IKKα and IKKβ Regulation of DNA Damage-Induced Cleavage of Huntingtin

Background

Proteolysis of huntingtin (Htt) plays a key role in the pathogenesis of Huntington''s disease (HD). However, the environmental cues and signaling pathways that regulate Htt proteolysis are poorly understood. One stimulus may be the DNA damage that accumulates in neurons over time, and the subsequent activation of signaling pathways such as those regulated by IκB kinase (IKK), which can influence neurodegeneration in HD.

Methodology/Principal Findings

We asked whether DNA damage induces the proteolysis of Htt and if activation of IKK plays a role. We report that treatment of neurons with the DNA damaging agent etoposide or γ-irradiation promotes cleavage of wild type (WT) and mutant Htt, generating N-terminal fragments of 80–90 kDa. This event requires IKKβ and is suppressed by IKKα. Elevated levels of IKKα, or inhibition of IKKβ expression by a specific small hairpin RNA (shRNA) or its activity by sodium salicylate, prevents Htt proteolysis and increases neuronal resistance to DNA damage. Moreover, IKKβ phosphorylates the anti-apoptotic protein Bcl-xL, a modification known to reduce Bcl-xL levels, and activates caspases that can cleave Htt. When IKKβ expression is blocked, etoposide treatment does not decrease Bcl-xL and activation of caspases is diminished. Similar to silencing of IKKβ, increasing the level of Bcl-xL in neurons prevents etoposide-induced caspase activation and Htt proteolysis.

Conclusions/Significance

These results indicate that DNA damage triggers cleavage of Htt and identify IKKβ as a prominent regulator. Moreover, IKKβ-dependent reduction of Bcl-xL is important in this process. Thus, inhibition of IKKβ may promote neuronal survival in HD as well as other DNA damage-induced neurodegenerative disorders.     

Cell migration to CXCL12 requires simultaneous IKKα and IKKβ-dependent NF-κB signaling

CXCL12 and its unique receptor CXCR4, is critical for the homing of a variety of cell lineages during both development and tissue repair. CXCL12 is particularly important for the recruitment of hemato/lymphopoietic cells to their target organs. In conjunction with the damage-associated alarmin molecule HMGB1, CXCL12 mediates immune effector and stem/progenitor cell migration towards damaged tissues for subsequent repair. Previously, we showed that cell migration to HMGB1 simultaneously requires both IKKβ and IKKα-dependent NF-κB activation. IKKβ-mediated activation maintains sufficient expression of HMGB1's receptor RAGE, while IKKα-dependent NF-κB activation ensures continuous production of CXCL12, which complexes with HMGB1 to engage CXCR4. Here using fibroblasts and primary mature macrophages, we show that IKKβ and IKKα are simultaneously essential for cell migration in response to CXCL12 alone. Non-canonical NF-κB pathway subunits RelB and p52 are also both essential for cell migration towards CXCL12, suggesting that IKKα is required to drive non-canonical NF-κB signaling. Flow cytometric analyses of CXCR4 expression show that IKKβ, but not IKKα, is required to maintain a critical threshold level of this CXCL12 receptor. Time-lapse video microscopy experiments in primary MEFs reveal that IKKα is required both for polarization of cells towards a CXCL12 gradient and to establish a basal level of velocity towards CXCL12. In addition, CXCL12 modestly up-regulates IKKα-dependent p52 nuclear translocation and IKKα-dependent expression of the CXCL12 gene. On the basis of our collective results we posit that IKKα is needed to maintain the basal expression of a critical protein co-factor required for cell migration to CXCL12.     

IKKα activation of NOTCH links tumorigenesis via FOXA2 suppression

Proinflammatory cytokine TNFα plays critical roles in promoting malignant cell proliferation, angiogenesis, and tumor metastasis in many cancers. However, the mechanism of TNFα-mediated tumor development remains unclear. Here, we show that IKKα, an important downstream kinase of TNFα, interacts with and phosphorylates FOXA2 at S107/S111, thereby suppressing FOXA2 transactivation activity and leading to decreased NUMB expression, and further activates the downstream NOTCH pathway and promotes cell proliferation and tumorigenesis. Moreover, we found that levels of IKKα, pFOXA2 (S107/111), and activated NOTCH1 were significantly higher in hepatocellular carcinoma tumors than in normal liver tissues and that pFOXA2 (S107/111) expression was positively correlated with IKKα and activated NOTCH1 expression in tumor tissues. Therefore, dysregulation of NUMB-mediated suppression of NOTCH1 by TNFα/IKKα-associated FOXA2 inhibition likely contributes to inflammation-mediated cancer pathogenesis. Here, we report a TNFα/IKKα/FOXA2/NUMB/NOTCH1 pathway that is critical for inflammation-mediated tumorigenesis and may provide a target for clinical intervention in human cancer.     

Integration of TNF-α signaling: crosstalk between caspasesl,IKK and JNK

The proinflammatory cytokine tumor necrosis factor-α (TNF-α) regulates immune responses, inflammation and programmed cell death. The ultimate fate of a cell exposed to TNF-αis determined by signal integration between its downstream effectors, including caspases, IKB kianse (IKK) and c-Jun N-terminal protein kinase (JNK). However, the molecular mechanisms are incompletely understood. We investigated this issue using genetic and biochemical approaches. We identified IKK β, a catalytic subunit of the IKK complex that is required for NF-KB activation and cell survival in response to TNF-α, was proteolyzed by casp-3-related caspases. This proteolysis eliminated IKK activity and promoted TNF-α, killing. Point     

TRAF2 Suppresses Basal IKK Activity in Resting Cells and TNFα Can Activate IKK in TRAF2 and TRAF5 Double Knockout Cells

Tumor necrosis factor receptor (TNFR)-associated factor 2 (TRAF2) and TRAF5 are adapter proteins involved in TNFα-induced activation of the c-Jun N-terminal kinase and nuclear factor κB (NF-κB) pathways. Currently, TNFα-induced NF-κB activation is believed to be impaired in TRAF2 and TRAF5 double knockout (T2/5 DKO) cells. Here, we report instead that T2/5 DKO cells exhibit high basal IκB kinase (IKK) activity and elevated expression of NF-κB-dependent genes in unstimulated conditions. Although TNFα-induced receptor-interacting protein 1 ubiquitination is indeed impaired in T2/5 DKO cells, TNFα stimulation further increases IKK activity in these cells, resulting in significantly elevated expression of NF-κB target genes to a level higher than that in wild-type cells. Inhibition of NIK in T2/5 DKO cells attenuates basal IKK activity and restores robust TNFα-induced IKK activation to a level comparable with that seen in wild-type cells. This suggests that TNFα can activate IKK in the absence of TRAF2 and TRAF5 expression and receptor-interacting protein 1 ubiquitination. In addition, both the basal and TNFα-induced expression of anti-apoptotic proteins are normal in T2/5 DKO cells, yet these DKO cells remain sensitive to TNFα-induced cell death, due to the impaired recruitment of anti-apoptotic proteins to the TNFR1 complex in the absence of TRAF2. Thus, our data demonstrate that TRAF2 negatively regulates basal IKK activity in resting cells and inhibits TNFα-induced cell death by recruiting anti-apoptotic proteins to the TNFR1 complex rather than by activating the NF-κB pathway.     

NIK prevents the development of hypereosinophilic syndrome-like disease in mice independent of IKKα activation

Immune cell-mediated tissue injury is a common feature of different inflammatory diseases, yet the pathogenetic mechanisms and cell types involved vary significantly. Hypereosinophilic syndrome (HES) represents a group of inflammatory diseases that is characterized by increased numbers of pathogenic eosinophilic granulocytes in the peripheral blood and diverse organs. On the basis of clinical and laboratory findings, various forms of HES have been defined, yet the molecular mechanism and potential signaling pathways that drive eosinophil expansion remain largely unknown. In this study, we show that mice deficient of the serine/threonine-specific protein kinase NF-κB-inducing kinase (NIK) develop a HES-like disease, reflected by progressive blood and tissue eosinophilia, tissue injury, and premature death at around 25-30 wk of age. Similar to the lymphocytic form of HES, CD4(+) T cells from NIK-deficient mice express increased levels of Th2-associated cytokines, and eosinophilia and survival of NIK-deficient mice could be prevented completely by genetic ablation of CD4(+) T cells. Experiments based on bone marrow chimeric mice, however, demonstrated that inflammation in NIK-deficient mice depended on radiation-resistant tissues, implicating that NIK-deficient immune cells mediate inflammation in a nonautonomous manner. Surprisingly, disease development was independent of NIK's known function as an IκB kinase α (IKKα) kinase, because mice carrying a mutation in the activation loop of IKKα, which is phosphorylated by NIK, did not develop inflammatory disease. Our data show that NIK activity in nonhematopoietic cells controls Th2 cell development and prevents eosinophil-driven inflammatory disease, most likely using a signaling pathway that operates independent of the known NIK substrate IKKα.     

IKKα Regulates the Mitotic Phase of the Cell Cycle by Modulating Aurora A Phosphorylation

The IKK complex includes two catalytic components, IKKα and IKKβ, in addition to the scaffold protein IKKγ/NEMO. Even though IKKα and IKKβ share significant sequence homology, they have distinct biological roles with IKKβ regulates the classical pathway of NF-κB activation and IKKα regulates the alternative pathways. In addition, it has been shown that the IKKs regulate the proliferation of both normal and tumor cells; however, the mechanisms by which the IKKs regulate the cell cycle remain to be further defined. Here, we demonstrate that IKKα, but not IKKβ, has role in regulating the M phase of the cell cycle. IKKα siRNA knock-down resulted in increased numbers of cells in the G2/M phase of the cell cycle as compared to control and IKKβ siRNA transfected HeLa cells. This effect was associated with upregulation of cyclin B1 and Plk1 protein levels and increased histone H3 phosphorylation, consistent with a potential role of IKKα in the regulation of M phase regulatory factors. IKKα was found to be associated with Aurora A in the centrosome and regulate Aurora A phosphorylation at threonine residue 288, a site which is important in modulating its kinase activity. Taken together, these data provide the evidence that IKKα regulates the M phase of the cell cycle by modulating Aurora A phosphorylation and activation leading to the regulation of the M phase of the cell cycle.     

IKKα的研究进展

IκB激酶(IKK复合体)是NF-κB信号转导途径成员之一,包括3个亚基:催化亚基IKKα、IKKβ和调节亚基IKKγ。无刺激时,NF-κB与抑制蛋白IκB家族的一个成员结合,或者与无活性前体(如p100)结合而以无活性形式存在。在外界信号如TNF-α或淋巴毒素β等刺激下,经过复杂的信号转导,IKK复合体被激活,导致IκB和(或)p100发生磷酸化,结果NF-κB被释放出来,进入细胞核内激活靶基因。最新研究发现在TNF-α刺激下,IKKα可直接进入细胞核内,通过催化组蛋白H3磷酸化进而激活特定NF-κB应答基因的表达。IKKα是首次发现的信号转导途径中直接进入细胞核内调节基因表达的上游成分,为NF-κB信号转导途径的研究开辟了新的道路。     

Mechanism of PP2A-mediated IKKβ dephosphorylation: a systems biological approach

Background  

Biological effects of nuclear factor-κB (NFκB) can differ tremendously depending on the cellular context. For example, NFκB induced by interleukin-1 (IL-1) is converted from an inhibitor of death receptor induced apoptosis into a promoter of ultraviolet-B radiation (UVB)-induced apoptosis. This conversion requires prolonged NFκB activation and is facilitated by IL-1 + UVB-induced abrogation of the negative feedback loop for NFκB, involving a lack of inhibitor of κB (IκBα) protein reappearance. Permanent activation of the upstream kinase IKKβ results from UVB-induced inhibition of the catalytic subunit of Ser-Thr phosphatase PP2A (PP2Ac), leading to immediate phosphorylation and degradation of newly synthesized IκBα.     

Regional susceptibility to TNF-α induction of murine brain inflammation via classical IKK/NF-κB signalling

It is becoming clear that inflammation plays a significant role in a number of neurological and psychiatric conditions. Post mortem brain samples in Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, schizophrenia and most recently autism spectrum condition, all exhibit neuroglial activation and inflammatory markers within the CSF. Many questions remain about the underlying molecular mechanisms. By adding the pro-inflammatory cytokine, TNF-α, to mouse brain tissue we demonstrated that the frontal lobes and temporal region, areas involved in higher functions such as memory and learning, are most susceptible to cytokine-induced inflammation via the NF-κB signalling pathway. We observed direct correlations between the volumetric increase and molecular expression indicating that therapeutic targets in these lobes may require different approaches when treating conditions with a central neuroinflammatory component.