Regulation of Down Syndrome Critical Region 1 expression by Nuclear Factor of Activated T cells in megakaryocytes

As precursors of platelets, megakaryocytes must fulfil the complex tasks of protein synthesis and platelet assembly. Megakaryocytic dysfunction can lead to neoplastic disorders, such as acute megakaryoblastic leukaemia, an entity with a 500-fold increased incidence in children with Down syndrome (DS). Down Syndrome Critical Region 1 ( DSCR1 ), a member of the calcipressin family of calcineurin inhibitors, is overexpressed in DS, and destabilization of the calcineurin/Nuclear Factor of Activated T cells (NFAT) pathway by overexpression of DSCR1 has been implicated in some of the pathophysiological features of the disease. The roles of NFAT and DSCR1 in megakaryocyte signalling and gene expression, however, are unknown. In this study, we show that calcineurin and NFAT are components of a calcium-induced signalling cascade in megakaryocytes. NFAT activation in megakaryocytes was induced by fibrillar collagen type I and was completely sensitive to the calcineurin inhibitor cyclosporin A. We established DSCR1 as a calcium-induced NFAT target gene in these cells and show that overexpression of DSCR1 in megakaryocytes strongly inhibits NFAT activation as well as NFAT-dependent expression of the Fas ligand gene ( FASLG ). These results suggest that DSCR1 acts as an endogenous feedback inhibitor of NFAT signalling in megakaryocytes, and may have implications for megakaryocytic gene expression in DS.     

Therapeutic Potential of VIVIT,a Selective Peptide Inhibitor of Nuclear Factor of Activated T cells,in Cardiovascular Disorders

Cardiovascular disease is the major cause of death in industrialized nations. Targeted intervention in calcineurin, a calmodulin‐dependent, calcium‐activated phosphatase and its substrate, nuclear factor of activated T cells (NFAT), was demonstrated to be effective in the treatment of cardiovascular diseases. Although effective in the disruption of calcineurin phosphatase activity, cyclosporin A (CsA) and FK506 also resulted in undesired side effects and toxicity, prompting the discovery of VIVIT, a novel peptide inhibitor. VIVIT selectively and potently inhibits calcineurin/NFAT interaction, but does not compromise calcineurin phosphatase activity and non‐NFAT‐mediated signaling. VIVIT displays a favorable therapeutic profile as a potential drug candidate and constitutes a useful tool in exploring calcineurin‐NFAT functionality. This review describes the development of VIVIT peptide as a selective NFAT inhibitor and its application as a therapeutic agent in cardiovascular disorders including cardiac hypertrophy, restenosis, atherosclerosis, and angiogenesis.     

CaN-NFAT信号传导通路参与EPCs凋亡作用的研究

目的:研究钙调神经磷酸酶-活化T细胞核因子(CaN-NFAT)信号传导通路参与内皮祖细胞(EPCs)的凋亡过程。方法:采用密度梯度离心法分离外周血单个核细胞,贴壁培养获得EPCs。实验分为4组:空白对照组、苯肾上腺素(PE)组、环孢素A(CsA)组、CsA+PE组。采用TUNEL染色测定EPCs凋亡状况。逆转录聚合酶链反应法(RT-PCR)检测Bax、Bcl-2、Caspase-3及NFAT4的mRNA的转录水平。免疫荧光染色检测NFAT4亚细胞水平定位。结果:(1)EPCs凋亡检测:与空白对照组及PE组比较,CsA组和CsA+PE组EPCs凋亡数明显增加[(3.41±0.89)比(4.39±1.92)比(23.68±1.14)比(25.92±2.62),P<0.05];(2)与空白对照组和PE组比较,CsA组和CsA+PE组的Bcl-2mRNA水平、Bcl-2/Bax mRNA比值明显下降(P均<0.05),Caspase-3mRNA水平明显升高(P<0.05)。NFAT4mRNA水平:与空白对照组比较,PE组的明显升高(P<0.05),CsA组的明显下降(P<0.05);与PE组比较,CsA组和CsA+PE组的NFAT4mRNA水平均明显下降(P<0.05)。四组之间Bax mRNA水平均无显著差异;(3)与空白对照组比较,PE组NFAT4核转移率明显增加[(25.33±2.08)%比(95±2)%,P<0.05];与空白对照组及PE组比较,CsA组和CsA+PE组NFAT4核转移率[(8.00±2.65)%、(7.00±1.73)%]明显下降(P均<0.05)。结论:CaN-NFAT信号传导通路参与EPCs凋亡的调节作用。     

Megakaryocyte growth and development factor-induced proliferation and differentiation are regulated by the mitogen-activated protein kinase pathway in primitive cord blood hematopoietic progenitors.

In several erythroleukemia cell lines, activation of mitogen-activated protein kinases (MAPK) by phorbol esters or megakaryocyte growth and development factor (MGDF) is required for induction of megakaryocytic phenotype and growth arrest. To support this model, we have examined the effect of a specific inhibitor of this pathway (PD98059) on human CD34(+) hematopoietic progenitors isolated from cord blood (CB), induced to differentiate along the megakaryocytic lineage in liquid cultures supplemented with rhuMGDF. RhuMGDF induced a sustained activation of MAPK in megakaryocytes and this activation was completely inhibited in the presence of low concentrations of PD98059 (6 to 10 micromol/L). At this concentration, PD98059 induced an increase in cell proliferation, resulting in accumulation of viable cells and a prolongation of the life time of the cultures. This increase correlated with an increase in DNA synthesis rather than with a reduction in apoptosis. This effect was combined with developmental changes indicative of delayed megakaryocytic differentiation: (1) PD98059-treated cells tended to retain markers of immature progenitors as shown by the increased proportion of both CD34(+) and CD41(+)CD34(+) cells. (2) PD98059-treated cultures were greatly enriched in immature blasts cells. (3) PD98059 increased megakaryocytic progenitors able to form colonies in semisolid assays. Thus, the MAPK pathway, although not required for megakaryocyte formation, seems to be involved in the transition from proliferation to maturation in megakaryocytes. Inhibition of MAPK activation also led to an increase in the number and size of erythroid colonies without affecting granulocyte/macrophage progenitor numbers suggesting that, in addition to the megakaryocytic lineage, the MAPK pathway could play a role in erythroid lineage differentiation.     

钙调神经磷酸酶-活化T细胞核因子信号传导通路对内皮祖细胞增殖作用的研究

目的研究钙调神经磷酸酶-活化T细胞核因子（CaN—NFAT）信号传导通路对内皮祖细胞（EPCs）增殖的作用。方法采用密度梯度离心法分离外周血单个核细胞,贴壁培养获得EPCs。比色法测定EPCs增殖能力;免疫印迹法检测EPCs内钙调神经磷酸酶Ap（cnAp）蛋白质水平的表达;逆转录聚合酶链反应法检测EPCs内cnAB和NFAT4的表达。观察不同干预因素24h和48h后对EPCs增殖能力的作用,干预因素48h后对EPCs内CnAβ蛋白质水平表达的影响及对EPCs内CnAB和NFAT4表达的影响。结果苯肾上腺素（PE）使EPCs增殖能力增高;环孢素A（CsA）不仅直接使EPCs增殖能力下降,而且可抑制PE引起的EPCs增殖能力增高。结论EPCs内存在CaN—NFAT信号传导通路,该通路受促进因素及抑制因素的调节;CaN—NFAT信号传导通路对EPCs的增殖有明显作用。     

NFAT control of innate immunity

The calcineurin/nuclear factor of activated T cells (NFAT) signaling pathway mediates multiple adaptive T-cell functions, but recent studies have shown that calcineurin/NFAT signaling also contributes to innate immunity and regulates the homeostasis of innate cells. Myeloid cells, including granulocytes and dendritic cells, can promote inflammation, regulate adaptive immunity, and are essential mediators of early responses to pathogens. Microbial ligation of pattern-recognition receptors, such as TLR4, CD14, and dectin 1, is now known to induce the activation of calcineurin/NFAT signaling in myeloid cells, a finding that has provided new insights into the molecular pathways that regulate host protection. Inhibitors of calcineurin/NFAT binding, such as cyclosporine A and FK506, are broadly used in organ transplantation and can act as potent immunosuppressive drugs in a variety of different disorders. There is increasing evidence that these agents influence innate responses as well as inhibiting adaptive T-cell functions. This review focuses on the role of calcineurin/NFAT signaling in myeloid cells, which may contribute to the various unexplained effects of immunosuppressive drugs already being used in the clinic.     

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 nucleotranslocation/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 NFAT2 is 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.     

Suppression of FasL expression in tumor cells and preventing TNF-induced apoptosis was better for immune cells survival

OBJECTIVE: To elucidate if Fas/FasL signal pathway participates in the immune escape of tumor cells, and if contemporarily preventing Fas/FasL and TNF-induced apoptosis is better for immune cells survival than just blocking Fas/FasL-induced apoptotic signal. METHODS: Suppression of FasL expression in mouse H22 hepatocellular cancer cells by siRNA technique. Wild-type Ad5 14.7K gene was amplified by PCR and transduced into Jurkat T cells. Detecting apoptosis of target Jurkat cells by Flow Cytometry. Detection of TNF-alpha in the culture supernatant of H22 cells by ELISA. FasL and 14.7K gene expression in stably transfected or transduced clones were determined by western blotting. RESULTS: FasL expression in H22 cells was down-regulated following stable transfection with a plasmid encoding antisense FasL cDNA. Down-regulation of FasL expression in H22 cells had no effect on tumor growth in vitro. There was an apparent decrease in the number of apoptotic Jurkat T cells following coculture with transfected H22 cells, relative to coculture with FasL-expressing untransfected cells. Compared with untransduced Jurkat cells, apoptotic rates in 14.7K transduced Jurkat cells were significantly reduced in three different E/T ratios (P < 0.01), respectively. CONCLUSIONS: Fas/FasL signal pathway participated in the immune escape of tumor cells by inducing immune cells apoptosis. Reducing the expression of FasL in tumor cells can decrease the apoptotic rate of immune cells. Further blocking of apoptotic signal pathway of immune cells by preventing TNF-induced apoptosis can increase the survival of immune cells.