Abstract 23: Disruption of TRAF2-TAK1-NF-κB Signaling Axis Triggers K-48 Linked Poly-Ubiquitylation of RIP1 and Necrotic Cell Death in Doxorubicin Cardiotoxicity
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The anthracycline doxorubicin (Dox) is a highly effective anti-tumour agent, however, its use is limited by its severe cardiotoxic effects that manifests as heart failure. The decline in cardiac performance induced by doxorubicin remains poorly defined. A critical survival role for the canonical IKKβ -mTOR-NF-κB signaling pathway has been demonstrated in ventricular myocytes. In this report, we demonstrate that, Dox impairs IKKβ-mTOR- NF-κB signaling in ventricular myocytes accompanied by mitochondrial perturbations including mPTP, loss of mitochondrial membrane potential and ROS production. IKKβ- NF-κB signaling involves TRAF 2 mediated ligation of K63- ubiquitin chains to RIP1 (Receptor Interacting Protein 1) which serves as scaffold for recruitment of ubiquitylated Tak1 complexes and phosphorylation-dependent activation of IKKβ -NF-kB signaling. Interestingly, ventricular myocytes treated with dox demonstrated reduction in expression levels of TRAF2 and TAK1, in vivo and in vitro. This was accompanied by a decline in K63- and concomitant increase in K-48 linked polyubiquitination on RIP1, impaired NF-kB activation and necrotic cell death of cardiac myocytes. Interestingly, inhibiting the kinase activity of RIP1 with Necrostatin-1, (Nec1) suppressed necrotic cell injury induced by dox but not NF-kB activation. Concordant with these findings was a marked increase in necrotic cell death in cardiac myocytes defective for IKKB signaling or MEF cells deficient for p65 treated with dox. Notably, mitochondrial perturbations, including PT-pore opening , ROS production, calcium uptake, LDH, Tn(T) and HMGB-1 release and necrotic cell injury induced by dox were completely abrogated by restoring NF-kB signaling in cardiac myocytes or Nec-1. Herein, we provide novel evidence that K-48 linked poly ubiquitylation of RIP1 provides a functional switch that impairs NF-kB activation and signals necrosis in cells treated with dox. Interventions that modulate NF-kB activity may prove beneficial in mitigating the cardiotoxic effects of dox.Keywords:
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NF-κB는 anti-apoptotic gene을 유도하는 전사인자로서 대부분의 세포의 생존에 필요하다. 그러나 NF-κB가 많은 종류의 암세포에서 지속적으로 과다 활성화됨이 알려지면서 NF-κB의 활성억제가 암의 예방과 치료에 유효하다는 점이 알려지게 되었다. 한편, Hsp70가 NF-κB의 활성을 조절한다는 사실이 알려지면서 Hsp70를 이용한 암예방과 치료가 주목받게 되었으나 아직 Hsp70에 의한 NF-κB의 활성조절기전은 명확하지 않다. 본 연구에서는 Hsp70에 의한 NF-κB의 활성조절과정에서 IKK complex의 구성성분인 IKKγ의 역할을 검토하였다. IKKγ의 wild type과 deletion mutants를 이용하여 Hsp70와 관련된 NF-κB의 활성조절을 연구한 결과 Hsp70는 NF-κB의 활성화를 억제하였으며, 이러한 억제효과는 IKKγ가 과발현되었을 때 더욱 증가하였다. 또한 IKKγ의 N-말단의 IKKβ 결합부위와 C-말단의 Leucine zipper 및 Zinc finger부위는 Hsp70와 연관된 NF-κB억제작용에 필요하지 않는 것으로 나타났으며, Hsp70와 IKKγ에 의한 NF-κB의 활성억제는 IκBα의 인산화와 분해를 저해함에 의해 일어나는 것으로 나타났다. 또한 RAW264.7 macrophage세포에서 LPS에 의한 COX-2의 발현유도는 Hsp70와 IKKγ가 동시에 발현 되었을 때 가장 효과적으로 억제되었다. 이상의 결과로부터 Hsp70에 의한 NF-κB의 활성억제작용은 IKKγ에 의해 상승됨을 알 수 있었으며, Hsp70와 IKKγ를 적절히 이용하면 NF-κB의 과다활성에 의해 발생하는 각종 질병의 예방과 치료에 도움을 줄 수 있을 것으로 기대된다. NF-κB acts as a critical transcription factor for the survival of cells via the induction of antiapoptotic genes. Constitutive activation of NF-κB in many types of solid tumors suggests that the inhibition of NF-κB might prevent or inhibit tumorigenesis. Although a number of studies demonstrated that Hsp70 regulated NF-κB activity, the exact mechanism is not clear. This study investigated the functional relationship of Hsp70 and IKKγ in the regulation of NF-κB activation using expression plasmids of components of the IKK complex. Wild-type and deletion mutants of IKKγ were expressed together with Hsp70, and the combined regulatory effect of Hsp70 and IKKγ on NF-κB activation was assayed. Hsp70 suppressed the activation of NF-κB in a reporter plasmid assay. Hsp70 also suppressed the phosphorylation and degradation of IκBα. The suppressive effect of Hsp70 on NF-κB activation was synergistically elevated by IKKγ. The N-terminal IKKβ binding site, C-terminal leucine zipper, and zinc finger domains of IKKγ were not necessary for the suppressive effect. Furthermore, Hsp70 and IKKγ synergistically suppressed the induction of COX-2 expression by lipopolysaccharides in RAW264.7 cells. These results suggest that overexpression of Hsp70 and IKKγ may be a strategic method for inhibition of NF-κB and related diseases.
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Cigarette smoke (CS), a major risk factor for developing lung cancer, is known to activate transcriptional activator nuclear factor kappa B (NF-κB). However, the underlying mechanism of this activation remains unclear because of conflicting reports. As NF-κB has a pivotal role in the generation and maintenance of malignancies, efforts were targeted towards understanding its activation mechanism using both ex vivo and in vivo studies. The results show that CS-induced NF-κB activation mechanism is different from that of other pro-inflammatory signals such as lipopolysaccharide (LPS). The NF-κB dimer that translocates to the nucleus upon stimulation with CS is predominantly composed of c-Rel/p50 and this translocation involves degradation of I-κBɛ and not I-κBα. This degradation of I-κBɛ depends on IKKβ activity, which preferentially targets I-κBɛ. Consistently, CS-activated form of IKKβ was found to be different from that involved in LPS activation as neither Ser177 nor Ser181 of IKKβ is crucial for CS-induced NF-κB activation. Thus, unlike other pro-inflammatory stimulations where p65 and I-κBα have a central role, the predominantly active signaling cascade in CS-induced NF-κB activation in the lung epithelial cells comprises of IKKβ-I-κBɛ-c-Rel/p50. Thus, this study uncovers a new axis of NF-κB activation wherein I-κBɛ and c-Rel have the central role.
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Abstract Background: Dimethyl itaconate (DMI), a membrane-permeable derivative of itaconate, was found to moderate IL-17-IκBζ-induced skin pathology including psoriasis in mouse experiments . TNF-α induced NF-κB pathway, which controls a variety of immune and inflammatory responses, was also proven to play a crucial role as mediator in psoriasis. However, whether DMI interacts with the TNF-α induced NF-κB pathway remains unclear. Results: Here we show that DMI inhibits TNF-α induced NF-κB transcriptional activities in dose-dependent manner in several human cell lines using dual luciferase assay and blocks the NF-κB nuclear entry. Moreover, DMI potently inhibits IKKβ dependent phosphorylation and degradation of IκBα in TNF-α induced activation of NF-κB pathway. We also demonstrate that DMI covalently binds to cysteine residue in IKKβ, a key regulator in NF-κB pathway, to suppress IKKβ activation and inhibit the canonical NF-κB pathway. Conclusion Our study presents a new mechanism for DMI as an anti-inflammatory agent that may have therapeutic potentials in treating NF-κB related human inflammatory diseases. Our results also suggest that itaconate produced by endogenous IRG1 may regulate NF-κB at post translation modification level, and the IRG1-itaconate-NF-κB axis could be targeted as a novel strategy for the treatment of IRG1-NF-κB mediated diseases.
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It is well documented that ionizing radiation (IR) activates the transcription factor (NF-κB) which is a trigger for resistance cancer cells to treatment. It is involved in activation of pro-survival signaling pathways and resulting in cancer development and progression. In unstimulated condition, NF-κB is sequestered in cytoplasm but after the cell exposure to IR, proteasomal degradation of IκB flowing phosphorylation via IKK, leads to aberrantly NF-κB activation and nuclear translocation. Therefore, interruption in IκB degradation, proteasome action, IKK phosphorylation and NF-κB nuclear translocation provide robust strategies for inhibiting adverse effect of IR induced NF-κB. In spite of uncompleted elucidation of NF-κB molecular mechanisms, different NF-κB inhibitors have been used in order to inhibiting the IR induced NF-κB. The aim of this review is to highlight the role of IR induced-NF-κB inhibitors such as MG132, bortezomib, curcumin, DHMEQ, naringin, sorafenib, genistein and parthenolide in suppression of IR induced NF-κB adverse effects. Moreover, their chemical, structural characteristics and molecular mechanisms will be discussed.
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NF-κB acts as a critical transcription factor for the survival of cells via the induction of antiapoptotic genes. Constitutive activation of NF-κB in many types of solid tumors suggests that the inhibition of NF-κB might prevent or inhibit tumorigenesis. Although a number of studies demonstrated that Hsp70 regulated NF-κB activity, the exact mechanism is not clear. This study investigated the functional relationship of Hsp70 and IKKγ in the regulation of NF-κB activation using expression plasmids of components of the IKK complex. Wild-type and deletion mutants of IKKγ were expressed together with Hsp70, and the combined regulatory effect of Hsp70 and IKKγ on NF-κB activation was assayed. Hsp70 suppressed the activation of NF-κB in a reporter plasmid assay. Hsp70 also suppressed the phosphorylation and degradation of IκBα. The suppressive effect of Hsp70 on NF-κB activation was synergistically elevated by IKKγ. The N-terminal IKKβ binding site, C-terminal leucine zipper, and zinc finger domains of IKKγ were not necessary for the suppressive effect. Furthermore, Hsp70 and IKKγ synergistically suppressed the induction of COX-2 expression by lipopolysaccharides in RAW264.7 cells. These results suggest that overexpression of Hsp70 and IKKγ may be a strategic method for inhibition of NF-κB and related diseases.
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Poxviruses have evolved unique proteins and mechanisms to counteract the nuclear factor κB (NF-κB) signaling pathway, which is an essential regulatory pathway of host innate immune responses. Here, we describe a NF-κB inhibitory virion protein of orf virus (ORFV), ORFV073, which functions very early in infected cells. Infection with ORFV073 gene deletion virus (OV-IA82Δ073) led to increased accumulation of NF-κB essential modulator (NEMO), marked phosphorylation of IκB kinase (IKK) subunits IKKα and IKKβ, IκBα and NF-κB subunit p65 (NF-κB-p65), and to early nuclear translocation of NF-κB-p65 in virus-infected cells (≤ 30 min post infection). Expression of ORFV073 alone was sufficient to inhibit TNFα induced activation of the NF-κB signaling in uninfected cells. Consistent with observed inhibition of IKK complex activation, ORFV073 interacted with the regulatory subunit of the IKK complex NEMO. Infection of sheep with OV-IA82Δ073 led to virus attenuation, indicating that ORFV073 is a virulence determinant in the natural host. Notably, ORFV073 represents the first poxviral virion-associated NF-κB inhibitor described, highlighting the significance of viral inhibition of NF-κB signaling very early in infection.
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The nuclear factor-κB (NF-κB) transcription factors control many physiological processes including inflammation, apoptosis, and angiogenesis. In our search for NF-κB inhibitors from natural resources, we identified 4′,6-dihydroxy-4-methoxyisoaurone (ISOA) as an inhibitor of NF-κB activation from the seeds of Trichosanthes kirilowii. However, the mechanism by which ISOA inhibits NF-κB activation is not fully understood. In the present study, we demonstrated the effect of ISOA on NF-κB activation in TNF-α-stimulated HeLa cells. This compound suppressed NF-κB activation through the inhibition of IκB kinase (IKK) activation. ISOA also has an influence on upstream signaling of IKK through the inhibition of expression of adaptor proteins, TNF receptor-associated factor 2 (TRAF2) and receptor interacting protein 1 (RIP1). Consequently, ISOA blocked the phosphorylation and degradation of the inhibitor of NF-κB alpha (IκBα), and subsequent phosphorylation and nuclear translocation of p65. The suppression of NF-κB activation by ISOA led to the down-regulation of target genes involved in inflammation, proliferation, as well as potentiation of TNF-α-induced apoptosis. Taken together, this study extends our understanding on the mechanisms underlying the anti-inflammatory and anti-cancer activities of ISOA. Our findings provide new insight into the molecular mechanisms and a potential application of ISOA for inflammatory diseases as well as certain cancers.
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