Structural basis for the intracellular regulation of ferritin degradation.

The interaction between nuclear receptor coactivator 4 (NCOA4) and the iron storage protein ferritin is a crucial component of cellular iron homeostasis. The binding of NCOA4 to the FTH1 subunits of ferritin initiates ferritinophagy-a ferritin-specific autophagic pathway leading to the release of the iron stored inside ferritin. The dysregulation of NCOA4 is associated with several diseases, including neurodegenerative disorders and cancer, highlighting the NCOA4-ferritin interface as a prime target for drug development. Here, we present the cryo-EM structure of the NCOA4-FTH1 interface, resolving 16 amino acids of NCOA4 that are crucial for the interaction. The characterization of mutants, designed to modulate the NCOA4-FTH1 interaction, is used to validate the significance of the different features of the binding site. Our results explain the role of the large solvent-exposed hydrophobic patch found on the surface of FTH1 and pave the way for the rational development of ferritinophagy modulators.

Steroid receptor coactivators in Treg and Th17 cell biology and function.

Steroid receptor coactivators (SRCs) are master regulators of transcription that play key roles in human physiology and pathology. SRCs are particularly important for the regulation of the immune system with major roles in lymphocyte fate determination and function, macrophage activity, regulation of nuclear factor κB (NF-κB) transcriptional activity and other immune system biology. The three members of the p160 SRC family comprise a network of immune-regulatory proteins that can function independently or act in synergy with each other, and compensate for - or moderate - the activity of other SRCs. Recent evidence indicates that the SRCs are key participants in governing numerous aspects of CD4+ T cell biology. Here we review findings that establish the SRCs as essential regulators of regulatory T cells (Tregs) and T helper 17 (Th17) cells, with a focus on their crucial roles in Treg immunity in cancer and Treg-Th17 cell phenotypic plasticity.

PM2.5-induced iron homeostasis imbalance triggers cardiac hypertrophy through ferroptosis in a selective autophagy crosstalk manner.

Exposure to PM2.5 is correlated with cardiac remodeling, of which cardiac hypertrophy is one of the main clinical manifestations. Ferroptosis plays an important role in cardiac hypertrophy. However, the potential mechanism of PM2.5-induced cardiac hypertrophy through ferroptosis remains unclear. This study aimed to explore the molecular mechanism of cardiac hypertrophy caused by PM2.5 and the intervention role of MitoQ involved in this process. The results showed that PM2.5 could induce cardiac hypertrophy and dysfunction in mice. Meanwhile, the characteristics of ferroptosis were observed, such as iron homeostasis imbalance, lipid peroxidation, mitochondrial damage and abnormal expression of key molecules. MitoQ treatment could effectively mitigate these alternations. After treating human cardiomyocyte AC16 with PM2.5, ferroptosis activator (Erastin) and inhibitor (Fer-1), it was found that PM2.5 could promote ferritinophagy and lead to lipid peroxidation, mitochondrial dysfunction as well as the accumulation of intracellular and mitochondrial labile iron. Subsequently, mitophagy was activated and provided an additional source of labile iron, enhancing the sensitivity of AC16 cells to ferroptosis. Furthermore, Fer-1 alleviated PM2.5-induced cytotoxicity and iron overload in the cytoplasm and mitochondria of AC16 cells. It was worth noting that during the process of PM2.5 caused ferroptosis, abnormal iron metabolism mediated the activation of ferritinophagy and mitophagy in a temporal order. In addition, NCOA4 knockdown reversed the iron homeostasis imbalance and lipid peroxidation caused by PM2.5, thereby alleviating ferroptosis. In summary, our study found that iron homeostasis imbalance-mediated the crosstalk of ferritinophagy and mitophagy played an important role in PM2.5-induced ferroptosis and cardiac hypertrophy.

Cadmium exposure induced neuronal ferroptosis and cognitive deficits via the mtROS-ferritinophagy pathway.

Exposure to environmental cadmium (Cd) is known to cause neuronal death and cognitive decline in humans. Ferroptosis, a novel iron-dependent type of regulated cell death, is involved in various neurological disorders. In the present study, Cd exposure triggered ferroptosis in the mouse hippocampus and in the HT22 murine hippocampal neuronal cell line, as indicated by significant increases in ferroptotic marker expression, intracellular iron levels, and lipid peroxidation. Interestingly, ferroptosis of hippocampal neurons in response to Cd exposure relied on the induction of autophagy since the suppression of autophagy by 3-methyladenine (3-MA) and chloroquine (CQ) substantially ameliorated Cd-induced ferroptosis. Furthermore, nuclear receptor coactivator 4 (NCOA4)-mediated degradation of ferritin was required for the Cd-induced ferroptosis of hippocampal neurons, demonstrating that NCOA4 knockdown decreased intracellular iron levels and lipid peroxidation and increased cell survival, following Cd exposure. Moreover, Cd-induced mitochondrial reactive oxygen species (mtROS) generation was essential for the ferritinophagy-mediated ferroptosis of hippocampal neurons. Importantly, pretreatment with the ferroptosis inhibitor ferrostatin-1 (Fer-1) effectively attenuated Cd-induced hippocampal neuronal death and cognitive impairment in mice. Taken together, these findings indicate that ferroptosis is a novel mechanism underlying Cd-induced neurotoxicity and cognitive impairment and that the mtROS-ferritinophagy axis modulates Cd-induced neuronal ferroptosis.

Regulation of NCOA4-mediated iron recycling ameliorates paraquat-induced lung injury by inhibiting ferroptosis.

Paraquat (PQ) is an irreplaceable insecticide in many countries for the advantage of fast-acting and broad-spectrum. However, PQ was classified as the most prevailing poisoning substance for suicide with no specific antidote. Therefore, it is imperative to develop more effective therapeutic agents for the treatment of PQ poisoning. In the present study, both the RNA-Seq and the application of various cell death inhibitors reflected that ferroptosis exerts a crucial regulatory role in PQ poisoning. Moreover, we found PQ strengthens lipid peroxidation as evidenced by different experimental approaches. Of note, pretreatment of iron chelation agent DFO could ameliorate the ferroptotic cell death and alleviate the ferroptosis-related events. Mechanistically, PQ treatment intensively impaired mitochondrial homeostasis, enhanced phosphorylation of AMPK, accelerated the autophagy flux and triggered the activation of Nuclear receptor coactivator 4-ferritin heavy chain (NCOA4-FTH) axis. Importantly, the activation of autophagy was observed prior to the degradation of ferritin, and inhibition of autophagy could inhibit the accumulation of iron caused by the ferritinophagy process. Genetic and pharmacological inhibition of ferritinophagy could alleviate the lethal oxidative events, and rescue the ferroptotic cell death. Excitingly, in the mouse models of PQ poisoning, both the administration of DFO and adeno-associated virus-mediated FTH overexpression significantly reduced PQ-induced ferroptosis and improved the pathological characteristics of pulmonary fibrosis. In summary, the current work provides an in-depth study on the mechanism of PQ intoxication, describes a framework for the further understanding of ferroptosis in PQ-associated biological processes, and demonstrates modulation of iron metabolism may act as a promising therapeutic agent for the management of PQ toxicity.

Nuclear receptor coactivator 6 is a critical regulator of NLRP3 inflammasome activation and gouty arthritis.

Transcriptional coactivators regulate the rate of gene expression in the nucleus. Nuclear receptor coactivator 6 (NCOA6), a coactivator, has been implicated in embryonic development, metabolism, and cancer pathogenesis, but its role in innate immunity and inflammatory diseases remains unclear. Here, we demonstrated that NCOA6 was expressed in monocytes and macrophages and that its level was increased under proinflammatory conditions. Unexpectedly, nuclear NCOA6 was found to translocate to the cytoplasm in activated monocytes and then become incorporated into the inflammasome with NLRP3 and ASC, forming cytoplasmic specks. Mechanistically, NCOA6 associated with the ATP hydrolysis motifs in the NACHT domain of NLRP3, promoting the oligomerization of NLRP3 and ASC and thereby instigating the production of IL-1ß and active caspase-1. Of note, Ncoa6 deficiency markedly inhibited NLRP3 hyperactivation caused by the Nlrp3R258W gain-of-function mutation in macrophages. Genetic ablation of Ncoa6 substantially attenuated the severity of two NLRP3-dependent diseases, folic-induced acute tubular necrosis and crystal-induced arthritis, in mice. Consistent with these findings, NCOA6 was highly expressed in macrophages derived from gout patients, and NCOA6-positive macrophages were significantly enriched in gout macrophages according to the transcriptome profiling results. Conclusively, NCOA6 is a critical regulator of NLRP3 inflammasome activation and is therefore a promising target for NLRP3-dependent diseases, including gout.

NCOA5 Haploinsufficiency in Myeloid-Lineage Cells Sufficiently Causes Nonalcoholic Steatohepatitis and Hepatocellular Carcinoma.

BACKGROUND & AIMS: The nuclear receptor coactivator 5 (NCOA5) is a putative type 2 diabetes susceptibility gene. NCOA5 haploinsufficiency results in the spontaneous development of nonalcoholic fatty liver disease (NAFLD), insulin resistance, and hepatocellular carcinoma (HCC) in male mice; however, the cell-specific effect of NCOA5 haploinsufficiency in various types of cells, including macrophages, on the development of NAFLD and HCC remains unknown. METHODS: Control and myeloid-lineage-specific Ncoa5 deletion (Ncoa5ΔM/+) mice fed a normal diet were examined for the development of NAFLD, nonalcoholic steatohepatitis (NASH), and HCC. Altered genes and signaling pathways in the intrahepatic macrophages of Ncoa5ΔM/+ male mice were analyzed and compared with those of obese human individuals. The role of platelet factor 4 (PF4) in macrophages and the underlying mechanism by which PF4 affects NAFLD/NASH were explored in vitro and in vivo. PF4 expression in HCC patient specimens and prognosis was examined. RESULTS: Myeloid-lineage-specific Ncoa5 deletion sufficiently causes spontaneous NASH and HCC development in male mice fed a normal diet. PF4 overexpression in Ncoa5ΔM/+ intrahepatic macrophages is identified as a potent mediator to trigger lipid accumulation in hepatocytes by inducing lipogenesis-promoting gene expression. The transcriptome of intrahepatic macrophages from Ncoa5ΔM/+ male mice resembles that of obese human individuals. High PF4 expression correlated with poor prognosis of HCC patients and increased infiltrations of M2 macrophages, regulatory T cells, and myeloid-derived suppressor cells in HCCs. CONCLUSIONS: Our findings reveal a novel mechanism for the onset of NAFLD/NASH and HCC initiated by NCOA5-deficient macrophages, suggesting the NCOA5-PF4 axis in macrophages as a potential target for developing preventive and therapeutic interventions against NAFLD/NASH and HCC.

Nrf2 protects against cartilage endplate degeneration through inhibiting NCOA4­mediated ferritinophagy.

Iron overload and ferroptosis are associated with intervertebral disc degeneration (IDD); however, the mechanism underlying the regulation of iron homeostasis remains to be elucidated. Nuclear factor erythroid 2­related factor 2 (Nrf2) has been reported to regulate cellular iron homeostasis; however, its impact on IDD pathology and the underlying mechanism of action requires further investigation. In the present study, immunohistochemistry analysis of Nrf2 expression in the cartilage endplate (CEP) was conducted and it was demonstrated that Nrf2 expression was increased in the CEP at the early stages of the development of IDD, whereas it was decreased at the late stages of the development of IDD. The results of western blot analysis indicated that the inadequate activation of Nrf2 may aggravate mitochondrial dysfunction and oxidative stress, thus promoting CEP chondrocyte degeneration and calcification. It was also revealed that Nrf2 was involved in TNF­α­induced CEP chondrocyte iron metabolism dysfunction and ferroptosis. Inhibition of Nrf2 expression using Nrf2 small interfering RNA could enhance the process of nuclear receptor coactivator 4 (NCOA4)­mediated ferritinophagy and increase ferrous ion content, which may promote CEP chondrocyte ferroptotic cell death and extracellular matrix degradation. Furthermore, a decrease in cellular iron concentration may inhibit CEP chondrocyte ferroptosis, and CEP degeneration and calcification. The present study highlights the role of the Nrf2/NCOA4 axis in chondrocyte ferroptosis and IDD pathogenesis, thus suggesting that activation of Nrf2 may be a promising strategy for IDD treatment.

NCOA4 requires a [3Fe-4S] to sense and maintain the iron homeostasis.

NCOA4 is a selective cargo receptor for ferritinophagy, the autophagic turnover of ferritin (FTH), a process critical for regulating intracellular iron bioavailability. However, how ferritinophagy flux is controlled through NCOA4 in iron-dependent processes needs to be better understood. Here, we show that the C-terminal FTH-binding domain of NCOA4 harbors a [3Fe-4S]-binding site with a stoichiometry of approximately one labile [3Fe-4S] cluster per NCOA4 monomer. By analyzing the interaction between NCOA4 and HERC2 ubiquitin ligase or NCOA4 and FTH, we demonstrate that NCOA4 regulates ferritinophagy by sensing the intracellular iron-sulfur cluster levels. Under iron-repletion conditions, HERC2 recognizes and recruits holo-NCOA4 as a substrate for polyubiquitination and degradation, favoring ferritin iron storage. Under iron-depletion conditions, NCOA4 exists in the form of apo-protein and binds ferritin to promote the occurrence of ferritinophagy and release iron. Thus, we identify an iron-sulfur cluster [3Fe-4S] as a critical cofactor in determining the fate of NCOA4 in favoring iron storage in ferritin or iron release via ferritinophagy and provide a dual mechanism for selective interaction between HERC2 and [3Fe-4S]-NCOA4 for proteasomal degradation or between ferritin and apo-NCOA4 for ferritinophagy in the control of iron homeostasis.

HECW1 induces NCOA4-regulated ferroptosis in glioma through the ubiquitination and degradation of ZNF350.

Tumor suppression by inducing NCOA4-mediated ferroptosis has been shown to be feasible in a variety of tumors, including gliomas. However, the regulatory mechanism of ferroptosis induced by NCOA4 in glioma has not been studied deeply. HECW1 and ZNF350 are involved in the biological processes of many tumors, but their specific effects and mechanisms on glioma are still unclear. In this study, we found that HECW1 decreased the survival rate of glioma cells and enhanced iron accumulation, lipid peroxidation, whereas ZNF350 showed the opposite effect. Mechanistically, HECW1 directly regulated the ubiquitination and degradation of ZNF350, eliminated the transcriptional inhibition of NCOA4 by ZNF350, and ultimately activated NCOA4-mediated iron accumulation, lipid peroxidation, and ferroptosis. We demonstrate that HECW1 induces ferroptosis and highlight the value of HECW1 and ZNF350 in the prognostic evaluation of patients with glioma. We also elucidate the mechanisms underlying the HECW1/ZNF350/NCOA4 axis and its regulation of ferroptosis. Our findings enrich the understanding of ferroptosis and provide potential treatment options for glioma patients.

Pseudolaric acid B triggers ferritinophagy and ferroptosis via upregulating NCOA4 in lung adenocarcinoma cells.

BACKGROUND: Ferroptosis is a novel subtype of programmed cell death caused by iron-dependent lipid peroxidation and excessive reactive oxygen species (ROS) production. Small-molecule ferroptotic drugs have the probability of selectively targeting the specific features of aggressive tumor cells. In particular, pseudolaric acid B (PAB) triggered ferroptosisin breast cancer cells. The aim of this study is to explore the antitumor effect of PAB on A549 cells and provide a theoretical basis for the further development and clinical application of PAB. METHODS: First, relevant databases were used to predict of target genes related to PAB, Then, EdU proliferation assay, colony formation and wound-healing assays were applied to calculate A549 cells proliferative abilities. Measurement of ferrous iron, lipid peroxidation, ROS, malondialdehyde (MDA) and glutathione (GSH) were utilized to explore the relevant mechanism. RESULTS: We showed that PAB decreased the viability of lung adenocarcinoma cells in vitro, which was accompanied by abnormally elevated levels of intracellular ferrous iron and overproduction of lipid reactive oxidate species (L-ROS). In turn, deferoxamine (DFO) significantly rescued PAB-induced lipid peroxidation. PAB also improved the intracellular labile iron pool by promoting ferritin autophagy via the upregulation of the nuclear receptor coactivator 4 (NCOA4). Moreover, silencing of NCOA4 alleviated PAB-inducedferroptotic death and reduced the levels of intracellular ferrous iron. CONCLUSIONS: In summary, PAB-triggered ferroptosis in lung adenocarcinoma cells by enhancing ferritinophagy. thus, PAB is a potential therapeutic agent for lung adenocarcinoma.

Puerarin Induces Ferroptosis in Colorectal Cancer Cells via Triggering NCOA4 Upregulation.

Puerarin shows promise as an anti-cancer compound, but its mechanism of action remains unclear. Here we explored whether and how it promotes ferroptosis in a colorectal cancer cell line. The level of ferroptosis and expression of autophagy proteins were compared between puerarin-treated HT-29 cells expressing normal or reduced levels of the autophagy protein ATG5 or the ferritinophagy protein nuclear receptor coactivator 4 (NCOA4). Puerarin increased lipid peroxidation and inhibited cell proliferation in a dose-dependent manner, indicating the induction of ferroptosis. These effects were partially reversed by ferrostatin-1, a scavenger of reactive oxygen species; by the iron chelator deferiprone; by repression of autophagy through administration of 3-methyladenine or knockdown of autophagy-related gene 5 (ATG5); or by repression of ferritinophagy through NCOA4 knockdown. Puerarin may induce the proliferative inhibition of colorectal cancer cells by triggering ferroptosis through a mechanism requiring NCOA4 ferritinophagy.

Schisandrin B promotes senescence of activated hepatic stellate cell via NCOA4-mediated ferritinophagy.

CONTEXT: Schisandrin B (Sch B), an active ingredient from Schisandrae chinensis (Turcz.) Baill. (Schisandraceae) Fructus, possesses diverse pharmacological activities including antitumor, anti-inflammation, and hepatoprotection. OBJECTIVE: To explore the effect of Sch B on activated HSCs senescence in hepatic fibrosis and the mechanisms implicated. MATERIALS AND METHODS: ICR mice with CCl4-induced hepatic fibrosis were supplemented with Sch B (40 mg/kg) for 30 d and LX2 cells were treated with Sch B (5, 10 and 20 µM) for 24 h. Cellular senescence was assessed by senescence-related indicators senescence-associated ß-galactosidase (SA-ß-gal) activity and the expression of p16, p21, p53, γ-H2AX, H3K9me3, TERT, TRF1, and TRF2. Ferric ammonium citrate (FAC) and NCOA4 siRNA were used to evaluate the mechanisms underlying Sch B's regulation of cellular senescence. RESULTS: Sch B (40 mg/kg) reduced serum levels of AST and ALT (53.2% and 63.6%), alleviated hepatic collagen deposition, and promoted activated HSCs senescence in mice. Treatment with Sch B (20 µM) decreased cell viability to 80.38 ± 4.87% and elevated SA-ß-gal activity, with the levels of p16, p21 and p53 increased by 4.5-, 2.9-, and 3.5-fold and the levels of TERT, TRF1 and TRF2 decreased by 2.4-, 2.7-, and 2.6-fold in LX2 cells. FAC (400 µM) enhanced Sch B's effect mentioned above. NCOA4 siRNA weakened the effects of Sch B on iron deposition and HSCs senescence. CONCLUSIONS: Sch B could ameliorate hepatic fibrosis through the promotion of activated HSCs senescence, which might be attributed to its induction of NCOA4-mediated ferritinophagy and subsequent iron overload.

Ferritinophagy in the etiopathogenic mechanism of related diseases.

Iron is an essential trace element that is involved in a variety of physiological processes. Ferritinophagy is selective autophagy mediated by nuclear receptor coactivator 4 (NCOA4), which regulates iron homeostasis in the body. Upon iron depletion or starvation, ferritinophagy is activated, releasing large amounts of Fe2+ and increasing reactive oxygen species (ROS), leading to ferroptosis. This plays a significant role in the etiopathogenesis of many diseases, such as metabolic diseases, neurodegenerative diseases, infectious diseases, tumors, cardiomyopathy, and ischemia-reperfusion ischemia-reperfusion injury. Here, we first review the regulation and functions of ferritinophagy and then describe its involvement in different diseases, with hopes of providing new understanding and insights into iron metabolism and iron disorder-related diseases and the therapeutic opportunity for targeting ferritinophagy.

SNS alleviates depression-like behaviors in CUMS mice by regluating dendritic spines via NCOA4-mediated ferritinophagy.

ETHNOPHARMACOLOGICAL RELEVANCE: Depression is one of the most common mood disturbances worldwide. The Si-ni-san formula (SNS) is a famous classic Traditional Chinese Medicine (TCM) widely used to treat depression for thousands of years in clinics. However, the mechanism underlying the therapeutic effect of SNS in improving depression-like behaviors following chronic unpredictable mild stress (CUMS) remains unknown. AIM OF THE STUDY: This study aimed to investigate whether SNS alleviates depression-like behaviors in CUMS mice by regulating dendritic spines via NCOA4-mediated ferritinophagy in vitro and in vivo. STUDY DESIGN AND METHODS: In vivo, mice were exposed to CUMS for 42 days, and SNS (4.9, 9.8, 19.6 g/kg/d), fluoxetine (10 mg/kg/d), 3-methyladenine (3-MA) (30 mg/kg/d), rapamycin(1 mg/kg/d), and deferoxamine (DFO) (200 mg/kg/d) were conducted once daily during the last 3 weeks of the CUMS procedure. In vitro, a depressive model was established by culture of SH-SY5Y cells with corticosterone, followed by treatment with different concentrations of freeze-dried SNS (0.001, 0.01, 0.1 mg/mL) and rapamycin (10 nM), NCOA4-overexpression, Si-NCOA4. After the behavioral test (open-field test (OFT), sucrose preference test (SPT), forced swimming test (FST) and tail suspension test (TST), dendritic spines, GluR2 protein expression, iron concentration, and ferritinophagy-related protein levels (P62, FTH, NCOA4, LC3-II/LC3-I) were tested in vitro and in vivo using immunohistochemistry, golgi staining, immunofluorescence, and Western blot assays. Finally, HEK-293T cells were transfected by si-NCOA4 or GluR2-and NCOA4-overexpression plasmid and treated with corticosterone(100 µM), freeze-dried SNS(0.01 mg/mL), rapamycin(25 nM), and 3-MA(5 mM). The binding amount of GluR2, NCOA4, and LC3 was assessed by the co-immunoprecipitation (CO-IP) assay. RESULTS: 3-MA, SNS, and DFO promoted depressive-like behaviors in CUMS mice during OFT, SPT, FST and TST, improved the amount of the total, thin, mushroom spine density and enhanced GluR2 protein expression in the hippocampus. Meanwhile, treatment with SNS decreased iron concentrations and inhibited NCOA4-mediated ferritinophagy activation in vitro and in vivo. Importantly, 3-MA and SNS could prevent the binding of GluR2, NCOA4 and LC3 in corticosterone-treated HEK-293T, and rapamycin reversed this phenomenon after treatment with SNS. CONCLUSION: SNS alleviates depression-like behaviors in CUMS mice by regulating dendritic spines via NCOA4-mediated ferritinophagy.

JNK-JUN-NCOA4 axis contributes to chondrocyte ferroptosis and aggravates osteoarthritis via ferritinophagy.

Interruption of iron homeostasis is correlated with cell ferroptosis and degenerative diseases. Nuclear receptor coactivator 4 (NCOA4)-mediated ferritinophagy has been reported as a vital mechanism to control cellular iron levels, but its impact on osteoarthritis (OA) pathology and the underline mechanism are unknown. Herein we aimed to investigate the role and regulatory mechanism of NCOA4 in chondrocyte ferroptosis and OA pathogenesis. We demonstrated that NCOA4 was highly expressed in cartilage of patients with OA, aged mice, post-traumatic OA mice, and inflammatory chondrocytes. Importantly, Ncoa4 knockdown inhibited IL-1ß-induced chondrocyte ferroptosis and extracellular matrix degradation. Contrarily, overexpression of NCOA4 promoted chondrocyte ferroptosis and the delivery of Ncoa4 adeno-associated virus 9 into knee joint of mice aggravated post-traumatic OA. Mechanistic study revealed that NCOA4 was upregulated in a JNK-JUN signaling-dependent manner in which JUN could directly bind to the promoter of Ncoa4 and initial the transcription of Ncoa4. NCOA4 could interact with ferritin and increase autophagic degradation of ferritin and iron levels, which caused chondrocyte ferroptosis and extracellular matrix degradation. In addition, inhibition of JNK-JUN-NCOA4 axis by SP600125, a specific inhibitor of JNK, attenuated development of post-traumatic OA. This work highlights the role of JNK-JUN-NCOA4 axis and ferritinophagy in chondrocyte ferroptosis and OA pathogenesis, suggesting this axis as a potential target for OA treatment.

The PTBP1­NCOA4 axis promotes ferroptosis in liver cancer cells.

Polypyrimidine tract­binding protein 1 (PTBP1) plays an important role in tumor immunity, cell proliferation, apoptosis, and autophagy by regulating RNA metabolism. However, the specific function and mechanism of PTBP1 in ferroptosis remain unclear. In the present study, it was investigated whether PTBP1 regulates ferroptosis and the exact mechanism. The iron, malondialdehyde (MDA), and GSH levels were detected in sorafenib (SF)­treated liver cancer cells. si­PTBP1 introduction into SF­treated liver cancer cells resulted in a significant reduction in the levels of MDA and iron. Additionally, a significant recovery of GSH levels was observed after silencing PTBP1. StarBase v2.0 database was used to predict potential transcripts that can physically interact with PTBP1 and nuclear receptor coactivator 4 (NCOA4) mRNA was identified as the most enriched binding partner in the PTBP1­RNA complex. A dual­luciferase assay then demonstrated that PTBP1 directly interacted with NCOA4. PTBP1 silencing did not affect NCOA4 stability following treatment with cycloheximide. A pull­down assay revealed that the PTBP1­binding region was in the 5'­UTR of the NCOA4 mRNA sequence. These results suggest that PTBP1 mediates ferroptosis in liver cancer cells by regulating NCOA4 translation. In vivo experiments reconfirmed the role of the PTBP1­NCOA4 axis in a xenograft transplantation model. It was observed that the mean tumor weight increased after PTBP1 knockout. In conclusion, silencing of PTBP1 decreased the sensitivity of liver cancer cells to ferroptosis after SF treatment and regulated ferritinophagy by mediating NCOA4 translation.

Melatonin inhibits ferroptosis and delays age-related cataract by regulating SIRT6/p-Nrf2/GPX4 and SIRT6/NCOA4/FTH1 pathways.

BACKGROUND: Cataracts are the main cause of reversible blindness worldwide. The ageing of the lens caused by ultraviolet B (UVB) radiation is mostly related to oxidative stress (OS). Little is known about whether OS induced by UVB enhances the sensitivity of lens epithelial cells to ferroptotic stress, which may be a new mechanism leading to age-related cataracts (ARCs). METHODS: Ferroptosis was detected by transmission electron microscopy (TEM), iron assay, lipid peroxidation (MDA) assay, real-time PCR, western blotting, and immunofluorescence. Genetic engineering technology was used to investigate the regulatory relationship among Sirtuin 6 (SIRT6), nuclear factor erythroid 2-related factor 2 (Nrf2), nuclear receptor coactivator 4 (NCOA4), glutathione peroxidase 4 (GPX4) and ferritin heavy chain (FTH1). Knockdown and overexpression of SIRT6 locally in vivo in rats were performed to probe the regulatory mechanism of SIRT6 in ferroptosis in ARCs. FINDINGS: Here, we observed that UVB can drastically induce ferroptosis in lens epithelial cells in vivo and in vitro. Surprisingly, inhibition of ferroptosis was the direct reason that melatonin rescued B-3, SRA01/04 and HEK-293 T cells survival; the pan-caspase inhibitor Z-Vad-FMK did not significantly reverse the death of UVB-irradiated cells compared with that in the UVB+DMSO group. SIRT6 was an upstream regulator of phosphorylated Nrf2 (p-Nrf2) and NCOA4 in B-3, SRA01/04 and HEK-293 T cells. Melatonin inhibited ferroptosis through the SIRT6/p-Nrf2/GPX4 and SIRT6/COA4/FTH1 pathways to neutralize lipid peroxidation toxicity, which protected cells against ferroptotic stress in vitro and delayed cataract formation caused by UVB exposure in rats. INTERPRETATION: Our findings reveal a novel causal role of melatonin in the pathogenesis of ARCs, which raises the possibility of selectively targeting the activation of SIRT6 and ferroptotic resistance as a latent antioxidative therapeutic strategy for ARCs.

Multiomics analyses and machine learning of nuclear receptor coactivator 6 reveal its essential role in hepatocellular carcinoma.

Nuclear receptor coactivator 6 (NCOA6), a coactivator of numerous nuclear receptors and transcription factors, regulates multiple critical cellular functions. Nuclear receptor coactivator 6 is dysregulated in various cancers, including hepatocellular carcinoma (HCC); however, its role remains largely unknown. Here we reported that NCOA6 was highly expressed in HCC compared to the adjacent liver tissue, and NCOA6 overexpression was significantly correlated with poor HCC prognosis. Experiments revealed that the knockdown of NCOA6 damaged the proliferation, migration, and invasion of HCC cells. Multiomics and immune infiltration analyses showed a close relationship between NCOA6 expression, multiple cancer-related malignant pathways, and the immunosuppressive microenvironment. Finally, we established an effective NCOA6-related microRNA (miRNA) signature to distinguish HCC from hepatitis\liver cirrhosis patients. To the best of our knowledge, this study is the first to provide a comprehensive analysis of NCOA6 expression in HCC. We found that NCOA6 plays an important role in HCC development and has a potential mechanism of action. Establishing an NCOA6-related miRNA signature will help develop novel diagnostic strategies for HCC patients.