NF-kappaB inhibition by an adenovirus expressed aptamer sensitizes TNFalpha-induced apoptosis

Prolonged activation of NF-kappaB is involved in the pathogenesis of chronic inflammatory diseases and associated cancers. NF-kappaB activation is considered to be a main mechanism opposing TNFalpha-induced apoptosis. We investigated whether inhibition of NF-kappaB could sensitize tumor and endothelial cells to TNFalpha-induced apoptosis. As such, we developed a novel H1 RNA polymerase III promoter driven adenoviral vector to express an RNA aptamer, Ad-A-p50, which selectively inhibits NF-kappaB activation in the nucleus. This event sensitizes human lung adenocarcinoma cells (A549) and human endothelial cells (HUVEC) to TNFalpha-induced apoptosis through the multiple pathways regulated by NF-kappaB, including Bcl-XL, HIF-1alpha, and VEGF. Our findings also suggest a new mechanism of HIF-1alpha regulation by NF-kappaB in the normoxic environment. RNA aptamer inhibition of NF-kappaB offers exciting opportunities for sensitizing inflammatory and tumor cells to TNFalpha-induced apoptosis.     

Expression of APOBEC2 is transcriptionally regulated by NF-kappaB in human hepatocytes

Apolipoprotein B mRNA-editing enzyme catalytic subunit 2 (APOBEC2) is a member of the nucleic-acid-editing enzymes. However, the physiological function of APOBEC2 remains unclear. We demonstrate that APOBEC2 expression is strongly enhanced in response to both tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta. Inhibition of NF-kappaB activation invariably blocks TNF-alpha-induced APOBEC2 expression. The promoter region of APOBEC2 contains functional NF-kappaB response elements in the 5' untranslated region of the gene at -625/-616. These results show that APOBEC2 expression is regulated by pro-inflammatory cytokines via NF-kappaB activation and suggest a possible role of APOBEC2 in the pathophysiology of hepatic inflammation.     

NF-kappaB/Egr-1/Gadd45 are sequentially activated upon UVB irradiation to mediate epidermal cell death

Chronic sun exposure can lead to severe skin disorders such as carcinogenesis. The cell death process triggered by ultraviolet B (UVB) irradiation is crucial because it protects the surrounding tissue from the emergence and the accumulation of cells that bear the risk of becoming transformed. Here, we show that repression of NF-kappaB and Egr-1 expression drastically inhibits UVB-mediated cell death. Furthermore, we demonstrate that Egr-1 is induced upon UVB irradiation through NF-kappaB activation and the binding of p65/RelA within the Egr-1 promoter. We show that Egr-1 contributes to the regulation of the Gadd45a and Gadd45b genes, which are involved in the control of cell cycle, DNA repair and apoptosis, by direct binding to their promoter. Our study demonstrates for the first time a signaling cascade involving sequential activation of NF-kappaB, Egr-1 and Gadd45 to induce UVB-mediated cell death. Failure in the induction of each protagonist of this pathway alters the UVB-mediated cell death process. Therefore, impairment of the cascade could be at the onset of skin carcinogenesis mediated by genotoxic stress.     

Identification of TNF-alpha-responsive NF-kappaB p65-binding element in the distal promoter of the mouse serine protease inhibitor SerpinE2

Serine protease inhibitor SerpinE2 is known as a cytokine-inducible gene. Here, we investigated whether tumor necrosis factor alpha-(TNF-alpha)-induced expression of SerpinE2 is mediated by the nuclear factor-kappaB (NF-kappaB) p65 subunit. Both steady state and TNF-alpha-induced expression of SerpinE2 mRNA were abrogated in p65-/- murine embryonic fibroblasts (MEFs). Reconstitution with wild-type p65 rescued SerpinE2 mRNA expression in an IkappaB kinase beta-dependent manner. Electrophoresis mobility shift assay and ChIP assay demonstrated that p65 bound to the kappaB-like DNA sequence located at approximately -9 kbp in the SerpinE2 promoter. In addition, TNF-alpha stimulated luciferase gene expression driven by the kappaB-like element in the reconstituted MEFs, but not in p65-/- MEFs. These results indicated that activation of NF-kappaB p65 plays an important role in TNF-alpha-induced expression of SerpinE2.     

Polycystin-1 promotes PKCalpha-mediated NF-kappaB activation in kidney cells

Polycystin-1 (PC1), the PKD1 gene product, is a membrane receptor which regulates many cell functions, including cell proliferation and apoptosis, both typically increased in cyst lining cells in autosomal dominant polycystic kidney disease. Here we show that PC1 upregulates the NF-kappaB signalling pathway in kidney cells to prevent cell death. Human embryonic kidney cell lines (HEK293(CTT)), stably expressing a PC1 cytoplasmic terminal tail (CTT), presented increased NF-kappaB nuclear levels and NF-kappaB-mediated luciferase promoter activity. This, consistently, was reduced in HEK293 cells in which the endogenous PC1 was depleted by RNA interference. CTT-dependent NF-kappaB promoter activation was mediated by PKCalpha because it was blocked by its specific inhibitor Ro-320432. Furthermore, it was observed that apoptosis, which was increased in PC1-depleted cells, was reduced in HEK293(CTT) cells and in porcine kidney LtTA cells expressing a doxycycline-regulated CTT. Staurosporine, a PKC inhibitor, and parthenolide, a NF-kappaB inhibitor, significantly reduced the CTT-dependent antiapoptotic effect. These data reveal, therefore, a novel pathway by which polycystin-1 activates a PKCalpha-mediated NF-kappaB signalling and cell survival.     

β-淀粉样蛋白对BV2小胶质细胞促炎作用的研究

目的探讨β-淀粉样蛋白(β-amyloid,Aβ)促进BV2小胶质细胞产生炎性因子IL-1β和TNFα的作用机制。方法体外培养BV2小胶质细胞,应用Aβ1-42作用于BV2小胶质细胞,或用吡咯烷二硫代氨基甲酸盐(PDTC)预孵育再给予Aβ1-42刺激,实时荧光定量反转录聚合酶链反应法(RT–PCR)检测IL-1β和TNFαmRNA表达;免疫印迹法(Western blot)检测胞核中NF-κB p65及其抑制蛋白胞浆中IkBα的表达。结果 Aβ1-42作用于BV2小胶质细胞后,Westernblot显示胞浆内IkBα表达下降,胞核内NF-κB p65表达明显增加,RT-PCR测定IL-1β和TNFαmRNA的表达增加;给予NF-κB信号通路特异阻断剂PDTC后,胞浆IkBα的下降和胞核内NF-κB p65的增加均被抑制,同时IL-1β和TNFαmRNA的表达亦受到抑制,PDTC的抑制效果呈剂量依赖性。结论 Aβ可通过激活小胶质细胞NF-κB信号通路促进IL-1β和TNFα的表达。     

IL-1beta induces MMP-9 via reactive oxygen species and NF-kappaB in murine macrophage RAW 264.7 cells

IL-1beta increased the production of proenzyme of MMP-9 (pro-MMP-9) in a time- and dose-dependent manner in murine macrophage RAW 264.7 cells. However, the production of MMP-2 was not significantly changed by IL-1beta treatment. The intracellular H(2)O(2) content, as determined with H(2)O(2)-sensitive probe 2('),7(')-dichlorodihydrofluorescein, also increased after IL-1beta treatment (5ng/ml). In addition, exogenous H(2)O(2) (50 microM) was found to increase the production of pro-MMP-9. Transient transfection study using a MMP-9 promoter-reporter construct showed that IL-1beta enhanced the MMP-9 promoter activity. Electrophoretic mobility shift assay and site-directed mutagenesis study on the consensus binding site for NF-kappaB revealed that the activation of NF-kappaB is required for the IL-1beta-induced activation of MMP-9 promoter. N-acetylcysteine, an antioxidant, could abrogate the production of pro-MMP-9, H(2)O(2) generation, and activation of NF-kappaB and MMP-9 promoter. These results suggest that IL-1beta upregulates the MMP-9 expression via production of reactive oxygen species and activation of NF-kappaB in RAW 264.7 cells.     

NF-κB、IκB、IKK在非小细胞肺癌中的表达及意义

目的检测非小细胞肺癌(non-small cell lung cancer,NSCLC)中NF-κB P65、p-IκBα(IκBα磷酸化)、p-IKKβ(IKKβ磷酸化)的表达情况及其与NSCLC临床特征的关系。方法采用免疫组化Elivision法检测NF-κB P65、p-IκBα、p-KKβ在56例NSCLC中表达情况,以20例癌旁组织作为对照。结果在NSCLC中NF-κB P65、p-IκBα、p-IKKβ的表达阳性率分别为83.9%(47/56)、55.7%(31/56)、69.6%(39/56),癌旁组织三者分别为20%(4/20)、25%(5/20)、30%(6/20),NF-κB P65、p-IκBα、p-IKKβ的表达与吸烟史、TNM分期、淋巴结转移相关,差异有统计学意义(P<0.05)。结论 NF-κB P65、p-IκBα、p-IKKβ高表达与NSCLC的发生、发展起着重要作用。     

Reduced cGMP signaling activates NF-kappaB in hypertrophied hearts of mice lacking natriuretic peptide receptor-A

Mice lacking natriuretic peptide receptor-A (NPRA) develop progressive cardiac hypertrophy and congestive heart failure. However, the mechanisms responsible for cardiac hypertrophic growth in the absence of NPRA signaling are not yet known. We sought to determine the activation of nuclear factor-kappaB (NF-kappaB) in Npr1 (coding for NPRA) gene-knockout (Npr1-/-) mice exhibiting cardiac hypertrophy and fibrosis. NF-kappaB binding activity was 4-fold greater in the nuclear extract of Npr1-/- mutant mice hearts as compared with wild-type (Npr1+/+) mice hearts. In parallel, inhibitory kappaB kinase-beta activity and IkappaB-alpha protein phosphorylation were also increased 3- and 4-fold, respectively, in hypertrophied hearts of mutant mice. cGMP levels were significantly reduced 5-fold in plasma and 10-fold in ventricular tissues of mutant mice hearts relative to wild-type controls. The present findings provide direct evidence that ablation of NPRA/cGMP signaling activates NF-kappaB binding activity associated with hypertrophic growth of mutant mice hearts.