RelA P65 Research Articles

NF-κB Transcription Factors: Their Distribution, Family Expansion, Structural Conservation, and Evolution in Animals.

The Nuclear Factor Kappa B (NF-κB) transcription factor family consists of five members: RelA (p65), RelB, c-Rel, p50 (p105/NF-κB1), and p52 (p100/NF-κB2). This family is considered a master regulator of classical biochemical pathways such as inflammation, immunity, cell proliferation, and cell death. The proteins in this family have a conserved Rel homology domain (RHD) with the following subdomains: DNA binding domain (RHD-DBD) and dimerization domain (RHD-DD). Despite the importance of the NF-κB family in biology, there is a lack of information with respect to their distribution patterns, evolution, and structural conservation concerning domains and subdomains in animals. This study aims to address this critical gap regarding NF-κB proteins. A comprehensive analysis of NF-κB family proteins revealed their distinct distribution in animals, with differences in protein sizes, conserved domains, and subdomains (RHD-DBD and RHD-DD). For the first time, NF-κB proteins with multiple RHD-DBDs and RHD-DDs have been identified, and in some cases, this is due to subdomain duplication. The presence of RelA/p65 exclusively in vertebrates shows that innate immunity originated in fishes, followed by amphibians, reptiles, aves, and mammals. Phylogenetic analysis showed that NF-κB family proteins grouped according to animal groups, signifying structural conservation after speciation. The evolutionary analysis of RHDs suggests that NF-κB family members p50/p105 and c-Rel may have been the first to emerge in arthropod ancestors, followed by RelB, RelA, and p52/p100.

G-Protein Coupled Receptor 1 Is Involved in Tetrachlorobisphenol A-Induced Inflammatory Response in Jurkat Cells.

Estrogens can affect the immune inflammatory response through estrogen receptor alpha (ERα), but the specific role of estrogen member receptor G-protein coupled receptor 1 (GPER1) in this process remains unclear. In this study, we evaluated the effects of tetrachlorobisphenol A (TCBPA), which has estrogen activity, on immune inflammatory-related indicators of Jurkat cells, as well as investigated the role of GPER1 in these effects. The results showed that TCBPA at lower concentrations significantly promoted the viability of Jurkat cells, whereas higher concentrations decreased cell viability. TCBPA at concentrations ranging from 1 to 25 μM increased the intracellular reactive oxygen species (ROS) levels. Additionally, treatment with 10 μM TCBPA increased the protein expression of ERα and GPER1, elevated the phosphorylation of protein kinase B (p-Akt), and upregulated the mRNA levels of GPER1, Akt, and phosphoinositide 3-kinase (PI3K) genes. Treatment with 10 μM TCBPA also upregulated the protein or gene expression of pro-inflammatory cytokines, such as interleukins (IL1β, IL2, IL6, IL8, IL12α) and tumor necrosis factor alpha (TNFα) in Jurkat cells. Furthermore, pretreatment with a GPER1 inhibitor G15 significantly reduced the mRNA levels of Akt induced by 10 μM TCBPA. Moreover, the upregulation of mRNA expression of RelA (p65), TNFα, IL6, IL8, and IL12α induced by 10 μM TCBPA was also significantly attenuated after G15 pretreatment. These findings suggest that TCBPA upregulates the expression of genes related to inflammatory responses by activating the GPER1-mediated PI3K/Akt signaling pathway. This study provides new insights into the mechanism of TCBPA-induced inflammatory response.

Saliva IL6 Upregulation As a Potential Indicator of Tobacco-Smoke Associated OSCC and Disease Progression and the Association of Saliva RELA(p65) Upregulation With OSCC Local Invasion in Non-Smokers

Saliva IL6 Upregulation As a Potential Indicator of Tobacco-Smoke Associated OSCC and Disease Progression and the Association of Saliva RELA(p65) Upregulation With OSCC Local Invasion in Non-Smokers

A positive feedback loop between PLD1 and NF-κB signaling promotes tumorigenesis of nasopharyngeal carcinoma

Phospholipase D (PLD) lipid-signaling enzyme superfamily has been widely implicated in various human malignancies, but its role and underlying mechanism remain unclear in nasopharyngeal carcinoma (NPC). Here, we analyze the expressions of 6 PLD family members between 87 NPC and 10 control samples through transcriptome analysis. Our findings reveal a notable upregulation of PLD1 in both NPC tumors and cell lines, correlating with worse disease-free and overall survival in NPC patients. Functional assays further elucidate the oncogenic role of PLD1, demonstrating its pivotal promotion of critical tumorigenic processes such as cell proliferation and migration in vitro, as well as tumor growth in vivo. Notably, our study uncovers a positive feedback loop between PLD1 and the NF-κB signaling pathway to render NPC progression. Specifically, PLD1 enhances NF-κB activity by facilitating the phosphorylation and nuclear translocation of RELA, which in turn binds to the promoter of PLD1, augmenting its expression. Moreover, RELA overexpression markedly rescues the inhibitory effects in PLD1-depleted NPC cells. Importantly, the application of the PLD1 inhibitor, VU0155069, substantially inhibits NPC tumorigenesis in a patient-derived xenograft model. Together, our findings identify PLD1/NF-κB signaling as a positive feedback loop with promising therapeutic and prognostic potential in NPC.

Hydrogen Peroxide Induces Ethanol-inducible CYP2E1 via the NFkB-classical Pathway: CYP2E1 mRNA Levels are not High in Alcoholic Hepatitis.

The aim of the present study is to elucidate the mechanism of CYP2E1 induction as a causative factor of alcoholic hepatitis (AH) and its relationship with inflammation. Chronic alcohol consumption induces CYP2E1, which is involved in the development of alcoholic hepatitis (AH). However, the mechanisms underlying the induction of CYP2E1 by alcohol remain unclear. Therefore, we herein investigated the induction of drug-metabolizing enzymes, particularly CYP2E1, by hydrogen peroxide (H2O2), the concentration of which is elevated under inflammatory conditions. The mechanisms underlying the induction of CYP2E1 by H2O2 were examined with a focus on Keap1, a target factor of H2O2. We assessed changes in the expression of drug-metabolizing enzymes in the human hepatoma cell line, Hep3B, following treatment with H2O2, and evaluated changes in the expression of the NFkB-related factor RelA(p65) after the knockdown of Keap1, a regulator of Nrf2 expression by reactive oxygen species. We also performed a promoter analysis using the upstream region of the CYP2E1 gene. We herein used the GSE89632 series for non-alcoholic hepatitis (NASH) and the GSE28619 series for AH. The induction of CYP2E1 by H2O2 was significantly stronger than that of other drugmetabolizing enzymes. On the other hand, the knockdown of Keap1, a target of H2O2, markedly increased RelA(p65), an NFkB factor. Furthermore, the overexpression of RelA(p65) strongly induced the expression of CYP2E1. Four candidate p65-binding sequences were identified upstream of the CYP2E1 gene, and promoter activity assays showed that the third sequence was responsive to the overexpression of RelA(p65). We used the GSE89632 series for NASH and the GSE28619 series for AH in the present study. The expression of CYP2E1 mRNA in the liver was significantly lower in AH patients than in HC patients, but was similar in HC patients and NASH patients. We herein demonstrated that the expression of CYP2E1 was induced by H2O2. The overexpression of RelA(p65) also induced CYP2E1 mRNA expression, whereas H2O2 did not after the knockdown of RelA. These results suggest that H2O2 acts on Keap1 to upregulate RelA (p65) in the NFkB system. One of the mechanisms underlying the induction of CYP2E1 was dependent on the H2O2-Keap1-RelA axis. The results of the database analysis revealed that the expression of CYP2E1 in the liver was significantly lower in AHH patients than in NASH patients, suggesting that CYP2E1 is not the main cause of AH; however, CYP2E1 may exacerbate the pathogenesis of AH.

The transcription factor BMI1 increases hypoxic signaling in oral cavity epithelia

The tongue epithelium is maintained by a proliferative basal layer. This layer contains long-lived stem cells (SCs), which produce progeny cells that move up to the surface as they differentiate. B-lymphoma Mo-MLV insertion region 1 (BMI1), a protein in mammalian Polycomb Repressive Complex 1 (PRC1) and a biomarker of oral squamous cell carcinoma, is expressed in almost all basal epithelial SCs of the tongue, and single, Bmi1-labelled SCs give rise to cells in all epithelial layers. We previously developed a transgenic mouse model (KrTB) containing a doxycycline- (dox) controlled, Tet-responsive element system to selectively overexpress Bmi1 in the tongue basal epithelial SCs. Here, we used this model to assess BMI1 actions in tongue epithelia. Genome-wide transcriptomics revealed increased levels of transcripts involved in the cellular response to hypoxia in Bmi1-overexpressing (KrTB+DOX) oral epithelia even though these mice were not subjected to hypoxia conditions. Ectopic Bmi1 expression in tongue epithelia increased the levels of hypoxia inducible factor-1 alpha (HIF1α) and HIF1α targets linked to metabolic reprogramming during hypoxia. We used chromatin immunoprecipitation (ChIP) to demonstrate that Bmi1 associates with the promoters of HIF1A and HIF1A-activator RELA (p65) in tongue epithelia. We also detected increased SC proliferation and oxidative stress in Bmi1-overexpressing tongue epithelia. Finally, using a human oral keratinocyte line (OKF6-TERT1R), we showed that ectopic BMI1 overexpression decreases the oxygen consumption rate while increasing the extracellular acidification rate, indicative of elevated glycolysis. Thus, our data demonstrate that high BMI1 expression drives hypoxic signaling, including metabolic reprogramming, in normal oral cavity epithelia.

296 Towards Nose-to-brain Inhibition of NFB: Activity of a Novel p65 Inhibitor in Glioblastoma and Feasibility of Intranasal Delivery With a Near-infrared Fluorescent HMC-Chitosan Nanoparticle

INTRODUCTION: Constitutive NFκB inflammatory signaling is a driving force of tumor progression, immune evasion, and therapeutic resistance in glioblastoma (GBM). CRL1101 is a novel small molecule inhibitor of RelA (p65), a transcription factor of NFκB-mediated gene expression. Previously, we have shown that heptamethine carbocyanine (HMC) conjugated nanoparticles target GBM tumor sites via organic anion transporter proteins. The activity of CRL1101 in GBM has not been explored. Poor blood-brain barrier (BBB) penetration and theoretical toxicities related to p65 inhibition pose challenges to its implementation in vivo. METHODS: GBM transcriptomic analysis was performed using Gene Profiling Interactive Analysis. In vitro cytotoxicity and migration assays were performed in U87 and GL261. Western blot was used to confirm the mechanism of CRL1101. Quantitative PCR was used to assess the relative expression of NFκB-regulated genes. HMC-chitosan-CRL1101 nanoparticles were synthesized by cross-linking chitosan with HMC and their uptake was visualized using immunocytochemistry. For survival analysis, we implanted GL261 cells into immunocompetent mice and treated intranasally with HMC-chitosan-CRL1101. RESULTS: RelA is overexpressed in GBM relative to normal brain tissue. CRL1101 alone effectively kills U87 cells (IC50 = 16.0 uM) and prevents migration in vitro (p < 0.0001) by disabling the nuclear translocation of RelA. Treatment with CRL1101 was associated with a decrease in IL-8 (p = 0.01) and PD-L1 (p = 0.44) expression. HMC-chitosan-CRL1101 demonstrated an IC50 of 25.23 uM in U87. Near-infrared imaging confirmed intranasal delivery of HMC-chitosan-CRL1101 across the BBB in vivo. CONCLUSIONS: RelA-targeted NFκB inhibition with CRL1101 is a promising immunotherapeutic strategy in GBM. Intranasal nanoparticle delivery is a practical route to bypass the BBB and minimize systemic toxicities in mouse models of GBM, with expected survival benefit in vivo.

Curcumin alleviates Alzheimer’s disease by inhibiting inflammatory response, oxidative stress and activating the AMPK pathway

BackgroundAlzheimer’s disease (AD) is a common degenerative brain disorder with limited therapeutic options. Curcumin (Cur) exhibits neuroprotective function in many diseases. We aimed to explore the role and mechanism of Cur in AD. Materials and MethodsFirstly, we established AD mice by injecting amyloid-β1–42 (Aβ1–42) solution into the hippocampus. Then, the AD mice received 150 mg/kg/d Cur for 10 consecutive days. The Morris water maze test was conducted to evaluate the cognitive function of the mice by hidden platform training and probe trials. To assess the spatial memory of the mice, spontaneous alternation behavior, the number of crossing the novel arm and the time spent in the novel arm during the Y-maze test was recorded. Hematoxylin and eosin (H&E) staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNAL) assay were performed to assess the pathological damage and apoptosis of brain tissues. The number of damaged neurons was inspected by Nissl staining. Immunohistochemical staining was then performed to detect Aβ1–42 deposition. The levels of tumor necrosis factor-α (TNF-a), interleukin-6 (IL-6) and interleukin-1β (IL-1β) in serum and hippocampus, the contents of super oxide dismutase (SOD) and malondialdehyde (MDA) in brain tissues were assessed by enzyme-linked immunosorbent assay (ELISA). Additionally, B-cell lymphoma-2 (Bcl-2), Bcl-2 associated X protein (Bax), RelA (p65) protein expressions and Adenosine 5′-monophosphate-activated protein kinase (AMPK) phosphorylation were tested using Western blot. ResultsCur not only improved cognitive function and spatial memory, but also alleviated the pathological damage and apoptosis of brain tissues for AD mice. Meanwhile, upon Cur treatment, the number of damaged neurons in AD mice was decreased, the level of Aβ1–42 in AD mice was significantly decreased. Furthermore, the AD mice treated with Cur exhibited lower TNF-a, IL-6, IL-1β and MDA levels and a higher SOD content. Besides, Cur also downregulated p65 expression and upregulated AMPK phosphorylation. ConclusionCur may improve AD via suppressing the inflammatory response, oxidative stress and activating the AMPK pathway, suggesting that Cur may be a potential drug for AD.

DDX5 (p68) orchestrates β-catenin, RelA and SP1 mediated MGMT gene expression in human colon cancer cells: Implication in TMZ chemoresistance

DDX5 (p68) upregulation has been linked with various cancers of different origins, especially Colon Adenocarcinomas. Similarly, across cancers, MGMT has been identified as the major contributor of chemoresistance against DNA alkylating agents like Temozolomide (TMZ). TMZ is an emerging potent chemotherapeutic agent across cancers under the arena of drug repurposing. Recent studies have established that patients with open MGMT promoters are prone to be innately resistant or acquire resistance against TMZ compared to its closed conformation. However, not much is known about the transcriptional regulation of MGMT gene in the context of colon cancer. This necessitates studying MGMT gene regulation which directly impacts the cellular potential to develop chemoresistance against alkylating agents. Our study aims to uncover an unidentified mechanism of DDX5-mediated MGMT gene regulation. Experimentally, we found that both mRNA and protein expression levels of MGMT were elevated in response to p68 overexpression in multiple human colon cancer cell lines and vice-versa. Since p68 cannot directly interact with the MGMT promoter, transcription factors viz., β-catenin, RelA (p65) and SP1 were also studied as reported contributors. Through co-immunoprecipitation and GST-pull-down studies, p68 was established as an interacting partner of SP1 in addition to β-catenin and NF-κB (p50-p65). Mechanistically, luciferase reporter and chromatin-immunoprecipitation assays demonstrated that p68 interacts with the MGMT promoter via TCF4-LEF, RelA and SP1 sites to enhance its transcription. To the best of our knowledge, this is the first report of p68 as a transcriptional co-activator of MGMT promoter and our study identifies p68 as a novel and master regulator of MGMT gene expression.

H3K18 lactylation of senescent microglia potentiates brain aging and Alzheimer's disease through the NFκB signaling pathway

Cellular senescence serves as a fundamental and underlying activity that drives the aging process, and it is intricately associated with numerous age-related diseases, including Alzheimer's disease (AD), a neurodegenerative aging-related disorder characterized by progressive cognitive impairment. Although increasing evidence suggests that senescent microglia play a role in the pathogenesis of AD, their exact role remains unclear. In this study, we quantified the levels of lactic acid in senescent microglia, and hippocampus tissues of naturally aged mice and AD mice models (FAD4T and APP/PS1). We found lactic acid levels were significantly elevated in these cells and tissues compared to their corresponding counterparts, which increased the level of pan histone lysine lactylation (Kla). We aslo identified all histone Kla sites in senescent microglia, and found that both the H3K18 lactylation (H3K18la) and Pan-Kla were significantly up-regulated in senescent microglia and hippocampus tissues of naturally aged mice and AD modeling mice. We demonstrated that enhanced H3K18la directly stimulates the NFκB signaling pathway by increasing binding to the promoter of Rela (p65) and NFκB1(p50), thereby upregulating senescence-associated secretory phenotype (SASP) components IL-6 and IL-8. Our study provides novel insights into the physiological function of Kla and the epigenetic regulatory mechanism that regulates brain aging and AD. Specifically, we have identified the H3K18la/NFκB axis as a critical player in this process by modulating IL-6 and IL-8. Targeting this axis may be a potential therapeutic strategy for delaying aging and AD by blunting SASP.

HOXA1 participates in VSMC-to-macrophage-like cell transformation via regulation of NF-κB p65 and KLF4: a potential mechanism of atherosclerosis pathogenesis

BackgroundMacrophage-like transformation of vascular smooth muscle cells (VSMCs) is a risk factor of atherosclerosis (AS) progression. Transcription factor homeobox A1 (HOXA1) plays functional roles in differentiation and development. This study aims to explore the role of HOXA1 in VSMC transformation, thereby providing evidence for the potential mechanism of AS pathogenesis.MethodsHigh fat diet (HFD)-fed apolipoprotein E knockout (ApoE−/−) mice were applied as an in vivo model to imitate AS, while 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POV-PC)-treated VSMCs were applied as an in vitro model. Recombinant adeno-associated-virus-1 (AAV-1) vectors that express short-hairpin RNAs targeting HOXA1, herein referred as AAV1-shHOXA1, were generated for the loss-of-function experiments throughout the study.ResultsIn the aortic root of AS mice, lipid deposition was severer and HOXA1 expression was higher than the wide-type mice fed with normal diet or HFD. Silencing of HOXA1 inhibited the AS-induced weight gain, inflammatory response, serum and liver lipid metabolism disorder and atherosclerotic plaque formation. Besides, lesions from AS mice with HOXA1 knockdown showed less trans-differentiation of VSMCs to macrophage-like cells, along with a suppression of krüppel-like factor 4 (KLF4) and nuclear factor (NF)-κB RelA (p65) expression. In vitro experiments consistently confirmed that HOXA1 knockdown suppressed lipid accumulation, VSMC-to-macrophage phenotypic switch and inflammation in POV-PC-treated VSMCs. Mechanism investigations further illustrated that HOXA1 transcriptionally activated RelA and KLF4 to participate in the pathological manifestations of VSMCs.ConclusionsHOXA1 participates in AS progression by regulating VSMCs plasticity via regulation of NF-κB p65 and KLF4. HOXA1 has the potential to be a biomarker or therapeutic target for AS.

EPEN-01. ZFTA-RELA FUSION PROGRAMS CONVERGE ON YAP PATHWAY ACTIVATION IN EPENDYMOMA

Abstract Approximately two thirds of supratentorial ependymoma are driven by the ZFTA-RELA gene fusion. ZFTA is a recently described gene with a role in DNA binding and transcriptional regulation, while RELA (p65) is a known principal mediator of the canonical NFkB pathway, a pathway activated many human malignancies. The underlying mechanism of NFkB pathway protein interaction with the ZFTA-RELA fusion to drive tumorgenesis has not yet been described. In this study, we utilized our autochthonous mouse tumor model using in utero electroporation (IUE) of the embryonic mouse brain to explore these questions. Using rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME) targeting HA-tagged ZFTA-RELA fusion in our IUE model, we demonstrate that both canonical and non-canonical NFkB proteins interact with ZFTA-RELA protein in ZFTA-RELA fusion ependymoma. Focusing on select key NFkB proteins, we performed chromatin immunoprecipitation sequencing (ChIP-Seq) targeting p50/NFkB1, a protein that partners with RELA/p65 in the canonical NFkB pathway, revealing that p50/NFkB1 regulatory programs define active genes that are both distinct from and shared with the ZFTA-RELA fusion protein. Functionally, we performed CRISPR-CAS9 knock out (KO) of p50/NFkB1 and identified no phenotypic effect, as KO lines demonstrated no growth impairment. Furthermore, NFkB targeting drugs did not impair tumor growth. Given these findings, suggesting redundant transcriptional programing, we identified that ZFTA and RELA intersect with TEAD transcription factor programs to activate YAP signaling and that p50 localizes to YAP-TEAD pathway members. Targeting TEAD 1 and 4 with ChIP-Seq, we found that TEAD 1 and 4 co-regulate p50/NFkB1 and ZFTA-RELA target genes. Finally, we demonstrate YAP pathway activation across all supratentorial ependymoma, including ZFTA-RELA ependymoma. Our pathway analysis consistently converged on YAP/TEAD pathways, suggesting collaboration of ZFTA-RELA fusion with YAP/TEAD in transcriptional regulation for these tumors. Targeting YAP/TEAD may therefore represent a promising therapeutic strategy for ZFTA-RELA fusion ependymoma.

Integrated molecular modeling and dynamics approaches revealed potential natural inhibitors of NF-κB transcription factor as breast cancer therapeutics

Breast cancer is a silent killer malady among women and a serious economic burden in health care management. A case of breast cancer is diagnosed among women every 19 s, and every 74 s, a woman dies of breast cancer somewhere in the world. Despite the pop-up of progressive research, advanced treatment approaches, and preventive measures, breast cancer remains amplifying ailment. The nuclear factor kappa B (NF-κB) is a key transcription factor that links inflammation with cancer and is demonstrated as being involved in the tumorigenesis of breast cancer. The NF-κB transcription factor family in mammals consists of five proteins; c-Rel, RelA(p65), RelB, NF-κB1(p50), and NF-κB2(p52). The antitumor effect of NF-κB has also been explored in breast cancer, however, the actual treatment for breast cancer is yet to be discovered. This study is attributed to the identification of novel drug targets against breast cancer by targeting c-Rel, RelA(p65), RelB, NF-κB1(p50), and NF-κB2(p52) proteins. To identify the putative active compounds, a structure-based 3D pharmacophore model to the protein active site cavity was generated followed by virtual screening, molecular docking, and molecular dynamics (MD) simulation. Initially, a library of 45000 compounds were docked against the target protein and five compounds namely Z56811101, Z653426226, Z1097341967, Z92743432, and Z464101066 were selected for further analysis. The relative binding affinity of Z56811101, Z653426226, Z1097341967, Z92743432, and Z464101066 with NF-κB1 (p50), NF-κB2 (p52), RelA (p65), RelB, and c-Rel proteins were −6.8, −8, −7.0, −6.9, and −7.2 kcal/mol, respectively which remained stable throughout the simulations of 200 ns. Furthermore, all of these compounds depict maximum drug-like properties. Therefore, the proposed compounds can be a potential candidate for patients with breast cancer, but, experimental validation is needed to ensure their safety. Communicated by Ramaswamy H. Sarma

Double knock-in reporter mice: a novel tool to study age-associated NF-κB dynamics in primary microglia

Abstract Microglia are immune sentinels in the brain that are capable of orchestrating potent inflammatory responses in aging and neurodegenerative diseases like Alzheimer’s and Parkinson’s diseases. NF-κB signaling pathway is commonly recognized as a significant regulator of inflammation and aging. Mapping the spatiotemporal complexity of NF-κB signaling is crucial to understand its impact and function in vivo, but the lack of tools to directly monitor NF-κB protein components has hindered such efforts. Our lab has generated reporter mice with the endogenous RelA (p65) and c-Rel labeled with distinct fluorescent proteins and a double knock-in line with both labeled subunits. To understand how aging affects microglial function, we isolated primary microglia from young and old animals. We cultured them in vitro and validated by P2RY12 +, CD11b +and CD45 int. Interestingly, we observed two different microglial populations, one motile, and the other forming clusters. Microglia from aged animals showed a higher prevalence of motile, free-roaming microglia. We also observed these distinct microglial populations in brain slices from the mice ex-vivo. Live-cell imaging of NF-kB dynamics in primary microglia from young and old mice revealed a shift towards c-Rel in old brains. Further, we stained for Ki67 as a proliferation marker to functionally characterize these microglia populations. We observed only the free-roaming microglia to be proliferative in nature. Furthermore, we are investigating the senescence pattern between the two populations to better understand the molecular mechanisms associated with age-dependent and inflammation-derived changes in young and old brains.

Investigating the mechanism of DNA binding enhancement by the RelA transcription activation domain.

The p50/RelA heterodimer of the NF-κB family of transcription factors activates genes involved in inflammation and immunity in response to cellular stressors. Both p50 and RelA (p65) contain a Rel homology domain which, when dimerized, binds to DNA. In addition, RelA contains a 230-residue intrinsically disordered transcription activation domain (TAD). The RelA TAD interacts with co-activator proteins and was previously believed to function independently from the DNA-binding Rel homology domain. However, we found that the presence of the RelA TAD enhances the DNA binding affinity of the p50/RelA heterodimer: full length p50/RelA binds to DNA with higher affinity than the truncated Rel homology domain. The strongest binding enhancement was observed for DNA sequences that do not match the κB consensus motif, leading to an overall decrease in sequence specificity when the RelA TAD is intact. We found that a proline-rich region consisting of the first 100 residues of the TAD is sufficient for this binding enhancement. We investigated the sequence features necessary for this phenomenon and the effects of the RelA TAD on the structure and dynamics of the p50/RelA heterodimer.

The Involvement of Canonical NFκB Pathway in Megakaryocyte Differentiation Induction by Nanocurcumin.

Background: Megakaryopoiesis is characterized by progressive polyploidization and the expression of megakaryocytic markers. Numerous transcription factors and physiological signaling pathways regulate this phenomenon. Megakaryocyte differentiation induction in the K562 cell line and hematopoietic stem cells via nanocurcumin drug has been identified in our previous study. K562 cells are typical Chronic Myelogenous Leukemia (CML) cells that are resistant to apoptosis and express the bcr-abl fusion gene. These cells have the potential to differentiate into erythrocytes and megakaryocytes. Curcumin is well known as a component with strong potential to alter NFκB activity in various cells. NFκB pathway regulates various genes such as apoptotic and immune response genes. The current study attempted to evaluate the possible role of nanocurcumin in NFκB pathway regulation during the megakaryopoiesis process in the K562 cell line. Materials and Methods: Megakaryocyte markers expression and phenotype alteration of nanocurcumin-treated K562 cells have been detected by flow cytometry and microscopy imaging. The nuclear level of the RelA (p65) subunit of NFκB was determined by western blot test in K562 cells during megakaryopoiesis induction via nanocurcumin treatment at different times. The expression of NFκB target genes including c-MYC, BAX, and NQO1 was also analyzed in nanocurcumin-treated K562 cells by quantitative RT-PCR assay at different times. Results: The study has shown that nanocurcumin causes an increase in NFκB activity transiently during megakaryocyte differentiation, followed by a change in the expression of c-MYC, BAX, and NQO1 target genes. Conclusion: The NFκB pathway can be considered a new pathway for inducing megakaryocyte differentiation by nanocurcumin in vitro and in vivo megakaryopoiesis experiments.

Protective effect of tanshinone ⅡA, MAPK1, and RELA on ischemic injury after stroke

Stroke ischemic injury has become one of the major diseases threatening human health and life, and reducing the secondary damage of neurons after cerebral ischemia is an important measure to treat this disease. Inflammatory factors, oxidative stress, and excessive activation of microglia lead to the aggravation of ischemic nerve damage, and if not stopped in time, it will cause large-scale infarction of brain tissue, resulting in permanent neurological deficits. Studies have found that the use of tanshinone II. A can improve the occurrence of the above reactions by inhibiting the two targets of MAPK1 and RELA (p65). This way of inhibiting specific targets to reduce cerebral ischemic damage has become one of the hot issues in today's research. This article mainly reviews the mechanism of tanshinone II. A by inhibiting MAPK and NF-κB signaling pathways, reducing the inflammatory response, oxidative stress, and regulating microglial polarization.

Lnc-ZEB2-19 Inhibits the Progression and Lenvatinib Resistance of Hepatocellular Carcinoma by Attenuating the NF-κB Signaling Pathway through the TRA2A/RSPH14 Axis.

Long non-coding RNAs have been reported to play a crucial role in tumor progression in hepatocellular carcinoma (HCC). Lnc-ZEB2-19 has been validated to be deficiently expressed in HCC. However, the capabilities and underlying mechanisms of lnc-ZEB2-19 remain uncertain. In this study, we verified that the downregulation of lnc-ZEB2-19 was prevalent in HCC and significantly correlated with the unfavorable prognosis. Further in vitro and in vivo verified that lnc-ZEB2-19 notably inhibited the proliferation, metastasis, stemness, and lenvatinib resistance (LR) of HCC cells. Mechanistically, lnc-ZEB2-19 inhibited HCC progression and LR by specifically binding to transformer 2α (TRA2A) and promoting its degradation, which resulted in the instability of RSPH14 mRNA, leading to the downregulation of Rela(p65) and p-Rela(p-p65). Furthermore, rescue assays showed that silencing RSPH14 partially restrained the effect of knockdown expression of lnc-ZEB2-19 on HCC cell metastatic ability and stemness. The findings describe a novel regulatory axis, lnc-ZEB2-19/TRA2A/RSPH14, downregulating the nuclear factor kappa B to inhibit HCC progression and LR.

Dickkopf1 fuels inflammatory cytokine responses

Many human diseases, including cancer, share an inflammatory component but the molecular underpinnings remain incompletely understood. We report that physiological and pathological Dickkopf1 (DKK1) activity fuels inflammatory cytokine responses in cell models, mice and humans. DKK1 maintains the elevated inflammatory tone of cancer cells and is required for mounting cytokine responses following ligation of toll-like and cytokine receptors. DKK1-controlled inflammation derives from cell-autonomous mechanisms, which involve SOCS3-restricted, nuclear RelA (p65) activity. We translate these findings to humans by showing that genetic DKK1 variants are linked to elevated cytokine production across healthy populations. Finally, we find that genetic deletion of DKK1 but not pharmacological neutralization of soluble DKK1 ameliorates inflammation and disease trajectories in a mouse model of endotoxemia. Collectively, our study identifies a cell-autonomous function of DKK1 in the control of the inflammatory response, which is conserved between malignant and non-malignant cells. Additional studies are required to mechanistically dissect cellular DKK1 trafficking and signaling pathways.

Double knockin mice show NF-κB trajectories in immune signaling and aging.

Invitro studies suggest that mapping the spatiotemporal complexity of nuclear factor κB (NF-κB) signaling is essential to understanding its function. The lack of tools to directly monitor NF-κB proteins invivo has hindered such efforts. Here, we introduce reporter mice with the endogenous RelA (p65) or c-Rel labeled with distinct fluorescent proteins and a double knockin with both subunits labeled. Overcoming hurdles in simultaneous live-cell imaging of RelA and c-Rel, we show that quantitative features of signaling reflect the identity of activating ligands, differ between primary and immortalized cells, and shift toward c-Rel in microglia from aged brains. RelA:c-Rel heterodimer is unexpectedly depleted in the nuclei of stimulated cells. Trajectories of subunit co-expression in immune lineages reveal a reduction at key cell maturation stages. These results demonstrate the power of these reporters in gaining deeper insights into NF-κB biology, with the spectral complementarity of the labeled NF-κB proteins enabling diverse applications.