Has-miR-199a-3p/RELA/SCD inhibits immune checkpoints in AMD and promotes macrophage-mediated inflammation and pathological angiogenesis through lipid metabolism pathway: A computational analysis.

More and more evidence shows that abnormal lipid metabolism leads to immune system dysfunction in AMD and promotes the occurrence of AMD by changing the homeostasis of ocular inflammation. However, the molecular mechanism underlying the effect of lipid metabolism on the phenotype and function of macrophages is still unclear, and the mechanism of association between AMD and cancer and COVID-19 has not been reported. The purpose of this study is to explore the interaction between lipid metabolism related genes, ferroptosis related genes and immunity in AMD, find out the key genes that affect the ferroptosis of AMD through lipid metabolism pathway and the molecular mechanism that mediates the action of macrophages, and find out the possible mechanism of lipid metabolism and potential co-therapeutic targets between AMD and cancer and COVID-19, so as to improve treatment decision-making and clinical results. For the first time, we have comprehensively analyzed the fatty acid molecule related genes, ferroptosis related genes and immune microenvironment of AMD patients, and determined that mast cells and M1 macrophages are the main causes of AMD inflammation, and found that SCD is the core gene in AMD that inhibits ferroptosis through lipid metabolism pathway, and verified the difference in the expression of SCD in AMD in a separate external data set. Based on the analysis of the mechanism of action of the SCD gene, we found for the first time that Has-miR-199a-3p/RELA/SCD is the core axis of action of lipid metabolism pathway to inhibit the ferroptosis of AMD. By inhibiting the immune checkpoint, we can enhance the immune cell activity of AMD and lead to the transformation of macrophages from M2 to M1, thereby promoting the inflammation and pathological angiogenesis of AMD. At the same time, we found that ACOX2 and PECR, as genes for fatty acid metabolism, may regulate the expression of SCD during the occurrence and development of COVID-19, thus affecting the occurrence and development of AMD. We found that FASD1 may be a key gene for the joint action of AMD and COVID-19, and SCD regulates the immune infiltration of macrophages in glioma and germ line tumors. In conclusion, our results can provide theoretical basis for the pathogenesis of AMD, help guide the treatment of AMD patients and their potentially related diseases and help to design effective drug targets.

Gasdermin E promotes translocation of p65 and c-jun into nucleus in keratinocytes for progression of psoriatic skin inflammation.

Gasdermin E (GSDME) has recently been identified as a critical executioner to mediate pyroptosis. While epidermal keratinocytes can initiate GSDME-mediated pyroptosis, the role of keratinocyte GSDME in psoriatic dermatitis remains poorly characterized. Through analysis of GEO datasets, we found elevated GSDME levels in psoriatic lesional skin. Additionally, GSDME levels correlated with both psoriasis severity and response to biologics treatments. Single-cell RNA sequencing (scRNA-seq) from a GEO dataset revealed GSDME upregulation in keratinocytes of psoriasis patients. In the imiquimod (IMQ)-induced psoriasis-like dermatitis mouse model, both full-length and cleaved forms of caspase-3 and GSDME were elevated in the epidermis. Abnormal proliferation and differentiation of keratinocytes and dermatitis were attenuated in Gsdme-/- mice and keratinocyte-specific Gsdme conditional knockout mice after IMQ stimulation. Exposure of keratinocytes to mixed cytokines (M5), mimicking psoriatic conditions, led to GSDME cleavage. Moreover, the interaction between GSDME-FL and p65 or c-jun was significantly increased after M5 stimulation. GSDME knockdown inhibited nuclear translocation of p65 and c-jun and decreased upregulation of psoriatic inflammatory mediators such as IL1ß, CCL20, CXCL1, CXCL8, S100A8, and S100A9 in M5-challenged keratinocytes. In conclusion, GSDME in keratinocytes contributes to the pathogenesis and progression of psoriasis, potentially in a pyroptosis-independent manner by interacting and promoting translocation of p65 and c-jun. These findings suggest that keratinocyte GSDME could serve as a potential therapeutic target for psoriasis treatment.

Moderate mechanical stress suppresses chondrocyte ferroptosis in osteoarthritis by regulating NF-κB p65/GPX4 signaling pathway.

Ferroptosis is a recently identified form of programmed cell death that plays an important role in the pathophysiological process of osteoarthritis (OA). Herein, we investigated the protective effect of moderate mechanical stress on chondrocyte ferroptosis and further revealed the internal molecular mechanism. Intra-articular injection of sodium iodoacetate (MIA) was conducted to induce the rat model of OA in vivo, meanwhile, interleukin-1 beta (IL-1ß) was treated to chondrocytes to induce the OA cell model in vitro. The OA phenotype was analyzed by histology and microcomputed tomography, the ferroptosis was analyzed by transmission electron microscope and immunofluorescence. The expression of ferroptosis and cartilage metabolism-related factors was analyzed by immunohistochemical and Western blot. Animal experiments revealed that moderate-intensity treadmill exercise could effectively reduce chondrocyte ferroptosis and cartilage matrix degradation in MIA-induced OA rats. Cell experiments showed that 4-h cyclic tensile strain intervention could activate Nrf2 and inhibit the NF-κB signaling pathway, increase the expression of Col2a1, GPX4, and SLC7A11, decrease the expression of MMP13 and P53, thereby restraining IL-1ß-induced chondrocyte ferroptosis and degeneration. Inhibition of NF-κB signaling pathway relieved the chondrocyte ferroptosis and degeneration. Meanwhile, overexpression of NF-κB by recombinant lentivirus reversed the positive effect of CTS on chondrocytes. Moderate mechanical stress could activate the Nrf2 antioxidant system, inhibit the NF-κB p65 signaling pathway, and inhibit chondrocyte ferroptosis and cartilage matrix degradation by regulating P53, SLC7A11, and GPX4.

The transcription factor NF-κB orchestrates nucleosome remodeling during the primary response to Toll-like receptor 4 signaling.

Inducible nucleosome remodeling at hundreds of latent enhancers and several promoters shapes the transcriptional response to Toll-like receptor 4 (TLR4) signaling in macrophages. We aimed to define the identities of the transcription factors that promote TLR-induced remodeling. An analysis strategy based on ATAC-seq and single-cell ATAC-seq that enriched for genomic regions most likely to undergo remodeling revealed that the transcription factor nuclear factor κB (NF-κB) bound to all high-confidence peaks marking remodeling during the primary response to the TLR4 ligand, lipid A. Deletion of NF-κB subunits RelA and c-Rel resulted in the loss of remodeling at high-confidence ATAC-seq peaks, and CRISPR-Cas9 mutagenesis of NF-κB-binding motifs impaired remodeling. Remodeling selectivity at defined regions was conferred by collaboration with other inducible factors, including IRF3- and MAP-kinase-induced factors. Thus, NF-κB is unique among TLR4-activated transcription factors in its broad contribution to inducible nucleosome remodeling, alongside its ability to activate poised enhancers and promoters assembled into open chromatin.

Inhibition of NF-κB-Mediated Proinflammatory Transcription by Ru(II) Complexes of Anti-Angiogenic Ligands in Triple-Negative Breast Cancer.

Nuclear factor kappa beta (NF-κB) plays a pivotal role in breast cancer, particularly triple-negative breast cancer, by promoting inflammation, proliferation, epithelial-mesenchymal transition, metastasis, and drug resistance. Upregulation of NF-κB boosts vascular endothelial growth factor (VEGF) expression, assisting angiogenesis. The Ru(II) complexes of methyl- and dimethylpyrazolyl-benzimidazole N,N donors inhibit phosphorylation of ser536 in p65 and translocation of the NF-κB heterodimer (p50/p65) to the nucleus, disabling transcription to upregulate inflammatory signaling. The methyl- and dimethylpyrazolyl-benzimidazole inhibit VEGFR2 phosphorylation at Y1175, disrupting downstream signaling through PLC-γ and ERK1/2, ultimately suppressing Ca(II)-signaling. Partial release of the antiangiogenic ligand in a reactive oxygen species-rich environment is possible as per our observation to inhibit both NF-κB and VEGFR2 by the complexes. The complexes are nontoxic to zebrafish embryos up to 50 µM, but the ligands show strong in vivo antiangiogenic activity at 3 µM during embryonic growth in Tg(fli1:GFP) zebrafish but no visible effect on the adult phase.

Co-imaging of RelA and c-Rel reveals features of NF-κB signaling for ligand discrimination.

Individual cell sensing of external cues has evolved through the temporal patterns in signaling. Since nuclear factor κB (NF-κB) signaling dynamics have been examined using a single subunit, RelA, it remains unclear whether more information might be transmitted via other subunits. Using NF-κB double-knockin reporter mice, we monitored both canonical NF-κB subunits, RelA and c-Rel, simultaneously in single macrophages by quantitative live-cell imaging. We show that signaling features of RelA and c-Rel convey more information about the stimuli than those of either subunit alone. Machine learning is used to predict the ligand identity accurately based on RelA and c-Rel signaling features without considering the co-activated factors. Ligand discrimination is achieved through selective non-redundancy of RelA and c-Rel signaling dynamics, as well as their temporal coordination. These results suggest a potential role of c-Rel in fine-tuning immune responses and highlight the need for approaches that will elucidate the mechanisms regulating NF-κB subunit specificity.

Anabolic Steroids Activate the NF-κB Pathway in Porcine Ovarian Putative Stem Cells Independently of the ZIP-9 Receptor.

Boldenone (Bdn) and nandrolone (Ndn) are anabolic androgenic steroids (AASs) that, as our previous studies have shown, may increase the risk of neoplastic transformation of porcine ovarian putative stem cells (poPSCs). The NF-κB pathway may be important in the processes of carcinogenesis and tumour progression. Therefore, in this work, we decided to test the hypothesis of whether Bdn and Ndn can activate the NF-κB pathway by acting through the membrane androgen receptor ZIP-9. For this purpose, the expression profiles of both genes involved in the NF-κB pathway and the gene coding for the ZIP-9 receptor were checked. The expression and localization of proteins of this pathway in poPSCs were also examined. Additionally, the expression of the ZIP-9 receptor and the concentration of the NF-κB1 and 2 protein complex were determined. Activation of the NF-κB pathway was primarily confirmed by an increase in the relative abundances of phosphorylated forms of RelA protein and IκBα inhibitor. Reduced quantitative profiles pinpointed not only for genes representing this pathway but also for unphosphorylated proteins, and, simultaneously, decreased concentration of the NF-κB1 and 2 complex may indicate post-activation silencing by negative feedback. However, the remarkably and sustainably diminished expression levels noticed for the SLC39A9 gene and ZIP-9 protein suggest that this receptor does not play an important role in the regulation of the NF-κB pathway.

Role of NF-κB p65/TNF-α in Cell Apoptosis in the Fetal Membranes of Pregnant Women with Preterm Premature Rupture of Membranes.

OBJECTIVE: This study aimed to investigate the roles of nuclear factor-kappa B p65 (NF-[Formula: see text]B p65) and tumor necrosis factor-α (TNF-α) in cell apoptosis occurring in the fetal membranes of pregnant women who experience preterm premature rupture of membranes (PPROM). METHODS: This was a case-control study involving 57 pregnant women who delivered in the obstetric department of Affiliated Loudi Hospital, Hengyang Medical School, University of South China, from June 2021 to June 2022. Samples of fetal membrane tissue were collected from pregnant women with PPROM (n=27) and pregnant women who had normal deliveries (control group; n=30). The membrane tissue morphology of both groups was observed, and the expression of NF-[Formula: see text]B p65, p-NF-[Formula: see text]B p65, TNF-α, and caspase-3 was detected. Apoptosis in fetal membranes was examined. RESULTS: Morphological evaluation of the fetal membrane tissues obtained from patients with PPROM revealed an abnormal structure with a thin collagen fiber layer and cells with a largely vacuolar cytoplasm. There was a positive correlation between the expression of p-NF-[Formula: see text]B p65/NF-[Formula: see text]B p65 and cell apoptosis (r1 =0.89, R2 =0.805, P=0.00). Furthermore, TNF-α was positively correlated with fetal membrane cell apoptosis (r2 =0.93, R2=0.881, P=0.00). CONCLUSION: NF-[Formula: see text]B p65 is involved in the occurrence of PPROM by promoting the expression of TNF-α, which upregulates caspase-3 to cause apoptosis of fetal membrane cells.

The NF-κB RelA transcription factor is not required for CD8+ T-cell function in acute viral infection and cancer.

CD8+ T cells are critical mediators of pathogen clearance and anti-tumor immunity. Although signaling pathways leading to the activation of NF-κB transcription factors have crucial functions in the regulation of immune responses, the CD8+ T cell-autonomous roles of the different NF-κB subunits, are still unresolved. Here, we investigated the function of the ubiquitously expressed transcription factor RelA in CD8+ T-cell biology using a novel mouse model and gene-edited human cells. We found that CD8+ T cell-specific ablation of RelA markedly altered the transcriptome of ex vivo stimulated cells, but maintained the proliferative capacity of both mouse and human cells. In contrast, in vivo experiments showed that RelA deficiency did not affect the CD8+ T-cell response to acute viral infection or transplanted tumors. Our data suggest that in CD8+ T cells, RelA is dispensable for their protective activity in pathological contexts.

Value of the NF-κB signalling pathway and the DNA repair gene PARP1 in predicting distant metastasis after breast cancer surgery.

The DNA repair gene PARP1 and NF-κB signalling pathway affect the metastasis of breast cancer by influencing the drug resistance of cancer cells. Therefore, this study focused on the value of the DNA repair gene PARP1 and NF-κB pathway proteins in predicting the postoperative metastasis of breast cancer. A nested case‒control study was performed. Immunohistochemical methods were used to detect the expression of these genes in patients. ROC curves were used to analyse the predictive effect of these factors on distant metastasis. The COX model was used to evaluate the effects of PARP1 and TNF-α on distant metastasis. The results showed that the expression levels of PARP1, IKKß, p50, p65 and TNF-α were significantly increased in the metastasis group (P < 0.001). PARP1 was correlated with IKKß, p50, p65 and TNF-α proteins (P < 0.001). There was a correlation between IKKß, p50, p65 and TNF-α proteins (P < 0.001). ROC curve analysis showed that immunohistochemical scores for PARP1 of > 6, IKKß of > 4, p65 of > 4, p50 of > 2, and TNF-α of > 4 had value in predicting distant metastasis (SePARP1 = 78.35%, SpPARP1 = 79.38%, AUCPARP1 = 0.843; Sep50 = 64.95%, Spp50 = 70.10%, AUCp50 = 0.709; SeTNF-α = 60.82%, SpTNF-α = 69.07%, AUCTNF-α = 0.6884). Cox regression analysis showed that high expression levels of PARP1 and TNF-α were a risk factor for distant metastasis after breast cancer surgery (RRPARP1 = 4.092, 95% CI 2.475-6.766, P < 0.001; RRTNF-α = 1.825, 95% CI 1.189-2.799, P = 0.006). Taken together, PARP1 > 6, p50 > 2, and TNF-α > 4 have a certain value in predicting breast cancer metastasis, and the predictive value is better when they are combined for diagnosis (Secombine = 97.94%, Spcombine = 71.13%).

Transcription factor RELA promotes hepatocellular carcinoma progression by promoting the transcription of m6A modulator METTL3.

OBJECTIVE: To explore the biological function of RELA proto-oncogene, NF-kB subunit (RELA) in hepatocellular carcinoma (HCC) progression, and its potential regulatory effects on the regulators of m6A modification. METHODS AND MATERIALS: GEPIA, UALCAN and Human Protein Atlas databases were applied to analyze the expression characteristics of RELA in HCC tissues and non-cancer liver tissues, and its relationship with clinicopathologic indicators and prognosis. Quantitative real-time PCR (qRT-PCR) was used to examine the expression level of RELA mRNA in HCC cells. Cell counting kit-8 (CCK-8) assay, EdU assay and flow cytometry were used to examine cell growth and apoptosis. PROMO database was applied to predict the binding sequence between RELA and methyltransferase like protein 3 (METTL3) promoter region, and this prediction was verified by dual luciferase reporter gene experiment and chromatin immunoprecipitation assay. The effect of RELA on METTL3 expression was examined by Western blot and qRT-PCT, and the regulatory effects of RELA on the other m6A regulators were evaluated by qRT-PCR. RESULTS: RELA was highly expressed in HCC tissues and cell lines, and was closely associated with adverse clinicopathologic indicators and poor prognosis of patients. Overexpression of RELA promoted the growth of HCC cells and inhibited apoptosis; Knocking down RELA had the opposite effects. Overexpression of RELA promoted METTL3 transcription. Knockdown or overexpression of METTL3 reversed the effects of overexpression or knockdown of RELA on HCC cell growth and apoptosis, respectively. RELA also promoted the expression of a series of m6A regulators at mRNA expression level in HCC cell lines. CONCLUSION: RELA promotes the transcription of METTL3 by binding to METTL3 promoter region, thus promoting the malignancy of HCC cells. This study suggests NF-κB signaling contributes the dysregulation of m6A modification in HCC tumorigenesis.

DDX6 is involved in the pathogenesis of inflammatory diseases via NF-κB activation.

The IL-6 amplifier was originally discovered as a mechanism for the enhanced activation of NF-κB in non-immune cells. In the IL-6 amplifier, IL-6-STAT3 and NF-κB stimulation is followed by an excessive production of IL-6, chemokines, and growth factors to develop chronic inflammation preceding the development of inflammatory diseases. Previously, using a shRNA-mediated genome-wide screening, we found that DEAD-Box Helicase 6 (DDX6) is a candidate positive regulator of the amplifier. Here, we investigate whether DDX6 is involved in the pathogenesis of inflammatory diseases via the IL-6 amplifier. We found that DDX6-silencing in non-immune cells suppressed the NF-κB pathway and inhibited activation of the IL-6 amplifier, while the forced expression of DDX6 enhanced NF-κB promoter activity independent of the RNA helicase activity of DDX6. The imiquimod-mediated dermatitis model was suppressed by the siRNA-mediated gene downregulation of DDX6. Furthermore, silencing DDX6 significantly reduced the TNF-α-induced phosphorylation of p65/RelA and IκBα, nuclear localization of p65, and the protein levels of IκBα. Mechanistically, DDX6 is strongly associated with p65 and IκBα, but not TRADD, RIP, or TRAF2, suggesting a novel function of DDX6 as an adaptor protein in the NF-κB pathway. Thus, our findings demonstrate a possible role of DDX6 beyond RNA metabolism and suggest DDX6 is a therapeutic target for inflammatory diseases.

Silent information regulator 2 deficiency exacerbates chronic cold exposure-induced colonic injury and p65 activation in mice.

Cold is a common stressor that threatens colonic health by affecting internal homeostasis. From the literature, Silent information regulator 2 (SIRT2) may have important roles during cold stress, but this conjecture requires investigation. To address this knowledge gap, we investigated the effects of SIRT2 on colonic injury in chronically cold-exposure mice. In a previous study, we showed that SIRT2 regulated p65 activation after cold exposure. In the current study, mice were exposed to 4 °C for 3 h/day for 3 weeks to simulate a chronic cold exposure environment. Chronic cold exposure shortened colon length, disrupted tight junctions in colonic epithelial tissue, and disordered colonic flora. Chronic cold exposure also increased p65 acetylation levels, promoted nuclear factor (NF)-κB activation, and increased the expression of its downstream pro-inflammatory factors, while SIRT2 knockdown aggravated the consequences of tissue structure disruption and increased inflammatory factors brought about by chronic cold exposure to some extent, but could alleviate the downregulation of colonic tight junction-related proteins to some extent. We also observed direct SIRT2 regulatory effects toward p65, and in Caco-2 cells treated with lipopolysaccharide (LPS), SIRT2 knockdown increased p65 acetylation levels and pro-inflammatory factor expression, while SIRT2 overexpression reversed these phenomena. Therefore, SIRT2 deletion exacerbated chronic cold exposure-induced colonic injury and p65 activation in mice. Mechanistically, p65 modification by SIRT2 via deacetylation may affect NF-κB signaling. These findings suggest that SIRT2 is a key target of colonic health maintenance under chronic cold exposure conditions.

Meet the authors: Vasty Osei Amponsa and Kylie J. Walters.

We talk to Vasty Osei Amponsa and Kylie J. Walters about their paper "hRpn13 shapes the proteome and transcriptome through epigenetic factors HDAC8, PADI4, and transcription factor NF-κB p50", their journeys across continents leading them to the NCI, and how Kylie tries to foster curiosity and a sense of belonging in her lab.

Gold Nanoparticles Downregulate IL-6 Expression/Production by Upregulating microRNA-26a-5p and Deactivating the RelA and NF-κBp50 Transcription Pathways in Activated Breast Cancer Cells.

MicroRNAs (miRNAs) are involved in the modulation of pathogenic genes by binding to their mRNA sequences' 3' untranslated regions (3'UTR). Interleukin-6 (IL-6) is known to promote cancer progression and treatment resistance. In this study, we aimed to explore the therapeutic effects of gold nanoparticles (GNP) against IL-6 overexpression and the modulation of miRNA-26a-5p in breast cancer (BC) cells. GNP were synthesized using the trisodium citrate method and characterized through UV-Vis spectroscopy, dynamic light scattering (DLS), and transmission electron microscopy (TEM). To predict the binding of miR-26a-5p in the IL-6 mRNA's 3'UTR, we utilized bioinformatics algorithms. Luciferase reporter clone assays and anti-miRNA-26a-5p transfection were employed to validate the binding of miR26a-5p in the IL-6 mRNA's 3'UTR. The activity of RelA and NF-κBp50 was assessed and confirmed using Bay 11-7082. The synthesized GNP were spherical with a mean size of 28.3 nm, exhibiting high stability, and were suitable for BC cell treatment. We found that miR-26a-5p directly regulated IL-6 overexpression in MCF-7 cells activated with PMA. Treatment of MCF-7 cells with GNP resulted in the inhibition of IL-6 overexpression and secretion through the increase of miR26a-5p. Furthermore, GNP deactivated NF-κBp65/NF-κBp50 transcription activity. The newly engineered GNP demonstrated safety and showed promise as a therapeutic approach for reducing IL-6 overexpression. The GNP suppressed IL-6 overexpression and secretion by deactivating NF-κBp65/NF-κBp50 transcription activity and upregulating miR-26a-5p expression in activated BC cells. These findings suggest that GNP have potential as a therapeutic intervention for BC by targeting IL-6 expression and associated pathways.

Alleviating effect of Nexrutine on mucosal inflammation in mice with ulcerative colitis: Involvement of the RELA suppression.

BACKGROUND: Nexrutine is an herbal extract derived from Phellodendron amurense, known for its anti-inflammatory, antidiarrheal, and hemostatic properties. However, its effect on ulcerative colitis (UC) remains unclear. METHODS: A mouse model of UC was induced by 3% dextran sulfate sodium, while human colonic epithelial cells NCM-460 were exposed to lipopolysaccharide. Both models were treated with Nexrutine at 300 or 600 mg/kg, with Mesalazine applied as a positive control regimen. The disease activity index (DAI) of mice was calculated, and the pathological injury scores were assessed through hematoxylin and eosin staining. The viability of NCM-460 cells was determined using the CCK-8 method. Inflammatory cytokines were detected using ELISA kits. Expression of mucin 3 (MUC3), Claudin-1, and tight junction protein (ZO-1) was detected to analyze mucosal barrier integrity. Target genes of Nexrutine were predicted using bioinformatics tools. Expression of RELA proto-oncogene (RELA) was analyzed using qPCR and western blot assays. RESULTS: The Nexrutine treatments significantly alleviated DAI of mice, mitigated pathological changes in their colon tissues, decreased the production of pro-inflammatory cytokines, enhanced the barrier integrity-related proteins, and increased NCM-460 cell viability in vitro. RELA, identified as a target gene of Nexrutine, showed elevated levels in UC models but was substantially suppressed by Nexrutine treatment. Adenovirus-mediated RELA upregulation in mice or the overexpression plasmid of RELA in cells counteracted the effects of Nexrutine treatments, exacerbating UC-related symptoms. CONCLUSION: This study demonstrates that Nexrutine alleviates inflammatory mucosal barrier damage in UC by suppressing RELA transcription.

GSK3ß/NF-κB -dependent transcriptional regulation of homeostatic hepatocyte Tnf production.

Maintenance of hepatocyte homeostasis plays an important role in mediating the pathogenesis of many diseases. A growing body of literature has established a critical role played by tumor necrosis factor-α (TNFα) in maintaining hepatocyte homeostasis; however, the transcriptional mechanisms underlying constitutive Tnf expression are unknown. Whole liver fractions and primary hepatocytes from adult control C57BL/6 mice and the murine hepatocyte cell line AML12 were assessed for constitutive Tnf expression. Impacts of glycogen synthase kinase-3 ß (GSK3ß) and nuclear factor κB (NF-κB) inhibition on constitutive Tnf expression were assessed in AML12 cells. Finally, AML12 cell proliferation following GSK3ß and NF-κB inhibition was evaluated. Constitutive Tnf gene expression is present in whole liver, primary hepatocytes, and cultured AML12 hepatocytes. Cytokine-induced Tnf gene expression is regulated by NF-κB activation. Pharmacological inhibition of GSK3ß resulted in a time- and dose-dependent inhibition of Tnf gene expression. GSK3ß inhibition decreased nuclear levels of the NF-κB subunits p65 and p50. We determined that NF-κB transcription factor subunit p65 binds to consensus sequence elements present in the murine TNFα promoter and inhibition of GSK3ß decreases binding and subsequent Tnf expression. Finally, AML12 cell growth was significantly reduced following GSK3ß and NF-κB inhibition. These results demonstrate that GSK3ß and NF-κB are essential for mediating Tnf expression and constitutive hepatocyte cell growth. These findings add to a growing body of literature on TNFα mediated hepatocyte homeostasis and identify novel molecular mechanisms involved in mediating response to various disease states in the liver.NEW & NOTEWORTHY Maintenance of hepatocyte homeostasis plays an important role in controlling the pathogenesis of many diseases. Our findings add to a growing body of literature on tumor necrosis factor-α (TNFα)-mediated hepatocyte homeostasis and identify novel molecular mechanisms involved in regulating this response.

Insulin-like growth factor 2 mRNA-binding protein 3 enhanced melanoma migration through regulation of AKT1 and RELA expression.

IMP-3 expression is a poor prognostic factor of melanomas and it promotes melanoma cell migration and invasion by a pathway modulating HMGA2 mRNA expression. We tried to identify other putative targets of IMP-3. We identified putative IMP-3-binding RNAs, including AKT1, MAPK3, RB1 and RELA, by RNA immunoprecipitation coupled with next-generation sequencing. IMP-3 overexpression increased AKT and RELA levels in MeWo cells. siRNAs against AKT1 and RELA inhibited MeWo/Full-length IMP-3 cell migration. IMP-3 knockdown of A2058 cells decreased AKT1 and RELA expression and lowered migration ability. Co-transfection of A2058 cells with AKT1- or RELA-expressing plasmids with IMP-3 siRNA restored the inhibitory effects of IMP-3 knockdown on migration. HMGA2 did not influence AKT1 and RELA expression in melanoma cells. Human melanoma samples with high IMP-3 levels also showed high HMGA2, AKT1 and RELA expression. Our results show that IMP-3 enhances melanoma cell migration through the regulation of the AKT1 and RELA axis.

Gene-to-gene coordinated regulation of transcription and alternative splicing by 3D chromatin remodeling upon NF-κB activation.

The NF-κB protein p65/RelA plays a pivotal role in coordinating gene expression in response to diverse stimuli, including viral infections. At the chromatin level, p65/RelA regulates gene transcription and alternative splicing through promoter enrichment and genomic exon occupancy, respectively. The intricate ways in which p65/RelA simultaneously governs these functions across various genes remain to be fully elucidated. In this study, we employed the HTLV-1 Tax oncoprotein, a potent activator of NF-κB, to investigate its influence on the three-dimensional organization of the genome, a key factor in gene regulation. We discovered that Tax restructures the 3D genomic landscape, bringing together genes based on their regulation and splicing patterns. Notably, we found that the Tax-induced gene-gene contact between the two master genes NFKBIA and RELA is associated with their respective changes in gene expression and alternative splicing. Through dCas9-mediated approaches, we demonstrated that NFKBIA-RELA interaction is required for alternative splicing regulation and is caused by an intragenic enrichment of p65/RelA on RELA. Our findings shed light on new regulatory mechanisms upon HTLV-1 Tax and underscore the integral role of p65/RelA in coordinated regulation of NF-κB-responsive genes at both transcriptional and splicing levels in the context of the 3D genome.

The NF-κB pathway is essential for coordinating gene expression in response to various stimuli, including viral infections. Most studies have focused on the role of NF-κB in transcriptional regulation. In the present study, the impact of the potent NF-κB activator HTLV-1 Tax oncoprotein on the three-dimensional organization of the genome was investigated. Tax-mediated NF-κB activation was found to restructure the 3D genomic landscape in cells and to bring genes together in multigene complexes that are coordinately regulated either transcriptionally or through alternative splicing by NF-κB. Induced coordinate changes in transcription and alternative splicing included the two master genes of NF-κB pathway NFKBIA and RELA. The findings have significant implications for understanding cell fate determination and disease development associated with HTLV-1 infection, as well as chronic NF-κB activation in various human inflammatory diseases and cancer.

Integrated Network Pharmacology and Experimental Approach to Investigate the Protective Effect of Jin Gu Lian Capsule on Rheumatoid Arthritis by Inhibiting Inflammation via IL-17/NF-κB Pathway.

Purpose: This study aimed to investigate the main pharmacological action and underlying mechanisms of Jin Gu Lian Capsule (JGL) against rheumatoid arthritis (RA) based on network pharmacology and experimental verification. Methods: Network pharmacology approaches were performed to explore the core active compounds of JGL, key therapeutic targets, and signaling pathways. Molecular docking was used to predict the binding affinity of compounds with targets. In vivo experiments were undertaken to validate the findings from network analysis. Results: A total of 52 targets were identified as candidate JGL targets for RA. Sixteen ingredients were identified as the core active compounds, including, quercetin, myricetin, salidroside, etc. Interleukin-1 beta (IL1B), transcription factor AP-1 (JUN), growth-regulated alpha protein (CXCL1), C-X-C motif chemokine (CXCL)3, CXCL2, signal transducer and activator of transcription 1 (STAT1), prostaglandin G/H synthase 2 (PTGS2), matrix metalloproteinase (MMP)1, inhibitor of nuclear factor kappa-B kinase subunit beta (IKBKB) and transcription factor p65 (RELA) were obtained as the key therapeutic targets. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that the efficacy of JGL was functionally involved in regulating immune-mediated inflammation, in which IL-17/NF-κB signaling was recommended as one of the main pathways. Molecular docking suggested that the core active compounds bound strongly to their respective targets. Experimentally, JGL treatment mitigated inflammation, showed analgesic activity, and ameliorated collagen-induced arthritis. Enzyme-linked immunosorbent assay showed that JGL effectively reduced the serum levels of cytokines, chemokines, and MMPs. Immunohistochemistry staining showed that JGL markedly reduced the expression of the targets in IL-17/NF-κB pathway including IL-17A, IL-17RA, NF-κB p65, C-X-C motif ligand 2, MMP1 and MMP13. Conclusion: This investigation provided evidence that JGL may alleviate RA symptoms by partially inhibiting the immune-mediated inflammation via IL-17/NF-κB pathway.