Mad2 Research Articles

Investigation of inflammatory responses of pulmonary microvascular endothelial cells induced by lipopolysaccharide and mechanism

Lipopolysaccharide (LPS) can activate pulmonary vascular endothelial cells (PMVECs) and induce inflammatory injury. Toll-like receptor-4 (TLR-4) is integrally involved in LPS signaling and has a requisite role in the activation of NF-κB. NF-κB is a key intercellular signaling event that mediates cell inflammatory responses. The aim of the study was to investigate in an in vitro model the inflammatory responses of PMVECs induced by LPS and the probable mechanism underlying the observed inflammatory responses. The present study was performed on isolated PMVECs from Sprague-Dawley rats. After being identified, PMVECs were divided into 2 groups: a control group, and a LPS (0.01, 0.1, 1, 10 mg/L) intervention group. ICAM-1, TNF-α and IL-8 were detected by ELISA or radioimmunological methods. The expression of TLR-4 mRNA was detected by real time PCR. The activation of NF-κB was detected by Western blot (proteins of I-κBα and NF-κB p65) and immunocytochemical staining (NF-κB p65). Compared with the control group, cytokines secreted from PMVECs-stimulated by LPS were increased in a dose-dependent manner. When stimulated with LPS 10 mg/L for 2, 6 and 12 h, cytokines measured were all increased. ICAM-1 and TNF-α were significantly increased and peaked after 2 h before gradually declining at 6 and 12 h. IL-8 was higher after 2 h, which continued up to 12 h. The expression of TLR-4 mRNA was significantly higher and peaked after 2 h and continued to 12 h (4.34 ± 1.42, 3.62 ± 1.45, 3.32 ± 1.36), which were all higher than that of the control group (1.00 ± 0.00, P < 0.05). Meanwhile, NF-κB was activated at 0.5, 2, 6 and 12 h indicated by the significant degradation of IκB-α and the significant increased release of NF-κB P65 and its subsequent translocation into the nucleus with approximately synchronized. Taken together, the results demonstrated that LPS was able to induce PMVECs inflammatory injury via activating TLR-4 and subsequently activating NF-κB.

Abstract 1396: Bradykinin enhances cell migration in human prostate carcinoma cells via bradykinin B2 receptor, PKCdelta, c-Src and NF-kappaB signaling pathways

Abstract Bradykinin (BK) is an inflammatory mediator, and shows elevated levels in regions of severe injury and inflammatory diseases. In addition, bradykinin has been found in many tumor cell lines and has been shown to be involved in carcinogenesis and cancer progression. Prostate cancer is the most common cancer excluding skin cancer in men and usually metastasizes to the particular organs in the late stages of cancer processes. The matrix metalloproteinase (MMP) play an important role in tissue remodeling associated with various physiological and pathological processes such as morphogenesis, angiogenesis, tissue repair, cirrhosis, arthritis and metastasis. In this study, we found that bradykinin increased the migration and the expression of MMP-9 in human prostate cancer cells (PC3,DU145 and LNCaP). Bradykinin-mediated migration and matrix MMP-9 up-regulation was attenuated by bradykinin B2 receptor antagonist (HOE140), protein kinase Cdelta (PKCdelta) inhibitor (Rottlerin), c-Src inhibitor (PP2), NF-kappaB inhibitor (PDTC), and IkappaB protease inhibitor (TPCK). Transfection of cells with bradykinin B2 receptor and protein kinase Cdelta (PKCdelta), siRNA or dominant-negative mutant of c-Src, IkappaBalpha kinase alpha(IKK alpha) and IkappaBalpha kinase beta(IKK beta), also inhibited the potentiating action of bradykinin. Stimulation of PC3 cells with bradykinin induced PKCdelta, c-Src, IKK alpha/beta, IkappaB and p65 phosphorylation, and kappaB-luciferase activity. In addition, BKR2 PCKdelta and c-Src pathways are involved in bradykinin-induced NFkappaB activation. Taken together, our results suggest that bradykinin enhances the migration of prostate carcinoma cells by increasing MMP9 expression through the bradykinin B2 receptor /PKCdelta/c-Src/NF-kappaB signal transduction pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1396. doi:10.1158/1538-7445.AM2011-1396

The Valais units in Savoy (France): a key area for understanding the palaeogeography and the tectonic evolution of the Western Alps

The Valais units in Savoy (Zone des Brèches de Tarentaise) have been re-mapped in great detail and are subject of combined stratigraphic, structural and petrological investigations summarized in this contribution. The sediments and rare relics of basement, together with Cretaceous age mafic and ultramafic rocks of the Valais palaeogeographical domain, represent the heavily deformed relics of the former distal European margin (External Valais units) and an ocean–continent transition (Internal Valais unit or Versoyen unit) that formed during rifting. This rifting led to the opening of the Valais ocean, a northern branch of the Alpine Tethys. Post-rift sediments referred to as “Valais trilogy” stratigraphically overlie both External and Internal Valais successions above an angular unconformity formed in Barremian to Aptian times, providing robust evidence for the timing of the opening of the Valais ocean. The Valais units in Savoy are part of a second and more external mid-Eocene high-pressure belt in the Alps that sutured the Briançonnais microcontinent to Europe. Top-N D1-deformation led to the formation of a nappe stack that emplaced the largely eclogite-facies Internal Valais unit (Versoyen) onto blueschist-facies External Valais units. The latter originally consisted of, from internal to external, the Petit St. Bernard unit, the Roc de l’Enfer unit, the Moûtiers unit and the Quermoz unit. Ongoing top-N D2-thrusting and folding substantially modified this nappe stack. Post 35 Ma D3 folding led to relatively minor modifications of the nappe stack within the Valais units but was associated with substantial top-WNW thrusting of the Valais units over the Dauphinois units along the Roselend thrust during W-directed indentation of the Adria block contributing to the formation of the arc of the Western Alps.

Protein–protein interactions involving IKKγ (NEMO) that promote the activation of NF‐κB

Inhibitor of kappaB kinase (IKK) gamma (IKKgamma), also referred to as nuclear factor kappaB (NF-kappaB) essential modulator (NEMO), is an important regulatory component of the IKK complex. The IKK complex is a signalosome that catalyzes the inducible phosphorylation of IkappaB proteins, which is a key step that leads to the activation of NF-kappaB. The exact functions of IKKgamma (NEMO) as part of the IKK complex have not yet been fully elucidated. This mini-review covers 16 proteins that have been reported to bind to IKKgamma and lead to the enhancement of the activities of the IKK complex, thus resulting in NF-kappaB activation. The major mechanisms by which these interactions are mediated involve the recognition of ubiquitinated upstream signaling components by IKKgamma or the modification of IKKgamma itself by ubiquitination. Additional mechanisms include the sumoylation or phosphorylation of IKKgamma and the modification of the tertiary or quaternary structure of IKKgamma.

IKKα and IKKβ Each Function to Regulate NF-κB Activation in the TNF-Induced/Canonical Pathway

BackgroundActivation of the transcription factor NF-κB by cytokines is rapid, mediated through the activation of the IKK complex with subsequent phosphorylation and degradation of the inhibitory IκB proteins. The IKK complex is comprised of two catalytic subunits, IKKα and IKKβ, and a regulatory protein known as NEMO. Using cells from mice that are genetically deficient in IKKβ or IKKα, or using a kinase inactive mutant of IKKβ, it has been proposed that IKKβ is critical for TNF-induced IκB phosphorylation/degradation through the canonical pathway while IKKα has been shown to be involved in the non-canonical pathway for NF-κB activation. These conclusions have led to a focus on development of IKKβ inhibitors for potential use in inflammatory disorders and cancer.MethodologyAnalysis of NF-κB activation in response to TNF in MEFs reveals that IKKβ is essential for efficient phosphorylation and subsequent degradation of IκBα, yet IKKα contributes to the NF-κB activation response in these cells as measured via DNA binding assays. In HeLa cells, both IKKα and IKKβ contribute to IκBα phosphorylation and NF-κB activation. A kinase inactive mutant of IKKβ, which has been used as evidence for the critical importance of IKKβ in TNF-induced signaling, blocks activation of NF-κB induced by IKKα, even in cells that are deficient in IKKβ.ConclusionsThese results demonstrate the importance of IKKα in canonical NF-κB activation, downstream of cytokine treatment of cells. The experiments suggest that IKKα will be a therapeutic target in inflammatory disorders.

DEXAMETHASONE INHIBITS THE INDUCTION OF iNOS GENE EXPRESSION THROUGH DESTABILIZATION OF ITS mRNA IN PROINFLAMMATORY CYTOKINE-STIMULATED HEPATOCYTES

In the inflamed liver, proinflammatory cytokines including TNF-alpha, IL-1beta, and IFN-gamma stimulate the induction of iNOS gene expression, leading to excess production of NO and resulting in liver injury. The induction of iNOS is regulated by transactivation of the iNOS promoter with transcription factors such as nuclear factor kappaB and by posttranscriptional modifications such as mRNA stabilization. The synthetic glucocorticoid dexamethasone has been reported to inhibit iNOS induction, which may contribute to its inflammation-reducing effects. The objective was to investigate the mechanisms involved in the down-regulation of iNOS gene expression by dexamethasone. Primary cultured rat hepatocytes were treated with IL-1beta (1 nM) in the presence or absence of dexamethasone. The induction of iNOS and its signal were analyzed. Dexamethasone (10-250 nM) inhibited the expression of iNOS mRNA and protein dose and time dependently, resulting in decreases in NO production. However, dexamethasone did not inhibit the up-regulation of type I IL-1 receptor stimulated by IL-1beta. Dexamethasone also had no effect on the degradation of IkappaB proteins and on the activation of nuclear factor kappaB. Transfection experiments with iNOS promoter-luciferase constructs revealed that dexamethasone had no effect on the transactivation of the iNOS promoter but decreased the stabilization of iNOS mRNA. In support of the latter observation, dexamethasone inhibited the expression of an iNOS gene antisense transcript, which stabilizes iNOS mRNA by interacting with its 3'-untranslated region and 3'-untranslated region-binding proteins. Dexamethasone may inhibit the induction of iNOS gene expression at the step of mRNA stabilization rather than promoter activation and may provide useful therapeutic effects in iNOS induction involved in liver injuries.

Piceatannol Inhibits Phorbol Ester-Induced NF-κ B Activation and COX-2 Expression in Cultured Human Mammary Epithelial Cells

There are multiple lines of evidence supporting that inflammation is causally linked to carcinogenesis. Abnormal upregulation of cyclooxygenase-2 (COX-2), a rate-limiting enzyme in the prostaglandin biosynthesis, has been implicated in carcinogenesis. Trans-3,4,3′,5′-tetrahydroxystilbene (piceatannol), a naturally occurring hydroxylated stilbene with potent anti-inflammatory and antioxidative activities, has been shown to inhibit the proliferation of several cancer cells by inducing apoptosis or blocking cell cycle progression. In this study, we examined the effect of piceatannol on activation of the nuclear transcription factor NF-κ B, one of the major transcription factors that regulate proinflammatory COX-2 gene transcription, in human mammary epithelial (MCF-10A) cells treated with the tumor promoter 12- O -tetradecanoylphorbol-13-acetate (TPA). When pretreated to MCF-10A cells, piceatannol markedly inhibited TPA-induced NF-κ B DNA binding to a greater extent than resveratrol and oxyresveratrol, stilbene analogs structurally related to piceatannol. Piceatannol also inhibited TPA-induced phosphorylation and degradation of Iκ Bα as well as nuclear translocation of the phosphorylated form of p65, the functionally active subunit of NF-κ B. Likewise, TPA-induced expression of COX-2 was abrogated by piceatannol pretreatment. The thiol reducing agent dithiothreitol abolished the inhibitory effects of piceatannol on NF-κ B DNA binding activity, suggesting that piceatannol may directly modify NF-κ B or its regulator through reaction with the cysteine thiol(s).

Metformin inhibits nuclear factor κB activation and decreases serum high-sensitivity C-reactive protein level in experimental atherogenesis of rabbits

Previous studies demonstrated that metformin has obvious antiatherogenic properties, but the exact mechanism remains unclear. Therefore, we established an atherosclerotic rabbit model in order to investigate the potential effects of metformin on transcription factor nuclear factor kappaB (NF-kappaB) and serum high-sensitivity C-reactive protein (hs-CRP) level, which had been regarded as proatherogenic factors. New Zealand rabbits were randomly divided into three groups: a control group (n = 8), an atherosclerotic group (AS group, n = 8), and a metformin treatment group (Met group, n = 8). The experimental atherosclerotic rabbit model was successfully established at the end of the 8th week. From the 9th week, rabbits in the Met group were administered with 150 mg/kg metformin daily by gavage. Blood samples were collected at days 0 and 8, and at 16 weeks to detect the level of blood lipid and serum glucose. At the end of the experiment, blood samples were withdrawn for determining serum hs-CRP. Aortic samples were harvested for histomorphometric analysis. Immunohistochemistry and Western blotting were used to detect the expression of NF-kappaB subunit p65 in nuclear extracts and phosphorylation of inhibitor of nuclear factor kappaB (IkappaB) in cytoplasmic extracts. An experimental atherosclerotic rabbit model was successfully established. The expression of nuclear NF-kappaB subunit p65 and cytoplasmic phosphorylation of IkappaB protein in the vessel wall was enhanced (P < 0.01, respectively) in the AS group, and serum hs-CRP level was significantly increased in the AS group compared with the control group (3.90 +/- 0.25 mg/l versus 1.36 +/- 0.14 mg/l, P < 0.01). Treatment with metformin significantly attenuated the progression of aortic atherosclerosis. In the Met group, there was a marked reduction in nuclear NF-kappaB subunit p65 and cytoplasmic phosphorylation of IkappaB protein expression (P < 0.01). Serum hs-CRP concentration was also significantly decreased (3.20 +/- 0.20 mg/l versus 3.90 +/- 0.25 mg/l, P < 0.05). Metformin inhibits the phosphorylation of IkappaB and the activation of NF-kappaB in the vessel wall of experimental atherogenesis of rabbits, as well as decreasing the serum level of hs-CRP, thus suggesting that metformin has vascular anti-inflammatory properties, which may be one of its antiatherogenic mechanisms.

Basal Cancer Cell Survival Involves JNK2 Suppression of a Novel JNK1/c-Jun/Bcl-3 Apoptotic Network

BackgroundThe regulation of apoptosis under basal (non-stress) conditions is crucial for normal mammalian development and also for normal cellular turnover in different tissues throughout life. Deficient regulation of basal apoptosis, or its perturbation, can result in impaired development and/or disease states including cancer. In contrast to stress-induced apoptosis the regulation of apoptosis under basal conditions is poorly understood. To address this issue we have compared basal- and stress-induced apoptosis in human epithelial cells of normal and cancerous origins. For this purpose we focussed our study on the opposing pro-apoptotic JNK/anti-apoptotic NFκB pathways.Methodology/Principal FindingsCombinatorial RNAi plus gene knockout were employed to access and map basal regulatory pathways of apoptosis. Follow-on, in-depth analyses included exogenous expression of phosphorylation mutants and chromatin immunoprecipitation. We demonstrate that basal apoptosis is constitutively suppressed by JNK2 in a range of human cancer cell lines. This effect was not observed in non-cancer cells. Silencing JNK2 by RNAi resulted in JNK1-dependent apoptosis of cancer cells via up-regulation of the AP-1 factor c-Jun. Unexpectedly we discovered that JNK1 and c-Jun promote basal apoptosis in the absence of “activating phosphorylations” typically induced by stress. Hypo-phosphorylated c-Jun accumulated to high levels following JNK2 silencing, auto-regulated its own expression and suppressed expression of Bcl-3, an unusual IκB protein and regulator of NFκB. Basal apoptosis was mediated by components of the TNFα response pathway but was mechanistically distinct from TNFα-induced apoptosis.Conclusions/SignificanceOur results demonstrate that mechanistically distinct pathways operate to regulate apoptosis in mammalian cells under basal (physiological) versus stress-induced conditions. We also describe a novel apoptotic network which governs the basal survival of cancer cells. Such information is crucial for understanding normal cellular turnover during mammalian development and subsequently throughout life. This information also opens new avenues for therapeutic intervention in human proliferative disease states including cancer.

NF-κB p100 limits TNF-induced bone resorption in mice by a TRAF3-dependent mechanism

TNF and RANKL mediate bone destruction in common bone diseases, including osteoarthritis and RA. They activate NF-kappaB canonical signaling directly in osteoclast precursors (OCPs) to induce osteoclast formation in vitro. However, unlike RANKL, TNF does not activate the alternative NF-kappaB pathway efficiently to process the IkappaB protein NF-kappaB p100 to NF-kappaB p52, nor does it appear to induce osteoclast formation in vivo in the absence of RANKL. Here, we show that TNF limits RANKL- and TNF-induced osteoclast formation in vitro and in vivo by increasing NF-kappaB p100 protein accumulation in OCPs. In contrast, TNF induced robust osteoclast formation in vivo in mice lacking RANKL or RANK when the mice also lacked NF-kappaB p100, and TNF-Tg mice lacking NF-kappaB p100 had more severe joint erosion and inflammation than did TNF-Tg littermates. TNF, but not RANKL, increased OCP expression of TNF receptor-associated factor 3 (TRAF3), an adapter protein that regulates NF-kappaB p100 levels in B cells. TRAF3 siRNA prevented TNF-induced NF-kappaB p100 accumulation and inhibition of osteoclastogenesis. These findings suggest that upregulation of TRAF3 or NF-kappaB p100 expression or inhibition of NF-kappaB p100 degradation in OCPs could limit bone destruction and inflammation-induced bone loss in common bone diseases.

The NF- B Family of Transcription Factors and Its Regulation

Nuclear factor-kappaB (NF-kappaB) consists of a family of transcription factors that play critical roles in inflammation, immunity, cell proliferation, differentiation, and survival. Inducible NF-kappaB activation depends on phosphorylation-induced proteosomal degradation of the inhibitor of NF-kappaB proteins (IkappaBs), which retain inactive NF-kappaB dimers in the cytosol in unstimulated cells. The majority of the diverse signaling pathways that lead to NF-kappaB activation converge on the IkappaB kinase (IKK) complex, which is responsible for IkappaB phosphorylation and is essential for signal transduction to NF-kappaB. Additional regulation of NF-kappaB activity is achieved through various post-translational modifications of the core components of the NF-kappaB signaling pathways. In addition to cytosolic modifications of IKK and IkappaB proteins, as well as other pathway-specific mediators, the transcription factors are themselves extensively modified. Tremendous progress has been made over the last two decades in unraveling the elaborate regulatory networks that control the NF-kappaB response. This has made the NF-kappaB pathway a paradigm for understanding general principles of signal transduction and gene regulation.

Redox Regulation of Nuclear Post-Translational Modifications During NF-κB Activation

The transcription factor NF-kappaB controls the expression of hundreds of genes involved in the regulation of the immune/inflammatory response, development, and apoptosis. In resting cells, NF-kappaB proteins are sequestered in the cytoplasm through their tight association with IkappaB proteins. NF-kappaB activation relies on the signal-induced IkappaB phosphorylation and degradation, thereby allowing the nuclear translocation of NF-kappaB proteins. In the nucleus, several post-translational modifications of NF-kappaB and chromatin remodeling of target genes are mandatory for NF-kappaB DNA binding and full transcription. Since 1991, reactive oxygen species (ROS) have been implicated in NF-kappaB activation. ROS enhance the cytoplasmic signaling pathways leading to NF-kappaB nuclear translocation, but reduction/oxidation (redox) also controls several key steps in the nuclear phase of the NF-kappaB program, including chromatin remodeling, recruitment of co-activators, and DNA binding. Here we describe the redox regulation of NF-kappaB activity in the nucleus.

A Structural Guide to Proteins of the NF- B Signaling Module

The prosurvival transcription factor NF-kappaB specifically binds promoter DNA to activate target gene expression. NF-kappaB is regulated through interactions with IkappaB inhibitor proteins. Active proteolysis of these IkappaB proteins is, in turn, under the control of the IkappaB kinase complex (IKK). Together, these three molecules form the NF-kappaB signaling module. Studies aimed at characterizing the molecular mechanisms of NF-kappaB, IkappaB, and IKK in terms of their three-dimensional structures have lead to a greater understanding of this vital transcription factor system.

Akt‐mediated regulation of NFκB and the essentialness of NFκB for the oncogenicity of PI3K and Akt

The serine/threonine kinase Akt (cellular homolog of murine thymoma virus akt8 oncogene), also known as PKB (protein kinase B), is activated by lipid products of phosphatidylinositol 3-kinase (PI3K). Akt phosphorylates numerous protein targets that control cell survival, proliferation and motility. Previous studies suggest that Akt regulates transcriptional activity of the nuclear factor-kappaB (NFkappaB) by inducing phosphorylation and subsequent degradation of inhibitor of kappaB (IkappaB). We show here that NFkappaB-driven transcription increases in chicken embryonic fibroblasts (CEF) transformed by myristylated Akt (myrAkt). Accordingly, both a dominant negative mutant of Akt and Akt inhibitors repress NFkappaB-dependent transcription. The degradation of the IkappaB protein is strongly enhanced in Akt-transformed cells, and the loss of NFkappaB activity by introduction of a super-repressor of NFkappaB, IkappaBSR, interferes with PI3K- and Akt-induced oncogenic transformation of CEF. The phosphorylation of the p65 subunit of NFkappaB at serine 534 is also upregulated in Akt-transformed cells. Our data suggest that the stimulation of NFkappaB by Akt is dependent on the phosphorylation of p65 at S534, mediated by IKK (IkappaB kinase) alpha and beta. Akt phosphorylates IKKalpha on T23, and this phosphorylation event is a prerequisite for the phosphorylation of p65 at S534 by IKKalpha and beta. Our results demonstrate two separate functions of the IKK complex in NFkappaB activation in cells with constitutive Akt activity: the phosphorylation and consequent degradation of IkappaB and the phosphorylation of p65. The data further support the conclusion that NFkappaB activity is essential for PI3K- and Akt-induced oncogenic transformation.

Bromelain inhibits COX-2 expression by blocking the activation of MAPK regulated NF-kappa B against skin tumor-initiation triggering mitochondrial death pathway

Chemoprevention impels the pursuit for either single targeted or cocktail of multi-targeted agents. Bromelain, potential agent in this regard, is a pharmacologically active compound, present in stems and fruits of pineapple ( Ananas cosmosus), endowed with anti-inflammatory, anti-invasive and anti-metastatic properties. Herein, we report the anti tumor-initiating effects of bromelain in 2-stage mouse skin tumorigenesis model. Pre-treatment of bromelain resulted in reduction in cumulative number of tumors (CNT) and average number of tumors per mouse. Preventive effect was also comprehended in terms of reduction in tumor volume up to a tune of ∼65%. Components of the cell signaling pathways, connecting proteins involved in cell death were targeted. Bromelain treatment resulted in upregulation of p53 and Bax and subsequent activation of caspase 3 and caspase 9 with concomitant decrease in Bcl-2. A marked inhibition in cyclooxygenase-2 (Cox-2) expression and inactivation of nuclear factor-kappa B (NF-κB) was recorded, as phosphorylation and consequent degradation of Iκ Bα was blocked by bromelain. Also, bromelain treatment curtailed extracellular signal regulated protein kinase (ERK1/2), p38 mitogen-activated protein kinase (MAPK) and Akt activity. The basis of anti tumor-initiating activity of bromelain was revealed by its time dependent reduction in DNA nick formation and increase in percentage prevention. Thus, modulation of inappropriate cell signaling cascades driven by bromelain is a coherent approach in achieving chemoprevention.

Expression of catalytic proteasome subunits in the gut of patients with Crohn’s disease

Background and purposeActivation of the transcription factor NF-κB by proteasomes and subsequent nuclear translocation of cytoplasmatic complexes play a crucial role in the intestinal inflammation. Proteasomes have a pivotal function in NF-κB activation by mediating degradation of inhibitory IκB proteins and processing of NF-κB precursor proteins. This study aims to analyze the expression of the human proteasome subunits in colonic tissue of patients with Crohn’s disease.Materials and methodsThirteen patients with Crohn’s disease and 12 control patients were studied. The expression of immunoproteasomes and constitutive proteasomes was examined by Western blot analysis, immunoflourescence and quantitative real-time PCR. For real-time PCR, AK2C was used as housekeeping gene.ResultsThe results indicate the influence of the intestinal inflammation on the expression of the proteasomes in Crohn’s disease. Proteasomes from inflamed intestine of patients with Crohn’s disease showed significantly increased expression of immunosubunits on both protein and mRNA levels. Especially, the replacement of the constitutive proteasome subunit β1 by inducible immunosubunit β1i was observed in patients with active Crohn’s disease. In contrast, relatively low abundance of immunoproteasomes was found in control tissue.ConclusionsOur data demonstrate that in contrast to normal colonic tissue, the expression of immunoproteasomes was evidently increased in the inflamed colonic mucosa of patients with Crohn’s disease. Thus, the chronic intestinal inflammation process in Crohn’s disease leads to significant alterations of proteasome subsets.

NS-398 Induces Apoptosis in Human Esophageal Cancer Cells Through Inhibition of NF-kappaB Downstream Regulation of Cyclooxygenase-2

Although non-steroidal anti-inflammatory drugs (NSAIDs) have been demonstrated to have cancer-preventive effects and induce apoptosis of cancer cells, the mechanism of their effects is not clearly known. We studied the mechanism in human esophageal cancer cell line TE13. The esophageal squamous cell carcinoma cell line TE-13 was cultured with NS-398 at different concentrations or for different times. Proliferation and apoptosis were measured by MTT reduction and flow cytometry. Prostaglandin F1α was determined with radioimmunoassay. Expression of COX-2 mRNA was measured by RT-PCR and COX-2 protein levels with Western blot analysis. Nuclear NF-kappaB and cytoplasmic IkappaB protein levels were determined by electrophoretic mobility shift assay and Western blot, respectively. NS-398 significantly inhibited cell proliferation and induced apoptosis at concentrations of 0.001, 0.01, 1, and 100 μ mol/L. NS-398 dose-dependently decreased the levels of COX-2 mRNA, COX-2 protein, nuclear NF-kappaB protein and production of PGF1α and increased the cytoplasmic IkappaB protein. In conclusion, NS-398 inhibits the proliferation of, and induced apoptosis in, the cultured TE-13 SCC cell line. These changes correlate with a reduction in COX-2 mRNA and protein expression, prostaglandin synthesis, an inhibition of NF-kappaB nuclear translocation, and an increase in cytoplasmic IkappaB.

Surfactin blocks NO production in lipopolysaccharide-activated macrophages by inhibiting NF-kappaB activation.

Surfactin is a natural biosurfactant derived from Bacillus subtilis and has various biological activities such as anticancer, antiplatelet, and anti-inflammatory effects. In this study, the inhibitory mechanism of surfactin in NO production from macrophages was examined. Surfactin downregulated LPSinduced NO production in RAW264.7 cells and primary macrophages with IC50 values of 31.6 and 22.4 microM, respectively. Immunoblotting analysis showed that surfactin strongly blocked the phosphorylation of IKK and IkBa and the nuclear translocation of NF-kappaB (p65). Therefore, these data suggest that surfactin may act as a bacterium-derived antiinflammatory agent with anti-NF-kappaB activity.

The Zinc Finger Protein Gfi1 Controls TLR4-Mediated Inflammatory Response by Directly Antagonizing NF-κB Transcription Factor

Inflammatory responses are complex and comprise multiple mediators including cytokines such as TNF-alpha (TNF-α) and IL-1beta. These cytokines are synthesized and secreted in response to signaling by plasma membrane receptors of the Toll-like receptor (TLR) family. A central downstream element of TLR-dependent signaling is the transcription factor NF-kappaB (NF-κB), which plays a pivotal role in controlling the proper sequence of events during an inflammatory response. In unstimulated cells, NF-κB is bound to inhibitory IkappaB (IκB) proteins and remains sequestered in the cytoplasm. Stimulation of TLRs triggers a signaling cascade that leads to phosphorylation and proteasomal degradation of IκB, resulting in the translocation of NF-κB to the nucleus, where it acts as a transcriptional activator of target genes. To keep the innate immune system under control, the TLR signaling cascade is under a tight control of many positive and negative regulators. We have previously shown that the transcription factor Growth Factor Independence 1 (Gfi1) represents a novel factor limiting the inflammatory immune response including TNF-α. Gfi1-deficient (Gfi1−/−) mice show a very strong systemic response to the TLR4 ligand and endotoxin LPS and die rapidly within 36 h with symptoms of septic shock. Here, we investigated the molecular mechanism of this exaggerated TNF-α production in the absence of Gfi1. It is known that endotoxin stimulation results in the activation of the transcription factor NF-κB through TLR4, leading to TNF-α production. This activation also resulted in rapid and de novo expression of Gfi1 in the nucleus in a time- and dose-dependent manner. The expression of Gfi1 was not due to feedback regulation from secreted TNF, since TNF-deficient macrophages were also able to upregulate Gfi1 mRNA following LPS stimulation. As expected, LPS stimulation of Gfi1−/− macrophages resulted in significantly higher levels of TNF-α mRNA, and secreted TNF-α cytokine. Strikingly and in contrast to most known negative regulators of TLRs, Gfi1 did not affect the activity or the expression levels of the cytoplasmic components of TLR signaling pathway. Additionally, NF-κB phosphorylation and nuclear translocation post- LPS treatment were intact in both Gfi1−/− and Gfi1+/+ macrophages. Immunoprecipitation analysis from cells endogenously expressing Gfi1 and NF-κB or over-expressing these two proteins post transfection, clearly revealed a direct interaction between Gfi1 and the p65 subunit of NF-κB. Immunofluorescence staining of macrophages post-LPS treatment confirmed direct interaction of these two proteins in the nucleus at the endogenous level. Gfi1 represses transcription by binding to DNA recognition sequences in target gene promoters. Thus, aiming to investigate the effect of Gfi1 expression on NF-κB nuclear signaling, we found that LPS treatment enhances NF-κB DNA binding activity in Gfi1−/− macrophages as compared to Gfi1+/+ cells. Furthermore, over expression of Gfi1 protein resulted in negative regulation of NF-κB mediated gene activation in a dose-dependent manner. Chromatin immune precipitation with anti-p65 antibodies from LPS stimulated Gfi1+/+ and Gfi1−/− macrophages revealed enhanced NF-κB promoter occupancy at the TNF gene in Gfi1−/− macrophages as compared to Gfi1+/+ cells. In conclusion, our findings reveal a novel function for Gfi1 in the innate immune response by directly antagonizing NF-κB function. This molecular perceptive of TNF-α regulation during inflammation may provide an attractive strategy for therapeutic intervention in chronic inflammatory diseases and certain cancers.

Oncogene Functions of FHL2 Are Independent from NF-κBIα in Gastrointestinal Cancer

Four and a half of LIM-only protein 2 (FHL2) is an adaptor protein that can interact with many transcription factors and thus plays a variety of biological functions. Previous studies by our group have demonstrated that suppression of FHL2 was capable of inducing tumor cell differentiation, and inhibiting the growth of experimental gastric and colon cancers. Therefore, FHL2 appears to function as an oncogene. In order to further explore the mechanisms of how FHL2 is involved in tumorigenesis, we attempted to test whether FHL2 has any direct association with nuclear factor (NF-kappaB), the most important transcription factor involved in apoptosis, inflammation, and carcinogenesis. Using an Yeast Two Hybrid (Y2H) screening system, we have shown that FHL2 may have an interaction with NF-kappaBIalpha, the coding gene for IkappaBalpha which is the most potent endogenous inhibitor for NF-kappaB activation. However, subsequent studies using co-immunoprecipitation and co-localization failed to confirm the Y2H finding. Down-regulation of FHL2 by FHL2-siRNA down-regulated the expression of NF-kappaB p65. We therefore concluded that under the physiological condition, FHL2 may activate NF-kappaB pathway, even though such an activation may not be mediated by a direct binding of FHL2 to NF-kappaB inhibitor protein IkappaB.