Induced NF-kappaB Activation Research Articles

Xanthoangelol D Isolated from the Roots of Angelica keiskei Inhibits Endothelin-1 Production through the Suppression of Nuclear Factor-.KAPPA.B

Transcription factor nuclear factor-kappa B (NF-kappaB) has been demonstrated to be important in regulating various gene expressions such as cytokines, adhesion molecules, and endothelin-1 (ET-1) in vascular endothelial cells. In the present study, we show the effects of xanthoangelol, xanthoangelol D, E, and F, which isolated from the root of Angelica keiskei KOIDZUMI (Umbelliferae), on NF-kappaB activation and ET-1 gene expression in cultured porcine aortic endothelial cells (PAECs). Treatments of xanthoangelol D but not xanthoangelol, xanthoangelol E and F markedly suppressed both of basal and tumor necrosis factor-alpha (TNF-alpha)-induced NF-kappaB activation in PAECs. To clarify the mechanism of xanthoangelol D-induced suppression on NF-kappaB activation, we evaluated the effects of xanthoangelol D on phosphorylation and degradation of IkappaBalpha, an inhibitory protein bound to NF-kappaB, and obtained evidence that xanthoangelol D selectively suppresses the phosphorylation of IkappaBalpha rather than the degradation of phosphorylated IkappaBalpha. In addition, xanthoangelol D significantly attenuated basal and TNF-alpha-induced prepro ET-1 mRNA expression in PAECs. These results suggest that xanthoangelol D may be useful for the treatment of various vascular diseases involved NF-kappaB activation.

INCA, a Novel Human Caspase Recruitment Domain Protein That Inhibits Interleukin-1β Generation

Using in silico methods for screening the human genome for new caspase recruitment domain (CARD) proteins, we have identified INCA (Inhibitory CARD) as a protein that shares 81% identity with the prodomain of caspase-1. The INCA gene is located on chromosome 11q22 between the genes of COP/Pseudo-ICE and ICEBERG, two other CARD proteins that arose from caspase-1 gene duplications. We show that INCA mRNA is expressed in many tissues. INCA is specifically upregulated by interferon-gamma in the monocytic cell lines THP-1 and U937. INCA physically interacts with procaspase-1 and blocks the release of mature IL-1beta from LPS-stimulated macrophages. Unlike COP/Pseudo-ICE and procaspase-1, INCA does not interact with RIP2 and does not induce NF-kappaB activation. Our data show that INCA is a novel intracellular regulator of procaspase-1 activation, involved in the regulation of pro-IL-1beta processing and its release during inflammation.

Clinical update: Novel targets in gynecologic malignancies

The proteasome inhibitor bortezomib has shown activity in chemotherapy-resistant tumors and is approved for treatment of multiple myeloma. The critical component of bortezomib's antitumor activity is the inhibition of nuclear factor-kappa B (NF-kappaB). Patients with ovarian cancer respond to initial platinum-based chemotherapy, such as cisplatin. However, these agents have been shown to induce tumor cell survival by inducing NF-kappaB activity. Phase I trials of bortezomib in solid tumors, including ovarian cancer, are summarized and examined to determine if the compound can overcome the impact of chemoresistance. In one trial of single-agent bortezomib in advanced malignancies, it was deemed a safe and manageable drug with potential efficacy in solid tumors. A second phase I trial explored inhibition of NF-kappaB with bortezomib to see if the drug rendered platinum agents more sensitive in ovarian cancer patients. Seven of the nine patients in the study had major responses to the combination of carboplatin and bortezomib. The two trials indicate promising results for bortezomib in patients with solid tumors and patients with recurrent ovarian cancer, but further investigation is warranted.

Fas Ligand Induces Cell-autonomous NF-κB Activation and Interleukin-8 Production by a Mechanism Distinct from That of Tumor Necrosis Factor-α

Fas ligand (FasL) has been well characterized as a death factor. However, recent studies revealed that FasL possesses inflammatory activity. Here we found that FasL induces production of the inflammatory chemokine IL-8 without inducing apoptosis in HEK293 cells. Reporter gene assays involving wild-type and mutated IL-8 promoters and NF-kappaB- and AP-1 reporter constructs indicated that an FasL-induced NF-kappaB and AP-1 activity are required for maximal promoter activity. FasL induced NF-kappaB activation with slower kinetics than did TNF-alpha, yet this response was cell autonomous and not mediated by secondary paracrine factors. The death domain of Fas, FADD, and caspase-8 were required for NF-kappaB activation by FasL. A dominant-negative mutant of IKKgamma inhibited the FasL-induced NF-kappaB activation. However, TRADD and RIP, which are essential for the TNF-alpha-induced NF-kappaB activation, were not involved in the FasL-induced NF-kappaB activation. Moreover, CLARP/FLIP inhibited the FasL- but not the TNF-alpha-induced NF-kappaB activation. These results show that FasL induces NF-kappaB activation and IL-8 production by a novel mechanism, distinct from that of TNF-alpha. In addition, we found that mouse FADD had a dominant-negative effect on the FasL-induced NF-kappaB activation in HEK293 cells, which may indicate a species difference between human and mouse in the FasL-induced NF-kappaB activation.

Nonsteroidal anti-inflammatory agents differ in their ability to suppress NF-kappaB activation, inhibition of expression of cyclooxygenase-2 and cyclin D1, and abrogation of tumor cell proliferation.

Nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin have been shown to suppress transcription factor NF-kappaB, which controls the expression of genes such as cyclooxygenase (COX)-2 and cyclin D1, leading to inhibition of proliferation of tumor cells. There is no systematic study as to how these drugs differ in their ability to suppress NF-kappaB activation and NF-kappaB-regulated gene expression or cell proliferation. In the present study, we investigated the effect of almost a dozen different commonly used NSAIDs on tumor necrosis factor (TNF)-induced NF-kappaB activation and NF-kappaB-regulated gene products, and on cell proliferation. Dexamethasone, an anti-inflammatory steroid, was included for comparison with NSAIDs. As indicated by DNA binding, none of the drugs alone activated NF-kappaB. All compounds inhibited TNF-induced NF-kappaB activation, but with highly variable efficacy. The 50% inhibitory concentration required was 5.67, 3.49, 3.03, 1.25, 0.94, 0.60, 0.38, 0.084, 0.043, 0.027, 0.024, and 0.010 mM for aspirin, ibuprofen, sulindac, phenylbutazone, naproxen, indomethacin, diclofenac, resveratrol, curcumin, dexamethasone, celecoxib, and tamoxifen, respectively. All drugs inhibited IkappaBalpha kinase and suppressed IkappaBalpha degradation and NF-kappaB-regulated reporter gene expression. They also suppressed NF-kappaB-regulated COX-2 and cyclin D1 protein expression in a dose-dependent manner. All compounds inhibited the proliferation of tumor cells, with 50% inhibitory concentrations of 6.09, 1.12, 0.65, 0.49, 1.01, 0.19, 0.36, 0.012, 0.016, 0.047, 0.013, and 0.008 mM for aspirin, ibuprofen, sulindac, phenylbutazone, naproxen, indomethacin, diclofenac, resveratrol, curcumin, dexamethasone, celecoxib, and tamoxifen, respectively. Overall these results indicate that aspirin and ibuprofen are least potent, while resveratrol, curcumin, celecoxib, and tamoxifen are the most potent anti-inflammatory and antiproliferative agents of those we studied.

NF-κB involvement in tumor–stroma interaction of squamous cell carcinoma

We investigated the interaction between tumor cells and stromal fibroblasts in tumor invasion of oral squamous cell carcinoma. Gelatin zymography showed that high levels of matrix metalloproteinase (MMP)-9 were present in the tissue of squamous cell carcinoma. When tumor cells and fibroblasts were isolated from the tissue and cultured separately, significant levels of MMP-9 were lost in the culture media of tumor cells as well as fibroblasts. When tumor cells and fibroblasts were cocultured in the presence of tumor necrosis factor alpha, high levels of MMP-9 were recovered in the culture media. The levels of MMP-9, which were secreted from tumor cells, but not fibroblasts, correlated with the number of cocultured fibroblasts. Cocultured fibroblasts, moreover, enhanced the induction of an active form of MMP-9, cell motility, and the activation of a transcription factor NF-kappaB in tumor cells. Stromal fibroblasts may induce NF-kappaB activation and promote the invasion of oral squamous cell carcinoma.

Taurodeoxycholate Stimulates Intestinal Cell Proliferation and Protects Against Apoptotic Cell Death Through Activation of NF- B

We hypothesized that the NF-kappaB pathway would be operative in the proliferative effect of bile salts on enterocytes. To determine this, we studied the effect of the bile salt taurodeoxycholate on cultured rat enterocyte proliferation and apoptosis and examined the role of NF-kappaB activation in these growth regulatory processes. Intestinal epithelial cells were grown for 6 days with or without taurodeoxycholate. Proliferation was measured. The cells were exposed to a known apoptotic stimulus, TNF-alpha and cyclohexamide. Apoptosis was quantified using cell number and the TUNEL stain. NF-kappaB activation was determined by an electrophoretic mobility shift assay. NF-kappaB activation was inhibited by an IkappaB superrepressor. Taurodeoxycholate stimulated cell proliferation (P < 0.01) and induced resistance to TNF-alpha induced apoptosis (P < 0.01). Taurodeoxycholate induced NF-kappaB activation. Inhibition of NF-kappaB prevented taurodeoxycholate-induced IEC-6 cell proliferation and rendered cells sensitive to TNF-alpha-induced apoptosis. Taurodeoxycholate stimulates intestinal epithelial cell proliferation and protects intestinal epithelial cells from TNF-alpha-induced apoptosis through NF-kappaB. These data support an important beneficial role of bile salts in regulation of mucosal growth and repair. Decreased enterocyte exposure to luminal bile salts, as occurs during starvation and parenteral nutrition, may have a detrimental effect on mucosal integrity.

A Novel Ubiquitin-like Domain in IκB Kinase β Is Required for Functional Activity of the Kinase

Activation of NF-kappaB requires two highly related kinases named IKKalpha and IKKbeta that share identity in the nature and positioning of their structural domains. Despite their similarity, the kinases are functionally divergent, and we therefore sought to identify any structural features specific for IKKalpha or IKKbeta. We performed bioinformatics analysis, and we identified a region resembling a ubiquitin-like domain (UBL) that exists only in IKKbeta and that we named the UBL-like domain (ULD). Deletion of the ULD rendered IKKbeta catalytically inactive and unable to induce NF-kappaB activity, and overexpression of only the ULD dose-dependently inhibited tumor necrosis factor-alpha-induced NF-kappaB activity. The ULD could not be functionally replaced within IKKbeta by ubiquitin or the corresponding region of IKKalpha, whereas deletion of the equivalent section of IKKalpha did not affect its catalytic activity against IkappaBalpha or its activation by NF-kappaB-inducing kinase. We identified five residues conserved among the larger family of UBL-containing proteins and IKKbeta, and alanine scanning revealed that the leucine at position 353 (Leu(353)) is absolutely critical for IKKbeta-induced NF-kappaB activation. Most intriguingly, the L353A mutant was catalytically active but, unlike wild-type IKKbeta, formed a stable complex with the NF-kappaB p65 subunit. Our findings therefore establish the ULD as a critical functional domain specific for IKKbeta that might play a role in dissociating IKKbeta from p65.

Genetic Deletion of Glycogen Synthase Kinase-3β Abrogates Activation of IκBα Kinase, JNK, Akt, and p44/p42 MAPK but Potentiates Apoptosis Induced by Tumor Necrosis Factor

Glycogen synthase kinase (GSK)-3beta is a constitutively active, proline-directed serine/threonine kinase that controls growth modulation and tumorigenesis through multiple intracellular signaling pathways. How GSK-3beta regulates signaling pathways induced by cytokines such as tumor necrosis factor (TNF) is poorly understood. In this study, we used fibroblasts derived from GSK-3beta gene-deleted mice to understand the role of this kinase in TNF signaling. TNF induced NF-kappaB activation as measured by DNA binding in wild-type mouse embryonic fibroblasts, but deletion of GSK-3beta abolished this activation. This inhibition was due to suppression of IkappaBalpha kinase activation and IkappaBalpha phosphorylation, ubiquitination, and degradation. TNF-induced NF-kappaB reporter gene transcription was also suppressed in GSK-3beta gene-deleted cells. NF-kappaB activation induced by lipopolysaccharide, interleukin-1beta, or cigarette smoke condensate was completely suppressed in GSK-3beta(-/-) cells. Deletion of GSK-3beta also abolished TNF-induced c-Jun N-terminal kinase and p44/p42 mitogen-activated kinase activation. Most surprisingly, TNF-induced Akt activation also required the presence of GSK-3beta. TNF induced expression of the NF-kappaB-regulated gene products cyclin D1, COX-2, MMP-9, survivin, IAP 1, IAP 2, Bcl-x(L), Bfl-1/A1, TRAF1, and FLIP in wild-type mouse embryonic fibroblasts but not in GSK-3beta(-/-) cells, and this correlated with potentiation of TNF-induced apoptosis as indicated by cell viability, annexin V staining, and caspase activation. Overall, our results indicate that GSK-3beta plays a critical role in TNF signaling and in the signaling of other inflammatory stimuli and that its suppression can be exploited as a potential target to inhibit angiogenesis, proliferation, and survival of tumor cells.

Accumulation of inhibitory kappaB-alpha as a mechanism contributing to the anti-inflammatory effects of surfactant protein-A.

The collectin surfactant protein (SP)-A has been implicated in multiple immunoregulatory functions of innate pulmonary host defense via modulating immune responses both in vitro and in vivo. The aim of the present study was to investigate mechanisms responsible for the anti-inflammatory effects of human (hu) SP-A on the inhibitory kappaB (IkappaB)/nuclear factor (NF)-kappaB signaling pathway in alveolar macrophages (AMs). Initial CD25 expression analysis by flow cytometry of CD14/hu Toll-like receptor 4-transfected Chinese hamster ovary reporter cells demonstrated that SP-A alone does not induce any NF-kappaB-dependent CD25 expression in these cells. In AMs, SP-A pretreatment caused a marked inhibition of lipopolysaccharide (LPS)-induced NF-kappaB activation independent of the LPS chemotype used as determined by electrophoretic mobility shift assay. Western blot analysis revealed that SP-A by itself increased the protein expression of IkappaB-alpha, the predominant regulator for rapidly induced NF-kappaB, in a dose- and time-dependent manner without enhancing IkappaB-alpha messenger RNA as determined by reverse transcription-polymerase chain reaction. SP-A did not interfere with LPS-induced serine(32) phosphorylation of IkappaB-alpha but significantly enhanced IkappaB-alpha abundance under LPS-coupled conditions. The data suggest that anti-inflammatory effects of SP-A on LPS-challenged AMs are associated with a SP-A-mediated direct modulation of the IkappaB-alpha turnover in these cells.

β-d-Glucoside Suppresses Tumor Necrosis Factor-induced Activation of Nuclear Transcription Factor κB but Potentiates Apoptosis

Mangiferin, a natural polyphenol is known to exhibit anti-inflammatory, antioxidant, and antiviral effects. However the molecular mechanism underlying these effects has not been well characterized. Because NF-kappaB plays an important role in these processes, it is possible that mangiferin modulates NF-kappaB activation. Our results show that mangiferin blocks tumor necrosis factor (TNF)-induced NF-kappaB activation and NF-kappaB-dependent genes like ICAM1 and COX2. The effect was mediated through inhibition of IKK activation and subsequent blocking of phosphorylation and degradation of IkappaBalpha. In addition, mangiferin inhibits TNF-induced p65 phosphorylation as well as translocation to the nucleus and also inhibits NF-kappaB activation induced by other inflammatory agents like PMA, ceramide, and SA-LPS. Mangiferin, similar to the other known antioxidants, NAC and PDTC, inhibits TNF-induced reactive oxygen intermediate (ROI) generation. Since intracellular glutathione (GSH) levels are known to modulate NF-kappaB levels, we measured the levels of GSH. Mangiferin enhances glutathione level by almost 2-fold more than other anti-oxidants, and at the same time it decreases the levels of GSSG and increases the activity of catalase. Depletion of GSH by buthionine sulfoximine led to a significant reversal of mangiferin effect. Hence mangiferin with its ability to inhibit NF-kappaB and increase the intracellular GSH levels may prove to be a potent drug for anti-inflammatory and antioxidant therapy. Mangiferin-mediated down-regulation of NF-kappaB also potentiates chemotherapeutic agent-mediated cell death, suggesting a role in combination therapy for cancer.

Inducible nuclear factor-kappaB activation contributes to chemotherapy resistance in gastric cancer.

Activation of nuclear factor-kappaB (NF-kappaB) inhibits chemotherapy-induced apoptosis in some cancer cell lines. Inhibition of NF-kappaB by adenoviral delivery of an IkappaBalpha superrepressor (Ad.IkappaBalpha-SR) should potentiate 5-fluorouracil (5-FU) and irinotecan chemotherapy in gastric cancer cells. NCI-N87 and AGS human gastric cancer cells were studied. Chemotherapy-induced NF-kappaB activation was assessed using a luciferase reporter assay. Inhibition of NF-kappaB was assessed by luciferase reporter assay and by electrophoretic mobility shift assay. Cells were pretreated for 1 hour with Ad.IkappaBalpha (25 MOI) and incubated with 5-FU or the active metabolite of irinotecan (SN-38). Cell growth was assessed by cell proliferation assay and induction of apoptosis was determined by flow cytometry and caspase 3/7 assay. 5-FU and SN-38 significantly induced NF-kappaB activation as measured by luciferase reporter assay (p < 0.001). Ad.IkappaBalpha-SR treatment inhibited NF-kappaB binding as demonstrated by electrophoretic mobility shift assay and by luciferase reporter assay. In AGS cells, pretreatment with Ad.IkappaBalpha-SR followed by 5-FU (0.005 mmol/L) or SN-38 (10 ng/mL) led to increased growth inhibition of 13% and 59%, respectively (p < 0.001). Similarly, growth inhibition in NCI cells was significantly increased by pretreatment with Ad.IkappaBalpha followed by 5-FU (0.001 mmol/L) or SN-38 (0.5 ng/mL) (p < 0.001). In both cell lines, Ad.IkappaBalpha-SR enhanced apoptosis by both flow cytometry and caspase 3/7 assay as compared with chemotherapy alone. NF-kappaB is activated in human gastric cancer in response to chemotherapy and may result in inducible chemoresistance. Inhibition of NF-kappaB by Ad.IkappaBalpha-SR enhances the antitumor effects of chemotherapy and has potential as a novel antineoplastic strategy.

Cyclooxygenase (COX)-2 inhibitor celecoxib abrogates activation of cigarette smoke-induced nuclear factor (NF)-kappaB by suppressing activation of IkappaBalpha kinase in human non-small cell lung carcinoma: correlation with suppression of cyclin D1, COX-2, and matrix metalloproteinase-9.

Cigarette smoke (CS) has been linked to cardiovascular, pulmonary, and malignant diseases. CS-associated malignancies including cancers of the larynx, oral cavity, and pharynx, esophagus, pancreas, kidney, bladder, and lung; all are known to overexpress the nuclear factor-kappaB (NF-kappaB)-regulated gene products cyclin D1, cyclooxygenase (COX)-2, and matrix metalloprotease-9. Whether the COX-2 inhibitor, celecoxib, approved for the treatment of colon carcinogenesis and rheumatoid arthritis, affects CS-induced NF-kappaB activation is not known, although the role of NF-kappaB in regulation of apoptosis, angiogenesis, carcinogenesis, and inflammation is established. In our study, in which we examined DNA binding of NF-kappaB in human lung adenocarcinoma H1299 cells, we found that cigarette smoke condensate (CSC)-induced NF-kappaB activation was persistent up to 24 h, and celecoxib suppressed CSC-induced NF-kappaB activation. Celecoxib was effective even when administered 12 h after CSC treatment. This effect, however, was not cell type-specific. The activation of inhibitory subunit of NF-kappaB kinase (IkappaB), as examined by immunocomplex kinase assay, IkappaB phosphorylation, and IkappaB degradation was also inhibited. Celecoxib also abrogated CSC-induced p65 phosphorylation and nuclear translocation and NF-kappaB-dependent reporter gene expression. CSC-induced NF-kappaB reporter activity induced by NF-kappaB inducing kinase and IkappaB alpha kinase but not that activated by p65 was also blocked by celecoxib. CSC induced the expression of NF-kappaB-regulated proteins, COX-2, cyclin D1, and matrix metalloproteinase-9, and celecoxib abolished the induction of all three. The COX-2 promoter that is regulated by NF-kappaB was activated by CSC, and celecoxib suppressed its activation. Overall, our results suggest that chemopreventive effects of celecoxib may in part be mediated through suppression of NF-kappaB and NF-kappaB-regulated gene expression, which may contribute to its ability to suppress inflammation, proliferation, and angiogenesis.

Monocyte-derived dendritic cells that capture dead tumor cells secrete IL-12 and TNF-alpha through IL-12/TNF-alpha/NF-kappaB autocrine loop.

This study focused on the question of how monocyte-derived dendritic cells (Mo-DCs) that capture dead tumor cells (Mo-DCs-Tum) secrete interleukin 12 (IL-12) and tumor necrosis factor alpha (TNF-alpha). Mo-DCs-Tum showed higher secretions of IL-12 and TNF-alpha than were shown by Mo-DCs. Enhanced nuclear factor-kappa B (NF-kappaB) activation was also induced in Mo-DCs-Tum within 6 h. The NF-kappaB inhibitor, pyrrolidine dithiocarbamate (PDTC), suppressed both IL-12 and TNF-alpha secretions from Mo-DCs-Tum. Administration of recombinant TNF-alpha or IL-12 enhanced IL-12 or TNF-alpha secretion respectively in Mo-DCs-Tum. Addition of anti-TNF-alpha or anti-IL-12 neutralizing antibody decreased NF-kappaB activation and IL-12 or TNF-alpha secretion in Mo-DCs-Tum. These results suggest that TNF-alpha or IL-12 secretion induces NF-kappaB activation, and it stimulates further TNF-alpha and IL-12 secretions, i.e., an IL-12/TNF-alpha/NF-kappaB autocrine loop, in Mo-DCs-Tum. Thus, Mo-DCs-Tum secrete a large amount of IL-12 and TNF-alpha through accelerated NF-kappaB activation induced by the IL-12/TNF-alpha/NF-kappaB autocrine loop.

P0877 HELICOBACTER PEPTIDOGLYCAN INDUCES NOD1-DEPENDENT PRO-INFLAMMATORY ACTIVATION

Introduction:Helicobacter pylori that harbor the cag pathogenicity island (cag PAI) induce NFkappaB activation and IL-8 synthesis in gastric epithelial cells and are responsible for the most severe inflammatory gastric lesions in humans. Since the intracellular receptor NOD1/CARD4 detects Gram-negative bacterial peptidoglycan, we studied the role of NOD1 in mediating the inflammatory response during H. pylori infection. Methods: Epithelial HEK293 cells were transfected with an NFkappaB-dependent luciferase reporter gene plus a NOD1 dominant negative construct or vector control. NFkappaB activation was determined after co-culture with different H. pylori strains or after intracellular delivery of bacterial compounds. IL-8 was measured by ELISA in infected gastric epithelial cells (AGS). The cellular distribution of H. pylori peptidoglycan was analyzed in AGS cells that were co-cultured with functional or non-functional cag PAI H. pylori in which the peptidoglycan had been tritium labeled. NOD1-/- and +/+ mice were infected by different Helicobacter strains and the bacterial loads were determined after 7 and 30 days post-infection. Results: Our results demonstrate that a functional cag PAI was necessary for H. pylori to activate NFkappaB and IL-8 synthesis in epithelial cells. A functionnal cag PAI was required for the translocation of peptidoglycan to the host cells. The transfec-tion of dominant negative NOD1 in HEK293 cells reduced NFkap-paB activation during H. pylori infection. Finally, NOD1 −/− mice were more sensitive to cag PAI-positive-H. pylori infection compared to wild-type controls. Conclusion: In conclusion, our findings suggest that H. pylori uses its cag PAI encoded secretion system to translocate peptidoglycan inside gastric epithelial cells where it can interact with NOD1 and thereby induce inflammatory signaling through NFkappaB activation. NOD1 is an intracellular receptor involved in the inflammatory response induced during the H. pylori infection and could hence participate in the ability of epithelial cells to differentiate between commensal flora and pathogens.

Cryopyrin-induced Interleukin 1β Secretion in Monocytic Cells: ENHANCED ACTIVITY OF DISEASE-ASSOCIATED MUTANTS AND REQUIREMENT FOR ASC

Several autoinflammatory disorders are associated with missense mutations within the nucleotide-binding oligomerization domain of cryopyrin. The mechanism by which cryopyrin mutations cause inflammatory disease remains elusive. To understand the molecular bases of these diseases, we generated constructs to express three common cryopyrin disease-associated mutations, R260W, D303N, and E637G, and compared their activity with that of the wild-type protein. All cryopyrin mutant proteins tested were found to induce potent NF-kappaB activity when compared with the wild-type protein. This activation was dependent on the expression of ASC, an adaptor protein previously suggested to mediate cryopyrin signaling. When the disease-associated mutants were expressed in monocytic THP-1 cells (which express endogenous ASC), each induced spontaneous IL-1beta secretion, whereas wild-type protein did not. In the absence of stimuli, wild-type cryopyrin was unable to bind to ASC, whereas the three mutants coimmunoprecipitated with ASC, suggesting a mechanism involved in the constitutive activation of mutant proteins. The induction of cryopyrin activity by enforced oligomerization in THP-1 cells resulted in ASC binding and the secretion of IL-1beta, an effect that was abolished by the inhibition of ASC expression with small interfering RNAs. Thus, cryopyrin-mediated IL-1beta secretion requires ASC in monocytic cells. Further, these results indicate that cryopyrin disease-associated mutants are constitutively active and able to induce NF-kappaB activation and IL-1beta secretion at least in part by an increased ability to interact with ASC.

Toll-Like Receptor 4 Mediates the Antitumor Host Response Induced by a 55-Kilodalton Protein Isolated fromAeginetia indicaL., a Parasitic Plant

A 55-kDa protein named AILb-A, isolated from the seed extract of Aeginetia indica L., a parasitic plant, induces a Th1-type T-cell response and elicits a marked antitumor effect in tumor-bearing mice. In the present study, we examined the role of Toll-like receptors (TLRs), which have been implicated in pathogen-induced cell signaling, in AILb-A-induced immune responses. In the luciferase assay using a nuclear factor (NF)-kappaB-dependent reporter plasmid, AILb-A induced NF-kappaB activation in the cells transfected with TLR4, but not with those transfected with the TLR2 gene, in a dose-dependent manner. TLR4-mediated NF-kappaB activation induced by AILb-A but not by lipopolysaccharide (LPS) was also observed under serum-free conditions. In in vitro experiments using human peripheral blood mononuclear cells, AILb-A-induced cytokine production was markedly inhibited by anti-TLR4 but not by anti-CD14 antibody, while LPS-induced, TLR4-mediated cytokine production was inhibited by anti-CD14 as well as anti-TLR4 antibodies. Cytokine production, killer cell activities, maturation of dendritic cells, phosphorylation of mitogen-activated protein kinases, and nuclear translocation of interferon-regulatory factor 3 induced by AILb-A were severely impaired in TLR4-deficient but not TLR2-deficient mice. Transfection of TLR4-deficient mouse-derived macrophages with the TLR4 expression plasmid led AILb-A to induce cytokines. Finally, the antitumor effect of AILb-A was also impaired in TLR4-deficient and TLR4-mutated mice. These findings suggest that TLR4 mediates antitumor immunity induced by the plant-derived protein AILb-A.

Apoptosis-inducing effect of chemotherapeutic agents is potentiated by soy isoflavone genistein, a natural inhibitor of NF-kappaB in BxPC-3 pancreatic cancer cell line.

Cancer chemotherapeutic strategies should be devised to provide higher tumor response and lower toxicity for combination chemotherapy. Genistein has been shown to inhibit the growth of various cancer cells in vitro and in vivo without toxicity to normal cells. The antitumor effects of genistein could be in part due to inactivation of NF-kappaB activity. In contrast, chemotherapeutic agents inadvertently induce NF-kappaB activity, which may lead to chemoresistance. In this study, we investigated whether the inactivation of NF-kappaB by genistein would enhance the efficacy of chemotherapeutic agents. BxPC-3 pancreatic cancer cells were pretreated with 30 micromol/L genistein for 24 hours and then exposed to lower concentrations of chemotherapeutic agents for an additional 24 hours. Cell growth inhibition assay, apoptosis assay, and NF-kappaB EMSA were performed. The combination of 30 micromol/L genistein with 1 nmol/L docetaxel or 100 nmol/L cisplatin elicited significantly greater inhibition of cell growth compared with either agent alone. The combination treatment induced more apoptosis in BxPC-3 cells compared with single agents. Moreover, the NF-kappaB activity was significantly increased within 2 hours of docetaxel or cisplatin treatment, and the NF-kappaB-inducing activity of these agents was completely abrogated in cells pretreated with genistein. These results clearly suggest that genistein pretreatment, which inactivates NF-kappaB activity, together with other cellular effects of genistein, may contribute to increased cell growth inhibition and apoptosis inducing effects of nontoxic doses of docetaxel and cisplatin, which could be a novel strategy for the treatment of pancreatic cancer.

DOUBLE-STRANDED RNA AND BACTERIAL LPS-INDUCED ACTIVATION OF VASCULAR SMOOTH MUSCLE CELLS: ROLE OF TOLL-LIKE RECEPTOR 3 AND TOLL-LIKE RECEPTOR 4

A growing body of evidence supports a central role of inflammation in the pathogenesis of atherosclerosis, however the triggers that initiate the inflammatory process have not been definitively identified. Infectious agents, including both bacteria and viruses, are among the proposed initiators of inflammation, yet the molecular mechanisms by which microbes activate cells in the vessel wall are unknown. Recent evidence supports a central role of transmembrane Toll-like receptors (TLRs) in cellular activation by microbial products. Ten different TLRs are activated by distinct microbial ligands, and in turn elicit differential cellular responses. To test the possible role of TLRs in vascular smooth muscle cell (VSMC) activation, we characterized the expression of TLR mRNAs in VSMC, and characterized the activation of VSMC by their respective microbial ligands. VSMC isolated from either human pulmonary artery or human aorta were found to express mRNA encoding TLR3, TLR4 and TLR6. TLR3 has recently emerged as an important receptor for poly ( IC), a mimic of double-stranded RNA that is produced by many viruses, whereas TLR4 is the receptor for bacterial lipopolysaccharide (LPS). Poly ( IC) was a potent stimulator of human VSMC, markedly stimulating the cellular release of monocyte-chemoattractant protein-1 (MCP-1), and the levels of cell-associated interleukin-1alpha ( IL-1alpha ), a potent SMC mitogen. LPS likewise stimulated MCP-1 release and IL-1alpha synthesis. Both poly ( IC) and LPS markedly induced NFkappaB activation in human VSMC, and in aortic SMC derived from wild-type mice that express functional TLR4. Poly ( IC) also activated NFkappaB in VSMC derived from mice that express a non-functional form of TLR4, whereas LPS was ineffective. Expression of a dominant negative form of TLR3 attenuated poly ( IC)-, but not LPSor IL-1alpha-induced NFkappaB activation in mouse aortic SMC. Poly ( IC ) also markedly stimulated proliferation in human VSMC and in mouse aortic VSMC derived from either wild-type or TLR4 mutant mice. In contrast, bacterial LPS did not affect proliferation either when added alone or in the presence of the LPS transfer factor CD14. These studies support the hypothesis that double-stranded RNA activates VSMC via TLR3, whereas bacterial LPS activates VSMC via TLR4, and that TLR3 and TLR4 appear to elicit differential effects on VSMC proliferation.

Activation of NF-kappaB in intestinal epithelial cells by E. coli strains isolated from the colonic mucosa of IBD patients.

The involvement of bacteria in the pathogenesis of inflammatory bowel disease has been discussed for several years. In this study we evaluated the ability of E. coli isolates from inflamed and noninflamed colonic mucosa to activate NF-kappaB. Fifteen bacterial strains from inflamed and six from noninflamed colonic tissues from IBD patients. Their ability to induce NF-kappaB activation was examined in vitro by gel-shift assays. The activation of the TNF-alpha promoter was determined by reporter gene assays. Bacterial isolates were characterized by invasion assays, electron microscopy, and PCR. Four of 15 E. coli bacterial isolates from inflamed IBD tissues induced NF-kappaB activity in intestinal epithelial cells as determined by gel-shift assays. NF-kappaB activation was only seen with living bacteria but not with heat-inactivated cells. Isolates from noninflamed tissues and a wild-type E. coli control strain induced a weaker or no activation. Reporter gene assays with a construct comprising a luciferase gene driven by the TNF-alpha promoter revealed that isolates from Crohn's disease patients induced a stronger activation of the TNF-alpha gene than isolates from ulcerative colitis patients. The isolated bacteria invaded HT-29 cells, although typical virulence genes for enteropathogenic, enterhemorrhagic, or enteroinvasive E. coli, i.e., eae, tir, EspA, Per (A-C), ipaC, were not detected in these cells. Bacterial invasion was additionally confirmed by electron microscopy examination. Our results indicate that E. coli strains can be found in the mucosa of some IBD patients which are able to activate NF-kappaB similar to known pathogenic strains. The absence of several virulence genes in these cells suggests that they are members of the luminal flora which acquire as yet unidentified virulence determinants and are therefore involved in the pathophysiology of IBD.