Expression of the serpin serine protease inhibitor 6 protects dendritic cells from cytotoxic T lymphocyte-induced apoptosis: differential modulation by T helper type 1 and type 2 cells

Dendritic cells (DCs) play a central role in the immune system as they drive activation of T lymphocytes by cognate interactions. However, as DCs express high levels of major histocompatibility complex class I, this intimate contact may also result in elimination of DCs by activated cytotoxic T lymphocytes (CTLs) and thereby limit induction of immunity. We show here that immature DCs are indeed susceptible to CTL-induced killing, but become resistant upon maturation with anti-CD40 or lipopolysaccharide. Protection is achieved by expression of serine protease inhibitor (SPI)-6, a member of the serpin family that specifically inactivates granzyme B and thereby blocks CTL-induced apoptosis. Anti-CD40 and LPS-induced SPI-6 expression is sustained for long periods of time, suggesting a role for SPI-6 in the longevity of DCs. Importantly, T helper 1 cells, which mature DCs and boost CTL immunity, induce SPI-6 expression and subsequent DC resistance. In contrast, T helper 2 cells neither induce SPI-6 nor convey protection, despite the fact that they trigger DC maturation with comparable efficiency. Our data identify SPI-6 as a novel marker for DC function, which protects DCs against CTL-induced apoptosis.     

Selenium nanoparticles inhibited H1N1 influenza virus-induced apoptosis by ROS-mediated signaling pathways

Influenza A (H1N1) viruses are distributed around the world and pose a threat to public health. Vaccination is the main treatment strategy to prevent influenza infection, but antiviral drugs also play an important role in controlling seasonal and pandemic influenza. Currently, as influenza viruses may be developing antiviral resistance, new agents with different modes of action are being investigated. Recently, selenium nanoparticles (SeNPs), which have antiviral effects, have attracted increasing attention in biomedical interventions. The appearance of nanotechnology has attracted great attention in the field of nanomedicine. SeNPs constitute an attractive vector platform for delivering a variety of drugs to action targets. SeNPs are being explored for potential therapeutic efficacy in a variety of oxidative stress and inflammation-mediated diseases, such as cancer, arthritis, diabetes, and kidney disease. SeNPs could inhibit infection of Madin–Darby canine kidney (MDCK) cells with H1N1 and prevent chromatin condensation and DNA fragmentation. ROS play a key role in physiological processes for apoptosis. SeNPs significantly inhibited the production of reactive oxygen species (ROS) in MDCK cells. Mechanistic investigation revealed that SeNPs inhibited the apoptosis induced by H1N1 virus infection in MDCK cells by improving the level of GPx1. Our results suggest that SeNPs are an effective selenium source and a promising H1N1 influenza antiviral candidate.

SeNPs inhibited the apoptosis induced by H1N1 virus infection in MDCK cells by improving the level of GPx1. Our results suggest that SeNPs are an effective selenium source and a promising H1N1 influenza antiviral candidate.     

The inhibition of H1N1 influenza induced apoptosis by sodium selenite through ROS-mediated signaling pathways

The high variability of influenza viruses has made it more difficult for people to cope with influenza. When antigen transformation occurs, even new influenza without preventive vaccines may be produced, which poses a great threat to human health. Selenium is an essential trace element in humans and mammals, and has many biological activities. It has attracted people''s research interest in recent years. In this study, MDCK cells were used as a model to observe the effect of sodium selenite on H1N1 influenza virus. Our research showed that sodium selenite (Na₂SeO₃) has an anti-influenza H1N1 virus effect, and the anti-viral effect of sodium selenite was further demonstrated by caspase-3, AKT, MAPK and p53 signaling pathways. The investigations of the mechanism revealed that the sodium selenite could block H1N1 influenza from infecting MDCK cells through inhibiting the production of ROS. The results demonstrate that selenium supplementation may provide a feasible approach to inhibit the infection of H1N1 influenza virus.

The high variability of influenza viruses has made it more difficult for people to cope with influenza.     

Differential roles of signaling pathways for proliferation and migration of rat pancreatic stellate cells

Activated pancreatic stellate cells (PSCs) have recently been implicated in the pathogenesis of pancreatic fibrosis and inflammation. Accumulation of PSCs is a fundamental feature of pancreatic fibrosis, and platelet-derived growth factor (PDGF)-BB is the most potent mitogen for PSCs. But, the molecular mechanisms responsible for PDGF's actions in PSCs are largely unknown. In hepatic stellate cells, it has been established that activation of both phosphatidylinositol (PI) 3-kinase and extracellular-signal regulated kinase (ERK) pathways is required for PDGF-BB-induced proliferation and migration. The aim of this study was to elucidate the signaling pathways mediating PDGF-BB's actions in PSCs. PSCs were isolated from rat pancreas tissue and used in their culture-activated, myofibroblast-like phenotype. Culture-activated PSCs expressed PDGF alpha- and beta-receptors. PDGF-BB induced autophosphorylation of its receptor, followed by the activation of PI 3-kinase, Akt, and ERK pathways. Activation of PI 3-kinase was not required for PDGF-BB-induced ERK activation. PDGF-BB induced approximately five-fold increase in proliferation and chemotaxis of PSCs. Inhibition of ERK pathway with PD98059 completely blocked proliferation, whereas PD98059 had a modest inhibitory effect on cell migration (approximately 50%). On the other hand, inhibition of PI 3-kinase pathway with wortmannin or LY294002 almost completely inhibited migration, but did not affect proliferation of PSCs. In conclusion, our results suggest that ERK pathway regulates proliferation and migration in response to PDGF-BB, whereas PI 3-kinase mediates cellular migration, but not proliferation of PSCs.     

Isoprenoids determine Th1/Th2 fate in pathogenic T cells, providing a mechanism of modulation of autoimmunity by atorvastatin

3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase is a critical enzyme in the mevalonate pathway that regulates the biosynthesis of cholesterol as well as isoprenoids that mediate the membrane association of certain GTPases. Blockade of this enzyme by atorvastatin (AT) inhibits the destructive proinflammatory T helper cell (Th)1 response during experimental autoimmune encephalomyelitis and may be beneficial in the treatment of multiple sclerosis and other Th1-mediated autoimmune diseases. Here we present evidence linking specific isoprenoid intermediates of the mevalonate pathway to signaling pathways that regulate T cell autoimmunity. We demonstrate that the isoprenoid geranylgeranyl-pyrophosphate (GGPP) mediates proliferation, whereas both GGPP and its precursor, farnesyl-PP, regulate the Th1 differentiation of myelin-reactive T cells. Depletion of these isoprenoid intermediates in vivo via oral AT administration hindered these T cell responses by decreasing geranylgeranylated RhoA and farnesylated Ras at the plasma membrane. This was associated with reduced extracellular signal-regulated kinase (ERK) and p38 phosphorylation and DNA binding of their cotarget c-fos in response to T cell receptor activation. Inhibition of ERK and p38 mimicked the effects of AT and induced a Th2 cytokine shift. Thus, by connecting isoprenoid availability to regulation of Th1/Th2 fate, we have elucidated a mechanism by which AT may suppress Th1-mediated central nervous system autoimmune disease.     

Anti-Fas/APO-1 antibody-mediated apoptosis of cultured human glioma cells. Induction and modulation of sensitivity by cytokines.

Fas/APO-1 is a transmembrane protein of the nerve growth factor/TNF alpha receptor family which signals apoptotic cell death in susceptible target cells. We have investigated the susceptibility of seven human malignant glioma cell lines to Fas/APO-1-dependent apoptosis. Sensitivity to Fas/APO-1 antibody-mediated cell killing correlated with cell surface expression of Fas/APO-1. Expression of Fas/APO-1 as well as Fas/APO-1-dependent cytotoxicity were augmented by preexposure of human malignant glioma cells to IFN gamma and TNF alpha. Further, pretreatment with TGF beta 2, IL1 and IL8 enhanced Fas/APO-1 antibody-induced glioma cell apoptosis whereas other cytokines including TNF beta, IL6, macrophage colony-stimulating factor, IL10 and IL13 had no such effect. None of the human malignant glioma cell lines was susceptible to TNF alpha-induced cytotoxicity. Fas/APO-1 antibody-sensitive glioma cell lines (n = 5), but not Fas/APO-1 antibody-resistant glioma cell lines (n = 2), became sensitive to TNF alpha when co-treated with inhibitors of RNA and protein synthesis. Resistance of human glioma cells to Fas/APO-1 antibody-mediated apoptosis was mainly related to low level expression of Fas/APO-1 and appeared not to be linked to overexpression of the anti-apoptotic protooncogene, bcl-2. Given the resistance of human malignant glioma to surgery, irradiation, chemotherapy and immunotherapy, we propose that Fas/APO-1 may be a promising target for a novel locoregionary approach to human malignant glioma. This strategy gains support from the demonstration of Fas/APO-1 expression in ex vivo human malignant glioma specimens and from the absence of Fas/APO-1 in normal human brain parenchyma.     

The modulation of CD40 ligand signaling by transmembrane CD28 splice variant in human T cells

The role of CD40 ligand (CD40L)/CD40 signaling in T cell-dependent B cell differentiation and maturation has been amply documented. The mechanism of CD40 signaling in B cells has been well established, whereas the signaling mechanism of CD40L in T cell costimulation remains unknown. In this study we show that CD28i, a transmembrane splice variant of CD28 costimulatory receptor, complexes with CD40L in human T cells. The cross-linking of CD40L resulted in the coendocytosis of CD28i with CD40L. The tyrosine phosphorylation of CD28i followed the cross-linking of CD40L, and the overexpression of CD28i augmented the c-Jun NH2-terminal kinase, p21-activated kinase 2, and nuclear factor kappaB activation. These data indicate that CD28i, by functioning as a signaling adaptor, transduces CD40L signaling as well as CD28 signaling in human T cells.     

反转录病毒载体介导Indian hedgehog信号蛋白在C3H10T1/2细胞中的表达及成骨诱导潜能

背景:目前大量研究证实Indian hedgehog(Ihh)信号通路在骨发育中发挥着重要的调控作用。目的:克隆Ihh重组反转录病毒载体,观察Ihh在C3H10T1/2细胞中的表达及其成骨诱导效果。方法:采用分子克隆技术将Ihh基因与增强型绿色荧光蛋白基因克隆到反转录病毒载体pSFG上,包装出假病毒转染C3H10T1/2细胞,在共聚焦显微镜下观察其绿色荧光蛋白强度及转染效率。运用Western blot方法检测Ihh蛋白的表达,观察C3H10T1/2细胞形态学变化,碱性磷酸酶染色检测成骨活性。结果与结论:共聚焦显微镜下观察到大量的绿色荧光表达,Western blot检测结果显示Ihh基因及增强型绿色荧光蛋白基因在C3H10T1/2中呈显著共表达。在体外培养过程中,Ihh能诱导C3H10T1/2细胞形态学发生改变,增加碱性磷酸酶活性,提示Ihh具有诱导C3H10T1/2细胞向骨细胞分化的潜能。     

Differential modulation of brainstem phosphatidylinositol 3-kinase/Akt and extracellular signal-regulated kinase 1/2 signaling underlies WIN55,212-2 centrally mediated pressor response in conscious rats

Our recent study demonstrated that central cannabinoid receptor 1 (CB?R) activation caused dose-related pressor response in conscious rats, and reported studies implicated the brainstem phosphatidylinositol 3-kinase (PI3K)/Akt-extracellular signal-regulated kinase 1/2 (ERK1/2) pathway in blood pressure control. Therefore, in this study, we tested the hypothesis that the modulation of brainstem PI3K/Akt-ERK1/2 signaling plays a critical role in the central CB(1)R-mediated pressor response. In conscious freely moving rats, the pressor response elicited by intracisternal (i.c.) (R)-(+)-[2,3-dihydro-5-methyl-3[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl) methanone mesylate salt (WIN55,212-2) (15 μg) was associated with significant increases in ERK1/2 phosphorylation in the rostral ventrolateral medulla (RVLM) and the nucleus tractus solitarius (NTS). In contrast, Akt phosphorylation was significantly reduced in the same neuronal pools. Pretreatment with the selective CB?R antagonist N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251) (30 μg i.c.) attenuated the neurochemical responses elicited by central CB?R activation. Furthermore, pretreatment with the ERK/mitogen-activated protein kinase kinase inhibitor 2'-amino-3'-methoxyflavone (PD98059) (5 μg i.c.) abrogated WIN55,212-2-evoked increases in blood pressure and neuronal ERK1/2 phosphorylation but not the reduction in Akt phosphorylation. On the other hand, prior PI3K inhibition with wortmannin (0.4 μg i.c.) exacerbated the WIN55,212-2 (7.5 and 15 μg i.c.) dose-related increases in blood pressure and ERK1/2 phosphorylation in the RVLM. The present neurochemical and integrative studies yield new insight into the critical role of two brainstem kinases, PI3K and ERK1/2, in the pressor response elicited by central CB?R activation in conscious rats.     

p38MAPK信号通路与应力介导成肌细胞的凋亡

背景：在体内条件下,细胞力学的功能研究因其所处生理环境的复杂性、实验条件的不易控制而很难得到满意结果。目的：在成功构建成肌细胞体外培养-力学刺激模型的基础上,研究p38MAPK信号通路在成肌细胞凋亡中的作用及其机制。方法：将体外培养的C2C12细胞分为对照组和SB203580组,SB203580组中加入20mmol/L的p38MAPK抑制剂SB203580。应用细胞应力加载装置FlecellStrainUnit-5000T给细胞提供15%的力值,分别施加0,6,12,24h的周期性张应力。每分钟10个循环,每循环包括3s牵张,3s松弛。Hoechst33258染色观察细胞的形态学变化;流式细胞仪检测细胞凋亡情况;RT-PCR法检测促凋亡基因baxmRNA的表达;Westernblot检测信号通路中p38MAPK和p-p38MAPK蛋白的表达。结果与结论：随着加力时间的延长,细胞逐渐出现核固缩及凋亡小体,凋亡率增加（P〈0.05）,baxmRNA表达增多（P〈0.05）;细胞p38MAPK和p-p38MAPK蛋白均在加力6h达到最低,此后逐渐升高。p38MAPK抑制剂SB203580可抑制加力引起的细胞凋亡,减少baxmRNA及p38MAPK和p-p38MAPK蛋白的表达（P〈0.05）。说明p38MAPK信号通路在应力介导的成肌细胞凋亡中起到重要的作用。     

Depsipeptide (FR901228) enhances the cytotoxic activity of TRAIL by redistributing TRAIL receptor to membrane lipid rafts.

TRAIL (TNF-related apoptosis-inducing ligand) induces apoptosis in various tumor cell types and is under investigation as a cancer therapeutic. The development of a recombinant adenovirus encoding the full-length human TRAIL gene (Ad5-TRAIL) replaces the need for large quantities of soluble TRAIL protein in tumor suppressive therapies. However, the full potential of Ad5-TRAIL has not yet been maximized. Recent investigation of a histone deacetylase inhibitor, depsipeptide (FR901228), has demonstrated that it increases cellular susceptibility to adenovirus infection and augments adenoviral transgene expression. Thus, studies were initiated to evaluate the ability of depsipeptide to enhance the cytotoxic activity of Ad5-TRAIL against human prostate tumor cells. In vitro, depsipeptide increased expression of coxsackie-adenovirus receptor, leading to increased adenoviral infection and transgene expression. Additionally, tumor cell killing by Ad5-TRAIL was higher following depsipeptide pretreatment. More surprisingly, depsipeptide also increased prostate tumor cell sensitivity to TRAIL-induced apoptosis. Investigation into the mechanism responsible for increased TRAIL responsiveness revealed increased levels of TRAIL-R1 and -R2 in membrane lipid rafts following depsipeptide treatment. These results indicate that depsipeptide is a potent agent for enhancing the activity of Ad5-TRAIL by multiple mechanisms, allowing for a more efficient use of Ad5-TRAIL as an antitumor therapy.     

p38MAPK信号通路与应力介导成肌细胞的凋亡(英文)

背景:在体内条件下,细胞力学的功能研究因其所处生理环境的复杂性、实验条件的不易控制而很难得到满意结果。目的:在成功构建成肌细胞体外培养-力学刺激模型的基础上,研究p38MAPK信号通路在成肌细胞凋亡中的作用及其机制。方法:将体外培养的C2C12细胞分为对照组和SB203580组,SB203580组中加入20mmol/L的p38MAPK抑制剂SB203580。应用细胞应力加载装置FlecellStrainUnit-5000T给细胞提供15%的力值,分别施加0,6,12,24h的周期性张应力。每分钟10个循环,每循环包括3s牵张,3s松弛。Hoechst33258染色观察细胞的形态学变化;流式细胞仪检测细胞凋亡情况;RT-PCR法检测促凋亡基因baxmRNA的表达;Westernblot检测信号通路中p38MAPK和p-p38MAPK蛋白的表达。结果与结论:随着加力时间的延长,细胞逐渐出现核固缩及凋亡小体,凋亡率增加(P<0.05),baxmRNA表达增多(P<0.05);细胞p38MAPK和p-p38MAPK蛋白均在加力6h达到最低,此后逐渐升高。p38MAPK抑制剂SB203580可抑制加力引起的细胞凋亡,减少baxmRNA及p38MAPK和p-p38MAPK蛋白的表达(P<0.05)。说明p38MAPK信号通路在应力介导的成肌细胞凋亡中起到重要的作用。     

骨形态发生蛋白2诱导C3H10T1/2细胞成骨分化中长链非编码RNA的作用

背景：人类长链非编码RNA是现今的研究热点,已有研究报道其在肿瘤发生中的作用机制,但其在成骨分化方面的机制并不明确。目的：观察人类长链非编码RNA在经骨形态发生蛋白2诱导小鼠C3H10T1/2细胞成骨分化中的作用,并探讨其分子机制。方法：首先进行碱性磷酸酶染色和成骨指标基因检测。对C3H10T1/2细胞在骨形态发生蛋白2诱导下的成骨分化过程长链非编码RNA表达变化进行芯片分析。采用高通量测序比较骨形态发生蛋白2诱导组和未经骨形态发生蛋白2诱导组的表达变化,筛选出表达下降的基因。过表达相应长链非编码RNA后观察对骨形态发生蛋白2诱导成骨分化的影响。结果与结论：骨形态发生蛋白2诱导C3H10T1/2细胞导致碱性磷酸酶活性增加。骨形态发生蛋白2诱导72 h后,碱性磷酸酶、Id1、骨钙素、Runx2、sp7表达上升（P〈0.05）。未诱导C3H10T1/2细胞与骨形态发生蛋白2诱导细胞芯片杂交后结果比较,下降达1.5倍的长链非编码RNAs有24条,其中只有AK035085有内含子。与未过表达AK035085的对照组相比,骨形态发生蛋白2诱导72 h后AK035085过表达的C3H10T1/2细胞碱性磷酸酶、Id1、骨钙素、Runx2、sp7表达均下降（P〈0.05）。提示骨形态发生蛋白2可刺激C3H10T1/2细胞发生成骨分化,AK035085可能对C3H10T1/2细胞的成骨分化存在抑制作用。     

Sulindac independently modulates extracellular signal-regulated kinase 1/2 and cyclic GMP-dependent protein kinase signaling pathways

Colorectal cancer is the second leading cause of cancer mortality in the United States. Substantial human and animal data support the ability of nonsteroidal anti-inflammatory drugs to cause regression of existing colon tumors and prevent new tumor formation. The mechanism by which the nonsteroidal anti-inflammatory drug sulindac prevents tumor growth is poorly understood and seems complex as sulindac can modulate several growth-related signaling pathways. Sulindac metabolites simultaneously (a) increase cellular cyclic GMP and subsequently activate cyclic GMP-dependent protein kinase (PKG); (b) activate c-jun NH2-terminal kinase (JNK); (c) inhibit extracellular signal-regulated kinase 1/2 (ERK1/2); and (d) decrease beta-catenin protein expression at times and doses consistent with apoptosis. The purpose of this study was to determine if PKG, ERK1/2, JNK, and beta-catenin are independent targets for sulindac in vitro. Pharmacologic activation of PKG with YC-1 increases JNK phosphorylation and induces apoptosis in colon cancer cells without modulating ERK1/2 phosphorylation or beta-catenin protein expression. Inhibition of ERK1/2 with U0126 induces apoptosis but fails to activate JNK phosphorylation or down-regulate beta-catenin protein expression. Cotreatment with U0126 and YC-1 synergistically increases apoptosis in colorectal cancer cells and recapitulates the effects of sulindac treatment on ERK1/2, JNK, and beta-catenin. These results indicate that sulindac metabolites modulate ERK1/2 and PKG pathways independently in colon cancer cells and suggest that the full apoptotic effect of sulindac is mediated by more than one pathway. Using similar combinatorial approaches in vivo may provide more effective, less toxic chemopreventive and chemotherapeutic strategies. Such therapies could dramatically reduce the incidence and death rate from colorectal cancer.     

Interleukin-10 production by effector T cells: Th1 cells show self control

Interleukin (IL)-10 is a cytokine that modulates both innate and adaptive immunity, primarily by exerting antiinflammatory effects. IL-10 was originally thought to be produced only by T helper (Th)2 cells, but is now known to be made by a variety of cell types. During many infections, CD4(+) T cells produce both interferon (IFN)-gamma, the signature Th1 cytokine, and IL-10. New data now show that the IL-10 produced by effector Th1 cells helps limit the collateral damage caused by exaggerated inflammation. But this control may also limit the effectiveness of the immune response, resulting in a failure to fully eliminate pathogens.     

Differential effects of low-dose docosahexaenoic acid and eicosapentaenoic acid on the regulation of mitogenic signaling pathways in mesangial cells

Although dietary fish oil supplementation has been used to prevent the progression of kidney disease in patients with IgA nephropathy, relatively few studies provide a mechanistic rationale for its use. Using an antithymocyte (ATS) model of mesangial proliferative glomerulonephritis, we recently demonstrated that fish oil inhibits mesangial cell (MC) activation and proliferation, reduces proteinuria, and decreases histologic evidence of glomerular damage. We therefore sought to define potential mechanisms underlying the antiproliferative effect of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), the predominant omega-3 polyunsaturated fatty acids found in fish oil, in cultured MC. DHA and EPA were administered to MC as bovine serum albumin fatty-acid complexes. Low-dose (10-50 micromol/L) DHA, but not EPA, inhibited basal and epidermal growth factor (EGF)-stimulated [(3)H]-thymidine incorporation in MCs. At higher doses (100 micromol/L), EPA and DHA were equally effective in suppressing basal and EGF-stimulated MC mitogenesis. Low-dose DHA, but not EPA, decreased ERK activation by 30% (P <.01), as assessed with Western-blot analysis using phosphospecific antibodies. JNK activity was increased by low-dose DHA but not by EPA. p38 activity was not significantly altered by DHA or EPA. Cyclin E activity, as assessed with a histone H1 kinase assay, was inhibited by low-dose DHA but not by EPA. DHA increased expression of the cell cycle inhibitor p21 but not p27; EPA had no effect on p21 or p27. We propose that the differential effect of low-dose DHA vs EPA in suppressing MC mitogenesis is related to down-regulation of ERK and cyclin E activity and to induction of p21.