Activation Of NF-kappa Research Articles

Expression of receptor activator of NF-kappa B ligand and osteoprotegerin in culture of human periodontal ligament cells.

The receptor activator of NF-kappa B ligand (RANKL) and its decoy receptor, osteoprotegerin (OPG), are the important proteins implicated in osteoclastogenesis. In this study, we investigated the expressions of RANKL and OPG in cultured human periodontal ligament (PDL) cells and their roles in osteoclastogenesis. Northern blotting revealed that the OPG mRNA was down-regulated remarkably by application of 10-8 m one-alpha, 25-dihydroxyvitamin D3[1,25-(OH)2D3] and 10-7 m dexamethasone (Dex). In contrast, RANKL mRNA was up-regulated by the same treatment. Western blotting demonstrated decrease of OPG by the application of 1,25-(OH)2D3 and Dex. Tartrate-resistant acid phosphatase-positive multinuclear cells were markedly induced when the PDL cells were cocultured with mouse bone marrow cells in the presence of an anti-OPG antibody together with 1,25-(OH)2D3 and Dex. These results indicate that PDL cells synthesize both RANKL and OPG and that inactivation of OPG may play a key role in the differentiation of osteoclasts.

RIP4 (DIK/PKK), a novel member of the RIP kinase family, activates NF-kappa B and is processed during apoptosis.

RIP1 and its homologs, RIP2 and RIP3, form part of a family of Ser/Thr kinases that regulate signal transduction processes leading to NF-kappa B activation. Here, we identify RIP4 (DIK/PKK) as a novel member of the RIP kinase family. RIP4 contains an N-terminal RIP-like kinase domain and a C-terminal region characterized by the presence of 11 ankyrin repeats. Overexpression of RIP4 leads to activation of NF-kappa B and JNK. Kinase inactive RIP4 or a truncated version containing the ankyrin repeats have a dominant negative (DN) effect on NF-kappa B induction by multiple stimuli. RIP4 binds to several members of the TRAF protein family, and DN versions of TRAF1, TRAF3 and TRAF6 inhibit RIP4-induced NF-kappa B activation. Moreover, RIP4 is cleaved after Asp340 and Asp378 during Fas-induced apoptosis. These data suggest that RIP4 is involved in NF-kappa B and JNK signaling and that caspase-dependent processing of RIP4 may negatively regulate NF-kappa B-dependent pro-survival or pro-inflammatory signals.

Epigallocatechin-3-gallate decreases VEGF production in head and neck and breast carcinoma cells by inhibiting EGFR-related pathways of signal transduction.

In a recent study on head and neck squamous cell carcinoma (HNSCC) cells we found that epigallocatechin-3-gallate (EGCG), a major biologically active component of green tea, inhibited activation of the epidermal growth factor receptor (EGFR) and related signaling pathways. Since activation of EGFR signaling pathways is associated with angiogenesis, we examined the effects of EGCG on vascular endothelial growth factor (VEGF) production by YCU-H891 HNSCC and MDA-MB-231 breast carcinoma cell lines, because we found that both of these cell lines display autocrine activation of transforming growth factor-alpha (TGF-alpha)/EGFR signaling and produce high levels of VEGF. Treatment with EGCG inhibited the constitutive activation of the EGFR, Stat3, and Akt in both cell lines. These changes were associated with inhibition of VEGF promoter activity and cellular production of VEGF. Mechanistic studies indicated that inhibition of Stat3, but not mitogen-activated protein kinase kinase (MEK)1 or phosphatidylinositol 3'-kinase (PI3K), significantly decreased VEGF promoter activity. However, the inhibitory effects of a dominant negative Stat3 on VEGF expression was not as strong as that produced by EGCG. An analysis of alternative pathways indicated that EGCG strongly inhibited the constitutive activation of NF-kappa B in both cell lines, and an NF-kappa B inhibitor strongly inhibited VEGF production. These results suggest that EGCG inhibits VEGF production by inhibiting both the constitutive activation of Stat3 and NF-kappa B, but not extracellular-signal-regulated kinase (ERK) or Akt, in these cells. Therefore, EGCG may be useful in treating HNSCC and breast carcinoma because it can exert both antiproliferative and antiangiogenic activities.

Association of the Adaptor TANK with the IκB Kinase (IKK) Regulator NEMO Connects IKK Complexes with IKKε and TBK1 Kinases

Canonical activation of NF-kappa B is mediated via phosphorylation of the inhibitory I kappa B proteins by the I kappa B kinase complex (IKK). IKK is composed of a heterodimer of the catalytic IKK alpha and IKK beta subunits and a presumed regulatory protein termed NEMO (NF-kappa B essential modulator) or IKK gamma. NEMO/IKK gamma is indispensable for activation of the IKKs in response to many signals, but its mechanism of action remains unclear. Here we identify TANK (TRAF family member-associated NF-kappa B activator) as a NEMO/IKK gamma-interacting protein via yeast two-hybrid analyses. This interaction is confirmed in mammalian cells, and the domains required are mapped. TANK was previously shown to assist NF-kappa B activation in a complex with TANK-binding kinase 1 (TBK1) or IKK epsilon, two kinases distantly related to IKK alpha/beta, but the underlying mechanisms remained unknown. Here we show that TBK1 and IKK epsilon synergize with TANK to promote interaction with the IKKs. The TANK binding domain within NEMO/IKK gamma is required for proper functioning of this IKK subunit. These results indicate that TANK can synergize with IKK epsilon or TBK1 to link them to IKK complexes, where the two kinases may modulate aspects of NF-kappa B activation.

Activation of the transcription factor NF-kappaB by Campylobacter jejuni.

Campylobacter jejuni is a food-borne pathogen responsible for infectious enterocolitis. The early-response transcription factor NF-kappa B triggers the expression of genes associated with cellular immune and inflammatory responses. Co-incubation of HeLa cells with viable C. jejuni leads to the activation of the transcription factor NF-kappa B as determined by specific induction of a cellular luciferase-based reporter. Boiled cell-free extracts of C. jejuni are also potent dose-dependent stimulators of NF-kappa B-dependent transcription, the levels of which can reach up to 1000-fold as compared with independent controls. Using both cultured HeLa cells and human colonic epithelial (HCA-7) cells, the activation of NF-kappa B by C. jejuni boiled extract has been monitored through the degradation of IKB alpha and DNA binding of the nuclear translocated p50/p65 heterodimer of NF-kappa B. These events are co-ordinated with elaboration of the pro-inflammatory cytokine interleukin-8. Fractionation of the boiled C. jejuni extract suggests that the majority of the bioactive component has a molecular mass of 3 kDa or less, which is insensitive to proteinase K treatment.

Nf-kappa B, chemokine gene transcription and tumour growth.

The constitutive expression of angiogenic and tumorigenic chemokines by tumour cells facilitates the growth of tumours. The transcription of these angiogenic and tumorigenic chemokine genes is modulated, in part, by the nuclear factor-kappa B (NF-kappa B) family of transcription factors. In some tumours, there is constitutive activation of the kinases that modulate the activity of inhibitor of NF-kappa B (I kappa B) kinase (IKK), which leads to the constitutive activation of members of the NF-kappa B family. This activation of NF-kappa B is associated with the dysregulation of transcription of genes that encode cytokines, chemokines, adhesion factors and inhibitors of apoptosis. In this review, I discuss the factors that lie upstream of the NF-kappa B cascade that are activated during tumorigenesis and the role of the putative NF-kappa B enhanceosome in constitutive chemokine gene transcription during tumorigenesis.

1,25(OH)2 vitamin D3-stimulated osteoclast formation in spleen-osteoblast cocultures is mediated in part by enhanced IL-1 alpha and receptor activator of NF-kappa B ligand production in osteoblasts.

We examined the ability of 1,25 (OH)(2) vitamin D(3) (Vit D) to stimulate osteoclast-like cell (OCL) formation in cocultures of spleen cells and primary calvarial osteoblasts from wild-type (WT) and IL-1R type 1-deficient (knockout; KO) mice. Vit D dose dependently increased OCL in cocultures containing WT osteoblasts. In contrast, there was a 90% reduction in OCL numbers in cocultures containing KO osteoblasts. In cocultures with either WT or KO osteoblasts, treatment with Vit D increased receptor activator of NF-kappaB ligand mRNA by 17-, 19-, or 3.5-fold, respectively. Vit D decreased osteoprotegerin mRNA to undetectable in all groups. Intracellular IL-1alpha protein increased after Vit D treatment in cocultures containing WT, but not KO osteoblasts. We also examined direct effects of Vit D, IL-1alpha, and their combination on gene expression in primary osteoblasts. In WT cells, Vit D and IL-1 stimulated receptor activator of NF-kappaB ligand mRNA expression by 3- and 4-fold, respectively, and their combination produced a 7-fold increase. Inhibition of osteoprotegerin mRNA in WT cells was partial with either agent alone and greatest with their combination. In KO cells, only Vit D stimulated a response. IL-1 alone increased IL-1alpha protein expression in WT osteoblasts. However, in combination with Vit D, there was a synergistic response (100-fold increase). In KO cultures, there were no effects of IL-1, Vit D, or their combination on IL-1alpha protein. These results demonstrate interactions between IL-1 and Vit D in primary osteoblasts that appear important in both regulation of IL-1alpha production and the ability of Vit D to support osteoclastogenesis.

Inhibition of JNK by Cellular Stress- and Tumor Necrosis Factor α-induced AKT2 through Activation of the NFκB Pathway in Human Epithelial Cells

Previous studies have demonstrated that AKT1 and AKT3 are activated by heat shock and oxidative stress via both phosphatidylinositol 3-kinase-dependent and -independent pathways. However, the activation and role of AKT2 in the stress response have not been fully elucidated. In this study, we show that AKT2 in epithelial cells is activated by UV-C irradiation, heat shock, and hyperosmolarity as well as by tumor necrosis factor alpha (TNFalpha) through a phosphatidylinositol 3-kinase-dependent pathway. The activation of AKT2 inhibits UV- and TNF alpha-induced c-Jun N-terminal kinase (JNK) and p38 activities that have been shown to be required for stress- and TNF alpha-induced programmed cell death. Moreover, AKT2 interacts with and phosphorylates I kappa B kinase alpha. The phosphorylation of I kappa B kinase alpha and activation of NF kappa B mediates AKT2 inhibition of JNK but not p38. Furthermore, phosphatidylinositol 3-kinase inhibitor or dominant negative AKT2 significantly enhances UV- and TNF alpha-induced apoptosis, whereas expression of constitutively active AKT2 inhibits programmed cell death in response to UV and TNFalpha -induced apoptosis by inhibition of stress kinases and provide the first evidence that AKT inhibits stress kinase JNK through activation of the NF kappa B pathway.

RIP2, a checkpoint in myogenic differentiation.

Using a subtractive cDNA library hybridization approach, we found that receptor interacting protein 2 (RIP2), a tumor necrosis factor receptor 1 (TNFR-1)-associated factor, is a novel early-acting gene that decreases markedly in expression during myogenic differentiation. RIP2 consists of three domains: an amino-terminal kinase domain, an intermediate domain, and a carboxy-terminal caspase activation and recruitment domain (CARD). In some cell types, RIP2 has been shown to be a potent inducer of apoptosis and an activator of NF-kappa B. To analyze the function of RIP2 during differentiation, we transduced C2C12 myoblasts with retroviral vectors to constitutively produce RIP2 at high levels. When cultured in growth medium, these cells did not show an enhanced rate of proliferation compared to controls. When switched to differentiation medium, however, they continued to proliferate, whereas control cells withdrew from the cell cycle, showed increased expression of differentiation markers such as myogenin, and began to differentiate into multinucleated myotubes. The complete RIP2 protein appeared to be necessary to inhibit myogenic differentiation, since two different deletion mutants lacking either the amino-terminal kinase domain or the carboxy-terminal CARD had no effect. A mutant deficient in kinase activity, however, had effects similar to wild-type RIP2, indicating that phosphorylation was not essential to the function of RIP2. Furthermore, RIP proteins appeared to be important during myogenic differentiation in vivo, as we detected a marked decrease in expression of the RIP2 homolog RIP in several muscle tissues of the dystrophic mdx mouse, a model for continuous muscle degeneration and regeneration. We conclude that RIP proteins can act independently of TNFR-1 stimulation by ligand to modulate downstream signaling pathways, such as activation of NF-kappa B. These results implicate RIP2 in a previously unrecognized role: a checkpoint for myogenic proliferation and differentiation.

PYPAF7, a Novel PYRIN-containing Apaf1-like Protein That Regulates Activation of NF-κB and Caspase-1-dependent Cytokine Processing

PYRIN-containing Apaf1-like proteins (PYPAFs) are members of the nucleotide-binding site/leucine-rich repeat (NBS/LRR) family of signal transduction proteins. We report here that PYPAF7 is a novel PYPAF protein that activates inflammatory signaling pathways. The expression of PYPAF7 is highly restricted to immune cells, and its gene maps to chromosome 19q13.4, a locus that contains a cluster of genes encoding numerous PYPAF family members. Co-expression of PYPAF7 with ASC results in the recruitment of PYPAF7 to distinct cytoplasmic loci and a potent synergistic activation of NF-kappa B. To identify other proteins involved in PYPAF7 and ASC signaling pathways, we performed a mammalian two-hybrid screen and identified pro-caspase-1 as a binding partner of ASC. Co-expression of PYPAF7 and ASC results in the synergistic activation of caspase-1 and a corresponding increase in secretion of interleukin-1 beta. In addition, PYPAF1 induces caspase-1-dependent cytokine processing when co-expressed with ASC. These findings indicate that PYPAF family members participate in inflammatory signaling by regulating the activation of NF-kappa B and cytokine processing.

Oligomerization State-dependent Activation of NF-κB Signaling Pathway by Adipocyte Complement-related Protein of 30 kDa (Acrp30)

Adipocyte complement-related protein of 30 kDa (Acrp30)/adiponectin is an adipocyte-derived hormone that affects lipid and glucose metabolism in muscle and liver, but its physical and biochemical properties are poorly characterized. Here we have used several approaches to show that Acrp30 expressed in and purified from Escherichia coli and human embryonic kidney 293T cells forms trimers and hexamers; 293T cells also produce a higher molecular weight species. Similar Acrp30 oligomers were found in mouse serum as well as in 3T3-L1 adipocyte-conditioned medium, although in different proportions. In parallel, we assessed whether Acrp30 is a signaling molecule by searching for promoter or enhancer elements that respond to Acrp30 or its isolated trimeric globular C-terminal domain, gAcrp30. Acrp30 addition to C2C12 myocytes or myotubes led to activation of NF-kappa B transcription factor in a manner dependent upon phosphorylation and degradation of I kappa B-alpha. Importantly, only hexameric and larger isoforms of Acrp30 activated NF-kappa B; trimeric Acrp30 or gAcrp30 could not activate NF-kappa B. Our data indicate that oligomerization of Acrp30 is important for at least some of its biological activities, and changes in the relative abundance of each oligomeric isoform in plasma may regulate Acrp30 activity.

Akt Is a Downstream Target of NF-κB

The ubiquitously expressed transcription factor NF-kappa B and the serine-threonine kinase Akt both are involved in the promotion of cell survival. Although initially believed to operate as components of distinct signaling pathways, several studies have demonstrated that the NF-kappa B and Akt signaling pathways can converge. Indeed, I kappa B kinase, the kinase involved in NF-kappa B activation, is a substrate of Akt, and activation of Akt therefore stimulates NF-kappa B activity. Although these results place Akt upstream of NF-kappa B activation in the sequence of signaling events, we report that this may not necessarily be the case and that Akt is a downstream target of NF-kappa B. Treatment of NIH3T3 cells with the NF-kappa B activators, tumor necrosis factor (TNF) alpha and lipopolysaccharide, results in the stimulation of Akt phosphorylation. The stimulation of Akt is, however, detected only after I kappa B-alpha degradation is induced by these agents. The nuclear translocation of p65 and increased DNA binding activity of NF-kappa B also precede Akt phosphorylation. Treatment with two pharmacological inhibitors of NF-kappa B, SN50 and N-tosyl-l-phenylalanine chloromethyl ketone (TPCK), blocks TNF-induced Akt activation. On the other hand TNF-mediated NF-kappa B activation is not reduced by the phosphoinositide-3 kinase inhibitors wortmannin and LY294002, although these inhibitors completely block the activation of Akt. These results suggest that NF-kappa B is required for TNF-mediated Akt activation and that it lies upstream of the stimulation of Akt. Consistent with this conclusion is the finding that overexpression of p65/RelA leads to Akt phosphorylation in the absence of extracellular stimulatory factors, whereas overexpression of I kappa B-alpha reduces Akt phosphorylation below basal levels. Interestingly, in addition to stimulating the phosphorylation of Akt, overexpression of p65 causes an increase in the expression of Akt mRNA and protein.

Activated rat pancreatic stellate cells express intercellular adhesion molecule-1 (ICAM-1) in vitro.

Activated pancreatic stellate cells (PSCs) have recently been implicated in the pathogenesis of pancreatic fibrosis. To examine the role of PSCs in pancreatic inflammation by determining whether activated PSCs express intercellular adhesion molecule-1 (ICAM-1). Culture-activated rat PSCs were treated with interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), ethanol, or acetaldehyde. ICAM-1 expression was analyzed by flow cytometry. The induction of mRNA was assessed by Northern blot analysis. The binding activity of transcription factors was examined by electrophoretic mobility shift assay. The activation of mitogen-activated protein kinases was assessed by Western blotting with use of antiphosphospecific antibodies. The adhesion of MOLT-4 cells to activated PSCs was also assessed. Culture-activated PSCs expressed ICAM-1. The expression was increased in response to IL-1 beta and TNF-alpha but not to alcohol, with comparable mRNA induction. IL-1 beta and TNF-alpha increased the nuclear factor-kappa B (NF-kappa B)-specific and activator protein-1-specific DNA binding activity, whereas the NF-IL6 activity was not altered. Alcohol did not increase NF-kappa B binding activity. IL-1 beta and TNF-alpha activated extracellular signal-regulated kinase 1/2, c-Jun N-terminal kinase/stress-activated protein kinase, and p38 mitogen-activated protein kinase. Inducible ICAM-1 expression was blocked by pyrrolidine dithiocarbamate, a specific inhibitor of NF-kappa B activation, but not by inhibitors of mitogen-activated protein kinase pathways. IL-1 beta and TNF-alpha increased the ICAM-1-mediated binding of MOLT-4 cells to activated PSCs, indicating a role of ICAM-1 in the adhesion of leukocytes to PSCs. Our results suggest that activated PSCs express ICAM-1 mainly through the activation of NF-kappa B, thus playing a role in the pathogenesis of pancreatic inflammation.

Transforming Growth Factor-β Induces Expression of Receptor Activator of NF-κB Ligand in Vascular Endothelial Cells Derived from Bone

Vascular endothelial cells in bone are thought to have significant roles on pathological bone resorption such as bone metastasis and hypercalcemia because this resorption is often seen where blood vessels are abundant. However, the detailed mechanisms have not yet been elucidated. Here, we focused on transforming growth factor-beta (TGF-beta) and studied its effects on vascular endothelial cells because TGF-beta is abundantly stored in bone matrix and is released and activated during bone resorption. We found that TGF-beta up-regulated the expression of receptor activator of NF-kappa B ligand (RANKL) mRNA and protein in bone marrow-derived endothelial cells and in primary vascular endothelial cells but not in osteoblasts. Further analysis revealed that TGF-beta promoted phosphorylation of cAMP response element-binding protein and p38. Protein kinase A inhibitor KT5720 and p38 inhibitor SB203580 significantly reduced the TGF-beta-induced RANKL expression. Moreover, we found two CRE-like domains in murine RANKL promoter region that were critical for TGF-beta-dependent RANKL expression. Therefore, protein kinase A and p38 signaling pathways are involved in TGF-beta-induced RANKL expression by stimulating transcription factors that bind to the CRE-like domains. Our findings indicate that TGF-beta stimulates osteoclastogenesis by promoting RANKL expression in endothelial cells under pathological conditions.

Inhibitory effects of the standardized extract (DA‐9601) of Artemisia asiatica Nakai on phorbol ester–induced ornithine decarboxylase activity, papilloma formation, cyclooxygenase‐2 expression, inducible nitric oxide synthase expression and nuclear transcription factor κB activation in mouse skin

Artemisia asiatica Nakai has been used in traditional Asian medicine for the treatment of inflammatory and other disorders. Previous studies have revealed that the formulated ethanol extract (DA-9601) of A. asiatica has pronounced antioxidative and antiinflammatory activities and exhibits cytoprotective effects against experimentally induced gastrointestinal, hepatic and pancreatic damage. In the present study, we assessed the inhibitory effect of DA-9601 on tumor promotion, which is closely linked to inflammatory tissue damage. As an initial approach to evaluating the possible antitumor-promoting potential of DA-9601, its effects on TPA-induced ear edema were examined in female ICR mice. Pretreatment of the inner surface of the mouse ear with DA-9601 30 min prior to topical application of TPA inhibited ear edema at 5 hr. TPA-stimulated expression of epidermal COX-2 and iNOS was also mitigated by topical application of the same extract. Moreover, DA-9601 abrogated the TPA-mediated activation of NF-kappa B/Rel and AP-1 in mouse epidermis. Suppression of epidermal NF-kappa B by DA-9601 appeared to be mediated in part through inhibition of I kappa B alpha degradation, thereby blocking the nuclear translocation of p65, the functional subunit of NF-kappa B. DA-9601 also significantly suppressed TPA-induced ODC activity and papilloma formation in mouse skin. Taken together, these findings suggest that DA-9601 derived from A. asiatica possesses potential chemopreventive activities.

Endoplasmic reticulum stress causes thyroglobulin retention in this organelle and triggers activation of nuclear factor-kappa B via tumor necrosis factor receptor-associated factor 2.

Perturbing the endoplasmic reticulum homeostasis of thyroid cell lines with thapsigargin, a specific inhibitor of the sarcoendoplasmic reticulum Ca(2+) adenosine triphosphatases, and tunicamycin, an inhibitor of the N-linked glycosylation, blocked Tg in the endoplasmic reticulum. This event was signaled outside the endoplasmic reticulum and resulted in activation of the c-Jun N-terminal kinase (JNK)/stress-activated protein kinase and nuclear factor-kappa B (NF-kappa B) stress response pathways. Activation of the JNK/stress-activated protein kinase signaling pathway was assessed by measuring the amount of phospho-JNK and the activity of JNK by kinase assays. Activation of the NF-kappa B signaling pathway was assessed by measuring the level of inhibitory subunit I kappa B alpha, DNA binding, and transcriptional activity of NF-kappa B. Cycloheximide treatment, at a dose able to profoundly inhibit protein synthesis in FRTL-5 cells, obliterated the decrease in the level of the inhibitory subunit I kappa B alpha produced by thapsigargin and tunicamycin. Therefore, protein synthesis was required to generate a signal from stressed endoplasmic reticulum. This substantiates the hypothesis that endoplasmic reticulum retention of newly synthesized Tg and other cargo (secretory and membrane) proteins functions upstream of signal activation. Dominant negative TNF receptor-associated factor 2 (TRAF2) inhibited activation of NF-kappa B, which was also inhibited in embryonic fibroblasts derived from TRAF2(-/-) mice, respect to their normal counterpart. These data extend the recent demonstration that TRAF2 mediated JNK activation in response to endoplasmic reticulum stress and strongly strengthened the idea that endogenous stress signals initiated in the endoplasmic reticulum proceed by a pathway similar to that initiated by plasma membrane receptors in response to extracellular signals.

P38 MAPK phosphorylation and NF-kappa B activation in human crescentic glomerulonephritis.

p38 mitogen-activated protein kinase (p38 MAPK) followed by the activation of NF-kappa B participates in the intracellular signal transduction and production of cytokines and chemokines. The pathophysiological roles of p38 MAPK and NF-kappa B in human glomerulonephritis, however, remain to be investigated. We investigated the phosphorylated p38 MAPK (p-p38 MAPK) and activated NF-kappa B immunohistochemically in the kidneys of 34 patients with crescentic glomerulonephritis and 26 control patients with thin basement membrane disease and minimal change nephrotic syndrome. We also explored the co-localization of p-p38 MAPK with CCR5, the signal of which leads to p38 MAPK activation. Furthermore, urinary levels of MIP-1 alpha, the cognate ligand for CCR5, were determined by enzyme-linked immunosorbent assay. p-p38 MAPK-positive cells and activated NF-kappa B-positive cells were mainly detected in crescentic lesions, tubular epithelial cells, and interstitial mononuclear infiltrates. The number of p-p38 MAPK-positive cells in patients with crescentic glomerulonephritis was higher than that in control patients. The number of p-p38 MAPK-positive cells in glomeruli was well correlated with the percentage of cellular crescents, the number of CD68-positive cells, and urinary MIP-1 alpha levels. In addition, the number of activated NF-kappa B-positive cells was well correlated with the number of p-p38 MAPK-positive cells in glomeruli. Dual staining revealed that most of CCR5-positive cells were positive for p-p38 MAPK. Finally, p-p38 MAPK-positive cells and activated NF-kappa B-positive cells decreased during glucocorticoid therapy-induced convalescence. We conclude that the phosphorylation of p38 MAPK associated with the activation of NF-kappa B may be involved in the upregulation of intrarenal MIP-1 alpha and the utilization of CCR5 signalling, which may result in human crescentic glomerulonephritis.

TNF-mediated inflammatory skin disease in mice with epidermis-specific deletion of IKK2.

The I kappa B kinase (IKK), consisting of the IKK1 and IKK2 catalytic subunits and the NEMO (also known as IKK gamma) regulatory subunit, phosphorylates I kappa B proteins, targeting them for degradation and thus inducing activation of NF-kappa B (reviewed in refs 1, 2). IKK2 and NEMO are necessary for NF-kappa B activation through pro-inflammatory signals. IKK1 seems to be dispensable for this function but controls epidermal differentiation independently of NF-kappa B. Previous studies suggested that NF-kappa B has a function in the growth regulation of epidermal keratinocytes. Mice lacking RelB or I kappa B alpha, as well as both mice and humans with heterozygous NEMO mutations, develop skin lesions. However, the function of NF-kappa B in the epidermis remains unclear. Here we used Cre/loxP-mediated gene targeting to investigate the function of IKK2 specifically in epidermal keratinocytes. IKK2 deficiency inhibits NF-kappa B activation, but does not lead to cell-autonomous hyperproliferation or impaired differentiation of keratinocytes. Mice with epidermis-specific deletion of IKK2 develop a severe inflammatory skin disease, which is caused by a tumour necrosis factor-mediated, alpha beta T-cell-independent inflammatory response that develops in the skin shortly after birth. Our results suggest that the critical function of IKK2-mediated NF-kappa B activity in epidermal keratinocytes is to regulate mechanisms that maintain the immune homeostasis of the skin.

Increased production of interleukin-8 in primary human monocytes and in human epithelial and endothelial cell lines after dengue virus challenge.

The more severe form of dengue virus infection, dengue hemorrhagic fever, is characterized by plasma leakage and derangements in hemostasis. As elevated interleukin-8 (IL-8) levels have been observed in sera from patients with more severe disease manifestations, a study was initiated to look at the effect of dengue virus infection in vitro on proinflammatory cytokine secretion and expression. A significant increase in IL-8 levels in the culture supernatant of primary human monocytes infected with dengue 2 virus (D2V) New Guinea C (NGC) was found by enzyme-linked immunosorbent assay. Additionally, by reverse transcriptase PCR, the mRNA was also augmented. Among the proinflammatory cytokines and their mRNAs measured (IL-6, IL-1 beta, IL-8, and tumor necrosis factor alpha), IL-8 showed the greatest change following D2V infection. Similarly, two cell lines, 293T (a human epithelial cell line) and ECV304 (an endothelial cell line), were permissive to D2V NGC and responded to the infection by increasing the synthesis of IL-8. Nuclear factor kappa B (NF-kappa B) and nuclear factor IL-6 (NFIL-6) are primary mediators of IL-8 expression. We studied the transcriptional regulation of IL-8 in the ECV304 and 293T cell lines and found that the induction of IL-8 gene expression involved the activation of NF-kappa B (P = 0.001) and, to a lesser extent, the activation of NFIL-6 in ECV304 cells only. We next observed by the chromatin immunoprecipitation procedure in vivo acetylation of core histones bound to the IL-8 promoter after D2V infection. IL-8 produced by infected monocytes and also IL-8 that may be produced by endothelial or other epithelial cells is associated with the hyperacetylation of histones bound to the IL-8 promoter in addition to the activation of transcription by NF-kappa B. We hypothesize that the overall increase in IL-8 synthesis observed in this in vitro study may play a role in the pathogenesis of the plasma leakage seen in dengue hemorrhagic fever and dengue shock syndrome.

Regulation of SRC-3 (pCIP/ACTR/AIB-1/RAC-3/TRAM-1) Coactivator activity by I kappa B kinase.

In the past few years, many nuclear receptor coactivators have been identified and shown to be an integral part of receptor action. The most frequently studied of these coactivators are members of the steroid receptor coactivator (SRC) family, SRC-1, TIF2/GRIP1/SRC-2, and pCIP/ACTR/AIB-1/RAC-3/TRAM-1/SRC-3. In this report, we describe the biochemical purification of SRC-1 and SRC-3 protein complexes and the subsequent identification of their associated proteins by mass spectrometry. Surprisingly, we found association of SRC-3, but not SRC-1, with the I kappa B kinase (IKK). IKK is known to be responsible for the degradation of I kappa B and the subsequent activation of NF-kappa B. Since NF-kappa B plays a key role in host immunity and inflammatory responses, we therefore investigated the significance of the SRC-3-IKK complex. We demonstrated that SRC-3 was able to enhance NF-kappa B-mediated gene expression in concert with IKK. In addition, we showed that SRC-3 was phosphorylated by the IKK complex in vitro. Furthermore, elevated SRC-3 phosphorylation in vivo and translocation of SRC-3 from cytoplasm to nucleus in response to tumor necrosis factor alpha occurred in cells, suggesting control of subcellular localization of SRC-3 by phosphorylation. Finally, the hypothesis that SRC-3 is involved in NF-kappa B-mediated gene expression is further supported by the reduced expression of interferon regulatory factor 1, a well-known NF-kappa B target gene, in the spleens of SRC-3 null mutant mice. Taken together, our results not only reveal the IKK-mediated phosphorylation of SRC-3 to be a regulated event that plays an important role but also substantiate the role of SRC-3 in multiple signaling pathways.