Kappa B-alpha Research Articles

NF-κB-independent sensitization of glioblastoma cells for TRAIL-induced apoptosis by proteasome inhibition

The transcription factor nuclear factor-kappaB (NF-kappaB) is a key regulator of stress-induced transcriptional activation and has been implicated in mediating primary or acquired apoptosis resistance in various cancers. In the present study, we therefore investigated the role of NF-kappaB in regulating apoptosis in malignant glioma, a prototypic tumor refractory to current treatment approaches. Here, we report that constitutive NF-kappaB DNA-binding activity was low or moderate in eight different glioblastoma cell lines compared to Hodgkin's lymphoma cells, known to harbor aberrant constitutive NF-kappaB activity. Specific inhibition of NF-kappaB by overexpression of inhibitor of kappaB (IkappaB)alpha superrepressor did not enhance spontaneous apoptosis of glioblastoma cells. Also, overexpression of IkappaBalpha superrepressor had no significant impact on apoptosis induced by two prototypic classes of apoptotic stimuli, that is, chemotherapeutic drugs or death-inducing ligands such as TNF-related apoptosis inducing ligand (TRAIL), which are known to trigger NF-kappaB activation as part of a cellular stress response. Similarly, inhibition of NF-kappaB by the proteasome inhibitor MG132 did not increase doxorubicin (Doxo)-induced apoptosis of glioblastoma cells, although it prevented DNA binding of NF-kappaB complexes in response to Doxo. Interestingly, proteasome inhibition significantly sensitized glioblastoma cells for TRAIL-induced apoptosis. These findings indicate that the characteristic antiapoptotic function of NF-kappaB reported for many cancers is not a primary feature of glioblastoma and thus, specific NF-kappaB inhibition may not be effective for chemosensitization of glioblastoma. Instead, proteasome inhibitors, which enhanced TRAIL-induced apoptosis in an NF-kappaB-independent manner, may open new perspectives to increase the efficacy of TRAIL-based regimens in glioblastoma, which warrants further investigation.

Clathrin heavy chain is required for TNF-induced inflammatory signaling

Tumor necrosis factor receptor I recruits tumor necrosis factor receptor-associated death domain (TRADD) and multiple kinases that ultimately phosphorylate inhibitor kappa B (IKB alpha). Degradation of phospho-IKB alpha (p-IKB alpha) frees nuclear factor kappa B (NFKB) to be active and phosphorylated. Many receptors require clathrin-mediated endocytosis to provide the scaffolds necessary for signaling. Therefore, we investigated the role of clathrin heavy chain (CHC) in tumor necrosis factor alpha (TNF-alpha)-induced IKB alpha phosphorylation and NFKB activation. We hypothesized that CHC was required for TNF-alpha-induced inflammatory signaling. We treated human pulmonary epithelial cells with small interfering RNA to knock down intracellular CHC (CHCsil). TRADD and scrambled (noncoding) small interfering RNA sequences were used as positive and negative controls, respectively. Treatment groups were exposed to 10 ng/mL of TNF-alpha. Total I kappaB alpha, p-I kappaB alpha, and phosphorylated P65 (a subunit of NFKB) were determined by immunoblot staining. Densitometry was normalized to controls for the analysis of the stains. TNF-alpha-induced release of monocyte chemoattractant protein 1 (MCP-1) was determined by enzyme-linked immunosorbent assay. Statistical analyses were determined by analysis of variance or paired t test as appropriate. TNF-alpha-induced I kappaB alpha phosphorylation and degradation at 5 and 30 minutes, respectively, and induced P65 phosphorylation. CHCsil diminished p-I kappaB alpha by 91% (P < .03); however, I kappaB alpha degradation was not affected. CHC knockdown caused a 66% decrease in P65 phosphorylation after 3 minutes of TNF-alpha. CHCsil decreased TNF-alpha-induced MCP-1 by 46% (P < .05), compared with control. CHCsil significantly impairs phosphorylation of both I kappaB alpha and P65. CHCsil also significantly decreased MCP-1 production. These data suggest that CHC is required for certain TNF-alpha-induced, inflammatory signaling pathways.

A role for Akt in the rapid regulation of inflammatory and apoptotic pathways in mouse bladder

Akt is linked to both inflammatory and neoplastic pathways. Akt activation is dependent on the phosphatidylinositol-3 kinase (PI3K) signaling pathways. Upon phosphorylation by PI3K, Akt can phosphorylate nuclear factor kappa B (NF-kappaB) and members of the forkhead family of transcription factors, which includes AFX. Our goal is to examine the effect of Escherichia coli lipopolysaccharide (LPS) on early cellular signaling in inflammatory (NF-kappaB) and apoptotic pathways (AFX) in a mouse-bladder model and in T-24 urothelial cancer cells. Female C57BL/6 mice were given an intraperitoneal (IP) injection of LPS or LPS free water and sacrificed 0-120 minutes later. Bladders were harvested, and immunohistochemistry (IHC) and/or immunoblotting performed using antibodies to PI3K, inhibitor kappa B-alpha (IkappaB-alpha), and total and phosphorylated Akt, NF-kappaB and AFX. Levels of IkappaB-alpha and total and phosphorylated Akt and NF-kappaB were determined in T-24 cells treated with LPS for 0-120 minutes. Bladders and T-24 cells were treated with PI3K inhibitors in some experiments. Protein amounts in different samples were normalized to immunoreactive actin. Phosphorylated and non-phosphorylated species of Akt, NF-kappaB, and AFX were localized to the urothelium. IP LPS injection rapidly (within 30 minutes) increased Akt phosphorylation. IP LPS injection decreased IkappaB-alpha levels, and increased NF-kappaB and AFX phosphorylation. Wortmannin effectively blocked phosphorylation of Akt in LPS-treated mice, and also reduced phosphorylation of AFX and, to a lesser extent, NF-kappaB. After treatment with LPS, Akt and NF-kappaB phosphorylation was rapidly increased in T-24 cells. Akt phosphorylation, and to a lesser extent NF-kappaB phosphorylation, were blocked by LY-294,002. LPS/PI3K/Akt is a cellular signaling pathway which rapidly activates downstream pathways of inflammation and neoplasia in bladder urothelium.

Acute alcohol inhibits the induction of nuclear regulatory factor kappa B activation through CD14/toll-like receptor 4, interleukin-1, and tumor necrosis factor receptors: a common mechanism independent of inhibitory kappa B alpha degradation?

Nuclear translocation and DNA binding of the nuclear factor kappaB (NF-kappaB) is an early event in inflammatory cell activation in response to stimulation with bacterial components or cytokines. Cell activation via different receptors culminates in a common pathway leading to NF-kappaB activation and proinflammatory cytokine induction. We have previously shown that acute alcohol inhibits NF-kappaB activation by lipopolysaccharide (LPS) in human monocytes. Here we investigated whether acute alcohol treatment of human monocytes also inhibits NF-kappaB when induced through activation of the interleukin (IL)-1 or tumor necrosis factor (TNF) receptors. Human peripheral blood monocytes were treated with LPS, TNFalpha, and IL-1beta in the presence or absence of 25mM alcohol for 1 hr. NF-kappaB activation was determined by electrophoretic mobility shift assays using nuclear extracts. Inhibitory kappaB(alpha) (IkappaB(alpha)) was estimated by Western blotting in cytoplasmic extracts. Chinese hamster ovary cells expressing human CD14 were treated with LPS in the presence or absence of alcohol to study NF-kappaB and IkappaB(alpha) regulation. Our results indicate that acute alcohol inhibits IL-1beta- and TNFalpha-induced NF-kappaB activation. We further show in CD14/toll-like receptor 4-expressing Chinese hamster ovary cells the specificity of alcohol-mediated inhibition of NF-kappaB via the toll-like receptor 4/CD14 receptors. Inhibition of NF-kappaB by acute alcohol was concomitant with decreased levels of the IkappaB(alpha) molecule in the cytoplasm of LPS, IL-1, and TNFalpha-activated monocytes. These data suggest a unique, IkappaB(alpha)-independent pathway for the inhibition of NF-kappaB activation by acute alcohol in monocytes. Universal inhibition of NF-kappaB by acute alcohol via these various receptor systems suggests a target for the effects of alcohol in the NF-kappaB activation cascade that is downstream from IkappaB(alpha) degradation. Further, these results demonstrate that acute alcohol is a potent inhibitor of NF-kappaB activation by mediators of early (LPS) or late (IL-1, TNF(alpha)) stages of inflammation in monocytes.

Glucose control with insulin results in reduction of NF‐κB‐binding activity in mononuclear blood cells of patients with recently manifested type 1 diabetes

Chronic elevated blood glucose levels are associated with the formation of advanced glycation end products (AGEs). Hyperglycaemia and AGEs have been shown to induce activation of the redox-sensitive transcription factor nuclear factor-kappaB (NF-kappaB). To validate the hypothesis that the maintenance of normal glucose levels results in the reduction of NF-kappaB-binding activity in vivo, the redox-sensitive transcription factor NF-kappaB was used as marker of hyperglycaemia-induced mononuclear cell activation in patients who recently developed type 1 diabetes. Twelve patients with recently manifested type 1 diabetes mellitus were examined in our study. After sampling blood for determination of baseline glucose values, the 12 patients were treated with insulin, and blood samples were taken 4 and 12 weeks later. Mononuclear cells were isolated and assayed in a tissue culture-independent electrophoretic mobility shift assay (EMSA)-based detection system for NF-kappaB-binding activity. Western blot analysis was used to determine nuclear and cytoplasmic localization of NF-kappaB-p65 and cytoplasmic content of inhibitor of kappa B-alpha (IkappaB-alpha). In addition, we determined the concentration of heme oxygenase-1 (HO-1) from cytoplasmic extract as a marker of oxidative stress. Normalization of blood glucose levels resulted in a highly significant reduction of NF-kappaB activation in EMSA. Before and after glucose normalization, there were no differences in binding by the members of the NF-kappaB family to the NF-kappaB consensus sequence oligonucleotide. Similar data were obtained by Western blot analysis showing NF-kappaB-p65 localization in the nucleus, while p65 levels increased in the cytoplasm. IkappaB-alpha increased in the cytoplasm after glucose normalization. HO-1 antigen consistently decreased, as expected from the decrease in NF-kappaB activation. Thus, we conclude that normalization of blood glucose levels results in the reduction of NF-kappaB activation and gene products controlled by this transcription factor.

THE SIGNAL TRANSDUCTION PATHWAYS INVOLVED IN HEPATIC CYTOCHROME P450 REGULATION IN THE RAT DURING A LIPOPOLYSACCHARIDE-INDUCED MODEL OF CENTRAL NERVOUS SYSTEM INFLAMMATION

It is well known that inflammatory and infectious conditions of the central nervous system (CNS) differentially regulate hepatic drug metabolism through changes in cytochrome P450 (P450); however, the pathways leading to this regulation remain unknown. We provide evidence delineating a signal transduction pathway for hepatic P450 gene expression down-regulation in an established rat model of CNS inflammation using lipopolysaccharide (LPS) injected (i.c.v.) directly into the lateral cerebral ventricle. Brain cytokine levels were elevated, and the expression of tumor necrosis factor alpha and inhibitor of kappaB alpha (IkappaBalpha) were increased in the liver following the i.c.v. administration of LPS, indicating the presence of an inflammatory response in the brain and liver. The expression of CYP2D1/5, CYP2B1/2, and CYP1A1 was down-regulated following CNS inflammation. The binding of several transcription factors [nuclear factor of the kappa enhancer in B cells (NF-kappaB), activator protein-1, cAMP response element binding protein, CCAAT-enhancer binding protein (C/EBP)] to responsive elements on P450 promoter regions was examined using electromobility shift assays. Binding of both NF-kappaB and C/EBP to the promoter regions of CYP2D5 and CYP2B1, respectively, was increased, indicating that they play an important role in the regulation of these two isoforms during inflammatory responses. Evidence is also provided suggesting that the rapid transfer of LPS from the CNS into the periphery likely accounts for the down-regulation of P450s in the liver.

HMG-CoA Reductase Inhibitors Reduce IκB Kinase Activity Induced by Oxidative Stress in Monocytes and Vascular Smooth Muscle Cells

Reactive oxygen species, such as superoxide anion (O2-) and hydrogen peroxide (H2O2), may act as second messengers of intracellular signaling and play a key role in the pathogenesis of atherosclerosis. The nuclear factor kappaB (NF-kappa B) is a redox-sensitive transcription factor that is involved in this process. The aim of the present study was to investigate the molecular mechanisms of action of statins on cultured vascular smooth muscle cells (VSMC) and monocytic cells (THP-1) under oxidative stress. In THP-1 and cultured VSMC, O2- caused an increase in NF-kappa B activation (P < 0.05) that was correlated with inhibitory I kappa B-alpha degradation. Atorvastatin or simvastatin decreased NF-kappa B activation induced by oxidative stress by around 50% in both cell types and was correlated with the I kappa B-alpha levels. In monocytes, O2- increased I kappa B kinase (IKK)-1 and IKK-2 activity (P < 0.05) and p38 and p42/44 activation and phosphorylation, which was reduced by statins. PD 98059 (p42/44 inhibitor) and SB20358 (p38 inhibitor) decreased NF-kappa B binding activity and prevented I kappa B-alpha degradation. However, we only observed a reduction in IKK-1 and IKK-2 activity with PD98059. Statins diminish NF-kappa B activation elicited by oxidative stress through the inhibition of IKK-1/-2, p38, and p42/44 activation. These data may help to further understand the molecular mechanisms of statins in cardiovascular disease.

Constitutive activation of PI3K-Akt and NF-κB during prostate cancer progression in autochthonous transgenic mouse model

Cancer progression is usually facilitated by independent growth signals that may lead to increased cell survival and evasion of apoptosis. Phosphatidylinositol 3'-OH kinase (P I3 K)-Akt and transcription factor NF-kappaB are important signaling molecules and key survival factors involved in the control of cell proliferation, apoptosis, and oncogenesis. Although P I3 K-Akt and NF-kappaB have been implicated in the development and progression of prostate cancer, expression of these molecules during progression of autochthonous disease has not been elucidated. Prostate cancer growth and progression in autochthonous transgenic adenocarcinoma of the mouse prostate (TRAMP) mice and male non-transgenic littermates were observed by magnetic resonance imaging (MRI). Expression patterns of P I3 K-Akt, NF-kappaB, I kappaB, and associated signaling molecules during different stages of cancer progression in these mice were examined by Western blot analysis, electrophoretic mobility shift assay (EMSA), enzyme-linked immunoabsorbent assay (ELISA), kinase assay, and immunohistochemistry. Sequential MRI and gross analysis of prostate gland exhibited increasing prostate volume associated with the development and progression of prostatic adenocarcinoma in TRAMP mice, compared to male non-transgenic littermates. Differential protein expression of P I3 K, phosphorylated-Akt (Ser 473), I kappa Balpha and its phosphorylation, IKK kinase activity, NF-kappaB/p65, p50, DNA binding, and transcriptional-regulated genes, viz., Bc l2, cyclin D1, MMP-9, and VEGF were observed during prostate cancer progression in TRAMP mice, compared to male non-transgenic littermates. Expressions of these molecules were significantly increased during cancer progression observed at 24 and 32 weeks of age. Differential expression pattern of P I3 K-Akt, NF-kappaB and I kappaB during prostate cancer progression in TRAMP mice suggest that these molecules represent potential molecular targets for prevention and/or therapeutic intervention.

Differential modulation of interleukin 8 by interleukin 4 and interleukin 10 in HepG2 cells treated with acetaldehyde

Pro-inflammatory cytokines and chemokines, such as interleukin (IL) 8, are important mediators of hepatic injury and repair following an insult. The purpose of this work was to study the regulation of IL-8 by IL-10 and IL-4 in HepG2 cells treated with acetaldehyde (Ac). HepG2 cells were pretreated with IL-10 or IL-4 before exposure to Ac, examining IL-8 expression by reverse transcription polymerase chain reaction and Western blot. Ac treatment produced an increment in IL-8 induction and secretion that was prevented by IL-4 pretreatment, while IL-10 pretreatment failed to decrease Ac-induced IL-8 production. Consistent with these findings Ac increased NF-kappa B and AP-1 activation that were prevented by IL-4 but not by IL-10, findings accompanied by greater I kappa B-alpha levels in IL-4 but not IL-10 pretreated cells. In contrast to the pro-inflammatory role of IL-10 in HepG2, IL-10 did not show any change in the activation of NF-kappa B by Ac in WRL-68 cells, a human fetal hepatic cell line. Moreover, IL-10 did not induce the degradation of I kappa B-alpha in cellular extract from rat primary cultured cells. While the present findings demonstrate the anti-inflammatory role of IL-4 in preventing the expression of IL-8 by Ac, the regulation of chemokines by anti-inflammatory cytokines is complex and depends on the cellular lineage.

Methanol Extract of &lt;i&gt;Xanthium strumarium&lt;/i&gt; L. Possesses Anti-inflammatory and Anti-nociceptive Activities

As an attempt to identify bioactive natural products with anti-inflammatory activity, we evaluated the effects of the methanol extract of the semen of Xanthium strumarium L. (MEXS) on lipopolysaccharide (LPS)-induced nitric oxide (NO), prostaglandin E2 (PGE2) and tumor necrosis factor-alpha (TNF-alpha) production in RAW 264.7 cells. Our data indicate that MEXS is a potent inhibitor of NO, PGE2 and TNF-alpha production. Consistent with these findings, the expression levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein and iNOS, COX-2 and TNF-alpha mRNA were down-regulated in a concentration-dependent manner. Furthermore, MEXS inhibited nuclear factor kappa B (NF-kappaB) DNA binding activity and the translocation of NF-kappaB to the nucleus by blocking the degradation of inhibitor of kappa B-alpha (IkappaB-alpha). We further evaluated the anti-inflammatory and anti-nociceptive activities of MEXS in vivo. MEXS (100, 200 mg/kg/d, p.o.) reduced acute paw edema induced by carrageenin in rats, and showed analgesic activities in an acetic acid-induced abdominal constriction test and a hot plate test in mice. Thus, our study suggests that the inhibitions of iNOS, COX-2 expression, and TNF-alpha release by the methanol extract of the semen of Xanthium strumarium L. are achieved by blocking NF-kappaB activation, and that this is also responsible for its anti-inflammatory effects.

All-Trans-Retinoic Acid Induces Interleukin-8 via the Nuclear Factor-κB and p38 Mitogen-Activated Protein Kinase Pathways in Normal Human Keratinocytes

Retinoic acid derivatives have been used successfully for the treatment of various dermatoses, such as psoriasis; however, topical application of these compounds often elicits skin irritation. We hypothesized that this irritation was as a result of the local production of interleukin-8 (IL-8). To test this hypothesis, we investigated whether all-trans-retinoic acid (ATRA) induced IL-8 production in normal human keratinocytes. Stimulation with 10(-7) M ATRA enhanced IL-8 mRNA expression and induced IL-8 production. We also studied the intracellular signaling mechanisms of ATRA-induced IL-8 production in keratinocytes. ATRA increased the expression of RelA (p65), RelB, nuclear factor (NF)-kappaB2 (p52), and NF-kappaB1 (p50), and elevated the DNA-binding activity of p65 and phosphorylation of inhibitor kappaB (IkappaB) alpha. Introduction of a dominant-negative mutant of IkappaBalpha completely abolished ATRA-induced IL-8 production, which indicates that this process is NF-kappaB-dependent. We also studied the role of the p38 mitogen-activated protein kinase (MAPK) pathway in this phenomenon. ATRA phosphorylated the p38 MAPK, and SB202180 inhibited ATRA-induced IL-8 production, which indicates that the p38 MAPK is also involved in ATRA-induced IL-8 production. In summary, ATRA induces IL-8 production in both NF-kappaB- and p38 MAPK-dependent manners in normal human keratinocytes.

EFFECTS OF ACETALDEHYDE AND TNFα ON THE INHIBITORY KAPPA B- α PROTEIN AND NUCLEAR FACTOR KAPPA B ACTIVATION IN HEPATIC STELLATE CELLS

Increased plasma tumour necrosis alpha (TNFalpha) and elevated monocyte nuclear factor kappa B (NF-kappaB) are associated with liver injury and inflammation in models of alcoholic liver disease and are found to be elevated in monocytes of patients with alcoholic hepatitis. Acetaldehyde enhances, whereas TNFalpha inhibits, transcription of the type I collagen promoters and type I collagen production. NF-kappaB, an inhibitor of the type I collagen promoters, is increased by both acetaldehyde and TNFalpha. This study determined the effects of acetaldehyde in comparison to the effects of TNFalpha on inhibitory kappa B-alpha (IkappaB-alpha) protein and NF-kappaB activation in hepatic stellate cells. Activated rat hepatic stellate cells in culture were exposed to acetaldehyde or TNFalpha for short periods of time, following which the cells were harvested for the determination of IkappaB-alpha protein, IkappaB-alpha kinase activity and nuclear NF-kappaB. Acetaldehyde increased IkappaB-alpha kinase activity and decreased IkappaB-alpha after 10 min of exposure, with recovery towards control levels at 20 min. In contrast, TNFalpha resulted in higher IkappaB-alpha kinase activity at 20 min than at 10 min, and similar low IkappaB-alpha at 10 and 20 min. Both acetaldehyde and TNFalpha enhanced nuclear NF-kappaB (p65), but acetaldehyde alone also increased NF-kappaB (p50). TNFalpha and acetaldehyde independently activate NF-kappaB by rapid enhancement of IkappaB-alpha kinase activity and degradation of IkB-alpha protein. Increased TNFalpha is the principal mechanism for the elevation of NF-kappaB in severe alcoholic hepatitis. The elevation of NF-kappaB due to TNFalpha enhance liver injury, but inhibit fibrogenesis. In contrast, the effect of acetaldehyde in activating NF-kappaB is associated with increases in both liver injury and fibrogenesis, indicating that the effects of acetaldehyde on fibrogenesis are mediated by cytokines and by trans-acting factors other than NF-kappaB.

Mechanism of concentration-dependent induction of heme oxygenase-1 by resveratrol in human aortic smooth muscle cells

Resveratrol-mediated heme oxygenase-1 (HO-1) induction has been shown to occur in primary neuronal cultures and is thought to have potential neuroprotective action. Further, antioxidant properties of resveratrol have been reported to protect against coronary heart disease. We attempted to examine resveratrol's HO-1 inducing potency and its induction regulation in human aortic smooth muscle cells (HASMC). We showed that resveratrol-mediated HO-1 induction occurred in concentration- and time-dependent manners, but only at low concentrations (1–10 μM), and that it was modulated at both the transcription and translation levels. Additionally, the results of our study showed that nuclear factor-kappa B (NF-κB) inhibitors eliminated resveratrol-mediated HO-1 induction and promoter activity, and that deletion of NF-κB binding sites in the HO-1 promoter region strongly reduced promoter activity, suggesting involvement of the NF-κB pathway in HO-1 induction by resveratrol. Suppression of NF-κB activity by resveratrol at high concentrations (≥20 μM) has been reported to be attributed to its anti-inflammatory and anti-oxidative properties. Likewise, we showed that resveratrol at concentrations of ≥20 μM blocked the activity of NF-κB through suppression of I kappa-B alpha (IκBα) phosphorylation, which caused inhibition of HO-1 induction. Conversely, resveratrol in a range of 1–10 μM enhanced the phosphorylation and degradation of IκBα, a key step in NF-κB activation, resulting in HO-1 induction. Collectively, we suggest that resveratrol-mediated HO-1 expression occurs, at least in part, through the NF-κB pathway, which might contribute to resveratrol's vascular-protective effect at physiological concentrations after moderate red wine consumption.

Adenovirus-mediated overexpression of inhibitor kappa B-alpha attenuates postinfarct remodeling in the rat heart?

The transcription factor nuclear factor kappa B (NF-kB) plays an important role in the inflammatory response following myocardial infarction. We hypothesized that NF-kB-blockade in an animal model of acute ischemia reduces the inflammatory response and therefore attenuates ventricular remodeling. Myocardial infarcts (MI) were produced in male Sprague-Dawley rats by ligation of the LAD and followed by adenovirus-mediated intramyocardial delivery of inhibitor kappa Balpha-gene (n=10), the physiological inhibitor of the transcription factor nuclear factor kappa B, respectively, of a beta-gal reporter-gene (n=11). Sham-operated animals (n=10) received neither ligation nor gene transfer. Five days after MI IkB-expression levels were determined by western blotting. Seven weeks after MI in vivo cardiac function was evaluated by transthoracic echocardiography. Based on left ventricular endsystolic and enddiastolic diameters ejection fraction and fractional shortening were calculated. Only animals with MI involving more than 30% of the left ventricle were included. Data are given as mean+/-SD. In IkBalpha-transfected hearts IkBalpha-levels were six-fold higher (P<0.05) than in beta-gal transfected hearts. Concerning in vivo hemodynamics IkBalpha-treated hearts showed reduced systolic and diastolic left ventricular dimensions compared to the beta-gal MI-group (systolic 48+/-4 vs. 66+/-3 mm; diastolic 67+/-5 vs. 84+/-6 mm; P<0.01). Consequently fractional shortening (27.8+/-1.5 vs. 20.4+/-4.0%; P<0.01) and ejection fraction (63.4+/-3.6 vs. 49.1+/-8.3%; P<0.05) were preserved in IkBalpha hearts compared to beta-gal MI-hearts. It can be concluded that overexpression of IkBalpha leads to an improved cardiac function thereby attenuating postinfarct remodeling.

Transcriptional response to circumscribed cortical brain ischemia: spatiotemporal patterns in ischemic vs. remote non‐ischemic cortex

Focal brain infarcts are surrounded by extended perilesional zones that comprise the partially ischemic penumbra but also completely non-ischemic cortex of the remote ipsilateral hemisphere. To delineate the impact of lesion-associated vs. remote processes on transcriptional programming after focal ischemia, we used cDNA array analysis, quantitative real-time polymerase chain reaction and immunohistochemistry in the photothrombosis model of circumscribed cortical ischemia in rats. At an early stage of 4 h after ischemia, gene induction occurred to a similar extent in the ischemic infarct and remote non-ischemic cortex of the ipsilateral hemisphere. Among the genes induced in non-ischemic cortex we found the NGF-inducible genes PC3, VGF and Arc, the transcriptional regulators I kappa B-alpha and Stat3, and the beta-chemokine MIP-1 alpha (CCL3). At 3 days, the spatial pattern of gene expression had changed dramatically with brain fatty acid-binding protein as the only gene significantly induced in non-ischemic ipsilateral cortex. In contrast, numerous genes were exclusively regulated at the lesion site, comprising genes involved in cell cycle regulation, proteolysis, apoptosis, lipid homeostasis and anti-inflammatory counter-regulation. Cortical spreading depression was identified as the main mechanism underlying gene induction in remote non-ischemic cortex. Our data demonstrate a dynamic spatiotemporal pattern of gene induction, which may contribute to delayed progression of damage or, alternatively, mediate neuroprotection, tissue remodeling and functional compensation.

Reduction of nuclear factor kappa b activity post myocardial infarction improves ventricular remodeling by attenuating the inflammatory response

Objective: The transcription factor nuclear factor kappa B (NF-kB) plays an important role in the inflammatory response following myocardial infarction (MI). We hypothesized that NF-kB- blockade by gene-therapeutic overexpression of its inhibitor Inhibitor KappaB alpha (IkB) in an animal model of acute ischemia reduces the inflammatory response and therefore reduces ventricular remodeling.

Inhibitory effect of Spirodela polyrhixa on the secretion of NO in LPS-stimulated macrophages.

Spirodela polyrhixa Schleid has been used in folk medicine to treat inflammatory diseases. Since nitric oxide (NO) is one of the major inflammatory parameters, we studied the effect of aqueous extracts of Spirodela polyrhixa (AESP) on NO production in lipopolysaccharide (LPS)-stimulated mouse peritoneal macrophages. AESP inhibited the secretion of NO in macrophages, without affecting cell viability. The protein level of inducible nitric oxide synthase (iNOS) in peritoneal macrophages was also decreased by AESP. Transient transfection assay of reporter plasmid and gel shift assay indicated that AESP blocked the activation of nuclear factor-kappa B (NF-kappa B), which was considered to be a potential transcription factor for iNOS expression. AESP also blocked the phosphorylation and degradation of inhibitory protein I kappa B-alpha (I kappa B-alpha). These results suggest that AESP could exert its anti-inflammatory actions by suppressing the synthesis of NO through inhibition of NF-kappa B activity.

Shear Stress Regulates Endothelial Nitric-oxide Synthase Promoter Activity through Nuclear Factor κB Binding

We have previously demonstrated that shear stress increases transcription of the endothelial nitric-oxide synthase (eNOS) by a pathway involving activation of the tyrosine kinase c-Src and extracellular signal-related kinase 1/2 (ERK1/2). In the present study sought to determine the events downstream of this pathway. Shear stress activated a human eNOS promoter chloramphenicol acetyl-CoA transferase chimeric construct in a time-dependent fashion, and this could be prevented by inhibition of the c-Src and MEK1/2. Studies using electromobility shift assays, promoter deletions, and promoter mutations revealed that shear activation of the eNOS promoter was due to binding of nuclear factor kappaB subunits p50 and p65 to a GAGACC sequence -990 to -984 base pairs upstream of the eNOS transcription start site. Shear induced nuclear translocation of p50 and p65, and activation of the eNOS promoter by shear could be prevented by co-transfection with a dominant negative I kappa Balpha. Exposure of endothelial cells to shear resulted in Ikappa kinase phosphorylation, and this was blocked by the MEK1/2 inhibitor PD98059 and the cSrc inhibitor PP1, suggesting these signaling molecules are upstream of NFkappaB activation. These experiments indicate that shear stress increases eNOS transcription by NFkappaB activation and p50/p65 binding to a GAGACC sequence present of the human eNOS promoter. While NFkappaB activation is generally viewed as a proinflammatory stimulus, the current data indicate that its transient activation by shear may increase expression of eNOS, which via production of nitric oxide could convey anti-inflammatory and anti-atherosclerotic properties.

An in vitro screening assay for inhibitors of proinflammatory mediators in herbal extracts using human synoviocyte cultures.

Tumor necrosis factor-alpha (TNF-alpha), cyclooxygenase (COX)-2, and prostaglandin (PG)E-2 play a critical role in the pathophysiology of arthritis. Tumor necrosis factor-alpha mediates induction of other cytokines, COX-2, PGs, and metalloproteinases, which leads to cartilage degradation. We developed an in vitro human synoviocyte assay system for screening inhibitors of proinflammatory mediators in herbal extracts. Synoviocytes (5 x 10(5) cells/well) obtained during primary knee replacement from osteoarthritic patients were incubated with: control media alone or ginger extract (hydroxy-methoxy-phenyl compounds [HAPC]: EV.EXT 77), 1 h before activation with 1 ng/ml TNF-alpha, 10 ng/ml interleukin-1beta, or control media alone at 5% carbon dioxide, 37 degrees C. Cell viability, TNF-alpha, COX-2, PGE-2, nuclear factor kappaB (NF-kappaB), and inhibitory subunit I kappa B-alpha (IkappaB-alpha) expression were analyzed by reverse transcriptase-polymerase chain reaction, enzyme-linked immunosorbent assay, electrophoretic mobility shift assay, and Western blots. Ginger extract-HAPC (100 microg/ml) significantly inhibited the activation of TNF-alpha and COX-2 expression in human synoviocytes as well as suppressed production of TNF-alpha and PGE-2. Inhibition of TNF-alpha and COX-2 activation was accompanied by suppression of NF-kappaB and IkappaB-alpha induction. Using our in vitro assay, we discovered that the ginger extract blocks activation of proinflammatory mediators and its transcriptional regulator suggesting its mode of action. These observations indicate that ginger extract-HAPC offers a complementary and alternative approach to modulate the inflammatory process involved in arthritis.

CXCL16 Signals via Gi, Phosphatidylinositol 3-Kinase, Akt, IκB Kinase, and Nuclear Factor-κB and Induces Cell-Cell Adhesion and Aortic Smooth Muscle Cell Proliferation

CXCL16, a recently discovered transmembrane chemokine, is expressed in human aortic smooth muscle cell (ASMC). It facilitates uptake of low density lipoproteins by macrophages, resulting in foam cell formation. However, it is not known whether ASMC express CXCR6, the receptor for CXCL16, or whether CXCL16 affects ASMC biology. To dissect the biological and signal transduction pathways elicited by CXCL16, human aortic smooth muscle cells (HASMC) were treated with pharmacological inhibitors or transiently transfected with pathway-specific dominant-negative or kinase-dead expression vectors prior to the addition of CXCL16. HASMC expressed CXCR6 at basal conditions. Exposure of HASMC to CXCL16 increased NF-kappa B DNA binding activity, induced kappa B-driven luciferase activity, and up-regulated tumor necrosis factor-alpha expression in an NF-kappa B-dependent manner. However, treatment with pertussis toxin (G(i) inhibitor), wortmannin or LY294002 (phosphatidylinositol 3-kinase (PI3K inhibitors)), or Akt inhibitor or overexpression of dominant-negative (dn) PI3K gamma, dnPDK-1, kinase-dead (kd) Akt, kdIKK-beta, dnIKK-gamma, dnI kappa B-alpha, or dnI kappa B-beta significantly attenuated CXCL16-induced NF-kappa B activation. Furthermore, CXCL16 increased cell-cell adhesion and induced cellular proliferation in an NF-kappa B-dependent manner. In conclusion, CXCL16 is a potent and direct activator of NF-kappaB and induces kappa B-dependent proinflammatory gene transcription. CXCL16-mediated NF-kappa B activation occurred via heterotrimeric G proteins, PI3K, PDK-1, Akt, and I kappa B kinase (IKK). CXCL16 induced I kappa B phosphorylation and degradation. Most importantly, CXCL16 increased cell-cell adhesion and induced kappa B-dependent ASMC proliferation, indicating that CXCL16 may play an important role in the development and progression of atherosclerotic vascular disease.