Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) promotes glioma cell invasion through induction of NF-κB-inducing kinase (NIK) and noncanonical NF-κB signaling

Background

High-grade gliomas are one of the most invasive and therapy-resistant cancers. We have recently shown that noncanonical NF-κB/RelB signaling is a potent driver of tumorigenesis and invasion in the aggressive, mesenchymal subtype of glioma. However, the relevant signals that induce activation of noncanonical NF-κB signaling in glioma and its function relative to the canonical NF-κB pathway remain elusive.

Methods

The ability of tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) to regulate NF-κB signaling and promote tumor progression was investigated in both established and primary high-grade glioma tumor lines using a three-dimensional (3-D) collagen invasion assay. The roles of specific NF-κB proteins in regulating glioma cell invasion and expression of Matrix Metalloproteinase 9 (MMP9) in response to TWEAK were evaluated using shRNA-mediated loss-of-function studies. The ability of NF-κB-inducing kinase (NIK) to promote glioma growth in vivo was investigated using an orthotopic xenograft mouse model.

Results

In glioma cells that display elevated noncanonical NF-κB signaling, loss of RelB attenuates invasion without affecting RelA expression or phosphorylation and RelB is sufficient to promote invasion in the absence of RelA. The cytokine TWEAK preferentially activates the noncanonical NF-κB pathway through induction of p100 processing to p52 and nuclear accumulation of both RelB and p52 without activating the canonical NF-κB pathway. Moreover, TWEAK, but not TNFα, significantly increases NIK mRNA levels. TWEAK also promotes noncanonical NFκB-dependent MMP9 expression and glioma cell invasion. Finally, expression of NIK is sufficient to increase gliomagenesis in vivo.

Conclusions

Our data establish a key role for NIK and noncanonical NF-κB in mediating TWEAK-induced, MMP-dependent glioma cell invasion. The findings also demonstrate that TWEAK induces noncanonical NF-κB signaling and signal-specific regulation of NIK mRNA expression. Together, these studies reveal the important role of noncanonical NF-κB signaling in regulating glioma invasiveness and highlight the therapeutic potential of targeting activation of NIK in this deadly disease.

Electronic supplementary material

The online version of this article (doi:10.1186/s12943-014-0273-1) contains supplementary material, which is available to authorized users.     

RelB在蛋白酶体抑制剂诱导的maspin表达中的作用研究

目的: 探讨前列腺癌细胞中,核转录因子NF-κB家族成员之一RelB在蛋白酶体抑制剂作用下maspin蛋白表达调控中的作用机制。方法: 采用Western blotting法检测前列腺癌细胞中内源性及蛋白酶体抑制剂MG-132作用下RelA、RelB和maspin的蛋白表达;采用免疫组化法检测前列腺癌组织中RelB和maspin的表达情况;通过转染小分子RNA至前列腺癌细胞中沉默RelB的表达;采用Western blotting法检测RelB沉默对MG-132诱导的maspin蛋白表达情况的影响;PI染色法结合流式细胞术检测细胞死亡率。结果: 雄激素非依赖型前列腺癌细胞DU145中RelB表达增强而maspin表达明显降低。在检测的10例前列腺癌组织样本中,恶性度高(Gleason评分4-5)的样本普遍同时存在胞核内RelB强染色和maspin表达缺失。MG-132作用DU145细胞24 h后RelB在胞浆和胞核中表达水平下调,伴随着maspin在胞核内的表达上调。MG-132处理RelB表达沉默的DU145细胞8和24 h后,maspin的表达无明显变化,而RelA蛋白的表达水平呈时间依赖性的下调。结论: 在雄激素非依赖型前列腺癌细胞和晚期前列腺癌组织中,maspin与RelB的表达呈负相关性。蛋白酶体抑制剂诱导maspin的表达上调过程中,RelB是重要的调控因子。     

沉默RelB表达诱导耐受性树突状细胞产生

目的 利用RelB短发卡RNA(shRNA)慢病毒转染树突状细胞(DC)诱导耐受性DC的产生.方法 将Lenti-RelB shRNA转染未成熟DC后,应用逆转录-聚合酶链反应(RT-PCR)和Western blot检测DC中RelB的表达；流式细胞仪检测DC凋亡以及表型分子人类主要组织相容性复合体(MHC)-Ⅱ、CD80、CD86、CD83的表达；酶联免疫吸附试验(ELISA)检测分泌的细胞因子白细胞介素(IL)-10、IL-12、转化生长因子-β1(TGF-β1)以及核因子(NF)-κB各亚基的DNA结合活性.结果 比较不成熟组、成熟组、空载体对照组DC 、Lenti-RelB shRNA-DC下调RelB的表达；而凋亡率同其余3组比较差异无统计学意义(P＞0.05)；同时下调DC表型分子MHC-Ⅱ(0.34±0.04)、CD80(0.38±0.03)、CD86(0.17 ±0.02)、CD83(0.21 ±0.02)的表达；转染后的DC低表达IL-12[(96.3±32.8)ng/L],高表达IL-10[(218.2 ±43.3) ng/L]；而RelB的DNA结合活性(0.28±0.06)较其他组显著下降,但其他亚基的DNA结合活性各组差异无统计学意义(P＞0.05).结论 利用LentiRelB shRNA沉默RelB表达可体外诱导耐受性DC的产生.     

RelB controls adaptive responses of astrocytes during sterile inflammation

In response to brain injury or infections, astrocytes become reactive, undergo striking morphological and functional changes, and secrete and respond to a spectrum of inflammatory mediators. We asked whether reactive astrocytes also display adaptive responses during sterile IL-1β-induced neuroinflammation, which may limit tissue injury associated with many disorders of the central nervous system. We found that astrocytes display days-to-weeks long specific tolerance of cytokine genes, which is coordinated by NF-κB family member, RelB. However, in contrast to innate immune cells, astrocytic tolerance does not involve epigenetic silencing of the cytokine genes. Establishment of tolerance depends on persistent higher levels of RelB in tolerant astrocytes and its phosphorylation on serine 472. Mechanistically, this phosphorylation prevents efficient removal of RelB from cytokine promoters by IκBα and helps to establish tolerance. Importantly, ablation of RelB from astrocytes in mice abolishes tolerance during experimental neuroinflammation in vivo.     

RelB基因沉默的髓源树突状细胞负载Tα146~162 对TAChR预致敏T细胞免疫反应的影响

目的：探讨RelB基因沉默的髓源树突状细胞(BMDC)负载电鳗乙酰胆碱受体(TAChR)优势肽段Tα146~162在TAChR预致敏C57BL/6小鼠中能否诱导免疫耐受。方法：制备产生RelB siRNA分子的重组慢病毒和对照病毒。慢病毒感染BMDC后,给予LPS刺激,相应的DC命名为DC-siRelB和DC-control。应用实时定量PCR和Western印迹分析细胞中RelB表达,流式细胞仪检测细胞表型,ELISA检测上清白细胞介素(IL)-12水平。36只C57BL/6小鼠随机分为A1,A2,A3,K1,K2,K3共6组。实验前1天,A1~A3组用TAChR+完全福氏佐剂(CFA)致敏;K1~K3组用钥孔戚血清蛋白(KLH)+CFA致敏。第7天,A2和K2组注射Tα146~162脉冲的DC-siRelB;A3和K3组注射Tα146~162脉冲的DC-control;A1和K1组给予PBS。第14天,3H-TdR掺入法检测淋巴细胞增殖反应。结果：成功构建了含RelBshRNA基因的重组慢病毒,RelB siRNA可显著下调DC中RelB的表达。与DC-control相比,DC-siRelB中CD80,CD86,MHCII表达及IL-12水平显著降低。与A1和A3组相比,A2组TAChR刺激下淋巴细胞增殖反应显著降低(P<0.05),ConA和KLH刺激下淋巴细胞增殖反应差异无统计学意义(P>0.05)。K1,K2和K3组在KLH刺激下淋巴细胞增殖反应差异均无统计学意义(P>0.05)。结论：慢病毒介导的RelB基因沉默的BMDC可抵制成熟诱导反应并能在TAChR预致敏的C57BL/6小鼠中诱导抗原特异性免疫耐受,为研究其用于治疗重症肌无力奠定了基础。     

miR-155调控前列腺癌细胞NF-κB通路机制研究

目的探讨miR-155调控前列腺癌细胞核因子转录κB(NF-κB)通路的分子机制。方法前列腺癌DU145和PC-3细胞株经Anti-miRTM miR-155抑制剂(anti-miR-155)转染后(实验组),采用蛋白印迹法(Western blot)和逆转录聚合酶链反应(RT-PCR)分别检测转染后细胞中RelA和RelB蛋白及mRNA表达水平,同时检测NF-κB活性及细胞因子水平。以转染AntimiR~(TM)阴性对照的DU145和PC-3细胞株为对照组。结果与对照组相比,实验组细胞RelA、白细胞介素(IL-6)、白细胞介素(IL-21)表达水平,以及NF-κB活性显著降低(P0.05),RelB表达水平比较差异无统计学意义(P0.05)。结论降低人前列腺癌细胞NF-κB活性和RelA表达水平,可能是miR-155参与调控前列腺癌生物学行为的分子机制之一。     

核转录因子RelB抑制途径对骨髓树突状细胞表面分子表达的影响研究

目的探讨核转录因子RelB抑制途径对小鼠骨髓树突状细胞（bone marrow dendritic cells）的表面分子表达的影响,为致耐受树突状细胞的研究提供新方法。方法 rmGM-CSF和rmIL-4联合诱导培养体系培养小鼠骨髓树突状细胞,免疫磁珠方法纯化;用慢病毒载体制备RelB shRNA慢病毒,与小鼠骨髓树突状细胞共培养,流式细胞术观察树突状细胞表面分子MHC-II、CD86和CD40的表达,设LPS-DC对照组、未处理组和LPSRNAi RelB DC组。结果核转录因子RelB抑制的树突状细胞表面分子MHC-II、CD86和CD40均低水平表达,显著低于成熟DC表面分子的表达（P〈0.05）,且经LPS刺激后（LPS RNAi RelB DC）DC表面上述三类分子的表达水平仍显著低于LPS-DC组（P〈0.05）,与未处理组（immature DC）表面分子表达水平相当。结论核转录因子RelB抑制的骨髓树突状细胞表面分子表达水平低,呈现出致耐受的树突状细胞的特点,是致耐受树突状细胞研究的一种新方法。     

小鼠骨髓成熟与不成熟树突状细胞中RelB基因的表达

目的:探讨体外分离培养的小鼠骨髓来源的成熟与未成熟DC中核转录因子RelB(avianreticuloendotheliosisviral(v-rel)oncogenerelatedB)基因的表达。方法:无菌从C57BL/6小鼠股骨和胫骨中取出骨髓细胞,利用rmGM-CSF和rmIL-4联合诱导骨髓前体细胞产生未成熟的DC,未成熟的DC在培养结束前18h经LPS刺激获得成熟的DC,用流式细胞术分析它们的表型,用RT-PCR和免疫荧光染色法检测成熟与未成熟DC中,RelBmRNA和其蛋白的表达。结果:流式细胞术分析显示未成熟的DC中MHC-Ⅱ类分子和共刺激分子(CD86和CD40)呈低水平表达;而成熟的DC则呈高水平表达。RT-PCR和免疫荧光染色法检测结果均显示,RelB基因在未成熟的DC中呈低水平表达;而在成熟的DC中呈高水平表达,两者比较具有统计学意义(P<0.01)。结论:RelB基因的表达与小鼠骨髓来源的DC的成熟状态密切相关。抑制DC中RelB基因的表达,有可能诱导产生具有耐受原性的未成熟的DC。     

RelB基因沉默对骨髓树突细胞生物学效应的影响

目的探讨核转录因子NFκ-B/RelB基因沉默对树突细胞(DC)生物学效应的影响。方法采用细胞因子诱导培养法体外扩增小鼠骨髓DC,并经免疫磁珠纯化,获得高纯度骨髓DC后,行RelB基因沉默,获得RNAi RelB-DC,扫描电镜和透射电镜观察细胞形态特点,并利用流式细胞术分析细胞表面分子的表达水平,同种异基因混合淋巴细胞反应(MLR)法分析RNAi RelB-DC刺激异基因T细胞增殖的能力。结果扫描电镜下RNAi RelB-DC细胞表面突起较少而短,细胞表面多皱褶;透射电镜下可见细胞内多吞噬泡样结构;流式细胞术分析显示细胞表面分子MHC-II、CD86和CD40的表达水平相对较低;MLR显示该DC刺激异基因T细胞增殖的能力相对较弱。结论RelB基因沉默DC的生物学效应表现为未成熟DC的特点,这种RNAi RelB-DC作为一种新型的致耐受DC,有望应用于免疫耐受的诱导研究。