p97/VCP promotes Cullin‐RING‐ubiquitin‐ligase/proteasome‐dependent degradation of IκBα and the preceding liberation of RelA from ubiquitinated IκBα

Cullin‐RING‐ubiquitin‐ligase (CRL)‐dependent ubiquitination of the nuclear factor kappa B (NF‐κB) inhibitor IκBα and its subsequent degradation by the proteasome usually precede NF‐κB/RelA nuclear activity. Through removal of the CRL‐activating modification of their cullin subunit with the ubiquitin (Ub)‐like modifier NEDD8, the COP9 signalosome (CSN) opposes CRL Ub‐ligase activity. While RelA phosphorylation was observed to mediate NF‐κB activation independent of Ub‐proteasome‐pathway (UPP)‐dependent turnover of IκBα in some studies, a strict requirement of the p97/VCP ATPase for both, IκBα degradation and NF‐κB activation, was reported in others. In this study, we thus aimed to reconcile the mechanism for tumour necrosis factor (TNF)‐induced NF‐κB activation. We found that inducible phosphorylation of RelA is accomplished in an IKK‐complex‐dependent manner within the NF‐κB/RelA‐IκBα‐complex contemporaneous with the phosphorylation of IκBα, and that RelA phosphorylation is not sufficient to dissociate NF‐κB/RelA from IκBα. Subsequent to CRL‐dependent IκBα ubiquitination functional p97/VCP is essentially required for efficient liberation of (phosphorylated) RelA from IκBα, preceding p97/VCP‐promoted timely and efficient degradation of IκBα as well as simultaneous NF‐κB/RelA nuclear translocation. Collectively, our data add new facets to the knowledge about maintenance of IκBα and RelA expression, likely depending on p97/VCP‐supported scheduled basal NF‐κB activity, and the mechanism of TNF‐induced NF‐κB activation.     

PMC,a potent hydrophilic α‐tocopherol derivative,inhibits NF‐κB activation via PP2A but not IκBα‐dependent signals in vascular smooth muscle cells

The hydrophilic α‐tocopherol derivative, 2,2,5,7,8‐pentamethyl‐6‐hydroxychromane (PMC), is a promising alternative to vitamin E in clinical applications. Critical vascular inflammation leads to vascular dysfunction and vascular diseases, including atherosclerosis, hypertension and abdominal aortic aneurysms. In this study, we investigated the mechanisms of the inhibitory effects of PMC in vascular smooth muscle cells (VSMCs) exposed to pro‐inflammatory stimuli, lipopolysaccharide (LPS) combined with interferon (IFN)‐γ. Treatment of LPS/IFN‐γ‐stimulated VSMCs with PMC suppressed the expression of inducible nitric oxide synthase (iNOS) and matrix metalloproteinase‐9 in a concentration‐dependent manner. A reduction in LPS/IFN‐γ‐induced nuclear factor (NF)‐κB activation was also observed in PMC‐treated VSMCs. The translocation and phosphorylation of p65, protein phosphatase 2A (PP2A) inactivation and the formation of reactive oxygen species (ROS) were significantly inhibited by PMC in LPS/IFN‐γ‐activated VSMCs. However, neither IκBα degradation nor IκB kinase (IKK) or ribosomal s6 kinase‐1 phosphorylation was affected by PMC under these conditions. Both treatments with okadaic acid, a PP2A‐selective inhibitor, and transfection with PP2A siRNA markedly reversed the PMC‐mediated inhibition of iNOS expression, NF‐κB‐promoter activity and p65 phosphorylation. Immunoprecipitation analysis of the cellular extracts of LPS/IFN‐γ‐stimulated VSMCs revealed that p65 colocalizes with PP2A. In addition, p65 phosphorylation and PP2A inactivation were induced in VSMCs by treatment with H₂O₂, but neither IκBα degradation nor IKK phosphorylation was observed. These results collectively indicate that the PMC‐mediated inhibition of NF‐κB activity in LPS/IFN‐γ‐stimulated VSMCs occurs through the ROS‐PP2A‐p65 signalling cascade, an IKK‐IκBα‐independent mechanism. Therapeutic interventions using PMC may therefore be beneficial for the treatment of vascular inflammatory diseases.     

Stromal cell‐derived factor‐1/CXCR4 enhanced motility of human osteosarcoma cells involves MEK1/2, ERK and NF‐κB‐dependent pathways

Osteosarcoma is characterized by a high malignant and metastatic potential. The chemokine stromal‐derived factor‐1α (SDF‐1α) and its receptor, CXCR4, play a crucial role in adhesion and migration of human cancer cells. Integrins are the major adhesive molecules in mammalian cells, and has been associated with metastasis of cancer cells. Here, we found that human osteosarcoma cell lines had significant expression of SDF‐1 and CXCR4 (SDF‐1 receptor). Treatment of osteosarcoma cells with SDF‐1α increased the migration and cell surface expression of αvβ3 integrin. CXCR4‐neutralizing antibody, CXCR4 specific inhibitor (AMD3100) or small interfering RNA against CXCR4 inhibited the SDF‐1α‐induced increase the migration and integrin expression of osteosarcoma cells. Pretreated of osteosarcoma cells with MAPK kinase (MEK) inhibitor PD98059 inhibited the SDF‐1α‐mediated migration and integrin expression. Stimulation of cells with SDF‐1α increased the phosphorylation of MEK and extracellular signal‐regulating kinase (ERK). In addition, NF‐κB inhibitor (PDTC) or IκB protease inhibitor (TPCK) also inhibited SDF‐1α‐mediated cell migration and integrin up‐regulation. Stimulation of cells with SDF‐1α induced IκB kinase (IKKα/β) phosphorylation, IκB phosphorylation, p65 Ser⁵³⁶ phosphorylation, and κB‐luciferase activity. Furthermore, the SDF‐1α‐mediated increasing κB‐luciferase activity was inhibited by AMD3100, PD98059, PDTC and TPCK or MEK1, ERK2, IKKα and IKKβ mutants. Taken together, these results suggest that the SDF‐1α acts through CXCR4 to activate MEK and ERK, which in turn activates IKKα/β and NF‐κB, resulting in the activations of αvβ3 integrins and contributing the migration of human osteosarcoma cells. J. Cell. Physiol. 221: 204–212, 2009. © 2009 Wiley‐Liss, Inc     

Influenza A virus‐encoded NS1 virulence factor protein inhibits innate immune response by targeting IKK

The IKK/NF‐κB pathway is an essential signalling process initiated by the cell as a defence against viral infection like influenza virus. This pathway is therefore a prime target for viruses attempting to counteract the host response to infection. Here, we report that the influenza A virus NS1 protein specifically inhibits IKK‐mediated NF‐κB activation and production of the NF‐κB induced antiviral genes by physically interacting with IKK through the C‐terminal effector domain. The interaction between NS1 and IKKα/IKKβ affects their phosphorylation function in both the cytoplasm and nucleus. In the cytoplasm, NS1 not only blocks IKKβ‐mediated phosphorylation and degradation of IκBα in the classical pathway but also suppresses IKKα‐mediated processing of p100 to p52 in the alternative pathway, which leads to the inhibition of nuclear translocation of NF‐κB and the subsequent expression of downstream NF‐κB target genes. In the nucleus, NS1 impairs IKK‐mediated phosphorylation of histone H3 Ser 10 that is critical to induce rapid expression of NF‐κB target genes. These results reveal a new mechanism by which influenza A virus NS1 protein counteracts host NF‐κB‐mediated antiviral response through the disruption of IKK function. In this way, NS1 diminishes antiviral responses to infection and, in turn, enhances viral pathogenesis.     

TNIP1‐mediated TNF‐α/NF‐κB signalling cascade sustains glioma cell proliferation

As a malignant tumour of the central nervous system, glioma exhibits high incidence and poor prognosis. Although TNIP1 and the TNF‐α/NF‐κB axis play key roles in immune diseases and inflammatory responses, their relationship and role in glioma remain unknown. Here, we revealed high levels of TNIP1 and TNF‐α/NF‐κB in glioma tissue. Glioma cell proliferation was activated with TNF‐α treatment and showed extreme sensitivity to the TNF receptor antagonist. Furthermore, loss of TNIP1 disbanded the A20 complex responsible for IκB degradation and NF‐κB nucleus translocation, and consequently erased TNFα‐induced glioma cell proliferation. Thus, our investigation uncovered a vital function of the TNIP1‐mediated TNF‐α/NF‐κB axis in glioma cell proliferation and provides novel insight into glioma pathology and diagnosis.     

NF‐κB regulates a cassette of immune/inflammatory genes in human pregnant myometrium at term

The onset of human labour resembles inflammation with increased synthesis of prostaglandins and cytokines. There is evidence from rodent models for an important role for nuclear factor‐κB (NF‐κB) activity in myometrium which both up‐regulates contraction‐associated proteins and antagonizes the relaxatory effects of progesterone. Here we show that in the human, although there are no differences in expression of NF‐κB p65, or IκB‐α between upper‐ or lower‐segment myometrium or before or after labour, there is nuclear localization of serine‐256‐phospho‐p65 and serine‐536‐phospho‐p65 in both upper‐ and lower‐segment myometrium both before and after the onset of labour at term. This shows that NF‐κB is active in both upper and lower segment prior to the onset of labour at term. To identify the range of genes regulated by NF‐κB we overexpressed p65 in myocytes in culture. This led to NF‐κB activation identical to that seen following interleukin (IL)‐1β stimulation, including phosphorylation and nuclear translocation of p65 and p50. cDNA microarray analysis showed that NF‐κB increased expression of 38 genes principally related to immunity and inflammation. IL‐1β stimulation also resulted in an increase in the expression of the same genes. Transfection with siRNA against p65 abolished the response to IL‐1β proving a central role for NF‐κB. We conclude that NF‐κB is active in myocytes in both the upper and lower segment of the uterus prior to the onset of labour at term and principally regulates a group of immune/inflammation associated genes, demonstrating that myocytes can act as immune as well as contractile cells.     

NF‐κB and estrogen receptor α interactions: Differential function in estrogen receptor‐negative and ‐positive hormone‐independent breast cancer cells

Estrogen receptor (ER)‐positive breast cancer cells have low levels of constitutive NF‐κB activity while ER negative (?) cells and hormone‐independent cells have relatively high constitutive levels of NF‐κB activity. In this study, we have examined the aspects of mutual repression between the ERα and NF‐κB proteins in ER+ and ER? hormone‐independent cells. Ectopic expression of the ERα reduced cell numbers in ER+ and ER? breast cancer cell lines while NF‐κB‐binding activity and the expression of several NF‐κB‐regulated proteins were reduced in ER? cells. ER overexpression in ER+/E2‐independent LCC1 cells only weakly inhibited the predominant p50 NF‐κB. GST‐ERα fusion protein pull downs and in vivo co‐immunoprecipitations of NF‐κB:ERα complexes showed that the ERα interacts with p50 and p65 in vitro and in vivo. Inhibition of NF‐κB increased the expression of diverse E2‐regulated proteins. p50 differentially associated directly with the ER:ERE complex in LCC1 and MCF‐7 cells by supershift analysis while p65 antibody reduced ERα:ERE complexes in the absence of a supershift. ChIP analysis demonstrated that NF‐κB proteins are present on an endogenous ERE. Together these results demonstrate that the ER and NF‐κB undergo mutual repression, which may explain, in part, why expression of the ERα in ER? cells does not confer growth signaling. Secondly, the acquisition of E2‐independence in ER+ cells is associated with predominantly p50:p50 NF‐κB, which may reflect alterations in the ER in these cells. Since the p50 homodimer is less sensitive to the presence of the ER, this may allow for the activation of both pathways in the same cell. J. Cell. Biochem. 107: 448–459, 2009. © 2009 Wiley‐Liss, Inc.     

CTGF enhances migration and MMP‐13 up‐regulation via αvβ3 integrin,FAK, ERK,and NF‐κB‐dependent pathway in human chondrosarcoma cells

Tumor malignancy is associated with several features such as proliferation ability and frequency of metastasis. Connective tissue growth factor (CTGF), a secreted protein that binds to integrins, modulates the invasive behavior of certain human cancer cells. However, the effect of CTGF on migration activity in human chondrosarcoma cells is mostly unknown. Here we found that CTGF increased the migration and expression of matrix metalloproteinase (MMP)‐13 in human chondrosarcoma cells (JJ012 cells). RGD peptide, αvβ3 monoclonal antibody (mAb) and MAPK kinase (MEK) inhibitors (PD98059 and U0126) but not RAD peptide inhibited the CTGF‐induced increase of the migration and MMP‐13 up‐regulation of chondrosarcoma cells. CTGF stimulation increased the phosphorylation of focal adhesion kinase (FAK) and extracellular signal‐regulated kinase (ERK). In addition, treatment of JJ012 cells with NF‐κB inhibitor (PDTC) or IκB protease inhibitor (TPCK) inhibited CTGF‐induced cell migration and MMP‐13 up‐regulation. Stimulation of JJ012 cells with CTGF also induced IκB kinase α/β (IKK α/β) phosphorylation, IκBα phosphorylation, p65 Ser⁵³⁶ phosphorylation, and κB‐luciferase activity. The CTGF‐mediated increases in κB‐luciferase activities were inhibited by RGD, PD98059, U0126 or FAK, and ERK2 mutant. Taken together, our results indicated that CTGF enhances the migration of chondrosarcoma cells by increasing MMP‐13 expression through the αvβ3 integrin, FAK, ERK, and NF‐κB signal transduction pathway. J. Cell. Biochem. 107: 345–356, 2009. © 2009 Wiley‐Liss, Inc.     

Protein–protein interactions involving IKKγ (NEMO) that promote the activation of NF‐κB

Inhibitor of κB kinase (IKK) gamma (IKKγ), also referred to as nuclear factor κB (NF‐κB) essential modulator (NEMO), is an important regulatory component of the IKK complex. The IKK complex is a signalosome that catalyzes the inducible phosphorylation of IκB proteins, which is a key step that leads to the activation of NF‐κB. The exact functions of IKKγ (NEMO) as part of the IKK complex have not yet been fully elucidated. This mini‐review covers 16 proteins that have been reported to bind to IKKγ and lead to the enhancement of the activities of the IKK complex, thus resulting in NF‐κB activation. The major mechanisms by which these interactions are mediated involve the recognition of ubiquitinated upstream signaling components by IKKγ or the modification of IKKγ itself by ubiquitination. Additional mechanisms include the sumoylation or phosphorylation of IKKγ and the modification of the tertiary or quaternary structure of IKKγ. J. Cell. Physiol. 223:558–561, 2010. © 2010 Wiley‐Liss, Inc.