Nuclear Translocation Activity Research Articles

Dihydrotanshinone I attenuates estrogen-deficiency bone loss through RANKL-stimulated NF-κB, ERK and NFATc1 signaling pathways

Postmenopausal osteoporosis, a chronic condition that predominantly affects postmenopausal women, presents a significant impediment to their overall well-being. The condition arises from estrogen deficiency, leading to enhanced osteoclast activity. Salvia miltiorrhiza, a well-established Chinese herbal medicine with a history of clinical use for osteoporosis treatment, contains diverse active constituents that have shown inhibitory effects on osteoclast formation and bone loss. Dihydrotanshinone I (DTI), a phenanthrenonequinone compound derived from the root of Salvia miltiorrhiza, has been identified as a potential therapeutic agent, although its mechanism of action on osteoclasts remains elusive.In this study, we aimed to elucidate the inhibitory potential of DTI on RANKL-induced osteoclastogenesis. We observed the ability of DTI to effectively impede the expression of key osteoclast-specific genes and proteins, as assessed by Real-time PCR and Western Blotting analyses. Mechanistically, DTI exerted its inhibitory effects on osteoclast formation by modulating critical signaling pathways including NF-κB, ERK, and calcium ion signaling. Notably, DTI intervention disrupted the nuclear translocation and subsequent transcriptional activity of the NFATc1, thus providing mechanistic insights into its inhibitory role in osteoclastogenesis. To further assess the therapeutic potential of DTI, we employed an ovariectomized osteoporosis animal model to examine its impact on bone loss. Encouragingly, DTI demonstrated efficacy in mitigating bone loss induced by estrogen deficiency.In conclusion, our investigation elucidates the ability of DTI to regulate multiple signaling pathways activated by RANKL, leading to the inhibition of osteoclast formation and prevention of estrogen-deficiency osteoporosis. Consequently, DTI emerges as a promising candidate for the treatment of osteoporosis.

Dysregulation of Histone Deacetylases Inhibits Trophoblast Growth during Early Placental Development Partially through TFEB-Dependent Autophagy-Lysosomal Pathway.

Dysregulated biological behaviors of trophoblast cells can result in recurrent spontaneous abortion (RSA)-whose underlying etiology still remains insufficient. Autophagy, a conserved intracellular physiological process, is precisely monitored throughout whole pregnancy. Although the exact mechanism or role remains elusive, epigenetic modification has emerged as an important process. Herein, we found that a proportion of RSA patients exhibited higher levels of autophagy in villus tissues compared to controls, accompanied with impaired histone deacetylase (HDAC) expression. The purpose of this study is to explore the connection between HDACs and autophagy in the pathological course of RSA. Mechanistically, using human trophoblast cell models, treatment with HDAC inhibitor (HDACI)-trichostatin A (TSA) can induce autophagy by promoting nuclear translocation and transcriptional activity of the central autophagic regulator transcription factor EB (TFEB). Specifically, overactivated autophagy is involved in the TSA-driven growth inhibition of trophoblast, which can be partially reversed by the autophagy inhibitor chloroquine (CQ) or RNA interference of TFEB. In summary, our results reveal that abnormal acetylation and autophagy levels during early gestation may be associated with RSA and suggest the potential novel molecular target TFEB for RSA treatment.

Nicotine promotes renal interstitial fibrosis via upregulation of XIAP in an alpha7-nAChR-dependent manner

Renal fibrosis, characterized by excessive accumulation of the extracellular matrix in the renal tubulointerstitium, can lead to chronic kidney disease (CKD), resulting in a heavy burden on families and society. Clinical studies have shown that smoking is closely associated with CKD deterioration in patients with diabetes, hypertension, polycystic kidney disease, and kidney transplantation. However, the mechanism of action of nicotine in renal fibrosis pathogenesis remains largely unknown. X-linked inhibitor of apoptosis (XIAP), a member of the inhibitor of apoptosis protein (IAP) family, is involved in apoptosis, necroptosis, autophagy, and immune response. Here, the upregulated expression of XIAP and α7 nicotine acetylcholine receptor (α7-nAChR) was determined in the kidneys of the CKD smoking group in human and animal studies. A significant positive correlation between XIAP and cotinine was observed. In addition, the nuclear translocation and transcriptional activity of SP1 were promoted when nicotine bound to α7-nAChR, resulting in XIAP overexpression and renal interstitial fibrosis progression. This phenotype can be reversed by the nicotine receptor subtype α7-nAChR antagonists methyllycaconitine. Our results revealed the complex underlying mechanism of nicotine in promoting renal fibrosis by altering SP1 nucleocytoplasmic translocation and regulating XIAP expression. These results provide novel insights into the pathogenesis and treatment of CKD.

Decidual lymphatic endothelial cell-derived granulocyte-macrophage colony-stimulating factor induces M1 macrophage polarization via the NF-κB pathway in severe pre-eclampsia.

Direct interactions between macrophages and lymphatic vessels have been shown previously.In pre-eclampsia (PE), macrophages are dominantly polarized into a proinflammatory M1 phenotype and lymphangiogenesis is defective in the decidua.Here, we investigated whether decidual lymphatic endothelial cells (dLECs) affect macrophage polarization in PE. THP-1 macrophages were cocultured with dLECs or cultured in the conditioned medium (CM) of dLECs. Macrophage polarization was measured using flow cytometry. Granulocyte-macrophage colony-stimulating factor (GM-CSF) expression in dLECs was measured using qRT-PCR and ELISA. The activation of nuclear translocation of nuclear factor-κ (NF-κB), an upstream signaling molecule of GM-CSF, was assessed by immunocytochemical localization of p65. Through GM-CSF knockdown and NF-κB inhibition in dLEC, we evaluated whether the GM-CSF/NF-κB pathway of PE dLEC affects decidual macrophage polarization. The ratio of inflammatory M1 macrophages with HLA-DR+ /CD80+ markers significantly increased following coculturing with PE dLECs or culturing in PE dLEC CM, indicating that the PE dLEC-derived soluble factor acts in a paracrine manner. GM-CSF expression was significantly upregulated in PE dLECs. Recombinant human GM-CSF induced macrophage polarization toward an M1-like phenotype, whereas its knockdown in PE dLECs suppressed it, suggesting PE dLECs induce M1 macrophage polarization by secreting GM-CSF. The NF-κB p65 significantly increased in PE dLECs compared to the control, and pretreatment with an NF-κB inhibitor significantly suppressed GM-CSF production from PE dLECs. In PE, dLECs expressing high levels of GM-CSF via the NF-κB-dependent pathway play a role in inducing decidual M1 macrophage polarization.

Sinomenine alleviates lipopolysaccharide-induced acute lung injury via a PPARβ/δ-dependent mechanism

Evidence is mounting that sinomenine and peroxisome proliferator-activated receptor β/δ (PPARβ/δ) are effective against lipopolysaccharide (LPS)-induced acute lung injury (ALI) via anti-inflammatory properties. However, it is unknown whether PPARβ/δ plays a role in the protective effect of sinomenine on ALI. Here, we initially observed that preemptive administration of sinomenine markedly alleviated lung pathological changes, pulmonary edema and neutrophil infiltration, accompanied by inhibition of the expression of the pro-inflammatory cytokines Tumor necrosis factor-α (TNF-α) and Interleukin-6 (IL-6), which were largely reversed following the addition of a PPARβ/δ antagonist. Subsequently, we also noticed that sinomenine upregulated adenosine A2A receptor expression in a PPARβ/δ-dependent manner in LPS-stimulated bone marrow-derived macrophages (BMDMs). Further investigation indicated that PPARβ/δ directly bound to the functional peroxisome proliferator responsive element (PPRE) in the adenosine A2A receptor gene promoter region to enhance the expression of the adenosine A2A receptor. Sinomenine was identified as a PPARβ/δ agonist. It could bind with PPARβ/δ, and promote the nuclear translocation and transcriptional activity of PPARβ/δ. In addition, combined treatment with sinomenine and an adenosine A2A receptor agonist exhibited synergistic effects and better protective roles than their single use against ALI. Taken together, our results reveal that sinomenine exerts advantageous effects on ALI by activating of PPARβ/δ, with the subsequent upregulation of adenosine A2A receptor expression, and provide a novel and potential therapeutic application for ALI.

RagD auto-activating mutations impair MiT/TFE activity in kidney tubulopathy and cardiomyopathy syndrome

Heterozygous mutations in the gene encoding RagD GTPase were shown to cause a novel autosomal dominant condition characterized by kidney tubulopathy and cardiomyopathy. We previously demonstrated that RagD, and its paralogue RagC, mediate a non-canonical mTORC1 signaling pathway that inhibits the activity of TFEB and TFE3, transcription factors of the MiT/TFE family and master regulators of lysosomal biogenesis and autophagy. Here we show that RagD mutations causing kidney tubulopathy and cardiomyopathy are “auto- activating”, even in the absence of Folliculin, the GAP responsible for RagC/D activation, and cause constitutive phosphorylation of TFEB and TFE3 by mTORC1, without affecting the phosphorylation of “canonical” mTORC1 substrates, such as S6K. By using HeLa and HK-2 cell lines, human induced pluripotent stem cell-derived cardiomyocytes and patient-derived primary fibroblasts, we show that RRAGD auto-activating mutations lead to inhibition of TFEB and TFE3 nuclear translocation and transcriptional activity, which impairs the response to lysosomal and mitochondrial injury. These data suggest that inhibition of MiT/TFE factors plays a key role in kidney tubulopathy and cardiomyopathy syndrome.

Statins improve endothelial function via suppression of epigenetic-driven EndMT.

The pleiotropic benefits of statins in cardiovascular diseases that are independent of their lipid-lowering effects have been well documented, but the underlying mechanisms remain elusive. Here we show that simvastatin significantly improves human induced pluripotent stem cell-derived endothelial cell functions in both baseline and diabetic conditions by reducing chromatin accessibility at transcriptional enhanced associate domain elements and ultimately at endothelial-to-mesenchymal transition (EndMT)-regulating genes in a yes-associated protein (YAP)-dependent manner. Inhibition of geranylgeranyltransferase (GGTase) I, a mevalonate pathway intermediate, repressed YAP nuclear translocation and YAP activity via RhoA signaling antagonism. We further identified a previously undescribed SOX9 enhancer downstream of statin-YAP signaling that promotes the EndMT process. Thus, inhibition of any component of the GGTase-RhoA-YAP-SRY box transcription factor 9 (SOX9) signaling axis was shown to rescue EndMT-associated endothelial dysfunction both in vitro and in vivo, especially under diabetic conditions. Overall, our study reveals an epigenetic modulatory role for simvastatin in repressing EndMT to confer protection against endothelial dysfunction.

Vortioxetine hydrobromide inhibits the growth of gastric cancer cells in vivo and in vitro by targeting JAK2 and SRC

Gastric cancer is the fourth leading cause of cancer deaths worldwide. Most patients are diagnosed in the advanced stage. Inadequate therapeutic strategies and the high recurrence rate lead to the poor 5-year survival rate. Therefore, effective chemopreventive drugs for gastric cancer are urgently needed. Repurposing clinical drugs is an effective strategy for discovering cancer chemopreventive drugs. In this study, we find that vortioxetine hydrobromide, an FDA-approved drug, is a dual JAK2/SRC inhibitor, and has inhibitory effects on cell proliferation of gastric cancer. Computational docking analysis, pull-down assay, cellular thermal shift assay (CETSA) and in vitro kinase assays are used to illustrate vortioxetine hydrobromide directly binds to JAK2 and SRC kinases and inhibits their kinase activities. The results of non-reducing SDS-PAGE and Western blotting indicate that vortioxetine hydrobromide suppresses STAT3 dimerization and nuclear translocation activity. Furthermore, vortioxetine hydrobromide inhibits the cell proliferation dependent on JAK2 and SRC and suppresses the growth of gastric cancer PDX model in vivo. These data demonstrate that vortioxetine hydrobromide, as a novel dual JAK2/SRC inhibitor, curbs the growth of gastric cancer in vitro and in vivo by JAK2/SRC-STAT3 signaling pathways. Our results highlight that vortioxetine hydrobromide has the potential application in the chemoprevention of gastric cancer.

Anti-nucleolin aptamer, iSN04, inhibits the inflammatory responses in C2C12 myoblasts by modulating the β-catenin/NF-κB signaling pathway

A myogenetic oligodeoxynucleotide, iSN04, is the 18-base single-stranded DNA that acts as an anti-nucleolin aptamer. iSN04 has been reported to restore myogenic differentiation by suppressing inflammatory responses in myoblasts isolated from patients with diabetes or healthy myoblasts exposed to cancer-releasing factors. Thus, iSN04 is expected to be a nucleic acid drug for the muscle wasting associated with chronic diseases. The present study investigated the anti-inflammatory mechanism of iSN04 in the murine myoblast cell line C2C12. Tumor necrosis factor-α (TNF-α) or Toll-like receptor (TLR) ligands (Pam3CSK4 and FSL-1) induced nuclear translocation and transcriptional activity of nuclear factor-κB (NF-κB), resulting in upregulated expression of TNF-α and interleukin-6. Pre-treatment with iSN04 significantly suppressed these inflammatory responses by inhibiting the nuclear accumulation of β-catenin induced by TNF-α or TLR ligands. These results demonstrate that antagonizing nucleolin with iSN04 downregulates the inflammatory effect mediated by the β-catenin/NF-κB signaling pathway in C2C12 cells. In addition, the anti-inflammatory effects of iSN04 were also observed in the rat smooth muscle cell line A10 and the murine adipocyte-like fibroblast cell line 3T3-L1, suggesting that iSN04 may be useful in preventing inflammation induced by metabolic disorders.

Abstract 1482: Critical role of OGT-mediated novel NF-κB o-glcNAcylation in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) has one of the highest mortalities of all malignancies, with a mere 5-year survival of ~10%. However, the current first-line treatments have poor patient outcomes, highlighting the urgent need for innovative therapeutics. The nuclear factor κB (NF-κB) is a crucial transcription factor frequently activated constitutively in PDAC. It mediates the transcription of oncogenic and inflammatory genes that facilitate multiple PDAC phenotypes. Thus, a better understanding of the mechanistic underpinnings of NF-κB activation holds substantial promise in PDAC diagnosis and new therapeutics. The purpose of this study is to identify novel regulation of NF-κB, with the aim of providing new diagnostic and therapeutic strategies for PDAC. Here, we report protein O-GlcNAc transferase (OGT) - mediated NF-κB activation through novel serine O-GlcNAcylation of its p65 subunit. We show that overexpression of serine-to-alanine (S-A) mutant at the O-GlcNAcylation site impaired NF-κB nuclear translocation and transcriptional activity in PDAC cells. Moreover, these S-A p65 mutants downregulate NF-κB-target genes important to major cancer hallmarks and inhibit cellular proliferation, migration, and anchorage-independent growth of PDAC cells compared to WT-p65. Interestingly, this modification happens downstream of the inhibitor of NF-κB (IκBα) degradation. Collectively, we have identified OGT-mediated serine O-GlcNAcylation of NF-κB and determined its mechanistic and cellular function in driving PDAC phenotypes. Thus, our study holds great significance as it uncovers a brand-new strategy for PDAC diagnosis and effective therapeutics development. Citation Format: Aishat Motolani, Matthew Martin, Benlian Wang, Guanglong Jiang, Yunlong Liu, Tao Lu. Critical role of OGT-mediated novel NF-κB o-glcNAcylation in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1482.

IKBA phosphorylation governs human sperm motility through ACC-mediated fatty acid beta-oxidation

The nuclear factor-κB (NF-κB) signaling pathway regulates specific immunological responses and controls a wide range of physiological processes. NF-κB inhibitor alpha (IKBA) is an NF-κB inhibitory mediator in the cytoplasm that modulates the nuclear translocation and DNA binding activities of NF-κB proteins. However, whether the upstream cascade of the canonical NF-κB signaling pathway has physiological roles independent of IKBA-mediated transcriptional activation remains unclear. Herein we investigated the function of IKBA in mature sperm in which transcriptional and translational events do not occur. IKBA was highly expressed in human sperm. The repression of IKBA phosphorylation by its inhibitor Bay117082 markedly enhanced sperm motility. On the contrary, lipopolysaccharide-stimulated IKBA phosphorylation significantly decreased sperm motility. Nevertheless, Bay117082 treatment did not affect the motility of IKBA-knockout sperm. Further, untargeted metabolomic analysis and pharmacological blocking assays revealed that the Bay117082-induced increase in sperm motility was attributable to fatty acid β-oxidation (FAO) enhancement. In addition, we found that IKBA phosphorylation inhibition resulted in a significant reduction of acetyl-CoA carboxylase levels in the FAO metabolic pathway. Our findings indicate that IKBA-mediated signaling orchestrates sperm motility program and improves our understanding of transcription-independent NF-κB signaling pathway in cells.

Genistein and Procyanidin B2 Reduce Carcinogen-Induced Reactive Oxygen Species and DNA Damage through the Activation of Nrf2/ARE Cell Signaling in Bronchial Epithelial Cells In Vitro.

Cancer is one of the leading causes of death worldwide. Chemotherapy and radiation therapy are currently providing the basis for cancer therapies, although both are associated with significant side effects. Thus, cancer prevention through dietary modifications has been receiving growing interest. The potential of selected flavonoids in reducing carcinogen-induced reactive oxygen species (ROS) and DNA damage through the activation of nuclear factor erythroid 2 p45 (NF-E2)-related factor (Nrf2)/antioxidant response element (ARE) pathway was studied in vitro. Dose-dependent effects of pre-incubated flavonoids on pro-carcinogen 4-[(acetoxymethyl)nitrosamino]-1-(3-pyridyl)-1-butanone (NNKAc)-induced ROS and DNA damage in human bronchial epithelial cells were studied in comparison to non-flavonoids. The most effective flavonoids were assessed for the activation of Nrf2/ARE pathway. Genistein, procyanidin B2 (PCB2), and quercetin significantly suppressed the NNKAc-induced ROS and DNA damage. Quercetin significantly upregulated the phosphorylated protein kinase B/Akt. PCB2 significantly upregulated the activation of Nrf2 and Akt through phosphorylation. Genistein and PCB2 significantly upregulated the phospho-Nrf2 nuclear translocation and catalase activity. In summary, genistein and PCB2 reduced the NNKAc-induced ROS and DNA damage through the activation of Nrf2. Further studies are required to understand the role of dietary flavonoids on the regulation of the Nrf2/ARE pathway in relation to carcinogenesis.

Nitration of cAMP-Response Element Binding Protein Participates in Myocardial Infarction-Induced Myocardial Fibrosis via Accelerating Transcription of Col1a2 and Cxcl12.

Aims: Myocardial fibrosis after myocardial infarction (MI) leads to heart failure. Nitration of protein can alter its function. cAMP-response element binding protein (CREB) is a key transcription factor involved in fibrosis. However, little is known about the role of nitrated CREB in MI-induced myocardial fibrosis. Meanwhile, downstream genes of transcription factor CREB in myocardial fibrosis have not been identified. This study aims to verify the hypothesis that nitrated CREB promotes MI-induced myocardial fibrosis via regulating the transcription of Col1a2 and Cxcl12. Results: Our study showed that (1) the level of nitrative stress was elevated and nitrated CREB was higher in the myocardium after MI. Tyr182, 307, and 336 were the nitration sites of CREB; (2) with the administration of peroxynitrite (ONOO-) scavengers, CREB phosphorylation, nuclear translocation, and binding activity to TORC2 (transducers of regulated CREB-2) were attenuated; (3) the expressions of extracellular matrix (ECM) proteins were upregulated and downregulated in accordance with the expression alteration of CREB both in vitro and in vivo; (4) CREB accelerated transcription of Col1a2 and Cxcl12 after MI directly. With the administration of ONOO- scavengers, ECM protein expressions were attenuated; meanwhile, the messenger RNA (mRNA) levels of Col1a2 and Cxcl12 were alleviated as well. Innovation and Conclusion: Nitration of transcription factor CREB participates in MI-induced myocardial fibrosis through enhancing its phosphorylation, nuclear translocation, and binding activity to TORCs, among which CREB transcripts Col1a2 and Cxcl12 directly. These data indicated that nitrated CREB might be a potential therapeutic target against MI-induced myocardial fibrosis. Antioxid. Redox Signal. 38, 709-730.

Skeletal Muscle DNA Methylation and mRNA Responses to a Bout of Higher versus Lower Load Resistance Exercise in Previously Trained Men.

We sought to determine the skeletal muscle genome-wide DNA methylation and mRNA responses to one bout of lower load (LL) versus higher load (HL) resistance exercise. Trained college-aged males (n = 11, 23 ± 4 years old, 4 ± 3 years self-reported training) performed LL or HL bouts to failure separated by one week. The HL bout (i.e., 80 Fail) consisted of four sets of back squats and four sets of leg extensions to failure using 80% of participants estimated one-repetition maximum (i.e., est. 1-RM). The LL bout (i.e., 30 Fail) implemented the same paradigm with 30% of est. 1-RM. Vastus lateralis muscle biopsies were collected before, 3 h, and 6 h after each bout. Muscle DNA and RNA were batch-isolated and analyzed using the 850k Illumina MethylationEPIC array and Clariom S mRNA microarray, respectively. Performed repetitions were significantly greater during the 30 Fail versus 80 Fail (p < 0.001), although total training volume (sets × reps × load) was not significantly different between bouts (p = 0.571). Regardless of bout, more CpG site methylation changes were observed at 3 h versus 6 h post exercise (239,951 versus 12,419, respectively; p < 0.01), and nuclear global ten-eleven translocation (TET) activity, but not global DNA methyltransferase activity, increased 3 h and 6 h following exercise regardless of bout. The percentage of genes significantly altered at the mRNA level that demonstrated opposite DNA methylation patterns was greater 3 h versus 6 h following exercise (~75% versus ~15%, respectively). Moreover, high percentages of genes that were up- or downregulated 6 h following exercise also demonstrated significantly inversed DNA methylation patterns across one or more CpG sites 3 h following exercise (65% and 82%, respectively). While 30 Fail decreased DNA methylation across various promoter regions versus 80 Fail, transcriptome-wide mRNA and bioinformatics indicated that gene expression signatures were largely similar between bouts. Bioinformatics overlay of DNA methylation and mRNA expression data indicated that genes related to "Focal adhesion," "MAPK signaling," and "PI3K-Akt signaling" were significantly affected at the 3 h and 6 h time points, and again this was regardless of bout. In conclusion, extensive molecular profiling suggests that post-exercise alterations in the skeletal muscle DNA methylome and mRNA transcriptome elicited by LL and HL training bouts to failure are largely similar, and this could be related to equal volumes performed between bouts.

PS-BPB09-3: CIGARETTE SMOKE EXTRACT INDUCES DNA DAMAGE AND TRIGGERS AN INNATE IMMUNE RESPONSE

Objective: Elevated blood pressure and tobacco smoking are causes of preventable mortality. The effects of tobacco smoking on blood pressure are complex and chronic effects are inconsistent. We have previously reported increased DNA damage in peripheral mononuclear cells of smokers compared to nonsmokers. In this study, we investigated the effects of cigarette smoke extract (CSE) on DNA damage and the physiological influence of DNA damage in endothelial cells. Design and method: The effect of CSE extracted from tobacco smoke was examined in human umbilical vein endothelial cells (HUVECs). CSE was added to HUVECs and the formation of DNA damage was quantified by fluorescent immunostaining. The mRNA expression of inflammatory cytokines was quantified by real-time RT PCR. Results: CSE increased double-strand breaks in HUVEC by 72 hours. CSE also increased oxidative DNA damage both in the nucleus and mitochondria, and decreased mitochondria membrane potential. Mitochondrial dysfunction partially induced mitochondrial outer membrane permeabilization, resulting in the partial activation of caspase-3 and nuclear translocation of caspase-activated deoxyribonuclease, which fragments DNA in the nucleus. It was suggested that this is one of the mechanisms of nuclear DNA double-strand breaks. CSE induced the accumulation of nuclear and mitochondrial DNA in the cytosol. We further examined whether accumulated cytosolic DNA activated cytosolic DNA-sensing pathways. The production of cGAMP, a second messenger in cGAS (a DNA-sensing receptor) signaling, was increased by CSE, followed by nuclear translocation of NF-kB and increased mRNA expression of IL-6. The increase in IL-6 expression was suppressed by si-cGAS. Conclusions: Our study revealed that continuous exposure to CSE induces DNA damages not only in the nucleus but also in the mitochondria, which leads to cytosolic DNA accumulation, and evokes chronic inflammation via the cGAS pathway.

AMPK activator decelerates osteoarthritis development by inhibition of β-catenin signaling in chondrocytes.

Osteoarthritis (OA) is a common degenerative joint disease with significant negative impact on the quality of life. It has been reported that abnormal upregulation of β-catenin signaling could lead to OA development; however, the upstream regulatory mechanisms of β-catenin signaling have not been determined. Primary rat chondrocytes and ATDC5 chondrocyte cell line were stimulated with AKT2 and treated with or without metformin, an adenosine 5'-monophosphate-activated protein kinase (AMPK) activator. Westerrn blot analysis, luciferase reporter assay and immunofluorescent (IF) staining were performed to examine changes in β-cateninS552 phosphorylation and β-catenin nuclear translocation in ATDC5 cells and in primary chondrocytes. We found that metformin inhibited β-cateninS552 phosphorylation in ATDC5 cells and in primary chondrocytes in a time-dependent manner. Metformin inhibited β-catenin nuclear translocation and β-catenin reporter activity. In addition, metformin also attenuated the expression of β-catenin downstream target genes. We also demonstrated that metformin inhibited β-cateninS552 phosphorylation in articular cartilage in mice. These findings suggest that metformin may exert its chondro-protective effect at least in part through the inhibition of β-catenin signaling in chondrocytes. This study demonstrated the interaction between AMPK and β-catenin signaling in chondrocytes and defined novel molecular targets for the treatment of OA disease.

Oleic acid from cancer-associated fibroblast promotes cancer cell stemness by stearoyl-CoA desaturase under glucose-deficient condition

BackgroundCancer-associated fibroblasts (CAFs) coordinate the malignancy of cancer cells via secretory materials. Reprogrammed lipid metabolism and signaling play critical roles in cancer biology. Oleic acid (OA) serves as a source of energy under glucose-deficient conditions, but its function in cancer progression remains unclear. The present study investigated that CAFs in xenografted tumors had higher amounts of fatty acids, particularly OA, compared to normal fibroblasts, and promoted the cancer cell stemness in lung adenocarcinoma cells under glucose-deficient condition.MethodsXenografts were established in immunodeficient mice by injection of NCI-H460 (H460) cells. Lipids and fatty acids were evaluated using the BODIPY staining and fatty-acid methyl esters analysis. The expression levels of markers for lipid metabolism and cancer stemness were determined by western blot, flow cytometry, and real-time PCR. Cancer cell subclones against stearoyl-CoA desaturase (SCD) were produced by lentiviral vector and CRISPR/cas9 systems. The expression of SCD was examined immunochemically in human adenocarcinoma tissues, and its clinical relevance to survival rate in lung adenocarcinoma patients was assessed by Kaplan–Meier analysis.ResultsTransferred CAF-derived OA through lipid transporter upregulated SCD in cancer cells under glucose-deficient conditions, resulting in enhanced lipid metabolism and autophagosome maturation. By OA treatment under glucose deficient condition, cancer cell stemness was significantly enhanced through sequential activation of SCD, F-actin polymerization and nuclear translocation of yes-associated protein. These findings were confirmed by experiments using chemical inhibitors, SCD-overexpressing cells and SCD-knockout (KO) cells. When xenografted, SCD-overexpressing cells produced larger tumors compared with parental cells, while SCD-KO cells generated much smaller tumors. Analysis of tumor tissue microarray from lung adenocarcinoma patients revealed that SCD expression was the marker for poor prognosis involving tumor grade, clinical stage and survival rate.ConclusionOur data indicate that CAFs-derived OA activated lipid metabolism in lung adenocarcinoma cells under glucose-deficient conditions, subsequently enhancing stemness and progression toward malignancy.

MEK1-dependent MondoA phosphorylation regulates glucose uptake in response to ketone bodies in colorectal cancer cells.

The Mondo family transcription factor MondoA plays a pivotal role in sensing metabolites, such as glucose, glutamine, and lactic acid, to regulate glucose metabolism and cell proliferation. Ketone bodies are important signals for reducing glucose uptake. However, it is unclear whether MondoA functions in ketone body-regulated glucose transport. Here we reported that ketone bodies promoted MondoA nuclear translocation and binding to the promoter of its target gene TXNIP. Ketone bodies reduced glucose uptake, increased apoptosis and decreased proliferation of colorectal cancer cells, which was impeded by MondoA knockdown. Moreover, we identified MEK1 as a novel component of the MondoA protein complex using a proteomic approach. Mechanistically, MEK1 interacted with MondoA and enhanced tyrosine 222, but not serine or threonine, phosphorylation of MondoA, inhibiting MondoA nuclear translocation and transcriptional activity. Ketone bodies decreased MEK1-dependent MondoA phosphorylation by blocking MondoA and MEK1 interaction, leading to MondoA nuclear translocation, TXNIP transcription, and inhibition of glucose uptake. Therefore, our study not only demonstrated that ketone bodies reduce glucose uptake, promote apoptosis, and inhibit cell proliferation in colorectal cancer cells by regulating MondoA phosphorylation but also identified MEK1-dependent phosphorylation as a new mechanism to manipulate MondoA activity.

The E3 ubiquitin ligase WWP2 regulates pro-fibrogenic monocyte infiltration and activity in heart fibrosis

Non-ischemic cardiomyopathy (NICM) can cause left ventricular dysfunction through interstitial fibrosis, which corresponds to the failure of cardiac tissue remodeling. Recent evidence implicates monocytes/macrophages in the etiopathology of cardiac fibrosis, but giving their heterogeneity and the antagonizing roles of macrophage subtypes in fibrosis, targeting these cells has been challenging. Here we focus on WWP2, an E3 ubiquitin ligase that acts as a positive genetic regulator of human and murine cardiac fibrosis, and show that myeloid specific deletion of WWP2 reduces cardiac fibrosis in hypertension-induced NICM. By using single cell RNA sequencing analysis of immune cells in the same model, we establish the functional heterogeneity of macrophages and define an early pro-fibrogenic phase of NICM that is driven by Ccl5-expressing Ly6chigh monocytes. Among cardiac macrophage subtypes, WWP2 dysfunction primarily affects Ly6chigh monocytes via modulating Ccl5, and consequentially macrophage infiltration and activation, which contributes to reduced myofibroblast trans-differentiation. WWP2 interacts with transcription factor IRF7, promoting its non-degradative mono-ubiquitination, nuclear translocation and transcriptional activity, leading to upregulation of Ccl5 at transcriptional level. We identify a pro-fibrogenic macrophage subtype in non-ischemic cardiomyopathy, and demonstrate that WWP2 is a key regulator of IRF7-mediated Ccl5/Ly6chigh monocyte axis in heart fibrosis.

Multi-target regulation of pro-inflammatory cytokine production by transcription factor Blimp-1

ObjectiveCytokine storm syndrome is a fatal condition related to infectious and autoimmune diseases. Here, we aim to investigate the regulatory mechanisms of Blimp-1 on multiple cytokine production.MethodsThe Blimp1 shRNA was transfected into RAW264.7 macrophages, followed by Toll-like receptor (TLR) ligand stimulation. The mRNA and protein levels of cytokines were detected by real-time PCR and flow cytometric bead array. The nuclear translocation of AP-1 and NF-κB p65 was measured by immunofluorescence staining. The transcriptional activity was detected by luciferase reporter assay with 5 × NF-κB reporter or with IL6 promoter reporter.ResultsBlimp-1 significantly inhibited the expression and secretion of IL-1β, IL-6, and IL-18 in macrophages during stimulation with a variety of TLR ligands. The immunofluorescence staining results showed that Blimp-1 strictly controlled the nuclear translocation of NF-κB p65 in LPS-challenged macrophages. Furthermore, Blimp-1 directly inhibited the transcriptional activity of NF-κB and the transcription of IL6 gene.ConclusionBlimp-1 represses the production of multiple pro-inflammatory cytokines by directly binding the genomic region and restricting the nuclear translocation and transcriptional activity of NF-κB. This finding may provide potential therapeutic strategies for the cytokine storm-related diseases.