MRTF-A-NF-κB/p65 axis-mediated PDL1 transcription and expression contributes to immune evasion of non-small-cell lung cancer via TGF-β.
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ABSTRACT:
SUBMITTER: Du F
PROVIDER: S-EPMC8492728 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundThe RING finger (RNF) proteins are a large group of ubiquitin ligases whose aberrant expression is often associated with disease progression. This study examines the function of RNF protein 182 (RNF182) in lung adenocarcinoma (LUAD) cells and its impact on p65 and programmed death ligand 1 (PDL1) regulation.MethodsExpression of RNF182, p65, and PDL1 in LUAD tissues and cells was measured using immunohistochemistry, reverse transcription quantitative polymerase chain reaction (RT-qPCR), and/or western blot (WB) assays. LUAD cells were induced to overexpress RNF182 and p65, followed by cell counting kit-8, colony formation, Transwell, and flow cytometry assays to evaluate the cells' malignant phenotype. Coimmunoprecipitation and WB assays were used to verify RNF182's effect on p65 ubiquitination. Chromatin immunoprecipitation-qPCR and luciferase assays were used to analyze p65's transcriptional regulation of PDL1. Coculture of LUAD with CD8+ cytotoxic T cells was performed to detect lactate dehydrogenase release and interferon-γ and interleukin-2 concentrations. LUAD cells were implanted in mice to analyze tumorigenicity.ResultsRNF182 was poorly expressed, while p65 and PDL1 were highly expressed in LUAD tissues and cells. RNF182 overexpression suppressed the malignant properties of LUAD cells, and it promoted p65 ubiquitination and protein degradation. p65 activated PDL1 transcription. Overexpression of RNF182 suppressed the PDL1 expression, increased the cytotoxicity in LUAD cells cocultured with CD8+ T cells, and suppressed the tumorigenesis of cancer cells in vivo. However, these tumor-suppressive effects of RNF182 on LUAD cells were blocked by p65 restoration.ConclusionThis research demonstrates that RNF182 induces p65 ubiquitination to suppress PDL1 transcription and immunosuppression in LUAD.
| S-EPMC10201958 | biostudies-literature
Project description:Although angiotensin II (AngII) is known to cause renal injury and fibrosis, the underlying mechanisms remain poorly characterized. Here we show that hypertensive nephropathy (HN) patients and AngII-infused mice exhibit elevated levels of circulating miR103a-3p. We observe a positive correlation between miR-103a-3p levels and AngII-induced renal dysfunction. miR-103a-3p suppresses expression of the sucrose non-fermentable-related serine/threonine-protein kinase SNRK in glomerular endothelial cells, and glomeruli of HN patients and AngII-infused mice show reduced endothelial expression of SNRK. We find that SNRK exerts anti-inflammatory effects by interacting with activated nuclear factor-κB (NF-κB)/p65. Overall, we demonstrate that AngII increases circulating miR-103a-3p levels, which reduces SNRK levels in glomerular endothelial cells, resulting in the over-activation of NF-κB/p65 and, consequently, renal inflammation and fibrosis. Together, our work identifies miR-103a-3p/SNRK/NF-κB/p65 as a regulatory axis of AngII-induced renal inflammation and fibrosis.
| S-EPMC6513984 | biostudies-literature
Project description:Caspase-3-mediated p65 cleavage is believed to suppress nuclear factor-kappa B (NF-κB)-mediated anti-apoptotic transactivation in cells undergoing apoptosis. However, only a small percentage of p65 is cleaved during apoptosis, not in proportion to the dramatic reduction in NF-κB transactivation. Here we show that the p65(1-97) fragment generated by Caspase-3 cleavage interferes with ribosomal protein S3 (RPS3), an NF-κB "specifier" subunit, and selectively retards the nuclear translocation of RPS3, thus dampening the RPS3/NF-κB-dependent anti-apoptotic gene expression. Our findings reveal a novel cell fate determination mechanism to ensure cells undergo programed cell death through interfering with RPS3/NF-κB-conferred anti-apoptotic transcription by the fragment from partial p65 cleavage by activated Caspase-3.
| S-EPMC4655178 | biostudies-literature
Project description:Intervertebral disc degeneration (IVDD) is a prevalent and debilitating condition characterized by chronic back pain and reduced quality of life. Strontium ranelate (SRR) is a compound traditionally used for treating osteoporosis via activating TGF-β1 signaling pathway. Recent studies have proved the anti-inflammatory effect of SRR on chondrocytes. Although the exact mechanism of IVDD remains unclear, accumulating evidences have emphasized the involvement of multifactorial pathogenesis including inflammation, oxidative stress damage, and etc. However, the biological effect of SRR on IVDD and its molecular mechanism has not been investigated. Firstly, this study proved the decreased expression of Transforming Growth Factor-beta 1(TGF-β1) in degenerated human intervertebral disc tissues. Subsequently, we confirmed for the first time that SRR could promote cell proliferation, mitigate inflammation and oxidative stress in human nucleus pulposus cells in vitro via increasing the expression of TGF-β1 and suppressing the Nuclear Factor Kappa-Light-Chain-Enhancer of Activated B Cells (NF-κB) pathway. The molecular docking result proved the interaction between SRR and TGF-β1 protein. To further verify this interaction, gain- and loss- of function experiments were conducted. We discovered that both TGF-β1 knockdown and overexpression influenced the activation of the NF-κB pathway. Taken together, SRR could mitigate IL-1β induced-cell dysfunction in human nucleus pulposus cells by regulating TGF-β1/NF-κB axis in vitro. Finally, the in vivo therapeutic effect of SRR on IVDD was confirmed. Our findings may contribute to the understanding of the complex interplay between inflammation and degenerative processes in the intervertebral disc and provide valuable insights into the development of targeted treatment-based therapeutics for IVDD.
| S-EPMC10620869 | biostudies-literature
Project description:BackgroundNumerous studies have demonstrated that autophagy plays a vital role in maintaining cellular homeostasis. Interestingly, several anticancer agents were found to exert their anticancer effects by triggering autophagy. Emerging data suggest that autophagy represents a novel mechanism that can be exploited for therapeutic benefit. Pharmacologically active natural compounds such as those from marine, terrestrial plants and animals represent a promising resource for novel anticancer drugs. There are several prominent examples from the past proving the success of natural products and derivatives exhibiting anticancer activity. Helenalin, a sesquiterpene lactone has been demonstrated to have potent anti-inflammatory and antitumor activity. Albeit previous studies demonstrating helenalin's multi modal action on cellular proliferative and apoptosis, the mechanisms underlying its action are largely unexplained.MethodsTo deduce the mechanistic action of helenalin, cancer cells were treated with the drug at various concentrations and time intervals. Using western blot, FACS analysis, overexpression and knockdown studies, cellular signaling pathways were interrogated focusing on apoptosis and autophagy markers.ResultsWe show here that helenalin induces sub-G1 arrest, apoptosis, caspase cleavage and increases the levels of the autophagic markers. Suppression of caspase cleavage by the pan caspase inhibitor, Z-VAD-fmk, suppressed induction of LC3-B and Atg12 and reduced autophagic cell death, indicating caspase activity was essential for autophagic cell death induced by helenalin. Additionally, helenalin suppressed NF-κB p65 expression in a dose and time dependent manner. Exogenous overexpression of p65 was accompanied by reduced levels of cell death whereas siRNA mediated suppression led to augmented levels of caspase cleavage, autophagic cell death markers and increased cell death.ConclusionsTaken together, these results show that helenalin mediated autophagic cell death entails inhibition of NF-κB p65, thus providing a promising approach for the treatment of cancers with aberrant activation of the NF-κB pathway.
| S-EPMC3464891 | biostudies-literature
Project description:IntroductionNovel plant DING proteins (full-length 38 kDa p38SJ, and 27 kDa p27SJ) exhibit phosphatase activity and modulate HIV-1 gene transcription. Previously, we demonstrated that DING regulates HIV-1 gene transcription by dephosphorylation and inactivation of CTD RNA polymerase II, the major elongating factor of HIV-1 Long Terminal Repeats (LTR). Because the transcription of HIV-1 is controlled by several viral and cellular factors, including p65/p50 subunits of NF-κB, we hypothesized that DING phosphatase can also affect the phosphorylation and activity of p65 NF-κB, in addition to C-terminal Domain (CTD) of RNA Polymerase II (RNAPII), to suppress HIV-1 gene transcription and inhibit HIV-1 infection.MethodsHere, we describe the inhibition of HIV-1 infection and the p65/p50 NF-κB phosphorylation by DING protein, analyzed by ELISA and northern-blot assays, western-blot assays, cell fractionation, and promoter-reporter assays in DING-expressing cells, using a pTet-on inducible system.ResultsResults from HIV-1 infection assays demonstrate a strong inhibition of HIV-1 and HIV-LTR RNA expression by DING protein, determined by p24 ELISA and by northern blot assay. Results from the western blot assays and cell fractionation assays show that there is an increase in the level of hypo-phosphorylated form of p65 NF-κB in DING-expressing cells. Both fractions of p65/p50, nuclear or cytoplasmic, are affected by DING phosphatase, but more cytoplasmic accumulation of p65 NF-κB was found in the presence of DING, suggesting that subsequent activation and nuclear import of active NF-κB is affected by DING. The major portion of nuclear p65 was dephosphorylated in DING-expressing cells. The promoter-reporter assay demonstrated that DING-mediated dephosphorylation and dysregulation of NF-κB p65 lead to the suppression of its binding to HIV-1 LTR, and resulted in the inhibition of p65-mediated activation of LTR transcription. Mapping of the region within LTR that was affected by DING revealed that both, NF-κB and CTD RNA Polymerase II binding sites were important, and cooperativity of these cellular factors was diminished by DING. In addition, mapping of the region within DING-p38SJ that affected LTR transcription, revealed that phosphate-binding domain is essential for this inhibitory activity.ConclusionWe have demonstrated the effect of DING phosphatases on HIV-1 infection, phosphorylation of p65 NF-κB, and transcription of HIV-1 LTR. Our studies suggest that one possible mechanism by which DING can regulate the expression of HIV-1 LTR can be through dysregulation of the transcription factor NF-κB p65 by preventing its phosphorylation and translocation to the nucleus and binding to the HIV-1 LTR, an action that could contribute to the utility of DING p38SJ as an antiviral agent. Importantly, DING not only inhibits HIV-1 LTR gene transcription in the presence of increased p65 NF-κB, but also suppresses HIV-1 infection. DING protein improved inhibitory effects of the known anti-retroviral drugs, Tenofovir (TFV) and Emtricitabine (FTS) on HIV-1, since in the combination with these drugs; the suppression of HIV-1 by DNG was significantly higher when it was in combination with these drugs, compared to controls or cases without DING. Thus, our data support the use of neuroprotective DING proteins as novel therapeutic antiviral drugs that suppress HIV-1 LTR transcription by interfering with the function of NF-κB.
| S-EPMC8296972 | biostudies-literature
Project description:Activating transcription factor 3 (ATF3) is induced by inflammatory responses, cell death, cytokines, and oxidative stress conditions. ATF3 is a negative regulator in the Toll-like receptor 4 signalling pathway. The principal molecule in this pathway is nuclear factor κB (NF-κB) that translocates into the nucleus to initiate the transcription of inflammatory mediators. However, scarce data are available regarding the interaction of ATF3 and p65, a part of the NF-κB dimer. Therefore, we studied the mechanism of regulation of p65 by ATF3 in RAW 264.7 cells. First, LPS-mediated NF-κB activation was confirmed, and then the direct interaction of ATF3 and p65 was observed through immunoprecipitation (IP). The presence of histone deacetylase 1 (HDAC1) was also detected in the complex. In ATF3 deficient cells, NF-κB activity was up-regulated and HDAC1 was not detected by IP. These observations suggest that p65 is attenuated by ATF3 such that ATF3 recruits HDAC1 to the ATF3/p65 complex and facilitates the deacetylation of p65. Likewise, inflammatory response genes were induced by translocated NF-κB in ATF3-deficient cells. Cumulatively, we uncovered a novel mechanism for the negative regulation of NF-κB by ATF3 via direct interaction with p65.
| S-EPMC4585983 | biostudies-other
Project description:Cardiac fibroblasts participate in the inflammatory process of heart diseases as sentinel cells of the cardiac tissue. In this study, we investigated the effect of the proinflammatory cytokine, interleukin 1β (IL-1β), on the expression of interleukin 8 (IL-8), which contributes to the induction of innate immunity via the activation and recruitment of innate immune cells, such as neutrophils, to the site of inflammation in canine cardiac fibroblasts. IL-1β mediates IL-8 mRNA expression and protein release in a dose- and time-dependent manner. The IL-β-mediated IL-8 protein release and mRNA expression were inhibited by 2-[(aminocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide, an inhibitor of the transcription factor, nuclear factor (NF)-κB. In cells treated with IL-1β, NF-κB p65 and p105 were transiently phosphorylated, indicating the activation of NF-κB. However, IL-1β failed to induce IL-8 mRNA expression in the cells transfected with p65 small interfering RNA (siRNA), but not in those transfected with p105 siRNA. These observations suggest that IL-1β induces IL-8 expression via the activation of NF-κB p65 in canine cardiac fibroblasts.
| S-EPMC9065446 | biostudies-literature
Project description:Understanding the regulatory mechanisms for the NF-κB transcription factor is key to control inflammation. IκBα maintains NF-κB in an inactive form in the cytoplasm of unstimulated cells, whereas nuclear NF-κB in activated cells is degraded by PDLIM2, a nuclear ubiquitin E3 ligase that belongs to a LIM protein family. How NF-κB activation is negatively controlled, however, is not completely understood. Here we show that PDLIM1, another member of LIM proteins, negatively regulates NF-κB-mediated signaling in the cytoplasm. PDLIM1 sequestered p65 subunit of NF-κB in the cytoplasm and suppressed its nuclear translocation in an IκBα-independent, but α-actinin-4-dependent manner. Consistently, PDLIM1 deficiency lead to increased levels of nuclear p65 protein, and thus enhanced proinflammatory cytokine production in response to innate stimuli. These studies reveal an essential role of PDLIM1 in suppressing NF-κB activation and suggest that LIM proteins comprise a new family of negative regulators of NF-κB signaling through different mechanisms.
| S-EPMC4683373 | biostudies-other
Project description:The interaction of transcription factors with specific DNA sequences is critical for activation of gene expression programs. In endothelial cells (EC), the transcription factor NF-κB is important in the switch from quiescence to activation, and is tightly controlled to avoid excessive inflammation and organ damage. Here we describe a novel mechanism that controls the activation of NF-κB in EC. The transcription factor Erg, the most highly expressed ETS member in resting EC, controls quiescence by repressing proinflammatory gene expression. Focusing on intercellular adhesion molecule 1(ICAM)-1 as a model, we identify two ETS binding sites (EBS -118 and -181) within the ICAM-1 promoter required for Erg-mediated repression. We show that Erg binds to both EBS -118 and EBS -181, the latter located within the NF-κB binding site. Interestingly, inhibition of Erg expression in quiescent EC results in increased NF-κB-dependent ICAM-1 expression, indicating that Erg represses basal NF-κB activity. Erg prevents NF-κB p65 from binding to the ICAM-1 promoter, suggesting a direct mechanism of interference. Gene set enrichment analysis of transcriptome profiles of Erg and NF-κB-dependent genes, together with chromatin immunoprecipitation (ChIP) studies, reveals that this mechanism is common to other proinflammatory genes, including cIAP-2 and IL-8. These results identify a role for Erg as a gatekeeper controlling vascular inflammation, thus providing an important barrier to protect against inappropriate endothelial activation.
| S-EPMC3320982 | biostudies-literature