TNFα-mediated NF-κB Activation Research Articles

Proteasomal Degradation of TRAF2 Mediates Mitochondrial Dysfunction in Doxorubicin-Cardiomyopathy.

Cytokines such as tumor necrosis factor-α (TNFα) have been implicated in cardiac dysfunction and toxicity associated with doxorubicin (DOX). Although TNFα can elicit different cellular responses, including survival or death, the mechanisms underlying these divergent outcomes in the heart remain cryptic. The E3 ubiquitin ligase TRAF2 (TNF receptor associated factor 2) provides a critical signaling platform for K63-linked polyubiquitination of RIPK1 (receptor interacting protein 1), crucial for nuclear factor-κB (NF-κB) activation by TNFα and survival. Here, we investigate alterations in TNFα-TRAF2-NF-κB signaling in the pathogenesis of DOX cardiotoxicity. Using a combination of in vivo (4 weekly injections of DOX 5 mg·kg-1·wk-1) in C57/BL6J mice and in vitro approaches (rat, mouse, and human inducible pluripotent stem cell-derived cardiac myocytes), we monitored TNFα levels, lactate dehydrogenase, cardiac ultrastructure and function, mitochondrial bioenergetics, and cardiac cell viability. In contrast to vehicle-treated mice, ultrastructural defects, including cytoplasmic swelling, mitochondrial perturbations, and elevated TNFα levels, were observed in the hearts of mice treated with DOX. While investigating the involvement of TNFα in DOX cardiotoxicity, we discovered that NF-κB was readily activated by TNFα. However, TNFα-mediated NF-κB activation was impaired in cardiac myocytes treated with DOX. This coincided with loss of K63- linked polyubiquitination of RIPK1 from the proteasomal degradation of TRAF2. Furthermore, TRAF2 protein abundance was markedly reduced in hearts of patients with cancer treated with DOX. We further established that the reciprocal actions of the ubiquitinating and deubiquitinating enzymes cellular inhibitors of apoptosis 1 and USP19 (ubiquitin-specific peptidase 19), respectively, regulated the proteasomal degradation of TRAF2 in DOX-treated cardiac myocytes. An E3-ligase mutant of cellular inhibitors of apoptosis 1 (H588A) or gain of function of USP19 prevented proteasomal degradation of TRAF2 and DOX-induced cell death. Furthermore, wild-type TRAF2, but not a RING finger mutant defective for K63-linked polyubiquitination of RIPK1, restored NF-κB signaling and suppressed DOX-induced cardiac cell death. Last, cardiomyocyte-restricted expression of TRAF2 (cardiac troponin T-adeno-associated virus 9-TRAF2) in vivo protected against mitochondrial defects and cardiac dysfunction induced by DOX. Our findings reveal a novel signaling axis that functionally connects the cardiotoxic effects of DOX to proteasomal degradation of TRAF2. Disruption of the critical TRAF2 survival pathway by DOX sensitizes cardiac myocytes to TNFα-mediated necrotic cell death and DOX cardiotoxicity.

Histone deacetylase 3 facilitates TNFα-mediated NF-κB activation through suppressing CTSB induced RIP1 degradation and is required for host defense against bacterial infection

BackgroundAs important enzymes regulating acetylation, histone deacetylases (HDACs) participate in a series of cell physiological process. However, the mechanisms responsible for individual HDAC family members in regulating innate immunity remained to be elucidated. Here we sought to reveal the mechanism of HDAC3 in regulating the inflammatory response of macrophages.MethodsRNAseq was done to detect the transcriptional influence of HDAC3 on macrophages. Kyoto Encyclopedia of Genes and Genomes was used to reveal the change of signaling pathways after HDAC3 knockout. CHIPseq was done to detect the deacetylation modification of HDAC3 on chromosome. Western blot, immunofluorescence, and real-time quantitative PCR were used to measure the change of genes and proteins’ levels. Mice were intratracheal instillation with lipopolysaccharide or Pseudomonas aeruginosa to determine the influence of HDAC3 on inflammatory response in vivo.ResultsHDAC3-deficient macrophages had increased expression of cathepsins resulting from elevated histone acetylation. Over-expressed cathepsins such as cathepsin B (CTSB) caused remarkable degradation of receptor (TNFRSF)-interacting serine-threonine kinase 1 (RIP1), which reduced TNFα mediated NF-κB activation and inflammatory response. Consistently, mice with macrophage specific knockout of HDAC3 were impaired in inflammatory response and thereby susceptible to Pseudomonas aeruginosa infection.ConclusionHDAC3 was required for protecting RIP1 from degrading by CTSB in macrophages. Decreased RIP1 in HDAC3 knockout macrophages impaired TNFα mediated NF-κB activation. Our studies uncovered important roles of HDAC3 in the regulation of cathepsin-mediated lysosomal degradation and RIP1-mediated inflammatory response in macrophages as well as in host defense against bacterial infection.

Single-Cell RNA Sequencing Analysis Reveals Mechanisms of Initiation and Progression in Chronic Myelomonocytic Leukemia

Despite advances in the genetic characterization of chronic myelomonocytic leukemia (CMML), the molecular mechanisms that drive the disease during its distinct phases remain unclear. To uncover vulnerabilities in CMML that could be therapeutically targeted to halt its evolution, we sought to dissect at the single-cell level the cellular and transcriptomic changes that occur in the hematopoietic system at the time of CMML's initiation and its progression after hypomethylating agent (HMA) therapy.To evaluate the molecular mechanisms underlying CMML maintenance, we performed single-cell RNA sequencing (scRNA-seq) analysis of lineage-negative (Lin -)CD34 + hematopoietic stem and progenitor cells (HSPCs) and bone marrow (BM) mononuclear cells (MNCs) isolated from untreated CMML patients (n=5 and 6, respectively) and age-matched healthy donors (HDs; n=2 and 3, respectively). Our integrated analysis revealed that CMML Lin -CD34 + HSPCs had a predominant granulomonocytic differentiation route with an increased frequency of early and committed myeloid-monocytic progenitors at the expense of HSCs and megakaryocyte/erythroid progenitors (Fig. 1a). Differential expression analysis among the clusters revealed that most transcriptomic differences occurred in CMML HSCs, which were characterized by the upregulation of genes involved in oxidative phosphorylation, type I interferon (IFN) and IFNγ response, myeloid development, and inflammatory signaling and had downregulated expression of genes involved in TNFα-mediated NF-κB activation (Fig. 1b). These data suggest that CMML HSCs undergo metabolic reprogramming and demand a higher level of mitochondrial activity to maintain activated monocytic differentiation in response to inflammatory signaling.Consistent with these results, scRNA-seq analysis of MNCs isolated from the same HD and CMML BM samples showed that monocytes were significantly increased at the expense of erythroid precursors and B cells in CMML (Fig. 1c). CMML monocytes had upregulated genes involved in IFNγ response, oxidative phosphorylation, MYC targets, NF-κB activation, and inflammation (e.g., S100A9, CCL3, IL1B). Interestingly, among the anti-apoptotic BCL2 family members, only the NF-κB transcriptional target BCL2A1 was significantly overexpressed.To investigate the mechanisms of resistance to HMA therapy, we performed integrated scRNA-seq analysis of sequential Lin -CD34 + cells and BM MNCs isolated from CMML patients at the time of disease initiation and progression after HMA therapy failure. CMML progression was driven by a significant expansion of lympho-myeloid progenitors (LMPPs) at the expense of earlier HSCs , which exacerbated myelomonocytic differentiation in the HSPC compartment (Fig. 1d). Expanded LMPPs were characterized by higher levels of IFNγ response, NF-κB survival signaling, and cell cycle regulators. Accordingly, scRNA-seq analysis of MNCs cells from the same patients showed significantly increased frequencies of monocytes and a reduction of naïve CD4 +/CD8 + T cells and effector memory CD8 + T cells. Differential expression analysis of the 2 sample groups in the monocyte population identified five different cellular clusters, one of which emerged only at progression (Fig. 1e). This population was characterized by high expression levels of inflammatory cytokines and the anti-apoptotic modulators MCL1 and BCL2A1. Together, these data suggest that CMML progression arises from immature myeloid progenitors at the stem cell level and that downstream monocytes undergo transcriptomic rewiring and acquire survival mechanisms that induce therapy resistance and further accelerate disease progression.In conclusion, our results elucidate the differentiation hierarchies and transcriptional programs associated with CMML's initiation and its progression after HMA therapy. Our data suggest that therapies targeting downstream effectors of NF-kB-mediated survival signaling could overcome treatment failure. [Display omitted] DisclosuresWei: Daiichi Sanko: Research Funding. Kantarjian: AbbVie: Honoraria, Research Funding; Immunogen: Research Funding; KAHR Medical Ltd: Honoraria; Jazz: Research Funding; Ipsen Pharmaceuticals: Honoraria; Astellas Health: Honoraria; NOVA Research: Honoraria; Pfizer: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Astra Zeneca: Honoraria; Ascentage: Research Funding; Aptitude Health: Honoraria; Daiichi-Sankyo: Research Funding; Amgen: Honoraria, Research Funding; BMS: Research Funding; Precision Biosciences: Honoraria; Taiho Pharmaceutical Canada: Honoraria.

The Deubiquitinating Enzyme USP20 Regulates the TNFα-Induced NF-κB Signaling Pathway through Stabilization of p62.

p62/sequestosome-1 is a scaffolding protein involved in diverse cellular processes such as autophagy, oxidative stress, cell survival and death. It has been identified to interact with atypical protein kinase Cs (aPKCs), linking these kinases to NF-κB activation by tumor necrosis factor α (TNFα). The diverse functions of p62 are regulated through post-translational modifications of several domains within p62. Among the enzymes that mediate these post-translational modifications, little is known about the deubiquitinating enzymes (DUBs) that remove ubiquitin chains from p62, compared to the E3 ligases involved in p62 ubiquitination. In this study, we first demonstrate a role of ubiquitin-specific protease USP20 in regulating p62 stability in TNFα-mediated NF-κB activation. USP20 specifically binds to p62 and acts as a positive regulator for NF-κB activation by TNFα through deubiquitinating lysine 48 (K48)-linked polyubiquitination, eventually contributing to cell survival. Furthermore, depletion of USP20 disrupts formation of the atypical PKCζ-RIPK1-p62 complex required for TNFα-mediated NF-κB activation and significantly increases the apoptosis induced by TNFα plus cycloheximide or TNFα plus TAK1 inhibitor. These findings strongly suggest that the USP20-p62 axis plays an essential role in NF-κB-mediated cell survival induced by the TNFα-atypical PKCζ signaling pathway.

Abstract 1109: CPAP promotes HCC angiogenesis and metastasis via interacting with and enhancing STAT3 signaling

Abstract Centrosomal P4.1-associated protein (CPAP) is a centrosomal protein and can as a transcriptional coactivator of STAT5 and NF-κB in cancer. Our previous studies indicated that CPAP is overexpressed in tumor tissue and can increase TNFα-mediated NF-κB activation in HCC. Here, we demonstrated that overexpressed CPAP increases tumor growth, angiogenesis, as well as metastasis ex vivo and in vivo. We found that CPAP increases these malignant abilities of tumor cells are through IL-6/STAT3 signaling. We demonstrated that CPAP directly interacts with C-terminal domain of STAT3 to increase STAT3 activity. Overexpression of CPAP enhances IL-6/STAT3/IL-8-mediated angiogenesis and other metastatic genes expression such as CD44 and MCAM. These results indicated that CPAP leads to HCC malignancy and metastasis by increasing STAT3 activity through directly interaction. Clinically, CPAP positively correlates with IL-8 in HCC with vascular invasion; and also positively correlates with CD44 and MCAM in HCC tissues. In summary, our findings shed light on the importance of CPAP to act as a potential therapeutic target for inhibiting the IL-6/STAT3-mediated angiogenesis pathway and treating metastatic HCC. Citation Format: Ruo Yu Chen, Liang-Yi Hung. CPAP promotes HCC angiogenesis and metastasis via interacting with and enhancing STAT3 signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1109.

Extracellular Superoxide Dismutase (SOD3) Links Tumor Necrosis Factor‐alpha Receptor 1 to Integrin Signaling

Tumor necrosis factor‐α (TNFα) signaling requires superoxide (O2•−) to be produced extracellularly by NADPH oxidase 1 (Nox1). Extracellular superoxide dismutase (ecSOD) can convert this O2•− to hydrogen peroxide (H2O2). ecSOD is anchored to the cell surface by interaction with either heparin sulfate proteoglycan via a heparin binding domain, or by binding to fibulin‐5 (Fib5), an extracellular matrix protein which interacts with integrin receptors. Fib5 binding does not activate integrins, but blocks interaction with other components of the extracellular matrix. The physiologic significance of ecSOD‐Fib5‐integrin interaction has not been well defined. We hypothesized that ecSOD regulates extracellular redox signaling of TNFα/Nox1 by modulating secondary effects on integrin signaling. We analyzed TNFα‐induced interleukin‐6 (IL‐6) production and NF‐κB activation in cultured vascular smooth muscle cells (VSMC). Knockdown of ecSOD by siRNA blocked TNFα‐mediated IL‐6 (siControl+TNFα 2922±354, si ecSOD+TNFα 952.5±99 pg/mL, p<0.05, n=8–9) and NF‐κB activation (siControl+TNFα 10.8±1.0, si ecSOD+TNFα 4.0±0.3 fold change, p<0.05, n=7). Similar inhibitory effects were observed following knockdown of Fib5 (IL‐6; siControl+TNFα 2372±123, siFib5+TNFα 1686±124 pg/mL, p<0.05, n=4. NF‐κB activation; siControl+TNFα 10.8±1.0, siFib5+TNFα 5.4±0.3 fold change, p<0.05, n=7). We next sought to determine how ecSOD affected integrin signaling. Focal adhesion kinase (FAK) and integrin‐linked kinase (ILK) are critical downstream effectors of integrin activation. TNFα induced phosphorylation of both FAK and Akt, which was used as a surrogate for ILK activation. This approach was validated by the ability of ILK inhibition (Cpd22; 3 μM) to prevent Akt phosphorylation. Both FAK and Akt phosphorylation in response to TNFα were inhibited by an anti‐integrin β1 antibody. Knockdown of ecSOD significantly enhanced TNFα‐induced phosphorylation of both FAK and Akt. FAK inhibition (FAK inhibitor 14; 2 μM) blocked while ILK inhibition (Cpd22; 3 μM) markedly potentiated NF‐κB activation. These results suggest that TNFα transactivates β1 integrins and therefore FAK and ILK in VSMCs. We speculate that ecSOD localizes H2O2 production to the vicinity of integrin receptors via physical association with Fib5 and provides tonic suppression of β1integrin receptor activation. Knockdown of either ecSOD or Fib5 would promote ILK‐dependent inhibition of NF‐κB activation and IL‐6 secretion which occurs via direct TNFR1‐dependent signaling.Support or Funding InformationAmerican Heart Association (16SDG30610002)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 3086: CPAP enhances diethylnitrosamine-induced hepatocarcinogenesis via STAT3 pathway

Abstract Chronic and persistent inflammation is a well-known carcinogenesis promoter. Hepatocellular carcinoma (HCC) is one of the most common inflammation-associated cancers; most HCC arises in the context of chronic inflammation and hepatic injury. Both NF-κB and STAT3 are important regulators for inflammation. Our previous studies indicated that CPAP (centrosomal P4.1-associated protein), a centrosomal protein that majorly participates in centrosome functions, is overexpressed in HCC and can increase TNFα-mediated NF-κB activation and IL-6-induced STAT3 activation. A hepatocyte-specific CPAP-expressing transgenic mouse was generated to investigate the physiological role of CPAP in hepatocarcinogenesis. An obvious inflammatory cell accumulation and fatty change were shown in the CPAP transgenic (Tg) mouse liver. The ALT level as well as the expression of inflammatory genes, such as IL-6, IL1β and TNF-α in CPAP Tg mouse are higher than in the wild type (WT) mouse. By high-dose and short-term diethylnitrosamine (DEN) treatment, the ALT level, pro-inflammatory genes and STAT3 activation are increased in CPAP Tg mouse; low-dose and long-term DEN treatment induces more severe liver tumor formation in CPAP Tg mouse than in WT mouse. In summary, these results suggest that CPAP may promote hepatocarcinogenesis through enhancing the inflammation pathway via activating the STAT3 pathway. Citation Format: Liang-Yi Hung. CPAP enhances diethylnitrosamine-induced hepatocarcinogenesis via STAT3 pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3086.

RNF8 negatively regulates NF-kappaB signaling by targeting IkappaB kinase: implications for the regulation of inflammation signaling

Persistent or excess activation of NF-κB leads to cancer, autoimmune and inflammatory diseases. Therefore, activated NF-κB needs to be terminated after induction, which highlights the physiological significance of NF-κB-negative regulators. However, the molecular mechanisms that negatively regulate NF-κB are not well understood. Here, we report that Ring Finger Protein 8 (RNF8), an E3 ubiquitin ligase, inhibits TNFα-mediated NF-κB activation by targeting IκB kinase (IKK). Upon TNFα stimulation, RNF8 binds to the catalytic subunits of IKK complex, resulting in inhibition of IKKα/β phosphorylation and subsequent NF-κB activation. RNF8 targets the IKK complex in a manner independent of its RING domain. We further provide evidence that the silencing of RNF8 results in enhanced TNFα-induced IKK activation, and an increase expression of NF-κB-induced inflammatory cytokine IL-8. Our study identifies a previously unrecognized role for RNF8 in the negative regulation of NF-κB activation by targeting and deactivating the IKK complex.

Inhibition of TNF-α-mediated NF-κB Activation by Ginsenoside Rg1 Contributes the Attenuation of Cardiac Hypertrophy Induced by Abdominal Aorta Coarctation.

Ginsenoside Rg1 (Rg1), a protopanaxadiol saponin extracted from Chinese medicine Panax ginseng C.A. Meyer, has been demonstrated to inhibit the cardiac hypertrophy. However, the molecular mechanisms underlying the inhibition remain poorly understood. Activation of nuclear factor-kappa B (NF-κB) mediated by tumor necrosis factor α (TNF-α) gets involved in the cardiac hypertrophy. This study is designed to investigate the effects and the potential mechanism of Rg1 on the abdominal aorta coarctation (AAC)-induced cardiac hypertrophy with focus on TNF-α/NF-κB signaling pathway. The results showed that oral administration of Rg1 dose-dependently improved the pathological changes, decreased the ratios of left ventricular weight/body weight (LVW/BW) and heart weight/BW (HW/BW), corrected the dysfunction of the cardiac hemodynamics by decreasing the left ventricular systolic pressure and left ventricular end-diastolic pressure and increasing the maximal rate of left ventricular systolic and diastolic pressure (±dp/dtmax) compared with the AAC alone. Rg1 also downregulated the atrial natriuretic peptide mRNA expression and decreased the mRNA and protein expression of TNF-α in the heart tissue of rats compared with the AAC alone. In addition, Rg1 and BAY, the specific inhibitor of NF-κB, decreased the protein content and downregulated the mRNA expression of atrial natriuretic peptide in neonatal rat ventricular myocytes treated with TNF-α. Furthermore, Rg1 increased the protein expression of p65, the subunit of NF-κB, in cytoplasm and decreased the expression p65 in nucleus of the heart tissue of rats undergoing the AAC and of neonatal rat ventricular myocytes treated with TNF-α. The results suggested that Rg1 attenuates the AAC-induced cardiac hypertrophy through inhibition of TNF-α/NF-κB signaling pathway.

Severe Hepatitis Promotes Hepatocellular Carcinoma Recurrence via NF-κB Pathway-Mediated Epithelial-Mesenchymal Transition after Resection.

Surgical resection is considered as a curative treatment modality for hepatocellular carcinoma; however, the incidence of postoperative tumor recurrence is high, leading to worse patient survival. Persistent hepatitis (inflammation) is one of the risk factors of tumor recurrence after surgical resection. The aim of this study is to investigate the underlying mechanisms linking liver inflammation to hepatocellular carcinoma progression. In this study, we used a cytokine array to identify important cytokines whose levels are increased in liver microenvironment with severe hepatitis. We evaluated the morphologic changes, migration and invasion ability, and signal transduction in hepatocellular carcinoma cells with or without inflammatory cytokine in vitro Finally, we analyzed the NF-κB signal pathway in tumor specimens from 232 patients with hepatocellular carcinoma by immunohistochemical staining. The proinflammatory cytokine TNFα was increased in the peritumoral microenvironment and contributed to tumor recurrence and metastasis. Specifically, TNFα promoted hepatocellular carcinoma cancer cell migration, invasion, and epithelial-mesenchymal transition (EMT) by upregulating the transcriptional regulator, Snail. We identified Snail as a direct target gene downstream of the TNFα-mediated canonical NF-κB activation. In addition, tumor recurrence-free survival of hepatocellular carcinoma patients correlated negatively with high p65 and Snail expression and positively with high E-cadherin expression. Our results establish a signaling axis that explains how inflammatory tumor microenvironment promotes hepatocellular carcinoma recurrence and metastasis. These findings suggest that controlling liver inflammation and/or targeting NF-κB-mediated Snail expression may be a potential therapeutic strategy to prevent hepatocellular carcinoma recurrence after hepatectomy.

Herpes simplex virus 1 DNA polymerase processivity factor UL42 inhibits TNF-α-induced NF-κB activation by interacting with p65/RelA and p50/NF-κB1

Herpes simplex virus 1 (HSV-1) is the archetypal member of the alphaherpesvirus with a large genome encoding over 80 viral proteins, many of which are involved in virus-host interactions and show immune modulatory capabilities. In this study, we demonstrated that the HSV-1 UL42 protein, a DNA polymerase processivity factor, was a novel antagonism of the canonical NF-κB signaling pathway. UL42 was shown to significantly suppress TNF-α mediated NF-κB activation. Co-immunoprecipitation experiment revealed that UL42 bound to the NF-κB subunits p65 and p50. Fluorescence microscopy demonstrated that UL42 abolished nuclear translocation of p65 and p50 upon TNF-α-stimulation. But the inhibiting capacity of UL42 2R/2A (R279A, R280A) and UL42 3R/3A (R113A, R279A and R280A) mutants were less than wild type UL42. Also UL42 bound to the Rel homology domain of the NF-κB subunit p65 and p50. Notably, the N-terminal of UL42 was sufficient to interact with p65 and p50 and abolished NF-κB reporter gene activity. Thus, it was first time we demonstrated that HSV-1 UL42 appeared to prevent NF-κB-dependent gene expression by retaining p65 and p50 in the cytoplasm, and UL42-dependent transcriptional activation were inherently coupled to promote HSV-1 lytic replication, which also may contribute to immune evasion and pathogenesis of HSV-1.

Oncrasin targets the JNK-NF-κB axis to sensitize glioma cells to TNFα-induced apoptosis

Resistance of glioblastoma multiforme (GBM) to tumor necrosis factor (TNF) α-induced apoptosis have been attributed to increased nuclear factor-kappaB (NF-κB) activation. As we have previously reported that certain anticancer chemotherapeutics can sensitize glioma cells to TNFα-induced apoptosis by abrogating NF-κB activation, we investigated the potential of oncrasin in sensitizing glioma cells to TNFα-induced apoptosis. Oncrasin reduced glioma cell viability, inhibited TNFα-mediated NF-κB activation and sensitized cells to TNFα-induced apoptosis. Apoptosis was accompanied by elevated Fas and Fas-associated death domain (FADD) levels, increased caspase-8 activation and formation of death-inducing signaling complex (DISC). Oncrasin also (i) affected expression of cell cycle regulators, (ii) triggered DNA damage response, (iii) induced G(2)/M cell cycle arrest, (iv) decreased telomerase activity, (v) abrogated STAT3 activation and (v) mediated extracellular release of high mobility group box 1 (HMGB1) along with its increased association with nucleosomes. Oncrasin-induced apoptosis did not involve mitochondria. Importantly, oncrasin increased c-jun N-terminal kinase (JNK) phosphorylation and pharmacological inhibition of JNK rescued oncrasin-induced apoptosis. JNK inhibition prevented oncrasin-induced decrease in TNFα-induced NF-κB activity and inhibition of NF-κB increased JNK phosphorylation in TNFα-treated cells. Oncrasin induced DISC formation and inhibited anchorage-independent growth of glioma cells in a JNK-dependent manner. By elucidating the existence of JNK-NF-κB cross-talk that regulates resistance to TNFα-induced apoptosis, this study has highlighted the importance of JNK in regulating viability of glioma cells.

An apoptosis-independent role of SMAC in tumor suppression.

Reduced expression of the pro-apoptotic protein SMAC (second mitochondria-derived activator of caspase) has been reported to correlate with cancer progression, while its significance and underlying mechanisms are poorly understood. In this study, we investigated the role of SMAC in intestinal tumorigenesis using both human samples and animal models. Decreased SMAC expression was found to correlate with increased cIAP2 expression and higher grades of human colon cancer. In mice, SMAC deficiency significantly increased the incidence and size of colon tumors induced by azoxymethane (AOM)/dextran sulfate sodium salt (DSS), and highly enriched β-catenin hot spot mutations. SMAC deficiency also significantly increased the incidence of spontaneous intestinal polyps in APC(Min/+) mice. Loss of SMAC in mice led to elevated levels of cIAP1 and cIAP2, increased proliferation and activation of the NF-κB p65 subunit in normal and tumor tissues. Unexpectedly, SMAC deficiency had little effect on the incidence of precursor lesions, or apoptosis induced by AOM or DSS, or in established tumors in mice. Furthermore, SMAC knockout enhanced TNFα-mediated NF-κB activation via cIAP2 in HCT 116 colon cancer cells. These results demonstrate an essential and apoptosis-independent function of SMAC in tumor suppression and provide new insights into the biology and targeting of colon cancer.

Suppression of Akt/Foxp3-mediated miR-183 expression blocks Sp1-mediated ADAM17 expression and TNFα-mediated NFκB activation in piceatannol-treated human leukemia U937 cells

To address the mechanism of piceatannol in inhibiting TNFα-mediated pathway, studies on piceatannol-treated human leukemia U937 cells were conducted. Piceatannol treatment reduced TNFα shedding and NFκB activation and decreased the release of soluble TNFα into the culture medium of U937 cells. Moreover, ADAM17 expression was down-regulated in piceatannol-treated cells. Over-expression of ADAM17 abrogated the ability of piceatannol to suppress TNFα-mediated NFκB activation. Piceatannol-evoked β-TrCP up-regulation promoted Sp1 degradation, thus reducing transcriptional level of ADAM17 gene in U937 cells. Piceatannol treatment induced p38 MAPK phosphorylation but inactivation of Akt and ERK. In contrast to p38 MAPK inhibitor or restoration of ERK activation, transfection of constitutive active Akt abolished the effect of piceatannol on β-TrCP, Sp1 and ADAM17 expression. Piceatannol-elicited down-regulation of miR-183 expression was found to cause β-TrCP up-regulation. Inactivation of Akt resulted in Foxp3 down-regulation and reduced miR-183 expression in piceatannol-treated cells. Knock-down of Foxp3 and chromatin immunoprecipitating revealed that Foxp3 genetically regulated transcription of miR-183 gene. Taken together, our data indicate that suppression of Akt/Foxp3-mediated miR-183 expression blocks Sp1-mediated ADAM17 expression in piceatannol-treated U937 cells. Consequently, piceatannol suppresses TNFα shedding, leading to inhibition of TNFα/NFκB pathway.

Inhibition of Casein kinase-2 induces p53-dependent cell cycle arrest and sensitizes glioblastoma cells to tumor necrosis factor (TNFα)-induced apoptosis through SIRT1 inhibition

Glioblastoma multiforme (GBM) are resistant to TNFα-induced apoptosis and blockade of TNFα-induced NF-κB activation sensitizes glioma cells to apoptosis. As Casein kinase-2 (CK2) induces aberrant NF-κB activation and as we observed elevated CK2 levels in GBM tumors, we investigated the potential of CK2 inhibitors (CK2-Is) - DRB and Apigenin in sensitizing glioma cells to TNFα-induced apoptosis. CK2-Is and CK2 small interfering RNA (siRNA) reduced glioma cell viability, inhibited TNFα-mediated NF-κB activation, and sensitized cell to TNFα-induced apoptosis. Importantly, CK2-Is activated p53 function in wild-type but not in p53 mutant cells. Activation of p53 function involved its increased transcriptional activation, DNA-binding ability, increased expression of p53 target genes associated with cell cycle progression and apoptosis. Moreover, CK2-Is decreased telomerase activity and increased senescence in a p53-dependent manner. Apoptotic gene profiling indicated that CK2-Is differentially affect p53 and TNFα targets in p53 wild-type and mutant glioma cells. CK2-I decreased MDM2-p53 association and p53 ubiquitination to enhance p53 levels. Interestingly, CK2-Is downregulated SIRT1 activity and over-expression of SIRT1 decreased p53 transcriptional activity and rescued cells from CK2-I-induced apoptosis. This ability of CK2-Is to sensitize glioma to TNFα-induced death via multiple mechanisms involving abrogation of NF-κB activation, reactivation of wild-type p53 function and SIRT1 inhibition warrants investigation.

Genome-wide siRNA screen for mediators of NF-κB activation

Although canonical NFκB is frequently critical for cell proliferation, survival, or differentiation, NFκB hyperactivation can cause malignant, inflammatory, or autoimmune disorders. Despite intensive study, mammalian NFκB pathway loss-of-function RNAi analyses have been limited to specific protein classes. We therefore undertook a human genome-wide siRNA screen for novel NFκB activation pathway components. Using an Epstein Barr virus latent membrane protein (LMP1) mutant, the transcriptional effects of which are canonical NFκB-dependent, we identified 155 proteins significantly and substantially important for NFκB activation in HEK293 cells. These proteins included many kinases, phosphatases, ubiquitin ligases, and deubiquinating enzymes not previously known to be important for NFκB activation. Relevance to other canonical NFκB pathways was extended by finding that 118 of the 155 LMP1 NF-κB activation pathway components were similarly important for IL-1β-, and 79 for TNFα-mediated NFκB activation in the same cells. MAP3K8, PIM3, and six other enzymes were uniquely relevant to LMP1-mediated NFκB activation. Most novel pathway components functioned upstream of IκB kinase complex (IKK) activation. Robust siRNA knockdown effects were confirmed for all mRNAs or proteins tested. Although multiple ZC3H-family proteins negatively regulate NFκB, ZC3H13 and ZC3H18 were activation pathway components. ZC3H13 was critical for LMP1, TNFα, and IL-1β NFκB-dependent transcription, but not for IKK activation, whereas ZC3H18 was critical for IKK activation. Down-modulators of LMP1 mediated NFκB activation were also identified. These experiments identify multiple targets to inhibit or stimulate LMP1-, IL-1β-, or TNFα-mediated canonical NFκB activation.

1, 25-Dihydroxyvitamin D Suppresses TNFα-Mediated NF-κB Activation in K562 and Human CD34+ Cells and Promotes Recovery of Erythroid Colony Formation in Human CD34+ Cells

Abstract 2097Anemia of inflammation (AI) has been widely associated with chronic rheumatologic, infectious, and cardiovascular disorders and comprises one-third of cases of anemia in the elderly. Although the antimicrobial peptide hepcidin is felt to be the prime regulator of AI, emerging evidence supports the premise that subsets of AI may be mediated via hepcidin-independent inflammatory pathways, such as those modulated by tumor necrosis factor alpha (TNFα), that directly suppress erythropoiesis. A better understanding of the pathophysiology of anemia subtypes is necessary if we are to develop more effective targeted, oral therapies for patients with inflammatory anemia. Our recent analysis of elderly participants in the Third National Health and Nutrition Examination Survey (NHANES III) revealed that vitamin D deficiency was strongly associated with AI in this population, with this subgroup exhibiting a rate of vitamin D deficiency nearly twice that of similarly aged non-anemic individuals. In the current study we aimed to evaluate whether vitamin D treatment may ameliorate TNFα-mediated erythroid colony suppression in humans. Human bone marrow-derived CD34+ cells from 3 healthy adults were first cultured in methylcellulose medium containing IL-3 (10ng/ml), EPO (1U/ml), SCF (50ng/ml) in the presence or absence of TNFα (25 ng/ml) and increasing doses of 1, 25-dihydroxyvitamin D3 [calcitriol, (0.01-1nM)], and erythroid burst-forming units (BFU-E) measured on day 14. The percentage of BFU-E colonies was reduced by 50% at day 14 in the presence of 25ng/ml TNFα (p < 0.01, t-test). TNFα-mediated suppression of erythropoiesis was reversed by the addition of vitamin D3 in a dose-dependent manner, with 1nM vitamin D3 resulting in a 50% recovery of BFU-E colony numbers at day 14 (p < 0.05, t-test). Vitamin D3 alone exhibited no significant effect on BFU-E formation at all concentrations tested. Based on studies showing that vitamin D receptor may serve as a negative regulator of NF-κB transcriptional activity, we next investigated whether vitamin D3 may alter TNFα-induced NF-κB activation in both K562 and human CD34+ cells. K562 cells pre-incubated with 1nM vitamin D3 for 3, 24, 48 and 72 hours were treated with 5 ng/ml TNFα for 30 minutes, and NF-κB activity in nuclear fraction was determined by an ELISA-format oligonucleotide binding assay. TNFα treatment alone markedly increased the oligonucleotide binding activity of the NF-κB p65 subunit, which was completely blocked by co-incubation with competitor oligonucleotide, confirming the assay specificity. Pre-incubation with vitamin D3 for 3 – 48 hours showed little effect on TNFα-mediated NF-κB activation, but pre-incubation for 72 hours resulted in suppression of TNFα-induced NF-κB activity by 46% (p < 0.02, t-test). Under similar conditions, human CD34+ cells pre-incubated with 1 nM vitamin D3 for 48 hours and 72 hours exhibited 38% and 84% suppression of TNFα-induced NF-κB activity, respectively (p < 0.01 for both conditions, t-test). Reduced NF-κB activity was correlated with the decreased nuclear translocation of NF-κB in K562 cells with no significant changes in IκB degradation, suggesting that vitamin D3 regulates the NF-κB nuclear translocation independent of IκB. Western analysis of whole cell lysates from both K562 and human CD34+ cells using antibodies recognizing known TNFα-associated signaling pathways revealed robust increases in phospho-p38, phospho-JNK, and phospho-ERK1/2 in response to TNFα stimulation compared with control, with no inhibition of these signaling pathways noted in response to vitamin D3 treatment at all doses tested, suggesting that vitamin D3 does not inhibit these TNFα-induced signaling cascades. Our study indicates that 1, 25-dihydroxyvitamin D3 significantly ameliorates TNFα-mediated suppression of erythroid colony development in human CD34+ cells via mechanisms that involve modulation of NF-κB signaling pathways and supports the design of future clinical trials examining whether vitamin D3 supplementation may prove to be an effective therapy for subgroups of patients with TNFα-mediated inflammatory anemia. Disclosures:No relevant conflicts of interest to declare.

Enterovirus 71 2C Protein Inhibits TNF-α–Mediated Activation of NF-κB by Suppressing IκB Kinase β Phosphorylation

Enterovirus 71 (EV71), a single, positive-stranded RNA virus, has been regarded as the most important neurotropic enterovirus after the eradication of the poliovirus. EV71 infection can cause hand, foot, and mouth disease or herpangina. Cytokine storm with elevated levels of proinflammatory and inflammatory cytokines, including TNF-α, has been proposed to explain the pathogenesis of EV71-induced disease. TNF-α-mediated NF-κB signaling pathway plays a key role in inflammatory response. We hypothesized that EV71 might also moderate host inflammation by interfering with this pathway. In this study, we tested this hypothesis and identified EV71 2C protein as an antagonist of TNF-α-mediated activation of NF-κB signaling pathway. Expression of 2C protein significantly reduced TNF-α-mediated NF-κB activation in 293T cells as measured by gene reporter and gel mobility shift assays. Furthermore, overexpression of TNFR-associated factor 2-, MEK kinase 1-, IκB kinase (IKK)α-, or IKKβ-induced NF-κB activation, but not constitutively active mutant of IKKβ (IKKβ SS/EE)-induced NF-κB activation, was inhibited by 2C protein. These data together suggested that the activation of IKKβ is most likely targeted by 2C; this notion was further strengthened by immunoblot detection of IKKβ phosphorylation and IκBα phosphorylation and degradation. Coimmunoprecipitation and colocalization of 2C and IKKβ expressed in mammalian cells provided compelling evidence that 2C interacts with IKKβ. Collectively, our data indicate that EV71 2C protein inhibits IKKβ activation and thus blocks NF-κB activation.

Resveratrol ameliorates TNFα‐mediated suppression of erythropoiesis in human CD34+ cells via modulation of NF‐κB signalling

Overexpression of pro-inflammatory cytokines, including tumour necrosis factor alpha (TNFα), has been implicated in the pathogenesis of anaemia of inflammation. TNFα suppresses erythroid colony formation via both direct and indirect effects on haematopoietic progenitors, often involving activation of nuclear factor (NF)-κB signalling resulting in downregulation of transcription factors critical for erythropoiesis. There is a dearth of effective and safe therapies for many patients with inflammatory anaemia. Resveratrol is a flavanol found in red wine grapes that possesses potent anti-inflammatory properties, but studies of its impact on human erythropoiesis have proven contradictory. We investigated whether resveratrol ameliorates TNFα-mediated suppression of erythropoiesis in human CD34(+) haematopoietic progenitors. We found that resveratrol partially reverses the erythroid suppressive effects of TNFα, leading to significant recovery in burst forming unit-erythroid colony formation in human CD34(+) cells. CD34(+) cells pre-incubated with resveratrol for 72 h in the presence of TNFα inhibited NF-κB activation via decreased NF-κB nuclear localization without altering total NF-κB protein levels and independent of IκB degradation. Resveratrol also significantly restored the baseline expression of erythroid transcription factors NFE2 and the GATA1/GATA2 ratio in CD34(+) cells treated with TNFα. In conclusion, resveratrol may inhibit TNFα-mediated NF-κB activation and promote erythropoiesis in primary human CD34(+) cells.

Adiponectin expression in patients with inflammatory cardiomyopathy indicates favourable outcome and inflammation control

Circulating adiponectin (APN) is an immunomodulatory, pro-angiogenic, and anti-apoptotic adipocytokine protecting against acute viral heart disease and preventing pathological remodelling after cardiac injury. The purpose of this study was to describe the regulation and effects of APN in patients with inflammatory cardiomyopathy (DCMi). Adiponectin expression and outcome were assessed in 173 patients with DCMi, 30 patients with non-inflammatory DCM, and 30 controls. Mechanistic background of these findings was addressed in murine experimental autoimmune myocarditis (EAM), a model of human DCMi, and further elucidated in vitro. Adiponectin plasma concentrations were significantly higher in DCMi compared with DCM or controls, i.e. 6.8 ± 3.9 µg/mL vs. 5.4 ± 3.6 vs. 4.76 ± 2.5 µg/mL (P< 0.05, respectively) and correlated significantly with cardiac mononuclear infiltrates (CD3+: r(2)= 0.025, P= 0.038; CD45R0+: r(2)= 0.058, P= 0.018). At follow-up, DCMi patients with high APN levels showed significantly increased left ventricular ejection fraction improvement, decreased left ventricular end-diastolic diameter, and reduced cardiac inflammatory infiltrates compared with patients with low APN levels. A multivariate linear regression analysis implicated APN as an independent prognostic factor for inhibition of cardiac inflammation. In accordance with these findings in human DCMi, EAM mice exhibited elevated plasma APN. Adiponectin gene transfer led to significant downregulation of key inflammatory mediators promoting disease. Mechanistically, APN acted as a negative regulator of T cells by reducing antigen specific expansion (P< 0.01) and suppressed TNFα-mediated NFκB activation (P< 0.01) as well as release of reactive oxygen species in cardiomyocytes. Our results implicate that APN acts as endogenously upregulated anti-inflammatory cytokine confining cardiac inflammation and progression in DCMi.