TNFα-induced NF-κB Signaling Research Articles

Hematopoietic stem cells with granulo-monocytic differentiation state overcome venetoclax sensitivity in patients with myelodysplastic syndromes

The molecular mechanisms of venetoclax-based therapy failure in patients with acute myeloid leukemia were recently clarified, but the mechanisms by which patients with myelodysplastic syndromes (MDS) acquire secondary resistance to venetoclax after an initial response remain to be elucidated. Here, we show an expansion of MDS hematopoietic stem cells (HSCs) with a granulo-monocytic-biased transcriptional differentiation state in MDS patients who initially responded to venetoclax but eventually relapsed. While MDS HSCs in an undifferentiated cellular state are sensitive to venetoclax treatment, differentiation towards a granulo-monocytic-biased transcriptional state, through the acquisition or expansion of clones with STAG2 or RUNX1 mutations, affects HSCs’ survival dependence from BCL2-mediated anti-apoptotic pathways to TNFα-induced pro-survival NF-κB signaling and drives resistance to venetoclax-mediated cytotoxicity. Our findings reveal how hematopoietic stem and progenitor cell (HSPC) can eventually overcome therapy-induced depletion and underscore the importance of using close molecular monitoring to prevent HSPC hierarchical change in MDS patients enrolled in clinical trials of venetoclax.

TSG101 Physically Interacts with Linear Ubiquitin Chain Assembly Complex (LUBAC) and Upregulates the TNFα-Induced NF-κB Activation.

Linear ubiquitin chain assembly complex (LUBAC) is a ubiquitin E3 ligase complex composed of HOIP, HOIL-1L, and SHARPIN that catalyzes the formation of linear/M1- linked ubiquitin chain. It has been shown to play a pivotal role in the nuclear factor (NF)-κB signaling induced by proinflammatory stimuli. Here, we found that tumor susceptibility gene (TSG101) physically interacts with HOIP, a catalytic component of LUBAC, and potentiates LUBAC activity. Depletion of TSG101 expression by RNA interference decreased TNFα-induced linear ubiquitination and the formation of TNFα receptor 1 signaling complex (TNFRSC). Furthermore, TSG101 facilitated the TNFα-induced stimulation of the NF-κB pathway. Thus, we suggest that TSG101 functions as a positive modulator of HOIP that mediates TNFα-induced NF-κB signaling pathway.

Divergent effect of Birinapant, and BV6 SMAC mimetic on TNFα induced NF-κB signaling and cell viability in activated hepatic stellate cells.

Tumor necrosis factor-α (TNFα) is a pleiotropic cytokine involved in nuclear factor kappa B (NF-κB) mediated cell survival as well as cell death. High serum TNFα levels correlate with liver fibrosis and enhance hepatic stellate cell (HSC) viability. However, the regulatory role of cellular inhibitor of apoptosis-1/2 (cIAP1/2) during TNFα induced NF-κB signaling in activated HSCs is largely unknown. Activated HSCs were treated with cIAP1/2 inhbitiors i.e., SMAC mimetic BV6, and Birinapant in the presence of TNFα and macrophage conditioned media. TNFα cytokine increased cIAP2 expression and enhanced cell viability through the canonical NF-κB signaling in activated HSCs. cIAP2 inhibition via BV6 decreased the TNFα induced canonical NF-κB signaling, and reduced cell viability in activated HSCs. SMAC mimetic, Birinapant alone did not affect the cell viability but treatment of TNFα sensitized HSCs with Birinapant induced cell death. While BV6 mediated cIAP2 ablation was able to decrease the TNFα induced canonical NF-κB signaling, this effect was not observed with Birinapant treatment. Secreted TNFα from M1 polarized macrophages sensitized activated HSCs to BV6 or Birinapant mediated cell death. However, M2 polarized macrophage conditioned medium rescued the activated HSCs from BV6 mediated cytotoxicity. In this study, we describe the regulatory role of cIAP2 in TNFα induced NF-κB signaling in activated HSCs. Targeting cIAP2 may be a promising approach for liver fibrosis treatment via modulating NF-κB signaling in activated HSCs.

TRIM56 positively regulates TNFα-induced NF-κB signaling by enhancing the ubiquitination of TAK1

Nuclear factor-κB (NF-κB) signaling participates in many biologic processes including immunity, inflammation, and cancer. Here we reported that tripartite motif-containing protein 56 (TRIM56), an E3 ligase enzyme, participated in TNFα-induced NF-κB signaling by interacting with TAK1. Overexpression of TRIM56 potentiated the activation of TNFα-induced NF-κB signaling, whereas knockdown of TRIM56 had an opposite effect. TRIM56 enhanced the ubiquitination of TAK1, specifically enhanced the M1-linked polyubiquitin chains to TAK1, leading to the tight interactions of the TAK1-IKKα complex. Consequently, the stimulation of TNFa and TRIM56 strengthened the interaction with TAK1. Furthermore, we found that the C terminal (CT) domain was the binding region of TRIM56, and the RING domain of TRIM56 was the E3 enzyme activity region which was important to the ubiquitination of TAK1. Together, these results reveal that TRIM56 positively regulates TNFα-induced NF-κB signaling by heightening the ubiquitination of TAK1 and provide new insight into the complicated mechanisms of the inflammatory and immune response.

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.

TRIM25 Promotes TNF-α-Induced NF-κB Activation through Potentiating the K63-Linked Ubiquitination of TRAF2.

As an important effector in response to various intracellular or extracellular stimuli, the NF-κB family extensively participates in a wide spectrum of biological events, and its dysregulation may result in many pathological conditions, such as microbial infection, tumor progression, and neurodegenerative disorders. Previous investigations showed that multiple types of ubiquitination play critical roles in the modulation of the NF-κB signaling pathway, yet the molecular mechanisms are still poorly understood. In the current study, we identified TRIM25, an E3 ubiquitin ligase, as a novel positive regulator in mediating NF-κB activation in human embryonic kidney 293T (HEK293T), HeLa cells, THP-1 cells, and PBMCs. The expression of TRIM25 promoted TNF-α-induced NF-κB signaling, whereas the knockdown had the opposite effect. Furthermore, TRIM25 interacted with TRAF2 and enhanced the K63-linked polyubiquitin chains attached to TRAF2. Moreover, TRIM25 bridged the interaction of TRAF2 and TAK1 or IKKβ. To our knowledge, our study has identified a previously unrecognized role for TRIM25 in the regulation of NF-κB activation by enhancing the K63-linked ubiquitination of TRAF2.

MST1 Negatively Regulates TNFα-Induced NF-κB Signaling through Modulating LUBAC Activity

The nuclear factor (NF)-κB pathway plays a central role in inflammatory and immune responses, with aberrant activation of NF-κB signaling being implicated in various human disorders. Here, we show that mammalian ste20-like kinase 1 (MST1) is a previously unrecognized component of the tumor necrosis factor α (TNFα) receptor 1 signaling complex (TNF-RSC) and attenuates TNFα-induced NF-κB signaling. Genetic ablation of MST1 in mouse embryonic fibroblasts and bone marrow-derived macrophages potentiated the TNFα-induced increase in IκB kinase (IKK) activity, as well as the expression of NF-κB target genes. TNFα induced the recruitment of MST1 to TNF-RSC and its interaction with HOIP, the catalytic component of the E3 ligase linear ubiquitin assembly complex (LUBAC). Furthermore, MST1 activated in response to TNFα stimulation mediates the phosphorylation of HOIP and thereby inhibited LUBAC-dependent linear ubiquitination of NEMO/IKKγ. Together, our findings suggest that MST1 negatively regulates TNFα-induced NF-κB signaling by targeting LUBAC.

CHIP regulates bone mass by targeting multiple TRAF family members in bone marrow stromal cells

Carboxyl terminus of Hsp70-interacting protein (CHIP or STUB1) is an E3 ligase and regulates the stability of several proteins which are involved in different cellular functions. Our previous studies demonstrated that Chip deficient mice display bone loss phenotype due to increased osteoclast formation through enhancing TRAF6 activity in osteoclasts. In this study we provide novel evidence about the function of CHIP. We found that osteoblast differentiation and bone formation were also decreased in Chip KO mice. In bone marrow stromal (BMS) cells derived from Chip−/− mice, expression of a panel of osteoblast marker genes was significantly decreased. ALP activity and mineralized bone matrix formation were also reduced in Chip-deficient BMS cells. We also found that in addition to the regulation of TRAF6, CHIP also inhibits TNFα-induced NF-κB signaling through promoting TRAF2 and TRAF5 degradation. Specific deletion of Chip in BMS cells downregulated expression of osteoblast marker genes which could be reversed by the addition of NF-κB inhibitor. These results demonstrate that the osteopenic phenotype observed in Chip−/− mice was due to the combination of increased osteoclast formation and decreased osteoblast differentiation. Taken together, our findings indicate a significant role of CHIP in bone remodeling.

A parapoxviral virion protein targets the retinoblastoma protein to inhibit NF-κB signaling.

Poxviruses have evolved multiple strategies to subvert signaling by Nuclear Factor κB (NF-κB), a crucial regulator of host innate immune responses. Here, we describe an orf virus (ORFV) virion-associated protein, ORFV119, which inhibits NF-κB signaling very early in infection (≤ 30 min post infection). ORFV119 NF-κB inhibitory activity was found unimpaired upon translation inhibition, suggesting that virion ORFV119 alone is responsible for early interference in signaling. A C-terminal LxCxE motif in ORFV119 enabled the protein to interact with the retinoblastoma protein (pRb) a multifunctional protein best known for its tumor suppressor activity. Notably, experiments using a recombinant virus containing an ORFV119 mutation which abrogates its interaction with pRb together with experiments performed in cells lacking or with reduced pRb levels indicate that ORFV119 mediated inhibition of NF-κB signaling is largely pRb dependent. ORFV119 was shown to inhibit IKK complex activation early in infection. Consistent with IKK inhibition, ORFV119 also interacted with TNF receptor associated factor 2 (TRAF2), an adaptor protein recruited to signaling complexes upstream of IKK in infected cells. ORFV119-TRAF2 interaction was enhanced in the presence of pRb, suggesting that ORFV119-pRb complex is required for efficient interaction with TRAF2. Additionally, transient expression of ORFV119 in uninfected cells was sufficient to inhibit TNFα-induced IKK activation and NF-κB signaling, indicating that no other viral proteins are required for the effect. Infection of sheep with ORFV lacking the ORFV119 gene led to attenuated disease phenotype, indicating that ORFV119 contributes to virulence in the natural host. ORFV119 represents the first poxviral protein to interfere with NF-κB signaling through interaction with pRb.

Uev1A-Ubc13 catalyzes K63-linked ubiquitination of RHBDF2 to promote TACE maturation

The TNFα-induced NF-κB signaling pathway plays critical roles in multiple biological processes. Extensive studies have explored the mechanisms regulating this signaling cascade, and identified an E2 complex, Uev1A-Ubc13, that mediates K63-linked poly-Ub chain formation and thus recruits NEMO to activate the signaling transduction. In this study, we demonstrate that the Uev1A-Ubc13 complex simultaneously serves as a repressor of the NF-κB pathway. It was found that cells overexpressing UEV1A silence the signal cascade earlier than control cells. Importantly, UEV1A overexpression enhances TACE maturation to shed the TNFα receptor. The Uev1A-Ubc13 complex interacts with RHBDF2, a key factor promoting TACE maturation, and inhibition of the Uev1A-Ubc13 activity interferes with RHBDF2-promoted TACE maturation. Furthermore, upon TNFα stimulation, the Uev1A-Ubc13 complex cooperates with CHIP to promote K63-linked ubiquitination of RHBDF2, enhancing its activity toward TACE maturation and subsequently blocking TNFα-induced NF-κB signaling.

Epithelial EZH2 serves as an epigenetic determinant in experimental colitis by inhibiting TNFα-mediated inflammation and apoptosis

Epithelial barrier disruption is a major cause of inflammatory bowel disease (IBD); however, the mechanism through which epigenetic regulation modulates intestinal epithelial integrity remains largely undefined. Here we show that EZH2, the catalytic subunit of polycomb repressive complex (PRC2), is indispensable for maintaining epithelial cell barrier integrity and homeostasis under inflammatory conditions. In accordance with reduced EZH2 expression in patients, the inactivation of EZH2 in IECs sensitizes mice to DSS- and TNBS-induced experimental colitis. Conversely, EZH2 overexpression in the intestinal epithelium renders mice more resistant to colitis. Mechanistically, the genes encoding TRAF2/5 are held in a finely tuned bivalent status under inflammatory conditions. EZH2 deficiency potentiates the expression of these genes to enhance TNFα-induced NF-κB signaling, thereby leading to uncontrolled inflammation. More importantly, we show that EZH2 depletion compromises the protective role of NF-κB signaling in cell survival by directly up-regulating ITCH, a well-known E3 ligase that degrades the c-FLIP protein. Thus, our findings highlight an epigenetic mechanism by which EZH2 integrates the multifaceted effects of TNFα signaling to promote the inflammatory response and apoptosis in colitis.

Therapeutic potential of Oroxylin A in rheumatoid arthritis

Excessive inflammation contributes greatly to the pathogenesis and development of rheumatoid arthritis (RA). Oroxylin A (OA) is a natural anti-inflammatory flavonoid compound. In this study, we investigated the effects of OA on collagen-induced arthritis (CIA) and human RA fibroblast-like synoviocytes (FLS). CIA was induced in DBA/1 mice and mice were intraperitoneally treated with OA (10mg/kg) for 10days. Arthritis severity was evaluated every day and the histopathologic examination of joints was done. Serum levels of anti-collagen II antibodies (anti-CII Abs) and cytokines were determined by ELISA. Frequency of regulatory T cells (Tregs) and Th17 cells in draining inguinal lymph nodes (ILN) was quantified by flow cytometry. FLS from patients with active RA were treated with varying doses of oroxylin A, followed by stimulation with tumor necrosis factor (TNF)-α (10ng/mL). The production of cytokines was measured by ELISA. Signal transduction proteins were examined by western blot. OA significantly diminished the arthritis and histological damage. Serum anti-CII Abs, IL-1β, IL-6, TNFα, and IL-17 were significantly diminished by OA treatment. Analysis of CD4+T cell populations in OA-treated mice showed an increase in Tregs and reduction in Th17 cells in the ILN. In vitro, OA decreased the secretion of IL-1β and IL-6 from TNFα-stimulated RA FLS in a dose-dependent manner. TNFα-induced p38 MAPK, ERK1/2 and NF-κB signaling pathways were suppressed by OA. Our results indicate that OA exerts an anti-inflammatory activity and may have therapeutic potential for human RA.

P62-ZZ Domain Inhibition Prevents MM Cell-Induced Epigenetic Changes at the Runx2 and C/EBPb Promoters

Multiple myeloma bone disease (MMBD) is a paradigm for uncoupled bone remodeling and is characterized by non-healing lytic bone lesions. Osteoblast (OB) function is highly suppressed or absent, persists in the absence of MM cells, and remains a significant cause of skeletal-related events. The persistence of OB suppression in MMBD suggests that MM cells induce repressive epigenetic changes at the Runx2 gene, the key transcription factor required for OB differentiation of bone marrow stromal cells (BMSC), which are preOB. We reported that TNFα is a major suppressor of OB in MMBD that reduces Runx2 levels by inducing Gfi1, a transcriptional repressor of Runx2. Using ChIP analysis of MM-exposed BMSC, we showed that Gfi1 directly binds the Runx2 promoter and recruits the chromatin corepressor HDAC1 to the Runx2 promoter in MM-exposed BMSC, reducing transcriptionally permissive euchromatin marks such as H3K9ac.Recently, we reported that p62 (sequestosome-1) in BMSC is critical for the formation of MM cell-induced signaling complexes that mediate OB suppression and IL-6 production. We found that XRK3F2, a novel inhibitor of the p62 ZZ domain (p62-ZZ), blunted MM cell-induced repression of Runx2 and induction of Gfi1 in BMSC, and induced new bone formation and remodeling in mice with established MMBD, but did not alter normal bone. In the current study, we further evaluated the specificity of XRK3F2's inhibition of p62-ZZ interactions and investigated the mechanism by which blocking p62-ZZ prevents MM-induced suppression of key osteogenic transcription factors in BMSC.We previously showed that TNFα and MM cell-induced IL-6 production by BMSC are both p62 dependent and independent. XRK3F2 blocked TNFα-induced NFκB signaling in BMSC and MM cells, and partially inhibited TNFα-induced IL-6 production by BMSC. We now report that XRK3F2 blunted the TNFα upregulation observed in BMSC after MM cell coculture but did not alter TNFα production in p62 knockout (p62KO) BMSC following coculture with MM cells. TNFα treatment of p62KO BMSCs resulted in minimal induction of IL-6, which was not altered by XRK3F2. Transfection of p62KO BMSC with full-length p62 constructs restored TNFα-induced IL-6 production and XRK3F2's capacity to reduce TNFα-induced IL-6. In contrast, XRK3F2 had no effect on TNFα-induced IL-6 production by p62KO BMSC transfected with p62 constructs lacking p62-ZZ.To further investigate XRK3F2's mechanism of action, we tested if XRK3F2 prevents Gfi1-induced epigenetic suppression of Runx2 by preventing Gfi1's upregulation and binding to Runx2. ChIP analysis of MM exposed BMSC treated with XRK3F2 demonstrated that XRK3F2 reduced MM-induced Gfi1 occupancy of the Runx2 promoter and prevented MM-induced reduction of H3K9ac. Since the region proximal to the Runx2 promoter contains putative C/EBPβ binding sites, we tested if MM-induced p62-ZZ signaling activates C/EBPβ, a transcription factor that regulates OB lineage maturation and IL-6 production in BMSC and plays a role in the upregulation of Gfi1 expression. MM-exposed BMSC had increased enrichment of C/EBPβ at both the Gfi1 gene and the C/EBPβ-regulated Il6 gene, and XRK3F2 reduced upregulation of MM-induced C/EBPβ binding at both the Gfi1 and Il6 genes. We conclude that XRK3F2's reduction of the MM-induced C/EBPβ binding in BMSC results in decreased Gfi1 mRNA expression, and thereby reduces MM-induced Gfi1 occupancy and HDAC1 recruitment at the Runx2 promoter. These results suggest that targeting p62-ZZ in MMBD may reverse C/EBPβ mediated Gfi1 activation, resulting in rescue of the MM cell-induced epigenetic suppression of Runx2 in BMSC, and that next-generation derivatives of XRK3F2 should impact BMSC-supported MM cell survival. DisclosuresSilbermann:Celgene: Research Funding; Amgen: Consultancy. Xie:Oxis Biotech: Consultancy, Membership on an entity's Board of Directors or advisory committees. Roodman:Eli Lilly: Research Funding; Amgen: Consultancy.

Sargahydroquinoic acid inhibits TNFα-induced AP-1 and NF-κB signaling in HaCaT cells through PPARα activation

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily of ligand-activated transcription factors and expressed in various cell types in the skin, including keratinocytes, fibroblasts and infiltrating immune cells. Thus, their ligands are targets for the treatment of various skin disorders, such as photo-aging and chronological aging of skin. Intensive studies have revealed that PPARα/γ functions in photo-aging and age-related inflammation by regulating matrix metalloproteinases (MMPs) via activator protein-1 (AP-1) and nuclear factor kappa B (NF-κB). However, the detailed mechanism of PPARα/γ’s role in skin aging has not yet been elucidated. In this study, we confirmed that sargahydroquinoic acid (SHQA) as a PPARα/γ ligand significantly decreased Tumor Necrosis Factor-alpha (TNFα)-induced MMP-2/-9 expression by downregulating TNFα-induced transcription factors, subsequently reducing IκBα degradation and blocking NF-κB p65 nuclear translocation in HaCaT human epidermal keratinocyte cells. Treatment of cells with SHQA and GW6471 (PPARα antagonist) not bisphenol A diglycidyl ether (PPARγ antagonists), reversed the effect on TNFα-induced inflammatory signaling pathway activation. Taken together, our data suggest that SHQA inhibit TNFα-induced MMP-2/-9 expression and age-related inflammation by suppressing AP-1 and NF-κB pathway via PPARα.

Abstract 5235: A model for TNFα-mediated NFκB signalling: A systems biology study on hepatocytes and liver cancer cells.

Abstract It is known that the development of hepatocellular carcinoma (HCC) is frequently associated to inflammation, hepatocytic inhibition of apoptosis, and compensatory liver regeneration. In this context, the classical κ-light-chain-enhancer of activated B cells (NFκB) signaling pathway is one central regulator of inflammatory responses and hepatocyte survival, which may promote HCC development. Upon stimulation, Kupffer cell-derived cytokine tumor necrosis factor (TNF)-α is one of the key factors during the priming phase of liver regeneration as well as in hepatocarcinogenesis mainly driving cell survival and proliferation by NFκB signalling pathway. In order to understand the impact of this pathway in the earliest stages of liver damage and tumorigenesis, mathematical modelling is necessary to describe the dynamic behaviour of this pathway in normal as well as in malignant-transformed cells. Several mathematical models have been developed for TNFα-induced NFκB signalling in immortalized fibroblasts and tumor cell lines; however, a model specifically analyzing and comparing hepatocytes and HCC cells is still missing. We here present a hepatocyte-specific ordinary differential equation (ODE) model for TNFα-induced NFκB signaling that considers experimental data of protein expression after TNFα stimulation (p65/pp65, IκBα/pIκBα), basal protein turnover (p65, IκBα), and IκBα mRNA. In order to sufficiently describe the pathway dynamics, an additional nuclear phosphorylation step of p65 was included in the model. Possible candidate kinases are the mitogen- and stress-activated protein kinase1 (MSK1) and the protein kinase C zeta (PKCζ). The established model was able to predict TNFα-induced p65/IκBα complex formation, which was experimentally confirmed using primary mouse hepatocytes cells. Finally, we compared the dynamics of TNFα-induced NF-kB pathway activation in different species and cell types (primary murine hepatocytes, human and mouse HCC lines, mouse liver tissue after partial hepatectomy). In conclusion, we here present a mathematical model for TNFα/NFκB signalling in primary hepatocytes cells, providing an important basis to quantitatively disentangle the complex processes factors in liver regeneration and tumorigenesis. Citation Format: Federico Pinna, Sven Sahle, Katharina Beuke, Michaela Bissinger, Selcan Tuncay, Lorenza D'Alessandro, Ralph Gauges, Andreas Raue, Jens Timmer, Ursula Klingmüller, Peter Schirmacher, Ursula Kummer, Kai Breuhahn. A model for TNFα-mediated NFκB signalling: A systems biology study on hepatocytes and liver cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5235. doi:10.1158/1538-7445.AM2013-5235

Saussurea lappaClarke-Derived Costunolide Prevents TNFα-Induced Breast Cancer Cell Migration and Invasion by Inhibiting NF-κB Activity

Saussurea lappa Clarke (SLC) has been used as a traditional medicine in Korea, China, and Japan for the treatment of abdominal pain and tenesmus. Costunolide, a sesquiterpene lactone isolated from SLC, has diverse medicinal effects. However, the anticancer effects of costunolide are still unclear in breast cancer. In this study, we demonstrate that costunolide suppresses tumor growth and metastases of MDA-MB-231 highly metastatic human breast cancer cells via inhibiting TNFα-induced NF-κB activation. Costunolide inhibited MDA-MB-231 tumor growth and metastases without affecting body weights in the in vivo mouse orthotopic tumor growth assays. In addition, costunolide inhibited in vitro TNFα-induced invasion and migration of MDA-MB-231 cells. Costunolide further suppressed TNFα-induced NF-κB signaling activation, resulting in a reduced expression of MMP-9, a well-known NF-κB-dependent gene to mediate breast cancer cell growth and metastases. Therefore, we conclude that SLC and its derivative costunolide suppress breast cancer growth and metastases by inhibiting TNFα-induced NF-κB activation, suggesting that costunolide as well as SLC may be promising anticancer drugs, especially for metastatic breast cancer.

Murine tumor necrosis factor α–induced adipose‐related protein (tumor necrosis factor α–induced protein 9) deficiency leads to arthritis via interleukin‐6 overproduction with enhanced NF‐κB, STAT‐3 signaling, and dysregulated apoptosis of macrophages

To elucidate the role of tumor necrosis factor α-induced adipose-related protein (TIARP; or tumor necrosis factor α-induced protein 9 [TNFAIP-9]) in the development and pathogenesis of arthritis. We generated TIARP-deficient (TIARP(-/-) ) mice and investigated several organs in aged mice. Peritoneal macrophages were collected and cultured with lipopolysaccharide (LPS) and TNFα, and then the production of cytokines and subsequent NF-κB signal transduction were analyzed. We also examined the susceptibility of young TIARP(-/-) mice to collagen-induced arthritis (CIA). Draining lymph nodes and splenocytes were isolated and cultured, and serum levels of anti-type II collagen (anti-CII) antibodies, interleukin-6 (IL-6), and TNFα on day 60 were measured. We further investigated the effects of anti-IL-6 receptor monoclonal antibody (mAb) on the development of arthritis in TIARP(-/-) mice. IL-6/STAT-3 signaling was also analyzed using TIARP(-/-) macrophages. TIARP(-/-) mice developed spontaneous enthesitis and synovitis, had high serum levels of IL-6, had increased CD11b+ cell counts in the spleen, and showed enhanced LPS- and TNFα-induced IL-6 expression in macrophages. Sustained degradation of IκBα with dysregulated apoptosis was also noted in TIARP(-/-) macrophages. CIA was clearly exacerbated in TIARP(-/-) mice, accompanied by marked neutrophil and macrophage infiltration in joints. The levels of anti-CII antibodies in serum were unchanged, whereas autoreactive Th1 cell and Th17 cell responses were higher in TIARP(-/-) mice. Treatment with anti-IL-6 receptor mAb prevented the development of CIA in TIARP(-/-) mice, and TIARP(-/-) macrophages showed increased IL-6-induced STAT-3 phosphorylation. These findings suggest that TIARP acts as a negative regulator of arthritis by suppressing IL-6 production, its signaling and TNFα-induced NF-κB signaling, resulting in enhanced apoptosis in macrophages.

Sulfated chitooligosaccharide II (SCOS II) suppress collagen degradation in TNF-induced chondrosarcoma cells via NF-κB pathway

Chitooligosaccharides (COS), the hydrolyzed product of chitosan and its derivatives, are known to have interesting pharmaceutical and medicinal applications due to its high solubility, non-toxicity, and increased functionality. Among them sulfated chitooligosaccharides (SCOSs) have been identified to possess enhanced biological activities. This study reports the effects of SCOSs with different molecular weights on the degradation of articular cartilage through unregulated collagenase expression. The results indicated that the SCOS II (3–5kDa) effectively inhibited the expressions of collagenases 1 and 3 and thereby prevented TNF-α induced degradation of collagen in human chondrosarcoma cells (SW-1353). Moreover, the signaling cascade responsible for this effect was found as SCOS II mediated suppression of NF-κB activation. Based on these data, it can be concluded that SCOS II prevented collagen degradation by inhibiting collagenases 1 and 3 via suppressing TNF-α induced NF-κB signaling. We suggest that SCOS II can be further studied as a potential candidate for the treatment of arthritis.

Hypoxia reduces the response of human adipocytes towards TNFα resulting in reduced NF-κB signaling and MCP-1 secretion

Obesity is associated with adipose tissue hypoxia, and is thought to be linked to the chronic low-grade inflammation of adipose tissue, although the precise mechanism has remained unclear. In this study, we investigated the effect of a prominent hypoxia on human primary adipocyte secretion and tumor necrosis factor alpha (TNFα)-induced nuclear factor-κB (NF-κB) signaling. Using cytokine array and ELISA analysis, we compared the secretion patterns of normoxic and hypoxic (1% O(2)) adipocytes and observed various alterations in adipokine release. We could reproduce known alterations like an induction of interleukin (IL)-6, vascular endothelial growth factor, leptin and a reduction in adiponectin release under hypoxia. Interestingly, we observed a significant reduction in the secretion of macrophage chemotactic protein (MCP)-1 and other NF-κB-related genes, such as growth-regulated oncogene-α, eotaxin and soluble TNF-Receptor1 (TNF-R1) under hypoxia. TNFα stimulation of hypoxic adipocytes resulted in a significantly reduced phosphorylation of NF-κB and its inhibitor IκBα compared with normoxic cells. Furthermore, chronic treatment of hypoxic adipocytes with TNFα resulted in an expected higher secretion of the chemokines MCP-1 and IL-8, but under hypoxia, the secretion level was substantially lower than that under normoxia. This reduction in protein release was accompanied by a reduced mRNA expression of MCP-1, whereas IL-8 mRNA expression was not altered. Additionally, we observed a significantly reduced expression of the TNF-receptor TNF-R1, possibly being one cause for the reduced responsiveness of hypoxic adipocytes towards TNFα stimulation. In conclusion, human primary adipocytes show a basal and TNFα-induced reduction of MCP-1 release under hypoxia. This effect may be due to a reduced expression of TNF-R1 and therefore attenuated TNFα-induced NF-κB signaling. These observations demonstrate a reduced responsiveness of hypoxic adipocytes towards inflammatory stimuli like TNFα, which may represent an adaptation process to maintain adipose tissue function under hypoxia and inflammatory conditions.

Abstract LB-235: SUMO-1 modification represses CPAP transcriptional co-activator activity in TNFα-induced NF-κB signaling pathway

Abstract The transcription factor nuclear factor-κB (NF-κB) is an important regulator in inflammation, immune responses, apoptosis, cell proliferation and differentiation. CPAP (Centrosomal protein P4.1-associated protein), which plays important roles on centrosomal functions, is previously identified as the transcriptional coactivator for NF-κB. Here, we have demonstrated that decreased expression of CPAP by siRNA can attenuate NF-κB-mediated genes expression under TNFα stimulus. On the contrary, overexpressed CPAP contributes to the NF-κB-driven transcriptional activation. Immunoflourescence data showed that the nuclear localization of CPAP is induced by TNFα stimulus. Chromatin immunoprecipitation assay further indicated that CPAP was recruited to COX-2 promoter region under TNF treatment. We found that the NF-κB signaling pathway is activated by CPAP through recruiting more IKKß to the inactivated NF-κB complex to enhance the degradation of IκB and the phosphorylation of p65. Importantly, CPAP was covalently modified by small ubiquitin-related modifier-1 (SUMO-1) protein under TNFα stimulus, and the specific acceptor sites were located in the C-terminal of CPAP. Interestingly, when overexpressing SUMO-deficient CPAP in A549 cells, the TNFα-induced NF-κB-mediated genes expression was up-regulated. Co-immunoprecipitation assay showed that the association of IKKß to NF-κB complex was augmented by SUMO-deficient CPAP than wild type CPAP. Taken together, these results suggest that there might be a mechanism for the SUMOylation of CPAP in modulating the activity of NF-κB. The detailed machinery for SUMOylated CPAP in the TNFα-induced NF-κB activation is currently under investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-235.