RORα Suppresses Cancer-Associated Inflammation by Repressing Respiratory Complex I-Dependent ROS Generation.
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ABSTRACT:
SUBMITTER: Mao W
PROVIDER: S-EPMC8509002 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Purpose Th17 cells and their cytokines are implicated in the pathogenesis of various autoimmune diseases. Retinoic acid-related orphan receptor alpha (RORα) is a transcription factor for the differentiation and the inflammatory functions of Th17 cells. In this study, we generated the nucleus-transducible form of transcription modulation domain of RORα (nt-RORα-TMD) to investigate the functional roles of RORα in vitro and in vivo under normal physiological condition without genetic alteration. Methods The functions of nt-RORα-TMD were analyzed in vitro through flow cytometry, luciferase assay, ELISA, and transcriptome sequencing. Finally, the in vivo therapeutic effects of nt-RORα-TMD were verified in dextran sulfate sodium (DSS)-induced colitis mice. Results nt-RORα-TMD was effectively delivered into the cell nucleus in a dose- and time-dependent manner without any cellular toxicity. nt-RORα-TMD competitively inhibited the RORα-mediated transcription but not RORγt-mediated transcription. Secretion of IL-17A from the splenocytes was suppressed by nt-RORα-TMD without affecting the secretion of Th1- or Th2-type cytokine and T cell activation events such as induction of CD69 and CD25. The differentiation potential of naïve T cells into Th17 cells, not into Th1, Th2, or Treg cells, was significantly blocked by nt-RORα-TMD. Consistently, mRNA sequencing analysis showed that nt-RORα-TMD treatment down-regulated the expression of the genes related to the differentiation and functions of Th17 cells. Treatment of DSS-induced colitis mice with nt-RORα-TMD improved the overall symptoms of colitis, such as body weight change, colon length, infiltration of inflammatory cells, and the level of inflammatory cytokines in the serum. In the mesenteric lymph node (MLN) of the nt-RORα-TMD-treated mice, the population of CD4+IL-17A+ Th17 cells was reduced, and the population of CD4+Foxp3+ Treg cells increased. Conclusion nt-RORα-TMD has a potential to be developed as a novel therapeutic reagent for treating various inflammatory diseases in which Th17 cells are the leading pathological player.
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Project description:Retinoid orphan nuclear receptor alpha (RORα) is a member of the orphan nuclear factor family and regulates gene expression by binding to ROR response elements (ROREs). RORα has been identified as a potential tumor suppressor; however, how downregulation of RORα promotes cancer progression is not fully understood. Here, we showed that protein levels of RORα were downregulated during the Snail-, Twist-, or transforming growth factor-β-induced epithelial-mesenchymal transition (EMT). We found that silencing of RORα induced expression of mesenchymal markers in MCF10A cells, accompanied by enhanced cell invasion, migration, and mammosphere formation. Furthermore, ectopic expression of RORα suppressed transforming growth factor-β-induced EMT processes in MCF10A and HMLE cells. These results indicate that downregulation of RORα is crucial for the induction of EMT in mammary epithelial cells. By analyzing gene expression profiles in control and RORα-expressing cells, we also identified Snail, a key regulator of EMT, as a potential target of RORα. We show that RORα expression significantly inhibits Snail transcription in breast cancer cells. Chromatin immunoprecipitation analysis demonstrated that RORα bound to the ROREs in promoter region of SNAI1 gene, and using the luciferase reporter assay, we showed that binding to the ROREs was critical for RORα to repress Snail transcription. Finally, rescue experiments substantiated that Snail mediates RORα function in suppressing EMT and mammosphere formation. These results reveal a novel function of RORα in suppressing EMT and identify Snail as a direct target of RORα in mammary epithelial cells.
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Project description:Pathologic ocular angiogenesis is a leading cause of blindness, influenced by both dysregulated lipid metabolism and inflammation. Retinoic-acid-receptor-related orphan receptor alpha (RORα) is a lipid-sensing nuclear receptor with diverse biologic function including regulation of lipid metabolism and inflammation; however, its role in pathologic retinal angiogenesis remains poorly understood. Using a mouse model of oxygen-induced proliferative retinopathy, we showed that RORα expression was significantly increased and genetic deficiency of RORα substantially suppressed pathologic retinal neovascularization. Loss of RORα led to decreased levels of proinflammatory cytokines and increased levels of antiinflammatory cytokines in retinopathy. RORα directly suppressed the gene transcription of suppressors of cytokine signaling 3 (SOCS3), a critical negative regulator of inflammation. Inhibition of SOCS3 abolished the antiinflammatory and vasoprotective effects of RORα deficiency in vitro and in vivo. Moreover, treatment with a RORα inverse agonist SR1001 effectively protected against pathologic neovascularization in both oxygen-induced retinopathy and another angiogenic model of very-low-density lipoprotein receptor (Vldlr)-deficient (Vldlr (-/-) ) mice with spontaneous subretinal neovascularization, whereas a RORα agonist worsened oxygen-induced retinopathy. Our data demonstrate that RORα is a novel regulator of pathologic retinal neovascularization, and RORα inhibition may represent a new way to treat ocular neovascularization.
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Project description:Acute liver failure (ALF) is characterized by loss of liver function in response to sustained augmentation of the acute-phase response (APR) in the liver, which can progress even to death. Although the inflammatory interleukin-6 (IL-6)-axis is a crucial factor that drives the hepatic APR by releasing diverse acute-phase proteins (APPs), therapeutic strategies to block the IL-6-STAT3-mediated APR are not well developed. Here, we show that the nuclear receptor retinoic acid-related orphan receptor α (RORα) limits APR-mediated liver injury by inhibiting the hepatic IL-6-STAT3 signaling pathway. Administration of JC1-40, an RORα activator, diminished diethylnitrosamine-induced acute liver injury and repressed transcriptional expression of APPs such as CXCL1 and LCN2 in mice. IL-6-mediated activation of STAT3 was repressed after RORα activation by either adenoviral infusion of RORα or JC1-40 treatment in primary hepatocytes. Activation of RORα decreased transcriptional expression of IL-6 receptor α, an upstream activator of STAT3, both in vitro and in vivo. This may be one mechanism underlying the RORα-mediated inhibition of STAT3. Taken together, our results suggest that RORα is a regulator of the hepatic IL-6-STAT3 signaling pathway and may be a new therapeutic target for treating APR-associated inflammatory ALF.
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Project description:ADP-ribosyltransferase diphtheria-toxin like 1/poly(ADP-ribose) polymerase 1 (ARTD1/PARP1) is a chromatin-associated protein in the nucleus and plays an important role in different cellular processes such as regulation of gene transcription. ARTD1 has been shown to coregulate the inflammatory response by modulating the activity of the transcription factor nuclear factor ?B (NF-?B), the principal regulator of interleukin 6 (IL-6), an important inflammatory cytokine implicated in a variety of diseases such as cancer. However, to what extent and how ARTD1 regulates IL-6 transcription has not been clear. Here, we show that ARTD1 suppresses lipopolysaccharide (LPS)-induced IL-6 expression in macrophages, without affecting the recruitment of the NF-?B subunit RelA to the IL-6 promoter and independent of its enzymatic activity. Interestingly, knockdown of ARTD1 did not alter H3 occupancy but increased LPS-induced trimethylation of histone 3 at lysine 4 (H3K4me3), a hallmark of transcriptionally active genes. We found that ARTD1 mediates its effect through the methyltransferase MLL1, by catalyzing H3K4me3 at the IL-6 promoter and forming a complex with NF-?B. These results demonstrate that ARTD1 modulates IL-6 expression by regulating the function of an NF-?B enhanceosome complex, which involves MLL1 and does not require ADP-ribosylation.
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Project description:Koumine (KM), one of the primary constituents of Gelsemium elegans, has been used for the treatment of inflammatory diseases such as rheumatoid arthritis, but whether KM impacts the activation of the NOD-like receptor protein 3 (NLRP3) inflammasome remains unknown. This study aimed to explore the inhibitory effect of KM on NLRP3 inflammasome activation and the underlying mechanisms both in vitro using macrophages stimulated with LPS plus ATP, nigericin or monosodium urate (MSU) crystals and in vivo using an MSU-induced peritonitis model. We found that KM dose-dependently inhibited IL-1β secretion in macrophages after NLRP3 inflammasome activators stimulation. Furthermore, KM treatment efficiently attenuated the infiltration of neutrophils and suppressed IL-1β production in mice with MSU-induced peritonitis. These results indicated that KM inhibited NLRP3 inflammasome activation, and consistent with this finding, KM effectively inhibited caspase-1 activation, mature IL-1β secretion, NLRP3 formation and pro-IL-1β expression in LPS-primed macrophages treated with ATP, nigericin or MSU. The mechanistic study showed that, KM exerted a potent inhibitory effect on the NLRP3 priming step, which decreased the phosphorylation of IκBα and p65, the nuclear localization of p65, and the secretion of TNF-α and IL-6. Moreover, the assembly of NLRP3 was also interrupted by KM. KM blocked apoptosis-associated speck-like protein containing a CARD (ASC) speck formation and its oligomerization and hampered the NLRP3-ASC interaction. This suppression was attributed to the ability of KM to inhibit the production of reactive oxygen species (ROS). In support of this finding, the inhibitory effect of KM on ROS production was completely counteracted by H2O2, an ROS promoter. Our results provide the first indication that KM exerts an inhibitory effect on NLRP3 inflammasome activation associated with blocking the ROS/NF-κB/NLRP3 signal axis. KM might have potential clinical application in the treatment of NLRP3 inflammasome-related diseases.
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Project description:Heterochromatin, characterized by histone H3 lysine 9 (H3K9) methylation, assembles on repetitive regions including centromeres. Although centromeric heterochromatin is important for correct segregation of chromosomes, its exact role in maintaining centromere integrity remains elusive. Here, we found in fission yeast that heterochromatin suppresses gross chromosomal rearrangements (GCRs) at centromeres. Mutations in Clr4/Suv39 methyltransferase increased the formation of isochromosomes, whose breakpoints were located in centromere repeats. H3K9A and H3K9R mutations also increased GCRs, suggesting that Clr4 suppresses centromeric GCRs via H3K9 methylation. HP1 homologs Swi6 and Chp2 and the RNAi component Chp1 were the chromodomain proteins essential for full suppression of GCRs. Remarkably, mutations in RNA polymerase II (RNAPII) or Tfs1/TFIIS, the transcription factor that facilitates restart of RNAPII after backtracking, specifically bypassed the requirement of Clr4 for suppressing GCRs. These results demonstrate that heterochromatin suppresses GCRs by repressing Tfs1-dependent transcription of centromere repeats.
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Project description:Hepatic fibrosis is the first stage of liver disease, and can progress to a chronic status, such as cirrhosis or hepatocellular carcinoma. Excessive production of extracellular matrix (ECM) components plays an important role in the development of fibrosis. Mechanistically, transforming growth factor beta (TGFβ)-induced phosphorylation of Smad is thought to be a key signaling pathway in the development of liver fibrosis. Although the natural isoquinoline alkaloid oxoglaucine (1,2,9,10-tetramethoxy-7H-dibenzo(de,g)quinolin-7-one) exerts numerous beneficial effects, including anti-cancer, anti-inflammatory, and anti-osteoarthritic effects in diverse cell types, the effects of oxoglaucine on liver fibrosis and fibrogenic gene expression have not been fully elucidated. The aim of this study is to evaluate the signaling pathway and antifibrotic activity of isoquinoline alkaloid oxoglaucine in TFGβ-induced hepatic fibrosis in vitro. Using Hepa1c1c7 cells and primary hepatocytes, we demonstrated that oxoglaucine treatment resulted in inhibition of the expression of fibrosis markers such as collagen, fibronectin, and alpha-SMA. Subsequent experiments showed that oxoglaucine suppressed TGFβ-induced phosphorylation of Smad2 and reactive oxygen species (ROS) generation, without altering cell proliferation. We further determined that the increase in Smad7 by oxoglaucine treatment is responsible for the inhibition of Smad2 phosphorylation and the anti-fibrogenic effects. These findings indicate that oxoglaucine plays a crucial role in suppression of fibrosis in hepatocytes, thereby making it a potential drug candidate for treatment of liver fibrosis.
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Project description:Neurodegenerative disorders, such as Alzheimer's disease, are a global public health burden with poorly understood aetiology. Neuroinflammation and oxidative stress (OS) are undoubtedly hallmarks of neurodegeneration, contributing to disease progression. Protein aggregation and neuronal damage result in the activation of disease-associated microglia (DAM) via damage-associated molecular patterns (DAMPs). DAM facilitate persistent inflammation and reactive oxygen species (ROS) generation. However, the molecular mechanisms linking DAM activation and OS have not been well-defined; thus targeting these cells for clinical benefit has not been possible. In microglia, ROS are generated primarily by NADPH oxidase 2 (NOX2) and activation of NOX2 in DAM is associated with DAMP signalling, inflammation and amyloid plaque deposition, especially in the cerebrovasculature. Additionally, ROS originating from both NOX and the mitochondria may act as second messengers to propagate immune activation; thus intracellular ROS signalling may underlie excessive inflammation and OS. Targeting key kinases in the inflammatory response could cease inflammation and promote tissue repair. Expression of antioxidant proteins in microglia, such as NADPH dehydrogenase 1 (NQO1), is promoted by transcription factor Nrf2, which functions to control inflammation and limit OS. Lipid droplet accumulating microglia (LDAM) may also represent a double-edged sword in neurodegenerative disease by sequestering peroxidised lipids in non-pathological ageing but becoming dysregulated and pro-inflammatory in disease. We suggest that future studies should focus on targeted manipulation of NOX in the microglia to understand the molecular mechanisms driving inflammatory-related NOX activation. Finally, we discuss recent evidence that therapeutic target identification should be unbiased and founded on relevant pathophysiological assays to facilitate the discovery of translatable antioxidant and anti-inflammatory therapeutics.
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Project description:Mediator complex subunit 1 (MED1) is a component of the mediator complex and functions as a coactivator involved in the regulated transcription of nearly all RNA polymerase II-dependent genes. Previously, we showed that MED1 in macrophages has a protective effect on atherosclerosis; however, the effect of MED1 on intimal hyperplasia and mechanisms regulating proinflammatory cytokine production after macrophage MED1 deletion are still unknown. In this study, we report that MED1 macrophage-specific knockout (MED1 ΔMac) mice showed aggravated neointimal hyperplasia, vascular smooth muscle cells (VSMCs), and macrophage accumulation in injured arteries. Moreover, MED1 ΔMac mice showed increased proinflammatory cytokine production after an injury to the artery. After lipopolysaccharide (LPS) treatment, MED1 ΔMac macrophages showed increased generation of reactive oxygen species (ROS) and reduced expression of peroxisome proliferative activated receptor gamma coactivator-1α (PGC1α) and antioxidant enzymes, including catalase and glutathione reductase. The overexpression of PGC1α attenuated the effects of MED1 deficiency in macrophages. In vitro, conditioned media from MED1 ΔMac macrophages induced more proliferation and migration of VSMCs. To explore the potential mechanisms by which MED1 affects inflammation, macrophages were treated with BAY11-7082 before LPS treatment, and the results showed that MED1 ΔMac macrophages exhibited increased expression of phosphorylated-p65 and phosphorylated signal transducer and activator of transcription 1 (p-STAT1) compared with the control macrophages, suggesting the enhanced activation of NF-κB and STAT1. In summary, these data showed that MED1 deficiency enhanced inflammation and the proliferation and migration of VSMCs in injured vascular tissue, which may result from the activation of NF-κB and STAT1 due to the accumulation of ROS.
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