Kappa B-alpha Research Articles

Inhibition of Helicobacter pylori-induced nuclear factor-kappa B activation and interleukin-8 gene expression by ecabet sodium in gastric epithelial cells.

Helicobacter pylori stimulates nuclear factor-kappa B (NF-kappa B) activation and chemokine interleukin-8 (IL-8) expression in gastric epithelial cells. Ecabet sodium (ecabet), a locally acting antiulcer drug, is known to have anti-H. pylori activity. However, there is little understanding of how ecabet induces anti-inflammatory activity in gastric epithelial cells infected with H. pylori. The aim of this study was to investigate the effects of ecabet on IL-8 gene expression and NF-kappa B activation in human gastric epithelial cells infected with H. pylori. After Hs746T, MKN-45, or SNU-5 gastric epithelial cell lines had been infected with cagA+cytotoxin+H. pylori in the presence of ecabet, IL-8 mRNA expression was assessed by quantitative reverse transcription-polymerase chain reaction, and IL-8 secretion was measured by enzyme-linked immunosorbent assay. NF-kappa B and inhibitory kappa B-alpha (I kappa B alpha) signals were assayed by electrophoretic mobility shift assay and Western blot, respectively. The activation of NF-kappa B and IL-8 reporter genes was determined by luciferase assay. Ecabet showed no antimicrobial activiy against Gram-positive or -negative bacteria. However, ecabet inhibited transcription of the IL-8 gene and secretion of IL-8 by gastric epithelial cells infected with H. pylori at a concentration of 5 micro g/ml. Moreover, ecabet inhibited the activation of NF- kappa B and the degradation of I kappa B alpha in gastric epithelial cells in response to H. pylori infection. In addition, the NF-kappa B signal inhibited by ecabet was comprised predominantly of heterodimers of p65/p50. Ecabet inhibited H. pylori-induced IL-8 gene transcription and secretion by suppressing the NF-kappa B signal. This inhibition might be one pathway by which ecabet exerts its anti-inflammatory effect on H. pylori-induced gastric inflammation.

Monitoring NF-kappa B transactivation potential via real-time PCR quantification of I kappa B-alpha gene expression.

Nuclear factor-kappa B (NF-kappa B) is an important transcription factor involved in the regulation of immune responses as well as in cell proliferation and survival. An abnormal and constitutive activation of NF-kappa B is observed in many pathological states as diverse as inflammation, neurological diseases, and cancer. Termination of NF-kappa B transcription is mediated through the NF-kappa B-dependent synthesis of the I kappa B-alpha inhibitory subunit. To quantify NF-kappa B activation we measured by real-time PCR the expression of I kappa B-alpha mRNA. The PCR data perfectly matched the results obtained by Northern blot or gene reporter analysis when Jurkat leukemic T cells or HeLa carcinoma cells were stimulated with various activators of NF-kappa B, such as the cytokine tumor necrosis factor (TNF)-alpha or the phorbol ester PMA. Constitutive NF-kappa B activation in Hodgkin's lymphoma cell line could also be evaluated by this approach. Kinetic experiments in HeLa cells show that TNF stimulation first induced NF-kappa B DNA binding within 30 minutes, followed by I kappa B-alpha gene transcription 30 minutes later. Removal of TNF after stimulation resulted in a faster decrease in both NF-kappa B DNA binding activity and I kappa B-alpha mRNA levels. No accumulation or stabilization of I kappa B-alpha mRNA was detected that could bias interpretation of the results. The sensitivity of the method allowed the detection of NF-kappa B activation in stimulated normal peripheral blood lymphocytes. The real-time PCR measure of I kappa B-alpha mRNA levels is a rapid, sensitive, and powerful method to quantify the transcriptional power of NF-kappa B. It can be easily used for clinical evaluation of NF-kappa B status.

Enhancement of lipopolysaccharide-stimulated JNK activity in rat aortic smooth muscle cells by pharmacological and adenovirus-mediated inhibition of inhibitory kappa B kinase signalling.

1. In rat aortic smooth muscle cells (RASMCs), the putative nuclear factor kappa B (NFkappaB) inhibitor Pyrrolidine dithiocarbamate (PDTC) was found to inhibit lipopolysaccharide (LPS)-stimulated NFkappaB DNA-binding. However, further investigation identified the site of inhibition as being at, or upstream of, the inhibitory kappa B kinases (IKKs) as their kinase activity was substantially reduced. 2. In addition, PDTC potentiated LPS-stimulated c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAP kinase) and MAP kinase-activated protein kinase-2 activity (the downstream target of p38 MAP kinase). 3. Another inhibitor of NFkappaB signalling, the serine protease inhibitor Nalphap-tosyl-L-lysine chloro-methylketone (TLCK), also inhibited LPS-stimulated IKK activity and potentiated JNK activity in response to LPS, suggesting that cross-talk may occur between the NFkappaB and stress-activated protein kinase pathways at the level of IKK or at a common point upstream. 4. Infection of RASMCs with an adenovirus encoding either inhibitory kappa Balpha or a dominant-negative IKKbeta potentiated LPS-stimulated JNK activity. 5. These studies therefore suggest that the loss of NFkappaB DNA-binding and resultant transcriptional activity, rather than the loss of IKK activity, is sufficient to cause an increase in JNK activity. This shows that either pharmacological or molecular inhibition of NFkappaB DNA-binding enhances JNK activation in vascular smooth muscle cells, an effect that may contribute to the pathophysiological effects of LPS.

Cardioprotection involves activation of NF-kappa B via PKC-dependent tyrosine and serine phosphorylation of I kappa B-alpha.

Previous studies indicated that activation of PKC and Src tyrosine kinases by ischemic preconditioning (PC) may participate in the activation of NF-kappa B. However, the molecular mechanisms underlying activation of NF-kappa B during ischemic PC remain unknown. In the hearts of conscious rabbits, it was found that ischemic PC (6 cycles of 4-min coronary occlusion and 4-min reperfusion) significantly induced both tyrosine (+226.9 +/- 42%) and serine (+137.0 +/- 36%) phosphorylation of the NF-kappa B inhibitory protein I kappa B-alpha, concomitant with increased activation of the I kappa B-alpha kinases IKK alpha (+255.0 +/- 46%) and IKK beta (+173.1 +/- 35%). Furthermore, both tyrosine and serine phosphorylation of I kappa B-alpha were blocked by pretreatment with either the nonreceptor tyrosine kinase inhibitor lavendustin-A (LD-A) or the PKC inhibitor chelerythrine (Che) (both given at doses previously shown to block ischemic PC). Interestingly, Che completely abolished PC-induced activation of IKK alpha/beta, whereas LD-A had no effect. In addition, I kappa B-alpha protein level did not change during ischemic PC. Together, these data indicate that ischemic PC-induced activation of NF-kappa B occurs through both tyrosine and serine phosphorylation of I kappa B-alpha and is regulated by nonreceptor tyrosine kinases and PKC.

Different short- and long-term effects of resveratrol on nuclear factor-κB phosphorylation and nuclear appearance in human endothelial cells

Resveratrol (a naturally occurring phytoalexin found in grapes and wine) has cardiovascular protective effects that suggest the antiatherogenic (ie, antiinflammatory) activities of the compound on endothelial cells. The antiinflammatory activity of resveratrol could be mediated by its interference with nuclear factor-kappaB (NF-kappaB)-dependent transcription. Thus, we studied the in vitro influence of physiologic concentrations of resveratrol (<or= 1 micromol/L) on the NF-kappaB signaling pathway after tumor necrosis factor alpha (TNF-alpha) stimulation of endothelial cells. The effects of a 30-min (acute) and an overnight incubation of resveratrol on the nuclear appearance of p50-NF-kappaB and p65-NF-kappaB on serine and tyrosine phosphorylation of the inhibitory subunit kappaB alpha (IkappaBalpha), cytoplasmic concentrations of IkappaBalpha, NF-kappaB phosphorylation or nitrosylation, the reduction of the mitotic inhibitor p21, and the activation of peroxisome proliferator-activated receptor alpha were evaluated. The nuclear appearance of p50-NFkappaB and p65-NFkappaB acutely induced by TNF-alpha was not modified by resveratrol but was increased after overnight incubation with resveratrol alone or in combination with TNF-alpha. Acute treatment with resveratrol did not modify TNF-alpha-induced cytoplasmic IkappaBalpha serine phosphorylation but did increase IkappaBalpha tyrosine phyophorylation. Resveratrol increased the tyrosine phosphorylation (but not nitrosylation) of immunoprecipitated NF-kappaB, did not decrease cellular p21, and did not increase peroxisome proliferator-activated receptor alpha activity. Acute resveratrol treatment does not inhibit the nuclear appearance of NF-kappaB in human umbilical vein endothelial cells, but overnight treatment does. The increase in tyrosine phosphorylation of IkappaBalpha, p50-NF-kappaB, and p65-NF-kappaB suggests the involvement of such alterations in the modulation of NF-kappaB transcription activity.

Fibrinogen-CD11b/CD18 interaction activates the NF-kappa B pathway and delays apoptosis in human neutrophils.

The regulation of neutrophil half-life by members of the coagulation cascade is critical for the resolution of the inflammatory response. We have demonstrated that soluble fibrinogen (sFbg) delays human neutrophil (PMN) apoptosis through a mechanism that involves CD11b interactions, and phosphorylation of focal adhesion kinase (FAK) and extracellular signal-regulated kinase 1/2 (ERK1/2). Since NF-kappa B is a key element in the regulation of apoptotic mechanisms in several immune cells, we investigated whether NF-kappa B is involved in the control of PMN survival by sFbg. We show that sFbg triggers inhibitor protein kappa B (I kappa B-alpha) degradation and NF-kappa B activation. Furthermore, pharmacological inhibition of NF-kappa B abrogates sFbg effects on apoptosis. In addition, specific inhibition of MAPK ERK1/2 significantly reduces NF-kappa B translocation by sFbg, suggesting a relationship between ERK1/2 and NF-kappa B activation. Similar results are obtained when granulocytic-differentiated HL-60 cells are treated with sFbg, making this model highly attractive for integrin-induced gene expression studies. It can be concluded that NF-kappa B participates in the prevention of apoptosis induced by sFbg with the participation of MAPK ERK1/2. These results shed light on the molecular mechanisms that control human granulocyte apoptosis, and suggest that NF-kappa B regulation may be of benefit for the resolution of the inflammatory response.

Secretory Component Is Cleaved by Neutrophil Serine Proteinases but its Epithelial Production Is Increased by Neutrophils through NF-κB– and p38 Mitogen-Activated Protein Kinase–Dependent Mechanisms

We previously showed that expression of polymeric immunoglobulin receptor (pIgR)/secretory component (SC), the epithelial receptor assuming transport of polymeric IgA in mucosal secretions, is strongly decreased in severe chronic obstructive pulmonary disease. Here, we evaluated in vitro the effects of polymorphonuclear neutrophil (PMN) mediators on pIgR/SC. On polyacrylamide gel electrophoresis analysis, soluble SC was rapidly cleaved by supernatants from phorbol-myristate-acetate-activated PMN, through a serine proteinase activity. Moreover, purified PMN serine proteinases also cleaved SC. Similarly, polymeric IgA was rapidly cleaved in monomers by neutrophil elastase, whereas secretory immunoglobulin A was relatively resistant to neutrophil elastase. Surface pIgR on human bronchial epithelial cells was also cleaved by serine proteinases, as shown by immunofluorescence. In contrast, pIgR/SC production by cultured epithelial cells (quantified by enzyme-linked immunosorbent assay) was significantly increased by supernatants from interleukin-8/formylmethionylleucylphenylalanine-activated PMN (122.6 +/- 17.3 versus 70.9 +/- 9 ng/mg protein, P < 0.01). Upregulation of pIgR/SC production by bronchial epithelial cells was abolished by nuclear factor kappa B- and p38 mitogen-activated protein kinase (MAPK) inhibitors. Moreover, supernatants from interleukin-8/formylmethionylleucylphenylalanine-activated PMN induced the phosphorylation of I kappa B-alpha and p38 MAPK in epithelial cells, independently of serine proteinases. Thus, PMN serine proteinases cleave pIgR/SC, whereas activated PMN induce an increased pIgR/SC expression through epithelial activation of nuclear factor kappa B and p38 MAPK pathways.

Measuring activated protein C in plasma to assess its role as a critical modulator of coagulation and inflammation

Measuring activated protein C in plasma to assess its role as a critical modulator of coagulation and inflammation

17β-estradiol Inhibits the Production of RANTES in Human Keratinocytes

A chemokine, regulated upon activation, normal T cell expressed and secreted (RANTES) attracts T helper-1 cells and macrophages. The production of RANTES is enhanced in keratinocytes of psoriatic skin lesions, which may contribute to the inflammatory infiltrate. It is known that estrogen regulates the natural course of psoriasis. We examined the in vitro effects of 17beta-estradiol on RANTES production by human keratinocytes. 17beta-estradiol inhibited tumor necrosis factor-alpha or interleukin-1beta-induced RANTES secretion, mRNA expression, and promoter activity in keratinocytes, and these effects of 17beta-estradiol were counteracted by estrogen receptor antagonist ICI 182 780. Two nuclear factor kappaB elements on RANTES promoter were required for tumor necrosis factor-alpha or interleukin-1beta-induced transcription and involved in the inhibition by 17beta-estradiol. 17beta-estradiol inhibited nuclear factor kappaB transcriptional activity, whereas it did not inhibit DNA binding of nuclear factor kappaB or phosphorylation or degradation of the inhibitor of nuclear factor kappaB alpha in tumor necrosis factor-alpha or interleukin-1beta-stimulated keratinocytes. 17beta-estradiol-induced inhibition of nuclear factor kappaB transcriptional activity and RANTES promoter activity was rescued by overexpression of a coactivator cyclic AMP response element-binding protein (CREB) or nuclear factor kappaB p65 but not by steroid receptor coactivator-1 or nuclear factor kappaB p50. The overexpression of CREB-binding protein rescued 17beta-estradiol-induced inhibition of transcription mediated by a chimeric protein, GAL4-p65286-551, which contained GAL4 DNA binding domain fused to C-terminal transactivating domain of p65 (amino acids 286-551). The transfection of estrogen receptor alpha or estrogen receptor beta into estrogen receptor-negative SKBR3 cells resulted in 17beta-estradiol-mediated inhibition of transcription via GAL4-p65286-551. These results suggest that 17beta-estradiol-bound estrogen receptor may inhibit nuclear factor kappaB-dependent transcription of RANTES gene by competing with p65 for limiting amounts of CREB-binding protein.

P1774 Postinfarct remodelling can be attenuated by intramyocardial adenovirus-mediated delivery of inhibitor kappa Balpha gene

P1774 Postinfarct remodelling can be attenuated by intramyocardial adenovirus-mediated delivery of inhibitor kappa Balpha gene

Tumour necrosis factor-alpha-induced expression of intercellular adhesion molecule-1 on human eosinophilic leukaemia EoL-1 cells is mediated by the activation of nuclear factor-kappaB pathway.

Intercellular adhesion molecule-1 (ICAM-1) has been shown to mediate the adhesion and migration of eosinophils to the site of allergic inflammation. However, molecular mechanisms regulating the expression of ICAM-1 in eosinophils are still being elucidated. We investigated the effect of tumour necrosis factor-alpha (TNF-alpha) on ICAM-1 expression of eosinophils. The surface expression of ICAM-1 on a human eosinophilic leukaemic cell line, EoL-1, was assessed by immunocytochemical staining. The phosphorylation of inhibitor kappa B-alpha (IkappaB-alpha) and p38 mitogen-activated protein kinase (MAPK) was detected by Western blot. Nuclear factor kappa-B (NF-kappaB) pathway-related genes were evaluated by the cDNA expression array system, whereas the activity of NF-kappaB was measured by electrophoretic mobility shift assay (EMSA). TNF-alpha was found to induce the cell surface expression of ICAM-1. A specific proteasome inhibitor N-cbz-Leu-Leu-leucinal (MG-132), but not a p38 MAPK inhibitor (SB 203580), was found to suppress the TNF-alpha-induced expression of ICAM-1 on EoL-1 cells. The gene expressions of ICAM-1, NF-kappaB and IkappaBalpha were up-regulated after the stimulation with TNF-alpha. Further, TNF-alpha was shown to induce IkappaB-alpha phosphorylation and degradation, thereby indicating the activation of NF-kappaB. In EMSA, there was a shifted NF-kappaB band on TNF-alpha-treated cells with or without SB 203580, but no shifted band was observed on MG-132-treated cells. In vitro studies of EoL-1 cells, an eosinophilic leukaemic cell line, confirmed that NF-kappaB plays an important role in the expression of ICAM-1 and recruitment of eosinophils in allergic inflammation.

Ultraviolet radiation modulates nuclear factor kappa B activation in human lens epithelial cells.

Exposure to ultraviolet radiation (UVR) is a known risk factor for cataract, but the molecular mechanisms involved have not been elucidated. We hypothesized that exposure to UVR would modulate the activation of nuclear factor kappa-B (NF-kappa B) within the human lens epithelium, since NF-kappa B is a key regulator of cellular responses to UVR stress in other cell types. Human lens epithelial (HLE) cells were exposed to acute physiological doses of ultraviolet A (UVAR), B (UVBR), C (UVCR) radiation, or interleukin-1 beta (IL-1 beta) and NF-kappa B activation was measured by electrophoretic shift assay (EMSA). Phosphorylation of I kappa B in response to UVAR was measured by Western blotting. Irradiation of HLE cells with UVAR (0-1100 J/m(2)) did not reduce cell survival, while UVBR (400-1600 J/m(2)) and UVCR (300-900 J/m(2)) significantly reduced HLE cell survival. EMSA analysis of HLE nuclear proteins indicated activation of NF-kappa B, but not activator protein-1 (AP-1), by UVAR. The effects of UVBR and UVCR were less pronounced. Exposure of HLE cells to UVAR (0-900 J/m(2)) followed by a 30-min incubation resulted in a dose-dependent activation of NF-kappa B. UVAR-induced NF-kappa B activation in HLE cells was evident 10 min postirradiation, maximal at 60 min and returned to control levels by 120 min. Western blot analysis of phosphorylation of the NF-kappa B inhibitory protein, I kappa B, revealed that UVAR activates NF-kappa B via a mechanism involving the phosphorylation of I kappa B-alpha; this effect was dose-dependent. Supershift analysis demonstrated that UVAR and IL-1 beta activate the transcriptionally active p65/p50 NF-kappa B dimer. These studies demonstrate that UVAR activates NF-kappa B in HLE cells in a time- and dose-dependent manner via signaling through I kappa B-alpha. The activation of NF-kappa B in HLE cells by UVAR may have implications for the development and progression of cataract and other related ocular disorders.

Phosphoinositide 3-kinase activity leads to silica-induced NF-kappaB activation through interacting with tyrosine-phosphorylated I(kappa)B-alpha and contributing to tyrosine phosphorylation of p65 NF-kappaB.

The role of the subunits of phosphoinositide (PI) 3-kinase in NF-kappaB activation in silica-stimulated RAW 264.7 cells was investigated. Results indicate that PI3-kinase activity was increased in response to silica. The p85alpha subunit of PI3-kinase interacted with tyrosine-phosphorylated I(kappa)B-alpha in silica-stimulated cells. PI3-kinase specific inhibitors, such as wortmannin and LY294003, substantially blocked both silica-induced PI3-kinase and NF-kappaB activation. The inhibition of NF-KB activation by PI3-kinase inhibitors was also observed in pervanadate-stimulated but not in LPS-stimulated cells. Furthermore, tyrosine phosphorylation of NF-kappaB p65 was enhanced in cells stimulated with silica, pervanadate or LPS, and wortmannin substantially inhibited the phosphorylation event induced by the first two stimulants but not LPS. Antioxidants, such as superoxide dismutase (SOD), N-acetylcysteine (NAC) and pyrrolidine dithiocarbamate (PDTC), blocked silica-induced PI3-kinase activation, suggesting that reactive oxygen species may be important regulatory molecules in NF-kappaB activation by mediating PI3-kinase activation. Our data suggest that p85 and p110 subunits of PI3-kinase play a role in NF-kappaB activation through interaction with tyrosine-phosphorylated I(kappa)B-alpha and contributing to tyrosine phosphorylation of p65 NF-kappaB.

Differential roles of hydrogen peroxide and superoxide in mediating IL-1-induced NF-kappa B activation and iNOS expression in bovine articular chondrocytes.

Our previous studies showed that reactive oxygen species (ROS) are required for the pro-inflammatory cytokine interleukin-1 beta (IL-1) to induce the activity of the Nuclear transcription Factor-kappa B (NF-kappa B) and the expression of the inducible isoform of the nitric oxide synthase (iNOS) in bovine articular chondrocytes. This study aimed at elucidating the role of hydrogen peroxide (H(2)O(2)) and the superoxide radical, two major ROS, in mediating those IL-1-induced responses. The results obtained show that chondrocytes produce both H(2)O(2) and superoxide radical in response to IL-1. Treatment of the chondrocyte cultures with H(2)O(2) alone did not induce NF-kappa B activation or iNOS expression. Addition of H(2)O(2) simultaneously with IL-1 did neither enhance nor inhibit NF-kappa B activation and iNOS expression, relatively to treatment with IL-1 alone. Accordingly, treatment with catalase did not inhibit those IL-1-induced responses. Treatment with superoxide dismutase, however, effectively prevented IL-1-induced I kappa B-alpha degradation and iNOS expression. Taken together, the results obtained indicate that superoxide mediates IL-1-induced I kappa B-alpha degradation and the consequent NF-kappa B activation and iNOS expression in chondrocytes, whereas H(2)O(2) does not seem to participate in those IL-1-induced responses. In conclusion, the present study identifies the superoxide radical as the ROS involved in mediating the IL-1-induced signaling pathway that leads to NF-kappa B activation and to the expression of NF-kappa B-dependent genes in bovine articular chondrocytes.

Critical role of protein kinase C ε for lipopolysaccharide-induced IL-12 synthesis in monocyte-derived dendritic cells

In the present study we have investigated the potential involvement of protein kinase C (PKC) in the maturation of human dendritic cells (DC) by bacterial lipopolysaccharide (LPS). LPS stimulation of DC derived from human monocytes resulted in PKC phosphorylation. Inhibition of PKC activation using bisindolylmaleimide (Bis), a pan-PKC inhibitor, was associated with a dose-dependent decrease of LPS-induced IL-12 production. In contrast, up-regulation of MHC class II, CD80 and CD86 was not altered. Consistent with the diminished IL-12 synthesis, DC stimulated with LPS in presence of Bis were deficient in the induction of IFN-gamma production by allogeneic CD4+ T cells. Furthermore, we found that PKC inhibition impaired LPS-induced I kappa B-alpha degradation and subsequent nuclear factor (NF)-kappa B activation in DC. LPS resulted in the phosphorylation of conventional alpha/beta and novel epsilon PKC isoforms in DC. Inhibition of LPS-induced PKC activity using pseudosubstrate peptides specific for PKC isoforms established that PKC epsilon but not PKC alpha/beta was involved in the production of IL-12 and TNF-alpha. Overall, these data provide evidence that PKC inhibition impairs LPS signaling in DC and identify PKC epsilon as a potential target for the inhibition of Toll-like receptor-4-mediated, IL-12-dependent Th1 type responses.

Doxorubicin inhibits the production of nitric oxide by colorectal cancer cells.

Doxorubicin (DOX) is an active and broad spectrum chemotherapeutic agent. Increased inducible nitric oxide synthase (NOS) expression and/or activity have been reported in several human tumors. While the relationship between DOX treatment and the enzymatic activity of endothelial NOS has been well characterized, little is known about the effects of DOX on the expression of iNOS in human cancer cells. In the present study, we characterized the effects of DOX on the nitric oxide (NO) production by colorectal cancer cells, DLD-1. IFN-gamma/IL-1beta (CM) increased the production of NO, whereas pretreatment of DOX inhibited the production of NO in response to CM in a dose dependent manner. The increased expressions of iNOS mRNA and protein by CM were completely blocked by DOX without affecting the iNOS mRNA stability. However, DOX activated nuclear factor-kappaB (NF-kappaB) in response to CM. Furthermore, the expression of inhibitor kappaB alpha was reduced by DOX in a dose dependent manner. Collectively, DOX inhibited the production of NO by DLD-1 cells, which is not linked to well known transcription factor, NF-kappaB. Therefore, further studies on the possible mechanisms of inhibitory effects of NO production by DOX would be worth pursuing.

The molecular mechanisms of arsenic-induced cell transformation and apoptosis.

Arsenic is a well-documented human carcinogen associated with cancers of the skin, lung, liver, and bladder. Interestingly, arsenic has also been used as an effective chemotherapeutic agent in the treatment of certain human cancers. However, the mechanisms by which arsenic induces proliferation of cancer cells or cancer cell death are not well understood. We found that exposure of JB6 P+ cells to low concentrations of arsenic induces cell transformation, whereas higher concentrations of arsenic induce cell apoptosis. Arsenite induces phosphorylation of extracellular signal-regulated protein kinases (Erks) and c-Jun NH(2)-terminal kinases (JNKs). Arsenite-induced Erk activation was markedly inhibited by introduction of dominant-negative Erk2 into cells, whereas expression of dominant-negative Erk2 did not inhibit JNKs or mitogen-activated protein kinase Erk kinase 1/2. Furthermore, arsenite-induced cell transformation was blocked in cells expressing dominant-negative Erk2. In contrast, overexpression of dominant-negative JNK1 increased cell transformation even though it inhibited arsenite-induced JNK activation. Arsenic also induced AP-1 and nuclear factor kappa B (NF-kappaB) activation. Blocking NF-kappaB activation by dominant-negative inhibitory kappa Balpha inhibited arsenic-induced apoptosis and enhanced arsenic-induced cell transformation. Arsenic induced activation of JNKs at a similar dose range that was effective for induction of apoptosis in JB6 cells. In addition, we found that arsenic did not induce p53-dependent transactivation. Similarly, apoptosis induction was not different between p53 wild-type (p53(+/+)) or p53-deficient (p53(-/-)) cells. In contrast, arsenic-induced apoptosis was almost totally blocked by expression of a dominant-negative mutant of JNK. Taken together with previous findings that p53 mutations are involved in approximately 50% of all human cancers and nearly all chemotherapeutic agents kill cancer cells mainly by apoptotic induction, we suggest that arsenic may be a useful agent for the treatment of cancers with p53 mutations. These results suggest that the activation of Erks is required for arsenic-induced cell transformation, whereas the activation of JNKs and NF-kappaB is involved in arsenic-induced apoptosis of JB6 cells.

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.

Synthetic Analogs of Green Tea Polyphenols as Proteasome Inhibitors

Animal, epidemiological and clinical studies have demonstrated the anti-tumor activity of pharmacological proteasome inhibitors and the cancer-preventive effects of green tea consumption. Previously, one of our laboratories reported that natural ester bond-containing green tea polyphenols (GTPs), such as (-)-epigallocatechin-3-gallate [(-)-EGCG] and (-)-gallocatechin-3-gallate [(-)-GCG], are potent and specific proteasome inhibitors. Another of our groups, for the first time, was able to enantioselectively synthesize (-)-EGCG as well as other analogs of this natural GTP. Our interest in designing and developing novel synthetic GTPs as proteasome inhibitors and potential cancer-preventive agents prompted our current study. GTP analogs, (+)-EGCG, (+)-GCG, and a fully benzyl-protected (+)-EGCG [Bn-(+)-EGCG], were prepared by enantioselective synthesis. Inhibition of the proteasome or calpain (as a control) activities under cell-free conditions were measured by fluorogenic substrate assay. Inhibition of intact tumor cell proteasome activity was measured by accumulation of some proteasome target proteins (p27, I kappa B-alpha and Bax) using Western blot analysis. Inhibition of tumor cell proliferation and induction of apoptosis by synthetic GTPs were determined by G(1) arrest and caspase activation, respectively. Finally, inhibition of the transforming activity of human prostate cancer cells by synthetic GTPs was measured by a colony formation assay. (+)-EGCG and (+)-GCG potently and specifically inhibit the chymotrypsin-like activity of purified 20S proteasome and the 26S proteasome in tumor cell lysates, while Bn-(+)-EGCG does not. Treatment of leukemic Jurkat T or prostate cancer LNCaP cells with either (+)-EGCG or (+)-GCG accumulated p27 and IkappaB-alpha proteins, associated with an increased G(1) population. (+)-EGCG treatment also accumulated the pro-apoptotic Bax protein and induced apoptosis in LNCaP cells expressing high basal levels of Bax, but not prostate cancer DU-145 cells with low Bax expression. Finally, synthetic GTPs significantly inhibited colony formation by LNCaP cancer cells. Enantiomeric analogs of natural GTPs, (+)-EGCG and (+)-GCG, are able to potently and specifically inhibit the proteasome both, in vitro and in vivo, while protection of the hydroxyl groups on (+)-EGCG renders the compound completely inactive.