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.     

Heat shock protein 70 negatively regulates the heat-shock-induced suppression of the IkappaB/NF-kappaB cascade by facilitating IkappaB kinase renaturation and blocking its further denaturation

Heat shock (HS) treatment has been previously shown to suppress the IkappaB/nuclear factor-kappaB (NF-kappaB) cascade by denaturing, and thus inactivating IkappaB kinase (IKK). HS is characterized by the induction of a group of heat shock proteins (HSPs). However, their role in the HS-induced suppression of the IkappaB/NF-kappaB cascade is unclear. Adenovirus-mediated HSP70 overexpression was found not to suppress the TNF-alpha-induced activation of the IkappaB/NF-kappaB pathway, thus suggesting that HSP70 is unlikely to suppress this pathway. When TNF-alpha-induced activation of the IkappaB/NF-kappaB pathway was regained 24 h after HS, HSP70 was found to be highly up-regulated. Moreover, blocking HSP70 induction delayed TNF-alpha-induced IkappaBalpha degradation and the resolubilization of IKK. In addition, HSP70 associated physically with IKK, suggesting that HSP70 is involved in the recovery process via molecular chaperone effect. Adenovirus-mediated HSP70 overexpression prior to HS blocked the IkappaBalpha stabilizing effect of HS by suppressing IKK insolubilization. Moreover, the up-regulation of endogenous HSP70 by preheating, suppressed this subsequent HS-induced IKK insolubilization, and this effect was abrogated by blocking HSP70 induction. These findings indicate that HSP70 accumulates during HS and negatively regulates the HS-induced suppression of the IkappaB/NF-kappaB cascade by facilitating the renaturation of IKK and blocking its further denaturation.     

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.     

The heat-shock-induced suppression of the IkappaB/NF-kappaB cascade is due to inactivation of upstream regulators of IkappaBalpha through insolubilization

Heat shock (HS) was found to suppress the IkappaB/NF-kappaB cascade via the inhibition of IkappaB kinase (IKK) activity; however, the mechanism has not been clear. This study was undertaken to elucidate the detail of the mechanism involved. TNF-alpha-induced activation of IKK was suppressed by HS in human bronchial epithelial cells, and this was associated with the absence of IKK in the immunoprecipitates. It was not due to a degradation of IKK, but due to a conversion of IKK from a soluble to an insoluble form. IKK lost its activity rapidly upon exposure to HS in vitro. The time course of the insolubilization of IKK coincided with the decrease in IKK activity. However, inhibition of IKK insolubilization by the induction of thermotolerance did not reverse the HS-induced suppression of IKK activation and IkappaBalpha degradation. Upstream activators of IKK, such as NF-kappaB-inducing kinase (NIK) and IL-1R-associated kinase (IRAK) were also insolubilized by HS. The HS-induced insolubilization of NIK was not blocked by the induction of thermotolerance. Overexpression of NIK resumed TNF-alpha-induced activation of IKK in thermotolerant cells. These results indicate that the loss of activity of NIK, IRAK, and IKK through insolubilization is responsible for the HS-induced suppression of IkappaB/NF-kappaB pathway.     

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.     

Two distinct ubiquitin-dependent mechanisms are involved in NF-kappaB p105 proteolysis

Generation of the p50 subunit of NF-kappaB is a rare case in which the ubiquitin system processes a longer precursor, p105, into a shorter active subunit: in the vast majority of cases, the target protein is completely degraded. The mechanisms involved in this process have remained elusive. It appears that a Gly rich region (GRR) in the middle of the molecule serves as a "processing stop signal", though under certain conditions, such as after stimulation, p105 can be completely degraded. Since NF-kappaB plays critical roles in a broad array of basic cellular processes, it is important to dissect the mechanisms that regulate its proteolysis-both destruction and processing. We have previously shown that signal-induced degradation of p105 requires ubiquitination on multiple lysines. Here we describe a novel region, a Processing Inhibitory Domain-PID, that upon its removal, the molecule is processed in high efficiency, which requires ubiquitination on a single, though non-specific, lysine.     

TAK1 is critical for IkappaB kinase-mediated activation of the NF-kappaB pathway

Cytokine treatment stimulates the IkappaB kinases, IKKalpha and IKKbeta, which phosphorylate the IkappaB proteins, leading to their degradation and activation of NF-kappaB regulated genes. A clear definition of the specific roles of IKKalpha and IKKbeta in activating the NF-kappaB pathway and the upstream kinases that regulate IKK activity remain to be elucidated. Here, we utilized small interfering RNAs (siRNAs) directed against IKKalpha, IKKbeta and the upstream regulatory kinase TAK1 in order to better define their roles in cytokine-induced activation of the NF-kappaB pathway. In contrast to previous results with mouse embryo fibroblasts lacking either IKKalpha or IKKbeta, which indicated that only IKKbeta is involved in cytokine-induced NF-kappaB activation, we found that both IKKalpha and IKKbeta were important in activating the NF-kappaB pathway. Furthermore, we found that the MAP3K TAK1, which has been implicated in IL-1-induced activation of the NF-kappaB pathway, was also critical for TNFalpha-induced activation of the NF-kappaB pathway. TNFalpha activation of the NF-kappaB pathway is associated with the inducible binding of TAK1 to TRAF2 and both IKKalpha and IKKbeta. This analysis further defines the distinct in vivo roles of IKKalpha, IKKbeta and TAK1 in cytokine-induced activation of the NF-kappaB pathway.     

X-linked inhibitor of apoptosis protein increases mitochondrial antioxidants through NF-kappaB activation

X chromosome-linked inhibitor of apoptosis protein is an endogenous inhibitor of caspases and is an important regulator of cell death. XIAP can also influence cell signaling, but downstream proteins affected are largely unknown. We show here using neuronal PC6.3 cells that XIAP increases the levels of antioxidants, particularly superoxide dismutase-2 that is localized to mitochondria. Studies using reporter constructs and NF-κB Rel-A deficient mouse embryonic fibroblasts showed that NF-κB signaling is required for the induction of Sod2 by XIAP. XIAP also reduced oxidative stress in the PC6.3 cells as shown by decreased production of reactive oxygen species. These findings disclose a novel role for XIAP in control of oxidative stress and mitochondrial antioxidants that may contribute to cell protection after various injuries.     

Identification of the RelA domain responsible for action of a new NF-kappaB inhibitor DHMEQ

A new NF-κB inhibitor dehydroxymethylepoxyquinomicin (DHMEQ) has a potential to be applied to clinical medicine as an anti-cancer and anti-inflammatory agent. DHMEQ inhibits localization of NF-κB in the nucleus and the inhibitory effect by DHMEQ is more potent on p50/RelA than on p50 homodimer. However, a molecular target of DHMEQ is unknown. In this study, we identified residues CEGRSAGSI, which appear in RelA (amino acids 38-46), c-Rel (28-36), and RelB (144-152), but not in p50 and p52, as a target of DHMEQ. As a possible mechanism, we propose that DHMEQ accesses CEGRSAGSI domain recognizing RSAGSI structure and directly binds to cysteine. This target domain appears to be unique among mammalian proteins. The results obtained in this study may provide better understanding of the action of DHMEQ and a key for developing a new NF-κB inhibitor with more potent activity.     

Participation of the E3-ligase TRIM13 in NF-κB p65 activation and NFAT-dependent activation of c-Rel upon T-cell receptor engagement

The nuclear factor κB (NF-κB) family members p65 and c-Rel chiefly orchestrate lymphocytes activation following T-cell receptor (TCR) engagement. In contrast to p65, which is rapidly mobilized, c-Rel activation occurs subsequently as it involves a nuclear factor of activated T-cells (NFAT)-dependent upregulation step. However, how TCR ligation drives p65 and c-Rel activation is not fully understood. Because several ubiquitylated components of NF-κB signaling cascade accumulate in close proximity to membranes, we screened a siRNA library against E3-ligases that contain transmembrane domains on TCR-mediated NF-κB activation. Here, we report the identification of the endoplasmic reticulum resident TRIM13 protein as an enhancer of NF-κB promoter activity. We found that knocking down TRIM13 by RNA interference reduced the activation of p65, while the translocation of c-Rel into the nucleus was blunted. We further observed that c-Rel induction was diminished without TRIM13, as NFAT activation was compromised. These results unveil that TRIM13 is a selective regulator of p65 and of c-Rel activation.     

IκBα缺失突变体真核表达载体的构建、表达及其生物活性检测

旨在构建缺失N端前36个氨基酸的IκBα突变体真核表达载体,并对其表达及生物学活性进行检测.从人源子宫颈癌细胞HeLa中提取总RNA,利用RT-PCR的方法获得IκBα缺失突变体的cDNA,将其克隆至真核表达载体pcDNA3.1/myc-His A中,构建重组载体pcDNA3.1-IκBαΔN.通过PCR方法、NcoⅠ酶切以及核酸测序分析对其进行鉴定;采用Western Blot检测IκBα缺失突变体蛋白在HeLa细胞中的表达.将pcDNA3.1-IκBαΔN和pNF-κB-Luc共转染 HeLa细胞,经TNF-α诱导后,利用萤光素酶报告系统来检测重组载体对NF-αB的抑制活性.结果表明,经PCR方法、NcoⅠ酶切鉴定及核酸测序分析后,证实成功构建了重组载体pcDNA3.1-IκBαΔN;IκBα缺失突变体蛋白在HeLa细胞中高效表达,并对NF-κB有显著的抑制活性(P＜0.01).因此,真核表达载体pcDNA3.1-IκBαΔN构建成功,为一步研究NF-κB信号传导通路及其相关疾病提供有效的分子工具.