Phosphoinositide 3-kinase activity leads to silica-induced NF-κB activation through interacting with tyrosine-phosphorylated IκB-α and contributing to tyrosine phosphorylation of p65 NF-κB

The role of the subunits of phosphoinositide (PI) 3-kinase in NF-B activation in silica-stimulated RAW 264.7 cells was investigated. Results indicate that PI3-kinase activity was increased in response to silica. The p85 subunit of PI3-kinase interacted with tyrosine-phosphorylated IB- in silica-stimulated cells. PI3-kinase specific inhibitors, such as wortmannin and LY294003, substantially blocked both silica-induced PI3-kinase and NF-B activation. The inhibition of NF-B activation by PI3-kinase inhibitors was also observed in pervanadate-stimulated but not in LPS-stimulated cells. Furthermore, tyrosine phosphorylation of NF-B p65 was enhanced in cells stimulated with silica, pervanadate or LPS, and wortmannin substantially inhibited the phosphorylation event induced by the first two stimulants but not LPS. Antioxidants, such as superoxide dismutase (SOD), N-acetylcysteine (NAC) and pyrrolidine dithiocarbamate (PDTC), blocked silica-induced PI3-kinase activation, suggesting that reactive oxygen species may be important regulatory molecules in NF-B activation by mediating PI3-kinase activation. Our data suggest that p85 and p110 subunits of PI3-kinase play a role in NF-B activation through interaction with tyrosine-phosphorylated IB- and contributing to tyrosine phosphorylation of p65 NF-B.     

CARMA3: A novel scaffold protein in regulation of NF-κB activation and diseases

CARD recruited membrane associated protein 3 (CARMA3) is a novel scaffold protein. It belongs to the CARMA protein family, and is known to activate nuclear factor (NF)-κB. However, it is still unknown which receptor functions upstream of CARMA3 to trigger NF-κB activation. Recently, several studies have demonstrated that CARMA3 serves as an indispensable adaptor protein in NF-κB signaling under some G protein-coupled receptors (GPCRs), such as lysophosphatidic acid (LPA) receptor and angiotensin (Ang) II receptor. Mechanistically, CARMA3 recruits its essential downstream molecules Bcl10 and MALT1 to form the CBM (CARMA3-Bcl10-MALT1) signalosome whereby it triggers NF-κB activation. GPCRs and NF-κB play pivotal roles in the regulation of various cellular functions, therefore, aberrant regulation of the GPCR/NF-κB signaling axis leads to the development of many types of diseases, such as cancer and atherogenesis. Recently, the GPCR/CARMA3/NF-κB signaling axis has been confirmed in these specific diseases and it plays crucial roles in the pathogenesis of disease progression. In ovarian cancer cell lines, knockdown of CARMA3 abolishes LPA receptor-induced NF-κB activation, and reduces LPA-induced ovarian cancer invasion. In vascular smooth cells, downregulation of CARMA3 substantially impairs Ang-II-receptor-induced NF-κB activation, and in vivo studies have confirmed that Bcl10-deficient mice are protected from developing Ang-II-receptor-induced atherosclerosis and aortic aneurysms. In this review, we summarize the biology of CARMA3, describe the role of the GPCR/CARMA3/NF-κB signaling axis in ovarian cancer and atherogenesis, and speculate about the potential roles of this signaling axis in other types of cancer and diseases. With a significant increase in the identification of LPA- and Ang-II-like ligands, such as endothelin-1, which also activates NF-κB via CARMA3 and contributes to the development of many diseases, CARMA3 is emerging as a novel therapeutic target for various types of cancer and other diseases.     

NEMO differentially regulates TCR and TNF-α induced NF-κB pathways and has an inhibitory role in TCR-induced NF-κB activation

NF-κB essential modulator (NEMO), the regulatory subunit of the IκB kinase (IKK) complex, is an essential adaptor both for inflammation stimuli and TCR-induced NF-κB activation. However, the exact mechanism of its function has not been fully understood. Here, we report that knockdown of NEMO by RNA interference in Jurkat E6.1 cells enhanced TCR-induced NF-κB report gene activity and IL-2 production by promotion of IκBα degradation and p65 nuclear translocation, whereas inhibited TNF-α and LPS-induced IκBα degradation without influencing the phosphorylation of MAPKs. In human primary T and Jurkat E6.1 cells, both CD3/CD28 and PMA/Ionomycin induced NF-κB activation showed a para-curve correlation with the dosage of small interfering RNA targeting NEMO (siNEMO): the NF-κB report gene activity was increased along with ascending doses of transfected siNEMO and reached the highest activity when knockdown about 70% of NEMO, then turned to decline and gradually be blocked once almost thoroughly knockdown of NEMO. Meanwhile, TNF-α induced NF-κB was always inhibited no matter how much NEMO was knockdown. Subcellular fractionation results suggested that upon CD3/CD28 costimulation, NEMO and IKKβ may not cotranslocate to cytoskeleton fraction as a conventional NEMO/IKK complex with a static stoichiometric ratio, instead the ratio of NEMO: IKKβ continuously shift from high to low. Depletion of NEMO accelerated TCR-induced cytoskeleton translocation of IKKβ. Altogether, this study suggests that NEMO may function as a rheostat exerting a negative action on TCR-induced NF-κB activation and differentially regulates TNF-α and TCR-induced NF-κB pathways.     

JAK2 is an important signal transducer in IL-33-induced NF-κB activation

IL-33, a member of the IL-1 family of cytokines, has been shown to activate NF-κB and MAP kinase family through the IL-1 receptor-related protein, ST2L. In this study, we found that IL-33 rapidly activated a tyrosine kinase, JAK2. Interestingly, we demonstrated the functional involvement of JAK2 in IL-33-induced IκBα degradation and NF-κB activation, since a JAK2 inhibitor, AG490, effectively inhibited this signaling pathway. Furthermore, IL-33 failed to induce IκBα degradation and NF-κB activation in JAK2-deficient MEFs expressing ST2L, compared with wild-type MEFs expressing ST2L. In addition, the introduction of wild-type JAK2 but not kinase dead JAK2 mutant (K882R) restored the IL-33-induced efficient activation of NF-κB in JAK2-deficient MEFs expressing ST2L, resulting in the induction of IL-6, CCL2/MCP-1 and CXCL1/KC expression. On the other hand, the activation of ERK, JNK and p38 was unaffected by JAK2 inhibition and JAK2 deficiency. Thus, these data demonstrate that JAK2 plays an important role in regulating IL-33-induced NF-κB activation.     

Suppression of NF-κB and NF-κB regulated oxidative stress and neuroinflammation by BAY 11-7082 (IκB phosphorylation inhibitor) in experimental diabetic neuropathy

Inflammation is an emerging patho-mechanism of diabetes and its complications. NF-κB pathway is one of the central machinery initiating and propagating inflammatory responses. The present study envisaged the involvement of NF-κB inflammatory cascade in the pathophysiology of diabetic neuropathy using BAY 11-7082, an IκB phosphorylation inhibitor. Streptozotocin was used to induce diabetes in Sprauge Dawley rats. BAY 11-7082 (1 &; 3 mg/kg) was administered to diabetic rats for 14 days starting from the end of six weeks post diabetic induction. Diabetic rats developed deficits in nerve functions and altered nociceptive parameters and also showed elevated expression of NF-κB (p65), IκB and p-IκB along with increased levels of IL-6 &; TNF-α and inducible enzymes (COX-2 and iNOS). Furthermore, there was an increase in oxidative stress and decrease in Nrf2/HO-1 expression. We observed that BAY 11-7082 alleviated abnormal sensory responses and deficits in nerve functions. BAY 11-7082 also ameliorated the increase in expression of NF-κB, IκB and p-IκB. BAY 11-7082 curbed down the levels of IL-6, TNF-α, COX-2 and iNOS in the sciatic nerve. Lowering of lipid peroxidation and improvement in GSH levels was also seen along with increased expression of Nrf2/HO-1. Thus it can be concluded that NF-κB expression and downstream expression of proinflammatory mediators are prominent features of nerve damage leading to inflammation and oxidative stress and BAY 11-7082 was able to ameliorate experimental diabetic neuropathy by modulating neuroinflammation and improving antioxidant defence.     

NF-κB activation in myeloid cells mediates ventilator-induced lung injury

Background

Although use of the mechanical ventilator is a life-saving intervention, excessive tidal volumes will activate NF-κB in the lung with subsequent induction of lung edema formation, neutrophil infiltration and proinflammatory cytokine/chemokine release. The roles of NF-κB and IL-6 in ventilator-induced lung injury (VILI) remain widely debated.

Methods

To study the molecular mechanisms of the pathogenesis of VILI, mice with a deletion of IкB kinase in the myeloid cells (IKKβ^△mye), IL-6^-/- to WT chimeric mice, and C57BL/6 mice (WT) were placed on a ventilator for 6 hr.WT mice were also given an IL-6-blocking antibody to examine the role of IL-6 in VILI.

Results

Our results revealed that high tidal volume ventilation induced pulmonary capillary permeability, neutrophil sequestration, macrophage drifting as well as increased protein in bronchoalveolar lavage fluid (BALF). IL-6 production and IL-1β, CXCR2, and MIP2 expression were also increased in WT lungs but not in those pretreated with IL-6-blocking antibodies. Further, ventilator-induced protein concentrations and total cells in BALF, as well as lung permeability, were all significantly decreased in IKKβ^△mye mice as well as in IL6^-/- to WT chimeric mice.

Conclusion

Given that IKKβ^△mye mice demonstrated a significant decrease in ventilator-induced IL-6 production, we conclude that NF-κB–IL-6 signaling pathways induce inflammation, contributing to VILI, and IкB kinase in the myeloid cells mediates ventilator-induced IL-6 production, inflammation, and lung injury.     

1,25-dihydroxyvitamin D3 impairs NF-κB activation in human naïve B cells

1α,25-dihydroxyvitamin D(3) (calcitriol), the bioactive metabolite of vitamin D, modulates the activation and inhibits IgE production of anti-CD40 and IL-4 stimulated human peripheral B cells. Engagement of CD40 results in NF-κB p50 activation, which is essential for the class switch to IgE. Herein, we investigated by which mechanism calcitriol modulates NF-κB mediated activation of human na?ve B cells. Na?ve B cells were predominantly targeted by calcitriol in comparison with memory B cells as shown by pronounced induction of the VDR target gene cyp24a1. Vitamin D receptor activation resulted in a strongly reduced p105/p50 protein and mRNA expression in human na?ve B cells. This effect is mediated by impaired nuclear translocation of p65 and consequently reduced binding of p65 to its binding site in the p105 promoter. Our data indicate that the vitamin D receptor reduces NF-κB activation by interference with NF-κB p65 and p105. Thus, the vitamin D receptor inhibits costimulatory signal transduction in na?ve B cells, namely by reducing CD40 signaling.     

Role of NF-κB activation in LPS-induced endothelial barrier breakdown

Endothelial barrier breakdown contributes to organ failure in sepsis. The key mechanism by which the potent sepsis inductor lipopolysaccharide (LPS) disrupts the endothelial barrier is controversial. Here, we tested the hypothesis that NF-κB activation is critically involved in endothelial barrier breakdown. Application of LPS to monolayers of porcine pulmonary artery endothelial cells (PAEC) and human dermal microvascular endothelial cells (HDMEC) induced a rapid and sustained activation of NF-κB as revealed by translocation of its subunit p65 into the nuclei in nuclear extraction assays and by immunostaining. Measurements of transendothelial electrical resistance (TER) and intercellular gap formation demonstrated significant breakdown of endothelial barrier properties following LPS treatment for 3?h. Interestingly, monolayers recovered spontaneously beginning after 10?h. Increased cAMP prevented LPS-induced loss of endothelial barrier properties, but did not block NF-κB activation. Application of the cell-permeable NEMO-binding domain (NBD) synthetic peptide was effective to prevent NF-κB activation, but did neither block LPS-induced loss of TER nor intercellular gap formation. NBD peptide alone did not alter endothelial barrier properties, but enhanced the barrier-compromising effects when applied in combination with LPS. Similarly, siRNA-mediated knock-down of p65 in HDMECs did not prevent LPS-induced barrier breakdown. Known targets of NF-κB-derived protein expression of caveolin or vasodilator-stimulated phosphoprotein (VASP) remained unaltered by LPS treatment of endothelial cells. In summary, our data indicate that NF-κB activation by LPS is not critically involved in disruption of endothelial barrier properties. Rather, our data suggest that NF-κB activation acts as a part of a rescue mechanism.