The Nuclear NF-κB Regulator IκBζ: Updates on Its Molecular Functions and Pathophysiological Roles.

More than a decade after the discovery of the classical cytoplasmic IκB proteins, IκBζ was identified as an additional member of the IκB family. Unlike cytoplasmic IκB proteins, IκBζ has distinct features, including its nuclear localization, preferential binding to NF-κB subunits, unique expression properties, and specialized role in NF-κB regulation. While the activation of NF-κB is primarily controlled by cytoplasmic IκB members at the level of nuclear entry, IκBζ provides an additional layer of NF-κB regulation in the nucleus, enabling selective gene activation. Human genome-wide association studies (GWAS) and gene knockout experiments in mice have elucidated the physiological and pathological roles of IκBζ. Despite the initial focus to its role in activated macrophages, IκBζ has since been recognized as a key player in the IL-17-triggered production of immune molecules in epithelial cells, which has garnered significant clinical interest. Recent research has also unveiled a novel molecular function of IκBζ, linking NF-κB and the POU transcription factors through its N-terminal region, whose role had remained elusive for many years.

Resveratrol attenuates non-steroidal anti-inflammatory drug-induced intestinal injury in rats in a high-altitude hypoxic environment by modulating the TLR4/NFκB/IκB pathway and gut microbiota composition.

INTRODUCTION: Non-steroidal anti-inflammatory drugs (NSAIDs) are currently the most widely used anti-inflammatory medications, but their long-term use can cause damage to the gastrointestinal tract(GIT). One of the risk factors for GIT injury is exposure to a high-altitude hypoxic environment, which can lead to damage to the intestinal mucosal barrier. Taking NSAIDs in a high-altitude hypoxic environment can exacerbate GIT injury and impact gut microbiota. The aim of this study is to investigate the mechanisms by which resveratrol (RSV) intervention alleviates NSAID-induced intestinal injury in a high-altitude hypoxic environment, as well as its role in regulating gut microbiota. METHODS: Aspirin was administered orally to rats to construct a rat model of intestinal injury induced by NSAIDs. Following the induction of intestinal injury, rats were administered RSV by gavage, and the expression levels of TLR4, NF-κB,IκB as well as Zonula Occludens-1 (ZO-1) and Occludin proteins in the different treatment groups were assessed via Western blot. Furthermore, the expression of the inflammatory factors IL-10, IL-1ß, and TNF-α was evaluated using Elisa.16sRNA sequencing was employed to investigate alterations in the gut microbiota. RESULTS: The HCk group showed elevated expression of TLR4/NF-κB/IκB pathway proteins, increased expression of pro-inflammatory factors IL-1ß and TNF-α, decreased expression of the anti-inflammatory factor IL-10, and expression of intestinal mucosal barrier proteins ZO-1 and Occludin. The administration of NSAIDs drugs in the plateau hypoxic environment exacerbates intestinal inflammation and damage to the intestinal mucosal barrier. After treatment with RSV intervention, the expression of TLR4/NF-κB/IκB signaling pathway proteins would be reduced, thereby lowering the expression of inflammatory factors in the HAsp group. The results of HE staining directly show the damage to the intestines and the repair of intestinal mucosa after RSV intervention. 16sRNA sequencing results show significant differences (P<0.05) in Ruminococcus, Facklamia, Parasutterella, Jeotgalicoccus, Coprococcus, and Psychrobacter between the HCk group and the Ck group. Compared to the HCk group, the HAsp group shows significant differences (P<0.05) in Facklamia, Jeotgalicoccus, Roseburia, Psychrobacter, and Alloprevotella. After RSV intervention, Clostridium_sensu_stricto bacteria significantly increase compared to the HAsp group. CONCLUSION: Resveratrol can attenuate intestinal damage caused by the administration of NSAIDs at high altitude in hypoxic environments by modulating the TLR4/NF-κB/IκB signaling pathway and gut microbiota composition.

CgIκB2 negatively regulates the expression of interferon-like protein by Rel/NF-κB signal in Crassostrea gigas.

Inhibitors of NF-κB (IκBs) have been implicated as major components of the Rel/NF-κB signaling pathway, playing an important negative regulatory role in host antiviral immunity such as in the activation of interferon (IFN) in vertebrates. In the present study, the immunomodulatory effect of IκB (CgIκB2) on the expression of interferon-like protein (CgIFNLP) was evaluated in Pacific oyster (Crassostrea gigas). After poly (I:C) stimulation, the mRNA expression level of CgIκB2 in haemocytes was significantly down-regulated at 3-12 h while up-regulated at 48-72 h. The mRNA expression of CgIκB2 in haemocytes was significantly up-regulated at 3 h after rCgIFNLP stimulation. In the CgIκB2-RNAi oysters, the mRNA expression of CgIFNLP, interferon regulatory factor-8 (CgIRF8) and NF-κB subunit (CgRel), the abundance of CgIFNLP and CgIRF8 protein in haemocytes, as well as the abundance of CgRel protein in nucleus were significantly increased after poly (I:C) stimulation. Immunofluorescence assay showed that nuclear translocation of CgIRF8 and CgRel protein was promoted in CgIκB2-RNAi oysters compared with that in EGFP-RNAi group. In the CgRel-RNAi oysters, the mRNA and protein expression level of CgIFNLP significantly down-regulated after poly (I:C) stimulation. The collective results indicated that CgIκB2 plays an important role in regulating CgIFNLP expression through its effects on Rel/NF-κB and IRF signaling pathways.

Mandarin fish von Hippel-Lindau protein regulates the NF-κB signaling pathway via interaction with IκB to promote fish ranavirus replication.

The von Hippel-Lindau tumor suppressor protein (VHL), an E3 ubiquitin ligase, functions as a critical regulator of the oxygen-sensing pathway for targeting hypoxia-inducible factors. Recent evidence suggests that mammalian VHL may also be critical to the NF-κB signaling pathway, although the specific molecular mechanisms remain unclear. Herein, the roles of mandarin fish ( Siniperca chuatsi) VHL ( scVHL) in the NF-κB signaling pathway and mandarin fish ranavirus (MRV) replication were explored. The transcription of scVHL was induced by immune stimulation and MRV infection, indicating a potential role in innate immunity. Dual-luciferase reporter gene assays and reverse transcription quantitative PCR (RT-qPCR) results demonstrated that scVHL evoked and positively regulated the NF-κB signaling pathway. Treatment with NF-κB signaling pathway inhibitors indicated that the role of scVHL may be mediated through scIKKα, scIKKß, scIκBα, or scp65. Co-immunoprecipitation (Co-IP) analysis identified scIκBα as a novel target protein of scVHL. Moreover, scVHL targeted scIκBα to catalyze the formation of K63-linked polyubiquitin chains to activate the NF-κB signaling pathway. Following MRV infection, NF-κB signaling remained activated, which, in turn, promoted MRV replication. These findings suggest that scVHL not only positively regulates NF-κB but also significantly enhances MRV replication. This study reveals a novel function of scVHL in NF-κB signaling and viral infection in fish.

Structural and biochemical analyses of the nuclear IκBζ protein in complex with the NF-κB p50 homodimer.

As part of the efforts to understand nuclear IκB function in NF-κB-dependent gene expression, we report an X-ray crystal structure of the IκBζ ankyrin repeat domain in complex with the dimerization domain of the NF-κB p50 homodimer. IκBζ possesses an N-terminal α helix that conveys domain folding stability. Affinity and specificity of the complex depend on a small portion of p50 at the nuclear localization signal. The model suggests that only one p50 subunit supports binding with IκBζ, and biochemical experiments confirm that IκBζ associates with DNA-bound NF-κB p50:RelA heterodimers. Comparisons of IκBζ:p50 and p50:κB DNA complex crystallographic models indicate that structural rearrangement is necessary for ternary complex formation of IκBζ and p50 with DNA.

Selective regulation of a defined subset of inflammatory and immunoregulatory genes by an NF-κB p50-IκBζ pathway.

The five NF-κB family members and three nuclear IκB proteins play important biological roles, but the mechanisms by which distinct members of these protein families contribute to selective gene transcription remain poorly understood, especially at a genome-wide scale. Using nascent transcript RNA-seq, we observed considerable overlap between p50-dependent and IκBζ-dependent genes in Toll-like receptor 4 (TLR4)-activated macrophages. Key immunoregulatory genes, including Il6, Il1b, Nos2, Lcn2, and Batf, are among the p50-IκBζ-codependent genes. IκBζ-bound genomic sites are occupied at earlier time points by NF-κB dimers. However, p50-IκBζ codependence does not coincide with preferential binding of either p50 or IκBζ, as RelA co-occupies hundreds of genomic sites with the two proteins. A common feature of p50-IκBζ-codependent genes is a nearby p50/RelA/IκBζ-cobound site exhibiting p50-dependent binding of both RelA and IκBζ. This and other results suggest that IκBζ acts in concert with RelA:p50 heterodimers. Notably, p50-IκBζ-codependent genes comprise a high percentage of genes exhibiting the greatest differential expression between TLR4-stimulated and tumor necrosis factor receptor (TNFR)-stimulated macrophages. Thus, our genome-centric analysis reveals a defined p50-IκBζ pathway that selectively activates a set of key immunoregulatory genes and serves as an important contributor to differential TNFR and TLR4 responses.

IκBε deficiency accelerates disease development in chronic lymphocytic leukemia.

The NFKBIE gene, which encodes the NF-κB inhibitor IκBε, is mutated in 3-7% of patients with chronic lymphocytic leukemia (CLL). The most recurrent alteration is a 4-bp frameshift deletion associated with NF-κB activation in leukemic B cells and poor clinical outcome. To study the functional consequences of NFKBIE gene inactivation, both in vitro and in vivo, we engineered CLL B cells and CLL-prone mice to stably down-regulate NFKBIE expression and investigated its role in controlling NF-κB activity and disease expansion. We found that IκBε loss leads to NF-κB pathway activation and promotes both migration and proliferation of CLL cells in a dose-dependent manner. Importantly, NFKBIE inactivation was sufficient to induce a more rapid expansion of the CLL clone in lymphoid organs and contributed to the development of an aggressive disease with a shortened survival in both xenografts and genetically modified mice. IκBε deficiency was associated with an alteration of the MAPK pathway, also confirmed by RNA-sequencing in NFKBIE-mutated patient samples, and resistance to the BTK inhibitor ibrutinib. In summary, our work underscores the multimodal relevance of the NF-κB pathway in CLL and paves the way to translate these findings into novel therapeutic options.

Selective Hypoxia-Sensitive Oxomer Formation by FIH Prevents Binding of the NF-κB Inhibitor IκBß to NF-κB Subunits.

Pharmacologic inhibitors of cellular hydroxylase oxygen sensors are protective in multiple preclinical in vivo models of inflammation. However, the molecular mechanisms underlying this regulation are only partly understood, preventing clinical translation. We previously proposed a new mechanism for cellular oxygen sensing: oxygen-dependent, (likely) covalent protein oligomer (oxomer) formation. Here, we report that the oxygen sensor factor inhibiting HIF (FIH) forms an oxomer with the NF-κB inhibitor ß (IκBß). The formation of this protein complex required FIH enzymatic activity and was prevented by pharmacologic inhibitors. Oxomer formation was highly hypoxia-sensitive and very stable. No other member of the IκB protein family formed an oxomer with FIH, demonstrating that FIH-IκBß oxomer formation was highly selective. In contrast to the known FIH-dependent oxomer formation with the deubiquitinase OTUB1, FIH-IκBß oxomer formation did not occur via an IκBß asparagine residue, but depended on the amino acid sequence VAERR contained within a loop between IκBß ankyrin repeat domains 2 and 3. Oxomer formation prevented IκBß from binding to its primary interaction partners p65 and c-Rel, subunits of NF-κB, the master regulator of the cellular transcriptional response to pro-inflammatory stimuli. We therefore propose that FIH-mediated oxomer formation with IκBß contributes to the hypoxia-dependent regulation of inflammation.

NF-κB as an Inducible Regulator of Inflammation in the Central Nervous System.

The NF-κB (nuclear factor K-light-chain-enhancer of activated B cells) transcription factor family is critical for modulating the immune proinflammatory response throughout the body. During the resting state, inactive NF-κB is sequestered by IκB in the cytoplasm. The proteasomal degradation of IκB activates NF-κB, mediating its translocation into the nucleus to act as a nuclear transcription factor in the upregulation of proinflammatory genes. Stimuli that initiate NF-κB activation are diverse but are canonically attributed to proinflammatory cytokines and chemokines. Downstream effects of NF-κB are cell type-specific and, in the majority of cases, result in the activation of pro-inflammatory cascades. Acting as the primary immune responders of the central nervous system, microglia exhibit upregulation of NF-κB upon activation in response to pathological conditions. Under such circumstances, microglial crosstalk with other cell types in the central nervous system can induce cell death, further exacerbating the disease pathology. In this review, we will emphasize the role of NF-κB in triggering neuroinflammation mediated by microglia.

Botulinum toxin type A inhibits M1 macrophage polarization by deactivation of JAK2/STAT1 and IκB/NFκB pathway and contributes to scar alleviation in aseptic skin wound healing.

The non-neuronal and non-muscular effects of botulinum toxin type A (BTXA) on scar reduction has been discovered. This study was designed to investigate the effects of BTXA on macrophages polarization during the early stage of skin repair. A skin defect model was established on the dorsal skin of SD rats. BTXA was intracutaneous injected into the edge of wound immediately as the model was established. Histological examinations were performed on scar samples. Raw 264.7 was selected as the cell model of recruited circulating macrophages, and was induced for M1 polarization by LPS. Identify the signaling pathways that primarily regulated M1 polarization and respond to BTXA treatment. Application of BTXA at early stage of injury significantly reduced the scar diameter without delaying wound closure. BTXA treatment improved fiber proliferation and arrangement, and inhibited angiogenesis in scar granular tissue. The number of M1 macrophages and the levels of pro-inflammation were decreased after treated with BTXA in scar tissues. LPS activated JAK2/STAT1 and IκB/NFκB pathways were downregulated by BTXA, as well as LPS induced M1 polarization. At early stage of skin wound healing, injection of BTXA effectively reduced the number of M1 macrophages and the levels of pro-inflammatory mediators which contributes to scar alleviation. BTXA resisted the M1 polarization of macrophages induced by LPS via deactivating the JAK2/STAT1 and IκB/NFκB pathways.

HOXA1 silencing inhibits cisplatin resistance of oral squamous cell carcinoma cells via IκB/NF-κB signaling pathway.

The resistance of oral squamous cell carcinoma (OSCC) cells to cisplatin remains a tough nut to crack in OSCC therapy. Homeobox A1 (HOXA1) overexpression has been detected in head and neck squamous carcinoma (HNSC). Accordingly, this study aims to explore the potential role and mechanism of HOXA1 on cisplatin resistance in OSCC. The expression of HOXA1 in HNSC and its role in overall survival (OS) rate of OSCC patients were analyzed by bioinformatic analysis. Following transfection as needed, OSCC cells were induced by different concentrations of cisplatin, and the cell viability and apoptosis were evaluated by cell counting kit-8 and flow cytometry assays. The mRNA and protein expression levels of HOXA1 and the phosphorylation of IκBα and p65 were determined by real-time quantitative PCR and western blot. HOXA1 expression level was upregulated in HNSC tissues and OSCC cells. Overexpressed HOXA1 was correlated with a low OS rate of OSCC patients. Cisplatin exerted an anti-cancer effect on OSCC cells. HOXA1 silencing or cisplatin suppressed OSCC cell viability, boosted the apoptosis, and repressed the phosphorylation of IκBα and p65. Intriguingly, the combination of HOXA1 silencing and cisplatin generated a stronger anti-cancer effect on OSCC cells than their single use. HOXA1 silencing attenuates cisplatin resistance of OSCC cells via IκB/NF-κB signaling pathway, hinting that HOXA1 is a biomarker associated with OSCC and HOXA1 silencing can enhance the sensitivity of OSCC cells to cisplatin.

Polyphenols Targeting NF-κB Pathway in Neurological Disorders: What We Know So Far?

Polyphenolic compounds have shown promising neuroprotective properties, making them a valuable resource for identifying prospective drug candidates to treat several neurological disorders (NDs). Numerous studies have reported that polyphenols can disrupt the nuclear factor kappa B(NF-κB) pathway by inhibiting the phosphorylation or ubiquitination of signaling molecules, which further prevents the degradation of IκB. Additionally, they prevent NF-κB translocation to the nucleus and pro-inflammatory cytokine production. Polyphenols such as curcumin, resveratrol, and pterostilbene had significant inhibitory effects on NF-κB, making them promising candidates for treating NDs. Recent experimental findings suggest that polyphenols possess a wide range of pharmacological properties. Notably, much attention has been directed towards their potential therapeutic effects in NDs such as Alzheimer's disease (AD), Parkinson's disease (PD), cerebral ischemia, anxiety, depression, autism, and spinal cord injury (SCI). Much preclinical data supporting the neurotherapeutic benefits of polyphenols has been developed. Nevertheless, this study has described the significance of polyphenols as potential neurotherapeutic agents, specifically emphasizing their impact on the NF-κB pathway. This article offers a comprehensive analysis of the involvement of polyphenols in NDs, including both preclinical and clinical perspectives.

Protective effect of olopatadine hydrochloride against LPS-induced acute lung injury: via targeting NF-κB signaling pathway.

BACKGROUND: Morbidity and mortality rates associated with acute lung injury/acute respiratory distress syndrome (ALI/ARDS) are high (30-40%). Nuclear factor-kappa B (NF-κB) is a transcription factor, associated with transcription of numerous cytokines leading to cytokine storm, and thereby, plays a major role in ALI/ARDS and in advanced COVID-19 syndrome. METHODS: Considering the role of NF-κB in ALI, cost-effective in silico approaches were utilized in the study to identify potential NF-κB inhibitor based on the docking and pharmacokinetic results. The identified compound was then pharmacologically validated in lipopolysaccharide (LPS) rodent model of acute lung injury. LPS induces ALI by altering alveolar membrane permeability, recruiting activated neutrophils and macrophages to the lungs, and compromising the alveolar membrane integrity and ultimately impairs the gaseous exchange. Furthermore, LPS exposure is associated with exaggerated production of various proinflammatory cytokines in lungs. RESULTS: Based on in silico studies Olopatadine Hydrochloride (Olo), an FDA-approved drug was found as a potential NF-κB inhibitor which has been reported for the first time, and considered further for the pharmacological validation. Intraperitoneal LPS administration resulted in ALI/ARDS by fulfilling 3 out of the 4 criteria described by ATS committee (2011) published workshop report. However, treatment with Olo attenuated LPS-induced elevation of proinflammatory markers (IL-6 and NF-κB), oxidative stress, neutrophil infiltration, edema, and damage in lungs. Histopathological studies also revealed that Olo treatment significantly ameliorated LPS-induced lung injury, thus conferring improvement in survival. Especially, the effects produced by Olo medium dose (1 mg/kg) were comparable to dexamethasone standard. CONCLUSION: In nutshell, inhibition of NF-κB pathway by Olo resulted in protection and reduced mortality in LPS- induced ALI and thus has potential to be used clinically to arrest disease progression in ALI/ARDS, since the drug is already in the market. However, the findings warrant further extensive studies, and also future studies can be planned to elucidate its role in COVID-19-associated ARDS or cytokine storm.

Autoantibodies against type I IFNs in humans with alternative NF-κB pathway deficiency.

Patients with autoimmune polyendocrinopathy syndrome type 1 (APS-1) caused by autosomal recessive AIRE deficiency produce autoantibodies that neutralize type I interferons (IFNs)1,2, conferring a predisposition to life-threatening COVID-19 pneumonia3. Here we report that patients with autosomal recessive NIK or RELB deficiency, or a specific type of autosomal-dominant NF-κB2 deficiency, also have neutralizing autoantibodies against type I IFNs and are at higher risk of getting life-threatening COVID-19 pneumonia. In patients with autosomal-dominant NF-κB2 deficiency, these autoantibodies are found only in individuals who are heterozygous for variants associated with both transcription (p52 activity) loss of function (LOF) due to impaired p100 processing to generate p52, and regulatory (IκBδ activity) gain of function (GOF) due to the accumulation of unprocessed p100, therefore increasing the inhibitory activity of IκBδ (hereafter, p52LOF/IκBδGOF). By contrast, neutralizing autoantibodies against type I IFNs are not found in individuals who are heterozygous for NFKB2 variants causing haploinsufficiency of p100 and p52 (hereafter, p52LOF/IκBδLOF) or gain-of-function of p52 (hereafter, p52GOF/IκBδLOF). In contrast to patients with APS-1, patients with disorders of NIK, RELB or NF-κB2 have very few tissue-specific autoantibodies. However, their thymuses have an abnormal structure, with few AIRE-expressing medullary thymic epithelial cells. Human inborn errors of the alternative NF-κB pathway impair the development of AIRE-expressing medullary thymic epithelial cells, thereby underlying the production of autoantibodies against type I IFNs and predisposition to viral diseases.

[Mechanism of dexamethasone combined with glutamine in reducing lung inflammation and pulmonary edema in rats with acute lung injury].

Objective To investigate the effect of dexamethasone (DEX) combined with glutamine (Gln) on lung inflammation and pulmonary edema in rats with acute lung injury induced by lipopolysaccharide (LPS) and its related mechanisms. Methods Fifty Wistar rats were randomly divided into control group, model group, dexamethasone group (DEX) and DEX combined with Gln group. Except for the control group, rats in other groups were injected with 6 mg/kg LPS intraperitoneally to induce an acute lung injury. The mRNA expression of p38 MAPK, NLRP3, and NF-κB in lung tissue were detected by real-time quantitative PCR. The protein expressions of p-p38 MAPK, NLRP3, phosphorylated inhibitor of nuclear factor κB (p-IκB), NF-κB p65, aquaporin 1 (AQP1) and AQP5 in lung tissue were detected by Western blot analysis. ELISA was used to detect the content of serum tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), interleukin 1ß (IL-1ß). Spectrophotometer was employed to detect the content of superoxide dismutase (SOD), malondialdehyde (MDA) and glutathione peroxidase (GSH-Px) in lung tissue. Results Compared with the control group, the lung index of the model group decreased, the content of the serum inflammatory factors TNF-α, IL-6 and IL-1ß significantly increased, and the protein expression of p38 MAPK, NLRP3, NF-κB mRNA, p-p38 MAPK, NLRP3, p-IκB and NF-κB p65 in the lung tissue significantly increased, while that of AQP1, AQP5 decreased, and the content of SOD and GSH-Px in lung tissue decreased, while that of MDA increased; Compared with the model group, the above mentioned symptoms and indicators in each treatment group were significantly improved, among which the DEX combined with Gln group was the most significant. Conclusion DEX combined with Gln can inhibit inflammation, resist oxidative damage, relieve pulmonary edema, and prevent acute lung injury. Its mechanism is related to inhibiting the activation of p38 MAPK, NLRP3, and NF-κB signaling pathways, promoting the expression of AQP1 and AQP5, and promoting the activity of antioxidant products.

Rabdosichuanin C inhibits productions of pro-inflammatory mediators regulated by NF-κB signaling in LPS-stimulated RAW264.7 cells.

Chronic pharyngitis (CP) is an inflammatory disease of the pharyngeal mucosa and its lymphatic tissues that is difficult to treat clinically. However, research on the exact therapeutic agents and molecular mechanisms of CP is still unclear. In this study, we investigated Rabdosichuanin C (RC) to attenuate lipopolysaccharide (LPS)-induced inflammatory damage in RAW264.7 cells by a combination of targeted virtual screening and in vitro activity assay and further clarified its molecular mechanism of action centering on the IκB/nuclear factor kappa B (NF-κB) pathway. Molecular docking and pharmacophore simulation methods were used to screen compounds with IκB inhibitory effects. Expression of genes and proteins related to the IκB/NF-κB signaling pathway by RC in LPS-induced inflammatory injury model of RAW264.7 cells was detected by PCR, enzyme-linked immunosorbent assay, and Western blot. The docking of RC with IκB protein showed good binding energy, and pharmacophore simulations further confirmed the active effect of RC in inhibiting IκB protein. RC intervention in LPS-induced RAW264.7 cells significantly reduced the expression levels of inflammatory factors tumor necrosis factor-α, interleukins-6, iNOS, and CD-86 at the messenger RNA and protein levels, downregulated IκB, p65 protein phosphorylation levels, and significantly inhibited IκB/NF-κB signaling pathway activation. Virtual screening provided us with an effective method to rapidly identify compounds RC that target inhibit the action of IκB, and the activity results showed that RC inhibits NF-κB signaling pathway activation. It is suggested that RC may play a role in the treatment of CP by inhibiting the IκB/NF-κB signaling pathway.

[Guizhi Fuling Capsule inhibits migration and induces apoptosis of human ovarian cancer cells by regulating the NF-κB signaling pathway].

OBJECTIVE: To study the inhibitory effect of Guizhi Fuling Capsule (GFC) on migration of human ovarian cancer cells and explore the possible mechanism. METHODS: Sixty Wistar rats were randomized into 4 groups for daily gavage of saline or 4, 8, or 16 g/kg GFC suspension for 5 days to prepare blank and low-, medium- and high-dose GFC-medicated sera. Cisplatinresistant ovarian cancer SKOV3/DDP cells were treated with these sera with nuclear factor-κB (NF-κB) inhibitor SN50 as the positive control, and the changes in migration ability and apoptosis of the cells were examined using scratch assay and flow cytometry, respectively; the changes in the mRNA and protein expressions of CDH1, CDH2, caspase 3 and NF- κB were detected using RT-qPCR and Western blotting. ATAC-seq was used to analyze the changes in expressions of CDH1, CDH2, caspase 3 and NF-κB genes in the open chromatin. RESULTS: Treatment with GFC-medicated sera dose-dependently inhibited the migration (P < 0.05), increased apoptosis (P < 0.01), inhibited CDH2 and NF-κB mRNA expression (P < 0.05), and enhanced caspase 3 and CDH1 mRNA expressions (P < 0.01) in SKOV3/DDP cells. The effects of high-dose GFC-medicated serum were comparable to those of SN50 (P>0.05), but its effect for enhancing DH1 protein expression was weaker than that of SN50 (P < 0.01). GFC-medicated sera significantly lowered the expressions of NF-κB and CDH2 and increased CDH1 expression in the open chromatin without obviously affecting caspase 3 expression. CONCLUSION: GFC- medicated sera inhibits the migration ability of SKOV3/DDP cells possibly by promoting cell apoptosis and caspase 3 and CDH1 expressions, inhibiting CDH2 and NF-κB expressions, and regulating the expressions of NF-κB, CDH2 and CDH1 in the open chromatin.

NF-κB: a mediator that promotes or inhibits angiogenesis in human diseases?

The nuclear factor of κ-light chain of enhancer-activated B cells (NF-κB) signaling pathway, which is conserved in invertebrates, plays a significant role in human diseases such as inflammation-related diseases and carcinogenesis. Angiogenesis refers to the growth of new capillary vessels derived from already existing capillaries and postcapillary venules. Maintaining normal angiogenesis and effective vascular function is a prerequisite for the stability of organ tissue function, and abnormal angiogenesis often leads to a variety of diseases. It has been suggested that NK-κB signalling molecules under pathological conditions play an important role in vascular differentiation, proliferation, apoptosis and tumourigenesis by regulating the transcription of multiple target genes. Many NF-κB inhibitors are being tested in clinical trials for cancer treatment and their effect on angiogenesis is summarised. In this review, we will summarise the role of NF-κB signalling in various neovascular diseases, especially in tumours, and explore whether NF-κB can be used as an attack target or activation medium to inhibit tumour angiogenesis.

Structure-Based Discovery of Receptor Activator of Nuclear Factor-κB Ligand (RANKL)-Induced Osteoclastogenesis Inhibitors.

Receptor activator of nuclear factor-κB ligand (RANKL) has been actively pursued as a therapeutic target for osteoporosis, given that RANKL is the master mediator of bone resorption as it promotes osteoclast differentiation, activity and survival. We employed a structure-based virtual screening approach comprising two stages of experimental evaluation and identified 11 commercially available compounds that displayed dose-dependent inhibition of osteoclastogenesis. Their inhibitory effects were quantified through TRAP activity at the low micromolar range (IC50 < 5 µΜ), but more importantly, 3 compounds displayed very low toxicity (LC50 > 100 µΜ). We also assessed the potential of an N-(1-aryl-1H-indol-5-yl)aryl-sulfonamide scaffold that was based on the structure of a hit compound, through synthesis of 30 derivatives. Their evaluation revealed 4 additional hits that inhibited osteoclastogenesis at low micromolar concentrations; however, cellular toxicity concerns preclude their further development. Taken together with the structure-activity relationships provided by the hit compounds, our study revealed potent inhibitors of RANKL-induced osteoclastogenesis of high therapeutic index, which bear diverse scaffolds that can be employed in hit-to-lead optimization for the development of therapeutics against osteolytic diseases.

Differential dysregulation of ß-TrCP1 and -2 by HIV-1 Vpu leads to inhibition of canonical and non-canonical NF-κB pathways in infected cells.

The HIV-1 Vpu protein is expressed late in the virus lifecycle to promote infectious virus production and avoid innate and adaptive immunity. This includes the inhibition of the NF-κB pathway which, when activated, leads to the induction of inflammatory responses and the promotion of antiviral immunity. Here we demonstrate that Vpu can inhibit both canonical and non-canonical NF-κB pathways, through the direct inhibition of the F-box protein ß-TrCP, the substrate recognition portion of the Skp1-Cul1-F-box (SCF)ß-TrCP ubiquitin ligase complex. There are two paralogues of ß-TrCP (ß-TrCP1/BTRC and ß-TrCP2/FBXW11), encoded on different chromosomes, which appear to be functionally redundant. Vpu, however, is one of the few ß-TrCP substrates to differentiate between the two paralogues. We have found that patient-derived alleles of Vpu, unlike those from lab-adapted viruses, trigger the degradation of ß-TrCP1 while co-opting its paralogue ß-TrCP2 for the degradation of cellular targets of Vpu, such as CD4. The potency of this dual inhibition correlates with stabilization of the classical IκBα and the phosphorylated precursors of the mature DNA-binding subunits of canonical and non-canonical NF-κB pathways, p105/NFκB1 and p100/NFκB2, in HIV-1 infected CD4+ T cells. Both precursors act as alternative IκBs in their own right, thus reinforcing NF-κB inhibition at steady state and upon activation with either selective canonical or non-canonical NF-κB stimuli. These data reveal the complex regulation of NF-κB late in the viral replication cycle, with consequences for both the pathogenesis of HIV/AIDS and the use of NF-κB-modulating drugs in HIV cure strategies. IMPORTANCE The NF-κB pathway regulates host responses to infection and is a common target of viral antagonism. The HIV-1 Vpu protein inhibits NF-κB signaling late in the virus lifecycle, by binding and inhibiting ß-TrCP, the substrate recognition portion of the ubiquitin ligase responsible for inducing IκB degradation. Here we demonstrate that Vpu simultaneously inhibits and exploits the two different paralogues of ß-TrCP by triggering the degradation of ß-TrCP1 and co-opting ß-TrCP2 for the destruction of its cellular targets. In so doing, it has a potent inhibitory effect on both the canonical and non-canonical NF-κB pathways. This effect has been underestimated in previous mechanistic studies due to the use of Vpu proteins from lab-adapted viruses. Our findings reveal previously unappreciated differences in the ß-TrCP paralogues, revealing functional insights into the regulation of these proteins. This study also raises important implications for the role of NF-κB inhibition in the immunopathogenesis of HIV/AIDS and the way that this may impact on HIV latency reversal strategies based on the activation of the non-canonical NF-κB pathway.