Expression Of NF-kappaB Target Genes Research Articles

Abstract A86: LRP16, interacting with PKR, prolongs NF-kappaB activation and drives colorectal cancer tumorigenesis

Abstract NF-kappaB frequently activated in colorectal cancer (CRC) is linked to rapid malignant progression of CRC and is also crucial for the maintenance of homeostasis and therapy resistance. However, the underlying mechanisms how NF-kappaB activation and the crosstalk of NF-kappaB with other transcription factors are controlled in CRC remain incompletely understood. Here, we show that LRP16 physically interacts with double-stranded RNA-dependent protein kinase (PKR) functions as a signal transducer in signaling pathways activated by different stimuli by a variety of effectors, including NF-kappaB. Overexpression of LRP16 confers PKR autophosphorylation leading to the phosphorylation and activation of the NF-kappaB complex. Modulation of NF-kappaB mediated gene expression in response to diverse signals is coordinated by PKR through the phosphorylation and activation of IKK complex and this process does not require PKR kinase activity. We identified PKR, an essential regulatory subunit of the IKK complex. Silencing PKR expression by RNA interference blocked LRP16 induced expression of NF-kappaB target genes. These cells were also not protected from apoptosis in response to chemotherapy. In vivo, overexpression of LRP16 promotes CRC tumorigenesis, in turn, inhibition of LRP16 or PKR blunts CRC tumorigenesis. These results demonstrated that LRP16 likely functions by recruiting PKR to the IKK complex and thus serves a regulatory function for NF-kappaB activation, suggesting that a novel role for LRP16 as mediator of chemotherapy resistance modulated in part through the protective effects of NF-kappaB activation and may be a novel strategy in the future to overcome chemotherapy resistance in CRC. Citation Format: Xiaolei Li, Zhiqiang Wu, Weidong Han. LRP16, interacting with PKR, prolongs NF-kappaB activation and drives colorectal cancer tumorigenesis. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A86.

Abstract B29: LRP16 is an essential mediator for DNA double-strand breaks induced NF-kappaB activation

Abstract DNA double-strand breaks (DSBs) are an intermediate in several important processes, such as V(D)J recombination, class-switch recombination, and meiosis; and a byproduct of a number of both normal or pathological conditions, such as metabolic respiration and inflammation. Thus, the determination of the proper fates of cells with this specific type of DNA damage, i.e., to repair and survive or to die, constitutes an important element in the homeostasis of individual mammals. The activation of NF-kappaB has emerged as an important mechanism for the modulation of the response to DSBs. The concomitant SUMOylation and phosphorylation of IKKgamma; by the SUMO E3 ligase protein inhibitor of activated STAT (PIASy) and the phosphorylation by ataxia telangiectasia mutated (ATM), respectively, is a key event in this mechanism. However, IKKgamma SUMOylation or phosphorylation can occur separately without activating NF-kappaB. Thus, the mechanism through which mammalian cells are able to accomplish these IKKgamma modifications in a timely and lesion-specific manner remains unclear. In this study, we demonstrate that LRP16 constitutively interacts with PARP1 and IKKgamma. This interaction is essential for efficient interactions among PARP1, IKKgamma, and PIASy, the modifications of IKKgamma, and the activation of NF-kappaB following DSB induction. The regulation of LRP16 in NF-kappaB activation is dependent on its poly (ADP-ribose) binding capability through the unique macro domain. The depletion of the DSB-specific sensor Ku80 resulted in a significant reduction in the physical interactions among LRP16, PARP1 and IKKgamma. Additionally, the knockdown of either endogenous Ku80 or Ku70 by siRNA markedly diminished DSB-induced NF-kappaB reporter gene activity and NF-kappaB target gene expression. These data strongly suggest that LRP16, through its constitutive interactions with PARP1 and IKKgamma, functions to facilitate the lesion-specific recruitment of PARP1 and IKKgamma and, ultimately, the concomitant recruitment of PIASy to IKKgamma in response to DSB damage. Therefore, the study has provided important new mechanistic insights concerning DSB-induced NF-kappaB activation. Citation Format: Zhiqiang Wu, Chunmeng Wang, Miaomiao Bai, Xiaolei Li, Qian Mei, Xiang Li, Yao Wang, Xiaobing Fu, Guangbin Luo, Weidong Han. LRP16 is an essential mediator for DNA double-strand breaks induced NF-kappaB activation. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr B29.

Bmi-1 promotes the aggressiveness of glioma via activating the NF-kappaB/MMP-9 signaling pathway

BackgroundThe prognosis of human glioma is poor, and the highly invasive nature of the disease represents a major impediment to current therapeutic modalities. The oncoprotein B-cell-specific Moloney murine leukemia virus integration site 1 protein (Bmi-1) has been linked to the development and progression of glioma; however, the biological role of Bmi-1 in the invasion of glioma remains unclear.MethodsA172 and LN229 glioma cells were engineered to overexpress Bmi-1 via stable transfection or to be silenced for Bmi-1 expression using RNA interfering method. Migration and invasiveness of the engineered cells were assessed using wound healing assay, Transwell migration assay, Transwell matrix penetration assay and 3-D spheroid invasion assay. MMP-9 expression and activity were measured using real-time PCR, ELISA and the gelatin zymography methods. Expression of NF-kappaB target genes was quantified using real-time PCR. NF-kappaB transcriptional activity was assessed using an NF-kappaB luciferase reporter system. Expression of Bmi-1 and MMP-9 in clinical specimens was analyzed using immunohistochemical assay.ResultsEctopic overexpression of Bmi-1 dramatically increased, whereas knockdown of endogenous Bmi-1 reduced, the invasiveness and migration of glioma cells. NF-kappaB transcriptional activity and MMP-9 expression and activity were significantly increased in Bmi-1-overexpressing but reduced in Bmi-1-silenced cells. The reporter luciferase activity driven by MMP-9 promoter in Bmi-1-overexpressing cells was dependent on the presence of a functional NF-kappaB binding site, and blockade of NF-kappaB signaling inhibited the upregulation of MMP-9 in Bmi-1 overexpressing cells. Furthermore, expression of Bmi-1 correlated with NF-kappaB nuclear translocation as well as MMP-9 expression in clinical glioma samples.ConclusionsBmi-1 may play an important role in the development of aggressive phenotype of glioma via activating the NF-kappaB/MMP-9 pathway and therefore might represent a novel therapeutic target for glioma.

Hypoxia-Inducible Factor 1α Determines Gastric Cancer Chemosensitivity via Modulation of p53 and NF-κB

BackgroundReduced chemosensitivity of solid cancer cells represents a pivotal obstacle in clinical oncology. Hence, the molecular characterization of pathways regulating chemosensitivity is a central prerequisite to improve cancer therapy. The hypoxia-inducible factor HIF-1α has been linked to chemosensitivity while the underlying molecular mechanisms remain largely elusive. Therefore, we comprehensively analysed HIF-1α's role in determining chemosensitivity focussing on responsible molecular pathways.Methodology and Principal FindingsRNA interference was applied to inactivate HIF-1α or p53 in the human gastric cancer cell lines AGS and MKN28. The chemotherapeutic agents 5-fluorouracil and cisplatin were used and chemosensitivity was assessed by cell proliferation assays as well as determination of cell cycle distribution and apoptosis. Expression of p53 and p53 target proteins was analyzed by western blot. NF-κB activity was characterized by means of electrophoretic mobility shift assay. Inactivation of HIF-1α in gastric cancer cells resulted in robust elevation of chemosensitivity. Accordingly, HIF-1α-competent cells displayed a significant reduction of chemotherapy-induced senescence and apoptosis. Remarkably, this phenotype was completely absent in p53 mutant cells while inactivation of p53 per se did not affect chemosensitivity. HIF-1α markedly suppressed chemotherapy-induced activation of p53 and p21 as well as the retinoblastoma protein, eventually resulting in cell cycle arrest. Reduced formation of reactive oxygen species in HIF-1α-competent cells was identified as the molecular mechanism of HIF-1α-mediated inhibition of p53. Furthermore, loss of HIF-1α abrogated, in a p53-dependent manner, chemotherapy-induced DNA-binding of NF-κB and expression of anti-apoptotic NF-κB target genes. Accordingly, reconstitution of the NF-κB subunit p65 reversed the increased chemosensitivity of HIF-1α-deficient cells.Conclusion and SignificanceIn summary, we identified HIF-1α as a potent regulator of p53 and NF-κB activity under conditions of genotoxic stress. We conclude that p53 mutations in human tumors hold the potential to confound the efficacy of HIF-1-inhibitors in cancer therapy.

Differential expression of NF-κB target genes in MALT lymphoma with and without chromosome translocation: insights into molecular mechanism

Mucosa-associated lymphoid tissue (MALT) lymphoma is characterized by t(11;18)(q21;q21)/API2-MALT1, t(1;14)(p22;q32)/BCL10-IGH and t(14;18)(q32;q21)/IGH-MALT1, which commonly activate the nuclear factor (NF)-kappaB pathway. Gastric MALT lymphomas harboring such translocations usually do not respond to Helicobacter pylori eradication, while most of those without translocation can be cured by antibiotics. To understand the molecular mechanism of these different MALT lymphoma subgroups, we performed gene expression profiling analysis of 21 MALT lymphomas (13 translocation-positive, 8 translocation-negative). Gene set enrichment analysis (GSEA) of the NF-kappaB target genes and 4394 additional gene sets covering various cellular pathways, biological processes and molecular functions have shown that translocation-positive MALT lymphomas are characterized by an enhanced expression of NF-kappaB target genes, particularly toll like receptor (TLR)6, chemokine, CC motif, receptor (CCR)2, cluster of differentiation (CD)69 and B-cell CLL/lymphoma (BCL)2, while translocation-negative cases were featured by active inflammatory and immune responses, such as interleukin-8, CD86, CD28 and inducible T-cell costimulator (ICOS). Separate analyses of the genes differentially expressed between translocation-positive and -negative cases and measurement of gene ontology term in these differentially expressed genes by hypergeometric test reinforced the above findings by GSEA. Finally, expression of TLR6, in the presence of TLR2, enhanced both API2-MALT1 and BCL10-mediated NF-kappaB activation in vitro. Our findings provide novel insights into the molecular mechanism of MALT lymphomas with and without translocation, potentially explaining their different clinical behaviors.

Cytosolic Hsp60 Is Involved in the NF-κB-Dependent Survival of Cancer Cells via IKK Regulation

Cytoplasmic presence of Hsp60, which is principally a nuclear gene-encoded mitochondrial chaperonin, has frequently been stated, but its role in intracellular signaling is largely unknown. In this study, we demonstrate that the cytosolic Hsp60 promotes the TNF-α-mediated activation of the IKK/NF-κB survival pathway via direct interaction with IKKα/β in the cytoplasm. Selective loss or blockade of cytosolic Hsp60 by specific antisense oligonucleotide or neutralizing antibody diminished the IKK/NF-κB activation and the expression of NF-κB target genes, such as Bfl-1/A1 and MnSOD, which thus augmented intracellular ROS production and ASK1-dependent cell death, in response to TNF-α. Conversely, the ectopic expression of cytosol-targeted Hsp60 enhanced IKK/NF-κB activation. Mechanistically, the cytosolic Hsp60 enhanced IKK activation via upregulating the activation-dependent serine phosphorylation in a chaperone-independent manner. Furthermore, transgenic mouse study showed that the cytosolic Hsp60 suppressed hepatic cell death induced by diethylnitrosamine in vivo. The cytosolic Hsp60 is likely to be a regulatory component of IKK complex and it implicates the first mitochondrial factor that regulates cell survival via NF-κB pathway.

HHQ and PQS, two Pseudomonas aeruginosa quorum‐sensing molecules, down‐regulate the innate immune responses through the nuclear factor‐κB pathway

To explore whether bacterial secreted 4-hydroxy-2-alkylquinolines (HAQs) can regulate host innate immune responses, we used the extracts of bacterial culture supernatants from a wild-type (PA14) and two mutants of Pseudomonas aeruginosa that have defects in making HAQs. Surprisingly, the extract of supernatants from the P. aeruginosa pqsA mutant that does not make HAQs showed strong stimulating activity for the production of innate cytokines such as tumour necrosis factor-alpha and interleukin-6 in the J774A.1 mouse monocyte/macrophage cell line, whereas the extract from the wild-type did not. The addition of 4-hydroxy-2-heptylquinoline (HHQ) or 2-heptyl-3,4-dihydroxyquinoline (PQS, Pseudomonas quinolone signal) to mammalian cell culture media abolished this stimulating activity of the extracts of supernatants from the pqsA mutant on the expression of innate cytokines in J774A.1 cells and in the primary bronchoalveolar lavage cells from C57BL/6 mice, suggesting that HHQ and PQS can suppress the host innate immune responses. The pqsA mutant showed reduced dissemination in the lung tissue compared with the wild-type strain in a mouse in vivo intranasal infection model, suggesting that HHQ and PQS may play a role in the pathogenicity of P. aeruginosa. HHQ and PQS reduced the nuclear factor-kappaB (NF-kappaB) binding to its binding sites and the expression of NF-kappaB target genes, and PQS delayed inhibitor of kappaB degradation, indicating that the effect of HHQ and PQS was mediated through the NF-kappaB pathway. Our results suggest that HHQ and PQS produced by P. aeruginosa actively suppress host innate immune responses.

Combination of ginsenoside Rg3 with docetaxel enhances the susceptibility of prostate cancer cells via inhibition of NF-κB

Ginsenoside Rg3 has been a subject of interest for use as a cancer preventive or therapeutic agent. Nuclear factor-kappa (NF-kappaB) is constitutively activated in prostate cancer, and gives cancer cells resistance to chemotherapeutic agents. To investigate whether Rg3 can suppress the activation of NF-kappaB, and thus increase susceptibility of prostate (LNCaP and PC-3, DU145) cells against chemotherapeutics, prostate cancer cell growth as well as activation of NF-kappaB was examined. We found that a combination treatment of Rg3 (50 microM) with a conventional agent docetaxel (5 nM) was more effective in the inhibition of prostate cancer cell growth and induction of apoptosis as well as G(0)/G(1) arrest accompanied with the significant inhibition of NF-kappaB activity than those by treatment of Rg3 or docetaxel alone. It was also found that NF-kappaB target gene expression of Bax, caspase-3, and caspase-9 was much more significantly enhanced, but the expression of Bcl-2, inhibitor of apoptosis protein (IAP-1) and X chromosome IAP (XIAP), and the expression of cell cycle regulatory proteins cyclin B, D1 and E, and cyclin dependent kinases 2 and 4 was also much more significantly inhibited by the combination treatment. The combination of Rg3 (50 microM) with cisplatin (10 microM) and doxorubicin (2 microM) was also more effective in the inhibition of prostate cancer cell growth and NF-kappaB activity than those by the treatment of Rg3 or chemotherapeutics alone. These results indicate that ginsenoside Rg3 inhibits NF-kappaB, and enhances the susceptibility of prostate cancer cells to docetaxel and other chemotherapeutics. Thus, ginsenoside Rg3 could be useful as an anti-cancer agent.

A Smac-mimetic sensitizes prostate cancer cells to TRAIL-induced apoptosis via modulating both IAPs and NF-kappaB

BackgroundAlthough tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising agent for human cancer therapy, prostate cancer still remains resistant to TRAIL. Both X-linked inhibitor of apoptosis (XIAP) and nuclear factor-kappaB function as key negative regulators of TRAIL signaling. In this study, we evaluated the effect of SH122, a small molecule mimetic of the second mitochondria-derived activator of caspases (Smac), on TRAIL-induced apoptosis in prostate cancer cells.MethodsThe potential of Smac-mimetics to bind XIAP or cIAP-1 was examined by pull-down assay. Cytotoxicity of TRAIL and/or Smac-mimetics was determined by a standard cell growth assay. Silencing of XIAP or cIAP-1 was achieved by transient transfection of short hairpin RNA. Apoptosis was detected by Annexin V-PI staining followed by flow cytometry and by Western Blot analysis of caspases, PARP and Bid. NF-kappaB activation was determined by subcellular fractionation, real time RT-PCR and reporter assay.ResultsSH122, but not its inactive analog, binds to XIAP and cIAP-1. SH122 significantly sensitized prostate cancer cells to TRAIL-mediated cell death. Moreover, SH122 enhanced TRAIL-induced apoptosis via both the death receptor and the mitochondrial pathway. Knockdown of both XIAP and cIAP-1 sensitized cellular response to TRAIL. XIAP-knockdown attenuated sensitivity of SH122 to TRAIL-induced cytotoxicity, confirming that XIAP is an important target for IAP-inhibitor-mediated TRAIL sensitization. SH122 also suppressed TRAIL-induced NF-kappaB activation by preventing cytosolic IkappaB-alpha degradation and RelA nuclear translocation, as well as by suppressing NF-kappaB target gene expression.ConclusionThese results demonstrate that SH122 sensitizes human prostate cancer cells to TRAIL-induced apoptosis by mimicking Smac and blocking both IAPs and NF-kappaB. Modulating IAPs may represent a promising approach to overcoming TRAIL-resistance in human prostate cancer with constitutively active NF-kappaB signaling.

Increased proteasome subunit protein expression and proteasome activity in colon cancer relate to an enhanced activation of nuclear factor E2-related factor 2 (Nrf2)

An elevated proteasome activity contributes to tumorigenesis, particularly by providing cancer cells with antiapoptotic protection and efficient clearance from irregular proteins. Still, the underlying mechanisms are poorly known. In this study, we report that in colon cancer patients, higher proteasome activity was detected in tumoral tissue compared with surrounding normal tissue, and also that increased levels of proteasomal subunit proteins, such as S5a/PSMD4 and alpha-5/PSMA5, could be detected. Colon tumors showed higher nuclear levels of nuclear factor E2-related factor 2 (Nrf2), a transcription factor supposed to be involved in the control of proteasomal subunit protein expression. The induction or overexpression of Nrf2 led to stronger S5a and alpha-5 expression in the human colon cancer cell lines, Colo320 and Lovo, as well as in NCM460 colonocytes along with higher proteasome activity. The small interfering RNA (siRNA)-mediated Nrf2 knockdown decreased S5a and alpha-5 expression and reduced proteasome activity. Additionally, Nrf2-dependent S5a and alpha-5 expression conferred protection from tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, an effect preceded by an increased nuclear factor (NF)-kappaB activation and higher expression of antiapoptotic NF-kappaB target genes. These findings point to an important role of Nrf2 in the gain of proteasome activity, thereby contributing to colorectal carcinogenesis. Nrf2 may therefore serve as a potential target in anticancer therapy.

Gene expression profiling of isolated tumour cells from anaplastic large cell lymphomas: insights into its cellular origin, pathogenesis and relation to Hodgkin lymphoma

Anaplastic large cell lymphoma (ALCL) is a main type of T-cell lymphomas and comprises three distinct entities: systemic anaplastic lymphoma kinase (ALK) positive, systemic ALK(-) and cutaneous ALK(-) ALCL (cALCL). Little is known about their pathogenesis and their cellular origin, and morphological and immunophenotypical overlap exists between ALK(-) ALCL and classical Hodgkin lymphoma (cHL). We conducted gene expression profiling of microdissected lymphoma cells of five ALK(+) and four ALK(-) systemic ALCL, seven cALCL and sixteen cHL, and of eight subsets of normal T and NK cells. The analysis supports a derivation of ALCL from activated T cells, but the lymphoma cells acquired a gene expression pattern hampering an assignment to a CD4(+), CD8(+) or CD30(+) T-cell origin. Indeed, ALCL display a down-modulation of many T-cell characteristic molecules. All ALCL types show significant expression of NFkappaB target genes and upregulation of genes involved in oncogenesis (e.g. EZH2). Surprisingly, few genes are differentially expressed between systemic and cALCL despite their different clinical behaviour, and between ALK(-) ALCL and cHL despite their different cellular origin. ALK(+) ALCL are characterized by expression of genes regulated by pathways constitutively activated by ALK. This study provides multiple novel insights into the molecular biology and pathogenesis of ALCL.

Invasion associated up‐regulation of nuclear factor κB target genes in colorectal cancer

: Colorectal cancer (CRC) displays intratumoral heterogeneity with less differentiated tumor cells at the invasive front (IF) than in the tumor center (TC). The authors previously observed that several genes were overexpressed at the IF of CRC with relations to inflammatory processes. Because nuclear factor kappaB (NF-kappaB), a dimeric transcription factor, is a major regulator of such processes, and because its target genes are involved in immune response, cell growth control, and cell survival, the expression of NF-kappaB target genes was investigated comparatively in CRC. : By using gene array profiling, NF-kappaB target gene expression was assessed in CRCs that expressed human mutL homolog 1 (hMLH1), hMSH2, and nuclear beta-catenin by comparing expression at the IF, in the TC, and in normal mucosa. In addition, 5 NF-kappaB target genes with high differential expression were validated by using immunohistochemistry. : The expression of NF-kappaB target genes in the TC, at the IF, and in normal mucosa was distinct; whereas, specifically at the IF, most differentially expressed NF-kappaB targets were up-regulated. Moreover, the results indicated that the expression diverged between epithelial tumor cells and inflammatory stromal cells. : Because the results demonstrated that inflammation and the activation of NF-kappaB signaling promoted CRC invasiveness, the current study provided further evidence that downstream targets of NF-kappaB signaling may be specifically relevant in invasion and progression of CRC. Finally, as has been suggested for colitis-associated cancer, the authors of this report concluded that the inhibition of NF-kappaB signaling also may be an additional option for the treatment of sporadic CRC. Cancer 2009. (c) 2009 American Cancer Society.

Inhibition of NF-κB by ginsenoside Rg3 enhances the susceptibility of colon cancer cells to docetaxel

Ginsenoside Rg3, the main constituent isolated from Panax ginseng, has been of interest for use as a cancer preventive or therapeutic agent. We investigated here whether Rg3 can inhibit the activity of NF-kappaB, a key transcriptional factor constitutively activated in colon cancer that confers cancer cell resistance to chemotherapeutic agents. To investigate whether RG3 can suppress activation of NF-kappaB, and thus inhibit cancer cell growth, we examined the susceptibility of colon cancer cells (SW620 and HCT116) to treatment with Rg3 (25, 50, 75, 100 microM) and RG3-induced activation of NF-kappaB. RG3 dose-dependently inhibited cancer cell growth through induction of apoptosis and decreased NF-kappaB activity. In a further study of compounds in colon cancer, we used half of the IC(50) dose, values in combined treatments of Rg3 (50 microM) with conventional agents - docetaxel (5 nM), paclitaxel (10 nM) cisplatin (10 microM) and doxorubicin (2 microM). Compared to treatment with Rg3 or chemotherapy alone, combined treatment was more effective (i.e., there were synergistic effects) in the inhibition of cancer cell growth and induction of apoptosis and these effects were accompanied by significant inhibition of NF-kappaB activity. NF-kappaB target gene expression of apoptotic cell death proteins (Bax, caspase-3, caspase-9) was significantly enhanced, but the expression of anti-apoptotic genes and cell proliferation marker genes (Bcl-2, inhibitor of apoptosis protein (IAP-1) and X chromosome IAP (XIAP), Cox-2, c-Fos, c-Jun and cyclin D1) was significantly inhibited by the combined treatment compared to Rg3 or docetaxel alone. These results indicate that ginsenoside Rg3 inhibits NF-kappaB, and enhances the susceptibility of colon cancer cells to docetaxel and other chemotherapeutics. Thus, ginsenoside Rg3 could be useful as an anti-cancer or adjuvant anti-cancer agent.

Phenanthrene-based tylophorine-1 (PBT-1) inhibits lung cancer cell growth through the Akt and NF-kappaB pathways.

Tylophorine and related natural compounds exhibit potent antitumor activities. We previously showed that PBT-1, a synthetic C9-substituted phenanthrene-based tylophorine (PBT) derivative, significantly inhibits growth of various cancer cells. In this study, we further explored the mechanisms and potential of PBT-1 as an anticancer agent. PBT-1 dose-dependently suppressed colony formation and induced cell cycle G2/M arrest and apoptosis. DNA microarray and pathway analysis showed that PBT-1 activated the apoptosis pathway and mitogen-activated protein kinase signaling. In contrast, PBT-1 suppressed the nuclear factor kappaB (NF-kappaB) pathway and focal adhesion. We further confirmed that PBT-1 suppressed Akt activation accelerated RelA degradation via IkappaB kinase-alpha and down-regulated NF-kappaB target gene expression. The reciprocal recruitment of RelA and RelB on COX-2 promoter region led to down-regulation of transcriptional activity. We conclude that PBT-1 induces cell cycle G2/M arrest and apoptosis by inactivating Akt and by inhibiting the NF-kappaB signaling pathway. PBT-1 may be a good drug candidate for anticancer chemotherapy.

Negative regulation of NF-κB action by Set9-mediated lysine methylation of the RelA subunit

Proper regulation of NF-kappaB activity is critical to maintain and balance the inflammatory response. Inactivation of the NF-kappaB complex relies in part on the proteasome-mediated degradation of promoter-bound NF-kappaB, but the detailed molecular mechanism initiating this process remains elusive. Here, we show that the methylation of the RelA subunit of NF-kappaB has an important function in this process. Lysine methyltransferase Set9 physically associates with RelA in vitro and in vivo in response to TNF-alpha stimulation. Mutational and mass spectrometric analyses reveal that RelA is monomethylated by Set9 at lysine residues 314 and 315 in vitro and in vivo. Methylation of RelA inhibits NF-kappaB action by inducing the proteasome-mediated degradation of promoter-associated RelA. Depletion of Set9 by siRNA or mutation of the RelA methylation sites prolongs DNA binding of NF-kappaB and enhances TNF-alpha-induced expression of NF-kappaB target genes. Together, these findings unveil a novel mechanism by which methylation of RelA dictates the turnover of NF-kappaB and controls the NF-kappaB-mediated inflammatory response.

Nuclear factor-kappa B in intestinal protection and destruction

Nuclear factor-kappa B (NF-kappaB) is a key transcriptional regulator of innate and adaptive immunity. This review highlights new insights into the functions of NF-kappaB in normal homeostasis and specific disease processes in the intestinal tract. Inflammatory bowel disease and experimental intestinal inflammation are characterized by NF-kappaB activation and increased expression of proinflammatory NF-kappaB target genes. Accordingly, NF-kappaB inhibition protects against chronic intestinal inflammation and necrotizing enterocolitis in animal models. However, recent findings suggest that NF-kappaB has not only proinflammatory but also tissue-protective functions. Thus, genetic ablation of the regulatory subunit, IkappaB kinase (IKK)gamma, of the central kinase complex required for NF-kappaB activation, IKK, or of both kinase subunits, IKKalpha and IKKbeta, in intestinal epithelial cells causes spontaneous murine colitis. Pharmacological inhibition of IKKbeta, and loss of IKKbeta or NF-kappaB p65 in the epithelium, sensitizes mice to acute inflammatory and injurious challenges. Deficiency in Toll-like receptor 5, a strong activator of NF-kappaB, results in spontaneous colitis and exacerbates mucosal inflammatory responses to Salmonella infection. Conversely, Toll-like receptor 5 stimulation confers radioprotection in the intestine. NF-kappaB has multiple, often opposing functions in the intestine. Antiapoptotic actions of NF-kappaB in intestinal epithelial cells dominate tissue responses to many acute inflammatory and injurious challenges, whereas proinflammatory and cell survival functions of NF-kappaB in macrophages and T cells govern chronic intestinal inflammation.

Two Modes of Transcriptional Activation at Native Promoters by NF-κB p65

The NF-κB family of transcription factors is crucial for the expression of multiple genes involved in cell survival, proliferation, differentiation, and inflammation. The molecular basis by which NF-κB activates endogenous promoters is largely unknown, but it seems likely that it should include the means to tailor transcriptional output to match the wide functional range of its target genes. To dissect NF-κB–driven transcription at native promoters, we disrupted the interaction between NF-κB p65 and the Mediator complex. We found that expression of many endogenous NF-κB target genes depends on direct contact between p65 and Mediator, and that this occurs through the Trap-80 subunit and the TA1 and TA2 regions of p65. Unexpectedly, however, a subset of p65-dependent genes are transcribed normally even when the interaction of p65 with Mediator is abolished. Moreover, a mutant form of p65 lacking all transcription activation domains previously identified in vitro can still activate such promoters in vivo. We found that without p65, native NF-κB target promoters cannot be bound by secondary transcription factors. Artificial recruitment of a secondary transcription factor was able to restore transcription of an otherwise NF-κB–dependent target gene in the absence of p65, showing that the control of promoter occupancy constitutes a second, independent mode of transcriptional activation by p65. This mode enables a subset of promoters to utilize a wide choice of transcription factors, with the potential to regulate their expression accordingly, whilst remaining dependent for their activation on NF-κB.

TRAF2-binding BIR1 domain of c-IAP2/MALT1 fusion protein is essential for activation of NF-κB

Marginal zone mucosa-associated lymphoid tissue (MALT) B-cell lymphoma is the most common extranodal non-Hodgkin lymphoma. The t(11;18)(q21;q21) translocation occurs frequently in MALT lymphomas and creates a chimeric NF-kappaB-activating protein containing the baculoviral IAP repeat (BIR) domains of c-IAP2 (inhibitor of apoptosis protein 2) fused with portions of the MALT1 protein. The BIR1 domain of c-IAP2 interacts directly with TRAF2 (TNFalpha-receptor-associated factor-2), but its role in NF-kappaB activation is still unclear. Here, we investigated the role of TRAF2 in c-IAP2/MALT1-induced NF-kappaB activation. We show the BIR1 domain of c-IAP2 is essential for NF-kappaB activation, whereas BIR2 and BIR3 domains are not. Studies of c-IAP2/MALT1 BIR1 mutant (E47A/R48A) that fails to activate NF-kappaB showed loss of TRAF2 binding, but retention of TRAF6 binding, suggesting that interaction of c-IAP2/MALT1 with TRAF6 is insufficient for NF-kappaB induction. In addition, a dominant-negative TRAF2 mutant or downregulation of TRAF2 achieved by small interfering RNA inhibited NF-kappaB activation by c-IAP2/MALT1 showing that TRAF2 is indispensable. Comparisons of the bioactivity of intact c-IAP2/MALT1 oncoprotein and BIR1 E47A/R48A c-IAP2/MALT1 mutant that cannot bind TRAF2 in a lymphoid cell line provided evidence that TRAF2 interaction is critical for c-IAP2/MALT1-mediated increases in the NF-kappaB activity, increased expression of endogenous NF-kappaB target genes (c-FLIP, TRAF1), and resistance to apoptosis.

Phenolic Constituents of Amorpha fruticosa That Inhibit NF-κB Activation and Related Gene Expression

NF-kappaB is known to play a crucial role in the regulation of genes controlling the immune system, apoptosis, tumor cell growth, and tissue differentiation. Bioassay-guided fractionation of the n-hexane-soluble fraction of a methanol extract of Amorpha fruticosa afforded four new compounds, 5, 7, 8, and 9, and eight known compounds. Their structures were elucidated by spectroscopic methods. All compounds inhibited NF-kappaB activity, and tephrosin (1), 11-hydroxytephrosin (2), and deguelin (3) were the most active, with IC50 values of 0.11, 0.19, and 0.22 microM, respectively, in TNF-alpha-stimulated HeLa cell-based reporter gene assays. Further investigations showed that compounds 1, 5, and 6 blocked NF-kappaB/DNA binding activity and suppressed the expression of NF-kappaB target genes.

Maintenance of constitutive IkappaB kinase activity by glycogen synthase kinase-3alpha/beta in pancreatic cancer.

Constitutive nuclear factor kappaB (NF-kappaB) activation is among the many deregulated signaling pathways that are proposed to drive pancreatic cancer cell growth and survival. Recent reports suggest that glycogen synthase kinase-3beta (GSK-3beta) plays a key role in maintaining basal NF-kappaB target gene expression and cell survival in pancreatic cancer cell lines. However, the mechanism by which GSK-3beta facilitates constitutive NF-kappaB signaling in pancreatic cancer remains unclear. In this report, we analyze the contributions of both GSK-3 isoforms (GSK-3alpha and GSK-3beta) in regulating NF-kappaB activation and cell proliferation in pancreatic cancer cell lines (Panc-1 and MiaPaCa-2). We show that GSK-3 isoforms are differentially required to maintain basal NF-kappaB DNA binding activity, transcriptional activity, and cell proliferation in Panc-1 and MiaPaCa-2 cells. Our data also indicate that IkappaB kinase (IKK) subunits are not equally required to regulate pancreatic cancer-associated NF-kappaB activity and cell growth. Importantly, we provide the first evidence that GSK-3 maintains constitutive NF-kappaB signaling in pancreatic cancer by regulating IKK activity. These data provide new insight into GSK-3-dependent NF-kappaB regulation and further establish GSK-3 and IKK as potential therapeutic targets for pancreatic cancer.