P65 Subunit Of NF-kappaB Research Articles

Glucosamine Sulfate Inhibits TNF-α and IFN-γ-Induced Production of ICAM-1 in Human Retinal Pigment Epithelial Cells In Vitro

Glucosamine sulfate (GS) is a naturally occurring sugar that possesses some immunosuppressive effects in vitro and in vivo, but its mechanism is unknown. We investigated whether GS could modulate the proinflammatory cytokine-induced expression of the gene for intercellular adhesion molecule (ICAM)-1, an inflammatory protein in human retinal pigment epithelial (RPE) cells. ARPE-19 cells were used as a model to determine the effects of GS on the expression of the ICAM-1 gene upregulated by TNF-alpha or IFN-gamma, by Western blot analysis and semiquantitative reverse transcription polymerase chain reaction (RT-PCR). The activation and nuclear translocation of the nuclear factors NF-kappaB and STAT1 were evaluated by immunocytochemistry, Western blot analysis, and electrophoretic mobility shift assay (EMSA). Both TNF-alpha and IFN-gamma increased the expression of ICAM-1 at the mRNA and protein levels in a time- and dose-dependent manner in ARPE-19 cells. GS effectively downregulated the TNF-alpha- or IFN-gamma-induced expression of ICAM-1 in the protein and mRNA level in a dose-dependent manner. GS further inhibited the nuclear translocation of p65 proteins in TNF-alpha and phosphorylated STAT1 in IFN-gamma-stimulated ARPE-19 cells. GS inhibits the expression of the ICAM-1 gene in ARPE-19 cell stimulated with TNF-alpha or IFN-gamma through blockade of NF-kappaB subunit p65 and nuclear translocation of STAT1. This study has demonstrated a potentially important property of GS in reducing ICAM-1 mediated inflammatory mechanisms in the eye.

Inhibition of Apolipoprotein AI Gene Expression by Tumor Necrosis Factor α: Roles for MEK/ERK and JNK Signaling

Plasma high-density lipoprotein and apolipoprotein AI (apoAI) levels are suppressed by tumor necrosis factor alpha. To determine the molecular mechanisms responsible for the effect of TNF alpha on the apoAI promoter activity, HepG2 cells were exposed to both genetic and pharmacological modulators of TNF alpha-mediated signaling in the presence or absence of TNF alpha. Exogenous ERK1 and ERK2 expression suppressed basal apoAI promoter activity; however, only ERK2 enhanced the ability of TNF alpha to suppress apoAI promoter activity. Exogenous expression of all three MEK isoforms (MEK1, MEK2A, and MEK2E) suppressed basal apoAI promoter activity and further aggravated TNF alpha-related apoAI promoter activity inhibition. Treatment with SB202190 (p38 MAP kinase inhibitor) alone significantly increased apoAI promoter activity; however, in the presence of TNF alpha, apoAI promoter activity was suppressed to an extent similar to that in cells not treated with SB202190. ApoAI promoter activity increased in cells treated with the specific JNK inhibitor SP600125, but unlike SB202190 treatment, the level of TNF alpha-related apoAI promoter inhibition was reduced by 50%. Similarly, the level of TNF alpha-related apoAI promoter inhibition was reduced in cells transfected with JNK1 siRNA. Finally, treatment of cells with the NF-kappaB inhibitors BAY and SN-50 or overexpression of NF-kappaB subunits p50 and p65 had no effect on the ability of TNF alpha to repress apoAI promoter activity. These results suggest that TNF alpha suppresses apoAI promoter activity through both the MEK/ERK and JNK pathways but is not mediated by either p38 MAP kinase activity or NF-kappaB activation.

Inhibition of Hepatocarcinogenesis by the Deletion of the p50 Subunit of NF-κB in Mice Administered the Peroxisome Proliferator Wy-14,643

Wy-14,643 (WY) is a hypolipidemic drug that induces hepatic peroxisome proliferation and tumors in rodents. We previously showed that peroxisome proliferators increase NF-kappaB DNA binding activity in rats, mice, and hepatoma cell lines, and that mice deficient in the p50 subunit of NF-kappaB had much lower cell proliferation in response to the peroxisome proliferator ciprofibrate. In this study we examined the promotion of hepatocarcinogenesis by WY in the p50 knockout (-/-) mice. The p50 -/- and wild type mice were first administered diethylnitrosamine (DEN) as an initiating agent. Mice were then fed a control diet or a diet containing 0.05% WY for 38 weeks. Wild-type mice receiving DEN only developed a low incidence of tumors, and the majority of wild-type mice receiving both DEN and WY developed tumors. However, no tumors were seen in any of the p50 -/- mice. Cell proliferation and apoptosis were measured in hepatocytes by BrdU labeling and the TUNEL assay, respectively. Treatment with DEN + WY increased both cell proliferation and apoptosis in both the wild-type and p50 -/- mice; DEN treatment alone has no effect. In the DEN/WY-treated mice, cell proliferation and apoptosis were slightly lower in the p50 -/- mice than in the wild-type mice. These data demonstrate that NF-kappaB is involved in the promotion of hepatic tumors by the peroxisome proliferator WY; however, the difference in tumor incidence could not be attributed to alterations in either cell proliferation or apoptosis.

Melittin Inhibits Vascular Smooth Muscle Cell Proliferation through Induction of Apoptosis via Suppression of Nuclear Factor-κB and Akt Activation and Enhancement of Apoptotic Protein Expression

In the present study, we have investigated the bee venom (BV) and melittin (a major component of BV)-mediated antiproliferative effect and defined its mechanisms of action in cultured rat aortic vascular smooth muscle cell(s) (VSMC). BV and melittin ( approximately 0.4-0.8 microg/ml) effectively inhibited 5% fetal bovine serum-induced and 50 ng/ml platelet-derived growth factor BB (PDGF-BB)-induced VSMC proliferation. The regulation of apoptosis has attracted much attention as a possible means of eliminating excessively proliferating VSMC. In the present study, the treatment of BV and melittin strongly induced apoptosis of VSMC. To investigate the antiproliferative mechanism of BV and melittin, we examined the effect of melittin on nuclear factor kappaB (NF-kappaB) activation, the PDGF-BB-induced IkappaBalpha phosphorylation, and its degradation were potently inhibited by melittin and whether DNA binding activity and nuclear translocation of NF-kappaB p50 subunit in response to the action of PDGF-BB were potently attenuated by melittin. In further investigations, melittin markedly inhibited the PDGF-BB-induced phosphorylation of Akt and weakly inhibited phosphorylation of extracellular signal-regulated kinase 1/2, upstream signals of NF-kappaB. Treatment of melittin also potently induced proapoptotic protein p53, Bax, and caspase-3 expression but decreased antiapoptotic protein Bcl-2 expression. These results suggest the antiproliferative effects of BV and melittin in VSMC through induction of apoptosis via suppressions of NF-kappaB and Akt activation and enhancement of apoptotic signaling pathway.

Ghrelin stimulates interleukin‐8 gene expression through protein kinase C‐mediated NF‐κB pathway in human colonic epithelial cells

Ghrelin, a newly identified gastric peptide, is known for its potent activity in growth hormone (GH) release and appetite. Although ghrelin is involved in several other responses such as stress and intestinal motility, its potential role in intestinal inflammation is not clear. Here, we show that expression of ghrelin and its receptor mRNA is significantly increased during acute experimental colitis in mice injected intracolonically with trinitrobenzene sulfate (TNBS). We found by PCR that ghrelin receptor mRNA is expressed in non-transformed human colonic epithelial NCM460 cells. Exposure of NCM460 cells stably transfected with ghrelin receptor mRNA to ghrelin, increased IkappaBalpha phosphorylation and its subsequent degradation. In addition, ghrelin stimulated NF-kappaB-binding activity and NF-kappaB p65 subunit phosphorylation, and induced IL-8 promoter activity and IL-8 protein secretion. Furthermore, our data show that ghrelin-induced IkappaBalpha and p65 phosphorylation was markedly reduced by pharmacological inhibitors of intracellular calcium mobilization (BAPTA/AM) and protein kinase C (GF 109203X). Pretreatment with BAPTA/AM or GF109203X also significantly attenuated ghrelin-induced IL-8 production. Together, our results strongly suggest that ghrelin may be a proinflammatory peptide in the colon. Ghrelin may participate in the pathophysiology of colonic inflammation by inducing PKC-dependent NF-kappaB activation and IL-8 production at the colonocyte level.

Analysis of DNA-Protein Interactions in Complexes of Transcription Factor NF-κB with DNA

We have applied bioinformatic analysis of X-ray 3D structures of complexes of transcription factor NF-kappaB with DNAs. We determined the number of possible Van der Waals contacts and hydrogen bonds between amino acid residues and nucleotides. Conservative contacts in the NF-kappaB dimer-DNA complex composed of p50 and/or p65 NF-kappaB subunit and DNA sequences like 5 -GGGAMWTTCC-3 were revealed. Based on these results, we propose a novel scheme for interactions between NF-kappaB p50 homodimer and the kappaB region of the immunoglobulin light chain gene enhancer (Ig-kappaB). We applied a chemical cross-linking technique to study the proximity of some Lys and Cys residues of NF-kappaB p50 subunit with certain reactive nucleotides into its recognition site. In all cases, the experimentally determined protein-DNA contacts were in good agreement with the predicted ones.

Proteasome subunit LMP2 is required for matrix metalloproteinase‐2 and ‐9 expression and activities in human invasive extravillous trophoblast cell line

The ubiquitin-proteasome pathway (UPP) is involved in the degradation of the extracellular matrix (ECM) and trophoblastic invasion during early pregnancy. Our previous studies demonstrated that inhibition of UPP suppresses expression of matrix metalloproteinase (MMP)-2 and -9. LMP2 is an important proteasome subunit that is critical for proteasome activity. This study investigated the regulatory mechanism of LMP2 on the expression and activities of MMP-2 and MMP-9. Our results showed that transfection of LMP2 siRNA plasmid into the human invasive extravillous trophoblast cell line (HTR8/Svneo) could significantly suppress expression of LMP2 mRNA and protein. The mRNA expression of MMP-2 and MMP-9 and their activities were markedly decreased in the LMP2-inhibited cells. Inhibition of LMP2 could also reduce IkappaBalpha mRNA level, although the expression of phosphorylated IkappaBalpha was increased. In the LMP2-inhibited cells, expression of mRNA encoding NF-kappaB subunits p50 and p65 remained normal, but the p50 protein level was significantly decreased in the cytosolic and nuclear extracts, while p65 protein was markedly reduced only in the nuclear extract. We also demonstrated that blockage of the NF-kappaB pathway by the NF-kappaB translocation inhibitor SN50 markedly reduced the expression of MMP-2 and MMP-9 in HTR8/Svneo cells, a result that is fully consistent with the results from the LMP2-inhibited HTR8/Svneo cells. These data suggest that LMP2 contributes to IkappaBalpha degradation and p50 generation, and that inhibition of LMP2 suppresses expression and activities of MMP-2 and MMP-9 by blocking the transfer of active NF-kappaB heterodimers into the nucleus.

Curcumin (Diferuloylmethane) Down-Regulates Expression of Cell Proliferation and Antiapoptotic and Metastatic Gene Products through Suppression of IκBα Kinase and Akt Activation

Curcumin (diferuloylmethane), an anti-inflammatory agent used in traditional medicine, has been shown to suppress cellular transformation, proliferation, invasion, angiogenesis, and metastasis through a mechanism not fully understood. Because several genes that mediate these processes are regulated by nuclear factor-kappaB (NF-kappaB), we have postulated that curcumin mediates its activity by modulating NF-kappaB activation. Indeed, our laboratory has shown previously that curcumin can suppress NF-kappaB activation induced by a variety of agents (J Biol Chem 270:24995-50000, 1995). In the present study, we investigated the mechanism by which curcumin manifests its effect on NF-kappaB and NF-kappaB-regulated gene expression. Screening of 20 different analogs of curcumin showed that curcumin was the most potent analog in suppressing the tumor necrosis factor (TNF)-induced NF-kappaB activation. Curcumin inhibited TNF-induced NF-kappaB-dependent reporter gene expression in a dose-dependent manner. Curcumin also suppressed NF-kappaB reporter activity induced by tumor necrosis factor receptor (TNFR)1, TNFR2, NF-kappaB-inducing kinase, IkappaB kinase complex (IKK), and the p65 subunit of NF-kappaB. Such TNF-induced NF-kappaB-regulated gene products involved in cellular proliferation [cyclooxygenase-2 (COX-2), cyclin D1, and c-myc], antiapoptosis [inhibitor of apoptosis protein (IAP)1, IAP2, X-chromosome-linked IAP, Bcl-2, Bcl-x(L), Bfl-1/A1, TNF receptor-associated factor 1, and cellular Fas-associated death domain protein-like interleukin-1beta-converting enzyme inhibitory protein-like inhibitory protein], and metastasis (vascular endothelial growth factor, matrix metalloproteinase-9, and intercellular adhesion molecule-1) were also down-regulated by curcumin. COX-2 promoter activity induced by TNF was abrogated by curcumin. We found that curcumin suppressed TNF-induced nuclear translocation of p65, which corresponded with the sequential suppression of IkappaBalpha kinase activity, IkappaBalpha phosphorylation, IkappaBalpha degradation, p65 phosphorylation, p65 nuclear translocation, and p65 acetylation. Curcumin also inhibited TNF-induced Akt activation and its association with IKK. Glutathione and dithiothreitol reversed the effect of curcumin on TNF-induced NF-kappaB activation. Overall, our results indicated that curcumin inhibits NF-kappaB activation and NF-kappaB-regulated gene expression through inhibition of IKK and Akt activation.

In situ glomerular expression of activated NF-κB in human lupus nephritis and other non-proliferative proteinuric glomerulopathy

Nuclear Factor-kappaB (NF-kappaB) has been suggested to play a role in the cellular and molecular mechanisms underlying glomerular injury. We investigated the potential role of NF-kappaB activation in the pathogenesis of glomerular injury in 31 patients with class III-V lupus nephritis (LN), 14 patients with non-proliferative proteinuric glomerulopathy and six normal controls. The expression of NF-kappaB subunits p65 and p50, and the NF-kappaB regulated proinflammatory mediators tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), interleukin-6 (IL-6) and intercellular adhesion molecule-1 (ICAM-1) as well as CD68 and synaptopodin was examined by Southwestern histochemistry (SWH) or immunohistochemistry. In contrast to non-proliferative glomerulopathy and normal controls, NF-kappaB activation (both p65 and p50) was enhanced in glomerular endothelial, mesangial cells or infiltrating cells in class IV LN, along with upregulation of TNF-alpha, IL-1beta, IL-6 and ICAM-1 expression. Glomerular endothelial and mesangial activation of NF-kappaB and mesangial ICAM-1 expression correlated with disease activity and the level of glomerular macrophage infiltration. Podocyte NF-kappaB overactivation (predominantly p65) paralleled podocyte expression of TNF-alpha and IL-1beta in patients with LN and non-proliferative glomerulopathy. Podocyte staining scores of NF-kappaB and p65 were positively correlated with the severity of proteinuria in LN and non-proliferative glomerulopathy. These results suggest a pathogenic role for NF-kappaB in glomerular injury by multiple mechanisms.

Roles of Endotoxin-related Signaling Molecules in the Progression of Acute Necrotizing Pancreatitis in Mice

To study the potential roles of lipopolysaccharide (LPS) signaling molecules, LPS-binding protein (LBP), CD14, and Toll-like receptor 4 (TLR4) in mice with acute necrotizing pancreatitis (ANP). The ANP model was made by 7 intraperitoneal injections of cerulein (50 mug/kg) at hourly intervals, challenged by LPS administration of a dose of 5 mg/kg intraperitoneally 5 hours after the first injection of cerulein. Fifty-nine Balb/C mice were divided into 4 groups: group A, ANP control group, n = 18, received physiological saline; group B, anti-LBP group, n = 18, received 200 mug anti-LBP antibody; group C, anti-CD14 group, n = 18, received 20 microg anti-CD14 antibody; group D, anti-TLR4 group, n = 5, received 20 mug anti-TLR4 antibody. All treatments were given at 15 minutes before LPS injection. Serum amylase and lactic dehydrogenase (LDH) were measured. Histologic alteration of the pancreas was evaluated. Expressions of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and intercellular adhesion molecular (ICAM-1) mRNA were determined using reverse transcription polymerase chain reaction (RT-PCR). Myeloperoxidase (MPO) activity in pancreas was also analyzed. Nuclear factor-kappaB (NF-kappaB) p65 subunit was measured by immunohistochemistry and Western blotting. Pretreatment of animals with anti-CD14 antibody resulted in a significant decrease in serum amylase and LDH levels, reduction of the severity of pancreatic injury, down-regulation of TNF-alpha, IL-1beta, and ICAM-1 mRNA expression, decrease in pancreatic MPO activity, and decrease in NF-kappaB expression compared with ANP control mice. In contrast, mice pretreated with anti-LBP antibody or anti-TLR4 antibody resulted in increasing of serum amylase and LDH levels, aggravation of the severity of necrosis and inflammation in pancreas, up-regulation of TNF-alpha, IL-1beta, and ICAM-1 mRNA expression, increasing of pancreatic MPO activity, and up-regulation of NF-kappaB expression compared with ANP control mice. (1) LBP per se possesses a protective effect on ANP. It could facilitate clearance of endotoxin. (2) CD14 plays a crucial intermediate role in the progression of ANP. (3) TLR4, the essential transducer of LPS responses, may act independently of LPS. The antibody against TLR4 may mimic endotoxin and induce activation of downstream cytokines. For implication, recombinant LBP, anti-CD14 antibody, or silent TLR4 might alleviate the progression of ANP.

Arsenic Trioxide Induces Apoptosis of Human Monocytes during Macrophagic Differentiation through Nuclear Factor-κB-Related Survival Pathway Down-Regulation

Arsenic trioxide (As(2)O(3)) is known to be toxic toward leukemia cells. In this study, we determined its effects on survival of human monocytic cells during macrophagic differentiation, an important biological process involved in the immune response. As(2)O(3) used at clinically relevant pharmacological concentrations induced marked apoptosis of human blood monocytes during differentiation with either granulocyte-macrophage colony-stimulating factor or macrophage colony-stimulating factor. Apoptosis of monocytes was associated with increased caspase activities and decreased DNA binding of p65 nuclear factor-kappaB (NF-kappaB); like As(2)O(3), the selective NF-kappaB inhibitor (E)-3-[(4-methylphenyl)-sulfonyl]-2-propenenitrile (Bay 11-7082) strongly reduced survival of differentiating monocytes. The role of NF-kappaB in arsenic toxicity was also studied in promonocytic U937 cells during phorbol 12-myristate 13-acetate-induced macrophagic differentiation. In these cells, As(2)O(3) first reduced DNA binding of p65 NF-kappaB and subsequently induced apoptosis. In addition, overexpression of the p65 NF-kappaB subunit, following stable infection with a p65 retroviral expressing vector, increased survival of As(2)O(3)-treated U937 cells. As(2)O(3) specifically decreased protein levels of X-linked inhibitor of apoptosis protein and FLICE-inhibitory protein, two NF-kappaB-regulated genes in both U937 cells and blood monocytes during their differentiations. Finally, As(2)O(3) was found to inhibit macrophagic differentiation of monocytic cells when used at cytotoxic concentrations; however, overexpression of the p65 NF-kappaB subunit in U937 cells reduced its effects toward differentiation. In contrast to monocytes, well differentiated macrophages were resistant to low concentrations of As(2)O(3). Altogether, our study demonstrates that clinically relevant concentrations of As(2)O(3) induced marked apoptosis of monocytic cells during in vitro macrophagic differentiation likely through inhibition of NF-kappaB-related survival pathways.

Corneal cell survival in adenovirus type 19 infection requires phosphoinositide 3-kinase/Akt activation.

Adenovirus type 19 is a major cause of epidemic keratoconjunctivitis, the only ocular adenoviral infection associated with prolonged corneal inflammation. In this study, we investigated the role of phosphoinositide 3-kinase (PI3K) and Akt and their downstream targets in adenovirus infection, and here we report the novel finding that adenovirus type 19 utilizes the PI3K/Akt pathway to maintain corneal fibroblast viability in acute infection. We demonstrate phosphorylation of GSK-3beta and nuclear translocation of the p65 subunit of NF-kappaB, both downstream targets of the PI3K/Akt pathway, in adenovirus-infected corneal fibroblasts in a PI3K-dependent manner. Inhibition of PI3K had no effect on early viral gene expression, suggesting normal viral internalization, but pretreatment with the PI3K inhibitor LY294002 or overexpression of dominant negative Akt induced early cytopathic effect and caspase-mediated cell death in adenovirus-infected cells. Early cell death could be circumvented despite LY294002 by overexpression of constitutively active Akt. Furthermore, we show an interaction between cSrc and the p85 regulatory subunit of PI3K in infected cells through a phosphorylation-dependent mechanism. The results presented in this paper provide the first direct evidence that PI3K-mediated Akt activation in adenovirus-infected corneal cells may contribute to viral pathogenesis by the prolongation of cell viability.

Tumor necrosis factor receptor I from patients with tumor necrosis factor receptor–associated periodic syndrome interacts with wild‐type tumor necrosis factor receptor I and induces ligand‐independent NF‐κB activation

To investigate the molecular consequences of expressing mutated forms of tumor necrosis factor receptor I (TNFRI) as found in patients with TNFR-associated periodic syndrome (TRAPS). We cloned and expressed full-length wild-type (WT) and T50K and P46L variants of TNFRI using a new tightly regulated doxycycline-dependent expression system. This system enabled the study of molecular interactions between these receptors at both physiologic and pathophysiologic levels of expression. We used chemical crosslinking on the cell surface to show that WT and mutant forms of TNFRI, derived from TRAPS patients, interact in the absence of TNF ligand. Doxycycline-controlled up-regulation of one TNFRI allele, either WT or mutant, caused down-regulation of the other allele, indicating dynamic control of cell surface assembly. We also demonstrated that increased expression of mutant TNFRI (T50K) was associated with a parallel increase in NF-kappaB p65 (RelA) subunit activation, which did not occur with increased expression of WT TNFRI. The T50K TRAPS-related variant is capable of sustaining inappropriate NF-kappaB activation, resulting in persistent auto-inflammation in target organs such as skin, synovial membrane, and the central nervous system. We conclude that some of the inflammatory processes seen in TRAPS do not involve direct interaction of TNF with its receptors, but that other proinflammatory mechanisms capable of up-regulating TNFRI expression may cause cellular activation through the NF-kappaB signaling pathway.

Blockade of NF-κB ameliorates myocardial hypertrophy in response to chronic infusion of angiotensin II

Nuclear factor (NF)-kappaB is a key transcription factor that regulates inflammatory processes. In the present study, we assessed the hypothesis that blockade of NF-kappaB may ameliorate ventricular hypertrophy in response to chronic infusion of angiotensin II. Mice with targeted disruption of the p50 subunit of NF-kappaB (KO) were used to block the activation of NF-kappaB. Male KO and age-matched wild-type (WT) mice were chronically infused with angiotensin II at the rate of 0.2 (low dose) or 2 microg/kg/min (high dose) for 4 weeks. High- but not low-dose angiotensin II significantly increased systemic blood pressure and left ventricular weight in WT mice. In contrast, although the pressor response was slightly but significantly augmented, the hypertrophic effect of angiotensin II was significantly attenuated in KO mice. The attenuated hypertrophic responsiveness was confirmed histologically (cross-sectional area) and transcriptionally (atrial natriuretic peptide). Echocardiography revealed no evidence of cardiac dysfunction in angiotensin II-treated KO mice. Although phosphorylation of MAPKs, including ERK, JNK, or p38-MAPK, was not affected after 4 weeks of angiotensin II treatment in WT mice, phosphorylation of JNK was specifically abrogated in KO mice. Angiotensin II increased myocardial expression of proinflammatory cytokines and chemokines in WT mice, while expression of TNF-alpha and RANTES was paradoxically augmented in KO mice. Blockade of NF-kappaB activation attenuated myocardial hypertrophy without deteriorating cardiac function. NF-kappaB may play an important role in cardiac hypertrophy and remodeling besides promoting inflammation.

Neuroprotection by stem cell factor in rat cortical neurons involves AKT and NFκB

Stem cell factor (SCF) is a highly expressed cytokine in the central nervous system. In the present study, we demonstrate a neuroprotective role for SCF and its tyrosine kinase receptor, c-kit, against camptothecin-induced apoptosis and glutamate excitotoxicity in rat cortical neurons. This protection was blocked by pharmacological or molecular inhibition of either the MEK/ERK or PI3K/Akt signaling pathways. The importance of these pathways was further confirmed by the activation of both ERK, in a MEK-dependent manner, and Akt, via PI3K. Activation of Akt increased the binding of the p50 and p65 subunits of NFkappaB, which was also important for neuroprotection. Akt inhibition prevented NFkappaB binding, suggesting a role for Akt in SCF-induced NFkappaB. Pharmacological inhibition of NFkappaB or dominant negative IkappaB also prevented neuroprotection by SCF. SCF up-regulated the anti-apoptotic genes, bcl-2 and bcl-xL in an NFkappaB-dependent manner. Together, these findings demonstrate a neuroprotective role for SCF in cortical neurons, an effect that was mediated by Akt and ERK, as well as NFkappaB-mediated gene transcription. SCF represents a novel therapeutic target in the treatment of neurodegenerative disease.

Activation of NF-κB by Extracellular Matrix Is Involved in Spreading and Glucose-stimulated Insulin Secretion of Pancreatic Beta Cells

Laminin-5-rich extracellular matrix derived from 804G cells (804G-ECM) engages beta1 integrins to induce spreading, improve glucose-stimulated insulin secretion (GSIS), and increase survival of pancreatic beta cells. The present study examines whether 804G-ECM activates the transcriptional activity of NF-kappaB and the involvement of NF-kappaB in those effects of 804G-ECM on pancreatic beta cells. 804G-ECM induces nuclear translocation and the DNA binding activity of the p65 subunit of NF-kappaB. 804G-ECM-induced nuclear translocation of NF-kappaB was weak as compared with that induced by interleukin-1beta. Transient 804G-ECM-induced DNA binding activity of NF-kappaB (peak at 2 h) and overexpression of NF-kappaB target genes IkappaB alpha and NF-kappaB1(p105) (peak at 4 h) were observed. When NF-kappaB was inhibited by an inhibitor of IkappaB alpha phosphorylation (Bay 11-7082) or by a recombinant adenovirus expressing the nonphosphorylatable form of IkappaB alpha, 804G-ECM-induced cell spreading and actin cytoskeleton organization were reduced. GSIS from cells on 804G-ECM was inhibited 5-fold, whereas cell survival was not affected. In summary, the results indicate that 804G-ECM induces a transient and moderate NF-kappaB activity. This study shows for the first time that ECM-induced NF-kappaB activity is necessary in maintaining GSIS, although it does not affect survival of pancreatic beta cells. The effects of ECM-induced NF-kappaB activity contrast with the deleterious effects of cytokine-induced NF-kappaB activity. It is proposed that transient and moderate NF-kappaB activity is essential for proper function of the pancreatic beta cell.

The Balance Between Expression of Intranuclear NF-κB and Glucocorticoid Receptor in Polymorphonuclear Leukocytes in SIRS Patients

We previously reported enhanced expression of nuclear factor kappa B (NF-kappaB) in activated polymorphonuclear leukocytes (PMNLs) from patients with systemic inflammatory response syndrome (SIRS). Inflammatory response, however, is not regulated only by stimulatory transcription factors. Glucocorticoid receptor (GR) has been recently reported to play an important role in anti-inflammatory signal transduction. The objective of our study was to evaluate the balance between expression of intranuclear NF-kappaB and GR in PMNLs from SIRS patients. In study 1, 29 patients with severe SIRS, who fulfilled the criteria for SIRS and had a serum C-reactive protein level of more than 10 mg/dL, were included. Expression of intranuclear NF-kappaB and GR in PMNLs was measured by flow cytometry with antibodies specific for NF-kappaB subunit p65 and GR. PMNL oxidative activity and serum cytokine levels were also measured. Study 2 included 13 patients with severe trauma (Injury Severity Score 24.6 +/- 12.2). We measured serial changes in expression of intranuclear NF-kappaB and GR in days 0 to 2, 3 to 6, and 7 to 14 after injury. In study 1, expression of both intranuclear NF-kappaB and GR in PMNLs was significantly higher in SIRS patients than in healthy controls. There was a strong correlation between expression of these two transcription factors (r = 0.78). Positive correlations were also found between PMNL oxidative activity and both transcription factors. In study 2, expression of both NF-kappaB and GR in PMNLs was markedly elevated on days 3 to 6 after injury and changed serially with strong mutual correlation. In activated PMNLs from SIRS patients, levels of both intranuclear NF-kappaB and GR were elevated and strongly correlated. In trauma patients, NF-kappaB and GR in PMNLs changed serially with strong mutual correlation. Further studies are needed to clarify the effect of the balance of NF-kappaB and GR on PMNL activation and systemic inflammatory process.

Articular chondrocytes express the receptor for advanced glycation end products: Potential role in osteoarthritis

The receptor for advanced glycation end products (RAGE) binds multiple ligands, including S100 proteins, high mobility group box chromosomal protein 1 (HMGB-1), and AGEs, all of which are present in articular cartilage. Stimulation of RAGE signaling can lead to MAP kinase activation and increased NF-kappaB activity. The objective of the present study was to determine if chondrocytes express functional RAGE. The presence of chondrocyte RAGE was analyzed by immunohistochemistry using normal and osteoarthritic (OA) cartilage from young and old monkeys and humans, immunoblotting of chondrocyte lysates and human cartilage extracts, and reverse transcription-polymerase chain reaction (RT-PCR) analysis of RNA from chondrocytes treated with interleukin-1 (IL-1) and fibronectin fragments. RAGE signaling was evaluated by stimulating chondrocytes with S100B and HMGB-1 and analyzing for activation of the ERK MAP kinase and NF-kappaB. The ability of S100B and HMGB-1 to stimulate matrix metalloproteinase 13 (MMP-13) production was also assessed. A pull-down assay using biotin-labeled S100B was used to demonstrate binding to RAGE. RAGE was detected in sections of monkey knee cartilage and human knee and ankle cartilage. Increased immunostaining for RAGE was noted in cartilage from older adult monkeys and humans and was further increased in OA tissue. RAGE was also detected by immunoblotting and by RT-PCR, where IL-1beta and fibronectin fragments were found to stimulate RAGE expression. Stimulation of chondrocytes with S100B or HMGB-1 increased phosphorylation of the ERK MAP kinase and the p65 subunit of NF-kappaB and increased the production of MMP-13. This signaling was inhibited in cells pretreated with soluble RAGE, and S100B was shown to bind to chondrocyte RAGE. Articular chondrocytes express functional RAGE. The increase in RAGE noted in OA cartilage and the ability of RAGE ligands to stimulate chondrocyte MAP kinase and NF-kappaB activity and to stimulate MMP-13 production suggests that chondrocyte RAGE signaling could play a role in OA.

PAR1‐mediated NFκB activation promotes survival of prostate cancer cells through a Bcl‐xL‐dependent mechanism

We have previously reported that protease-activated receptor 1 (PAR1 or thrombin receptor) is over-expressed in metastatic prostate cancer cell lines compared to prostate epithelial cells. In this study, we examined 1,074 prostate biopsies by tissue microarray analysis and demonstrated that PAR1 expression is significantly increased in prostate cancer compared to normal prostate epithelial cells and benign prostatic hyperplasia. We hypothesized that PAR1 activation contributed to prostate cancer cell progression. We demonstrated that stimulation of PAR1 by thrombin or thrombin receptor activating peptide (TRAP6), in androgen-independent DU145 and PC-3 cells resulted in increased DNA binding activity of the NFkappaB p65 subunit. IL-6 and IL-8 levels were also elevated in conditioned media by at least two-fold within 4-6 h of PAR1 activation. This induction of cytokine production was abrogated by pretreatment of cells with the NFkappaB inhibitor caffeic acid phorbol ester. The p38 and ERK1/2 MAPK signaling cascades were also activated by PAR1 stimulation, whereas the SAPK/JNK pathway was unaffected. Inhibition of p38 and ERK1/2 by SB-203589 and PD-098059, respectively, did not abrogate NFkappaB activity, suggesting an independent induction of NFkappaB by PAR1 stimulation. Furthermore, TUNEL assay showed that activation of PAR1 attenuated docetaxel induced apoptosis through the upregulation of the Bcl-2 family protein Bcl-xL. Akt activation was not observed, suggesting that drug resistance induced by PAR1 was independent of PI3K signaling pathway. Because thrombin and PAR1 are over-expressed in prostate cancer patients, targeting the inhibition of their interaction may attenuate NFkappaB signaling transduction resulting in decreased drug resistance and subsequent survival of prostate cancer cells.

IKKβ phosphorylates p65 at S468 in transactivaton domain 2

The transcription factor nuclear factor-kappa B (NF-kappaB) subunit p65 is phosphorylated by IkappaB kinase (IKK) at S536 in transactivation domain (TAD) 1. In this study, we investigate the presence of IKK sites in TAD2 of p65. Recombinant IKKbeta, but not IKKalpha, phosphorylated a GST-p65 substrate in which TAD1 was deleted. Mutational analysis revealed S468 as the only IKK site in TAD2. S468 phosphorylation occurred rapidly after TNF-alpha and IL-1beta in T cell, B cell, cervix carcinoma, hepatoma, breast cancer, and astrocytoma lines and in primary hepatic stellate cells as well as peripheral blood mononuclear cells. S468-phosphorylated p65 coimmunoprecipitated with IkappaBalpha, indicating that p65 is phosphorylated while bound to IkappaBalpha. Dominant negative IKKbeta or pharmacological IKK inhibition blocked S468 phosphorylation after TNF-alpha or IL-1beta, whereas dominant negative IKKalpha or inhibitors of MEK, p38, JNK, PI-3 kinase, or GSK-3 had no effect. p65S468A-reconstituted p65-/- mouse embryonic fibroblasts (MEFs) showed a small, but significant, elevation of NF-kappaB-driven luciferase activity and RANTES mRNA levels after TNF-alpha and IL-1beta in comparison to wtp65-reconstituted MEFs. p65 nuclear translocation was not altered in p65S468A-expressing MEFs. In conclusion, our results indicate that 1) IKKbeta phosphorylates multiple p65 sites, 2) IKKbeta phosphorylates p65 in an IkappaB-p65 complex, and 3) S468 phosphorylation slightly reduces TNF-alpha- and IL-1beta-induced NF-kappaB activation.