Antinociceptive Effect of Tetrandrine on LPS-Induced Hyperalgesia via the Inhibition of IKKβ Phosphorylation and the COX-2/PGE2 Pathway in Mice

Tetrandrine (TET) is a bisbenzylisoquinoline alkaloid that is isolated from the Stephania Tetrandra. It is known to possess anti-inflammatory and immunomodulatory effects. We have shown that TET can effectively suppress the production of bacterial lipopolysaccharide (LPS)-induced inflammatory mediators, including cyclooxygenases (COXs), in macrophages. However, whether TET has an antinociceptive effect on LPS-induced hyperalgesia is unknown. In the present study, we investigated the potential antinociceptive effects of TET and the mechanisms by which it elicits its effects on LPS-induced hyperalgesia. LPS effectively evoked hyperalgesia and induced the production of PGE₂ in the sera, brain tissues, and cultured astroglia. TET pretreatment attenuated all of these effects. LPS also activated inhibitor of κB (IκB) kinase β (IKKβ) and its downstream components in the IκB/nuclear factor (NF)-κB signaling pathway, including COX-2; the increase in expression levels of these components was significantly abolished by TET. Furthermore, in primary astroglia, knockdown of IKKβ, but not IKKα, reversed the effects of TET on the LPS-induced increase in IκB phosphorylation, P65 phosphorylation, and COX-2. Our results suggest that TET can effectively exert antinociceptive effects on LPS-induced hyperalgesia in mice by inhibiting IKKβ phosphorylation, which leads to the reduction in the production of important pain mediators, such as PGE₂ and COX-2, via the IKKβ/IκB/NF-κB pathway.     

Pentacyclic Triterpenoids Inhibit IKKβ Mediated Activation of NF-κB Pathway: In Silico and In Vitro Evidences

Pentacyclic Triterpenoids (PTs) and their analogues as well as derivatives are emerging as important drug leads for various diseases. They act through a variety of mechanisms and a majority of them inhibit the nuclear factor kappa-beta (NF-κB) signaling pathway. In this study, we examined the effects of the naturally occurring PTs on IκB kinase-β (IKKβ), which has great scientific relevance in the NF-κB signaling pathway. On virtual screening, 109 PTs were screened through the PASS (prediction of activity spectra of substances) software for prediction of NF-κB inhibitory activity followed by docking on the NEMO/IKKβ association complex (PDB: 3BRV) and testing for compliance with the softened Lipinski’s Rule of Five using Schrodinger (LLC, New York, USA). Out of the projected 45 druggable PTs, Corosolic Acid (CA), Asiatic Acid (AA) and Ursolic Acid (UA) were assayed for IKKβ kinase activity in the cell free medium. The UA exhibited a potent IKKβ inhibitory effect on the hotspot kinase assay with IC50 of 69 μM. Whereas, CA at 50 μM concentration markedly reduced the NF-κB luciferase activity and phospho-IKKβ protein expressions. The PTs tested, attenuated the expression of the NF-κB cascade proteins in the LPS-stimulated RAW 264.7 cells, prevented the phosphorylation of the IKKα/β and blocked the activation of the Interferon-gamma (IFN-γ). The results suggest that the IKKβ inhibition is the major mechanism of the PTs-induced NF-κB inhibition. PASS predictions along with in-silico docking against the NEMO/IKKβ can be successfully applied in the selection of the prospective NF-κB inhibitory downregulators of IKKβ phosphorylation.     

Regulatory subunit NEMO promotes polyubiquitin-dependent induction of NF-κB through a targetable second interaction with upstream activator IKK2

Canonical NF-κB signaling through the inhibitor of κB kinase (IKK) complex requires induction of IKK2/IKKβ subunit catalytic activity via specific phosphorylation within its activation loop. This process is known to be dependent upon the accessory ubiquitin (Ub)-binding subunit NF-κB essential modulator (NEMO)/IKKγ as well as poly-Ub chains. However, the mechanism through which poly-Ub binding serves to promote IKK catalytic activity is unclear. Here, we show that binding of NEMO/IKKγ to linear poly-Ub promotes a second interaction between NEMO/IKKγ and IKK2/IKKβ, distinct from the well-characterized interaction of the NEMO/IKKγ N terminus to the “NEMO-binding domain” at the C terminus of IKK2/IKKβ. We mapped the location of this second interaction to a stretch of roughly six amino acids immediately N-terminal to the zinc finger domain in human NEMO/IKKγ. We also showed that amino acid residues within this region of NEMO/IKKγ are necessary for binding to IKK2/IKKβ through this secondary interaction in vitro and for full activation of IKK2/IKKβ in cultured cells. Furthermore, we identified a docking site for this segment of NEMO/IKKγ on IKK2/IKKβ within its scaffold-dimerization domain proximal to the kinase domain–Ub-like domain. Finally, we showed that a peptide derived from this region of NEMO/IKKγ is capable of interfering specifically with canonical NF-κB signaling in transfected cells. These in vitro biochemical and cell culture–based experiments suggest that, as a consequence of its association with linear poly-Ub, NEMO/IKKγ plays a direct role in priming IKK2/IKKβ for phosphorylation and that this process can be inhibited to specifically disrupt canonical NF-κB signaling.     

VRK2 activates TNFα/NF-κB signaling by phosphorylating IKKβ in pancreatic cancer

NF-κB signaling is active in more than 50% of patients with pancreatic cancer and plays an important role in promoting the progression of pancreatic cancer. Revealing the activation mechanism of NF-κB signaling is important for the treatment of pancreatic cancer. In this study, the regulation of TNFα/NF-κB signaling by VRK2 (vaccinia-related kinase 2) was investigated. The levels of VRK2 protein were examined by immunohistochemistry (IHC). The functions of VRK2 in the progression of pancreatic cancer were examined using CCK8 assay, anchorage-independent assay, EdU assay and tumorigenesis assay. The regulation of VRK2 on the NF-κB signaling was investigated by immunoprecipitation and invitro kinase assay. It was discovered in this study that the expression of VRK2 was upregulated in pancreatic cancer and that the VRK2 expression level was significantly correlated with the pathological characteristics and the survival time of patients. VRK2 promoted the growth, sphere formation and subcutaneous tumorigenesis of pancreatic carcinoma cells as well as the organoid growth derived from the pancreatic cancer mouse model. Investigation of the molecular mechanism indicated that VRK2 interacts with IKKβ, phosphorylating its Ser177 and Ser181 residues and thus activating the TNFα/NF-κB signaling pathway. An IKKβ inhibitors abolished the promotive effect of VRK2 on the growth of organoids. The findings of this study indicate that VRK2 promotes the progression of pancreatic cancer by activating the TNFα/NF-κB signaling pathway, suggesting that VRK2 is a potential therapeutic target for pancreatic cancer.     

Liver Kinase B1 Suppresses Lipopolysaccharide-induced Nuclear Factor κB (NF-κB) Activation in Macrophages

Liver kinase B1 (LKB1), a serine/threonine kinase, is a tumor suppressor and metabolic regulator. Recent data suggest that LKB1 is essential in regulating homeostasis of hematopoietic cells and immune responses. However, its role in macrophages and innate immune system remains unclear. Here we report that macrophage LKB1 inhibits pro-inflammatory signaling in response to LPS. LPS-induced pro-inflammatory cytokines and pro-inflammatory enzymes were monitored in bone marrow-derived macrophages isolated from myeloid cell-specific LKB1 knock out mice and their wild type littermate control mice. LPS induced higher levels of pro-inflammatory cytokines and pro-inflammatory enzymes in bone marrow-derived macrophages from LKB1 KO than those from wild type mice. Consistently, LPS induced higher levels of NF-κB activation in LKB1-deficient macrophages than those in wild type. Further, LPS stimulation significantly increased LKB1 phosphorylation at serine 428, which promoted its binding to IκB kinaseβ (IKKβ), resulting in the inhibition of NF-κB. Finally, LPS injection caused higher levels of cytokine release and more severe tissue injury in the lung tissues of LKB1 KO mice than in those of control mice. We conclude that LKB1 inhibits LPS-induced NF-κB activation in macrophages.     

Vitamin D Receptor Inhibits Nuclear Factor κB Activation by Interacting with IκB Kinase β Protein

1,25-Dihydroxyvitamin D (1,25(OH)₂D₃) is known to suppress NF-κB activity, but the underlying mechanism remains poorly understood. Here we show that the vitamin D receptor (VDR) physically interacts with IκB kinase β (IKKβ) to block NF-κB activation. 1,25(OH)₂D₃ rapidly attenuates TNFα-induced p65 nuclear translocation and NF-κB activity in a VDR-dependent manner. VDR overexpression inhibits IKKβ-induced NF-κB activity. GST pull-down assays and coimmunoprecipitation experiments demonstrated that VDR physically interacts with IKKβ and that this interaction is enhanced by 1,25(OH)₂D₃. Protein mapping reveals that VDR-IKKβ interaction occurs between the C-terminal portions of the VDR and IKKβ proteins. Reconstitution of VDR^−/− cells with the VDR C terminus restores the ability to block TNFα-induced NF-κB activation and IL-6 up-regulation. VDR-IKKβ interaction disrupts the formation of the IKK complex and, thus, abrogates IKKβ phosphorylation at Ser-177 and abolishes IKK activity to phosphorylate IκBα. Consequently, stabilization of IκBα arrests p65/p50 nuclear translocation. Together, these data define a novel mechanism whereby 1,25(OH)₂D₃-VDR inhibits NF-κB activation.     

A Splicing Variant of NME1 Negatively Regulates NF-κB Signaling and Inhibits Cancer Metastasis by Interacting with IKKβ

IKKβ functions as a principal upstream activator of the canonical NF-κB pathway by phosphorylating IκB, leading to its proteasomal degradation. Because IKKβ is considered a therapeutic target, understanding its regulation may facilitate the design of efficient regulators of this molecule. Here, we report a novel IKKβ-interacting molecule, NME1L, a splicing variant of the NME1 protein. NME1 has attracted attention in cancer research because of its antimetastatic activity and reduced expression in multiple aggressive types of cancer. However, the effect was just moderate but not dramatic in anti-cancer activities. We found that only NME1L interacts with IKKβ. Exogenous expression of NME1L resulted in a potent decrease in TNFα-stimulated NF-κB activation, whereas knockdown of NME1/NME1L with shRNA enhanced activity of NF-κB. NME1L down-regulates IKKβ signaling by blocking IKKβ-mediated IκB degradation. When NME1L was introduced into highly metastatic HT1080 cells, the mobility was efficiently inhibited. Furthermore, in a metastasis assay, NME1L-expressing cells did not colonize the lung. Based on these results, NME1L is a potent antimetastatic protein and may be a useful weapon in the fight against cancers.     

Probing the Solution Structure of IκB Kinase (IKK) Subunit γ and Its Interaction with Kaposi Sarcoma-associated Herpes Virus Flice-interacting Protein and IKK Subunit β by EPR Spectroscopy

Viral flice-interacting protein (vFLIP), encoded by the oncogenic Kaposi sarcoma-associated herpes virus (KSHV), constitutively activates the canonical nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) pathway. This is achieved through subversion of the IκB kinase (IKK) complex (or signalosome), which involves a physical interaction between vFLIP and the modulatory subunit IKKγ. Although this interaction has been examined both in vivo and in vitro, the mechanism by which vFLIP activates the kinase remains to be determined. Because IKKγ functions as a scaffold, recruiting both vFLIP and the IKKα/β subunits, it has been proposed that binding of vFLIP could trigger a structural rearrangement in IKKγ conducive to activation. To investigate this hypothesis we engineered a series of mutants along the length of the IKKγ molecule that could be individually modified with nitroxide spin labels. Subsequent distance measurements using electron paramagnetic resonance spectroscopy combined with molecular modeling and molecular dynamics simulations revealed that IKKγ is a parallel coiled-coil whose response to binding of vFLIP or IKKβ is localized twisting/stiffening and not large-scale rearrangements. The coiled-coil comprises N- and C-terminal regions with distinct registers accommodated by a twist: this structural motif is exploited by vFLIP, allowing it to bind and subsequently activate the NF-κB pathway. In vivo assays confirm that NF-κB activation by vFLIP only requires the N-terminal region up to the transition between the registers, which is located directly C-terminal of the vFLIP binding site.