Modeling oscillatory control in NF-κB, p53 and Wnt signaling

Oscillations are commonly observed in cellular behavior and span a wide range of timescales, from seconds in calcium signaling to 24 hours in circadian rhythms. In between lie oscillations with time periods of 1-5 hours seen in NF-κB, p53 and Wnt signaling, which play key roles in the immune system, cell growth/death and embryo development, respectively. In the first part of this article, we provide a brief overview of simple deterministic models of oscillations. In particular, we explain the mechanism of saturated degradation that has been used to model oscillations in the NF-κB, p53 and Wnt systems. The second part deals with the potential physiological role of oscillations. We use the simple models described earlier to explore whether oscillatory signals can encode more information than steady-state signals. We then discuss a few simple genetic circuits that could decode information stored in the average, amplitude or frequency of oscillations. The presence of frequency-detector circuit downstream of NF-κB or p53 would be a strong clue that oscillations are important for the physiological response of these signaling systems.     

Cyclophilin A (CypA) Interacts with NF-κB Subunit,p65/RelA,and Contributes to NF-κB Activation Signaling

Background

Peptidyl-prolyl isomerase cyclophilin A (CypA) plays important roles in signaling, protein translocation, inflammation, and cancer formation. However, little is known about the mechanisms by which CypA exerts its effects. C57BL/6 Ppia (encoding CypA)-deficient embryonic fibroblasts show reduced activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), the p65/RelA subunit, suggesting that CypA may mediate modulation of NF-κB activity to exert its biological effects.

Methodology

Western blotting and qRT-PCR analyses were used to evaluate the association of CypA deficiency with reduced activation of NF-κB/p65 at the protein level. GST pull-down and co-immunoprecipitation were used to examine interactions between CypA and p65/RelA. Truncation mutants and site-directed mutagenesis were used to determine the sequences of p65/RelA required for interactions with CypA. Enhancement of p65/RelA nuclear translocation by CypA was assessed by co-transfection and immunofluorescent imaging. Treatment of cells with cycloheximide that were harvested at various time points for Western blot analyses was carried out to evaluate p65/RelA protein stability. The functional activity of NF-κB was assessed by electrophoretic mobility-shift assays (EMSA), luciferase assays, and changes in expression levels of target genes.

Results

GST pull-down assays in vitro and co-immunoprecipitation analyses in vivo provided evidence for protein-protein interactions. These interactions were further supported by identification of a CypA-binding consensus-like sequence within NF-κB subunit p65 at the N-terminal 170–176 amino acid residues. Significantly, CypA provided stability for NF-κB p65 and promoted NF-κB p65 nuclear translocation, resulting in increased nuclear accumulation and enhanced NF-κB activity.

Conclusions

Our findings revealed important mechanisms that regulate NF-κB activation, and offer new insights into the role of CypA in aberrant activation of NF-κB-mediated signaling for altered expression of its target genes, resulting in pathological effects in various diseases.     

Coordinate Regulation of TPL-2 and NF-κB Signaling in Macrophages by NF-κB1 p105

The role of IκB kinase (IKK)-induced proteolysis of NF-κB1 p105 in innate immune signaling was investigated using macrophages from Nfkb1(SSAA/SSAA) mice, in which the IKK target serines on p105 are mutated to alanines. We found that the IKK/p105 signaling pathway was essential for TPL-2 kinase activation of extracellular signal-regulated kinase (ERK) mitogen-activate protein (MAP) kinase and modulated the activation of NF-κB. The Nfkb1(SSAA) mutation prevented the agonist-induced release of TPL-2 from its inhibitor p105, which blocked activation of ERK by lipopolysaccharide (LPS), tumor necrosis factor (TNF), CpG, tripalmitoyl-Cys-Ser-Lys (Pam(3)CSK), poly(I · C), flagellin, and R848. The Nfkb1(SSAA) mutation also prevented LPS-induced processing of p105 to p50 and reduced p50 levels, in addition to decreasing the nuclear translocation of RelA and cRel. Reduced p50 in Nfkb1(SSAA/SSAA) macrophages significantly decreased LPS induction of the IκBζ-regulated Il6 and Csf2 genes. LPS upregulation of Il12a and Il12b mRNAs was also impaired although specific blockade of TPL-2 signaling increased expression of these genes at late time points. Activation of TPL-2/ERK signaling by IKK-induced p105 proteolysis, therefore, induced a negative feedback loop to downregulate NF-κB-dependent expression of the proinflammatory cytokine interleukin-12 (IL-12). Unexpectedly, TPL-2 promoted soluble TNF production independently of IKK-induced p105 phosphorylation and its ability to activate ERK, which has important implications for the development of anti-inflammatory drugs targeting TPL-2.     

1,8-Cineol inhibits nuclear translocation of NF-κB p65 and NF-κB-dependent transcriptional activity

Natural plant-derived products are commonly applied to treat a broad range of human diseases, including cancer as well as chronic and acute airway inflammation. In this regard, the monoterpene oxide 1,8-cineol, the active ingredient of the clinically approved drug Soledum®, is well-established for the therapy of airway diseases, such as chronic sinusitis and bronchitis, chronic obstructive pulmonary disease and bronchial asthma. Although clinical trials underline the beneficial effects of 1,8-cineol in treating inflammatory diseases, the molecular mode of action still remains unclear.Here, we demonstrate for the first time a 1,8-cineol-depending reduction of NF-κB-activity in human cell lines U373 and HeLa upon stimulation using lipopolysaccharides (LPS). Immunocytochemistry further revealed a reduced nuclear translocation of NF-κB p65, while qPCR and western blot analyses showed strongly attenuated expression of NF-κB target genes. Treatment with 1,8-cineol further led to increased protein levels of IκBα in an IKK-independent matter, while FRET-analyses showed restoring of LPS-associated loss of interaction between NF-κB p65 and IκBα. We likewise observed reduced amounts of phosphorylated c-Jun N-terminal kinase 1/2 protein in U373 cells after exposure to 1,8-cineol. In addition, 1,8-cineol led to decreased amount of nuclear NF-κB p65 and reduction of its target gene IκBα at protein level in human peripheral blood mononuclear cells.Our findings suggest a novel mode of action of 1,8-cineol through inhibition of nuclear NF-κB p65 translocation via IκBα resulting in decreased levels of proinflammatory NF-κB target genes and may therefore broaden the field of clinical application of this natural drug for treating inflammatory diseases.     

突变型p53与NF-κB在肿瘤发生中的相互作用

p53是人体内重要的肿瘤抑制因子。但超过50%人类肿瘤携带突变型p53(mutant p53,mutp53)而失去功能。核转录因子NF-κB作为调节炎症反应的关键因子,不仅参与免疫应答,还可促进组织或器官由慢性炎症向肿瘤的恶性转化。同时,p53和NF-κB的异常激活与肿瘤的预后不良以及化疗耐受性密切相关。在炎症和肿瘤中,mutp53与NF-κB两者之间存在相互调控关系。因此,mutp53和NF-κB均可作为肿瘤治疗的潜在靶点。该文总结了在炎症和肿瘤中mutp53和NF-κB蛋白之间相互作用的分子机制,以及两者相互作用对肿瘤进程的影响,从而为进一步研究两者间的相互作用及相关抗肿瘤策略设计提供思路。     

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。     

1,6-O,O-diacetylbritannilactones inhibits IκB kinase β-dependent NF-κB activation

To determine the chemical constituents responsible for pharmacological effects of Inula britannica-F., three specific sesquiterpene lactones in Inula britannica were isolated from chloroform extract and identified, including britannilactone (BL), 1-O-acetylbritannilactone (ABLO), and 1,6-O,O-diacetylbritannilactone (ABLOO). Electrophoretic mobility shift assay (EMSA) was performed to detect the nuclear translocation of nuclear factor-κB (NF-κB) p65. The expressions of IκBα, pIκBα, inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), IκB kinase α/β (IKKα/β) and NF-κB kinase (NIK) were detected by Western blot and RT-PCR. We found that acetyl side groups enhanced the inhibitory action of the agents on LPS/IFN-γ-induced iNOS and COX-2 expression. Their inhibiting activity was positive correlation with the acetyl side group number. The effects of LPS/IFN-γ were reversed by ABLOO, and BL without acetyl side groups showed only a weak inhibitory action. Further study indicated that ABLOO markedly inhibited the phosphorylation of IKKβ down to based level, but not IKKα, corresponding with decreased in IκBα degradation and phosphorylation induced by LPS/IFN-γ, resulting in the suppression of NF-κB nuclear translocation and activity. These results suggest that the acetyl moieties add to the lipophilicity, and consequently enhance cellular penetration, so that ABLOO possess the most anti-inflammatory effect and may be a potent lead structure for the development of therapeutic and cytokine-suppressing remedies valuable for the treatment of various inflammatory diseases.     

NF-κB p65在皮肤血管瘤不同时期的表达

目的探讨NF-κB p65在血管瘤发生、发展及退化过程中的表达状况及其意义.方法采用免疫组织化学方法(S-P法)检测人皮肤血管瘤增生期、退化期及正常皮肤组织及内皮中NF-κB p65的表达水平.利用计算机图像分析技术测量不同时期血管瘤组织和正常皮肤组织NF-κB p65表达的平均光密度和平均阳性面积.结果增生期血管瘤内皮细胞NF-κB p65表达水平高于退化期,差异有显著性(P＜0.01),退化期血管瘤内皮细胞NF-κB p65表达水平与正常皮肤组织相比,差异无显著性(P＞0.05).结论 NF-κB p65可能通过正向调控血管生成因子的表达而促进血管瘤的形成.     

Endoplasmic Reticulum Stress Stimulates p53 Expression through NF-κB Activation

Background

Induction of apoptosis by endoplasmic reticulum (ER) stress is implicated as the major factor in the development of multiple diseases. ER stress also appears to be a potentially useful major response to many chemotherapeutic drugs and environmental chemical compounds. A previous study has indicated that one major apoptotic regulator, p53, is significantly increased in response to ER stress, and participates in ER stress-induced apoptosis. However, the regulators of p53 expression during ER stress are still not fully understood.

Principal Findings

In this report, we demonstrate that induction of p53 expression is mediated through NF-κB signaling pathways during ER stress in MCF-7 cells. Tunicamycin or brefeldin A, two ER stress inducers, increased p53 expression in MCF-7 and Hela cells. We found p53 nuclear localization, activity, and phosphorylation at serine 15 on p53 increased during ER stress. Nuclear translocation of NF-κB and activity of NF-κB were also observed during ER stress. ER stress-induced p53 expression was significantly inhibited by coincubation with the NF-κB inhibitor, Bay 11-7082 and downregulation of NF-κB p65 expression. The role of p53 in mediating Brefeldin A-induced apoptosis was also investigated. Induction of p53 expression by Brefeldin A was correlated to Brefeldin A-induced apoptosis. Furthermore, downregulation of p53 expression by p53 siRNA significantly reduced Brefeldin A-induced apoptosis in MCF-7 cells.

Significance

Taken together, NF-κB activation and induction of p53 expression is essential for ER stress-induced cell death which is important for therapeutic effects of clinical cancer drugs. Our results may provide insight into the mechanism of cancer chemotherapy efficacy that is associated with induction of ER stress.     

NF-κB的生物学功能

NF—κB因其广泛参与机体的免疫及其它应激反应而受到人们的关注,其常见的形式是由p50和p65组成的异源二聚体。细胞受到外部因素刺激后,NF-κB由细胞质转移到细胞核中,并发生磷酸化和乙酰化,启动相关基因的表达。目前研究表明受NF-κB调节的基因有200多种,其激活因子不少于150种,所以对NF-κB在各种条件下的激活过程及信号传导网络的研究具有重要的意义。本文综合了当前关于NF—κB研究的最新进展,着重阐述了由TNF-α、IL-Ⅰ及LPS刺激而引起NF—κB激活的信号传导通路,并进一步阐述了其在人类某些疾病当中的生物学功能。     

Non-canonical NF-κB signaling pathway

The non-canonical NF-κB pathway is an important arm of NF-κB signaling that predominantly targets activation of the p52/RelB NF-κB complex. This pathway depends on the inducible processing of p100, a molecule functioning as both the precursor of p52 and a RelB-specific inhibitor. A central signaling component of the non-canonical pathway is NF-κB-inducing kinase (NIK), which integrates signals from a subset of TNF receptor family members and activates a downstream kinase, IκB kinase-α (IKKα), for triggering p100 phosphorylation and processing. A unique mechanism of NIK regulation is through its fate control: the basal level of NIK is kept low by a TRAF-cIAP destruction complex and signal-induced non-canonical NF-κB signaling involves NIK stabilization. Tight control of the fate of NIK is important, since deregulated NIK accumulation is associated with lymphoid malignancies.