NCOA4-mediated Ferritinophagy Research Articles

L-Citrulline Supplementation Restrains Ferritinophagy-Mediated Ferroptosis to Alleviate Iron Overload-Induced Thymus Oxidative Damage and Immune Dysfunction.

L-citrulline (L-cit) is a key intermediate in the urea cycle and is known to possess antioxidant and anti-inflammation characteristics. However, the role of L-cit in ameliorating oxidative damage and immune dysfunction against iron overload in the thymus remains unclear. This study explored the underlying mechanism of the antioxidant and anti-inflammation qualities of L-cit on iron overload induced in the thymus. We reported that L-cit administration could robustly alleviate thymus histological damage and reduce iron deposition, as evidenced by the elevation of the CD8+ T lymphocyte number and antioxidative capacity. Moreover, the NF-κB pathway, NCOA4-mediated ferritinophagy, and ferroptosis were attenuated. We further demonstrated that L-cit supplementation significantly elevated the mTEC1 cells' viability and reversed LDH activity, iron levels, and lipid peroxidation caused by FAC. Importantly, NCOA4 knockdown could reduce the intracellular cytoplasmic ROS, which probably relied on the Nfr2 activation. The results subsequently indicated that NCOA4-mediated ferritinophagy was required for ferroptosis by showing that NCOA4 knockdown reduced ferroptosis and lipid ROS, accompanied with mitochondrial membrane potential elevation. Intriguingly, L-cit treatment significantly inhibited the NF-κB pathway, which might depend on restraining ferritinophagy-mediated ferroptosis. Overall, this study indicated that L-cit might target ferritinophagy-mediated ferroptosis to exert antioxidant and anti-inflammation capacities, which could be a therapeutic strategy against iron overload-induced thymus oxidative damage and immune dysfunction.

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TRIM7 modulates NCOA4-mediated ferritinophagy and ferroptosis in glioblastoma cells

ObjectiveGlioblastoma is one of the most common intracranial malignant tumors with an unfavorable prognosis, and iron metabolism as well as ferroptosis are implicated in the pathogenesis of glioblastoma. The present study aims to decipher the role and mechanisms of tripartite motif-containing protein 7 (TRIM7) in ferroptosis and glioblastoma progression. MethodsStable TRIM7-deficient or overexpressing human glioblastoma cells were generated with lentiviral vectors, and cell survival, lipid peroxidation and iron metabolism were evaluated. Immunoprecipitation, protein degradation and ubiquitination assays were performed to demonstrate the regulation of TRIM7 on its candidate proteins. ResultsTRIM7 expression was elevated in human glioblastoma cells and tissues. TRIM7 silence suppressed growth and induced death, while TRIM7 overexpression facilitated growth and inhibited death of human glioblastoma cells. Meanwhile, TRIM7-silenced cells exhibited increased iron accumulation, lipid peroxidation and ferroptosis, which were significantly reduced by TRIM7 overexpression. Mechanistically, TRIM7 directly bound to and ubiquitinated nuclear receptor coactivator 4 (NCOA4) using K48-linked chains, thereby reducing NCOA4-mediated ferritinophagy and ferroptosis of human glioblastoma cells. Moreover, we found that TRIM7 deletion sensitized human glioblastoma cells to temozolomide therapy. ConclusionWe for the first time demonstrate that TRIM7 modulates NCOA4-mediated ferritinophagy and ferroptosis in glioblastoma cells, and our findings provide a novel insight into the progression and treatment for human glioblastoma.

NCOA4-mediated ferritinophagy is involved in ionizing radiation-induced ferroptosis of intestinal epithelial cells

Ferroptosis is a newly recognized form of regulated cell death that is characterized by severe lipid peroxidation initiated by iron overload and the generation of reactive oxygen species (ROS). However, the role of iron in ionizing radiation (IR)-induced intestinal injury has not been fully illustrated yet. In this study, we found that IR induced ferroptosis in intestinal epithelial cells, as indicated by the increase in intracellular iron levels and lipid peroxidation, upregulation of prostaglandin-endoperoxide synthase 2 (PTGS2) mRNA, reduced glutathione peroxidase 4 (GPX4) mRNA and glutathione (GSH) levels, and significant mitochondrial damage. In addition, the iron chelator deferoxamine (DFO) attenuated IR-induced ferroptosis and intestinal injury in vitro and in vivo. Intriguingly, pharmacological inhibition of autophagy with 3-methyladenine (3-MA) mitigated IR-induced ferritin downregulation, iron overload and ferroptosis. IR increased the levels of nuclear receptor coactivator 4 (NCOA4) mRNA and protein. NCOA4 knockdown significantly inhibited the reduction of ferritin, decreased the level of intracellular free iron, and mitigated ferroptosis induced by IR in HIEC cells, indicating that NCOA4-mediated autophagic degradation of ferritin (ferritinophagy) was required for IR-induced ferroptosis. Furthermore, cytoplasmic iron further activated mitoferrin2 (Mfrn2) on the mitochondrial membrane, which in turn increased iron transport into the mitochondria, resulting in increased ROS production and ferroptosis. In addition, mice fed with an iron-deficient diet for 3 weeks showed a significant reversal in the intestinal injury induced by abdominal IR exposure. Taken together, ferroptosis is a novel mechanism of IR-induced intestinal epithelial cytotoxicity, and is dependent on NCOA4-mediated ferritinophagy.

Tetrandrine Citrate Suppresses Breast Cancer via Depletion of Glutathione Peroxidase 4 and Activation of Nuclear Receptor Coactivator 4-Mediated Ferritinophagy.

Tetrandrine citrate (TetC), a novel tetrandrine salt with high water solubility, demonstrates a potent antitumor activity in chronic myeloid leukemia. Studies have indicated an important role of ferroptosis in breast cancer (BC). However, whether TetC inhibits BC progression via ferroptosis has never been explored. In the present study, we showed that TetC had a significant inhibitory effect on the proliferation and migration of MCF7 and MDA-MB-231 cells. Then, we combined TetC with different inhibitors to determine which form of cell death could be driven by TetC. MTT assay showed that ferrostatin (Fer-1) demonstrated the most potent effect on improving TetC-induced cell death in contrast to other inhibitors. TetC was also shown to significantly increase the mRNA level of prostaglandin-endoperoxide synthase 2 (Ptgs2), a ferroptosis marker. Further studies showed that TetC significantly suppressed the expression of glutathione peroxidase 4 (GPX4) and ferritin heavy chain 1 (FTH1) but increased the expression of nuclear receptor coactivator 4 (NCOA4) in MCF7 and MDA-MB-231 cells even in the presence of erastin or Ras-selective lethal 3 (RSL3). Collectively, we showed novel data that ferroptosis was a major form of TetC-induced cell death. Moreover, TetC-induced ferroptotic cell death was achieved via suppressing GPX4 expression and activating NCOA4-mediated ferritinophagy in BC cells.

Iron Administration Overcomes Resistance to Erastin-Mediated Ferroptosis in Ovarian Cancer Cells.

ObjectivesDeveloping novel therapeutic approaches to defeat chemoresistance is the major goal of ovarian cancer research. Induction of ferroptosis has shown promising antitumor effects in ovarian cancer cells, but the existence of still undefined genetic and metabolic determinants of susceptibility has so far limited the application of ferroptosis inducers in vivo.MethodsErastin and/or the iron compound ferlixit were used to trigger ferroptosis in HEY, COV318, PEO4, and A2780CP ovarian cancer cell lines. Cell viability and cell death were measured by MTT and PI flow cytometry assay, respectively. The “ballooning” phenotype was tested as ferroptosis specific morphological feature. Mitochondrial dysfunction was evaluated based on ultrastructural changes, mitochondrial ROS, and mitochondrial membrane polarization. Lipid peroxidation was tested through both C11-BODIPY and malondialdehyde assays. VDAC2 and GPX4 protein levels were quantified as additional putative indicators of mitochondrial dysfunction or lipid peroxidation, respectively. The effect of erastin/ferlixit treatments on iron metabolism was analyzed by measuring intracellular labile iron pool and ROS. FtH and NCOA4 were measured as biomarkers of ferritinophagy.ResultsHere, we provide evidence that erastin is unable to induce ferroptosis in a series of ovarian cancer cell lines. In HEY cells, provided with a high intracellular labile iron pool, erastin treatment is accompanied by NCOA4-mediated ferritinophagy and mitochondrial dysfunction, thus triggering ferroptosis. In agreement, iron chelation counteracts erastin-induced ferroptosis in these cells. COV318 cells, with low baseline intracellular labile iron pool, appear resistant to erastin treatment. Notably, the use of ferlixit sensitizes COV318 cells to erastin through a NCOA4-independent intracellular iron accumulation and mitochondrial dysfunction. Ferlixit alone mimics erastin effects and promotes ferroptosis in HEY cells.ConclusionThis study proposes both the baseline and the induced intracellular free iron level as a significant determinant of ferroptosis sensitivity and discusses the potential use of ferlixit in combination with erastin to overcome ferroptosis chemoresistance in ovarian cancer.

Modulation of the HIF2α-NCOA4 axis in enterocytes attenuates iron loading in a mouse model of hemochromatosis

AbstractIntestinal iron absorption is activated during increased systemic demand for iron. The best-studied example is iron deficiency anemia, which increases intestinal iron absorption. Interestingly, the intestinal response to anemia is very similar to that of iron overload disorders, as both the conditions activate a transcriptional program that leads to a hyperabsorption of iron via the transcription factor hypoxia-inducible factor 2α (HIF2α). However, pathways for selective targeting of intestine-mediated iron overload remain unknown. Nuclear receptor coactivator 4 (NCOA4) is a critical cargo receptor for autophagic breakdown of ferritin and the subsequent release of iron, in a process termed ferritinophagy. Our work demonstrates that NCOA4-mediated intestinal ferritinophagy is integrated into systemic iron demand via HIF2α. To demonstrate the importance of the intestinal HIF2α/ferritinophagy axis in systemic iron homeostasis, whole-body and intestine-specific NCOA4−/− mouse lines were generated and assessed. The analyses revealed that the intestinal and systemic response to iron deficiency was not altered after disruption of intestinal NCOA4. However, in a mouse model of hemochromatosis, ablation of intestinal NCOA4 was protective against iron overload. Therefore, NCOA4 can be selectively targeted for the management of iron overload disorders without disrupting the physiological processes involved in the response to systemic iron deficiency.

NCOA4-Mediated Ferritinophagy: A Vicious Culprit in COVID-19 Pathogenesis?

Coronavirus disease 2019 (COVID-19) is a global pandemic that has caused widespread loss of life. Notably, in this disease, severe inflammatory reactions characterized by cytokine storms are caused by severe acute respiratory syndrome coronavirus 2. The cytokine storms may promote hyper-ferritinemia which can further intensify the inflammation. Moreover, elevated ferritin levels trigger nuclear receptor coactivator 4 (NCOA4)-mediated ferritinophagy, in which ferritin is degraded and iron is released. Excess iron released from ferritinophagy can promote ferroptosis and cellular damage. Therefore, we propose that NCOA4-mediated ferritinophagy can be targeted to limit the ferroptosis and prevent the multi-organ damage and severity in COVID-19 patients.

Inhibition of DNMT-1 alleviates ferroptosis through NCOA4 mediated ferritinophagy during diabetes myocardial ischemia/reperfusion injury

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.

The Macrophage Nuclear Receptor Coactivator 4 Counteracts Iron-Deficiency Anemia

The Nuclear Receptor Coactivator 4 (NCOA4) protein has been recently recognized as a novel player in iron metabolism contributing to regulation of cell and systemic iron homeostasis. NCOA4 is a cargo receptor that promotes selectively autophagy of the iron storage ferritin in conditions of iron deficiency, facilitating iron recovery from cellular stores. Inactivation of NCOA4 in cells increases ferritin aggregates [Mancias JD et al., Nature 2014; Dowdle WE et al., Nat Cell Biol 2014; Mancias JD et al., Elife 2015] and C57Bl/6 Ncoa4-ko mice show ferritin and iron accumulation in several organs, in particular spleen [Dowdle WE et al., Nat Cell Biol 2014; Bellelli R et al., Cell Rep 2016]. In these animals, iron retention in tissue ferritin reduces iron availability for erythropoiesis causing mild microcytic anaemia in iron-balanced (IB) conditions. In addition, compared to wt animals, Ncoa4-ko mice fed an iron-deficient (ID) diet develop a more severe anemia [Bellelli R et al., Cell Rep 2016].Given the important impact of mouse strain on iron metabolism, we evaluated the ability of Ncoa4-ko mice on a different background to respond to variation of dietary iron content. To this aim wt and null mice on Sv129/J background were fed an IB or an ID diet for 6 months. The ID diet induced almost no changes in hemoglobin levels of wt mice (IB=14.0±1.9 g/dL; ID=13.3±2.9 g/dL), while Ncoa4-ko developed severe microcytic anemia (Hb: IB=13.5±2.0 g/dL, ID=6.7±2.9 g/dL, p<0.05; MCV: IB=47.8±1.3 fL, ID=36.5±4.8 fL, p<0.05). These results are consistent with wt mice on Sv129/J strain being able to counteract ID by using the large reserves of iron present in their tissues. Notwithstanding such high iron deposits, Sv129/J Ncoa4 -ko fail this compensation. To better evaluate the bone marrow (BM)-derived cells (erythroid precursors, BM and spleen macrophages) contribution to the inability of Ncoa4-ko mice to respond to variation of dietary iron in comparison to other tissues (eg. liver and duodenum), we performed BM transplantation experiments in Ncoa4-ko using Sv129/J background. Lethally irradiated Ncoa4-ko mice were transplanted with wt or Ncoa4-ko BM, generating chimeric null animals with wt (Ncoa4-kowt-BM) or Ncoa4-ko BM cellsas control (Ncoa4-koko-BM). Previously published results obtained in vitro and in vivo in zebrafish suggest an essential role for NCOA4 in erythroid cells development and hemoglobinization [Mancias JD et al., Elife 2015; Ryu MS et al., JCI 2017]. However, we observed that Ncoa4-ko BM cells were able to reconstitute an almost normal erythropoiesis in lethally irradiated mice, indicating that in vivo the loss of Ncoa4 has a limited, if any, effect on the erythroid lineage differentiation. In addition, Ncoa4-ko mice with wt bone marrow have normal hematological parameters, suggesting that the mild microcytosis observed in Ncoa4-ko animals in basal conditions is due to a defect of BM-derived cells. Theoretically this effect may be due to cells belonging either to the erythroid or to the macrophage lineage; based on our findings we suggest a prevalent role for macrophages. In line with this hypothesis, Ncoa4-koko-BM mice fed an ID diet for 3 months developed a more severe anemia than Ncoa4-kowt-BM (Hb= 10.7±0.6 g/dL vs 11.7±1.0 g/dL; p=0.06), accompanied by comparable liver (LIC: Ncoa4-koko-BM=174.8±50.4 μg/g; Ncoa4-kowt-BM=157.1±49.5 μg/g) but higher spleen iron concentration (SIC: Ncoa4-koko-BM=719.0±221.3 μg/g; Ncoa4-kowt-BM=176.1±74.6 μg/g; p<0.05), consistent with decreased iron recycling by spleen macrophages of Ncoa4-koko-BM animals.Over all, our results suggest that NCOA4-mediated ferritinophagy plays a central role in macrophages in counteracting dietary ID, promoting iron release and ensuring efficient erythropoiesis. DisclosuresCamaschella:Vifor Pharma: Membership on an entity's Board of Directors or advisory committees.

Ferritinophagy is involved in the zinc oxide nanoparticles-induced ferroptosis of vascular endothelial cells

ABSTRACT 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.

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

Nuclear receptor coactivator 4 (NCOA4)-mediated ferritinophagy has been implicated in the ferroptosis in cancer cells and hematopoiesis in the bone marrow. However, the role of iron metabolism, especially NCOA4-mediated degradation of ferritin, has not been explored in the proliferation of mesenchymal stem cells. The present study was designed to explore the role of NCOA4-mediated ferritinophagy in hypoxia-treated dental pulp stem cells (DPSCs). Hypoxia treatment increased ROS generation, boosted cytosolic labile iron pool, increased expression of transferrin receptor 1 and NCOA4. Moreover, colocalization of LC3B with NCOA4 and ferritin was observed in hypoxia-treated DPSCs, indicating the development of ferritinophagy. Hypoxia promoted the proliferation of DPSCs, but not ferroptosis, under normal serum supplement and serum deprivation. NCOA4 knock-down reduced ferritin degradation and inhibited proliferation of DPSCs under hypoxia. Furthermore, the activation of hypoxia inducible factor 1α and p38 mitogen-activated protein kinase signaling pathway was involved in the upregulation of NCOA4 in hypoxia. Therefore, our present study suggested that NCOA4-mediated ferritinophagy promoted the level of labile iron pool, leading to enhanced iron availability and elevated cell proliferation of DPSCs. Our present study uncovered a physiological role of ferritinophagy in the proliferation and growth of mesenchymal stem cells under hypoxia.

Dexmedetomidine reverses MTX-induced neurotoxicity and inflammation in hippocampal HT22 cell lines via NCOA4-mediated ferritinophagy.

The incidence of chemotherapy-induced cognitive impairment (CICI) has attracted massive attention. Some studies have demonstrated the neuroprotective effects of dexmedetomidine (DEX). Here, alterations in nuclear receptor coactivator 4 (NCOA4)-mediated ferritinophagy were investigated as the possible causes of DEX’s neuroprotection of HT22 cells against methotrexate (MTX)-induced neurotoxicity. We used various concentrations of DEX and NCOA4-siRNA to treat MTX-induced neurotoxicity and inflammation in HT22 cells. The biomarkers of HT22 cells viability, apoptosis and inflammatory were tested. The expression of ferritinophagy markers were detected in the HT22 cells by using western blot and Immunofluorescence. We found that 10 and 50 ng/mL of DEX alleviated MTX-induced hippocampal neuronal inflammatory injuries. Meanwhile, DEX also reversed MTX-induced iron and ROS overproduction. Increasing DEX concentrations caused significant falls in the expression of ferritin heavy chain 1 (FTH1). DEX also increased vital ferritinophagy markers, NCOA4 and LC3II. NCOA4-siRNA transfection annulled the neuroprotective effects of DEX on MTX-induced inflammation in HT22 cells. Additionally, because NCOA4-siRNA disrupted ferritinophagy, DEX’s inhibitory impact on MTX-induced iron and ROS overproduction in HT22 cells was also annihilated. DEX weakened MTX-provoked neurontoxicity in HT22 cells, possibly by improving NCOA4-mediated ferritinophagy. Our discoveries present further mechanisms for understanding the protective effects of DEX against MTX-induced cognitive impairment.

NCOA4-mediated ferritinophagy promoted inflammatory responses in periodontitis.

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.

The Role of NCOA4-Mediated Ferritinophagy in Ferroptosis.

Nuclear receptor coactivator 4 (NCOA4) is a selective cargo receptor that mediates the autophagic degradation of ferritin, the cytosolic iron storage complex, in a process known as ferritinophagy. NCOA4-mediated ferritinophagy is required to maintain intracellular and systemic iron homeostasis and thereby iron-dependent physiologic processes such as erythropoiesis. Given this role of ferritinophagy in regulating iron homeostasis, modulating NCOA4-mediated ferritinophagic flux alters sensitivity to ferroptosis, a non-apoptotic iron-dependent form of cell death triggered by peroxidation of polyunsaturated fatty acids (PUFAs). A role for ferroptosis has been established in the pathophysiology of cancer and neurodegeneration; however, the importance of ferritinophagy in these pathologies remains largely unknown. Here, we review the available evidence on biochemical regulation of NCOA4-mediated ferritinophagy and its role in modulating sensitivity to innate and induced ferroptosis in neurodegenerative diseases and cancer. Finally, we evaluate the potential of modulating ferritinophagy in combination with ferroptosis inducers as a therapeutic strategy.

NCOA4-mediated ferritinophagy promotes ferroptosis induced by erastin, but not by RSL3 in HeLa cells

Ferroptosis is a regulated cell death characterized by a lethal accumulation of lipid peroxides due to an increase of intracellular iron and a decrease of antioxidant capacity. The reduction of antioxidant activity is obtained by using chemical agents, such as erastin and RSL3, the first one inhibiting the transmembrane cystine-glutamate antiporter causing a cysteine and glutathione depletion and the second one inactivating directly the glutathione peroxidase 4 (GPX4) respectively. The role of iron and its related proteins in supporting the formation of lipid peroxides, is not completely understood hence to try to shed light on it we generated HeLa clones with altered ferritinophagy, the ferritin degradation process, by knocking-out or overexpressing Nuclear Receptor Coactivator 4 (NCOA4), the ferritin autophagic cargo-receptor. NCOA4 deficiency abolished ferritinophagy increasing ferritin level and making the cells more resistant to erastin, but unexpectedly more sensitive to RSL3. Interestingly, we found that erastin promoted ferritinophagy in HeLa cells expressing NCOA4, increasing the free iron, lipid peroxidation and the sensitivity to ferroptosis. In contrast, RSL3 did not modulate ferritinophagy, while NCOA4 overexpression delayed RSL3-induced cell death suggesting that RSL3 mechanism of action is independent of ferritin degradation process. Therefore, the ferritin-iron release in the execution of ferroptosis seems to depend on the inducing compound, its target and downstream pathway of cell death activation.

Periodontitis-level butyrate-induced ferroptosis in periodontal ligament fibroblasts by activation of ferritinophagy

Loss of periodontal ligament fibroblasts (PDLFs) is one critical issue for regenerating lost periodontal tissues. A wide variety of regulated cell death pathways, such as apoptosis, pyroptosis, and necroptosis have been proposed in the periodontitis development. The aim of the present study was to explore whether long-term periodontitis-level butyrate may trigger ferroptosis, a newly characterized iron-dependent regulated cell death in PDLFs. Here, we showed that long-term treatment of butyrate, an important short-chain fatty acid in the periodontal pocket, induces the cargo receptor nuclear receptor coactivator 4 (NCOA4)-mediated ferritinophagy and ferroptosis in PDLFs. Butyrate-induced iron accumulation, reactive oxygen species (ROS) generation, glutathione depletion and lipid peroxidation in PDLFs, and the butyrate-induced ferroptosis can be blocked by the lipid peroxide scavenger ferrostatin-1. The NCOA4-mediated ferritinophagy is dependent on p38/hypoxia inducible factor-1α (HIF-1α) pathway activation as well as Bromodomain-containing protein (BRD) 4 and cyclin-dependent kinase 9 (CDK9) coordination. These lines of evidence provide a new mechanistic insight into the mechanism of loss of PDLFs during periodontitis development, showing that periodontitis-level butyrate disrupted iron homeostasis by activation of NCOA4-mediated ferritinophagy, leading to ferroptosis in PDLFs.

NCOA4-mediated ferritinophagy in macrophages is crucial to sustain erythropoiesis in mice.

Nuclear receptor coactivator 4 (NCOA4) promotes ferritin degradation and Ncoa4-ko mice in a C57BL/6 background show microcytosis and mild anemia, aggravated by iron deficiency. To understand tissue-specific contributions of NCOA4-mediated ferritinophagy we explored the effect of Ncoa4 genetic ablation in the iron-rich Sv129/J strain. Increased body iron content protects these mice from anemia and, in basal conditions, Sv129/J Ncoa4-ko mice show only microcytosis; nevertheless, when fed a low-iron diet they develop a more severe anemia compared to that of wild-type animals. Reciprocal bone marrow (BM) transplantation from wild-type donors into Ncoa4-ko and from Ncoa4-ko into wild-type mice revealed that microcytosis and susceptibility to iron deficiency anemia depend on BM-derived cells. Reconstitution of erythropoiesis with normalization of red blood count and hemoglobin concentration occurred at the same rate in transplanted animals independently of the genotype. Importantly, NCOA4 loss did not affect terminal erythropoiesis in iron deficiency, both in total and specific BM Ncoa4-ko animals compared to controls. On the contrary, upon a low iron diet, spleen from wild-type animals with Ncoa4-ko BM displayed marked iron retention compared to (wild-type BM) controls, indicating defective macrophage iron release in the former. Thus, erythropoietin administration failed to mobilize iron from stores in Ncoa4-ko animals. Furthermore, Ncoa4 inactivation in thalassemic mice did not worsen the hematologic phenotype. Overall our data reveal a major role for NCOA4-mediated ferritinophagy in macrophages to favor iron release for erythropoiesis, especially in iron deficiency.

Polybrominated Diphenyl Ethers Quinone Induces NCOA4-Mediated Ferritinophagy through Selectively Autophagic Degradation of Ferritin.

Polybrominated diphenyl ethers (PBDEs) have been detected ubiquitously in biological and environmental samples. Growing epidemiological data suggested the obvious correlation of PBDEs exposure with adverse health outcomes toward human beings, but exact molecular mechanism(s) are limited. Especially, the toxicological information regarding PBDEs metabolites is missing. Thereafter, this study intends to explore unidentified cell death modalities caused by PBDEs reactive quinone-type metabolite, PBDEQ. We found that PBDEQ induces autophagy in an ROS-dependent manner. Interestingly, the results indicated that PBDEQ degraded ferritin and activated a selective autophagy (termed as ferritinophagy) by using NCOA4 as its cargo receptor. These processes may further promote the release of iron and ROS. These results suggested the incidence of ferritinophagy induced by PBDEQ, which may contribute to PBDE exposure-caused diseases and dysfunctions.

NCOA4-Mediated Ferritinophagy Is Essential for HT22 Neuronal Cell Survival During Iron Deficiency (OR15-07-19)

ObjectivesIron is essential for proper cell function and development. However, mishandled iron may lead to ferroptotic cell death. NCOA4 manages the cellular labile iron pool by controlling the release of ferritin iron via ferritinophagy. The present studies examined the capacity of hippocampal cells in handling iron fluctuation, with particular focus on NCOA4 and ferritin. MethodsHT22 mouse hippocampal cells were treated with ferric ammonium citrate (FAC) and deferoxamine (Dfo) to produce cellular iron overload and deprivation, respectively. Ferroptosis was determined by measures of ferrostatin-1 effects and Ptgs2 mRNA. For ferritinophagy studies, Ncoa4 was silenced by siRNA transfections. Functional impacts of impaired ferritinophagy were assessed via CCK-8 cell viability assays and western and qPCR analyses of iron-related genes. ResultsHT22 cells were highly susceptible to cellular iron overload. FAC-treated cells featured acute morphological changes, decreased viability, and elevated Ptgs2 mRNA abundance. Iron effects were prevented by ferrostatin-1, indicating ferroptosis by cellular iron overload. Dfo alone had minimal impact on cell morphology and viability. NCOA4 protein, but not mRNA, levels were acutely upregulated by Dfo treatments. Ferritin turnover by iron deficiency was impaired in NCOA4-depleted cells, presumably due to impaired ferritinophagy. Moreover, HT22 cells became sensitive to iron deficiency by loss of NCOA4. ConclusionsOur studies demonstrate iron can induce ferroptosis in neuronal cells. We also identify NCOA4-mediated ferritinophagy as an integral process for neuronal cell survival during iron deficiency. Further investigation using multi-omics approaches are in progress to determine the mechanisms by which NCOA4 depletion leads to cell death when extracellular iron supply is limited. Funding SourcesSupported by the NIFA, USDA, Hatch project under MIN-18–118 and intramural support to M-S.R.