A dominant negative protein kinase C zeta subspecies blocks NF-kappa B activation.

Nuclear factor kappa B (NF-kappa B) plays a critical role in the regulation of a number of genes. NF-kappa B is a heterodimer of 50- and 65-kDa subunits sequestered in the cytoplasm complexed to inhibitory protein I kappa B. Following stimulation of cells, I kappa B dissociates from NF-kappa B, allowing its translocation to the nucleus, where it carries out the transactivation function. The precise mechanism controlling NF-kappa B activation and the involvement of members of the protein kinase C (PKC) family of isotypes have previously been investigated. It was found that phorbol myristate acetate, (PMA) which is a potent stimulant of phorbol ester-sensitive PKC isotypes, activates NF-kappa B. However, the role of PMA-sensitive PKCs in vivo is not as apparent. It has recently been demonstrated in the model system of Xenopus laevis oocytes that the PMA-insensitive PKC isotype, zeta PKC, is a required step in the activation of NF-kappa B in response to ras p21. We demonstrate here that overexpression of zeta PKC is by itself sufficient to stimulate a permanent translocation of functionally active NF-kappa B into the nucleus of NIH 3T3 fibroblasts and that transfection of a kinase-defective dominant negative mutant of zeta PKC dramatically inhibits the kappa B-dependent transactivation of a chloramphenicol acetyltransferase reporter plasmid in NIH 3T3 fibroblasts. All these results support the notion that zeta PKC plays a decisive role in NF-kappa B regulation in mammalian cells.     

Human T-lymphotropic virus type I tax activates I-kappa B kinase by inhibiting I-kappa B kinase-associated serine/threonine protein phosphatase 2A

I-kappa B kinase (IKK) is a serine/threonine kinase that phosphorylates I-kappa B alpha and I-kappa B beta and targets them for polyubiquitination and proteasome-mediated degradation. IKK consists of two highly related catalytic subunits, alpha and beta, and a regulatory gamma subunit, which becomes activated after serine phosphorylation of the activation loops of the catalytic domains. The human T-lymphotropic retrovirus type-I trans-activator, Tax, has been shown to interact directly with IKK gamma and activates IKK via a mechanism not fully understood. Here we demonstrate that IKK binds serine/threonine protein phosphatase 2A (PP2A), and via a tripartite protein-protein interaction, Tax, IKK gamma, and PP2A form a stable ternary complex. In vitro, PP2A down-regulates active IKK prepared from Tax-producing MT4 cells. In the presence of Tax, however, the ability of PP2A to inactivate IKK is diminished. Despite their interaction with IKK gamma, PP2A-interaction-defective Tax mutants failed to activate NF-kappa B. Our data support the notion that IKK gamma-associated PP2A is responsible for the rapid deactivation of IKK, and inhibition of PP2A by Tax in the context of IKK x PP2A x Tax ternary complex leads to constitutive IKK and NF-kappa B activation.     

Cobrotoxin inhibits NF-kappa B activation and target gene expression through reaction with NF-kappa B signal molecules

Cobrotoxin is known to bind with cysteine residues of biological molecules such as nicotine acetylcholine receptor. Cobrotoxin may modify IKKs and p50 through protein-protein interaction since cysteine residues are present in the kinase domains of IKKalpha and IKKbeta and in the p50 of NF-kappaB. Our surface plasmon resonance analysis showed that cobrotoxin directly binds to p50 (K(d) = 1.54 x 10(-)(5) M), IKKalpha (K(d) = 3.94 x 10(-)(9) M) and IKKbeta (K(d) = 3.4 x 10(-)(8) M) with high binding affinity. Moreover, these protein-protein interactions suppressed the lipopolysaccharide (LPS, 1 microg/mL)- and the sodium nitroprusside (SNP, 200 microM)-induced DNA binding activity of NF-kappaB and NF-kappaB-dependent luciferase activity in astrocytes and Raw 264.7 macrophages. These inhibitory effects were correlated with the inhibition of IkappaB release and p50 translocation. Inhibition of NF-kappaB by cobrotoxin resulted in reductions in the LPS-induced expressions of COX-2, iNOS, cPLA(2), IL-4, and TNF-alpha in astrocytes and in COX-2 expression induced by SNP, LPS, and TNF-alpha in astrocytes. Moreover, these inhibitory effects of cobrotoxin were reversed by adding reducing agents, dithiothreitol and glutathione. In addition, cobrotoxin did not have any inhibitory effect on NF-kappaB activity in cells carrying mutant p50 (C62S), IKKalpha (C178A), and IKKbeta (C179A), with the exception of IKKbeta (K44A) mutant plasmid. Confocal microscopic analysis showed that cobrotoxin is uptaken into the nucleus of cells. These results demonstrate that cobrotoxin directly binds to the sulfhydryl groups of p50 and IKKs, and that this results in reduced IkappaB release and the translocation of p50, thereby inhibiting the activation of NF-kappaB.     

Curcumin prevents alcohol-induced liver disease in rats by inhibiting the expression of NF-kappa B-dependent genes

Induction of NF-kappaB-mediated gene expression has been implicated in the pathogenesis of alcoholic liver disease (ALD). Curcumin, a phenolic antioxidant, inhibits the activation of NF-kappaB. We determined whether treatment with curcumin would prevent experimental ALD and elucidated the underlying mechanism. Four groups of rats (6 rats/group) were treated by intragastric infusion for 4 wk. One group received fish oil plus ethanol (FE); a second group received fish oil plus dextrose (FD). The third and fourth groups received FE or FD supplemented with 75 mg. kg(-1). day(-1) of curcumin. Liver samples were analyzed for histopathology, lipid peroxidation, NF-kappaB binding, TNF-alpha, IL-12, monocyte chemotactic protein-1, macrophage inflammatory protein-2, cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and nitrotyrosine. Rats fed FE developed fatty liver, necrosis, and inflammation, which was accompanied by activation of NF-kappaB and the induction of cytokines, chemokines, COX-2, iNOS, and nitrotyrosine formation. Treatment with curcumin prevented both the pathological and biochemical changes induced by alcohol. Because endotoxin and the Kupffer cell are implicated in the pathogenesis of ALD, we investigated whether curcumin suppressed the stimulatory effects of endotoxin in isolated Kupffer cells. Curcumin blocked endotoxin-mediated activation of NF-kappaB and suppressed the expression of cytokines, chemokines, COX-2, and iNOS in Kupffer cells. Thus curcumin prevents experimental ALD, in part by suppressing induction of NF-kappaB-dependent genes.     

Activation of interleukin-6 gene expression through the NF-kappa B transcription factor.

The promoter region of the interleukin-6 (IL-6) gene has a putative NF-kappa B-binding site. We found that a fragment of the IL-6 promoter containing the site specifically binds highly purified NF-kappa B protein and the NF-kappa B protein in nuclear extracts of phorbol ester-induced Jurkat cells. Mutations of the NF-kappa B site abolished complex formation with both purified NF-kappa B and the nuclear extract protein. Transient expression of chloramphenicol acetyltransferase (CAT) plasmids containing the IL-6 promoter revealed very little activity of the promoter in U-937 monocytic cells and in HeLa cells before stimulation. However, stimulation of U-937 and HeLa cells by inducers of NF-kappa B led to a dramatic increase in CAT activity. Mutations in the NF-kappa B-binding site abolished inducibility of IL-6 promoter-cat constructs in U-937 cells by lipopolysaccharide, tumor necrosis factor alpha, the double-stranded RNA poly(IC), or phytohemagglutinin and in HeLa cells by tumor necrosis factor alpha and drastically reduced but did not completely eliminate inducibility in HeLa cells stimulated by double-stranded RNA poly(IC) or phorbol 12-myristate 13-acetate. These results suggest that NF-kappa B is an important mediator for activation of the IL-6 gene by a variety of IL-6 inducers in both U-937 and HeLa cells and that alternative inducible enhancer elements contribute in a cell-specific manner to IL-6 gene induction. Because NF-kappa B is involved in the control of a variety of genes activated upon inflammation, NF-kappa B may play a central role in the inflammatory response to infection and tissue injury.     

Bruton's tyrosine kinase mediates NF-kappa B activation and B cell survival by B cell-activating factor receptor of the TNF-R family

Loss of Bruton's tyrosine kinase (Btk) function results in mouse Xid disease characterized by a reduction in mature B cells and impaired humoral immune responses. These defects have been mainly attributed to impaired BCR signaling including reduced activation of the classical NF-kappaB pathway. In this study we show that Btk also couples the receptor for B cell-activating factor (BAFF) of the TNF family (BAFF-R) to the NF-kappaB pathway. Loss of Btk results in defective BAFF-mediated activation of both classical and alternative NF-kappaB pathways. Btk appears to regulate directly the classical pathway in response to BAFF such that Btk-deficient B cells exhibit reduced kinase activity of IkappaB kinase gamma-containing complexes and defective IkappaBalpha degradation. In addition, Btk-deficient B cells produce reduced levels of NF-kappaB2 (p100) basally and in response to stimulation via the BCR or BAFF-R, resulting in impaired activation of the alternative NF-kappaB pathway by BAFF. These results suggest that Btk regulates B cell survival by directly regulating the classical NF-kappaB pathway under both BCR and BAFF-R, as well as by inducing the expression of the components of alternative pathway for sustained NF-kappaB activation in response BAFF. Thus, impaired BCR- and BAFF-induced signaling to NF-kappaB may contribute to the observed defects in B cell survival and humoral immune responses in Btk-deficient mice.     

Tyrosine phosphorylation of I-kappa B kinase alpha/beta by protein kinase C-dependent c-Src activation is involved in TNF-alpha-induced cyclooxygenase-2 expression

The signaling pathway involved in TNF-alpha-induced cyclooxygenase-2 (COX-2) expression was further studied in human NCI-H292 epithelial cells. A protein kinase C (PKC) inhibitor (staurosporine), tyrosine kinase inhibitors (genistein and herbimycin A), or a Src kinase inhibitor (PP2) attenuated TNF-alpha- or 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced COX-2 promoter activity. TNF-alpha- or TPA-induced I-kappaB kinase (IKK) activation was also blocked by these inhibitors, which reversed I-kappaBalpha degradation. Activation of c-Src and Lyn kinases, two Src family members, was inhibited by the PKC, tyrosine kinase, or Src kinase inhibitors. The dominant-negative c-Src (KM) mutant inhibited induction of COX-2 promoter activity by TNF-alpha or TPA. Overexpression of the constitutively active PKCalpha (PKCalpha A/E) or wild-type c-Src plasmids induced COX-2 promoter activity, and these effects were inhibited by the dominant-negative c-Src (KM), NF-kappaB-inducing kinase (NIK) (KA), or IKKbeta (KM) mutant. The dominant-negative PKCalpha (K/R) or c-Src (KM) mutant failed to block induction of COX-2 promoter activity caused by wild-type NIK overexpression. In coimmunoprecipitation experiments, IKKalpha/beta was found to be associated with c-Src and to be phosphorylated on its tyrosine residues after TNF-alpha or TPA treatment. Two tyrosine residues, Tyr(188) and Tyr(199), near the activation loop of IKKbeta, were identified to be crucial for NF-kappaB activation. Substitution of these residues with phenylalanines attenuated COX-2 promoter activity and c-Src-dependent phosphorylation of IKKbeta induced by TNF-alpha or TPA. These data suggest that, in addition to activating NIK, TNF-alpha also activates PKC-dependent c-Src. These two pathways cross-link between c-Src and NIK and converge at IKKalpha/beta, and go on to activate NF-kappaB, via serine phosphorylation and degradation of IkappaB-alpha, and, finally, to initiate COX-2 expression.     

Curcumin (diferuloylmethane) inhibits receptor activator of NF-kappa B ligand-induced NF-kappa B activation in osteoclast precursors and suppresses osteoclastogenesis

Numerous studies have indicated that inflammatory cytokines play a major role in osteoclastogenesis, leading to the bone resorption that is frequently associated with cancers and other diseases. Gene deletion studies have shown that receptor activator of NF-kappaB ligand (RANKL) is one of the critical mediators of osteoclastogenesis. How RANKL mediates osteoclastogenesis is not fully understood, but an agent that suppresses RANKL signaling has potential to inhibit osteoclastogenesis. In this report, we examine the ability of curcumin (diferuloylmethane), a pigment derived from turmeric, to suppress RANKL signaling and osteoclastogenesis in RAW 264.7 cells, a murine monocytic cell line. Treatment of these cells with RANKL activated NF-kappaB, and preexposure of the cells to curcumin completely suppressed RANKL-induced NF-kappaB activation. Curcumin inhibited the pathway leading from activation of IkappaBalpha kinase and IkappaBalpha phosphorylation to IkappaBalpha degradation. RANKL induced osteoclastogenesis in these monocytic cells, and curcumin inhibited both RANKL- and TNF-induced osteoclastogenesis and pit formation. Curcumin suppressed osteoclastogenesis maximally when added together with RANKL and minimally when it was added 2 days after RANKL. Whether curcumin inhibits RANKL-induced osteoclastogenesis through suppression of NF-kappaB was also confirmed independently, as RANKL failed to activate NF-kappaB in cells stably transfected with a dominant-negative form of IkappaBalpha and concurrently failed to induce osteoclastogenesis. Thus overall these results indicate that RANKL induces osteoclastogenesis through the activation of NF-kappaB, and treatment with curcumin inhibits both the NF-kappaB activation and osteoclastogenesis induced by RANKL.     

IL-2 gene expression and NF-kappa B activation through CD28 requires reactive oxygen production by 5-lipoxygenase.

Activation of the CD28 surface receptor provides a major costimulatory signal for T cell activation resulting in enhanced production of interleukin-2 (IL-2) and cell proliferation. In primary T lymphocytes we show that CD28 ligation leads to the rapid intracellular formation of reactive oxygen intermediates (ROIs) which are required for CD28-mediated activation of the NF-kappa B/CD28-responsive complex and IL-2 expression. Delineation of the CD28 signaling cascade was found to involve protein tyrosine kinase activity, followed by the activation of phospholipase A2 and 5-lipoxygenase. Our data suggest that lipoxygenase metabolites activate ROI formation which then induce IL-2 expression via NF-kappa B activation. These findings should be useful for therapeutic strategies and the development of immunosuppressants targeting the CD28 costimulatory pathway.     

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.     

Oleic acid induces endothelin-1 expression through activation of protein kinase C and NF-kappa B

This study investigated the effect of oleic acid on the expression levels of endothelin-1 (ET-1) and on the signaling pathways mediating it in human aortic endothelial cells (HAECs). ET-1 mRNA expression was significantly increased by oleic acid in a dose- and time-dependent manner. Elevation of ET-1 expression in response to oleic acid was inhibited by the protein kinase C (PKC) inhibitor, GF109203X, or the NF-kappa B inhibitor, pyrrolidine dithiocarbamate. In addition, both PKC and NF-kappa B activities were significantly increased by oleic acid. Immunoblot analysis revealed that conventional PKCs (PKC-alpha and -beta II isoforms) were significantly increased in the membranous fractions of HAECs treated with oleic acid. PKC inhibitor completely abolished oleic acid-induced NF-kappa B activation, suggesting that PKC activation is upstream of NF-kappa B activation in oleic acid-induced ET-1 expression. These data suggest that elevated plasma oleic acid levels observed in obese, insulin-resistant subjects result in endothelial dysfunction, at least in part, through an increase in ET-1 expression.     

Inhibition of protein kinase C zeta subspecies blocks the activation of an NF-kappa B-like activity in Xenopus laevis oocytes.

Nuclear factor kappa B (NF-kappa B) plays a critical role in the regulation of a large variety of cellular genes. However, the mechanism whereby this nuclear factor is activated remains to be determined. In this report, we present evidence that in oocytes from Xenopus laevis, (i) ras p21- and phospholipase C (PLC)-mediated phosphatidylcholine (PC) hydrolysis activates NF-kappa B and (ii) protein kinase C zeta subspecies is involved in the activation of NF-kappa B in response to insulin/ras p21/PC-PLC. Thus, the microinjection of either ras p21 or PC-PLC, or the exposure of oocytes to insulin, promotes a significant translocation to the nucleus of an NF-kappa B-like activity. This effect is not observed when oocytes are incubated with phorbol myristate acetate or progesterone, both of which utilize a ras p21-independent pathway for oocyte activation. These data strongly suggest a critical role of the insulin/ras p21/PC-PLC/protein kinase C zeta pathway in the control of NF-kappa B activation.