Prevented NF-kappaB Activation Research Articles

Diosmectite inhibits the interaction between SARS-CoV-2 and human enterocytes by trapping viral particles, thereby preventing NF-kappaB activation and CXCL10 secretion

SARS-CoV-2 enters the intestine by the spike protein binding to angiotensin-converting enzyme 2 (ACE2) receptors in enterocyte apical membranes, leading to diarrhea in some patients. Early treatment of COVID-19-associated diarrhea could relieve symptoms and limit viral spread within the gastrointestinal (GI) tract. Diosmectite, an aluminomagnesium silicate adsorbent clay with antidiarrheal effects, is recommended in some COVID-19 management protocols. In rotavirus models, diosmectite prevents pathogenic effects by binding the virus and its enterotoxin. We tested the trapping and anti-inflammatory properties of diosmectite in a SARS-CoV-2 model. Trapping effects were tested in Caco-2 cells using spike protein receptor-binding domain (RBD) and heat-inactivated SARS-CoV-2 preparations. Trapping was assessed by immunofluorescence, alone or in the presence of cells. The effect of diosmectite on nuclear factor kappa B (NF-kappaB) activation and CXCL10 secretion induced by the spike protein RBD and heat-inactivated SARS-CoV-2 were analyzed by Western blot and ELISA, respectively. Diosmectite bound the spike protein RBD and SARS-CoV-2 preparation, and inhibited interaction of the spike protein RBD with ACE2 receptors on the Caco-2 cell surface. Diosmectite exposure also inhibited NF-kappaB activation and CXCL10 secretion. These data provide direct evidence that diosmectite can bind SARS-CoV-2 components and inhibit downstream inflammation, supporting a mechanistic rationale for consideration of diosmectite as a management option for COVID-19-associated diarrhea.

The Establishment of Bortezomib Resistant Myeloma Cell Line KM3/BTZ and Explore the Resistance Mechanism

Background: Multiple myeloma (MM) is an incurable B-cell malignancy resulting in significant morbidity and mortality, the incidence of second place in hematological malignancies. The proteasome inhibitor bortezomib inhibits IkappaB degradation, prevents NF-kappaB activation, and induces apoptosis in MM cells, has become first-line drug to MM. Despite its promising activity in traditional chemoresistant myeloma patients, however, some patients are resistant or become refractory to bortezomib. Chauhan D et al reported that Blockade of Hsp27 overcomes bortezomib resistance in lymphoma cells (Chauhan D et al., Cancer Re, 2003). However, bortezomib resistance mechanisms in MM remain controversial, the molecular basis of this reduced responsiveness is currently not fully understood.Objective: To establish a bortezomib-resistant cell line KM3 /BTZ of MM, then search for genes related to bortezomib resistance phenotype by analyzing the differential gene expression patterns with cDNA microarray,and explain the mechanism of bortezomib-resistant multiple myeloma.Methods: Bortezomib-resistant KM3/BTZ cell line was generated by increasing concentration of bortezomib to induce KM3 cells in vitro. MTT assay was employed to detect the cytotoxic effect on KM3 and KM3/BTZ cells. The cells apoptosis were analyzed by flow cytometry. We detect the gene expression profile changes between KM3/BTZ and its parent cell line KM3 cells by CDNA microarray. Combining of molecular annotation system MAS3.0 software and detailed analysis of documented resistance genes were used to analyze the data. Specific differentially-expressed genes were chosen for further verification using real-time RT-PCR. The expression of multi-drug resistance 1(MDR-1) gene mRNA was determined by RT-PCR.Results: The KM3/BTZ cell line was established successfully. Bortezomib resistance index values were significantly higher in KM3/BTZ (IC50 =351.2±3.51ng /ml) when compared to KM3 cells (IC50 = 17.8±1.03ng /ml),and the resistance index is 19.7 (P< 0.05). Through gene expression profiling filtering, compared with KM3,770 significantly differently expressed genes were screened out in the KM3/BTZ cell line, of which up and down regulated genes were 287 and 383 respectively, in which heat shock protein family gene HSPB2 was significantly up-regulated (Ratio=14.7455). Downregulated gene list in KM3/BTZ cell line included transcriptional regulators like ZNF family proteins (Tab 1). Upregulated genes in KM3/BTZ cell line included signal transduction related genes like MS4A family protein (Tab 1) also transcriptional regulators like ZNF family proteins (Tab 1). The expression trend of eight genes for further verification was almost consistent with the microarray, excepting gene of JUN (Fig 1). The expression of MDR-1 mRNA was not observed in either resistant or parental cells (Fig 2).Conclusion: We have successfully established bortezomib-resistant cell line KM3/BTZ. MDR-1 was not involved in bortezomib-resistant multiple myeloma cells.HSPB2, ZNF and MS4A family genes are probably related to bortezomib resistance in KM3/BTZ cells. Our results suggest that they may be new targets to overcome resistance to bortezomib in patients with MM.Table 1ZNF and MS4A family gene expression in KM3/BTZ and KM3 cellsGeneB signal*B detection*A signal*A detection*BvsA signal log ratio*BvsA change*ZNF71185.08P206.16P0.41269DZNF926.75P14.57P0.46328DZNF70410.7A26.78P0.39955DZNF4927.08P3.48P2.03448IZNF53287.93P43.54A2.01952IZNF59410.25P4.63P2.21382IZNF789269.79P97.3P2.77276IZNF506330.36P149.48P2.21006IMS4A31721.58P145.3P11.84845IMS4A4A2688.23P100.8P26.66895IMS4A6A141.96P56.47M2.513901IMS4A149.02P53.11P0.92299NC PHigh expression; A: Low expression; M:Marginally D: Decrease; I: Increase; NC: No change*: A:KM3; B: KM3/BTZ [Display omitted] [Display omitted] MDNA Marker; 1-2: K562/A cell line; 3-4: KM3cell line; 5-6: KM3/BTZ cell line DisclosuresNo relevant conflicts of interest to declare.

Critical role of glutathione in melatonin enhancement of tumor necrosis factor and ionizing radiation‐induced apoptosis in prostate cancer cells in vitro

The role of antioxidants in reducing cancer initiation and progression has been highlighted in recent years. Not only antioxidants limit cancer cell growth but also, in some situations, they promote the effectiveness of conventional treatments. Melatonin, an endogenously synthesized antioxidant, reduces cell growth of several tumor types both in vivo and in vitro. Additionally, the indole limits the collateral damage induced by many chemotherapeutic agents. By using a cellular model of human prostate cancer, we studied the ability of melatonin to enhance apoptosis induced by tumor necrosis factor or gamma radiation. It has been reported that melatonin reduces prostate cancer cell growth and, more recently, it promotes cell differentiation. In this work, we also show that melatonin elevates p21 protein levels and increases antioxidant capacity of prostate cancer cells. In addition, melatonin significantly enhances hrTNFalpha induced cell death by decreasing NFkappaB activation. Bcl-2 and survivin down-regulation appears to be associated to apoptosis stimulation under NFkappaB inhibition. On the contrary, melatonin does not promote irradiation-induced cell death due to an increment in intracellular glutathione content. In conclusion, prevention of NFkappaB activation by melatonin enhances the effectiveness of cytokine treatment in prostate cancer cells but it is not sufficient to enhance cell death triggered by other therapies which generate free radicals. A crucial role of glutathione in survival mechanisms of prostate cancer cells should be carefully considered.

Docosahexaenoic acid sensitizes Ramos cells to Gamma-irradiation-induced apoptosis through involvement of PPAR-γ activation and NF-κB suppression

Gamma-irradiation (Gamma-IR) resistance is a character of many malignant cells that decreases the efficacy of radiotherapy. Although ionizing radiation activates multiple cellular factors that vary depending on dose and tissue specificity, the activation of nuclear factor-kappa B appears to be a well-conserved response in tumor cells exposed to Gamma-IR which can lead to the inhibition of radiation-induced apoptosis. Thus, inhibition of NF-kappaB activation is an important strategy to abolish radioresistance. Recently, we have demonstrated that docosahexaenoic acid (DHA; 22:6 n-3 polyunsaturated fatty acids)-induced apoptosis may occur via ligand-dependent transcription factor, peroxisome proliferator-activated receptors-gamma. Moreover, many reports described that activation of PPAR-gamma can lead to the induction of apoptosis through NF-kappaB inhibition. Therefore, we addressed the mechanism that NF-kappaB is a downstream target of DHA and may be involved in the process of radiosensitization. Ramos cells are a highly radiation-resistant and p53-deficient Burkitt's lymphoma cell line. The results of present study showed that cotreatment of Ramos cells with low doses of DHA and Gamma-IR leads to marked phosphorylation of IkappaB and translocation of p65/NF-kappaB to nucleus in parallel with increase in apoptosis. Preincubation of the cells with GW9662, a selective antagonist for PPAR-gamma, significantly prevented NF-kappaB activation profile. Taken together, these results suggest that low concentration of DHA inhibited Gamma-IR-induced activation of NF-kappaB and sensitized Ramos cells to IR-induced cytotoxicity. Pretreatment of Ramos cells with GW9662 abrogated the ability of DHA to inhibit Gamma-IR-induced activation of NF-kappaB and diminished the DHA radiosensitizing effect indicating that PPAR-gamma may act as a mediator of DHA in inhibition of NF-kappaB. Taken together, these results suggest that low concentration of DHA inhibited Gamma-IR-induced activation of NF-kappaB and sensitized Ramos cells to IR-induced cytotoxicity. Pretreatment of Ramos cells with GW9662 abrogated the ability of DHA to inhibit Gamma-IR-induced activation of NF-kappaB and diminished the DHA radiosensitizing effect indicating that PPAR-gamma may act as a mediator of DHA in inhibition of NF-kappaB.

Role of NF-κB-dependent Caveolin-1 Expression in the Mechanism of Increased Endothelial Permeability Induced by Lipopolysaccharide

We investigated the role of NF-kappaB activation by the bacterial product lipopolysaccharide (LPS) in inducing caveolin-1 (Cav-1) expression and its consequence in contributing to the leakiness of the endothelial barrier. We observed that LPS challenge of human lung microvascular endothelial cells induced concentration- and time-dependent increases in expression of Cav-1 mRNA and protein. The NEMO (NF-kappaB essential modifier binding domain)-binding domain peptide (IkB kinase (IKK)-NEMO-binding domain (NBD) peptide), which prevents NF-kappaB activation by inhibiting the interaction of IKKgamma with the IKK complex, blocked LPS-induced Cav-1 mRNA and protein expression. Knockdown of NF-kappaB subunit p65/RelA expression with small interfering RNA also prevented LPS-induced Cav-1 expression. Caveolae open to the apical and basal plasmalemma of endothelial cells increased 2-4-fold within 4 h of LPS exposure. IKK-NBD peptide markedly reduced the LPS-induced increase in the number of caveolae as well as transendothelial albumin permeability. These observations were recapitulated in mouse studies in which IKK-NBD peptide prevented Cav-1 expression and interfered with the increase in lung microvessel permeability induced by LPS. Thus, LPS mediates NF-kappaB-dependent Cav-1 expression that results in increased caveolae number and thereby contributes to the mechanism of increased transendothelial albumin permeability.

Development of Cell-selective Targeting Systems of NFκB Decoy for Inflammation Therapy

NFkappaB regulate several inflammatory related molecules and evoke immune and inflammatory response by several stimuli, therefore inhibition of NFkappaB activation would be a novel therapeutic strategy. To date, there are many conventional drugs including nonsteroldal or steroldal anti-inflammatory drugs or immune suppressors etc. were known to inhibit NFkappaB activation, however, several side effects were also reported. Recently, double stranded oligonucleotide including NFkappaB binding sequence, called NFkappaB decoy, was developed to prevent NFkappaB activation, which is powerful tool in a new class of anti-gene strategy for molecular therapy with low side effect. However, NFkappaB decoy is easily degraded by nuclease and rapidly excreted to urine, therefore it is necessary to develop carrier for NFkappaB decoy therapy. Here, we shall review delivery system for NFkappaB decoy and introduce our cell-selective delivery system for NFkappaB decoy using sugar decorated cationic liposomes.

Role of nuclear IκB proteins in the regulation of host immune responses

A variety of microbial components activate a transcription factor called nuclear factor-kappaB (NF-kappaB) that plays an essential role in the optimal activation of host immune systems. The transcriptional activity of NF-kappaB is tightly regulated at multiple steps in immune signaling pathways, because excessive activation is detrimental to the host. One mechanism to prevent NF-kappaB activation is mediated by cytoplasmic IkappaB family proteins. Although cytoplasmic IkappaBs interact with NF-kappaB subunits in the cytoplasm of unstimulated cells, IkappaBs are rapidly degraded on stimulation, allowing free NF-kappaB to translocate into the nucleus and activate the transcription of genes encoding various immune mediators. After the translocation of NF-kappaB from the cytoplasm to the nucleus, nuclear proteins that are structurally similar to cytoplasmic IkappaBs take part in the regulation of NF-kappaB transcriptional activity, as activators or inhibitors, by associating with NF-kappaB subunits. Therefore, the regulatory IkappaB-like nuclear molecules are described as "nuclear IkappaB proteins." In this review, the in vivo function of the nuclear IkappaB proteins, Bcl-3, IkappaBzeta, and IkappaBNS in the context of host immune responses and diseases will be discussed.

Prevention of Deoxycholate-Induced Gastric Apoptosis by Aspirin: Roles of NF-κB and PKC Signaling

Apoptosis is a major mechanism of gastric cell death induced by deoxycholate (DC) and aspirin (ASA), and the caspase cascade and protein kinase C (PKC) signaling play key roles in this process. The transcription factor kappa B (NF-kappaB) has been shown to modulate apoptosis by regulating the transcription of numerous pro- and anti-apoptotic genes. The aim of this study was to investigate the effect of DC and ASA on NF-kappaB signaling, and determine its role in programmed cell death in a human gastric carcinoma cell line.Cells were incubated with DC in the presence or absence of ASA or proteasome inhibitors (PI- I, lactacystin, and MG-132). Cell lysates were evaluated by Western blotting. NF-kappaB (p65) was measured in the cytosol and nuclear fractions.DC induced a translocation of NF-kappaB into the nuclear compartment that was completely blocked by proteasome inhibitors. Although, ASA itself had no effect on the NF-kappaB pathway, nor did it reduce DC-induced NF-kappaB translocation, it did prevent DC-induced caspase-3, -6 and -9 activation, poly (ADP-ribose) polymerase and lamin A processing, DNA degradation, and PKC signaling, all indices of apoptosis. In contrast, proteasome inhibitors had no effect on DC-induced apoptosis.Deoxycholate activates NF-kappaB at the same time that it induces apoptosis in gastric epithelial cells. Prevention of NF-kappaB activation does not alter DC-induced apoptosis, indicating that in our experimental conditions, NF-kappaB is not essential for apoptosis to proceed. In contrast, the ability of aspirin to restore the alterations in PKC isoforms induced by DC and at the same time prevent caspase cascade activation suggests the importance of the PKC signaling system in this process.

Parthenolide Inhibits IκB Kinase, NF-κB Activation, and Inflammatory Response in Cystic Fibrosis Cells and Mice

Cystic fibrosis (CF) is characterized by prolonged and excessive inflammatory responses in the lung and increased activation of NF-kappaB. Parthenolide is a sesquiterpene lactone derived from the plant feverfew, which has been used in folk medicine for anti-inflammatory activity. Several studies suggest that this compound inhibits the NF-kappaB pathway, but the exact site is controversial. We hypothesized that parthenolide might ameliorate the excessive inflammatory response in CF models by inhibiting activation of NF-kappaB. This was tested in vitro, using two pairs of cell lines with defective versus normal CF transmembrane conductance regulator (CFTR) (antisense/sense transfected 16 HBE and IB-3/S9), and in vivo, using CFTR-knockout (KO) mice. All cell lines were pretreated with parthenolide and then stimulated with IL-1beta and/or TNF. Parthenolide significantly inhibited IL-8 secretion induced by these cytokines and prevented NF-kappaB activation, IkappaBalpha degradation, and IkappaB Kinase complex activity. CFTR-KO and wild-type mice were pretreated with parthenolide or vehicle alone then challenged intratracheally with LPS. Bronchoalveolar lavage was performed 3, 6, and 8 h later. Parthenolide pretreatment inhibited PMN influx as well as cytokine and chemokine production. This was also associated with inhibition of IkappaBalpha degradation and NF-kappaB activation. We thus conclude that parthenolide inhibits IkappaB kinase, resulting in stabilization of cytoplasmic IkappaBalpha, which in turn leads to inhibition of NF-kappaB translocation and attenuation of subsequent inflammatory responses. IkappaB kinase may be a good target, and parthenolide and/or feverfew might be promising treatments for the excessive inflammation in CF.

Maintenance of colonic homeostasis by distinctive apical TLR9 signalling in intestinal epithelial cells

The mechanisms by which commensal bacteria suppress inflammatory signalling in the gut are still unclear. Here, we present a cellular mechanism whereby the polarity of intestinal epithelial cells (IECs) has a major role in colonic homeostasis. TLR9 activation through apical and basolateral surface domains have distinct transcriptional responses, evident by NF-kappaB activation and cDNA microarray analysis. Whereas basolateral TLR9 signals IkappaBalpha degradation and activation of the NF-kappaB pathway, apical TLR9 stimulation invokes a unique response in which ubiquitinated IkappaB accumulates in the cytoplasm preventing NF-kappaB activation. Furthermore, apical TLR9 stimulation confers intracellular tolerance to subsequent TLR challenges. IECs in TLR9-deficient mice, when compared with wild-type and TLR2-deficient mice, display a lower NF-kappaB activation threshold and these mice are highly susceptible to experimental colitis. Our data provide a case for organ-specific innate immunity in which TLR expression in polarized IECs has uniquely evolved to maintain colonic homeostasis and regulate tolerance and inflammation.

Role for IKK2 in muscle: waste not, want not

Activation of transcription factor NF-kappaB, the major regulator of the inflammatory response, depends on the inhibitor of NF-kappaB kinase (IKK) complex, which is composed of 2 catalytic subunits, IKK1 and IKK2 (also known as IKKalpha and IKKbeta), and a regulatory subunit, IKKgamma (also known as NEMO). In this issue of the JCI, Mourkioti et al. show that muscle-specific disruption in mice of the gene encoding IKK2 prevents NF-kappaB activation in response to denervation or toxin-induced injury (see the related article beginning on page 2945). Importantly, this genetic manipulation prevents muscle wasting, thereby providing strong evidence in support of a major pathogenic role for inflammation in a variety of muscular dystrophies characterized by progressive muscle fiber degeneration.

Ethanol beverages containing polyphenols decrease nuclear factor kappa-B activation in mononuclear cells and circulating MCP-1 concentrations in healthy volunteers during a fat-enriched diet

Aims Different epidemiological studies have demonstrated that some ethanol containing beverages intake could be associated with a reduction of cardiovascular mortality, effect attributed in part to its antioxidant properties. Nuclear factor-kappa B (NF-kappaB) is a redox sensitive transcription factor implicated in the pathogenesis of atherosclerosis. We have examined the effect of four different ethanol containing beverages on the activation of NF-kappaB in peripheral blood mononuclear cells (PBMC) and circulating concentrations of monocyte chemoattractant protein-1 (MCP-1) in healthy volunteers receiving a fat-enriched diet. Methods and results Sixteen volunteers received 16 g/m 2 of ethanol in form of red wine, spirits (vodka, rum, and brandy) or no ethanol intake along with a fat-enriched diet during 5 days and all of them took all alcohols at different periods. NF-kappaB activation (electrophoretic mobility shift assay) and circulating MCP-1 levels (ELISA) were examined in blood samples taken before and after 5 days of ethanol intake. Subjects receiving a fat-enriched diet had increased NF-kappaB activation in PBMC at day 5. Furthermore, MCP-1 levels were increased in plasma at day 5. Red wine intake and some ethanol beverages containing polyphenols (brandy and rum) prevented NF-kappaB activation and decreased MCP-1 release. Conclusion Consumption of moderate amounts of alcoholic drinks containing polyphenols decreases NF-kappaB activation in PBMCs and MCP-1 plasma levels during a fat-enriched diet. Our results provide additional evidence of the anti-inflammatory effects of some ethanol containing beverages, further supporting the idea that its moderate consumption may help to reduce overall cardiovascular mortality.

Perfluorocarbons Decrease Chlamydophila pneumoniae–Mediated Inflammatory Responses of Rat Type II Pneumocytes In Vitro

Chlamydophila pneumoniae alter the expression of Toll-like receptor (TLR) 4 in alveolar type II (ATII)-cells. Subsequently nuclear factor kappaB (NF-kappaB) is activated and tumor necrosis factor-alpha (TNF-alpha) and macrophage inflammatory protein 2 (MIP-2) are produced. Perfluorocarbons (PFC) are beneficial in animals with bacterial pneumonia and reduce production of TNF-alpha. Using isolated ATII-cells, it was studied whether PFC prevent C. pneumoniae-induced TNF-alpha and MIP-2 release and what the underlying pathway is. PF5080 preincubation prevented C. pneumoniae-induced secretion of TNF-alpha (43 +/- 10 versus 661 +/- 41 pg/mL) and MIP-2 (573 +/- 41 versus 4786 +/- 502 pg/mL). The C. pneumoniae-induced 2.2-fold increase of TNF-alpha Receptor 1 expression was reduced by PF5080. C. pneumoniae reduced cytoplasmatic IkappaBalpha (3.7 +/- 0.3 versus 14 +/- 1) and increased NF-kappaB p65 (31 +/- 7.5 versus 3.6 +/- 1.1) compared with control. PF5080 prevented NF-kappaB activation. TLR4 expression was 1.5-fold higher after C. pneumoniae incubation, but remained at control levels after PF5080 pretreatment. After 24 h of C. pneumoniae incubation, in 88 +/- 6% of cells bacteria were found in the perinuclear region and in 50% of these cells bacteria adhered to cellular surface. After PF5080 preincubation, C. pneumoniae were in 32 +/- 4% attached to and in 5 +/- 1% internalized in ATII-cells. Since PF5080 was found in ATII-cell membranes, PF5080 effect could be explained by an alteration of the cellular membrane, preventing activation of the inflammatory cascade.

Sulfasalazine prevents apoptosis in spermatogenic cells after experimental testicular torsion/detorsion

To determine whether sulfasalazine can prevent apoptosis in spermatogenic cells by preventing the activation of NF-kappaB in spermatogenic epithelium in experimental testicular torsion. Thirty-two adult male Sprague-Dawley rats were subjected to unilateral 720 degree testicular torsion for durations of 0 h and 2 h, then the torsion was relieved. The ischemic/reperfused testes were collected for the detection of NF-kappaB expression with Western blotting and immunohistochemistry techniques, and detection of apoptosis with TUNEL techniques. The NF-kappaB coefficient of spermatogenic epithelium and the apoptosis index of spermatogenic cells were significantly different in the operation and the sham-operation groups after experimental testicular torsion (P<0.01). NF-kappaB activation of spermatogenic epithelium is related to apoptosis of spermatogenic cells. Sulfasalazine can prevent apoptosis in spermatogenic cells after the experimental testicular torsion through prevention of NF-kappaB activation.

An evaluation of the efficacy of topical application of salicylic acid for the treatment of familial cylindromatosis

Familial cylindromatosis is a rare genetic disorder, giving rise to neoplasms of the skin appendages. We have recently shown that loss of the cylindromatosis tumour suppressor gene leads to activation of NF-kappaB, a transcription factor having antiapoptotic activity. This provides a possible explanation for the deregulated growth of cylindromas. In cell-based assays, salicylate can prevent NF-kappaB activation caused by loss of the cylindromatosis gene, suggesting that salicylic acid application might be a potential treatment for cylindromatosis. To assess the effectiveness of topical application of salicylic acid on familial cylindromas. Cylindromas in five patients from four different cylindromatosis families were treated with twice daily and then once daily topical salicylic acid. Clinical response was determined by serial tumour measurements. In total 17 cylindromas in five patients were studied: 12 target lesions and five control lesions. The median size of the cylindromas was 1.0 cm (range, 0.6-2.8 cm). Two of the 12 cylindromas showed a complete remission. Another eight lesions showed some response, but not sufficient to qualify as partial remission. The control lesions remained stable or increased in size. Salicylic acid is a well-tolerated and potential new treatment for cylindromatosis.

Host response to the attenuated poxvirus vector NYVAC: upregulation of apoptotic genes and NF-kappaB-responsive genes in infected HeLa cells.

NYVAC has been engineered as a safe, attenuated vaccinia virus (VV) vector for use in vaccination against a broad spectrum of pathogens and tumors. Due to the interest in NYVAC-based vectors as vaccines and current phase I/II clinical trials with this vector, there is a need to analyze the human host response to NYVAC infection. Using high-density cDNA microarrays, we found 368 differentially regulated genes after NYVAC infection of HeLa cells. Clustering of the regulated genes identified six discrete gene clusters with altered expression patterns. Clusters 1 to 3 represented 47.5% of the regulated genes, with three patterns of gene activation kinetics, whereas clusters 4 to 6 showed distinct repression kinetics. Quantitative real-time reverse transcription-PCR analysis of selected genes validated the array data. Upregulated transcripts correlated with genes implicated in immune responses, including those encoding interleukin-1 receptor 2 (IL-1R2), IL-6, ISG-15, CD-80, and TNFSF7. NYVAC upregulated several intermediates of apoptotic cascades, including caspase-9, correlating with its ability to induce apoptosis. NYVAC infection also stimulated the expression of NF-kappaB1 and NF-kappaB2 as well as that of NF-kappaB target genes. Expression of the VV host range K1L gene during NYVAC infection prevented NF-kappaB activation, but not the induction of apoptosis. This study is the first overall analysis of the transcriptional response of human cells to NYVAC infection and provides a framework for future functional studies to evaluate this vector and its derivatives as human vaccines.

Ethanol‐Induced Sensitization to Endotoxin in Kupffer Cells Is Dependent upon Oxidative Stress

Activation of Kupffer cells by gut-derived endotoxin plays a pivotal role in alcoholic liver injury. On the other hand, it was reported that acute ethanol administration reduced activation of Kupffer cells. We found that Kupffer cells isolated from rat treated only once with ethanol were sensitized to endotoxin 24 hrs later correlatively with CD14 expression. Moreover, it was shown that Kupffer cell activation by endotoxin via Toll-like receptor (TLR)-4 is involved in alcohol-induced liver injury and ethanol-induced oxidative stress is important in the regulation of transcription factor NFkappaB activation and cytokine production by Kupffer cells. Here, we show that IRAK, one of signaling molecules of TLR-4, regulates tolerance and sensitization to LPS and acute ethanol increases in IRAK expression through a mechanism dependent upon oxidant production. Female C57BL/6 mice were given ethanol (5 g/kg) intragastrically, and LPS was injected 1 or 21 hrs later. Serum transaminase levels were measured. Moreover, some mice were treated with NADPH oxidase inhibitor diphenyleneiodonium sulfate (DPI, 1 mg/kg/day) or infected with adenovirus (1 x 10 plaque-forming units, intravenously) containing IkappaB superrepressor gene, which prevent NFkappaB activation of Kupffer cells, for three days. Kupffer cells were isolated from mice 1 hr and 21 hrs after ethanol treatment. After the addition of LPS, TNF-alpha in the media was measured using ELISA, Electrophoretic mobility shift assay (EMSA) was performed to analyze DNA binding activity of NFkappaB. Further, expression of Interleukin-1 receptor-associated kinase (IRAK) was evaluated by Western blotting. LPS-induced increases in transaminases were blunted in mice treated with ethanol before 1 hr. However, ethanol treatment 21 hrs earlier augmented LPS-increased transaminases three-fold over controls. Pretreatment with nonabsorbable antibiotics blocked these effects of ethanol. LPS-induced TNF-alpha production by Kupffer cells isolated from mice 1 hr after ethanol was reduced to about 60% of values from control Kupffer cells, while LPS-induced TNF-alpha production by Kupffer cells isolated from mice treated with ethanol 21 hrs earlier increased 1.5-fold over control Kupffer cells. In Kupffer cells from mice 1 hr after ethanol treatment, expression of IRAK was decreased, and LPS-induced activation of NFkappaB was decreased correlatively. In contrast, ethanol treatment to mice increased expression of IRAK in Kupffer cells 21hrs later and LPS-induced activation of NFkappaB was elevated significantly. On the other hands, DPI treatment for three days prior to ethanol did not prevent decreases in IRAK expression due to ethanol treatment for 1 hr. However, DPI treatment blunted ethanol-induced increases in IRAK expression. Additionally, inhibition of NKkappaB activation with dominant-negative IkappaBalpha blunted ethanol-induced increase in IRAK expression. Contrary, inhibition of NKkappaB did not affect decrease of IRAK expression due to ethanol treatment for 1 hr. Ethanol causes tolerance in the early phase after ethanol consumption, while sensitization was observed later. Both tolerance and sensitization were induced by gut-derived endotoxin. These findings indicate that ethanol-induced both tolerance and sensitization of Kupffer cells to endotoxin involve IRAK expression. Further, NADPH oxidase plays a pivotal role in the increase in IRAK expression due to ethanol via activation of NFkappaB signaling pathway. In conclusion, these data indicate that acute ethanol causes sensitization to endotoxin through mechanisms dependent upon oxidative stress.

Stress-Activated Signaling Pathways Mediate the Stimulation of Pregnancy-Associated Plasma Protein-A Expression in Cultured Human Fibroblasts

Pregnancy-associated plasma protein-A (PAPP-A) is an IGF binding protein protease that appears to function as a posttranslational modulator of IGF bioavailability in response to injury. A previous study indicated that the proinflammatory cytokines, TNFalpha and IL-1beta, were potent stimulators of PAPP-A expression in cultured human fibroblasts. In this study, we investigated the intracellular signaling pathways mediating cytokine-stimulated PAPP-A expression. Treatment of human fibroblasts with TNFalpha and IL-1beta (1 nm) had little or no effect on phosphatidylinositol 3-kinase and Erk1/2 activation, pathways commonly associated with proliferation. On the other hand, TNFalpha and IL-1beta induced p38, c-Jun N-terminal kinase (JNK), and nuclear factor (NF)kappaB activation, pathways more closely related to stress response. An inhibitor of p38 activation (SB203580) had no effect on TNFalpha- or IL-1beta-stimulated PAPP-A expression. The JNK inhibitor, SP600125, had no effect on IL-1beta- or TNFalpha-stimulated PAPP-A mRNA expression. However, SP600125 effectively inhibited IL-1beta-induced PAPP-A protein expression. MG-132, a proteasome inhibitor that blocked degradation of the intrinsic NFkappaB inhibitor, IkappaB, and thereby prevented NFkappaB activation, was a potent inhibitor of both TNFalpha- and IL-1beta-stimulated PAPP-A mRNA and protein expression and IGF binding protein-4 protease activity. MG-132 had no effect on JNK phosphorylation or p38 activation, and SB203580 and SP600125 had no effect on IkappaB degradation, documenting inhibitor specificity. BAY11-7082, another inhibitor of NFkappaB activation, also inhibited TNFalpha- and IL-1beta-stimulated PAPP-A expression and IGF binding protein-4 protease activity. These data indicate that NFkappaB activation is the primary mediator of cytokine-stimulated PAPP-A expression in human fibroblasts.

Decreased apoptosis in polyamine depleted IEC-6 cells depends on Akt-mediated NF-κB activation but not GSK3β activity

The PI3-kinase/Akt pathway promotes cell survival in many different cell types including intestinal epithelial cells. Increased AKT activation in polyamine depleted intestinal epithelial cells correlated well with the decrease in TNF-alpha-induced apoptosis. Increased Akt activation and GSK3beta (Ser 9) phosphorylation without significant effect on Bad (Ser136) phosphorylation indicate that Akt-mediated protection is independent of Bad phosphorylation but may depend on GSK3beta. Pretreatment of polyamine-depleted cells with LY294002 increased caspase-9 and caspase-3 activation and decreased basal levels of GSK-3beta phosphorylation. Inhibition of GSK3beta activity using AR-A014418 or lithium chloride or siRNA-mediated downregulation of its expression had no effect on apoptosis. Inhibition of PI3-kinase and over-expression of dominant negative Akt (DN-AKT), significantly increased apoptosis in polyamine depleted cells. DN-Akt expression reversed the protective effect of polyamine depletion on apoptosis. DN-Akt, as well as the PI3-kinase inhibitors, prevented Akt activation and subsequent translocation of NF-kappaB to the nucleus. Constitutively active Akt (CA-AKT) expression increased resistance to TNF-alpha-induced apoptosis. Constitutively active-Akt expression increased nuclear staining of NF-kappaB. Moreover, polyamine depletion of DN-Akt cells prevented basal and TNF-alpha-induced IkappaBalpha phosphorylation. Prevention of NF-kappaB activation in DN-IkappaBalpha-transfected cells increased apoptosis in control cells and restored it in polyamine-depleted cells to control levels. These data indicate that Akt regulates the mitochondrial pathway, preventing activation of caspase-9 and thereby caspase-3 via NF-kappaB and these effects are independent of GSK-3beta activity.

Activated AKT regulates NF-κB activation, p53 inhibition and cell survival in HTLV-1-transformed cells

AKT activation enhances resistance to apoptosis and induces cell survival signaling through multiple downstream pathways. We now present evidence that AKT is activated in HTLV-1-transformed cells and that Tax activation of AKT is linked to NF-kappaB activation, p53 inhibition and cell survival. Overexpression of AKT wild type (WT), but not a kinase dead (KD) mutant, resulted in increased Tax-mediated NF-kappaB activation. Blocking AKT with the PI3K/AKT inhibitor LY294002 or AKT SiRNA prevented NF-kappaB activation and inhibition of p53. Treatment of C81 cells with LY294002 resulted in an increase in the p53-responsive gene MDM2, suggesting a role for AKT in the Tax-mediated regulation of p53 transcriptional activity. Further, we show that LY294002 treatment of C81 cells abrogates in vitro IKKbeta phosphorylation of p65 and causes a reduction of p65 Ser-536 phosphorylation in vivo, steps critical to p53 inhibition. Interestingly, blockage of AKT function did not affect IKKbeta phosphorylation of IkappaBalpha in vitro suggesting selective activity of AKT on the IKKbeta complex. Finally, AKT prosurvival function in HTLV-1-transformed cells is linked to expression of Bcl-xL. We suggest that AKT plays a role in the activation of prosurvival pathways in HTLV-1-transformed cells, possibly through NF-kappaB activation and inhibition of p53 transcription activity.