Activation Of NF-kappa Research Articles

Cytoplasmic Aggregation of TRAF2 and TRAF5 Proteins in the Hodgkin-Reed-Sternberg Cells

We previously reported that ligand-independent signaling by highly expressed CD30 in Hodgkin-Reed-Sternberg (H-RS) cells is responsible for constitutive activation of NF-kappa B. In the present study, we characterize the intracellular localization of tumor necrosis factor (TNF) receptor associated factor (TRAF) proteins in H-RS cells. Confocal immunofluorescence microscopy of cell lines derived from H-RS cells and HEK293 transformants highly expressing CD30 revealed aggregation of TRAF2 and TRAF5 in the cytoplasm as well as clustering near the cell membrane. In contrast, TRAF proteins were diffusely distributed in the cytoplasm in cell lines unrelated to Hodgkin's disease (HD) and control HEK293 cells. Furthermore, the same intracellular distribution of TRAF proteins was demonstrated in H-RS cells of lymph nodes of HD, but not in lymphoma cells in lymph nodes of non-Hodgkin's lymphoma. Dominant-negative TRAF2 and TRAF5 suppressed cytoplasmic aggregation along with constitutive NF-kappa B activation in H-RS cell lines. Confocal immunofluorescence microscopy also revealed co-localization of IKK alpha, NIK, and I kappa B alpha with aggregated TRAF proteins in H-RS cell lines. These results suggest involvement of TRAF protein aggregation in the signaling process of highly expressed CD30 and suggest they function as scaffolding proteins. Thus, cytoplasmic aggregation of TRAF proteins appears to reflect constitutive CD30 signaling which is characteristic of H-RS cells.

Accumulation of an inactive form of p53 protein in cells treated with TNF alpha.

In MCF-7 cells, TNF alpha induces a G1 arrest with an increased expression of p21/Waf1, an activation of NF-kappa B and an accumulation of p53. NF-kappa B and p53 are two transcriptional factors known to activate p21/Waf1 gene expression. Here we show that p53 inhibition has no effect on p21/Waf1 mRNA accumulation following TNF alpha treatment. In contrast, inactivation of NF-kappa B inhibits p21/Waf1 expression without affecting G1 arrest. The fact that p21/Waf1 gene expression is still stimulated when p53 is inactivated strongly suggests that TNF alpha induces accumulation of an inactive form of p53 protein. This assumption was further supported by the following observations: (i) the p53 DNA-binding activity to its consensus sequence was not stimulated following TNF alpha treatment, (ii) phosphorylation at Ser-15, -20 or -392 was not detected in response to TNF alpha, (iii) the transcription rate of Ddb2, another p53 target gene, was not stimulated by TNF alpha. Finally, the accumulation of p53 in the nuclei of TNF alpha-treated MCF-7 cells was concomitant with an increase in p53 mRNA level, suggesting a regulation at the transcription level.

Sphingosine Kinase Interacts with TRAF2 and Dissects Tumor Necrosis Factor-α Signaling

Tumor necrosis factor-alpha (TNF) receptor-associated factor 2 (TRAF2) is one of the major mediators of TNF receptor superfamily transducing TNF signaling to various functional targets, including activation of NF-kappa B, JNK, and antiapoptosis. We investigated how TRAF2 mediates differentially the distinct downstream signals. We now report a novel mechanism of TRAF2-mediated signal transduction revealed by an association of TRAF2 with sphingosine kinase (SphK), a lipid kinase that is responsible for the production of sphingosine 1-phosphate. We identified a TRAF2-binding motif of SphK that mediated the interaction between TRAF2 and SphK resulting in the activation of the enzyme, which in turn is required for TRAF2-mediated activation of NF-kappa B but not JNK. In addition, by using a kinase inactive dominant-negative SphK and a mutant SphK that lacks TRAF2-binding motif we show that the interaction of TRAF2 with SphK and subsequent activation of SphK are critical for prevention of apoptosis during TNF stimulation. These findings show a role for SphK in the signal transduction by TRAF2 specifically leading to activation of NF-kappa B and antiapoptosis.

A Novel Zinc Finger Protein That Inhibits Osteoclastogenesis and the Function of Tumor Necrosis Factor Receptor-associated Factor 6

A variety of surface receptors eliciting diverse cellular responses have been shown to recruit tumor necrosis factor receptor-associated factor (TRAF) adaptor molecules. However, a few TRAF-interacting intracellular proteins that serve as downstream targets or regulators of TRAF function have been identified. In search of new intracellular molecules that bind TRAF6, we carried out a yeast two-hybrid cDNA library screening with an N-terminal segment of TRAF6 as the bait. A novel human C(2)H(2)-type zinc finger family protein was identified, which when coexpressed with TRAF6 led to a suppression of TRAF6-induced activation of NF-kappa B and c-Jun N-terminal kinase. This novel protein was designated TIZ (for TRAF6-inhibitory zinc finger protein). TIZ expression also inhibited the signaling of RANK (receptor activator of NF-kappa B), which together with TRAF6 has been shown to be essential for osteoclastogenesis. Furthermore, the expression level of TIZ appeared to be regulated during the differentiation of human peripheral blood monocytes into osteoclasts. More significantly, transfection of TIZ into the monocyte/macrophage cell line Raw264.7 reduced the RANK ligand-induced osteoclastogenesis of this cell line. Our findings suggest that the novel zinc finger protein TIZ may play a role during osteoclast differentiation by modulating TRAF6 signaling activity.

Enhancement of 1 alpha,25-dihydroxyvitamin D(3)-induced differentiation of human leukaemia HL-60 cells into monocytes by parthenolide via inhibition of NF-kappa B activity.

1. Transcription factors such as NF-kappa B provide powerful targets for drugs to use in the treatment of cancer. In this report parthenolide (PT), a sesquiterpene lactone of herbal remedies such as feverfew (Tanacetum parthenium) with NF-kappa B inhibitory activity, markedly increased the degree of human leukaemia HL-60 cell differentiation when simultaneously combined with 5 nM 1 alpha,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)). PT by itself did not induce HL-60 cell differentiation. 2. Cytofluorometric analysis indicated that PT stimulated 1,25-(OH)(2)D(3)-induced differentiation of HL-60 cells predominantly into monocytes. 3. Pretreatment of HL-60 cells with PT before the 1,25-(OH)(2)D(3) addition also potentiated the 1,25-(OH)(2)D(3)-induced HL-60 cell differentiation in both a dose- and a time-dependent manner, in which the enhanced levels of cell differentiation closely correlated with the inhibitory levels of NF-kappa B binding activity by PT. 4. In contrast, santonin, a sesquiterpene lactone without an inhibitory activity of NF-kappa B binding to the kappa B sites, did not enhance the 1,25-(OH)(2)D(3)-induced HL-60 cell differentiation. 5. In transfection experiments, PT enhanced 1,25-(OH)(2)D(3)-induced VDRE-dependent promoter activity. Furthermore, PT restored 1,25-(OH)(2)D(3)-induced VDRE-dependent promoter activity inhibited by TNF-alpha, an activator of NF-kappa B signalling pathway. 6. These results indicate that PT strongly potentiates the 1,25-(OH)(2)D(3)-induced HL-60 cell differentiation into monocytes via the inhibition of NF-kappa B activity and provide evidence that inhibition of NF-kappa B activation can be a pre-requisite to the efficient entry of promyelocytic leukaemia cells into a differentiation pathway.

Modulation of matrix metalloproteinase-1, its tissue inhibitor, and nuclear factor-kappa B by losartan in hypercholesterolemic rabbits.

Upregulation of angiotensin II receptor, may be involved in the initiation and progression of atherosclerosis. To examine the contribution of AT1 receptor in the expression of matrix metalloproteinase-1 (MMP-1) and its tissue inhibitor (TIMP-2) in lipid-deposited arterial tissues, New Zealand white rabbits were given high-cholesterol chow (with losartan 25 mg/d or vehicle) for 10 weeks. Losartan reduced the areas of sudanophilia in the aorta of rabbits fed high-cholesterol diet (p < 0.01 vs. control). Losartan also significantly decreased the enhanced mRNA expression of MMP-1 and TIMP-2 in aortas of rabbits with high-cholesterol diet. Losartan-treated rabbits revealed a reduction in immunohistochemical expression of MMP-1, whereas TIMP-2 expression became localized to the intima. In addition, losartan treatment reduced the activation of NF-kappa B by inhibiting the degradation of its inhibitor I kappa-B alpha. These observations demonstrate that AT1 receptor blockade with losartan reduces lipid deposition and exerts potent inhibitory effects on NF-kappa B activation and modulates the expression of MMP-1 and TIMP-2 in hypercholesterolemic rabbits.

Chronic antioxidant supplementation attenuates nuclear factor-kappa B activation and preserves endothelial function in hypercholesterolemic pigs.

Hypercholesterolemia (HC), a pro-oxidant condition, activates nuclear factor-kappa beta (NF-kappa B) and is associated with coronary endothelial dysfunction. The physiological significance of in vivo chronic antioxidant intervention on HC-induced NF-kappa B activation and coronary endothelial function remains unclear. Four groups of pigs were studied after 12 weeks of normal diet, normal diet with concomitant antioxidant intervention (100 IU/kg of vitamin E and 1 g of vitamin C daily), 2% HC diet, or HC diet+antioxidant supplementation. NF-kappa B activation and the nitric oxide (NO) pathway were investigated by Western blotting and immunohistochemistry, while oxidative stress was evaluated by coronary artery tissue radical scavenger activity and levels of vitamin E and C. Endothelial function was studied in vitro by coronary vasoreactivity to bradykinin and substance P. HC animals had increased activation of NF-kappa B, decreased endothelial NO synthase expression, and decreased radical scavenger system activity, associated with impaired coronary endothelial function. Antioxidant supplementation in HC normalized NF-kappa B activation and NO bioactivity, and preserves coronary endothelial function. This study demonstrates for the first time that in vivo chronic interruption of the endogenous oxidative stress cascade reduces HC-induced NF-kappa B activation and normalizes NO bioactivity in association with preservation of coronary endothelial function. This study suggests a role for increased oxidative stress and NF-kappa B activation in early atherosclerosis.

A Novel NF-κB-inducing Kinase-MAPK Signaling Pathway Up-regulates NF-κB Activity in Melanoma Cells

Constitutive activation of NF-kappa B is an emerging hallmark of various types of tumors including breast, colon, pancreatic, ovarian, and melanoma. In melanoma cells, the basal expression of the CXC chemokine, CXCL1, is constitutively up-regulated. This up-regulation can be attributed in part to constitutive activation of NF-kappa B. Previous studies have shown an elevated basal I kappa B kinase (IKK) activity in Hs294T melanoma cells, which leads to an increased rate of I kappa B phosphorylation and degradation. This increase in I kappa B-alpha phosphorylation and degradation leads to an approximately 19-fold higher nuclear localization of NF-kappa B. However, the upstream IKK kinase activity is up-regulated by only about 2-fold and cannot account for the observed increase in NF-kappa B activity. We now demonstrate that NF-kappa B-inducing kinase (NIK) is highly expressed in melanoma cells, and IKK-associated NIK activity is enhanced in these cells compared with the normal cells. Kinase-dead NIK blocked constitutive NF-kappa B or CXCL1 promoter activity in Hs294T melanoma cells, but not in control normal human epidermal melanocytes. Transient overexpression of wild type NIK results in increased phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), which is inhibited in a concentration-dependent manner by PD98059, an inhibitor of p42/44 MAPK. Moreover, the NF-kappa B promoter activity decreased with overexpression of dominant negative ERK expression constructs, and EMSA analyses further support the hypothesis that ERK acts upstream of NF-kappa B and regulates the NF-kappa B DNA binding activity. Taken together, our data implicate involvement of I kappa B kinase and MAPK signaling cascades in NIK-induced constitutive activation of NF-kappa B.

The Fas-associated death domain protein suppresses activation of NF-kappa B by LPS and IL-1 beta.

Activation of NF-kappa B by bacterial LPS promotes the upregulation of proinflammatory cytokines that contribute to the pathogenesis of Gram-negative septic shock. LPS activation of NF-kappa B is dependent upon the interaction of two death domain-containing (DD-containing) proteins, MyD88 and IL-1 receptor-associated kinase IRAK. Another DD-containing protein, Fas-associated death domain (FADD), also binds MyD88 through respective DD-DD interactions. Although FADD has been classically described as a proapoptotic signaling molecule, several reports have implicated a role for FADD in mediating NF-kappa B activation. In the present report, we investigated whether FADD could mediate LPS activation of NF-kappa B. Overexpression of FADD blocked LPS-induced NF-kappa B activation, whereas absence of FADD enhanced activation of NF-kappa B by LPS. Further, LPS-induced expression of two NF-kappa B-dependent gene products, IL-6 and KC, was enhanced in FADD(-/-) mouse embryo fibroblasts (MEFs) compared with wild-type. This increase in NF-kappa B activity correlated with enhanced I kappa B degradation. FADD(-/-) MEFs were also resistant to NF-kappa B activation induced by IL-1 beta. Finally, reconstitution of full-length FADD in the FADD(-/-) MEFs completely reversed the enhanced activation of NF-kappa B elicited by either LPS or IL-1 beta. Together, these data indicate that FADD negatively regulates LPS- and IL-1 beta-induced NF-kappa B activation and that this regulation occurs upstream of I kappa B degradation.

Interleukin-4 Reversibly Inhibits Osteoclastogenesis via Inhibition of NF-κB and Mitogen-activated Protein Kinase Signaling

To define the molecular mechanism(s) by which interleukin (IL)-4 reversibly inhibits formation of osteoclasts (OCs) from bone marrow macrophages (BMMs), we examined the capacity of this T cell-derived cytokine to impact signals known to modulate osteoclastogenesis, which include those initiated by macrophage colony-stimulating factor (M-CSF), receptor for activation of NF-kappa B ligand (RANKL), tumor necrosis factor (TNF), and IL-1. We find that although pretreatment of BMMs with IL-4 does not alter M-CSF signaling, it reversibly blocks RANKL-dependent activation of the NF-kappa B, JNK, p38, and ERK signals. IL-4 also selectively inhibits TNF signaling, while enhancing that of IL-1. Contrary to previous reports, we find that MEK inhibitors dose-dependently inhibit OC differentiation. To identify more proximal signals mediating inhibition of OC formation by IL-4, we used mice lacking STAT6 or SHIP1, two adapter proteins that bind the IL-4 receptor. IL-4 fails to inhibit RANKL/M-CSF-induced osteoclastogenesis by BMMs derived from STAT6-, but not SHIP1-, knockout mice. Consistent with this observation, the inhibitory effects of IL-4 on RANKL-induced NF-kappa B and mitogen-activated protein kinase activation are STAT6-dependent. We conclude that IL-4 reversibly arrests osteoclastogenesis in a STAT6-dependent manner by 1) preventing I kappa B phosphorylation and thus NF-kappa B activation, and 2) blockade of the JNK, p38, and ERK mitogen-activated protein kinase pathways.

Inhibition of nuclear factor kappa B (NF-B): an emerging theme in anti-inflammatory therapies.

The application of anti-inflammatory therapies began thousands of years ago with the use of readily available natural resources. It is only recently, however, that the cellular and molecular mechanisms of inflammation have been appreciated sufficiently to design anti-inflammatory strategies with limited side effects. For example, salicylates and glucocorticoids, two widely used anti-inflammatory drug classes, are now known to inhibit the activation of NF-kappa B, a transcription factor that regulates the inducible expression of a wide range of proinflammatory mediators. New generations of NF-kappa B-targeting anti-inflammatory agents that are specific, efficacious, and cost-effective may therefore complement or replace current therapies. In this review, we describe various classes of NF-kappa B inhibitors and discuss important unresolved issues regarding their use.

Crystal Structure of the Extracellular Domain of Mouse RANK Ligand at 2.2-Å Resolution

Bone remodeling involves the resorption of bone by osteoclasts and the synthesis of bone matrix by osteoblasts. Receptor activator of NF-kappa B ligand (RANKL, also known as ODF and OPGL), a member of the tumor necrosis factor (TNF) family, triggers osteoclastogenesis by forming a complex with its receptor, RANK. We have determined the crystal structure of the extracellular domain of mouse RANKL at 2.2-A resolution. The structure reveals that the RANKL extracellular domain is trimeric, which was also shown by analytical ultracentrifugation, and each subunit has a beta-strand jellyroll topology like the other members of the TNF family. A comparison of RANKL with TNF beta and TNF-related apoptosis-inducing ligand (TRAIL), whose structures were determined to be in the complex form with their respective receptor, reveals conserved and specific features of RANKL in the TNF superfamily and suggests the presence of key residues of RANKL for receptor binding.

Human periodontal ligament cells derived from deciduous teeth induce osteoclastogenesis in vitro

The receptor activator of NF-kappa B ligand (RANKL) and its decoy receptor, osteoprotegerin (OPG), are the important proteins involved in osteoclastogenesis. In this study, we investigated the expressions of RANKL and OPG in cultured human periodontal ligament cells derived from deciduous teeth (DPDL cells) and their roles in osteoclastogenesis. Northern blotting revealed that the OPG mRNA was down-regulated by application of 10−8M 1 alpha, 25(OH)2 vitamin D3 [1,25-(OH)2D3] and 10−7M dexamethasone (Dex). In contrast, RANKL mRNA was up-regulated by the same treatment. Western blotting demonstrated a decrease in OPG following application of 1, 25-(OH)2D3 and Dex. Tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells (MNCs) were induced when DPDL cells were co-cultured with mouse bone marrow cells in the presence of 1,25-(OH)2D3 and Dex. TRAP-positive MNCs increased significantly when the DPDL cells were co-cultured with bone marrow cells in the presence of anti-human OPG antibody together with 1, 25-(OH)2D3 and Dex. These results indicate that PDL cells derived from deciduous teeth synthesize both RANKL and OPG and could regulate the differentiation of osteoclasts.

Ethanol-mediated regulation of transcription factors in immunocompetent cells

The immunomodulatory effects of acute and chronic alcohol use are characterized by impaired antigen-specific immune activation and by increased susceptibility to infections due to alterations in innate immune responses and inflammatory mediator production. The central feature of cellular responses to inflammatory and stress signals is the activation of the nuclear regulatory kappa B/Rel family of transcriptional factors via various surface receptor systems in immunocompetent cells. Activation of NF-kappa B, however, is regulated at multiple levels including I-kappa B degradation, nuclear translocation, and by interaction of NF-kappa B/Rel with other transcription factors. Data from our and other laboratories demonstrate that acute alcohol treatment inhibits activation and nuclear binding of the p65/p50 NF-kappa B functional heterodimer in human monocytes, a mechanism likely contributing to inhibition of pro-inflammatory cytokine production. Here we show that acute alcohol-mediated inhibition of NF-kappa B activation in various monocytic cells including human monocytes and murine macrophages. Inhibition of NF-kappa B activation by alcohol in monocytic cells was independent of I-kappa B alpha degradation. These acute-alcohol-induced changes in monocytic cells were different compared to T lymphocytes, both in Jurkat CD4 cells and peripheral human T cells, acute alcohol had a biphasic effect on TNF-alpha-induced NF-kappa B activation via an I-kappa B alpha-dependent mechanism. Inhibition of NF-kappa B activation by acute alcohol in LPS-activated human monocytes was associated with an increase in nuclear glucocorticoid receptor (GR) levels and reduced GR binding to the glucocorticoid response element (GRE). Together these findings support the hypothesis that in the presence of alcohol, nuclear interaction of NF-kappa B (p65) with glucocorticoid receptor and/or other transcription factors may contribute to the reduced NF-kappa B activation. In contrast to the inhibitory effects of acute alcohol on NF-kappa B activation in monocytic cells, chronic alcohol use and alcoholic hepatitis result in an augmentation of NF-kappa B activation and pro-inflammatory cytokine induction. These results suggest that the complex interactions of the NF-kappa B/Rel and related transcription factors including GR and heat-shock responses determine the level of activation of the immunocompetent cells in response to the challenge of acute and chronic alcohol use at the single cell level.

Ethanol-mediated regulation of transcription factors in immunocompetent cells

The immunomodulatory effects of acute and chronic alcohol use are characterized by impaired antigen-specific immune activation and by increased susceptibility to infections due to alterations in innate immune responses and inflammatory mediator production. The central feature of cellular responses to inflammatory and stress signals is the activation of the nuclear regulatory kappa B/Rel family of transcriptional factors via various surface receptor systems in immunocompetent cells. Activation of NF-kappa B, however, is regulated at multiple levels including I-kappa B degradation, nuclear translocation, and by interaction of NF-kappa B/Rel with other transcription factors. Data from our and other laboratories demonstrate that acute alcohol treatment inhibits activation and nuclear binding of the p65/p50 NF-kappa B functional heterodimer in human monocytes, a mechanism likely contributing to inhibition of pro-inflammatory cytokine production. Here we show that acute alcohol-mediated inhibition of NF-kappa B activation in various monocytic cells including human monocytes and murine macrophages. Inhibition of NF-kappa B activation by alcohol in monocytic cells was independent of I-kappa B alpha degradation. These acute-alcohol-induced changes in monocytic cells were different compared to T lymphocytes, both in Jurkat CD4 cells and peripheral human T cells, acute alcohol had a biphasic effect on TNF-alpha-induced NF-kappa B activation via an I-kappa B alpha-dependent mechanism. Inhibition of NF-kappa B activation by acute alcohol in LPS-activated human monocytes was associated with an increase in nuclear glucocorticoid receptor (GR) levels and reduced GR binding to the glucocorticoid response element (GRE). Together these findings support the hypothesis that in the presence of alcohol, nuclear interaction of NF-kappa B (p65) with glucocorticoid receptor and/or other transcription factors may contribute to the reduced NF-kappa B activation. In contrast to the inhibitory effects of acute alcohol on NF-kappa B activation in monocytic cells, chronic alcohol use and alcoholic hepatitis result in an augmentation of NF-kappa B activation and pro-inflammatory cytokine induction. These results suggest that the complex interactions of the NF-kappa B/Rel and related transcription factors including GR and heat-shock responses determine the level of activation of the immunocompetent cells in response to the challenge of acute and chronic alcohol use at the single cell level.

Mechanisms involved in bone resorption.

Osteoclasts, which are present only in bone, are multinucleated giant cells with the capacity to resorb mineralized tissues. These osteoclasts are derived from hemopoietic progenitors of the monocyte-macrophage lineage. Osteoblasts or bone marrow-derived stromal cells are involved in osteoclastogenesis through a mechanism involving cell-to-cell contact with osteoclast progenitors. Experiments on the osteopetrotic op/op mouse model have established that a product of osteoblasts, macrophage colony-stimulating factor (M-CSF), regulates differentiation of osteoclast progenitors into osteoclasts. Recent discovery of osteoclast differentiation factor (ODF)/receptor activator of NF-kappa B ligand (RANKL) allowed elucidation of the precise mechanism by which osteoblasts regulate osteoclastic bone resorption. Treatment of osteoblasts with bone-resorbing factors up-regulated expression of RANKL mRNA. In contrast, TNF alpha stimulates osteoclast differentiation in the presence of M-CSF through a mechanism independent of the RANKL system. IL-1 also directly acts on mature osteoclasts as a potentiator of osteoclast activation. In addition, TGF-beta super family members, such as bone morphogenetic proteins (BMPs) strikingly enhanced osteoclast differentiation from their progenitors and survival of mature osteoclasts induced by RANKL. These results suggest that BMP-mediated signals cross-communicate with RANKL-mediated ones in inducing osteoclast differentiation and function.

Immunolocalization of osteoclast differentiation factor in rat periodontium

The aim here was to observe the immunohistochemical localization of osteoclast differentiation factor (ODF)/receptor activator of NF kappa B ligand (RANKL) in the rat periodontium. Rat mandibles were demineralized and embedded in paraffin, and horizontal and frontal sections were prepared for immunohistochemical analysis. In horizontal sections, immunolocalization of RANKL was marked in the distal area of the periodontium of molars in which osteoclasts appeared, due to physiological tooth drift. In frontal sections, RANKL immunoreactivity was localized on spindle-shaped mesenchymal cells around blood vessels near the bone surface in the periodontium. In addition, immunoreaction for RANKL was detected on structures that appeared to be elongated cell processes near blood vessels in frontal sections. Immunohistochemical examination for the general antigen of nerve-specific protein suggested a similarity between these structures and nerve fibres.

Sphingosine 1-Phosphate Activates Nuclear Factor-κB through Edg Receptors: ACTIVATION THROUGH Edg-3 AND Edg-5, BUT NOT Edg-1, IN HUMAN EMBRYONIC KIDNEY 293 CELLS

Sphingosine 1-phosphate (S1P) exerts a variety of actions as a second messenger or as an agonist that binds to one or more members of the Edg family of G protein-coupled receptors. By using human embryonic kidney 293 cells, we show that S1P activates nuclear factor-kappa B (NF-kappa B) in a receptor-dependent fashion. Edg-3 and Edg-5, which are coupled to G(i), G(q), and G(13), affect activation of NF-kappa B, whereas Edg-1, which is coupled to G(i) alone, does not. We find that the activation of NF-kappa B requires protein kinase C and Ca(2+), probably downstream of G(q), but that the activation of Rho alone by S1P, whether through G(q) or G(13), does not translate into the activation of NF-kappa B. G beta gamma has little effect of its own but potentiates the activation of NF-kappa B achieved through other G proteins. We conclude that the activation of NF-kappa B by S1P is a receptor-mediated process that relies primarily on the activation of a phospholipase C by G(q) and secondarily on effector regulation through other G proteins.

Role of nuclear factor-kappa B (NF-kappa B) in inflammation, periodontitis, and atherogenesis.

Atherosclerosis, the major cause of death and disability in the United States, is a chronic disease with inflammatory components. The first objective of this review is to explain how activation of NF-kappa B contributes to atherosclerosis. The second objective is to describe a potential link between inflammation, activation of NF-kappa B, and periodontitis. The nuclear transcription factor NF-kappa B controls the expression of many genes linked to atherogenesis including those involved with inflammation. We hypothesize that one unifying mechanism in this complex disease is the activation of NF-kappa B. The mechanism(s) that activates NF-kappa B in atherogenesis is unknown and the effect of inhibiting NF-kappa B activation on atherogenesis is untested. Periodontal disease has now been established as a risk factor for atherosclerosis and its thrombotic complications. It is unknown if periodontal disease contributes to the initiation or progression of atherosclerosis. We hypothesize that the chronic and intense inflammatory response accompanying periodontal disease produces an excess burden of circulating mediators of inflammation that initiate or exacerbate the inflammatory components of atherogenesis. Further understanding of the mechanisms involved in the activation of NF-kappa B in atherosclerosis could lead to important therapeutic applications especially as it relates to the impact of periodontitis.

Anti-Fas Induces Hepatic Chemokines and Promotes Inflammation by an NF-κB-independent, Caspase-3-dependent Pathway

Agonistic antibodies against the Fas receptor, when administered to mice in vivo, cause significant apoptosis in the liver. In this study we show that anti-Fas antibody not only causes apoptosis of liver cells but also provokes hepatic inflammation. Two hours after injection of anti-Fas, when mice displayed evidence of caspase-3 activation and apoptosis, we found significant hepatic induction of the CXC chemokines macrophage inflammatory protein-2 and KC. Coincident with the chemokine induction was infiltration of the hepatic parenchyma by neutrophils. Neutralization experiments identified that chemokines were the cause of Fas-induced hepatic inflammation, with KC having the predominant effect. Chemokine induction in the livers of anti-Fas-treated mice was not associated with activation of NF-kappa B. Instead, it coincided with nuclear translocation of activator protein-1 (AP-1). AP-1 activation in liver was detected 1-2 h after anti-Fas treatment, suggesting a connection to the onset of apoptosis. When apoptosis was prevented by pretreating mice with a caspase-3 inhibitor, AP-1 activation and hepatic chemokine production were both significantly reduced. Hepatic inflammation was also reduced by 70%. Taken together, these findings indicate that Fas ligation can induce inflammation in the liver in vivo. Inflammation does not arise from Fas-mediated signaling through NF-kappa B; rather, it represents an indirect effect, requiring activation of caspase-3 and nuclear translocation of AP-1.