Aberrant activation of nuclear factor of activated T cell 2 in lamina propria mononuclear cells in ulcerative colitis

AIM:To investigate the role of nuclear factor of activated T cell 2(NFAT2),the major NFAT protein in peripheral T cells,in sustained T cell activation and intractable inflammation in human ulcerative colitis(UC). METHODS:We used two-dimensional gel-electrophoresis, immunohistochemistry,double immunohistochemical staining,and confocal microscopy to inspect the expression of NFAT2 in 107,15,48 and 5 cases of UC, Crohn's disease(CD),non-specific colitis,and 5 healthy individuals,respectively. RESULTS:Up-regulation with profound nucleo- translocation/activation of NFAT2 of lamina propria mononuclear cells(LPMC)of colonic mucosa was found specifically in the affected colonic mucosa from patients with UC,as compared to CD or NC(P<0.001,Kruskal- Wallis test).Nucleo-translocation/activation of NFAT2 primarily occurred in CD8+T,but was less prominent in CD4+T cells or CD20+B cells.It was strongly associated with the disease activity,including endoscopic stage (τ=0.2145,P=0.0281)and histologic grade(τ=0.4167, P<0.001). CONCLUSION:We disclose for the first time the nucleo-translocation/activatin of NFAT2 in lamina propria mononuclear cells in ulcerative colitis.Activation of NFAT2 was specific for ulcerative colitis and highly associated with disease activity.Since activation of NFAT2is implicated in an auto-regulatory positive feedback loop of sustained T-cell activation and NFAT proteins play key roles in the calcium/calcineurin signaling pathways,our results not only provide new insights into the mechanism for sustained intractable inflammation,but also suggest the calcium-calcineurin/NFAT pathway as a new therapeutic target for ulcerative colitis.

NFAT2 is implicated in corticosterone-induced rat Leydig cell apoptosis

Aim： To investigate the activation of the nuclear factor of activated T cells （NFAT） and its function in the corticosterone （CORT）-induced apoptosis of rat Leydig cells. Methods： NFAT in rat Leydig cells was detected by Western blotting and immunohistochemical staining. Cyclosporin A （CsA） was used to evaluate potential involvement of NFAT in the CORT-induced apoptosis of Leydig cells. Intracellular Ca^2＋ was monitored in CORT-treated Leydig cells using Fluo-3/AM. After the Leydig cells were incubated with either CORT or CORT plus CsA for 12 h, the levels of NFAT2 in the nuclei and in the cytoplasm were measured by semi-quantitative Western blotting. The role of NFAT2 in CORT- induced Leydig cell apoptosis was further evaluated by observing the effects of NFAT2 overexpression and the inhibition of NFAT2 activation by CsA on FasL expression and apoptosis. Results： We found that NFAT2 was the predominant isoform in Leydig cells. CsA blocked the CORT-induced apoptosis of the Leydig cells. The intracellular Ca^2＋ level in the Leydig cells was significantly increased after the CORT treatment. The CORT increased the level of NFAT2 in the nuclei and decreased its level in the cytoplasm. CsA blocked the CORT-induced nuclear translocation of NFAT2 in the Leydig cells. Both CORT-induced apoptosis and FasL expression in the rat Leydig cells were enhanced by the overexpression of NFAT2 and antagonized by CsA. Conclusion： NFAT2 was activated in CORT-induced Leydig cell apoptosis. The effects of NFAT2 overexpression and the inhibition of NFAT2 activation suggest that NFAT2 may potentially play a pro-apoptotic role in CORT-induced Leydig cell apoptosis through the up-regulation of FasL.

Cav3.2 channel regulates cerebral ischemia/reperfusion injury:a promising target for intervention

Calcium influx into neurons triggers neuronal death during cerebral ischemia/reperfusion injury.Various calcium channels are involved in cerebral ischemia/reperfusion injury.Cav3.2 channel is a main subtype of T-type calcium channels.T-type calcium channel blockers,such as pimozide and mibefradil,have been shown to prevent cerebral ischemia/reperfusion injury-induced brain injury.However,the role of Cav3.2 channels in cerebral ischemia/reperfusion injury remains unclear.Here,in vitro and in vivo models of cerebral ischemia/reperfusion injury were established using middle cerebral artery occlusion in mice and high glucose hypoxia/reoxygenation exposure in primary hippocampal neurons.The results showed that Cav3.2 expression was significantly upregulated in injured hippocampal tissue and primary hippocampal neurons.We further established a Cav3.2 gene-knockout mouse model of cerebral ischemia/reperfusion injury.Cav3.2 knockout markedly reduced infarct volume and brain water content,and alleviated neurological dysfunction after cerebral ischemia/reperfusion injury.Additionally,Cav3.2 knockout attenuated cerebral ischemia/reperfusion injury-induced oxidative stress,inflammatory response,and neuronal apoptosis.In the hippocampus of Cav3.2-knockout mice,calcineurin overexpression offset the beneficial effect of Cav3.2 knockout after cerebral ischemia/reperfusion injury.These findings suggest that the neuroprotective function of Cav3.2 knockout is mediated by calcineurin/nuclear factor of activated T cells 3 signaling.Findings from this study suggest that Cav3.2 could be a promising target for treatment of cerebral ischemia/reperfusion injury.

Combination of carboxyamidotriazole and 1-Methyl-L-tryptophan has synergistic inhibtory effects on programmed death 1 expression

OBJECTIVE To evaluate whether the IDO1 inhibitor 1-methyl-L-tryptophan(1-MT)combine calcium influx inhibitor carboxyamidotriazole(CAI)could further enhance the suppression of programmed death 1(PD-1)in CD8^+T cells and investigate the curative effect of the combined use.METHODS CD8^+T cells were isolated from normal mice spleen by negative selection using magnetic cell separation.The isolated CD8^+T cells were cultured in RPMI 1640 medium containing 10%FBS and 100 U·mL^(-1)IL-2 and activated by the addition of anti-CD3 and anti-CD28(1 g·L^(-1) each mabs).CD8^+T cells were pretreated for 48 h with drug and the fluo-3 as a marker of intracellular calcium concentration was detected by flow cytometry.The calcineurin(Ca N)levels were assayed with ELISA in CD8^+T cells after 48 h incubation with 10μm CAI.The nuclear translocations of NFAT and AHR were detected by immunofluorescent staining after 48 h of drug treatment.The expression of PD-1 in CD8^+T cells was analyzed by flow cytometry.RESULTS Intracellular fluorescent intensity was markedly debase due to CAI treatment(P<0.01).Meanwhile,the changes of CaN content had a resembled correlation(P<0.01).Immunofluorescence experiment showed that after combination therapy the transfer of NFAT and AHR in nuclear substantially reduced.Flow cytometry revealed that after the combination caused a significant decrease in PD-1 expression in CD8^+T cells.CONCLUSION CAI and 1-MT could inhibit markedly the expression of PD-1 in CD8^+T cells by inhibiting the nuclear translocation of NFAT and AHR,respectively and the combination of them has synergetic effect.

Expression of T cell factor-4 in non-small-cell lung cancer

Background T cell factor 4 (TCF 4) plays an important role in development and carcinogenesis Recently, the role of TCF 4 has been described in colon cancer and other cancers However, whether TCF 4 plays a similar role in lung cancer is unknown To answer this question, we studied the expression of TCF 4 protein and mRNA in nonsmallcell lung cancer (NSCLC) and the relation of TCF 4 expression pattern to histological type and cell differentiation Methods Tissue samples from sixty cases of pathologically diagnosed NSCLC and eight normal tissue samples were obtained between September 2001 and March 2003 Immunohistochemistry was used to investigate the distribution of TCF 4 protein The staining patterns of the tumors were divided into 4 categories: nuclear staining alone or nuclear staining greater than cytoplasmic staining; cytoplasmic staining or cytoplasmic staining greater than nuclear staining; equal nuclear and cytoplasmic staining; no nuclear staining or cytoplasmic staining The integrated optical density (OD) values of all sections were analyzed by UIC MetaMorph image analysis software The expression of TCF 4 mRNA was detected by onestep reverse transcriptionpolymerase chain reaction (RTPCR) The integrated density values of the PCR products were analyzed semiquantitatively Results Immunohistochemistry showed that there was no expression of TCF 4 in normal tissue However, TCF 4 was expressed in 86.7% (52/60) of NSCLC samples, mainly in the nuclei of tumor cells Furthermore, there was a significant difference in TCF 4 localization patterns between squamous cell carcinomas and adenocarcinomas (P<005) The integrated OD values of TCF 4 expression was significantly higher in tumors with moderatepoor cell differentiation than in well differentiated tumors (5163±667 vs 4613±1231, P<001) There was no TCF 4 mRNA expression in normal tissue However, 63.9% (23/36) of carcinoma samples expressed TCF 4 mRNA TCF 4 mRNA expression was significantly higher in tumors with moderatepoor cell differentiation than in well differentiated tumors (P<005) There were no significant differences in mRNA expression in comparison with histological type Conclusions The subcellular distribution of TCF 4 may correlate with NSCLC histological type High expression of TCF 4 mRNA and protein may be associated with the degree of cell differentiation in NSCLC

Liuwei Dihuang pill suppresses metastasis by regulating the wnt pathway and disrupting β-catenin/T cell factor interactions in a murine model of triple-negative breast cancer

OBJECTIVE:To investigate if the Liuwei Dihuang pill(LWDHP)can inhibit metastasis to the liver and lungs in mice bearing triple-negative breast cancer(TNBC),and the molecular mechanism underpinning this action.METHODS:Ninety-nine TNBC bearing-mice were distributed randomly to five groups:control(Con),paclitaxel(PTX),low-dose LWDHP(LLP,2.3 g·kg^-1·d^-1),middle-dose LWDHP(MLP,4.6 g·kg^-1·d^-1)and high-dose LWDHP(HLP,9.2 g·kg^-1·d^-1).The LWDHP were administered(p.o.)to the agonal stage.The morphology of BC cells was observed by hematoxylin&eosin staining.Expression of axin-2,β-catenin,T cell factor(TCF),cyclin-D1 and vascular endothelial growth factor(VEGF)was detected by western blotting or immunofluorescence.β-catenin/TCF-1 interaction was measured using a co-immunoprecipitation assay.RESULTS:After LWDHP treatment,metastasis of BC cells to the lungs and liver was inhibited,expression of axin-2 was increased,expression of TCF-1,β-catenin,cyclin-D1 and VEGF was decreased,andβ-catenin/TCF-1 interaction was disrupted.CONCLUSION:The LWDHP could inhibit metastasis of BC cells to the liver and lungs.The molecular mechanism underlying this action may be regulation of protein expression andβ-catenin/TCF-1 interactions in the Wnt pathway.

Role of the Ca^2＋-Calcineurin-Nuclear Factor of Activated T cell Pathway in Mitofusin-2-Mediated Immune Function of Jurkat Cells

Background：Mitofusin-2 （MFN2）,a well-known mitochondrial fusion protein,has been shown to participate in innate immunity,but its role in mediating adaptive immunity remains poorly characterized.In this study,we explored the potential role of MFN2 in mediating the immune function of T lymphocytes.Methods：We manipulated MFN2 gone expression in Jurkat cells via lentiviral transduction of MFN2 small interfering RNA （siRNA） or full-length MFN2.After transduction,the immune response and its underlying mechanism were determined in Jurkat cells.One-way analysis of variance and Student＇s t-test were performed to determine the statistical significance between the groups.Results：Overexpression of MFN2 enhanced the immune response of T lymphocytes by upregulating Ca2＋ （359.280 ± 10.130 vs.266.940 ± 10.170,P =0.000）,calcineurin （0.513 ± 0.014 vs.0.403 ± 0.020 nmol/L,P =0.024）,and nuclear factor of activated T cells （NFATs） activation （1.040 ± 0.086 vs.0.700 ± 0.115,P =0.005）,whereas depletion of MFN2 impaired the immune function ofT lymphocytes by downregulating Ca2＋ （141.140 ± 14.670 vs.267.060 ± 9.230,P =0.000）,calcineurin （0.054 ± 0.030 nmol/L vs.0.404 ± 0.063 nmol/L,P =0.000）,and NFAT activation （0.500 ± 0.025 vs.0.720 ± 0.061,P =0.012）.Furthermore,upregulated calcineurin partially reversed the negative effects ofMFN2 siRNA on T cell-mediated immunity evidenced by elevations in T cell proliferation （1.120 ± 0.048 vs.0.580 ± 0.078,P =0.040）,interleukin-2 （IL-2） production （473.300 ± 24.100 vs.175.330 ± 12.900 pg/ml,P =0.000）,and the interferon-γ/IL-4 ratio （3.080 ± 0.156 vs.0.953 ± 0.093,P =0.000）.Meanwhile,calcineurin activity inhibitor depleted the positive effects of overexpressed MFN2 on T cells function.Conclusions：Our findings suggest that MFN2 may regulate T cell immune functions primarily through the Ca2＋-calcineurin-NFAT pathway.MFN2 may represent a potential therapeutic target for T cell immune dysfunction-related diseases.

Characterization of Adapter Protein NRBP as a Negative Regulator of T Cell Activation

Adapter proteins can regulate the gene transcriptions in disparate signaling pathway by interacting with multiple signaling molecules, including T cell activation signaling. Nuclear receptor binding protein （NRBP）, a novel adapter protein, represents a small family of evolutionarily conserved proteins with homologs in Caenorhabditis elegans （C. elegans）, Drosophila melanogaster （i）. melanogaster）, mouse and human. Here, we demonstrated that overexpression of NRBP in Jurkat TAg cells specifically impairs T cell receptor （TCR） or phorbol myristate acetate （PMA） /ionomycin-mediated signaling leading to nuclear factor of activated T cells （NFAT） promoter activation. Furthermore, the N-terminal of NRBP is necessary for its regulation of NFAT activation. Finally, we showed that NRBP has minimal effect on both TCR- and PMA-induced CD69 up-regulation in Jurkat TAg cells, which suggests that NRBP may function downstream of protein kinase C （PKC） /Ras pathway.