I-kappa B kinase beta is critical for B cell proliferation and antibody response.

The NF-kappaB proteins are critical in the regulation of the immune and inflammatory response. Stimulation of the NF-kappaB pathway leads to increases in I-kappaB kinase beta (IKKbeta) kinase activity to result in the enhanced phosphorylation and degradation of I-kappaB and the translocation of the NF-kappaB proteins from the cytoplasm to the nucleus. In this study, a dominant-negative IKKbeta mutant expressed from the IgH promoter was used to generate transgenic mice to address the role of IKKbeta on B cell function. Although these transgenic mice were defective in activating the NF-kappaB pathway in B cells, they exhibited no defects in B lymphocyte development or basal Ig levels. However, they exhibited defects in the cell cycle progression and proliferation of B cells in response to treatment with LPS, anti-CD40, and anti-IgM. Furthermore, selective defects in the production of specific Ig subclasses in response to both T-dependent and T-independent Ags were noted. These results suggest that IKKbeta is critical for the proliferation of B cells and the control of some aspects of the humoral response.     

Lipopolysaccharide promotes the survival of osteoclasts via Toll-like receptor 4, but cytokine production of osteoclasts in response to lipopolysaccharide is different from that of macrophages

Lipopolysaccharide is a pathogen that causes inflammatory bone loss. Monocytes and macrophages produce proinflammatory cytokines such as IL-1, TNF-alpha, and IL-6 in response to LPS. We examined the effects of LPS on the function of osteoclasts formed in vitro in comparison with its effect on bone marrow macrophages, osteoclast precursors. Both osteoclasts and bone marrow macrophages expressed mRNA of Toll-like receptor 4 (TLR4) and CD14, components of the LPS receptor system. LPS induced rapid degradation of I-kappaB in osteoclasts, and stimulated the survival of osteoclasts. LPS failed to support the survival of osteoclasts derived from C3H/HeJ mice, which possess a missense mutation in the TLR4 gene. The LPS-promoted survival of osteoclasts was not mediated by any of the cytokines known to prolong the survival of osteoclasts, such as IL-1beta, TNF-alpha, and receptor activator of NF-kappaB ligand. LPS stimulated the production of proinflammatory cytokines such as IL-1beta, TNF-alpha, and IL-6 in bone marrow macrophages and peritoneal macrophages, but not in osteoclasts. These results indicate that osteoclasts respond to LPS through TLR4, but the characteristics of osteoclasts are quite different from those of their precursors, macrophages, in terms of proinflammatory cytokine production in response to LPS.     

Tollip regulates proinflammatory responses to interleukin-1 and lipopolysaccharide

Activation of interleukin-1 (IL-1) receptor (IL-1R), Toll-like receptor 2 (TLR2), and TLR4 triggers NF-kappaB and mitogen-activated protein kinase (MAPK)-dependent signaling, thereby initiating immune responses. Tollip has been implicated as a negative regulator of NF-kappaB signaling triggered by these receptors in in vitro studies. Here, deficient mice were used to determine the physiological contribution of Tollip to immunity. NF-kappaB, as well as MAPK, signaling appeared normal in Tollip-deficient cells stimulated with IL-1beta or the TLR4 ligand lipopolysaccharide (LPS). Similarly, IL-1beta- and TLR-driven activation of dendritic cells and lymphocytes was indistinguishable from wild-type cells. In contrast, the production of the proinflammatory cytokines, IL-6 and tumor necrosis factor alpha was significantly reduced after IL-1beta and LPS treatment at low doses but not at lethal doses of LPS. Tollip therefore controls the magnitude of inflammatory cytokine production in response to IL-1beta and LPS.     

Inhibition of farnesyltransferase prevents collagen-induced arthritis by down-regulation of inflammatory gene expression through suppression of p21(ras)-dependent NF-kappaB activation

Farnesylation of p21(ras) is an important step in the intracellular signaling pathway of growth factors, hormones, and immune stimulants. We synthesized a potent and selective farnesyltransferase inhibitor (LB42708) with IC(50) values of 0.8 nM in vitro and 8 nM in cultured cells against p21(ras) farnesylation and examined the effects of this inhibitor in the settings of inflammation and arthritis. LB42708 suppressed NF-kappaB activation and iNOS promoter activity by suppressing the I-kappaB kinase activity and I-kappaBalpha degradation. The inhibitor suppressed the expression of inducible NO synthase, cyclooxygenase-2, TNF-alpha, and IL-1beta and the production of NO and PGE(2) in immune-activated macrophages and osteoblasts as well as LPS-administrated mice. Furthermore, in vivo administration of LB42708 significantly decreased the incidence and severity of arthritis as well as mRNA expression of inducible NO synthase, cyclooxygenase-2, TNF-alpha, and IL-1beta in the paws of collagen-induced arthritic mice compared with controls. These observations indicate that the anti-inflammatory and antiarthritic effects of the farnesyltransferase inhibitor may be ascribed to the inhibition of I-kappaB kinase activity and subsequent suppression of NF-kappaB-dependent inflammatory gene expression through the suppression of p21(ras) farnesylation. Together, these findings reveal that the inhibitory effect of LB42708 on p21(ras)-dependent NF-kappaB activation may have potential therapeutic value for arthritis and other inflammatory diseases.     

Effect of heat shock preconditioning on NF-kappaB/I-kappaB pathway during I/R injury of the rat liver

Hepatic ischemia-reperfusion (I/R) injury continues to be a fatal complication after liver surgery. Heat shock (HS) preconditioning is an effective strategy for protecting the liver from I/R injury, but its exact mechanism is still unclear. Because the activation of nuclear factor-kappaB (NF-kappaB) is an important event in the hepatic I/R-induced inflammatory response, the effect of HS preconditioning on the pathway for NF-kappaB activation was investigated. In the control group, NF-kappaB was activated 60 min after reperfusion, but this activation was suppressed in the HS group. Messenger RNA expressions of proinflammatory mediators during reperfusion were also reduced with HS preconditioning. Concomitant with NF-kappaB activation, NF-kappaB inhibitor I-kappaB proteins were degraded in the control group, but this degradation was suppressed in the HS group. This study shows that HS preconditioning protected the liver from I/R injury by suppressing the activation of NF-kappaB and the subsequent expression of proinflammatory mediators through the stabilization of I-kappaB proteins.     

beta-TrCP binding and processing of NF-kappaB2/p100 involve its phosphorylation at serines 866 and 870

Processing of the NF-kappaB2 precursor protein p100 is a major step in noncanonical NF-kappaB signaling. This signaling step requires the NF-kappaB inducing kinase (NIK) and its downstream kinase, IkappaB kinase alpha (IKKalpha). We show here that p100 undergoes phosphorylation at serines 866, 870, and possibly 872, in cells stimulated with noncanonical NF-kappaB stimuli or transfected with NIK and IKKalpha. Phosphorylation of this serine cluster creates a binding site for beta-TrCP, the receptor subunit of the beta-TrCP(SCF) ubiquitin ligase. Mutation of either serine 866 or serine 870 abolishes the beta-TrCP recruitment and ubiquitination of p100. The functional significance of p100 phosphorylation is further supported by the finding that this molecular event occurs in a NIK- and IKKalpha-dependent manner. Additionally, induction of p100 phosphorylation can be blocked by a protein synthesis inhibitor, suggesting the requirement of de novo protein synthesis. These data suggest that p100 processing involves its phosphorylation at specific terminal serines, which form a binding site for beta-TrCP thereby regulating p100 ubiquitination.     

Complement factor B gene regulation: synergistic effects of TNF-alpha and IFN-gamma in macrophages

Complement factor B (Bf) plays an important role in activating the alternative complement pathway. The inflammatory cytokines, in particular TNF-alpha and IFN-gamma, are critical in the regulation of Bf gene expression in macrophages. In this study, we investigated the mechanisms of Bf gene regulation by TNF-alpha and IFN-gamma in murine macrophages. Northern analysis revealed that Bf mRNA expression was synergistically up-regulated by TNF-alpha and IFN-gamma in MH-S cells. Truncations of the 5' Bf promoter identified a region between -556 and -282 bp that mediated TNF-alpha responsiveness as well as the synergistic effect of TNF-alpha and IFN-gamma on Bf expression. Site-directed mutagenesis of a NF-kappaB-binding element in this region (-433 to -423 bp) abrogated TNF-alpha responsiveness and decreased the synergistic effect of TNF-alpha and IFN-gamma on Bf expression. EMSAs revealed nuclear protein binding to this NF-kappaB cis-binding element on TNF-alpha stimulation. Supershift analysis revealed that both p50 and p65 proteins contribute to induction of Bf by TNF-alpha. An I-kappaB dominant negative mutant blocked Bf induction by TNF-alpha and reduced the synergistic induction by TNF-alpha and IFN-gamma. In addition, the proteasome inhibitor MG132, which blocks NF-kappaB induction, blocked TNF-alpha-induced Bf promoter activity and the synergistic induction of Bf promoter activity by TNF-alpha and IFN-gamma. LPS was found to induce Bf promoter activity through the same NF-kappaB cis-binding site. These findings suggest that a NF-kappaB cis-binding site between -433 and -423 bp is required for TNF-alpha responsiveness and for TNF-alpha- and IFN-gamma-stimulated synergistic responsiveness of the Bf gene.     

Thrombin-induced autoinhibitory factor, Down syndrome critical region-1, attenuates NFAT-dependent vascular cell adhesion molecule-1 expression and inflammation in the endothelium

Activation and dysfunction of the endothelium underlie many vascular disorders including atherosclerosis, tumor growth, and inflammation. We recently reported that thrombin and vascular endothelial growth factor, but not tumor necrosis factor-alpha, results in dramatic up-regulation of Down syndrome critical region (DSCR)-1 gene in endothelial cells, a negative feedback regulator of calcineurin-NFAT signaling. Constitutive expression of DSCR-1 in activated endothelial cells markedly impaired NFAT nuclear localization, proliferation, tube formation, and tumor growth. The goal of the present study was to elucidate the relative roles of NFAT/DSCR-1 and NF-kappaB/I-kappaB in mediating thrombin-responsive gene expression in endothelial cells. DNA microarrays of thrombin-treated human umbilical vein endothelial cells overexpressing DSCR-1 or constitutive active IkappaBalpha revealed genes that were dependent on NFAT and/or NF-kappaB activity. Vascular cell adhesion molecule-1 was inhibited both by DSCR-1 and I-kappaB at the level of mRNA, protein, promoter activity, and function (monocyte adhesion). Using a combination of transient transfections, electrophoretic mobility shift assays, and chromatin immunoprecipitation, thrombin was shown to induce time-dependent coordinate binding of RelA and NFATc to a tandem NF-kappaB element in the upstream promoter region of vascular cell adhesion molecule-1. Together, these findings suggest that thrombin-mediated activation of endothelial cells involves an interplay between NFAT and NF-kappaB signaling pathways and their negative feedback inhibitors, DSCR-1 and I-kappaB, respectively. As natural brakes in the inflammatory process, DSCR-1 and I-kappaB may lend themselves to therapeutic manipulation in vasculopathic disease states.     

Astaxanthin inhibits nitric oxide production and inflammatory gene expression by suppressing I(kappa)B kinase-dependent NF-kappaB activation

Astaxanthin, a carotenoid without vitamin A activity, has shown anti-oxidant and anti-inflammatory activities; however, its molecular action and mechanism have not been elucidated. We examined in vitro and in vivo regulatory function of astaxanthin on production of nitric oxide (NO) and prostaglandin E2 (PGE2) as well as expression of inducible NO synthase (iNOS), cyclooxygenase-2, tumor necrosis factor-alpha (TNF-alpha), and interleukin-1beta (IL-1beta). Astaxanthin inhibited the expression or formation production of these proinflammatory mediators and cytokines in both lipopolysaccharide (LPS)-stimulated RAW264.7 cells and primary macrophages. Astaxanthin also suppressed the serum levels of NO, PGE2, TNF-alpha, and IL-1beta in LPS-administrated mice, and inhibited NF-kappaB activation as well as iNOS promoter activity in RAW264.7 cells stimulated with LPS. This compound directly inhibited the intracellular accumulation of reactive oxygen species in LPS-stimulated RAW264.7 cells as well as H2O2-induced NF-kappaB activation and iNOS expression. Moreover, astaxanthin blocked nuclear translocation of NF-kappaB p65 subunit and I(kappa)B(alpha) degradation, which correlated with its inhibitory effect on I(kappa)B kinase (IKK) activity. These results suggest that astaxanthin, probably due to its antioxidant activity, inhibits the production of inflammatory mediators by blocking NF-kappaB activation and as a consequent suppression of IKK activity and I(kappa)B-alpha degradation.     

The 15-deoxy-delta12,14-prostaglandin J2 inhibits the inflammatory response in primary rat astrocytes via down-regulating multiple steps in phosphatidylinositol 3-kinase-Akt-NF-kappaB-p300 pathway independent of peroxisome proliferator-activated receptor gamma

Ligands for peroxisome proliferator-activated receptor gamma (PPARgamma), such as 15-deoxy-12,14-PGJ2 (15d-PGJ2), have been proposed as a new class of anti-inflammatory compounds because 15d-PGJ2 was able to inhibit the induction of inflammatory response genes such as inducible NO synthase (iNOS) and TNF (TNF-alpha) in a PPAR-dependent manner in various cell types. In primary astrocytes, the anti-inflammatory effects (inhibition of TNF-alpha, IL-1beta, IL-6, and iNOS gene expression) of 15d-PGJ2 are observed to be independent of PPARgamma. Overexpression (wild-type and dominant-negative forms) of PPARgamma and its antagonist (GW9662) did not alter the 15d-PGJ2-induced inhibition of LPS/IFN-gamma-mediated iNOS and NF-kappaB activation. The 15d-PGJ2 inhibited the inflammatory response by inhibiting IkappaB kinase activity, which leads to the inhibition of degradation of IkappaB and nuclear translocation of p65, thereby regulating the NF-kappaB pathway. Moreover, 15d-PGJ2 also inhibited the LPS/IFN-gamma-induced PI3K-Akt pathway. The 15d-PGJ2 inhibited the recruitment of p300 by NF-kappaB (p65) and down-regulated the p300-mediated induction of iNOS and NF-kappaB luciferase reporter activity. Coexpression of constitutive active Akt and PI3K (p110) reversed the 15d-PGJ2-mediated inhibition of p300-induced iNOS and NF-kappaB luciferase activity. This study demonstrates that 15d-PGJ2 suppresses inflammatory response by inhibiting NF-kappaB signaling at multiple steps as well as by inhibiting the PI3K/Akt pathway independent of PPARgamma in primary astrocytes.