Nuclear Factor Of Activated T Cell 1 Expression Research Articles

Suppression of bone resorption by Mori Radicis Cortex through NFATc1 and c-Fos signaling-mediated inhibition of osteoclast differentiation.

Mori Radicis Cortex (MRC) is the root bark of the mulberry family as Morus alba L. In Korea, it is known as "Sangbaegpi". Although MRC has demonstrated anti-inflammatory and antioxidant effects, its specific mechanisms of action and impact on osteoporosis remain poorly understood. To investigate the antiosteoporosis effect of MRC, we examined the level of osteoclast differentiation inhibition in receptor activator of nuclear factor kappa-Β ligand (RANKL)-induced-RAW 264.7 cells and animal models of ovariectomy (OVX) with MRC. Serum analysis in OVX animals was investigated by enzyme-linked immunosorbent assay (ELISA), and bone density analysis was confirmed by micro-computed tomography (micro-CT). The expression analysis of nuclear factor of activated T cells 1 (NFATc1) was confirmed by immunohistochemistry (IHC) in femur tissue. In addition, osteoclast differentiation inhibition was measured using tartrate-resistant acid phosphatase (TRAP). mRNA analysis was performed using reverse transcription-polymerase chain reaction (RT-PCR), and the protein expression analysis was investigated by western blot. Micro-CT analysis showed that MRC effectively inhibited bone loss in the OVX-induced rat model. MRC also inhibited the expression of alkaline phosphatase (ALP) and TRAP in serum. Histological analysis showed that MRC treatment increased bone density and IHC analysis showed that MRC significantly inhibited the expression of NFATc1. In RANKL-induced-RAW 264.7 cells, MRC significantly reduced TRAP activity and actin ring formation. In addition, MRC significantly inhibited the expression of NFATc1 and c-Fos, and suppressed the mRNA expression. Based on micro-CT, serum and histological analysis, MRC effectively inhibited bone loss in an OVX-induced rat model. In addition, MRC treatment suppressed the expression of osteoclast differentiation, fusion, and bone resorption markers through inhibition of NFATc1/c-Fos expression in RANKL-induced RAW 264.7 cells, ultimately resulting in a decrease in osteoclast activity. These results demonstrate that MRC is effective in preventing bone loss through inhibiting osteoclast differentiation and activity.

Hydroxychavicol Inhibits In Vitro Osteoclastogenesis via the Suppression of NF-κB Signaling Pathway.

Hydroxychavicol, a primary active phenolic compound of betel leaves, previously inhibited bone loss in vivo by stimulating osteogenesis. However, the effect of hydroxychavicol on bone remodeling induced by osteoclasts is unknown. In this study, the anti-osteoclastogenic effects of hydroxychavicol and its mechanism were investigated in receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclasts. Hydroxychavicol reduced the number of tartrate resistance acid phosphatase (TRAP)-positive multinucleated, F-actin ring formation and bone-resorbing activity of osteoclasts differentiated from RAW264.7 cells in a concentration-dependent manner. Furthermore, hydroxychavicol decreased the expression of osteoclast-specific genes, including cathepsin K, MMP-9, and dendritic cell-specific transmembrane protein (DC-STAMP). For mechanistic studies, hydroxychavicol suppressed RANKL-induced expression of major transcription factors, including the nuclear factor of activated T-cells 1 (NFATc1), c-Fos, and c-Jun. At the early stage of osteoclast differentiation, hydroxychavicol blocked the phosphorylation of NF-κB subunits (p65 and Iκβα). This blockade led to the decrease of nuclear translocation of p65 induced by RANKL. In addition, the anti-osteoclastogenic effect of hydroxychavicol was confirmed by the inhibition of TRAP-positive multinucleated differentiation from human peripheral mononuclear cells (PBMCs). In conclusion, hydroxychavicol inhibits osteoclastogenesis by abrogating RANKL-induced NFATc1 expression by suppressing the NF-κB signaling pathway in vitro.

Role of nuclear factor of activated T Cells-1 in Sepsis-induced behavioral deficits in mice

IntroductionThe nuclear factor of activated T cells-1 (NFAT1) is involved in both neuroinflammation and cognitive dysfunction. In this study, we examined the role of NFAT1 in sepsis-induced cognitive impairment in a mouse model. MethodsSepsis was established in adult mice by cecal ligation and puncture (CLP). Novel object recognition tests on days 14–21 and fear conditioning tests on days 22–23 post-surgery showed that CLP impaired both behaviors. BV2 microglia cells exposed to lipopolysaccharide (LPS) were used to examine the effects of short interfering RNA targeting NFAT1 on autophagy and inflammatory cytokines. ResultsCLP increased the expression of NFAT1 in hippocampal microglia and induced hippocampal autophagy by downregulating p62, upregulating beclin-1 and autophagy-related gene-5, and increasing the ratio of microtubule-associated protein 1 light chain 3-I (LC3-I) to LC3-II. In addition, CLP shifted microglial polarization from M2 to M1 and the production of inflammatory cytokines, similar to the effects of lipopolysaccharide on BV2 microglia cells. Conversely, NFAT1 knockdown or the autophagy inhibitor 3-methyladenine attenuated the effects of CLP on autophagy and inflammation in vitro and in vivo, while rapamycin partially reversed the protective effects of NFAT1 inhibition. ConclusionThis study suggests that NFAT1 downregulation attenuates sepsis-induced behavioral deficits by inhibiting autophagy, microglia polarization, and neuroinflammation..

Low-density lipoprotein receptor deficiency reduced bone mass in mice via the c-fos/NFATc1 pathway

AimThe low-density lipoprotein receptor (LDLR) plays a crucial role in regulating lipid metabolism. However, whether LDLR deficiency affects bone mass and morphology remains controversial. This study aimed to analyze the bone phenotypes of LDLR knockout (LDLR−/−) mice. Main methodsEight-week-old LDLR−/− and wild-type (WT) mice were subjected to microcomputed tomography to detect bone phenotypes. Enzyme-linked immunosorbent assay kits were used to detect the serum estrogen levels and matrix metalloproteinase 9 (MMP-9) levels in tissue homogenates. Von Kossa, toluidine blue, tartrate-resistant acid phosphatase (TRAP) staining, and calcein labeling were performed to explore bone turnover parameters. In vitro, osteoclastogenesis was induced in bone marrow cells from LDLR−/− mice and WT mice in the presence or absence of 17β-estradiol. The microphotographs and number of osteoclasts were validated using TRAP staining. Relative gene expression during osteoclast differentiation and maturation was determined by quantitative real-time polymerase chain reaction. Key findingsLDLR deficiency results in reduced bone mineral density of the tibial cancellous bone, indicating bone loss to some extent in LDLR−/− mice. LDLR deficiency significantly increased the number of osteoclasts, but not osteoblasts. In vitro, bone marrow cells from LDLR−/− mice displayed enhanced osteoclastic potential along with increased expression of TRAP, cathepsin K, nuclear factor of activated T-cells 1 (NFATc1), c-fos, and MMP-9 and inhibited dendritic cell-specific transmembrane protein expression. Moreover, 17β-estradiol treatment can inhibit osteoclastogenesis in vitro. SignificanceOur data demonstrated that LDLR deficiency promoted osteoclastogenesis by upregulating c-fos and NFATc1 expression, reducing cancellous bone mass in LDLR−/− mice.

PD-1+ CD4 T cell immune response is mediated by HIF-1α/NFATc1 pathway after P. yoelii infection.

The morbidity and mortality of malaria are still high. Programmed cell death-1(PD-1) is an important co-inhibitory factor and CD8 T cells with PD-1 were reported to be exhausted cells. It remains unknown what the role of CD4 T cells expressing PD-1 is and what the upstream regulating molecules of PD-1 in CD4 T cells are. The C57BL/6 mice were injected with Plasmodium yoelii (P. yoelii) in this study. Expressions of PD-1, activation markers, and cytokines were tested. The differentially expressed genes between PD-1+/- CD4 T cells were detected by microarray sequencing. Western blot, chromatin immunoprecipitation (ChIP), siRNA, hypoxia inducible factor-1α (HIF-1α) inducer and inhibitor were used to explore PD-1’s upstream molecules, respectively. The proportions of PD-1+ CD4 T cells increased post P. yoelii infection. PD-1+ CD4 T cells expressed more activated surface markers and could produce more cytokines. Nuclear factor of activated T cells 1 (NFATc1) was found to be a key transcription factor to induce PD-1 expression after infection. Both the inducer and the inhibitor of HIF-1α could change the expressions of NFATc1 and PD-1 in vivo and in vitro, respectively. Taken together, P. yoelii infection induced NFATc1 expression by HIF-1α. The highly expressed NFATc1 entered the nucleus and initiated PD-1 expression. PD-1+ CD4 T cells appeared to be more activated and could secrete more cytokines to regulate the host’s immune responses against malaria.

The role of NFATc1/c-myc/PKM2/IL-10 axis in activating cervical cancer tumor-associated M2 macrophage polarization to promote cervical cancer progression

Nuclear factor of activated T cells 1 (NFATc1) is mainly expressed in tumor microenvironment, especially in macrophages. However, whether NFATc1 is involved in the polarization of tumor associated macrophages (TAMs) and tumor progression in cervical cancer (CC) remains unclear. Immunofluorescence staining was used to detect the expression of CD68 and NFATc1 in CC tissues or adjacent normal tissues of patients. RT-qPCR, flow cytometry, ELISA, and inhibitors treatment were used to observe the effect of NFATc1 on TAMs polarization. Clonal formation, scratch, and transwell assays were used to examine the effects of NFATc1-transfected macrophages or NFATc1-transfected TAM on tumor proliferation, migration, and invasion. Further, a xenograft model was established to confirm the roles of NFATc1+ TAM in CC tumorigenesis. NFATc1+CD68+/CD68+ TAMs ratio was significantly increased in CC tissues compared with the normal tissue, and NFATc1+ TAM showed an M2-like TAM subtype. NFATc1 induced macrophages to secrete IL-10, which further induced M2 polarization of macrophages. Mechanically, the c-myc-PKM2 pathway mediated the expression of IL-10 in NFATc1-induced macrophages. Functionally, NFATc1 induced M2 macrophages promoted the proliferation, migration, and invasion of CC cells, and the knockout of NFATc1 in TAMs significantly inhibited the tumor-promoting function of TAMs. Further, the tumorigenesis test in nude mice confirmed that NFATc1+ TAM promoted the tumorigenicity of CC cells in vivo. In conclusion, NFATc1 mediated IL-10 secretion by regulating the c-myc/PKM2 pathway, thereby inducing M2 polarization of TAMs and promoting the progression of CC.

Inhibitory Effects of Astaxanthin on CML-HSA-Induced Inflammatory and RANKL-Induced Osteoclastogenic Gene Expression in RAW 264.7 Cells.

Objective: Elevated levels of serum Nε-carboxymethyllysine (CML), a well-known advanced glycation end-product (AGE), were observed in patients with inflammation or osteoporosis. Astaxanthin was reported to possess anti-inflammatory and antioxidant effects. In the present study, we investigated the effects of commercially available dietary supplement AstaReal ACTR (ASR) capsule content as astaxanthin on CML-HSA-induced inflammatory and receptor activator of nuclear factor-kappa-Β ligand (RANKL)-induced osteoclastogenic gene expression. Methods: RAW 264.7 murine macrophage cells were stimulated with CML-HSA to trigger inflammatory gene expression and treated with either a vehicle control or varied concentrations of astaxanthin. Inflammatory gene expression was measured using an enzyme-linked immunosorbent assay (ELISA) or qPCR. We triggered osteoclastogenesis using RANKL, and osteoclastogenic gene expression was measured through tartrate-resistant acid phosphatase (TRAP) activity, staining, immunofluorescence, and qPCR analyses. Results: CML-HSA showed a stimulatory effect on inflammatory gene expression, and astaxanthin reduced the expression by at least two-fold. The levels of autoinflammatory gene expression were reduced by astaxanthin. The RANKL-induced osteoclastogenesis was significantly inhibited by astaxanthin, with reductions in the activation of nuclear factor-κB (NF-κB), the expression of NFATc1 (nuclear factor of activated T cells 1), multinucleated cell formation, and the expression of mature osteoclast marker genes. Conclusion: Astaxanthin has potential as a remedy for CML-HSA-induced inflammation and RANKL-induced excessive bone loss.

Chromatin-Independent Interplay of NFATc1 and EZH2 in Pancreatic Cancer.

Background: The Nuclear Factor of Activated T-cells 1 (NFATc1) transcription factor and the methyltransferase Enhancer of Zeste Homolog 2 (EZH2) significantly contribute to the aggressive phenotype of pancreatic ductal adenocarcinoma (PDAC). Herein, we aimed at dissecting the mechanistic background of their interplay in PDAC progression. Methods: NFATc1 and EZH2 mRNA and protein expression and complex formation were determined in transgenic PDAC models and human PDAC specimens. NFATc1 binding on the Ezh2 gene and the consequences of perturbed NFATc1 expression on Ezh2 transcription were explored by Chromatin Immunoprecipitation (ChIP) and upon transgenic or siRNA-mediated interference with NFATc1 expression, respectively. Integrative analyses of RNA- and ChIP-seq data was performed to explore NFATc1-/EZH2-dependent gene signatures. Results: NFATc1 targets the Ezh2 gene for transcriptional activation and biochemically interacts with the methyltransferase in murine and human PDAC. Surprisingly, our genome-wide binding and expression analyses do not link the protein complex to joint gene regulation. In contrast, our findings provide evidence for chromatin-independent functions of the NFATc1:EZH2 complex and reveal posttranslational EZH2 phosphorylation at serine 21 as a prerequisite for robust complex formation. Conclusion: Our findings disclose a previously unknown NFATc1-EZH2 axis operational in the pancreas and provide mechanistic insights into the conditions fostering NFATc1:EZH2 complex formation in PDAC.

Nanog/NFATc1/Osterix signaling pathway-mediated promotion of bone formation at the tendon\u2013bone interface after ACL reconstruction with De-BMSCs transplantation

BackgroundBone formation plays an important role in early tendon–bone healing after anterior cruciate ligament reconstruction (ACLR). Dedifferentiated osteogenic bone marrow mesenchymal stem cells (De-BMSCs) have enhanced osteogenic potential. This study aimed to investigate the effect of De-BMSCs transplantation on the promotion of bone formation at the tendon–bone interface after ACLR and to further explore the molecular mechanism of the enhanced osteogenic potential of De-BMSCs.MethodsBMSCs from the femurs and tibias of New Zealand white rabbits were subjected to osteogenic induction and then cultured in medium without osteogenic factors; the obtained cell population was termed De-BMSCs. De-BMSCs were induced to undergo osteo-, chondro- and adipo-differentiation in vitro to examine the characteristics of primitive stem cells. An ACLR model with a semitendinosus tendon was established in rabbits, and the animals were divided into a control group, BMSCs group, and De-BMSCs group. At 12 weeks after surgery, the rabbits in each group were sacrificed to evaluate tendon–bone healing by histologic staining, micro-computed tomography (micro-CT) examination, and biomechanical testing. During osteogenic differentiation of De-BMSCs, an siRNA targeting nuclear factor of activated T-cells 1 (NFATc1) was used to verify the molecular mechanism of the enhanced osteogenic potential of De-BMSCs.ResultsDe-BMSCs exhibited some properties similar to BMSCs, including multiple differentiation potential and cell surface markers. Bone formation at the tendon–bone interface in the De-BMSCs group was significantly increased, and biomechanical strength was significantly improved. During the osteogenic differentiation of De-BMSCs, the expression of Nanog and NFATc1 was synergistically increased, which promoted the interaction of NFATc1 and Osterix, resulting in increased expression of osteoblast marker genes such as COL1A, OCN, and OPN.ConclusionsDe-BMSCs transplantation could promote bone formation at the tendon–bone interface after ACLR and improve the biomechanical strength of the reconstruction. The Nanog/NFATc1/Osterix signaling pathway mediated the enhanced osteogenic differentiation efficiency of De-BMSCs.

NFATc1 promotes epithelial-mesenchymal transition and facilitates colorectal cancer metastasis by targeting SNAI1

Metastatic recurrence remains a major cause of colorectal cancer (CRC) mortality. In this study, we investigated the mechanistic role of nuclear factor of activated T cells 1 (NFATc1) in CRC metastasis. First, we explored the potential role of NFATc1 in CRC using bioinformatics and hypothesized that NFATc1 might play different roles at different stages of CRC development. Then, we examined the relative expression of NFATc1 in 25 CRC tissues and adjacent normal tissues, and further analyzed the correlation between NFATc1 expression levels and clinical stages in 120 CRC patients. The role of NFATc1 in CRC metastasis and the molecular mechanisms were investigated in both in vitro and in vivo models. Our results showed that the expression of NFATc1 was increased in metastatic CRC tissues and positively associated with clinical stages (stage I vs. stage II, III or IV) of CRC. Overexpression of NFATc1 promoted CRC cell migration, invasion, and epithelial-mesenchymal transition (EMT). Moreover, SNAI1 was verified as the direct transcriptional target of NFATc1 and interacted with SLUG to promote EMT. Remarkably, our lung and liver metastasis mouse model demonstrated that NFATc1 overexpression accelerated CRC metastasis, and treatment with FK506, a calcineurin-NFAT pathway inhibitor, could suppress CRC metastasis in vivo. Taken together, our findings suggest that NFATc1 could transcriptionally activate SNAI1, which in turn interacts with SLUG to mediate EMT to promote CRC metastasis. Thus, making NFATc1 a promising therapeutic target in the treatment of metastatic CRC.

RNA-binding protein CELF2 inhibits breast cancer cell invasion and angiogenesis by downregulating NFATc1.

Breast cancer constitutes a major cause of morbidity and mortality among women in China and worldwide. The aim of the present study was to investigate whether CUGBP Elav-like family member 2 (CELF2) could inhibit breast cancer cell invasion and angiogenesis by downregulating nuclear factor of activated T cells 1 (NFATc1) expression. The expression of CELF2 and NFATc1 in breast cancer cells and tissues was detected by reverse transcription-quantitative PCR analysis. H&E staining was used to assess the number of microvessels in tumor tissue. The expression of proteins associated with invasion and angiogenesis and NFATc1 in tumor tissues and transfected cells was examined by western blotting. RNA pull-down assay was used to verify the interaction between CELF2 and NFATc1. Cell proliferation, invasion and tube-forming ability was analyzed using Cell Counting Kit-8, Transwell and HUVEC tube formation assays, respectively. CELF2 expression was found to be decreased in breast cancer cells, whereas CELF2 overexpression suppressed the proliferation and invasion of breast cancer cells and inhibited tumor growth and angiogenesis. Furthermore, CELF2 overexpression decreased the expression of N-cadherin (N-cad), CD34 and NFATc1 in tumor tissues, whereas NEAFc1 overexpression increased the expression of N-cad and NFATc1 in MCF cells transfected with OverExp-CELF2. CELF2 was found to be inversely associated with NFATc1, and NFATc1 overexpression reversed the effects of CELF2 overexpression. In conclusion, the findings of the present study demonstrated that CELF2 may inhibit breast cancer cell invasion and angiogenesis by downregulating NFATc1.

Oxymatrine Attenuates Osteoclastogenesis via Modulation of ROS-Mediated SREBP2 Signaling and Counteracts Ovariectomy-Induced Osteoporosis.

Osteoporosis, mainly caused by osteoclast-induced bone resorption, has become a major health problem in post-menopausal women and the elderly. Growing evidence indicates that inhibiting osteoclastogenesis is an efficient approach to develop alternative therapeutic agents for treating osteoporosis. In this study, we identified the potential regulating role of Oxymatrine (OMT), a quinazine alkaloid extracted from Sophora flavescens with various therapeutic effects in many diseases, on osteoclastogenesis for the first time. We found that OMT attenuated RANKL-induced osteoclast formation in both time- and dose-dependent manners. Further, OMT significantly suppressed RANKL-induced sterol regulatory element-binding protein 2 (SREBP2) activation and the expression of the nuclear factor of activated T cells 1 (NFATc1). Moreover, OMT inhibited the generation of RANKL-induced reactive oxygen species (ROS), and the upregulation of ROS could rescue the inhibition of SREBP2 by OMT. More importantly, ovariectomy (OVX) mouse model showed that OMT could effectively improve ovariectomy (OVX)-induced osteopenia by inhibiting osteoclastogenesis in vivo. In conclusion, our data demonstrated that OMT impaired ROS mediated SREBP2 activity and downstream NFATc1 expression during osteoclastogenesis, suppressed OVX-induced osteopenia in vivo, which suggested that OMT could be a promising compound for medical treatment against osteoporosis.

Knockdown of Tcirg1 inhibits large-osteoclast generation by down-regulating NFATc1 and IP3R2 expression.

The TCIRG1 gene encodes the a3 isoform of vacuolar H+-ATPase (V-ATPase), which forms a proton transport channel in osteoclasts. Defects in this gene lead to functional impairment of osteoclasts and increased bone mass; however, the molecular mechanisms of TCIRG1 loss have not been fully elucidated. In the current study, we transfected mouse bone marrow-derived monocytes with control or Tcirg1-knockdown lentiviruses to further investigate the mechanisms of TCIRG1. Our results demonstrate that knockdown of Tcirg1 inhibits large-osteoclast (>100 μm) generation by decreasing the expression of nuclear factor of activated T-cells 1 (NFATc1) and inositol-1,4,5-trisphosphate receptor 2 (IP3R2). The decreased IP3R2 reduces intracellular calcium levels, which limits the nuclear translocation of NFATc1 in RANKL-induced mouse bone marrow-derived monocytes. These findings provide a mechanism to explain the effects of TCIRG1 impairment, with potential implications for the development of therapies for osteopetrosis.

Abstract 3142: Stress regulated role of lncRNA Malat1 in colorectal cancer progression and metastasis

Abstract Background: Colorectal carcinoma (CRC) is the second leading cause of cancer related deaths in the United States. The five-year survival rate of patients diagnosed with distant stages declines to 14%, which is of concern when compared to 90% for localized-stage and 71% for regional stage disease. This necessitates the need for early diagnostic biomarkers, to minimize poor drug response/resistance, recurrence, metastasis and mortality. Disproportionate biochemical stressors increase the risk of developing CRC and its progression by influencing molecular drivers within the coding and noncoding parts of the genome. Thus, understanding the biological mechanism of these stress factors on the molecular drivers of this disease can provide pivotal information pertinent to CRC development and progression. Recently, our laboratory has identified a novel long noncoding RNA (lncRNA) namely, Metastasis Associated Lung Adenocarcinoma Transcript 1 (MALAT1), which is highly over-expressed in CRC and is involved in its pathogenesis and is regulated by transcription factor Nuclear Factor of Activated T cell 1 (NFATc1). Methods: Archived human CRC tissues were stained using a recently standardized Z-probe technology. TCGA database of ~600 CRC patients was also analyzed using the bioinformatic approach. CRC cell lines were profiled for MALAT1 expression using RT-PCR. Lentiviral constructs were used to generate stable lncRNA MALAT1 expressing cell lines. CRISPR/Cas9 constructs were used to knockdown lncRNA MALAT1 and NFATc1. Mouse model was used to verify the stress induced expression of MALAT1 and NFATc1. ReCLP (Reversible Cross-Linked Precipitation) and iRAP (invitro RNA Antisense Proteomics) studies are in progress to identify the associated proteins and complexes. Results: RNAScope analysis showed MALAT1 to be highly over-expressed in human CRC tissues. MALAT1 expression increased with stage and negatively correlated with the tumor size. TCGA database analysis confirmed our findings. Multiple NFATc1 binding site were identified by ChIPseq database. Overexpression of transcription factor NFATc1 upregulated lncRNA MALAT1 expression. CRISPR/Cas9 based knockdown of NFATc1 downregulated NFATC1 and lncRNA MALAT1, but vice versa was not true, indicating NFATc1 to be upstream of lncRNA MALAT1. Also, expression of MALAT1, NFATc1 and IL-6 were highly upregulated by biochemical stressor cortisol in mouse colonic tissues. Further studies are in progress for direct association of NFATc1 on MALAT1 promoter and mechanism by which the stress factor regulate NFATc1 expression and hence lncRNA MALAT1 expression. Conclusion: This study helps to understand influence of biochemical stress factors on long noncoding RNA MALAT1 and transcription factor NFATc1 etiology. Early diagnosis of these molecular markers will help in designing novel preventive/therapeutic strategies to reduce CRC progression, metastasis and hence mortality. Citation Format: Kyle Doxtater, Chidi Zacheaus, Radhika Sekhri, Utkarsh K. Mishra, Zachary E. Stiles, Nitish Mishra, Chittibabu Guda, Nadeem Zafar, Mahul Amin, Pradeep Shukla, Murali M. Yallapu, Meena Jaggi, Subhash C. Chauhan, Manish K. Tripathi. Stress regulated role of lncRNA Malat1 in colorectal cancer progression and metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3142.

MiR‐506‐3p alleviates uncontrolled osteoclastogenesis via repression of RANKL/NFATc1 signaling pathway

Bone erosion is the major cause of deformities in autoimmune disease conditions such as osteoporosis and rheumatoid arthritis. Aberrant receptor activator of nuclear factor kappa B ligand (RANKL) secretion in bone disorders have been implicated to promote uncontrolled osteoclast differentiation through the regulation of nuclear factor of activated T cells 1 (NFATc1) transcription factor. This phenomenon is governed by several molecular factors including microRNAs, which are under-expressed during disease progression. This report focuses on elucidating the molecular mechanism of miR-506-3p towards the RANKL/NFATc1 pathway. miR-506-3p showed high binding affinity towards NFATc1 (ΔG = -22.4 kcal/mol). Bone marrow-derived macrophages (BMMs) isolated from rats stimulated with RANKL (100 ng/ml) showed active expression of NFATc1 which differentiated into mature osteoclasts. Moreover, NFATc1 activation resulted in downstream secretion of various bone resorptive enzymes (cathepsin K, carbonic anhydrase II, tartarate acid phosphatase, and matrix metalloproteinase 9) which lead to active bone resorption. However, transfection of miR-506-3p resulted in selective repression of NFATc1 inside the cells. This further resulted in the diminished release of bone resorptive enzymes that were essential for the degradation of the bone. Overall, we predict that miR-506-3p can be used as a molecular intervention for RANKL/NFATc1 mediated osteoclastogenesis.

The elevated serum levels of calcineurin and nuclear factor of activated T-cells 1 in children with Kawasaki disease

BackgroundThe calcineurin and nuclear factor of activated T-cells (CaN-NFAT) signaling pathway had been found to be associated with Kawasaki disease (KD) susceptibility and coronary artery aneurysm formation as a contributor. To evaluate serum calcineurin (CaN) and nuclear factor of activated T-cells 1(NFAT1) levels in patients with Kawasaki disease (KD).MethodsSerum levels of CaN and NFAT1 were measured by enzyme-linked immunosorbent assay method in 66 healthy children and 74 KD patients at acute, afebrile and subacute stage.ResultsThe serum levels of CaN and NFAT1 increased significantly in the acute stage, and decreased progressively in the afebrile and subacute stage, along with the reduction of C-reactive protein, white blood cells and neutrophil counts. And in the acute stage, the afebrile stage and the subacute stage, the expression of CaN and NFAT1 was upregulated significantly in KD patients compared to that in the healthy control. After the IVIG treatment, the serum levels of CaN and NFAT1 declined significantly in IVIG responders. However, the CaN and NTAT1 levels in the IVIG non-responders declined slowly. And in the afebrile stage, the NFAT1 levels were lower in KD patients with coronary artery lesions (CALs) (268.82 ± 11.96 ng/ml) than those without CALs (285.84 ± 25.13 ng/ml). However, the serum levels of CaN in KD patients with CALs had no significant difference with those in KD patients without CALs.ConclusionsThe specific regulation of CaN and NFAT1 serum levels in the course of KD was suggested that both of them were related in the development of KD.

Rev-erbα Negatively Regulates Osteoclast and Osteoblast Differentiation through p38 MAPK Signaling Pathway.

The circadian clock regulates various physiological processes, including bone metabolism. The nuclear receptors Rev-erbs, comprising Rev-erbα and Rev-erbβ, play a key role as transcriptional regulators of the circadian clock. In this study, we demonstrate that Rev-erbs negatively regulate differentiation of osteoclasts and osteoblasts. The knockdown of Rev-erbα in osteoclast precursor cells enhanced receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation, as well as expression of nuclear factor of activated T cells 1 (NFATc1), osteoclast-associated receptor (OSCAR), and tartrate-resistant acid phosphatase (TRAP). The overexpression of Rev-erbα leads to attenuation of the NFATc1 expression via inhibition of recruitment of c-Fos to the NFATc1 promoter. The overexpression of Rev-erbα in osteoblast precursors attenuated the expression of osteoblast marker genes including Runx2, alkaline phosphatase (ALP), bone sialoprotein (BSP), and osteocalcin (OC). Rev-erbα interfered with the recruitment of Runx2 to the promoter region of the target genes. Conversely, knockdown of Rev-erbα in the osteoblast precursors enhanced the osteoblast differentiation and function. In addition, Rev-erbα negatively regulated osteoclast and osteoblast differentiation by suppressing the p38 MAPK pathway. Furthermore, intraperitoneal administration of GSK4112, a Rev-erb agonist, protects RANKL-induced bone loss via inhibition of osteoclast differentiation in vivo. Taken together, our results demonstrate a molecular mechanism of Rev-erbs in the bone remodeling, and provide a molecular basis for a potential therapeutic target for treatment of bone disease characterized by excessive bone resorption.

Osteoclastogenesis inhibition by mutated IGSF23 results in human osteopetrosis.

ObjectivesOsteopetrosis is a rare inherited skeletal disease characterized by increased bone mineral density due to the loss of osteoclast function or differentiation potential.Materials and MethodsThe study involved a Chinese patient with osteopetrosis (the proband) and her immediate family members and 180 controls without osteopetrosis. Bone density of the femoral neck, lumbar spine and total body was measured using dual‐energy x‐ray absorptiometry. Osteoclast differentiation by the participants’ peripheral blood mononuclear cells (PBMCs) was investigated using tartrate‐resistant acid phosphatase (TRAP) staining. Osteoblast differentiation was examined with Alizarin Red S staining. Reverse transcription‐quantitative PCR was used to amplify immunoglobulin superfamily member 23 (IGSF23), c‐FOS and nuclear factor of activated T cells 1 (NFATC1).ResultsWe found a homozygous mutation (c.295C>T) in the IGSF23 gene in two osteopetrosis samples. The mutation led to the formation of a stop codon, causing loss of the immunoglobulin‐like domain and the whole transmembrane domain. PBMCs from the proband (IGSF23 −/−) exhibited poor ability for differentiating into mature osteoclasts in vitro. Overexpression of IGSF23 rescued the ability of IGSF23 −/− PBMCs to differentiate into osteoclasts. Moreover, knockdown of IGSF23 reversed the bone loss in OVX mice by injecting AAV‐shIGSF23 into mice femoral bone marrow cavity. Furthermore, we also found that the IGSF23 mutation led to decreased c‐Fos and NFATC1 expression levels by inhibiting the mitogen‐activated protein kinase signalling pathways.ConclusionsIGSF23‐mediated osteoclast differentiation of PBMCs may serve as a potential target in osteoporosis therapy.

Role of the Btk-PLCγ2 Signaling Pathway in the Bone Destruction of Apical Periodontitis.

Chronic apical periodontitis is characterized by alveolar bone absorption in the apical region and is the result of the participation of various inflammatory mediators. Studies have shown that the Bruton tyrosine kinase- (Btk-) phospholipase Cγ2 (PLCγ2) signaling pathway plays an important role in bone absorption, but it is unknown whether it plays a role in apical periodontitis bone destruction. Therefore, this study verified the role of Btk and PLCγ2 in bone resorption of apical periodontitis by in vivo and in vitro experiments. In the in vivo experiment, a mice model of apical periodontitis was established; apical bone resorption was confirmed by the numbers of osteoclasts and HE staining. Btk, PLCγ2, and nuclear factor of activated T-cells 1 (NFATc-1) were detected by immunohistochemical staining. In the in vitro experiment, lipopolysaccharides (LPS) were used to stimulate osteoclast precursor cell RAW264.7 to establish an inflammatory microenvironment and detect osteoclast differentiation. By silencing Btk, the expression of Btk, PLCγ2, and NFATc-1 was detected by real-time qPCR and Western blot, and osteoclastogenesis was detected by enzyme histochemical staining to further confirm the role of Btk in bone resorption. It was found that the expression of Btk, PLCγ2, and NFATc-1 changed significantly with the progression of inflammation and bone destruction, indicating that Btk and PLCγ2 may be involved in the progression of inflammation in apical periodontitis and bone absorption. In vitro experiments confirmed that the differentiation of osteoclasts and the expression of PLCγ2 and NFATc-1 were significantly inhibited after silencing Btk expression, but osteoclast precursor cells could be differentiated due to the proinflammatory factor lipopolysaccharide. This study demonstrates that Btk and PLCγ2 are key factors involved in the apical inflammatory response and bone destruction.

NFAT1-Mediated Regulation of NDEL1 Promotes Growth and Invasion of Glioma Stem-like Cells.

Glioma stem-like cells (GSC) promote tumor generation and progression. However, the mechanism of GSC induction or maintenance is largely unknown. We previously demonstrated that the calcium-responsive transcription factor nuclear factor of activated T cells-1 (NFAT1) is activated in glioblastomas and regulates the invasion of tumor cells. In this study, we further explored the role of NFAT1 in GSC. We found that NFAT1 expression was associated with an aggressive phenotype and predicted poor survival in gliomas. Compared with normal glioma cells, NFAT1 was upregulated in GSC. NFAT1 knockdown reduced GSC viability, invasion, and self-renewal in vitro and inhibited tumorigenesis in vivo, whereas NFAT1 overexpression enhanced the growth and invasion of GSCs. RNA sequencing showed that NFAT1 depletion was associated with reduced neurodevelopment protein 1-like 1 (NDEL1, a potential downstream target of NFAT1) expression, whereas NFAT1 overexpression induced NDEL1 expression. In addition, NFAT1 regulated the promoter activities of NDEL1, whereas rescue of NDEL1 in NFAT1-silenced GSC partially restored tumor growth and invasion. Upregulation of NFAT1-NDEL1 signaling elevated Erk activation, increased protein levels of stemness markers in GSC, and resulted in de-differentiation of normal neuronal cells and astrocytes. Our results indicate that NFAT1 controls the growth and invasion of GSC partially through regulation of NDEL1. Targeting the NFAT1-NDEL1 axis therefore might be of potential benefit in the treatment of patients with glioma. SIGNIFICANCE: NFAT1 controls the growth and invasion of GSCs, partially by regulating NDEL1. Targeting the NFAT1-NDEL1 axis might provide opportunities in treating patients with glioma.