Heat shock protein 27 association with the I kappa B kinase complex regulates tumor necrosis factor alpha-induced NF-kappa B activation

Heat shock protein 27 (Hsp27) is a ubiquitously expressed member of the heat shock protein family that has been implicated in various biological functions including the response to heat shock, oxidative stress, and cytokine treatment. Previous studies have demonstrated that heat shock proteins are involved in regulating signal transduction pathways including the NF-kappa B pathway. In this study, we demonstrated that Hsp27 associates with the I kappa B kinase (IKK) complex and that this interaction was stimulated by tumor necrosis factor alpha treatment. Phosphorylation of Hsp27 by the kinase mitogen-activated protein kinase-activated protein kinase 2, a downstream substrate of the mitogen-activated protein kinase p38, enhanced the association of Hsp27 with IKK beta to result in decreased IKK activity. Consistent with these observations, treatment of cells with a p38 inhibitor reduced the association of Hsp27 with IKK beta and thus resulted in increased IKK activity. These studies indicate that Hsp27 plays a negative role in down-regulating IKK signaling by reducing its activity following tumor necrosis factor alpha stimulation.     

I kappa B kinase complex alpha kinase activity controls chemokine and high endothelial venule gene expression in lymph nodes and nasal-associated lymphoid tissue

The lymphotoxin (LT) beta receptor plays a critical role in secondary lymphoid organogenesis and the classical and alternative NF-kappaB pathways have been implicated in this process. IKKalpha is a key molecule for the activation of the alternative NF-kappaB pathway. However, its precise role and target genes in secondary lymphoid organogenesis remain unknown, particularly with regard to high endothelial venules (HEV). In this study, we show that IKKalpha(AA) mutant mice, who lack inducible kinase activity, have hypocellular lymph nodes (LN) and nasal-associated lymphoid (NALT) tissue characterized by marked defects in microarchitecture and HEV. In addition, IKKalpha(AA) LNs showed reduced lymphoid chemokine CCL19, CCL21, and CXCL13 expression. IKKalpha(AA) LN- and NALT-HEV were abnormal in appearance with reduced expression of peripheral node addressin (PNAd) explained by a severe reduction in the HEV-associated proteins, glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1), and high endothelial cell sulfotransferase, a PNAd-generating enzyme that is a target of LTalphabeta. In this study, analysis of LTbeta(-/-) mice identifies GlyCAM-1 as another LTbeta-dependent gene. In contrast, TNFRI(-/-) mice, which lose classical NF-kappaB pathway activity but retain alternative NF-kappaB pathway activity, showed relatively normal GlyCAM-1 and HEC-6ST expression in LN-HEV. In addition, in this communication, it is demonstrated that LTbetaR is prominently expressed on LN- and NALT-HEV. Thus, these data reveal a critical role for IKKalpha in LN and NALT development, identify GlyCAM-1 and high endothelial cell sulfotransferase as new IKKalpha-dependent target genes, and suggest that LTbetaR signaling on HEV can regulate HEV-specific gene expression.     

Selective I kappa B kinase expression in airway epithelium generates neutrophilic lung inflammation

To determine whether NF-kappaB activation is sufficient to generate lung inflammation in vivo, we selectively expressed a constitutively active form of IkappaB kinase 1 (cIKK1) or IkappaB kinase 2 (cIKK2) in airway epithelium. After intratracheal administration of adenoviral vectors expressing cIKK1 or cIKK2 to transgenic reporter mice that express Photinus luciferase under the control of an NF-kappaB-dependent promoter, we detected significantly increased luciferase activity over time (up to 96 h). Compared with control mice treated with adenoviral vectors expressing beta-galactosidase, lung bioluminescence and tissue luciferase activity were increased in NF-kappaB reporter mice treated with adenovirus (Ad)-cIKK1 or Ad-cIKK2. NF-kappaB activation in lungs of Ad-cIKK1- and Ad-cIKK2-treated mice was confirmed by immunoblots for RelA and EMSA from lung nuclear protein extracts. Mice treated with Ad-cIKK1 or Ad-cIKK2 showed induction of mRNA expression of several chemokines and cytokines in lung tissue. In lung lavage fluid, mice treated with Ad-cIKK1 or Ad-cIKK2 showed elevated concentrations of NF-kappaB-dependent chemokines macrophage-inflammatory protein 2 and KC and increased numbers of neutrophils. Coadministration of adenoviral vectors expressing a transdominant inhibitor of NF-kappaB with Ad-cIKK1 or Ad-cIKK2 resulted in abrogated NF-kappaB activation and other parameters of lung inflammation, demonstrating that the observed inflammatory effects of Ad-cIKK1 and Ad-cIKK2 were dependent on NF-kappaB activation by these kinases. These data show that selective expression of IkappaB kinases in airway epithelium results in NF-kappaB activation, inflammatory mediator production, and neutrophilic lung inflammation. Therapies targeted to NF-kappaB in lung epithelium may be beneficial in treating inflammatory lung diseases.     

Regulation of I(kappa)B kinase complex by phosphorylation of (gamma)-binding domain of I(kappa)B kinase (beta) by Polo-like kinase 1

IkappaB kinase (IKK) complex is a key regulator of NF-kappaB pathways. Signal-induced interaction of the IKKgamma (NEMO) subunit with the C-terminal IKKgamma/NEMO-binding domain (gammaBD) of IKKbeta is an essential interaction for IKK regulation. Underlying regulatory mechanism(s) of this interaction are not known. Phosphorylation of gammaBD has been suggested to play a regulatory role for IKK activation. However, a kinase that phosphorylates gammaBD has not been identified. In this study, we used a C-terminal fragment of IKKbeta as substrate and purified Polo-like kinase 1 (Plk1) from HeLa cell extracts by standard chromatography as a gammaBD kinase. Plk1 phosphorylates serines 733, 740, and 750 in the gammaBD of IKKbeta in vitro. Phosphorylating gammaBD with Plk1 decreased its affinity for IKKgamma in pulldown assay. We generated phosphoantibodies against serine 740 and showed that gammaBD is phosphorylated in vivo. Expressing a constitutively active Plk1 in mammalian cells reduced tumor necrosis factor (TNF)-induced IKK activation, resulting in decreased phosphorylation of endogenous IkappaBalpha and reduced NF-kappaB activation. To activate endogenous Plk1, cells were treated with nocodazole, which reduced TNF-induced IKK activation, and increased the phosphorylation of gammaBD. Knocking down Plk1 in mammalian cells restored TNF-induced IKK activation in nocodazole-treated cells. Activation of Plk1 inhibited TNF-induced expression of cyclin D1. In cells in which Plk1 was knocked down, TNFalpha increased expression of cyclin D1 and the proportion of cells in the S phase of the cell cycle. Taken together, this study shows that phosphorylation regulates the interaction of gammaBD of IKKbeta with IKKgamma and therefore plays a critical role for IKK activation. Moreover, we identify Plk1 as a gammaBD kinase, which negatively regulates TNF-induced IKK activation and cyclin D1 expression, thereby affecting cell cycle regulation. Untimely activation of cyclin D1 by TNFalpha can provide a potential mechanism for an involvement of TNFalpha in inflammation-induced cancer.     

Attachment of Staphylococcus aureus is required for activation of nuclear factor kappa B in human osteoblasts

Nuclear factor kappa B (NF-κB) plays a prominent role in the pathogenesis of infectious diseases. Staphylococcus aureus (S. aureus), which can attach to and invade human osteoblasts, is the most common causative agent of osteomyelitis. To determine whether S. aureus can activate NF-κB in human osteoblasts and explore the possible factors of activation in response to infection, we used flow cytometry, enzyme-linked immunosorbent assay, immunoblots, and electrophoretic mobility shift assays to quantify the invasion of bacteria, to measure the interleukin-6 (IL-6) of culture supernatants, and to investigate the IκBα degradation and NF-κB activation in human osteoblasts. Moreover, we explored the possible factors responsible for the activation of NF-κB by preventing S. aureus from physically touching human osteoblasts or inhibiting the invasion of S. aureus into human osteoblasts under co-culture conditions, by incubating proteinase K-treated or ultraviolet-killed S. aureus with human osteoblasts and by treating human osteoblasts with peptidoglycan (PGN) or lipoteichoic acid (LTA). We found that S. aureus induced the IκBα degradation and NF-κB activation, which could regulate IL-6 secretion in the culture supernatants of human osteoblasts in response to infection. In addition, the maximal IκBα degradation and NF-κB activation in human osteoblasts occurred prior to the maximal invasion of S. aureus. It was the attachment not invasion or the secreted soluble factor(s), PGN, LTA of S. aureus, that could induce the IκBα degradation and NF-κB activation in human osteoblasts. These results indicated that S. aureus can activate NF-κB in human osteoblasts and that the attachment of S. aureus is required for this activation in response to infection.     

Differential phosphorylation of the signal-responsive domain of I kappa B alpha and I kappa B beta by I kappa B kinases

NF-kappa B activity is regulated by its association with the inhibitory I kappa B proteins, among which I kappa B alpha and I kappa B beta are the most abundant. I kappa B proteins are widely expressed in different cells and tissues and bind to similar combinations of NF-kappa B proteins. The degradation of I kappa B proteins allows nuclear translocation of NF-kappa B and hence plays a critical role in NF-kappa B activation. Previous studies have demonstrated that, although both I kappa B proteins are phosphorylated by the same I kappa B kinase (IKK) complex, and their ubiquitination and degradation following phosphorylation are carried out by the same ubiquitination/degradation machinery, their kinetics of degradation are quite different. To better understand the underlying mechanism of the differences in degradation kinetics, we have carried out a systematic, comparative analysis of the ability of the IKK catalytic subunits to phosphorylate I kappa B alpha and I kappa B beta. We found that, whereas IKK alpha is a weak kinase for the N-terminal serines of both I kappa B isoforms, IKK beta is an efficient kinase for those residues in I kappa B alpha. However, IKK beta phosphorylates the N-terminal serines of I kappa B beta far less efficiently, thereby providing an explanation for the slower rate of degradation observed for I kappa B beta. Mutational analysis indicated that the regions around the two N-terminal serines collectively influence the relative phosphorylation efficiency, and no individual residue is critical. These findings provide the first systematic analysis of the ability of I kappa B alpha and I kappa B beta to serve as substrates for IKKs and help provide a possible explanation for the differential degradation kinetics of I kappa B alpha and I kappa B beta.