Ferritin heavy chain protects the developing wing from reactive oxygen species and ferroptosis.
ABSTRACT: The interplay between signalling pathways and metabolism is crucial for tissue growth. Yet, it remains poorly understood. Here, we studied the consequences of modulating iron metabolism on the growth of Drosophila imaginal discs. We find that reducing the levels of the ferritin heavy chain in the larval wing discs leads to drastic growth defects, whereas light chain depletion causes only minor defects. Mutant cell clones for the heavy chain lack the ability to compete against Minute mutant cells. Reactive oxygen species (ROS) accumulate in wing discs with reduced heavy chain levels, causing severe mitochondrial defects and ferroptosis. Preventing ROS accumulation alleviates some of the growth defects. We propose that the increased expression of ferritin in hippo mutant cells may protect against ROS accumulation.
Project description:Ferroptosis, a recently discovered form of iron-dependent cell death, requires an increased level of lipid-reactive oxygen species (ROS). Ferritinophagy, a ferritin degradation pathway, depends on a selective autophagic cargo receptor (NCOA4). By screening various types of natural compounds, formosanin C (FC) was identified as a novel ferroptosis inducer, characterized by attenuations of FC-induced viability inhibition and lipid ROS formation in the presence of ferroptosis inhibitor. FC also induced autophagic flux, evidenced by preventing autophagic marker LC3-II degradation and increasing yellow LC3 puncta in tandem fluorescent-tagged LC3 (mRFP-GFP) reporter plasmid (ptfLC3) transfected cells when combined with autophagic flux inhibitor. It is noteworthy that FC-induced ferroptosis and autophagic flux were stronger in HepG2 cells expressing higher NCOA4 and lower ferritin heavy chain 1 (FTH1) levels, agreeing with the results of gene expression analysis using CTRP and PRISM, indicating that FTH1 expression level exhibited a significant negative correlation with the sensitivity of the cells to a ferroptosis inducer. Confocal and electron microscopy confirmed the pronounced involvement of ferritinophagy in FC-induced ferroptosis in the cells with elevated NCOA4. Since ferroptosis is a non-apoptotic form of cell death, our data suggest FC has chemotherapeutic potential against apoptosis-resistant HCC with a higher NCOA4 expression via ferritinophagy.
Project description:The proper number of cells in developing tissues is achieved by coordinating cell division with apoptosis. In Drosophila, the adult wing is derived from wing imaginal discs, which undergo a period of growth and proliferation during larval stages without much programmed cell death. In this report, we demonstrate that the Drosophila casein kinase Iepsilon/delta, known as Discs overgrown (Dco), is required for maintaining this low level of apoptosis. Expression of dco can suppress the apoptotic activity of Head involution defective (Hid) in the developing eye. Loss of dco in the wing disc results in a dramatic reduction in expression of the caspase inhibitor DIAP1 and a concomitant activation of caspases. The regulation of DIAP1 by Dco occurs by a post-transcriptional mechanism that is independent of hid. Mutant clones of dco are considerably smaller than controls even when apoptosis is inhibited, suggesting that Dco promotes cell division/growth in addition to its role in cell survival. The dco phenotype cannot be explained by defects Wingless (Wg) signaling. We propose that Dco coordinates tissue size by stimulating cell division/growth and blocking apoptosis via activation of DIAP1 expression.
Project description:Protein ser/thr phosphatase 2A family members (PP2A, PP4, and PP6) are implicated in the control of numerous biological processes, but our understanding of the in vivo function and regulation of these enzymes is limited. In this study, we investigated the role of Tap42, a common regulatory subunit for all three PP2A family members, in the development of Drosophila melanogaster wing imaginal discs. RNAi-mediated silencing of Tap42 using the binary Gal4/UAS system and two disc drivers, pnr- and ap-Gal4, not only decreased survival rates but also hampered the development of wing discs, resulting in a remarkable thorax cleft and defective wings in adults. Silencing of Tap42 also altered multiple signaling pathways (HH, JNK and DPP) and triggered apoptosis in wing imaginal discs. The Tap42(RNAi)-induced defects were the direct result of loss of regulation of Drosophila PP2A family members (MTS, PP4, and PPV), as enforced expression of wild type Tap42, but not a phosphatase binding defective Tap42 mutant, rescued fly survivorship and defects. The experimental platform described herein identifies crucial roles for Tap42•phosphatase complexes in governing imaginal disc and fly development.
Project description:Cellular growth, function, and protection require proper iron management, and ferritin plays a crucial role as the major iron sequestration and storage protein. Ferritin is a 24 subunit spherical shell protein composed of both light (FTL) and heavy chain (FTH1) subunits, possessing complimentary iron-handling functions and forming three-fold and four-fold pores. Iron uptake through the three-fold pores is well-defined, but the unloading process somewhat less and generally focuses on lysosomal ferritin degradation although it may have an additional, energetically efficient pore mechanism. Hereditary Ferritinopathy (HF) or neuroferritinopathy is an autosomal dominant neurodegenerative disease caused by mutations in the FTL C-terminal sequence, which in turn cause disorder and unraveling at the four-fold pores allowing iron leakage and enhanced formation of toxic, improperly coordinated iron (ICI). Histopathologically, HF is characterized by iron deposition and formation of ferritin inclusion bodies (IBs) as the cells overexpress ferritin in an attempt to address iron accumulation while lacking the ability to clear ferritin and its aggregates. Overexpression and IB formation tax cells materially and energetically, i.e., their synthesis and disposal systems, and may hinder cellular transport and other spatially dependent functions. ICI causes cellular damage to proteins and lipids through reactive oxygen species (ROS) formation because of high levels of brain oxygen, reductants and metabolism, taxing cellular repair. Iron can cause protein aggregation both indirectly by ROS-induced protein modification and destabilization, and directly as with mutant ferritin through C-terminal bridging. Iron release and ferritin degradation are also linked to cellular misfunction through ferritinophagy, which can release sufficient iron to initiate the unique programmed cell death process ferroptosis causing ROS formation and lipid peroxidation. But IB buildup suggests suppressed ferritinophagy, with elevated iron from four-fold pore leakage together with ROS damage and stress leading to a long-term ferroptotic-like state in HF. Several of these processes have parallels in cell line and mouse models. This review addresses the roles of ferritin structure and function within the above-mentioned framework, as they relate to HF and associated disorders characterized by abnormal iron accumulation, protein aggregation, oxidative damage, and the resulting contributions to cumulative cellular stress and death.
Project description:Pacman/Xrn1 is a highly conserved exoribonuclease known to play a critical role in gene regulatory events such as control of mRNA stability, RNA interference and regulation via miRNAs. Although Pacman has been well studied in Drosophila tissue culture cells, the biologically relevant cellular pathways controlled by Pacman in natural tissues are unknown. This study shows that a hypomorphic mutation in pacman (pcm (5)) results in smaller wing imaginal discs. These tissues, found in the larva, are known to grow and differentiate to form wing and thorax structures in the adult fly. Using microarray analysis, followed by quantitative RT-PCR, we show that eight mRNAs were increased in level by>2-fold in the pcm5 mutant wing discs compared with the control. The levels of pre-mRNAs were tested for five of these mRNAs; four did not increase in the pcm (5) mutant, showing that they are regulated at the post-transcriptional level and, therefore, could be directly affected by Pacman. These transcripts include one that encodes the heat shock protein Hsp67Bc, which is upregulated 11.9-fold at the post-transcriptional level and 2.3-fold at the protein level. One miRNA, miR-277-3p, is 5.6-fold downregulated at the post-transcriptional level in mutant discs, suggesting that Pacman affects its processing in this tissue. Together, these data show that a relatively small number of mRNAs and miRNAs substantially change in abundance in pacman mutant wing imaginal discs. Since Hsp67Bc is known to regulate autophagy and protein synthesis, it is possible that Pacman may control the growth of wing imaginal discs by regulating these processes.
Project description:Programmed cell death, or apoptosis, is a highly conserved cellular process that is crucial for tissue homeostasis under normal development as well as environmental stress. Misregulation of apoptosis is linked to many developmental defects and diseases such as tumour formation, autoimmune diseases and neurological disorders. In this paper, we show a novel role for the exoribonuclease Pacman/Xrn1 in regulating apoptosis. Using Drosophila wing imaginal discs as a model system, we demonstrate that a null mutation in pacman results in small imaginal discs as well as lethality during pupation. Mutant wing discs show an increase in the number of cells undergoing apoptosis, especially in the wing pouch area. Compensatory proliferation also occurs in these mutant discs, but this is insufficient to compensate for the concurrent increase in apoptosis. The phenotypic effects of the pacman null mutation are rescued by a deletion that removes one copy of each of the pro-apoptotic genes reaper, hid and grim, demonstrating that pacman acts through this pathway. The null pacman mutation also results in a significant increase in the expression of the pro-apoptotic mRNAs, hid and reaper, with this increase mostly occurring at the post-transcriptional level, suggesting that Pacman normally targets these mRNAs for degradation. Our results uncover a novel function for the conserved exoribonuclease Pacman and suggest that this exoribonuclease is important in the regulation of apoptosis in other organisms.
Project description:UNLABELLED:Ferroptosis is a recently recognized form of regulated cell death caused by an iron-dependent accumulation of lipid reactive oxygen species. However, the molecular mechanisms regulating ferroptosis remain obscure. Here, we report that nuclear factor erythroid 2-related factor 2 (NRF2) plays a central role in protecting hepatocellular carcinoma (HCC) cells against ferroptosis. Upon exposure to ferroptosis-inducing compounds (e.g., erastin, sorafenib, and buthionine sulfoximine), p62 expression prevented NRF2 degradation and enhanced subsequent NRF2 nuclear accumulation through inactivation of Kelch-like ECH-associated protein 1. Additionally, nuclear NRF2 interacted with transcriptional coactivator small v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog proteins such as MafG and then activated transcription of quinone oxidoreductase-1, heme oxygenase-1, and ferritin heavy chain-1. Knockdown of p62, quinone oxidoreductase-1, heme oxygenase-1, and ferritin heavy chain-1 by RNA interference in HCC cells promoted ferroptosis in response to erastin and sorafenib. Furthermore, genetic or pharmacologic inhibition of NRF2 expression/activity in HCC cells increased the anticancer activity of erastin and sorafenib in vitro and in tumor xenograft models. CONCLUSION:These findings demonstrate novel molecular mechanisms and signaling pathways of ferroptosis; the status of NRF2 is a key factor that determines the therapeutic response to ferroptosis-targeted therapies in HCC cells.
Project description:Cellular iron, at the physiological level, is essential to maintain several metabolic pathways, while an excess of free iron may cause oxidative damage and/or provoke cell death. Consequently, iron homeostasis has to be tightly controlled. Under hypoxia these regulatory mechanisms for human macrophages are not well understood. Hypoxic primary human macrophages reduced intracellular free iron and increased ferritin expression, including mitochondrial ferritin (FTMT), to store iron. In parallel, nuclear receptor coactivator 4 (NCOA4), a master regulator of ferritinophagy, decreased and was proven to directly regulate FTMT expression. Reduced NCOA4 expression resulted from a lower rate of hypoxic NCOA4 transcription combined with a micro RNA 6862-5p-dependent degradation of NCOA4 mRNA, the latter being regulated by c-jun N-terminal kinase (JNK). Pharmacological inhibition of JNK under hypoxia increased NCOA4 and prevented FTMT induction. FTMT and ferritin heavy chain (FTH) cooperated to protect macrophages from RSL-3-induced ferroptosis under hypoxia as this form of cell death is linked to iron metabolism. In contrast, in HT1080 fibrosarcome cells, which are sensitive to ferroptosis, NCOA4 and FTMT are not regulated. Our study helps to understand mechanisms of hypoxic FTMT regulation and to link ferritinophagy and macrophage sensitivity to ferroptosis.
Project description:RNA-seq on 120hr L3 larval wing imaginal discs. 3 replicates of dis3L2 null mutant, and 3 replicates of control wing discs. rRNA depleted, Illumina TruSeq libraries. Paired-end sequencing on a HiSeq 3000.
Project description:Background:Ferroptosis is a form of iron-dependent non-apoptotic cell death, with characteristics of loss of the activity of the lipid repair enzyme, glutathione (GSH) peroxidase 4 (GPX4), and accumulation of lethal reactive lipid oxygen species. The mechanism of ferroptosis in myelodysplastic syndrome (MDS) is unclear. Methods:Cell viability assay, reactive oxygen species (ROS) assay, GSH assay, and GPX activity assay were performed to study the regulation of ferroptosis in MDS cells obtained from MDS patients, the iron overload model mice, and cell lines. Results:The growth-inhibitory effect of decitabine could be partially reversed by ferrostatin-1 and iron-chelating agent [desferrioxamine (DFO)] in MDS cell lines. Erastin could increase the cytotoxicity of decitabine on MDS cells. The level of GSH and the activity of GPX4 decreased, whereas the ROS level increased in MDS cells upon treatment with decitabine, which could be reversed by ferrostatin-1. The concentration of hemoglobin in peripheral blood of iron overload mice was negatively correlated with intracellular Fe2+ level and ferritin concentration. Iron overload (IO) led to decreased viability of bone marrow mononuclear cells (BMMNCs), which was negatively correlated with intracellular Fe2+ level. Ferrostatin-1 partially reversed the decline of cell viability in IO groups. The level of GSH and the activity of GPX4 decreased, whereas the ROS level increased in BMMNCs of IO mice. DFO could increase the level of GSH. Ferrostatin-1 and DFO could increase the GPX4 activity of BMMNCs in IO mice. Ferrostatin-1 could significantly reverse the growth-inhibitory effect of decitabine in MDS patients. Decitabine could significantly increase the ROS level in MDS groups, which could be inhibited by ferrostatin-1 or promoted by erastin. Ferrostatin-1 could significantly reverse the inhibitory effect of decitabine on GSH levels in MDS patients. Erastin combined with decitabine could further reduce the GSH level. Erastin could further decrease the activity of GPX4 compared with the decitabine group. Conclusion:Ferroptosis may account for the main mechanisms of how decitabine induced death of MDS cells. Decitabine-induced ROS raise leads to ferroptosis in MDS cells by decreasing GSH level and GPX4 activity.