Regulation of activity and function of the p52 NF-κB subunit following DNA damage

We have previously shown that after DNA-damage, the p52 NF-kB subunit can function cooperatively with the p53 tumor suppressor to both repress and induce Skp2 expression. However, the wider role and activation of p52 after DNA-damage has not been determined. Activation of NF-kB in response to DNA break inducers can be mediated by ATM (ataxia telangiectasia mutated)-dependent phosphorylation of NEMO (NF-kB essential modulator), resulting in IKKβ mediated induction of the classical NF-kB pathway, leading to the induction of RelA(p65)/p50 dimers. Here, we show that DNA damage also induces p100 (NF-kB2) processing to generate active p52. We further demonstrate that p52 generation is dependent not only on IKKα but also on atypical activation by NEMO/ATM. Moreover, we identify a post-DNA damage, positive feedback loop of p52 activation through induction of NF-kB2 gene expression, involving both the classical and alternative NF-kB pathways. Gene expression and chromatin immunoprecipitation analyses indicated DNA damage induced p52 dimer recruitment on multiple, p53 dependent and independent, target genes associated with promoting cell cycle arrest and cell death. These results demonstrate an important role for the alternative, p52 NF-kB pathway after DNA-damage distinct from its functions as a regulator of adaptive immunity.     

Involvement of nuclear factor-kappa B in bcl-xL-induced interleukin 8 expression in glioblastoma

We recently reported that bcl-xL regulates interleukin 8 (CXCL8) protein expression and promoter activity in glioblastoma cells. In this paper we demonstrate that CXCL8 induction by bcl-xL is mediated through a nuclear factor-kappa B (NF-kB)-dependent mechanism. Mutational studies on the CXCL8 promoter showed that NF-kB binding site was required for bcl-xL-induced promoter activity and an enhanced nuclear expression of NF-kB subunits p65 and p50 was observed after bcl-xL over-expression. Electrophoretic mobility shift assay showed an increased DNA-binding activity of NF-kB in bcl-xL over-expressing cells and the use of specific antibodies confirmed the involvement of p65 and p50 in NF-kB activity on CXCL8 promoter sequence. NF-kB activity regulation by bcl-xL involved IkBalpha and IKK complex signaling pathway. In fact, bcl-xL over-expression induced a decrease of cytoplasmic expression of the IkBalpha protein, paralleled by an increase in the phosphorylation of the same IkBalpha and IKKalpha/beta. Moreover, the down-regulation of the ectopic or endogenous bcl-xL expression through RNA interference confirmed the ability of bcl-xL to modulate NF-kB pathway, and the transient expression of a degradation-resistant form of the cytoplasmic NF-kB inhibitor IkBalpha in bcl-xL transfectants confirmed the involvement of that inhibitor in bcl-xL-induced CXCL8 expression and promoter activity. In conclusion, our results demonstrate the role of NF-kB as the mediator of bcl-xL-induced CXCL8 up-regulation in glioblastoma cells.     

Role of Toll-like receptors and defensins in antimicrobial protection of urogenital tract in females

Levels of expression of hBD-1 gene (beta-defensin 1) and Toll-like receptors (TLR1, TLR2, TLR6) in cells of cervical mucosa in healthy nonpregnant and healthy pregnant women as well as in pregnant women with urogenital infection was measured by developed RT-PCR systems. During normal pregnancy compared with nonpregnant women, increase of TLRs genes expression which was correlated with increase of hBD-1 gene expression was observed. During urogenital infection in pregnant women compared with healthy pregnant, 10- fold and 50-fold increase of TLR1 and TLR2 genes expression respectively was associated with 2.5-fold decrease of hBD-1 gene expression in cervical mucosa. In group of women with untrauterine infection more marked increase of TLRs genes expression was observed. Thus significant changes (TLRs, antimicrobial peptides, cytokines etc.) in cells of cervical mucosa can be used as prognostic criteria for development of intrauterine infection.     

Association of cytokines in hepatitis E with pregnancy outcome

AimsThe aim of this study was to evaluate tumour necrosis factor-alpha (TNF-α), interleukin (IL)-6, interferon gamma (IFN-γ) and transforming growth factor-beta1 (TGF-β1) in hepatitis E infection during pregnancy and its relation with pregnancy outcome.MethodsA total of 272 pregnant and 219 non-pregnant women with hepatitis and 262 age and gestational age matched healthy pregnant women and 208 age matched, healthy non-pregnant women were evaluated on the basis of history, clinical examination, liver function profile. Serological tests of hepatitis A, B, C and E and cytokines using commercially available (ELISA) kits. The patients with hepatitis E were further evaluated for viral load by Real Time PCR. All these were followed till delivery for pregnancy outcome.ResultsHEV viral load in acute viral hepatitis (AVH) and fulminant hepatic failure (FHF) of pregnant women were comparatively higher than non-pregnant women. Significantly higher levels of TNF-α, IL-6, IFN-γ and TGF-β1 were present in HEV infected pregnant women compared to non-pregnant women and controls. TNF-α, IL-6 and IFN-γ had significant positive correlation with viral load, serum bilirubin and prothrombin time in pregnant women. Higher levels of all four cytokines were found in pregnant women with HEV infection having adverse pregnancy outcome compared to that of pregnant women with non-HEV infection and controls.ConclusionIn conclusion, severity of HEV infection and associated adverse pregnancy outcome might be mediated by cytokine in pregnancy.     

NIK and Cot cooperate to trigger NF-kappaB p65 phosphorylation

The serine/threonine kinase Cot triggers NF-κB-dependent transactivation and activation of various MAPKinases. Here we identify Cot as a novel p65 interacting protein kinase. Cot expression induces p65 phosphorylation at serines 536 and 468 in dependence from its kinase function. Accordingly, shRNA-mediated knockdown of Cot expression interferes with TNF-induced NF-κB-dependent gene expression. Also the C-terminally truncated, oncogenic form of Cot is able to trigger p65 phosphorylation. In vitro kinase assays and dominant negative mutants revealed that NIK functions downstream of Cot to mediate p65 phosphorylation.