Humans ; Sarcoma ; NFATC Transcription Factors ; RNA-Binding Protein EWS

OBJECTIVETo study the expression of Foxp3 and NFAT1 protein in peripheral blood (PB) in children with aplastic anemia (AA) and their roles in the pathogenesis of AA.

METHODSThe expression levels of Foxp3 and NFAT1 protein of mononuclear cells in PB were measured by Western blot in 68 children with AA before and after treatment and in 60 normal children (control group). The correlation between Foxp3 and NFAT1 protein expression and the correlation of the Foxp3 and NFAT1 protein expression with blood Hb, WBC and platelet levels were analyzed.

RESULTSThe expression levels of Foxp3 and NFAT1 protein in PB in the acute phase in the AA group were significantly lower than in the control group (P<0.05). After treatment (recovery phase) the expression levels of Foxp3 and NFAT1 protein increased obviously compared with those in the acute phase (P<0.05). The Foxp3 protein level was positively correlated with the NFAT1 protein level (r=0.812, P<0.05). Both the Foxp3 and NFAT1 protein levels were positively correlated with blood Hb, WBC and platelet levels in children with AA in the recovery phase (r=0.537, 0.579, 0.655 respectively; P<0.05).

CONCLUSIONSThe Foxp3 and NFAT1 protein levels in PB are reduced in children with AA, suggesting that they are involved in the pathogenesis of AA. The measurement of Foxp3 and NFAT1 protein levels may be useful in the severity evaluation of AA.

Adolescent ; Anemia, Aplastic ; blood ; etiology ; Child ; Child, Preschool ; Female ; Forkhead Transcription Factors ; blood ; Humans ; Male ; NFATC Transcription Factors ; blood

PURPOSE: In order to determine whether the Tonicity responsive enhancer binding protein (TonEBP) is expressed by hypertonic and hyperosmolar stress, TonEBP expression was investigated in the retinal ganglion cell (RGC) line, RGC-5 cells. METHODS: After RGC-5 cells were cultured by Staurosporine, TonEBP expression was measured with Western immunoblotting analysis and real-time reverse transcription-polymerase chain reaction in 50 mM NaCl, 100 mM mannitol, 50 mM glucose, or 100 mM glucose at 3, 6, 12, and 24 hours after exposure to each environment. RESULTS: In this study, the protein expression of TonEBP was determined to be statistically significantly checked in 50 mM NaCl after 3, and 6 hours, in 100 mM mannitol after 6 hours, and in 100 mM glucose after 3, and 6 hours. TonEBP messenger Ribonucleic acid (mRNA) expression was determined to be statistically significantly checked in 50 mM NaCl after 3 hours, in 100 mM mannitol after 3, and 24 hours, and in 50 mM glucose after 3, and 24 hours. CONCLUSIONS: These results suggested that TonEBP was expressed by hypertonic and hyperosmolar stress at the protein and mRNA levels. Further studies are nedded to determine the role of TonEBP and the mechanism of expression and regulation of TonEBP.
Blotting, Western ; Glucose ; Mannitol ; NFATC Transcription Factors ; Osmotic Pressure ; Retinal Ganglion Cells ; RNA ; RNA, Messenger ; Staurosporine

Calcineurin ; metabolism ; Cardiovascular System ; growth & development ; metabolism ; Humans ; NFATC Transcription Factors ; metabolism ; Signal Transduction

OBJECTIVE: To explore the mechanism by which simvastatin (SIM) regulates osteoclast apoptosis. METHODS: Murine macrophage RAW264.7 cells were divided into 5 groups, namely group A (control group), group B (sRANKL+ M-CSF), group C (SIM+sRANKL+M-CSF), group D (VIVIT peptide+sRANKL+ M-CSF), and group E (SIM+VIVIT peptide+sRANKL+M-CSF). WST-1 assay was used to assess the effects of simvastatin on the proliferation activity of the osteoclasts, and flow cytometry was performed to analyze the effects of SIM and VIVIVIT peptide (a NFATc1 pathway inhibitor) on apoptosis of the osteoclasts. The translocation of NFATc1 into the nucleus was investigated using immunofluorescence assay, and Western blotting was employed to assess the effect of SIM on the phosphorylation of NFATc1 in the nucleus. RESULTS: WST-1 assay showed that SIM (1×10 mol/L) treatment for 24 and 48 h significantly inhibited the proliferation of the osteoclasts (=0.039 and 0.022, respectively). Compared with the control group, the SIM-treated osteoclasts exhibited significantly reduced cell percentage in G0/G1 phase (=0.041) and increased cells in sub-G1 phase (=0.028) with obvious cell apoptosis. DAPI staining and flow cytometry showed that both SIM and VIVIVIT peptide alone significantly promoted osteoclast apoptosis (=0.002 and 0.015, respectively), and their combination produced a similar pro-apoptosis effect (=0.08). Immunofluorescence and Western blotting showed that SIM significantly inhibited the intranuclear translocation of NFATc1 and the phosphorylation of NFATc1 pathway protein (=0.013). CONCLUSIONS SIM promotes osteoclast apoptosis through NFATc1 signaling pathway.
Animals ; Apoptosis ; Cell Differentiation ; Mice ; NFATC Transcription Factors ; Osteoclasts ; RANK Ligand ; Simvastatin

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

This study examined the anti-osteoclastogenic effects of baicalin on receptor activator of NF-kB ligand (RANKL)-induced RAW264.7 cells. Baicalin is a flavonoid that is produced by Scutellaria baicalensis and is known to have multiple biological properties, including antibacterial, anti-inflammatory and analgesic effects. The effects of baicalin on osteoclasts were examined by measuring 1) cell viability; 2) the formation of tartrate-resistant acid phosphatase (TRAP) (+) multinucleated cells; 3) RANK/RANKL signaling pathways and 4) mRNA levels of osteoclast-associated genes. Baicalin inhibited the formation of RANKL-stimulated TRAP (+) multinucleated cells and also suppressed the RANKL-stimulated activation of p-38, ERK, cSrc and AKT signaling. Baicalin also inhibited the RANKL-stimulated degradation of IkappaB in RAW264.7 cells. In addition, the RANKL-stimulated induction of NFATc1 transcription factors was found to be abrogated by this flavonoid. Baicalin was further found to decrease the mRNA expression of osteoclast-associated genes, including carbonic anhydrase II, TRAP and cathepsin K in the RAW264.7 cells. Our data thus demonstrate that baicalin inhibits osteoclastogenesis by inhibiting the RANKL-induced activation of signaling molecules and transcription factors in osteoclast precursors.
Acid Phosphatase ; Carbonic Anhydrase II ; Cathepsin K ; Flavonoids ; Isoenzymes ; NF-kappa B ; NFATC Transcription Factors ; Osteoclasts ; RNA, Messenger ; Scutellaria baicalensis ; Transcription Factors

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

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

OBJECTIVETo investigate the role of nuclear factor of activated T cells -2 (NFAT2) in release of high mobility protein box-1 (HMGB1) from human monocytic THP-1 cells in vitro.

METHODSThe level of HMGB1 release from THP-1 cells in response to lipopolysaccharide (LPS) stimulation was examined by Western blotting and enzyme-linked immunosorbent assay (ELISA). The effect of LPS stimulation on NFAT2 and HMGB1 interaction in the cytoplasm was observed by immunoprecipitation assay. HMGB1 production and release was detected in cells with specific small interfering RNA (siRNA)-mediated suppression of NFAT2 expression.

RESULTSLPS stimulated HMGB1 release from THP-1 cells. As LPS stimulation prolonged, HMGB1 concentration increased in the cell culture supernatant and decreased in the cytoplasm, and the binding between NFAT2 and HMGB1 was not detected in the cell nuclei. NFAT2 suppression by the siRNA plasmid resulted in increased HMGB1 level in the cell culture supernatant.

CONCLUSIONNFAT2 can inhibit HMGB1 release from THP-1 cells in vitro.

Blotting, Western ; Cell Line ; Cell Nucleus ; Cytoplasm ; metabolism ; Enzyme-Linked Immunosorbent Assay ; HMGB1 Protein ; metabolism ; Humans ; Lipopolysaccharides ; Monocytes ; metabolism ; NFATC Transcription Factors ; metabolism