesis has not been investigated to date. Here, we report that SOX2 protein and mRNA expression was upregulated during osteoclast differentiation. The overexpression or knockdown of SOX2 in osteoclast precursor cells enhanced or suppressed, respectively, receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclast differentiation and migration, and nuclear factor of activated T-cell c1 (NFATc1) and factor-associated suicide ligand (FASL) expression. In addition, epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinase (ERK) activation were regulated by SOX2 expression; both EGFR and ERK inhibitors abrogated the SOX2 overexpression-induced increase in osteoclast differentiation and NFATc1 expression under RANKL stimulation. Overall, these results suggest SOX2 as a positive regulatory factor during osteoclast differentiation partly through the EGFR and ERK signaling pathways, highlighting a new potential target for restoring abnormal osteoclast activation.

Fluid Shear Stress Suppresses Osteoclast Differentiation in RAW264.7 Cells through Extracellular Signal-Regulated Kinase 5 (ERK5) Signaling Pathway.

BackgroundAlthough extracellular signal-regulated kinase 5 (ERK5) is known to be critical for osteoclast differentiation, there are few studies on how fluid shear stress (FSS) regulates osteoclast differentiation through the ERK5 signaling pathway. We examined the expression of nuclear factor of activated T cells c1 (NFATc1) in RAW264.7 cells and its downstream factors, including cathepsin K (CTSK), tartrate-resistant acid phosphatase (TRAP), matrix metalloproteinases-9 (MMP-9) and their relationship with ERK5.Material/MethodsRAW264.7 cells were treated with RANKL, XMD8-92 (ERK5 inhibitor), and then loaded onto 12 dyn/cm2 FSS for 4 days. Endpoints measured were osteoclast differentiation, bone resorption, and TRAP activity. Cell viability was detected by using the Cell Counting Kit-8 (CCK-8) assay. Western blot was used to analyze protein expression of phosphorylated-ERK5 (p-ERK5), NFATc1, CTSK, TRAP, and MMP-9.ResultsFSS inhibited osteoclast differentiation and expression of NFATc1, CTSK, TRAP, and MMP-9; cell viability was not affected. ERK5 expression increased by FSS but not by RANKL, and it was blocked by XMD8-92. Furthermore, FSS suppressed osteoclast differentiation in RAW264.7 cells through ERK5 pathway.ConclusionsOur findings demonstrated that FSS inhibited osteoclast differentiation in RAW264.7 cells via the ERK5 pathway through reduced NFATc1 expression and its downstream factors MMP-9, CTSK, and TRAP.

The expression of nuclear factor of activated T cell c1 and receptor activator of nuclear factor kappa β induced by Enterococcus faecalis in osteoclastogenesis (laboratory experiment on Wistar rats)

Background: Enterococcus faecalis (E. faecalis) is the most common bacteria species in persistent endodontic infection of teeth undergoing root canal treatment at a prevalence of 38%. The virulence factor of this bacterium is Lipoteichoic acid (LTA) which can be recognized by Toll-like receptors-4 (TLR-4) that produce a stimulus and provoke an immune response. Inflammation results in bone defects that feature multiple cytokines and interactions between different cell types. Bone loss within a periapical tooth is characterized by osteoclast formation (osteoclastogenesis) in the bone. Purpose: This study aimed to determine the expression of nuclear factor of activated T cell c1 (NFATc1) and receptor activator of nuclear factor kappa β (RANK) which played a role in osteoclastogenesis at different time intervals. Methods: 36 upper molar teeth of the research subjects were induced with 106 CFU Enterococcus faecalis and subsequently observed for 7 and 21 days with the NFATc1 and RANK being counted microscopically at 1000X magnification across 20 viewing fields. Thereafter, the data was examined and analyzed by means of an independent T test using SPSS. Results: NFATc1 and RANK expression were higher in the group including E. faecalis on days 7 and 21 than in the control group. There were significant differences between the treatment group and control group with regard to NFATc1 and RANK expression (p<0.05). Conclusion: The study showed that the expression of NFATc1 and RANK, which plays a role in osteoclastogenesis, was higher in periapical bone defects in Wistar rats induced by E. faecalis than those which were not induced.

MiR-338 regulates NFATc1 expression and inhibits the proliferation and epithelial-mesenchymal transition of human non-small-cell lung cancer cells.

BackgroundIt is well known that nuclear factor of activated T cells c1 (NFATc1) expression is closely associated with progression of many cancers. And we found that miR‐338 could directly target the NFATc1. However, the precise mechanisms of miR‐338 in non‐small‐cell lung cancer (NSCLC) have not been well clarified. Our study aimed to explore the interaction between NFATc1 and miR‐338 in NSCLC.MethodsQuantitative RT‐PCR was utilized to determine the expressions of NFATc1 and miR‐338 in NSCLC tissues and cell lines. And the cell proliferation and epithelial‐mesenchymal transition (EMT) were assessed to determine the functional roles of miR‐338 and NFATc1 in NSCLC cells. NFATc1 expression was detected using quantitative RT‐PCR and western blotting, respectively. Luciferase reporter assays were performed to validate NFATc1 as a target of miR‐338 in NSCLC cells.ResultsIn this study, our results showed that NFATc1 expression was significantly up‐regulated in NSCLC tissues and cell lines, and the miR‐338 level was dramatically down‐regulated. Moreover high NFATc1 expression was closely associated with low miR‐338 level in NSCLC tissues. Moreover introduction of miR‐338 significantly inhibited proliferation and EMT of NSCLC cells. Bioinformatics analysis predicted that the NFATc1 was a potential target gene of miR‐338. We demonstrated that miR‐338 could directly target NFATc1 by using luciferase reporter assay. Besides, knockdown of NFATc1 had the similar effects with miR‐338 overexpression on NSCLC cells. Up‐regulation of NFATc1 in NSCLC cells partially abolished the inhibitory effects of miR‐338 mimic.ConclusionsOverexpression of miR‐338 inhibited cell proliferation and EMT of NSCLC cells by directly down‐regulating NFATc1 expression.

Berberine Suppresses RANKL-Induced Osteoclast Differentiation by Inhibiting c-Fos and NFATc1 Expression.

Osteoporosis is a common disorder of bone remodeling, marked by excessive osteoclast formation. Recent studies indicated that berberine (BBR) is a potential natural drug for the treatment of various bone diseases. However, it still needs to be further studied for the treatment of osteoporosis. The current study investigated the inhibitory effects of BBR on receptor activator of nuclear factor- B ligand (RANKL)-induced osteoclast differentiation in vitro and in vivo. Cell-based assays were performed using osteoclasts generated in cultures of murine bone marrow-derived macrophages (BMMs) treated with RANKL and M-CSF. The effects of BBR on in vivo lipopolysaccharide (LPS)-mediated bone loss were evaluated using ICR mice. BBR significantlyinhibited TRAP-positive osteoclast formation induced by RANKL. BBR also inhibited RANKL-induced Akt, p38 and ERK phosphorylation and I B degradation, and suppressed RANKL-induced expression of c-Fos and nuclear factor of activated T cells c1 (NFATc1), which is a key transcription factors for osteoclast formation. BBR reduced the mRNA levels of osteoclast markers, including TRAP, osteoclast-associated receptor (OSCAR), cathepsin K, and ATPase H transporting V0 subunit d2 (ATP6v0d2).Moreover, BBR prevented LPS-mediated bone loss in vivo. We suggest BBR as a natural compound that can be a potential therapeutic agent for osteoclast-related bone diseases.

Neogambogic Acid Suppresses Receptor Activator of Nuclear Factor κB Ligand (RANKL)-Induced Osteoclastogenesis by Inhibiting the JNK and NF-κB Pathways in Mouse Bone Marrow-Derived Monocyte/Macrophages.

BackgroundNeogambogic acid (NGA) is used in traditional Chinese medicine. The aim of this study was to investigate the effects of NGA on gene signaling pathways involved in osteoclastogenesis in mouse bone marrow-derived monocyte/macrophages (BMMs) and on bone resorption in vitro.Material/MethodsPrimary mouse BMMs were cultured with increasing concentrations of NGA. Real-time polymerase chain reaction was used to study the expression of mRNAs corresponding to gene products specific to receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation, including tartrate-resistant acid phosphatase (TRAP), calcitonin receptor (CTR), cathepsin K (CTSK), and nuclear factor of activated T cells c1 (NFATc1). A cell counting kit-8 assay was used to evaluate cell proliferation. Western blotting and confocal immunofluorescence microscopy were used to investigate the signaling pathways. A bone resorption model was used to quantify bone resorption.ResultsAn NGA dose of ≤0.4 μg/ml had no significant effect on the proliferation of mouse BMMs in vitro (P>0.05); concentrations of between 0.1–0.4 μg/ml significantly inhibited RANKL-induced osteoclastogenesis (P<0.01) in a dose-dependent manner. Compared with the control group, NGA significantly reduced RANKL-induced bone resorption in vitro (P <0.01), and downregulated the expression of osteoclast-related mRNAs of TRAP, CTR, CTSK, and NFATc1. NGA suppressed the activation of JNK but not the p38 signaling pathway and significantly reduced NF-κB p65 phosphorylation and the nuclear transport of NF-κB molecules, which inhibited NFATc1 expression.ConclusionsNGA suppressed RANKL-induced osteoclastogenesis by inhibiting the JNK and NF-κB pathways in mouse BMMs in vitro and reduced osteoclastic bone resorption.

Resolvin E1 Inhibits Osteoclastogenesis and Bone Resorption by Suppressing IL-17-induced RANKL Expression in Osteoblasts and RANKL-induced Osteoclast Differentiation

Resolvin E1 (RvE1) derived from the ω-3 polyunsaturated fatty acid eicosapentaenoic acid is known to be a potent pro-resolving lipid mediator that prevents chronic inflammation and osteoclastogenesis. We investigated the inhibitory effects of RvE1 on osteoclastogenesis and bone resorption to clarify its therapeutic potential for rheumatoid arthritis (RA). Receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation was assessed with tartrate-resistant acid phosphatase staining. RANKL-induced bone resorption was assessed by the measurement of pit formation using calcium phosphate-labeled fluorescent polyanionic molecules in RAW264.7 cells as osteoclast precursors. The effects of RvE1 on the RANKL-induced mRNA expression of osteoclast-specific genes and transcriptional factors such as c-fos and nuclear factor of activated T cells c1 (NFATc1) in RAW264.7 cells were measured by quantitative real-time PCR. The distribution of NFATc1 induced by RANKL was evaluated by immunofluorescence staining in RAW264.7 cells. To analyze the mechanism of the inhibitory effect of RvE1 on osteoclastogenesis, we measured IL-17-induced RANKL mRNA expression in MC3T3-E1 osteoblast cells treated with RvE1 using quantitative real-time PCR and determined the level of prostaglandin E2 (PGE2) production by enzyme-linked immunosorbent assay. RvE1 significantly suppressed RANKL-induced osteoclast differentiation and bone resorption. RvE1 inhibited the RANKL-induced mRNA expression of osteoclast-specific genes along with the transcription factors NFATc1 and c-fos. Moreover, NFATc1 translocation from the cytoplasm to the nucleus of RAW264.7 cells was suppressed following RvE1 treatment. RvE1 also inhibited IL-17-induced RANKL mRNA expression and PGE2 production in MC3T3-E1 cells. RvE1 inhibited osteoclastogenesis and bone resorption by suppressing RANKL-induced NFATc1 and c-fos expression in osteoclasts and IL-17-induced RANKL expression through the autocrine action of PGE2 in osteoblasts. Our data suggest RvE1 as a new therapeutic target of RA.

3',4',7,8-Tetrahydroxyflavone inhibits RANKL-induced osteoclast formation and bone resorption.

Osteoclasts, which are specialized bone multinuclear cells, are responsible for bone lytic diseases such as osteoporosis. 3',4',7,8-tetrahydroxyflavone is a flavonoid from Acacia confusa. In the present study, we found that 3',4',7,8-tetrahydroxyflavone markedly inhibited receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclastic differentiation from mouse bone marrow-derived macrophages (BMMs). 3',4',7,8-tetrahydroxyflavone also reduced the mRNA expression levels of osteoclastic marker genes including the calcitonin receptor (CTR) and cathepsin K. In addition, 3',4',7,8-tetrahydroxyflavone decreased the bone resorption activity of osteoclasts on dentin slices. We found that 3',4',7,8-tetrahydroxyflavone inhibited RANKL-induced expression of c-Fos and nuclear factor of activated T cells c1 (NFATc1), a key transcription factor of osteoclast differentiation. Furthermore, ectopic overexpression of a constitutively active form of NFATc1 completely rescued the anti-osteoclastogenic effect of 3',4',7,8-tetrahydroxyflavone, suggesting that the anti-osteoclastogenic effect was mainly attributed to the reduction in NFATc1 expression. Taken together, our data suggest that 3',4',7,8-tetrahydroxyflavone inhibits osteoclast differentiation and bone loss and may therefore be considered a promising drug candidate for treating or preventing bone-lytic diseases.

CD137-CD137L signaling promotes angiogenesis in atherosclerosis plaque of mice through activating nuclear factor of activated T cells c1

Objective: To explore whether CD137-CD137L signaling can promote angiogenesis in atherosclerosis plaque via activating nuclear factor of activated T cells c1 (NFATc1). Methods: Apolipoprotein E knock out mice were divided into the following groups: control group (n=5), CD137 activated group(n=5)and CD137 inhibited group (n=5). Immunohistochemistry was performed to detect the expression of CD31 in aortic plaque. Endothelial cells (bEnd.3) were purchased from ATCC and divided into the following groups: control group, IgG isotype control group, CD137 activated group and CD137 inhibited group. Western blot was used to determine total protein and nucleoprotein expression of NFATc1. The expression level of CD137 protein on the surface of endothelial cells was detected by flow cytometry(FCM) and CD137 protein of lysate of endothelial cells was detected by enzyme-linked immunosorbent assay (ELISA). Transwell assay was used to observe the migration ability of endothelial cells.Matrigel tube formation ability of endothelial cells were tested in the following groups: control group, CD137 activated group, silent NFATc1 + CD137 activated group, CD137 inhibited group, and over expressed NFATc1+ CD137 inhibited group. Results: (1) In vivo, the expression level of CD31 was significantly higher in the aortic plaque of CD137 activated group than in control group(1 191±187 vs. 115±30, P<0.05), while which was significantly downregulated in CD137 inhibited group(450±92, P<0.05). (2) The level of nucleoprotein(3.07±0.03 vs. 1.00±0.00, P<0.05) and total protein(2.18±0.30 vs. 1.00±0.00, P<0.05) of NFATc1 were significantly higher in CD137 activated group than in IgG isotype control group. The level of nucleoprotein(0.82±0.04) and total protein(0.84 ± 0.09) of NFATc1 were significantly lower in CD137 inhibited group than in CD137 activated group(both P<0.05). (3) FCM results showed that the fluorescence intensity of CD137 on the cell membrane was significantly higher in endothelial cells stimulated by TNF-α than in normal endothelial cells(5 163±329 vs. 1 660±162, P<0.05). (4) ELISA examination showed that the level of CD137 protein was significantly higher in endothelial cells stimulated by TNF-α than in normal endothelial cells ((573.4±23.7)pg/mg vs.(69.5±16.7)pg/mg, P<0.05). (5) Migration cell number was remarkably higher in CD137 activated group than in IgG isotype control group(1.19±0.13 vs. 1.00±0.00, P<0.05) and significantly lower in CD137 inhibited group(0.82±0.06)than in control group (P<0.05). (6) Values of the formation of the tube length ((5.76±0.18)mm vs. (4.21±0.11)mm, P<0.05) and branch number (29.38±1.28 vs. 21.13±0.96, P<0.05) were both significantly higher in CD137 activated group than in the control group. The formation of the tube length ((1.90±0.11)mm) and branch number(8.91±0.72)were significantly lower in silent NFATc1 + CD137 activated group than in the CD137 activated group (both P<0.05). The formation of the tube length((1.28±0.34)mm) and branch number(5.07±0.35)were also significantly decreased in the CD137 inhibited group compared with the CD137 activated group (both P<0.05). Compared with the CD137 inhibited group, the formation of the tube length((4.82±0.09)mm) and branch number(24.44±1.05) in the over expressed NFATc1+ CD137 inhibited group was increased (both P<0.05). Conclusion: CD137 can promote the angiogenesis in atherosclerosis plaque by activating NFATc1.

CCAAT/Enhancer-binding Protein α (C/EBPα) Is Important for Osteoclast Differentiation and Activity

CCAAT/enhancer-binding protein (C/EBPα) can appoint mouse bone marrow (MBM) cells to the osteoclast (OC) lineage for osteoclastogenesis. However, whether C/EBPα is also involved in OC differentiation and activity is unknown. Here we demonstrated that C/EBPα overexpression in MBM cells can promote OC differentiation and strongly induce the expression of the OC genes encoding the nuclear factor of activated T-cells, c1 (NFATc1), cathepsin K (Cstk), and tartrate-resistant acid phosphatase 5 (TRAP) with receptor activator of NF-κB ligand-evoked OC lineage priming. Furthermore, while investigating the specific stage of OC differentiation that is regulated by C/EBPα, our gene overexpression studies revealed that, although C/EBPα plays a stronger role in the early stage of OC differentiation, it is also involved in the later stage. Accordingly, C/EBPα knockdown drastically inhibits osteoclastogenesis and markedly abrogates the expression of NFATc1, Cstk, and TRAP during OC differentiation. Consistently, C/EBPα silencing revealed that, although lack of C/EBPα affects all stages of OC differentiation, it has more impact on the early stage. Importantly, we showed that ectopic expression of rat C/EBPα restores osteoclastogenesis in C/EBPα-depleted MBM cells. Furthermore, our subsequent functional assays showed that C/EBPα exhibits a dispensable role on actin ring formation by mature OCs but is critically involved in bone resorption by stimulating extracellular acidification and regulating cell survival. We revealed that C/EBPα is important for receptor activator of NF-κB ligand-induced Akt activation, which is crucial for OC survival. Collectively, these results indicate that C/EBPα functions throughout osteoclastogenesis as well as in OC function. This study provides additional understanding of the roles of C/EBPα in OC biology.

NFATc1 regulates cell proliferation, migration, and invasion of ovarian cancer SKOV3 cells in vitro and in vivo

NFATc1 (nuclear factor of activated T‑cells c1) is associated with malignancy in several cancer models. However, the expression and function of NFATc1 in ovarian cancer remain elusive. In the present study, we investigated the role of NFATc1 in human epithelial ovarian cancer (EOC) using human ovarian adenocarcinoma SKOV3 cells and patient characteristics. NFATc1 expression was silenced by siRNA in the SKOV3 ovarian cancer cell line and in human ovarian cancer nude mouse xenografts. Real‑time PCR, western blotting, immunohistochemical staining, MTT, flow cytometry, transwell, erasion trace and mouse assays were used to detect NFATc1 expression, cell proliferation, apoptosis, cell invasion and migration, tumor growth and angiogenesis. Survival analysis was performed to assess the correlation between NFATc1 expression and survival. NFATc1 was overexpressed in the SKOV3 ovarian cancer cell line and in human serous/mucinous ovarian cancer tissues. The silencing of NFATc1 expression by siRNA reduced cell proliferation and migration and promoted apoptosis invitro and decreased the ovarian cancer cell tumorigenesis invivo in nude mice. NFATc1 overexpression in high‑grade serous ovarian carcinomas was an independent prognostic factor of poor overall survival and of early relapse (P<0.01) in a univariate analysis. Our present data provide evidence that NFATc1 is overexpressed in human serous/mucinous ovarian cancer and is associated with a poor prognosis. NFATc1 silencing regulates the cell cycle, apoptosis, invasion and migration. NFATc1 thus has the potential to be a therapeutic target and to be used in EOC diagnosis and prognosis.

Physiological Activity of Coffee Beans and Roasted Black Beans (Rhynchosia nulubilis) Mixture Extracts for Coffee Alternative Beverage Development

This study was designed to develop and to qualify a coffee alternative beverage using a mixture of coffee beans and roasted black beans (Rhynchosia nulubilis). Therefore, the total isoflavone content (TIC), total phenol content (TPC), antioxidant activity, anti-inflammatory activity, NFATc1 (Nuclear factor of activated T-cells c1) expression in RANKL (receptor activator of nuclear factor kappa-B ligand)-stimulated RAW264.7 cells and sensory evaluation were measured for 5 different Cb (coffee bean)-RoS (roasted seomoktae) mixture extracts (Cb100RoS0, Cb75RoS25, Cb50RoS50, Cb25RoS75, and Cb0RoS100). Cb0RoS100 had the highest TIC (<TEX>$516.83{\pm}36.61mg/100g$</TEX>) and TPC (<TEX>$18.11{\pm}1.77mg$</TEX> TAE/100 g) along with the highest antioxidant activity as measured by DPPH radical scavenging activity (<TEX>$73.55{\pm}8.11%$</TEX>) and ABTS radical scavenging activity (<TEX>$63.27{\pm}7.27%$</TEX>). Also, Cb0RoS100 showed the highest anti-inflammatory activity as measured by NO production (<TEX>$13.57{\pm}2.21{\mu}M$</TEX>) and PGE2 production (<TEX>$3.25{\pm}0.21ng/mL$</TEX>). The more the RoS ratio was increased in the mixtures of Cb-RoS, the more the NFATc1 protein expression was decreased in RANKL-stimulated RAW264.7 cells. In case of sensory evaluation, Cb50RoS50 had the highest scores for flavor, delicate flavor and overall quality, which were similar to those in Cb alone (Cb100RoS0). We suggest that the use of RoS replacement instead of Cb in/as a coffee alternative beverage may help to reduce the risk of caffeine-related bone loss and/or bone disease by effectively blocking NFATc1 expression in RANKL-stimulated RAW264.7 cells compared with Cb alone.

The effect of NFATc1 on vascular generation and the possible underlying mechanism in epithelial ovarian carcinoma.

We investigated the effect of nuclear factor of activated T cells c1 (NFATc1) on the growth and vascular generation of human ovarian carcinoma SKOV3 cell-transplanted tumors in nude mice and explored the possible underlying mechanism. NFATc1 siRNA was transfected into the SKOV3 cells, which were then subjected to immunofluorescence tests and real-time reverse transcription polymerase chain reaction (RT-PCR) to determine the transfection-induced inhibition rate. The tumor volumes in the nude mice in all groups were measured to determine the in vivo antitumor effect of NFATc1 siRNA. Immunohistochemical (IHC) methods were employed to detect NFATc1 expression in tumor tissue, combined with cytokeratin (CK) staining to label the epithelial origin of the tumor tissue. CD34 and podoplanin were used as markers for labeling microvessels and microlymphatic vessels, respectively. The densities of microvessels and microlymphatic vessels in each group were calculated and statistically analyzed. RT-PCR and western blotting were performed to detect the protein and mRNA expression levels of NFATc1, the ELR+ CXC chemokine interleukin (IL)-8, fibroblast growth factor-2 (FGF-2), and platelet-derived growth factor BB (PDGF BB) in xenografted tumor tissue in all groups. NFATc1 was highly expressed in tumor tissue in the control groups. The intervention group exhibited a tumor growth inhibition rate of 57.08% and presented a lower tumor weight and volume compared with the two control groups. In the control groups, the microvessel densities were 12.00 ± 1.65 and 11.47 ± 0.32, respectively, and the microlymphatic vessel densities were 10.03 ± 0.96 and 9.95 ± 1.12; these values were significantly higher than in the intervention group. RT-PCR and western blot shows that NFATc1 siRNA could markedly suppress the expression of IL-8, FGF-2 and PDGF BB at the mRNA and the protein level. In conclusion, it was shown that NFATc1 siRNA significantly suppresses the growth and vascular generation of SKOV3 human ovarian carcinoma cell-transplanted tumors subcutaneously xenografted into nude mice. The downregulation of the expression of IL-8, FGF-2 and PDGF BB may be one of the mechanisms underlying the above inhibitory effects.

IL-1R/TLR2 through MyD88 Divergently Modulates Osteoclastogenesis through Regulation of Nuclear Factor of Activated T Cells c1 (NFATc1) and B Lymphocyte-induced Maturation Protein-1 (Blimp1)

Toll-like receptors (TLR) and the receptor for interleukin-1 (IL-1R) signaling play an important role in bacteria-mediated bone loss diseases including periodontitis, rheumatoid arthritis, and osteomyelitis. Recent studies have shown that TLR ligands inhibit the receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation from un-committed osteoclast precursors, whereas IL-1 potentiates RANKL-induced osteoclast formation. However, IL-1R and TLR belong to the same IL-1R/TLR superfamily, and activate similar intracellular signaling pathways. Here, we investigate the molecular mechanisms underlying the distinct effects of IL-1 and Porphyromonas gingivalis lipopolysaccharide (LPS-PG) on RANKL-induced osteoclast formation. Our results show that LPS-PG and IL-1 differentially regulate RANKL-induced activation of osteoclast genes encoding Car2, Ctsk, MMP9, and TRAP, as well as expression of NFATc1, a master transcription factor of osteoclastogenesis. Regulation of osteoclast genes and NFATc1 by LPS-PG and IL-1 is dependent on MyD88, an important signaling adaptor for both TLR and IL-1R family members. Furthermore, LPS-PG and IL-1 differentially regulate RANKL-costimulatory receptor OSCAR (osteoclast-associated receptor) expression and Ca(2+) oscillations induced by RANKL. Moreover, LPS-PG completely abrogates RANKL-induced gene expression of B lymphocyte-induced maturation protein-1 (Blimp1), a global transcriptional repressor of anti-osteoclastogenic genes encoding Bcl6, IRF8, and MafB. However, IL-1 enhances RANKL-induced blimp1 gene expression but suppresses the gene expression of bcl6, irf8, and mafb. Our study reveals the involvement of multiple signaling molecules in the differential regulation of RANKL-induced osteoclastogenesis by TLR2 and IL-1 signaling. Understanding the signaling cross-talk among TLR, IL-1R, and RANK is critical for identifying therapeutic strategies to control bacteria-mediated bone loss.

Adseverin mediates RANKL-induced osteoclastogenesis by regulating NFATc1

Adseverin is a Ca2+-dependent actin filament-severing protein that has been reported to regulate exocytosis via rearrangements of the actin cytoskeleton in secretory cells. However, the role of adseverin in bone cells has not yet been well characterized. Here, we investigated the role of adseverin in osteoclastogenesis using primary osteoclast precursor cells. Adseverin expression was upregulated during RANKL (receptor activator of nuclear factor-κB ligand)-induced osteoclast differentiation. Moreover, genetic silencing of adseverin decreased the number of osteoclasts generated by RANKL. Adseverin knockdown also suppressed the RANKL-mediated induction of nuclear factor of activated T-cell c1 (NFATc1), which is a key transcription factor in osteoclastogenesis. In addition, adseverin knockdown impaired bone resorption and the secretion of bone-degrading enzymes from osteoclasts. These effects were accompanied by decreased NFATc1 expression and the activation of nuclear factor-κB. Collectively, our results indicate that adseverin has a crucial role in osteoclastogenesis by regulating NFATc1.

Inhibition of calcineurin/NFAT pathway plays an essential role in renoprotective effect of tropisetron in early stage of diabetic nephropathy

Recent studies have shown that calcineurin plays a central role in hypertrophy and extracellular matrix (ECM) accumulation in glomeruli at the early stages of diabetic nephropathy. Tropisetron is an effective antiemetic drug which also can potently inhibit calcineurin. The aim of this study was to investigate whether tropisetron can prevent glomerular hypertrophy and ECM expansion in early diabetic nephropathy. Streptozotocin (STZ)-induced diabetic rats were treated with tropisetron and cyclosporine A, a pharmacological calcineurin inhibitor, and the renal function and the expression of calcineurin and fibronectin were then assessed as well as nuclear localization of nuclear factor of activated T-cell c1 (NFATc1). 2 weeks after diabetes induction, all STZ-treated rats showed hyperglycemia, polyuria, body weight loss and renal dysfunction, as evidenced by increased glomerular filtration rate (GFR), along with a marked pathological changes in kidney. Calcineurin expression was increased in association with increased nuclear localization of the calcineurin substrate NFATc1 and fibronectin expression in glomeruli of diabetic rats. In parallel, the diabetic glomeruli became hypertrophic with an increase in kidney weight. Tropisetron, as potent as cyclosporine A, significantly ameliorated the early nephropathy symptoms, potentially through suppression of calcineurin expression, nuclear localization of NFATc1 and accumulation of fibronectin, and thereby reduced hypertrophy in glomeruli of diabetic rats.In conclusion, our results showed that tropisetron could ameliorate kidney injury in the early stage of diabetic nephropathy in rats. The renoprotective effects of tropisetron can be attributed, at least in part, to the suppression of diabetes-induced increases in calcineurin expression in kidney tissue.

CD137 signaling regulates the expression of nuclear factor of activated T cells c1 through miR-145a-5p in ApoE(-)/(-) mice

To investigate if miR-145a-5p participates the modulation process of CD137 signaling on the expression of nuclear factor of activated T cells c1 (NFATc1) in ApoE(-)/(-) mice. Atherosclerotic plaque model was produced by perivascular carotid collar placement in ApoE(-)/(-) mice. After surgery, the mice were randomly divided into the following groups: CD137 activated group (CD137 group, n = 6), CD137 inhibited group (anti-CD137 group, n = 6) and control group (n = 6). The mRNA expression of miR-145a-5p in plaque and cells was measured by real-time quantitative PCR (RT-PCR). Immunofluorescence was used to observe the distribution of NFATc1 in plaque and the expression of NFATc1 at mRNA and protein levels were detected by qRT-PCR, Western blot, respectively. The mouse vascular smooth muscle cells (VSMCs) were isolated and transfected with miR-145a-5p mimics or inhibitors by Lipofectamine. The eukaryotic expression vector and luciferase vector including p3xFLAG-NFATc1, p3xFLAG-NFATc1-3'UTR, psicheck2-NFATc1, psicheck2-NFATc1-Mut were constructed through molecular cloning and homologous recombination techniques, 293T cells were transfected with the miR-145a-5p mimics or inhibitors and the protein level and fluorescence intensity were then measured, respectively. In vivo or in vitro, the level of miR-145a-5p was significantly decreased (0.21 ± 0.06 vs. 1.00 ± 0.00, P < 0.05, 0.22 ± 0.07 vs. 0.50 ± 0.12, P < 0.05) while the opposite effects were observed in anti-CD137 group. NFATc1 expression was decreased or increased in VSMCs transfected with miR-145a-5p mimics or inhibitors, respectively (all P < 0.05). miR-145a-5p mimics decreased the expression of p3xFLAG-NFATc1-3'UTR and the fluorescence intensity (0.56 ± 0.08 vs. 1.00 ± 0.00, P < 0.05). CD137 signaling participates the regulation process on the expression of NFATc1 through miR-145a-5p in ApoE(-)/(-) mice.