Nitrated Nuclear Receptor Coactivator 4 Contributes to Ferritinophagy in Hyperhomocysteinemia-Induced Liver Injury.

Ferritinophagy-derived iron causes protein nitration and mitochondrial dysfunction in acetaminophen-induced liver injury.

NCOA4 Acts in Hepatocytes to Regulate Ferritin Subunit Levels and Hepatic Iron Storage

NCOA4 Links Iron Bioavailability to DNA Metabolism

Background: Deoxynivalenol (DON) contamination, pervasive throughout all stages of food production and processing, presents a significant threat to human health. The degradation of ferritin mediated by nuclear receptor coactivator 4 (NCOA4), termed ferritinophagy, plays a crucial role in maintaining iron homeostasis and regulating ferroptosis. Aim: This study aims to elucidate the role of ferritinophagy and ferroptosis in DON-induced liver injury. Methods: Male mice and AML12 cells were subjected to varying doses of DON, serving as in vivo and in vitro models, respectively. Protein expression was assessed by using immunofluorescence and western blot techniques. Co-immunoprecipitation was employed to investigate the protein-protein interactions. Results: Our findings demonstrate that DON triggers hepatocyte ferroptosis in a ferritinophagy-dependent manner. Specifically, DON impedes the activation of the mammalian target of rapamycin complex 1 (mTORC1) by inhibiting RAC1's binding to mTOR, thereby ultimately inducing autophagy. Concurrently, DON amplifies NCOA4's affinity for ferritin by facilitating NCOA4 phosphorylation through the ataxia-telangiectasia mutated kinase (ATM), thus promoting the autophagy-dependent degradation of ferritin. Both autophagy inhibition and NCOA4 expression suppression ameliorate DON-induced ferroptosis. Conclusion: Our study concludes that DON facilitates NCOA4-mediated ferritinophagy via the ATM-NCOA4 pathway, subsequently inducing ferroptosis in the liver.

Iron homeostasis plays a crucial role in the combat against pathogen invasion. Ferrous iron can trigger generous production of reactive oxygen species (ROS) by Fenton reaction. Nuclear receptor coactivator 4 (NCOA4), a selectivecargoreceptor to deliver ferritin to lysosome, may trigger release of ferritin-bound iron into the cytosol. The aim of the present study was to explore whether NCOA4-mediated ferritinophagy participated in the pathogenesis of periodontitis, and its role in promoting the periodontal inflammation. Inflamed and healthy periodontal tissues were harvested for immunobiological staining of ferritinophagy-related genes in the periodontal tissues, while real-time quantitative PCR (qPCR) was utilized to detect mRNA transcription. Periodontal ligament fibroblasts (PDLFs) were isolated and infected with Porphyromonas gingivalis. The mRNA transcription and protein expression of genes involved in the iron metabolism, including NCOA4, transferrin receptor 1 (TFR1), and ferroportin (SLC40A1) were detected by qPCR and western blot. Levels of labile iron pool and ROS production were detected by flow cytometry and confocal endoscopy. Small interference RNA was utilized to knock down NCOA4. Elevated expression of NCOA4, ferritin heavy chain, and light chain were observed in the diseased periodontal tissues. P. gingivalis infection promoted expression of TFR1, NCOA4, and microtubule-associated protein 1-light chain 3 B (LC3B), enhanced levels of intracellular labile iron pool and ROS production. NCOA4 knockdown reduced ROS generation in PDLFs in response to P. gingivalis and mitigated production of pro-inflammatory monocyte chemoattractant protein-1 and interleukin 6. P. gingivalis triggered activation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase signaling pathway. In addition, inhibitors of JNK, SP600125, and inhibitors of p38, SB203580 blocked NCOA4 transcription. NCOA4-ferritinophagy participated in the progress of periodontitis progression. P. gingvalis-triggered ferritinophagy aggravated production of ROS and inflammatory responses in PDLFS. These findings suggest iron homeostasis plays an important role in the pathogenesis of periodontitis.

STAT3 signaling promotes cardiac injury by upregulating NCOA4-mediated ferritinophagy and ferroptosis in high-fat-diet fed mice.

Nuclear receptor coactivator 4 (NCOA4) protein is a selective cargo receptor that plays a crucial role in ferritinophagy by targeting and delivering the ferritin iron storage protein to lysosomes for degradation and releasing iron. TRIM7 overexpression inhibits ferroptosis in glioblastoma cells by ubiquitinating NCOA4 protein.

NCOA4 is regulated by HIF and mediates mobilization of murine hepatic iron stores after blood loss

Background/Objectives: Atherosclerosis (AS)-related cardiovascular diseases are a major global health threat, with vascular smooth muscle cells (VSMCs) phenotypic switching, abnormal proliferation, and migration as key progression drivers. Nuclear receptor coactivator 4 (NCOA4), a core ferritinophagy mediator overexpressed in AS plaques, may promote VSMCs ferroptosis by perturbing mitochondrial iron metabolism and ROS homeostasis, but precise mechanisms remain unclear. The classic Chinese herbal combination "Gualou-Xiebai" (GLXB) has anti-AS effects, yet how it modulates NCOA4-mediated ferroptosis to inhibit VSMCs' functions is unknown. This study addresses this gap to advance GLXB's therapeutic potential and identify AS targets. Methods: An AS model was established in ApoE-/- mice by 12-week high-fat diet feeding, with model validation confirmed via ultrasound monitoring and H&E staining. NCOA4 was genetically modulated (knockdown and overexpression) to assess its role in plaque formation and lipid deposition using H&E staining, aortic imaging, immunofluorescence, and Western blotting. In vitro, VSMCs were stimulated with ox-LDL to induce proliferation and migration. NCOA4 was silenced using siRNA to examine associated ferroptosis levels and molecular mechanisms. Protein interactions between NCOA4 and the mitochondrial iron storage protein FTMT were evaluated by Co-IP and GST pull-down assays, while mitochondrial ROS (mitoROS) levels were measured to explore functional relationships. The extent of ferroptosis and the underlying regulatory mechanisms were assessed following treatment with GLXB-containing serum or transfection with small interfering RNA targeting LOX-1 (si-LOX-1). Results: NCOA4 knockdown reduced aortic lipid deposition, plaque burden, VSMC proliferation/migration, and mitochondrial ferroptosis. NCOA4 bound and suppressed FTMT, inducing mitochondrial iron overload, ROS accumulation, membrane depolarization, and ferroptosis. Combining NCOA4 silencing with FTMT inhibition elevated mitoROS, confirming the axis's role in iron homeostasis. GLXB attenuated VSMCs dysregulation in vivo and in vitro, an effect abrogated by LOX-1 overexpression. Conclusions: NCOA4 promotes AS by binding FTMT, disrupting mitochondrial iron homeostasis, and triggering VSMCs ferroptosis. GLXB inhibits LOX-1-mediated NCOA4 expression, mitigating ferroptosis and VSMCs dysregulation, supporting its potential as a targeted anti-AS therapy.

Zinc oxide nanoparticles (ZnONPs) hold great promise for biomedical applications. Previous studies have revealed that ZnONPs exposure can induce toxicity in endothelial cells, but the underlying mechanisms have not been fully elucidated. In this study, we report that ZnONPs can induce ferroptosis of both HUVECs and EA.hy926 cells, as evidenced by the elevation of intracellular iron levels, lipid peroxidation and cell death in a dose- and time-dependent manner. In addition, both the lipid reactive oxygen species (ROS) scavenger ferrostatin-1 and the iron chelator deferiprone attenuated ZnONPs-induced cell death. Intriguingly, we found that ZnONPs-induced ferroptosis is macroautophagy/autophagy-dependent, because the inhibition of autophagy with a pharmacological inhibitor or by ATG5 gene knockout profoundly mitigated ZnONPs-induced ferroptosis. We further demonstrated that NCOA4 (nuclear receptor coactivator 4)-mediated ferritinophagy (autophagic degradation of the major intracellular iron storage protein ferritin) was required for the ferroptosis induced by ZnONPs, by showing that NCOA4 knockdown can reduce the intracellular iron level and lipid peroxidation, and subsequently alleviate ZnONPs-induced cell death. Furthermore, we showed that ROS originating from mitochondria (mtROS) probably activated the AMPK-ULK1 axis to trigger ferritinophagy. Most importantly, pulmonary ZnONPs exposure caused vascular inflammation and ferritinophagy in mice, and ferrostatin-1 supplementation significantly reversed the vascular injury induced by pulmonary ZnONPs exposure. Overall, our study indicates that ferroptosis is a novel mechanism for ZnONPs-induced endothelial cytotoxicity, and that NCOA4-mediated ferritinophagy is required for ZnONPs-induced ferroptotic cell death. Abbreviations: 3-MA: 3-methyladenine; ACTB: Actin beta; AMPK: AMP-activated protein kinase; ATG: Autophagy-related; BafA1: Bafilomycin A1; CQ: Choloroquine; DFP: Deferiprone; FACS: Fluorescence-activated cell sorting; Fer-1: Ferrostatin-1; FTH1: Ferritin heavy chain 1; GPX4: Glutathione peroxidase 4; GSH: Glutathione; IREB2/IRP2: Iron responsive element binding protein 2; LIP: Labile iron pool; MAP1LC3B/LC3B: Microtubule associated protein 1 light chain 3 beta; MTOR: Mechanistic target of rapamycin kinase; NCOA4: Nuclear receptor coactivator 4; NFE2L2/NRF2: Nuclear factor, erythroid 2 like 2; PGSK: Phen Green™ SK; ROS: Reactive oxygen species; siRNA: Small interfering RNA; SQSTM1/p62: Sequestosome 1; TEM: Transmission electron microscopy; ULK1: Unc-51 like autophagy activating kinase 1; ZnONPs: Zinc oxide nanoparticles.

IntroductionThis study investigates the molecular mechanisms by which excessive autophagy exacerbates post-myocardial infarction heart failure (post-MI HF) through nuclear receptor coactivator 4 (NCOA4)-mediated ferritinophagy and ferroptosis.Material and methodsWe developed a post-MI heart failure model in Sprague-Dawley rats via coronary artery ligation, alongside an in vitro heart failure model using hypoxia/reoxygenation-stimulated H9C2 cells. Intervention with rapamycin (autophagy activator), 3-methyladenine (autophagy inhibitor), desferrioxamine, and ferredoxin-1 (ferroptosis inhibitors) was performed. Various techniques, including echocardiography, immunofluorescence colocalization, C11 BODIPY 581/591 staining, flow cytometry, transmission electron microscopy, western blotting, and RT-qPCR, were employed.ResultsIn vivo analyses revealed that NCOA4-mediated ferritinophagy and ferroptosis are significant in post-MI HF. Manipulating autophagy through rapamycin and 3-methyladenine influenced the expression of NCOA4 and glutathione peroxidase 4 (GPX4), subsequently affecting ferroptosis and modulating heart failure severity. Our in vitro experiments corroborated these findings, demonstrating that heightened autophagy amplifies NCOA4 expression, which in turn fosters ferroptosis and exacerbates myocardial injury. Interestingly, silencing of NCOA4 partially mitigated autophagy-induced iron deficiency, indicating a crucial intersection between autophagy and iron metabolism. Moreover, the cardioprotective effects observed following NCOA4 silencing were negated by concurrent GPX4 silencing.ConclusionsOur findings show that autophagy precedes NCOA4 in its regulatory pathway and directly influences ferritinophagy. Enhanced autophagy augments intracellular free iron and unstable iron pools, triggering lipid peroxidation through ferritinophagy, which promotes ferroptosis and impairs cardiac function. These insights offer a novel scientific basis for developing therapeutic strategies for post-MI HF.

NCOA4-mediated ferritinophagy participates in cadmium-triggered ferroptosis in spermatogonia

The purpose of this study was to investigate whether inhibition of DNA (cytosine-5)-methyltransferase 1 (DNMT-1) alleviated ferroptosis through nuclear receptor coactivator 4 (NCOA4)-mediated ferritinophagy during diabetes myocardial (DM) ischemia/reperfusion (I/R) injury (IRI). Rat DM + sham (DS), I/R, and DM + I/R (DIR), H9c2 cell high glucose (HG), hypoxia reoxygenation (H/R), and high-glucose hypoxia reoxygenation (HH/R) models were established. DNMT-1 inhibitor 5-Aza-2’-deoxycytidine (5-aza-CdR) was administered to rat and cell models. The protein level of DNMT-1, NCOA4, FTH, GPX4, Beclin-1, and P62 was detected by western blotting. Compared with normal sham (NS) group, myocardial tissue was injured in DS and I/R models. The level of DNMT-1, NCOA4, and ferroptosis was increased. Moreover, the cell injury was more serious in rat DIR or HH/R model. 5-Aza-CdR could reduce NCOA4-mediated ferritinophagy and myocardial injury in DIR and HH/R models. Moreover, the siRNA for NCOA4 could also reduce the level of ferritinophagy and cell injury in HH/R model. 5-Aza-CdR enhanced the protective effect for NCOA4-siRNA in the process of cell injury. Inhibition of DNMT-1 could reduce ferroptosis during DIR, which the NCOA4-mediated ferritinophagy might be regulated.

Nuclear receptor coactivator 4-mediated ferritinophagy drives proliferation of dental pulp stem cells in hypoxia

Oxidative medicine and cellular longevity the role and mechanism of NCOA4 in ferroptosis induced by intestinal ischemia reperfusion