OBJECTIVETo determine the effects of noncoding repressor of NFAT (NRON) overexpression or silencing on human umbilical vein endothelial cells (HUVECs) functions.

METHODSStable HUVECs cell lines with NRON overexpression and short hairpin RNA (shRNA) interference were obtained. HUVECs, the empty vector pBABE-cell line and the empty vector pSuper-cell line served as controls. Cell proliferations of these cell lines were tested using MTS method, tube formation capacity and migration function were also examined.

RESULTSMTS experiments evidenced dose-dependent cells proliferations in all cell lines after 48 h culture with fetal bovine serum (HUVECs, r = 0.91;pBABE empty vectors cell-line, r = 0.88;NRON overexpression cell-line, r = 0.89;pSuper empty vectors cell-line, r = 0.95;shRNA infererence cell-line, r = 0.97). Proliferation capacity was lower in NRON overexpressed HUVECs and was higher in NRON silencing HUVECs compared with pBABE empty vectors treated and normal HUVECs (all P < 0.05). Tube formation and migration functions were also reduced in NRON overexpressed HUVECs [(8.33 ± 0.12) roots, (1857 ± 65) cells] and increased in shRNA infererence of NRON treated HUVECs [(36.00 ± 0.51) roots, (6987 ± 50) cells] compared with pBABE empty vectors treated HUVECs [(19.67 ± 1.42) roots, (4411 ± 117) cells], pSuper empty vectors treated HUVECs [(17.33 ± 2.93) roots, (3883 ± 109) cells] and normal HUVECs [(23.33 ± 3.01) roots, (5145 ± 72) cells, all P < 0.05].

CONCLUSIONNRON overexpression could reduce and NRON silencing could increase proliferation, tube formation and migration capacities of HUVECs.

Cell Line ; Cell Proliferation ; Human Umbilical Vein Endothelial Cells ; cytology ; Humans ; NFATC Transcription Factors ; genetics ; RNA, Long Noncoding ; genetics

OBJECTIVE: To construct a luciferase reporter gene vector carrying human nuclear factor of activated T cells 2 (NFATc2) gene promoter and examine the effects of metformin and lipopolysaccharide (LPS) on the transcriptional activity of NFATc2 gene. METHODS: The promoter sequence of human NFATc2 gene was acquired from UCSC website for PCR amplification. NFATc2 promoter fragment was inserted into pGL3-basic plasmid double cleaved with Kpn Ⅰ and Hind Ⅲ. The resultant recombinant plasmid pGL3-NFATC2-promoter was co-transfected with the internal reference plasmid pRL-TK in 293F cells, and luciferase activity in the cells was detected. Reporter gene vectors of human NFATc2 gene promoter with different fragment lengths were also constructed and assayed for luciferase activity. The changes in transcription activity of NFATc2 gene were assessed after treatment with different concentrations of metformin and LPS for 24 h. We also examined the effect of mutation in RUNX2-binding site in NFATC2 gene promoter on the regulatory effects of metformin and LPS on NFATc2 transcription. RESULTS: We successfully constructed pGL3-NFATc2-promoter plasmids carrying different lengths (2170 bp, 2077 bp, 1802 bp, 1651 bp, 1083 bp, 323 bp) of NFATc2 promoter sequences as verified by enzymatic digestion and sequencing. Transfection of 293F cells with the plasmid carrying a 1651 bp NFATc2 promoter (pGL3-1651 bp) resulted in the highest transcriptional activity of NFATc2 gene, and the luciferase activity was approximately 3.3 times that of pGL3-2170 bp (1.843 ± 0.146 vs 0.547 ± 0.085). Moderate (5 mmol/L) and high (10 mmol/L) concentrations of metformin significantly upregulated the transcriptional activity of pGL3-1651 bp by up to 2.5 and 3 folds, respectively. LPS at different doses also upregulated the transcriptional activity of pGL3-1651 bp by at least 1.6 folds. The mutation in the RUNX2 binding site on pGL3-1651 bp obviously reduced metformin- and LPS-induced enhancement of pGL3-1651bp transcription by 1.7 and 2 folds, respectively. CONCLUSION pGL3-NFATc2-promoter can be transcribed and activated in 293F cells, and LPS and metformin can activate the transcription of pGL3- NFATc2-promoter in a RUNX2-dependent manner.
Core Binding Factor Alpha 1 Subunit/genetics* ; Humans ; Lipopolysaccharides/pharmacology* ; Luciferases/genetics* ; Metformin/pharmacology* ; NFATC Transcription Factors/genetics* ; Promoter Regions, Genetic ; T-Lymphocytes ; Transcription, Genetic/drug effects* ; Transfection

BACKGROUNDInterleukin-13 (IL-13) is recognized to be a key modulator in the pathogenesis of Th2-induced allergic inflammation. Transcription factors GATA3 and NFAT1 have been both implicated in the regulation of Th2 cytokines. We previously demonstrated the GATA3-NFAT1 association during human T cell activation. However, the function of the GATA3-NFAT1 complex in Th2 cytokines regulation is still unknown. Small interference RNA (siRNA) was constructed to knock down GATA3 expression in Hut-78 cells to investigate the possible role of GATA3-NFAT1 complex in IL-13 transcription.

METHODSCells were stimulated with anti-CD3 plus anti-CD28 antibodies to mimic in vivo antigen-mediated co-stimulation; the expression of IL-13 mRNA was determined by real-time PCR; chromation immunoprecipitation (CHIP) assay was employed to investigate the NFAT1 binding to IL-13 promoter.

RESULTSGATA3 siRNA suppressed the expression of GATA3 both in mRNA and protein levels in Hut-78 cells. The binding of NFAT1 to IL-13 promoter was inhibited by GATA3 siRNA in activated T cells, which was followed by the reduction of IL-13 transcription.

CONCLUSIONGATA3-NFAT1 complex may play an important role in the regulation of IL-13 transcription in human T cells.

Cells, Cultured ; GATA3 Transcription Factor ; antagonists & inhibitors ; genetics ; Humans ; Interleukin-13 ; genetics ; NFATC Transcription Factors ; metabolism ; Promoter Regions, Genetic ; RNA, Small Interfering ; genetics ; T-Lymphocytes ; metabolism ; Transfection

OBJECTIVETo study the effect of ultra high molecular weight polyethylene (UHMWPE) particles on macrophages and evaluate the expression of NFAT2, a key transcriptional factor for osteoclast differentiation.

METHODSFrom November 2004 to February 2005, macrophages were co-cultured with UHMWPE particles. When observed at different times, the proliferation activity of macrophages was analyzed by MTT and the expression of calcineurin (CaN) and NFAT2 by immunohistochemical and RT-PCR method respectively.

RESULTSThe macrophages phagocytosed UHMWPE particles in an early time, the expression of CaN and NFAT2 was increased, while the proliferation activity was not enhanced.

CONCLUSIONSUHMWPE particles can stimulate macrophages to phagocytose significantly, and enhance the expression of the transcriptional factor NFAT2.

Animals ; In Vitro Techniques ; Joint Prosthesis ; Macrophages ; drug effects ; physiology ; Mice ; NFATC Transcription Factors ; biosynthesis ; genetics ; Phagocytosis ; drug effects ; Polyethylenes ; pharmacology ; RNA, Messenger ; genetics

OBJECTIVETo explore whether hepatitis C virus core protein (HCV C) regulates the expression of NFAT1 to participate in the progression and malignant biological behavior of intrahepatic cholangiocarcinoma cells.

METHODSThe recombinant plasmid pEGFP-N(3)-HCV C and the empty vector pEGFP-N(3) were cotransfected with enhanced green fluorescent protein (EGFP) into RBE cells using liposome. Real-time PCR and Western blotting were used to examine the expression of NFAT1 mRNA and protein in the transfected RBE cells. MTT assay was used to evaluate the changes in the cell proliferation, and the cell cycle changes were analyzed by flow cytometry.

RESULTSHCV C transfection significantly enhanced the expressions of NFAT1 mRNA and protein in RBE cells (P<0.05) and promoted the progression of cell cycle into G(2)/M phase to accelerate the cell proliferation.

CONCLUSIONTransfection with HCV C gene up-regulates NFAT1 expression and promotes the cell cycle progression and proliferation of intrahepatic cholangiocarcinoma cells, suggesting the involvement of HCV C in the progression of intrahepatic cholangiocarcinoma.

Bile Duct Neoplasms ; pathology ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Cholangiocarcinoma ; pathology ; Gene Expression ; Humans ; NFATC Transcription Factors ; genetics ; Plasmids ; Transfection ; Viral Core Proteins ; genetics

OBJECTIVEThis study investigated the role and mechanism of calcineurin (CaN)-nuclear factor of activated T cells (NFAT) pathway in the myoblast apoptosis induced by cyclic tensile strain.

METHODSMyoblasts were cultured using an in vitro-mechanical stimulation model and imposed with tension for different hours with a multi-channel cell stress loading system. Cyclosporine (CsA) was used as CaN inhibitor to clarify the role of CaN in the apoptosis induced by cyclic stress. Hochest 33258 staining and flow cytometry detection were performed to detect the apoptotic cells. Real-time polymerase chain reaction was conducted to detect the mRNA expression of CaN and NFAT. Protein levels of NFAT3 were evaluated by Western blot.

RESULTSThe apoptosis rate increased with the extension of loading time. The mRNA expression of the CaN subunits, CnA and CnB, and the protein levels of NFAT3 also increased. When the myoblasts were incubated with CsA, the apoptosis rate decreased, the mRNA expression of CnA and NFAT3 significantly decreased, and the NFAT3 protein expression levels became significantly lower than those of the groups without CsA.

CONCLUSIONContinuous cyclic tensile stress can induce myoblast apoptosis. The CaN-NFAT signaling pathway may be involved in the cyclic stretch-induced apoptosis of myoblasts.

Apoptosis ; Calcineurin ; genetics ; Cyclosporine ; Flow Cytometry ; Myoblasts ; physiology ; NFATC Transcription Factors ; metabolism ; Real-Time Polymerase Chain Reaction ; Signal Transduction ; T-Lymphocytes

OBJECTIVETo investigate the effect of OX40/OX40L interaction on the nuclear factor of activated T cells c1 (NFATc1) in ApoE-/- mice.

METHODSLymphocytes were prepared from mouse spleens after Collar-treated Surgery, then incubated with a range of agonistic anti-OX40 mAbs and inhibitory anti-OX40L mAb to stimulate or inhibit OX40-OX40L interaction in vitro. The expression of NFATc1 mRNA and protein in lymphocytes of ApoE-/- mice was measured by Real Time PCR and flow cytometry, respectively.

RESULTS(1) After stimulating OX40-OX40L signal pathway, the expression of NFATc1 mRNA and protein in leukocytes of ApoE-/- mice was significantly increased, with maximal effect occurring at 20 µg/ml anti-OX40 mAb-stimulated, and peaked at 24 h at any concentration (P < 0.01). (2) Anti-OX40L mAb significantly suppressed the expression of NFATc1 in leukocytes of ApoE-/- mice, with maximal effect occurring at 20 µg/ml anti-OX40L mAb, and peaked at 24 h (P < 0.001).

CONCLUSIONSOX40-OX40L interaction can regulate the mRNA and protein expressions of NFATc1 in lymphocytes of ApoE-/- mice.

Animals ; Apolipoproteins E ; genetics ; Atherosclerosis ; metabolism ; pathology ; Female ; Lymphocyte Activation ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; NFATC Transcription Factors ; metabolism ; Receptors, OX40 ; metabolism ; T-Lymphocytes ; metabolism

To screen NFAT antagonistic drugs and research signal transduction pathway related to NFAT. Four recombinant vectors were constructed. Each consists of three tandem copies of the human IL-2 distal NFAT-AP1 binding site in the context of the minimal IL-2 enhancer, either the sequence from -326 - +46 or the sequence from -89 - +46 (containing only the TATA box), driving a luciferase reporter gene or a destabilized enhanced green fluorescence protein (d2EGFP) reporter gene, respectively. Transient transfection of Jurkat cells was achieved by electroporation with 5 - 10 microg of the above plasmid and one pulse at 200V, 65ms. Plasmid pEFBos-mNFAT1 constitutively expressing murine full length NFAT1 protein was used for transient cotransfection. The results showed that neither of non-stimulation nor PMA or ionomycin stimulation alone could activate the reporter gene except PMA plus ionomycin costimulation. Furthermore, overexpressed murine NFAT1 augmented the activation of either IL-2 promoter or NFAT-AP1 enhancer drived reporter gene compared to the endogenous did. However, the reporter gene expression was nearly completely inhibited by pretreatment for 1h with FK506 at 5 microg/mL and then stimulation for 6-12h with PMA plus ionomycin in the presence of FK506. These findings indicated that such a transient Jurkat cell model offered a potential platform for preliminary screening of FK506 or CsA-like immunosuppressive agents.
Animals ; Drug Evaluation, Preclinical ; Enhancer Elements, Genetic ; genetics ; Green Fluorescent Proteins ; genetics ; Humans ; Immunosuppressive Agents ; pharmacology ; Interleukin-2 ; genetics ; Jurkat Cells ; Luciferases ; genetics ; metabolism ; Mice ; Models, Biological ; NFATC Transcription Factors ; genetics ; metabolism ; Promoter Regions, Genetic ; Signal Transduction ; Tacrolimus ; pharmacology

Animals ; Calcineurin ; genetics ; physiology ; Cardiomegaly ; genetics ; physiopathology ; prevention & control ; Cyclosporine ; pharmacology ; therapeutic use ; Gene Expression ; Humans ; Immunosuppressive Agents ; pharmacology ; therapeutic use ; NFATC Transcription Factors ; genetics ; physiology ; Signal Transduction ; drug effects ; genetics ; physiology ; Tacrolimus ; pharmacology ; therapeutic use

BACKGROUNDHow the transcriptional factors regulated the innate and adaptive immune system in pregnancy and pre-eclampsia are less understood. Nevertheless, what the plasma work in the development of this disease was not sure. The present study was design to evaluate what the transcriptional factors change in innate and adaptive immune system and what the plasma do in this filed.

METHODSPeripheral blood mononuclear cells (PBMC) from non-pregnant women (n = 18), women with clinically normal pregnancies (n = 23) and women with pre-eclampsia (n = 20) were separated from peripheral blood to isolate monocytes and T cells. The purity of monocytes and T cells were analysed by flow cytometry. Monocytes and T cells were stimulated in either lipopolysaccharides (LPS) or phorbol-myristate-acetate (PMA), respectively. Transcription Factor Arrays were used to screen the transcription factors of interest in comparing of different groups. PBMC were isolated from another 8 non-pregnant samples were co-incubated with different groups of plasma. Polymerase chain reaction (PCR) was performed using whole cell extractions of the samples.

RESULTSNuclear factor of activated T-cells-1 (NFAT-1), signal transducers and activators of transcription-1 (STAT-1) and activator protein-1 (AP-1) are up-regulated in monocytes in pregnancy and more so in pre-eclampsia. On the the contrary, NFAT-1, STAT-1 and AP-1 are down-regulated in T cells in pregnancy and more so in pre-eclampsia. A reduction was observed in interferon (IFN)-γ, interleukin (IL)-12 and IL-4 expression in T cells incubated with pre-eclamptic plasma. An elevation was observed in tumor necrosis factor (TNF)-α, IL-1 and IL-12 expression in monocytes incubated with pre-eclamptic plasma.

CONCLUSIONSInnate immunity is over activated and adaptive immunity is over suppressed in the development of pre-eclampsia. NFAT-1, STAT-1 and AP-1 might be the central transcription factors in the pathogenesis of pre-eclampsia. They induced some changes in plasma and "educate" the monocytes and T cells for relevant cytokine production. Successful completion of this study will enhance our understanding of pre-eclampsia and will discover new knowledge beyond pregnancy. The work will inform future therapies for the treatment of a wide range of condition such as transplantation immunology and a wide range of immune and inflammatory conditions.

Adult ; Female ; Humans ; Immunity, Innate ; physiology ; Interferon-gamma ; metabolism ; Interleukin-12 ; metabolism ; Interleukin-4 ; metabolism ; Leukocytes, Mononuclear ; metabolism ; Male ; NFATC Transcription Factors ; genetics ; metabolism ; Pre-Eclampsia ; immunology ; metabolism ; Pregnancy ; STAT1 Transcription Factor ; genetics ; metabolism ; Transcription Factor AP-1 ; genetics ; metabolism ; Transcription Factors ; genetics ; metabolism ; Young Adult