香鱼(Plecoglossus altivelis)NFκB抑制因子α基因PaIκBα克隆及表达

核转录因子κB抑制因子α(IκBα)是NFκB/IκB信号传导通路的重要成员,参与机体抗细菌感染等多种免疫反应,可通过蛋白质间的相互作用结合核转录因子NFκB,从而调控生物体多种免疫基因表达。该研究采用RACE技术从香鱼中克隆得到核转录因子κB抑制因子PaIκBα基因的cDNA全长序列(1341bp,GenBank Accession No.JN801027),开放阅读框ORF为936bp,编码311个氨基酸,5’非编码区为64bp,3’非编码区为341bp。生物信息学分析表明,香鱼IκBα蛋白的序列中包含5个保守的锚蛋白重复序列,N末端含有信号诱导蛋白,C末端含有PEST序列。同源性比对结果表明,香鱼IκBα蛋白与胡瓜鱼IκBα的同源性最高,为95%;其次是大西洋鲑、虹鳟、尼罗罗非鱼和鳜鱼等,同源性分别为76%、75%、70%和68%。系统进化树分析表明,香鱼IκBα蛋白与胡瓜鱼、虹鳟、尼罗罗非鱼、鳜鱼和大西洋鲑等亲缘关系最近。RT-PCR分析表明,PaIκBα基因在香鱼肝脏、肾脏、脾脏和鳃中表达水平较高,其次是肠、脑和肌肉,在心脏中表达极少。嗜水气单胞菌(Aeromonas hydrophil)感染香鱼后,PaIκBα基因表达增强,感染24h达最大值,表明PaIκBα基因在香鱼受到嗜水气单胞菌刺激的免疫过程中可能发挥着重要作用。     

Tetrandrine suppresses LPS-induced astrocyte activation via modulating IKKs-IκBα-NF-κB signaling pathway

Astrocyte activation has been implicated in the pathogenesis of many neurological diseases. These reactive astrocytes are capable of producing a variety of proinflammatory mediators and potentially neurotoxic compounds, such as nitric oxide (NO), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and interleukin-1beta (IL-1beta). In this study, we examined the suppressive effects of Tetrandrine (TET) on astrocyte activation induced by lipopolysaccharide (LPS) in vitro. We found that TET decreased the release of NO, TNF-alpha, IL-6 and IL-1beta in LPS-activated astrocytes. Also mRNA expression levels of inducible nitric oxide synthase (iNOS), macrophage inflammatory protein-1alpha (MIP-1alpha) and vascular cell adhesion molecule-1 (VCAM-1) were inhibited in TET pretreated astrocytes. Such suppressive effects might be resulted from the inhibition of nuclear factor kappa B (NF-kappaB) activation through downregulating IkappaB kinases (IKKs) phosphoration, which decreased inhibitor of nuclear factor-kappaB-alpha (IkappaBalpha) phosphoration and degradation. Our results suggest that TET acted to regulate astrocyte activation through inhibiting IKKs-IkappaBalpha-NF-kappaB signaling pathway.     

Activation of NF-κB by alloferon through down-regulation of antioxidant proteins and IκBα

Alloferon is a 13-amino acid peptide isolated from the bacteria-challenged larvae of the blow fly Calliphora vicina. The pharmaceutical value of the peptide has been well demonstrated by its capacity to stimulate NK cytotoxic activity and interferon (IFN) synthesis in animal and human models, as well as to enhance antiviral and antitumor activities in mice. Antiviral and the immunomodulatory effectiveness of alloferon have also been supported clinically proved in patients suffering with herpes simplex virus (HSV) and human papilloma virus (HPV) infections. To elucidate molecular response to alloferon treatment, we initially screened a model cell line in which alloferon enhanced IFN synthesis upon viral infection. Among the cell lines tested, Namalva was chosen for further proteomic analysis. Fluorescence difference gel electrophoresis (DIGE) revealed that the levels of a series of antioxidant proteins decreased after alloferon treatment, while at least three glycolytic enzymes and four heat-shock proteins were increased in their expression levels. Based on the result of our proteomic analysis, we speculated that alloferon may activate the NF-kappaB signaling pathway. IkappaB kinase (IKK) assay, Western blot analysis on IkappaBalpha and its phosphorylated form at Ser 32, and an NF-kappaB reporter assay verified our proteomics-driven hypothesis. Thus, our results suggest that alloferon potentiates immune cells by activating the NF-kappaB signaling pathway through regulation of redox potential. Since NF-kappaB activation is involved in IFN synthesis, our results provide further clues as to how the alloferon peptide may stimulate IFN synthesis.     

Enterovirus 71 2C protein inhibits TNF-α-mediated activation of NF-κB by suppressing IκB kinase β phosphorylation

Enterovirus 71 (EV71), a single, positive-stranded RNA virus, has been regarded as the most important neurotropic enterovirus after the eradication of the poliovirus. EV71 infection can cause hand, foot, and mouth disease or herpangina. Cytokine storm with elevated levels of proinflammatory and inflammatory cytokines, including TNF-α, has been proposed to explain the pathogenesis of EV71-induced disease. TNF-α-mediated NF-κB signaling pathway plays a key role in inflammatory response. We hypothesized that EV71 might also moderate host inflammation by interfering with this pathway. In this study, we tested this hypothesis and identified EV71 2C protein as an antagonist of TNF-α-mediated activation of NF-κB signaling pathway. Expression of 2C protein significantly reduced TNF-α-mediated NF-κB activation in 293T cells as measured by gene reporter and gel mobility shift assays. Furthermore, overexpression of TNFR-associated factor 2-, MEK kinase 1-, IκB kinase (IKK)α-, or IKKβ-induced NF-κB activation, but not constitutively active mutant of IKKβ (IKKβ SS/EE)-induced NF-κB activation, was inhibited by 2C protein. These data together suggested that the activation of IKKβ is most likely targeted by 2C; this notion was further strengthened by immunoblot detection of IKKβ phosphorylation and IκBα phosphorylation and degradation. Coimmunoprecipitation and colocalization of 2C and IKKβ expressed in mammalian cells provided compelling evidence that 2C interacts with IKKβ. Collectively, our data indicate that EV71 2C protein inhibits IKKβ activation and thus blocks NF-κB activation.     

RNA干扰NF-κB p65对缺血再灌注损伤鼠肺NF-κB和TNF-α的影响

为了研究RNA干扰NF-κBp65对鼠肺缺血再灌注损伤肺组织中NF-κB、TNF-α表达的影响,并探讨减轻鼠肺损伤的保护机制。本研究构建了体外靶向NF-κBp65的短发夹RNA (short hairpin RNA, shRNA)重组表达载体,并采用QPCR检测NF-κBp65的沉默结果。供试的16只SD大鼠随机分为4组:假手术组4只、缺血再灌注+生理盐水组4只,缺血再灌注+NF-κBp65 sh RNA组4只,缺血再灌注+空载组4只,假手术组无缺血再灌注损伤,开胸游离左肺门180 min,缺血再灌注+生理盐水组左肺门阻断前术前24 h给予生理盐水处理,开胸游离左肺门,左肺缺血60 min,再灌注120 min,缺血再灌注+NF-κBp65 shRNA组左肺门阻断前术前24 h滴鼻给予NF-κB shRNA腺病毒(1×1010pfu, 100μL/只),开胸游离左肺门,左肺缺血60 min,再灌注120 min,缺血再灌注+空载组左肺门阻断前术前24 h滴鼻给予空载shRNA腺病毒(1×10~(10)pfu, 100μL/只),开胸游离左肺门,左肺缺血60 min,再灌注120 min。实验结束后每组分别留取左肺组织,留取左肺上叶肺组织测定肺湿/干重比(W/D),部分肺组织光镜下观察病理变化,ELISA法测量NF-κB和TNF-α的表达含量。研究结果表明:成功构建重组NF-κBp65载体,并获得稳定转染的NF-κBp65 shRNA细胞,与转染空载体的阴性对照组(negative control, NC)比较,NF-κBp65 sh RNA能明显使NF-κBp65基因沉默(p0.05),肺组织NF-κB、TNF-α的表达量及W/D值与假手术组比较,缺血再灌注+生理盐水组和缺血再灌注+空载组有明显升高(p0.05);而与缺血再和灌注+生理盐水组和缺血再灌注+空载组比较,缺血再灌注+NF-κBp65 shRNA组明显降低(p0.05),病理学检查表明:假手术组肺组织无明显的炎症损伤;缺血再灌注+NF-κBp65 shRNA组肺炎性损伤较缺血再灌注+生理盐水组和缺血再灌注+空载组明显减轻。本研究结果初步说明,RNA干扰NF-κBp65能明显减轻早期肺移植损伤,其机制可能与抑制NF-κB和TNF-α的表达从而减轻肺组织炎症损伤有关。     

乳腺癌中NF-κB蛋白表达及其与TNF-α相关性研究

目的：研究乳腺癌中细胞核因子-κB（nuclear factor-kappa B,NF-κB）的表达及其与肿瘤坏死因子-α（tumor necrosis factorα,TNF-α）的相关性。方法：采用免疫组织化学的方法检测67例原发乳腺癌组织中NF-κB和TNF-α的表达,检测NF-κB在癌旁正常组织、乳腺增生和乳腺癌中的表达情况,并分析NF-κB的表达与乳腺癌临床病理特征的关系以及NF-κB与TNF-α的相关性。结果：1）NF-κB在癌旁正常组织、乳腺增生和乳腺癌中表达的阳性率分别为20%（3/15）、33.3%（5/15）、79.1%（53/67）;2）NF-κB的表达与年龄、肿瘤大小、组织学类型均无相关性（P〉0.05）,与淋巴转移的有无及组织学分期有显著相关性（P〈0.05）;3）NF-κB的表达与TNF-α呈显著正相关（P〈0.05）。结论：乳腺癌中NF-κB的表达显著增加且与其淋巴转移的有无及肿瘤组织学分期有显著相关性,与TNF-α呈显著正相关,对乳腺癌良恶性判断、诊断、靶向治疗及预后提供重要参考依据。     

TNF-α诱导的NF-κB慢病毒报告基因系统的构建

目的:构建一种肿瘤坏死因子α(TNF-α)诱导的NF-κB慢病毒报告基因系统。方法:以NF-κB信号通路为基础,设计并构建含有NF-κB响应元件和mCherry报告基因序列的表达载体,并进一步建立受NF-κB调控表达mCherry的细胞株,最后使用TNF-α进行刺激验证。结果:质粒经过双酶切鉴定,得到约1500 bp的目的片段,符合预计大小且测序分析正确,表明质粒构建成功;在TNF-α刺激实验中,NF-κB信号通路的刺激物TNF-α作用于构建的NF-κB调控表达mCherry荧光蛋白的细胞株后出现特异性荧光反应,TNF-α诱导组的细胞有较强的mCherry表达,阳性率约为90%,而未加TNF-α组细胞mCherry阳性表达率约为10%。结论:构建了受NF-κB调控稳定表达mCherry的慢病毒报告基因系统,可用于对NF-κB活化效果的检测及筛选,具有普适性的应用价值。     

Nitric Oxide Potentiates TNF-α-Induced Neurotoxicity Through Suppression of NF-κB

Modulation of enzyme activity through nitrosylation has recently been identified as a new physiological activity of nitric oxide (NO). We hypothesized that NO enhances the TNF-α-induced death of retinal neurons through a suppression of nuclear factor-κB (NF-κB) by nitrosylation. In this study, cells from the RGC-5 line were exposed to different concentrations (2.0, 10, and 50 ng/ml) of TNF-α, and the degree of TNF-α-induced cell death was determined by the WST-8 assay and by flow cytometric measurements of the externalization of phosphatidylserine. The effects of etanercept, a soluble TNFR-Fc fusion protein, and S-nitroso-N-penicillamine (SNAP), an NO donor, on the toxicity were determined. Experiments were also performed to determine whether nitric oxide synthase (NOS) was associated with the toxicity of TNF-α. The activation of NF-κB was determined by the detection of the p65 subunit in the nuclear extracts. Our results showed that exposure of RGC-5 cells to different concentrations of TNF-α significantly decreased the number of living cells in a dose-dependent way. The death was partially due to apoptosis with an externalization of phosphatidylserine, and the death was suppressed by etanercept. Exposure to TNF-α increased the activation of NF-κB and the expression of iNOS. Although NF-κB inhibitors suppressed the increase of iNOS, they also potentiated the TNF-α-induced death. Both L-NAME and aminoguanidine, both NOS inhibitors, rescued the cells from death. In contrast, addition of SNAP caused nitrosylation of the inhibitory κB kinase, and suppressed the NF-κB activation and potentiated the TNF-α-induced neurotoxicity. These results indicate that NO potentiates the neurotoxicity of TNF-α by suppressing NF-κB.     

The RelA nuclear localization signal folds upon binding to IκBα

The nuclear localization signal (NLS) polypeptide of RelA, the canonical nuclear factor-κB family member, is responsible for regulating the nuclear localization of RelA-containing nuclear factor-κB dimers. The RelA NLS polypeptide also plays a crucial role in mediating the high affinity and specificity of the interaction of RelA-containing dimers with the inhibitor IκBα, forming two helical motifs according to the published X-ray crystal structure. In order to define the nature of the interaction between the RelA NLS and IκBα under solution conditions, we conducted NMR and isothermal titration calorimetry studies using a truncated form of IκBα containing residues 67-206 and a peptide spanning residues 293-321 of RelA. The NLS peptide, although largely unfolded, has a weak tendency toward helical structure when free in solution. Upon addition of the labeled peptide to unlabeled IκBα, the resonance dispersion in the NMR spectrum is significantly greater, providing definitive evidence that the RelA NLS polypeptide folds upon binding IκBα. Isothermal titration calorimetry studies of single-point mutants reveal that residue F309, which is located in the middle of the more C-terminal of the two helices (helix 4) in the IκBα-bound RelA NLS polypeptide, is critical for the binding of the RelA NLS polypeptide to IκBα. These results help to explain the role of helix 4 in mediating the high affinity of RelA for IκBα.     

Interactions between NFκB and Its Inhibitor iκB: Biophysical Characterization of a NFκB/iκB-α Complex

The N-terminal domain (1–318 amino acids) of mouse NFB (p65) has been purified to homogeneity from the soluble fraction of Escherichia coli cells expressing this protein. Its complex with a full-length iB- (MAD3, 1–317 amino acids) molecule was generated by binding the E. coli-derived iB- to the purified NFB and purifying the complex by sequential chromatography. The stoichiometry of NFB to iB in the complex was determined to be 2 to 1 by light scattering and SDS–polyacrylamide gel electrophoresis. The secondary structure of the NFB (p65) determined by Fourier-transform infrared (FTIR) spectroscopy is in good agreement with that of the p50 in the crystal structure of the p50/DNA complex, indicating that no significant structural change in NFB occurs upon binding of DNA. The FTIR spectrum of the NFB/iB complex indicates that its secondary structure is composed of 17% -helix, 39% -strand, 18% irregular structures, and 26% -turns and loops. By comparing these data to the FTIR data for NFB alone, it is concluded that the iB (MAD3) in the complex contains 35% -helix, 27% -strand, 22% irregular structures, and 16% -turns and loops. Circular dichroism (CD) analysis of a shorter form of iB (pp40) indicates that it contains at least 20% -helix and that the iB subunit accounts for nearly all of the -helix present in the NFB/iB complex, consistent with the FTIR results. The stabilities of NFB, iB, and their complex against heat-induced denaturation were investigated by following changes in CD signal. The results indicate that the thermal stability of iB is enhanced upon the formation of the NFB/iB complex.     

The IκB kinase complex in NF-κB regulation and beyond

The IκB kinase (IKK) complex is the signal integration hub for NF-κB activation. Composed of two serine-threonine kinases (IKKα and IKKβ) and the regulatory subunit NEMO (also known as IKKγ), the IKK complex integrates signals from all NF-κB activating stimuli to catalyze the phosphorylation of various IκB and NF-κB proteins, as well as of other substrates. Since the discovery of the IKK complex components about 15 years ago, tremendous progress has been made in the understanding of the IKK architecture and its integration into signaling networks. In addition to the control of NF-κB, IKK subunits mediate the crosstalk with other pathways, thereby extending the complexity of their biological function. This review summarizes recent advances in IKK biology and focuses on emerging aspects of IKK structure, regulation and function.     

核因子κB研究进展

核因子κB(nuclear factor κB,NF-κB)是一种广泛存在于各种细胞、具有多种调节作用的转录因子。它在正常情况下在胞浆内与抑制蛋白(IκB)结合而呈非活性状态。当细胞受到各种刺激原如紫外辐射、细胞因子(如TNF-α、IL-1)、活性氧作用时,NF-κB与IκB解离并进入细胞核内,与特定的启动子结合,从而调控各种基因的表达,如细胞因子、炎症因子、黏附分子等。NF-κB在炎症发生时复杂的细胞因子网络中起着中心调节作用。在细胞增殖、分化和凋亡及肿瘤发生中NF-κB也扮演着重要角色。以NF-κB作为药物作用的靶点,通过调节NF-κB的活性,可改善某些疾病的治疗效果。     

Lipopolysaccharide-Induced NF-κB Activation in Human Endothelial Cells Involves Degradation of IκBα but Not IκBβ

We studied the signal transduction pathways involved in NF-κB activation and the induction of the cytoprotective A20 gene by lipopolysaccharide (LPS) in human umbilical vein endothelial cells (HUVEC). LPS induced human A20 mRNA expression with a maximum level 2 h after stimulation. The proteasome inhibitorN-acetyl-leucinyl-leucinyl-norleucinal-H (ALLN) and the tyrosine kinase inhibitor herbimycin A (HMA) blocked A20 mRNA expression and partially inhibited NF-κB DNA-binding activity induced by LPS treatment. LPS induced IκBα degradation at 30–60 min after treatment, but did not induce IκBβ degradation up to 120 min. In contrast, TNF-α rapidly induced IκBα degradation within 5 min and IκBβ degradation within 15 min. Cycloheximide did not prevent LPS-induced IκBα degradation, indicating that newly synthesized proteins induced by LPS were not involved in LPS-stimulated IκBα degradation. LPS-induced IκBα degradation was inhibited by ALLN, confirming that ALLN inhibits NF-κB activation by preventing IκBα degradation. Of note, HMA also inhibited LPS-induced IκBα degradation. However, tyrosine phosphorylation of IκBα itself was not elicited by LPS stimulation, suggesting that tyrosine phosphorylation of a protein(s) upstream of IκBα is required for subsequent degradation. We conclude that in HUVEC, LPS induces NF-κB-dependent genes through degradation of IκBα, not IκBβ, and propose that this degradation is induced in part by HMA-sensitive kinase(s) upstream of IκBα.