Search Results (43)

The IκB kinase (IKK) complex plays a central role in many signaling pathways that activate NF-κB, which turns on a battery of genes important for immune response, inflammation, and cancer development. Ubiquitination is one of the most prevalent post-translational modifications of proteins and is best known for targeting substrates for proteasomal degradation. The investigations of NF-κB signaling pathway primed the unveiling of the non-degradative roles of protein ubiquitination. The NF-κB-essential modulator (NEMO) is the IKK regulatory subunit that is essential for IKK activation by diverse intrinsic and extrinsic stimuli. The studies centered on NEMO as a polyubiquitin-binding protein have remarkably advanced understandings of how NEMO transmits signals to NF-κB activation and have laid a foundation for determining the molecular events demonstrating non-degradative ubiquitination as a major driving element in IKK activation. Furthermore, these studies have largely solved the enigma that IKK can be activated by diverse pathways that employ distinct sets of intermediaries in transmitting signals. NEMO and NEMO-related proteins that include optineurin, ABIN1, ABIN2, ABIN3, and CEP55, as non-degradative ubiquitin chain receptors, play a key role in sensing and transmitting ubiquitin signals embodied in different topologies of polyubiquitin chains for a variety of cellular processes and body responses. Studies of these multifaceted proteins in ubiquitin sensing have promoted understanding about the functions of non-degradative ubiquitination in intracellular signaling, protein trafficking, proteostasis, immune response, DNA damage response, and cell cycle control. In this review, I will also discuss how dysfunction in the NEMO family of protein-mediated non-degradative ubiquitin signaling is associated with various diseases, including immune disorders, neurodegenerative diseases, and cancer, and how microbial virulence factors target NEMO to induce pathogenesis or manipulate host response. A profound understanding of the molecular bases for non-degradative ubiquitin signaling will be valuable for developing tailored approaches for therapeutic purposes. Full article

Jacaranone derived from Senecio scandens, a traditional Chinese medicine used for centuries, has been documented to exhibit anti-inflammatory and antiproliferative properties in various tumor cell lines. However, the mechanism of action and relationship between inflammation and apoptosis induced by jacaranone remain inadequately elucidated. In this study, the targets of jacaranone and cancer were identified from various databases, while potential targets and pathways were predicted through the analysis of the protein–protein interactions (PPI) network and pathway enrichment. Through a comprehensive network pharmacology analysis and corroborating experimental findings, we revealed that jacaranone induces tumor cell death by fine-tuning the tumor necrosis factor receptor 1 (TNFR1) downstream signaling pathway. TNFR1 serves as a key node that assembles into complexes I and II, regulating pathways including the nuclear factor (NF)-κB signaling pathway and the cell apoptosis pathway, which play crucial roles in cellular life activities. Jacaranone successfully guides survival signaling pathways to apoptotic mechanisms by inhibiting the assembly of complex I and promoting the formation of complex II. In particular, the main action mechanism of jacaranone lies in inducing the degradation of the inhibitor of apoptosis protein (cIAP)-2. cIAP-2 serves as an E3 ubiquitin ligase that ubiquitinates receptor-interacting serine/threonine-protein kinase 1 (RIPK1), thereby hindering the formation of complex I and effectively reducing the phosphorylation of Inhibitor of κB kinase (IKK) β. When the deubiquitylation process of RIPK1 is triggered, it may promote the formation of complex II, which ultimately leads to cell apoptosis. This fully demonstrates the key role of jacaranone in regulating TNFR1 complexes, especially through the degradation of cIAP-2. Taken together, jacaranone hinders the assembly of TNFR1 complex I and promotes the formation of complex II to induce apoptosis of cancer cells. Our findings unveil a novel mechanism underlying jacaranone, while also presenting a fresh approach for the development of new pharmaceuticals. Full article

Background: Lutein, a carotenoid, exhibits various biological activities such as maintaining the health of the eye, skin, heart, and bone. Recently, we found that lutein has dual roles in suppressing bone resorption and promoting bone formation. In this study, we examined the effects of lutein in a disuse-induced osteoporosis model using hindlimb-unloaded (HLU) mice. Methods: Osteoclast differentiation was assessed by coculturing mouse primary osteoblasts and bone marrow cells or culturing a mouse osteoclast precursor cell line. The bone-resorbing activity was determined by mouse calvarial organ cultures. An in situ docking simulation was conducted to reveal the interaction of lutein and IκB kinase (IKK) β protein. HLU mice were fed a 1% lutein-containing diet for two weeks, and the femoral bone mass was measured by μCT. Results: Osteoclast differentiation is significantly inhibited by lutein, astaxanthin, and β-cryptoxanthin. In contrast, only lutein promoted osteoblastic calcified bone nodule formation. To elucidate the molecular role of lutein, we functionally analyzed the NF-κB complex, a molecule involved in bone metabolism, especially in osteoclasts. Docking simulations showed that lutein binds to IKK, thus inhibiting the activation of NF-κB. In a cell culture analysis, the phosphorylation of p65, the active form of NF-κB in osteoblasts, was suppressed by lutein treatment. In vivo, a μCT analysis of the bone microarchitecture showed that lutein improves several bone parameters while maintaining bone mass. Conclusions: Lutein is effective in maintaining bone mass by controlling both bone resorption and formation, which is applied to prevent disuse-induced osteoporosis. Full article

Gastrointestinal cancers account for over a quarter of all cancer cases and are associated with poor prognosis and high mortality rates. The IKK complex (the canonical I kappa B kinase), comprising the CHUK, IKBKB, and IKBKG genes, plays a crucial role in activating the NF-kB signaling pathway. This study aimed to analyze publicly available bioinformatics data to elucidate the oncogenic role of IKK genes in selected gastrointestinal cancers. Our findings reveal that IKBKB and IKBKG are significantly upregulated in all examined cancers, while CHUK is upregulated in esophageal carcinoma and stomach adenocarcinoma. Additionally, the expression of IKK genes varies with histological grade and nodal metastases. For instance, in stomach adenocarcinoma, CHUK and IKBKB are upregulated in higher histological grades and greater lymph node infiltration. Lower expression levels of CHUK, IKBKB, and IKBKG in stomach adenocarcinoma and IKBKB in esophageal squamous cell carcinoma correlate with shorter overall survival. Conversely, in esophageal adenocarcinoma, reduced IKBKG expression is linked to longer overall survival, while higher IKBKB expression in colon adenocarcinoma is associated with longer overall survival. Given the significant role of IKK genes in the development and progression of selected gastrointestinal cancers, they hold potential as prognostic markers and therapeutic targets, offering valuable insights for clinical practice. Full article

Stress granules (SGs) are cytoplasmic membraneless compartments that can form in stressed cells. There is an intricate relationship between SGs and innate immune signaling pathways. A previous study reported that the innate immune signaling mediated by Toll-like receptors (TLRs) can inhibit SGs induced by endoplasmic reticulum stress (ER stress) in bone-marrow-derived macrophages (BMDMs) and the chemotherapy drug oxaliplatin in B16 melanoma cells. We wanted to test if this observation can be generalized to other cell types. First, we recapitulated the results from the previous study showing TLR signaling-mediated inhibition of SGs in BMDMs induced by ER stress. However, SGs formed in response to ER stress were either not inhibited or only very weakly inhibited by TLR4 stimulation in human lung cancer-derived A549 cells, murine immortalized mouse lung fibroblasts (iMLFs) and primary murine mouse lung fibroblasts. This correlated with a weak induction of IKK complex kinase activity by TLR4 stimulation in these cells. SGs formed by sodium arsenite treatment also remained unaffected by TLR4 signaling. Our results indicate that the innate immune signaling-mediated inhibition of SGs is cell-type-dependent, thus opening a new avenue for mechanistic studies of the crosstalk between innate immune and stress signaling pathways. Full article

Polysaccharides have been assessed as a potential natural active component in Chinese herbal medicine with anti-inflammatory properties. However, the complex and indefinite structures of polysaccharides limit their applications. This study explains the structures and anti-inflammatory potentials of three neutral polysaccharides, RIP-A1 (M_w 1.8 × 10⁴ Da), RIP-B1 (M_w 7.4 × 10⁴ Da) and RIP-B2 (M_w 9.3 × 10⁴ Da), which were isolated from the roots of Isatis indigotica Fort. with sequenced ultrafiltration membrane columns, DEAE-52 and Sephadex G-100. The planar structures and microstructures of RIP-A1, RIP-B1 and RIP-B2 were further determined by HPGPC, GC–MS, methylation analysis, FT-IR, SEM and AFM, in which the structure of RIP-A1 was elucidated in detail using 1D/2D NMR. The Raw 264.7 cells were used for the anti-inflammatory activity in vitro. The results showed that RIP-A1, RIP-B1 and RIP-B2 are all neutral polysaccharides, with RIP-A1 having the smallest M_w and the simplest monosaccharide composition of the three. RIP-A1 is mainly composed of Ara and Gal, except for a small quantity of Rha. Its main structure is covered with glycosidic linkages of T-α-Araf, 1,2-α-Rhap, 1,5-α-Araf, T-β-Galp, 1,2,4-α-Rhap, 1,3,5-α-Araf and 1,6-β-Galp with 0.33:0.12:1.02:0.09:0.45:11.41:10.23. RIP-A1 significantly inhibited pro-inflammatory cytokines (NO, TNF-α, IL-6 and IL-1β) and increased anti-inflammatory cytokines (IL-4) in LPS-stimulated RAW 264.7 cells. Moreover, RIP-A1 could significantly inhibit the mRNA expression of TNF-α, IL-6 and L-1β. It could also activate IKK, p65 and IκBα (the components of the NF-κB signaling pathway). In conclusion, the above results show the structural characterization and anti-inflammatory potentials of RIP-A1 as an effective natural anti-inflammatory drug. Full article

Ocular exposure to particulate matter (PM) causes local inflammation; however, the influence of neutrophils on PM-induced ocular inflammation is still not fully understood. In this study, we constructed a system to investigate the role of PM in ocular inflammation using a co-culture of human corneal epithelial cells (HCE-T) and differentiation-induced neutrophils (dHL-60). To investigate whether HCE-T directly endocytosed PM, we performed a holographic analysis, which showed the endocytosis of PM in HCE-T. The cytokines and chemokines produced by HCE-T were measured using an ELISA. HCE-T treated with PM produced IL-6 and IL-8, which were inhibited by N-Acetyl-L-cysteine (NAC), suggesting the involvement of ROS. Their co-culture with dHL-60 enhanced their production of IL-6, IL-8, and MCP-1. This suggests an inflammatory loop involving intraocular corneal epithelial cells and neutrophils. These cytokines and chemokines are mainly regulated by NF-κB. Therefore, this co-culture system was examined in the presence of an IKK inhibitor known to downregulate NF-κB activity. The IKK inhibitor dramatically suppressed the production of these factors in co-culture supernatants. The results suggest that the inflammatory loop observed in the co-culture is mediated through ROS and the transcription factor NF-κB. Thus, the co-culture system is considered a valuable tool for analyzing complex inflammations. Full article

IκB kinases (IKKs), specifically IKKα and IKKβ, have long been recognized for their pivotal role in the NF-κB pathway, orchestrating immune and inflammatory responses. However, recent years have unveiled their dual role in cancer, where they can act as both promoters and suppressors of tumorigenesis. In addition, the interplay with pathways such as the MAPK and PI3K pathways underscores the complexity of IKK regulation and its multifaceted role in both inflammation and cancer. By exploring the molecular underpinnings of these processes, we can better comprehend the complex interplay between IKKs, tumor development, immune responses, and the development of more effective therapeutics. Ultimately, this review explores the dual role of IκB kinases in cancer, focusing on the impact of phosphorylation events and crosstalk with other signaling pathways, shedding light on their intricate regulation and multifaceted functions in both inflammation and cancer. Full article

The NF-κB-signaling pathway plays a crucial role in cancer progression, including muscle-derived cancers such as rhabdomyosarcoma or sarcoma. Several natural compounds have been studied for their ability to alter NF-κB signaling in these types of cancers. This review paper summarizes the current knowledge on the effects of natural compounds, including curcumin, resveratrol, quercetin, epigallocatechin-3-gallate, and berberine, on NF-κB signaling in muscle-derived cancers. These compounds have been shown to inhibit NF-κB signaling in rhabdomyosarcoma cells through various mechanisms, such as inhibiting the activation of the IKK complex and the NF-κB transcription factor. These findings suggest that natural compounds could be potential therapeutic agents for muscle-derived cancers. However, further research is needed to fully understand their mechanisms of action and potential clinical applications. Full article

Non-alcoholic steatohepatitis (NASH) is a metabolic disorder that often leads to other severe liver diseases, yet treatment options are limited. Endoplasmic reticulum (ER) stress is an important pathogenetic mechanism of NASH and plays a key role in tandem steatosis as well as liver inflammation. This study aims to develop a progressive NASH model through sustained lipid accumulation and to elucidate its molecular mechanism through IRE1α/TRAF2 complex. Male SD rats were fed a high-fat diet (HFD) for 4, 8, and 12 weeks to induce progressive NASH. MRNA sequencing and PPI analysis were used to screen core genes. Transmission electron microscopy, immunofluorescence staining, ELISA, qRT-PCR, and Western blotting were used at each time point to compare differences between each index of progressive NASH at 4, 8, and 12 weeks. Sustained lipid accumulation led to structural disruption of the ER, a reduction in ER number, and an increase of lipid droplet aggregation in hepatocytes. Persistent lipid accumulation led to a persistent increase in mRNA and protein expression of the IRE1α/TRAF2 complex, IKK/IκB/NF-κB signaling pathway and ASK1/JNK1 signaling pathway, and TNF-α, IL-1β, and IL-6 also continued to increase. Persistent lipid accumulation led to a persistent exacerbation of ER stress and inflammation in progressive NASH via the IRE1α/TRAF2 complex. Full article

Vacuolar ATPases (V-ATPases) are multi-subunit ATP-dependent proton pumps necessary for cellular functions, including pH regulation and membrane fusion. The evidence suggests that the V-ATPase a-subunit’s interaction with the membrane signaling lipid phosphatidylinositol (PIPs) regulates the recruitment of V-ATPase complexes to specific membranes. We generated a homology model of the N-terminal domain of the human a4 isoform (a4NT) using Phyre2.0 and propose a lipid binding domain within the distal lobe of the a4NT. We identified a basic motif, K²³⁴IKK²³⁷, critical for interaction with phosphoinositides (PIP), and found similar basic residue motifs in all four mammalian and both yeast a-isoforms. We tested PIP binding of wildtype and mutant a4NT in vitro. In protein lipid overlay assays, the double mutation K234A/K237A and the autosomal recessive distal renal tubular-causing mutation K237del reduced both PIP binding and association with liposomes enriched with PI(4,5)P₂, a PIP enriched within plasma membranes. Circular dichroism spectra of the mutant protein were comparable to wildtype, indicating that mutations affected lipid binding, not protein structure. When expressed in HEK293, wildtype a4NT localized to the plasma membrane in fluorescence microscopy and co-purified with the microsomal membrane fraction in cellular fractionation experiments. a4NT mutants showed reduced membrane association and decreased plasma membrane localization. Depletion of PI(4,5)P₂ by ionomycin caused reduced membrane association of the WT a4NT protein. Our data suggest that information contained within the soluble a4NT is sufficient for membrane association and that PI(4,5)P₂ binding capacity is involved in a4 V-ATPase plasma membrane retention. Full article

Head and neck squamous cell carcinoma (HNSCC) remains a prevalent diagnosis with current treatment options that include radiotherapy and immune-mediated therapies, in which tumor necrosis factor-α (TNFα) is a key mediator of cytotoxicity. However, HNSCC and other cancers often display TNFα resistance due to activation of the canonical IKK–NFκB/RELA pathway, which is activated by, and induces expression of, cellular inhibitors of apoptosis proteins (cIAPs). Our previous studies have demonstrated that the IAP inhibitor birinapant sensitized HNSCC to TNFα-dependent cell death in vitro and radiotherapy in vivo. Furthermore, we recently demonstrated that the inhibition of the G2/M checkpoint kinase WEE1 also sensitized HNSCC cells to TNFα-dependent cell death, due to the inhibition of the pro-survival IKK-NFκB/RELA complex. Given these observations, we hypothesized that dual-antagonist therapy targeting both IAP and WEE1 proteins may have the potential to synergistically sensitize HNSCC to TNFα-dependent cell death. Using the IAP inhibitor birinapant and the WEE1 inhibitor AZD1775, we show that combination treatment reduced cell viability, proliferation and survival when compared with individual treatment. Furthermore, combination treatment enhanced the sensitivity of HNSCC cells to TNFα-induced cytotoxicity via the induction of apoptosis and DNA damage. Additionally, birinapant and AZD1775 combination treatment decreased cell proliferation and survival in combination with radiotherapy, a critical source of TNFα. These results support further investigation of IAP and WEE1 inhibitor combinations in preclinical and clinical studies in HNSCC. Full article

The inhibition of Glycogen Synthase Kinase 3 β (GSK3β) by Ser⁹ phosphorylation affects many physiological processes, including the immune response. However, the consequences of GSK3β inhibition by alternative Ser³⁸⁹ phosphorylation remain poorly characterized. Here we have examined neuroinflammation in GSK3β Ser³⁸⁹ knock-in (KI) mice, in which the phosphorylation of Ser³⁸⁹ GSK3β is impaired. The number of activated microglia/infiltrated macrophages, astrocytes, and infiltrated neutrophils was significantly higher in these animals compared to C57BL/6J wild-type (WT) counterparts, which suggests that the failure to inactivate GSK3β by Ser³⁸⁹ phosphorylation results in sustained low-grade neuroinflammation. Moreover, glial cell activation and brain infiltration of immune cells in response to lipopolysaccharide (LPS) failed in GSK3β Ser³⁸⁹ KI mice. Such effects were brain-specific, as peripheral immunity was not similarly affected. Additionally, phosphorylation of the IkB kinase complex (IKK) in response to LPS failed in GSK3β Ser³⁸⁹ KI mice, while STAT3 phosphorylation was fully conserved, suggesting that the NF-κB signaling pathway is specifically affected by this GSK3β regulatory pathway. Overall, our findings indicate that GSK3β inactivation by Ser³⁸⁹ phosphorylation controls the brain inflammatory response, raising the need to evaluate its role in the progression of neuroinflammatory pathologies. Full article

HIV latent infection may be associated with disrupted viral RNA sensing, interferon (IFN) signaling, and/or IFN stimulating genes (ISG) activation. Here, we evaluated the use of compounds selectively targeting at the inhibitor of nuclear factor-κB (IκB) kinase (IKK) complex subunits and related kinases (TBK1) as a novel pathway to reverse HIV-1 latency in latently infected non-clonal lymphoid and myeloid cell in vitro models. IKK inhibitors (IKKis) triggered up to a 1.8-fold increase in HIV reactivation in both, myeloid and lymphoid cell models. The best-in-class IKKis, targeting TBK-1 (MRT67307) and IKKβ (TCPA-1) respectively, were also able to significantly induce viral reactivation in CD4+ T cells from people living with HIV (PLWH) ex vivo. More importantly, although none of the compounds tested showed antiviral activity, the combination of the distinct IKKis with ART did not affect the latency reactivation nor blockade of HIV infection by ART. Finally, as expected, IKKis did not upregulate cell activation markers in primary lymphocytes and innate immune signaling was blocked, resulting in downregulation of inflammatory cytokines. Overall, our results support a dual role of IKKis as immune modulators being able to tackle the HIV latent reservoir in lymphoid and myeloid cellular models and putatively control the hyperinflammatory responses in chronic HIV-1 infection. Full article

Age-related macular degeneration (AMD) is an irreversible chronic degenerative pathology that affects the retina. Despite therapeutic advances thanks to the use of anti-vascular endothelial growth factor (VEGF) agents, resistance mechanisms have been found to accentuate the visual deficit. In the present study, we explored whether a nutraceutical formulation composed of omega-3 fatty acids and resveratrol, called Resvega^®, was able to disrupt VEGF-A secretion in human ARPE-19 retina cells. We found that Resvega^® inhibits VEGF-A secretion through decreases in both the PI3K-AKT-mTOR and NFκB signaling pathways. In NFκB signaling pathways, Resvega^® inhibits the phosphorylation of the inhibitor of NFκB, IκB, which can bind NFκB dimers and sequester them in the cytoplasm. Thus, the NFκB subunits cannot migrate to the nucleus where they normally bind and stimulate the transcription of target genes such as VEGF-A. The IκB kinase complex (IKK) is also affected by Resvega^® since the nutraceutical formulation decreases both IKKα and IKKβ subunits and the IKKγ subunit which is required for the stimulation of IKK. Very interestingly, we highlight that Resvega^® could prolong the anti-angiogenic effect of Avastin^®, which is an anti-VEGF agent typically used in clinical practice. Our results suggest that Resvega^® may have potential interest as nutritional supplementation against AMD. Full article