Mammalian Target of Rapamycin Coordinates Iron Metabolism with Iron-Sulfur Cluster Assembly Enzyme and Tristetraprolin

Nutrition 30 (2014) 968–974

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Nutrition

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Review Mammalian target of rapamycin coordinates iron metabolism with iron-sulfur cluster assembly enzyme and tristetraprolin

Peng Guan Ph.D. a, Na Wang M.D. a,b,* a Key Laboratory of Animal Physiology, Biochemistry and Molecular Biology of Hebei Province, Hebei Normal University, Hebei Province, China b School of Basic Medical Sciences, Hebei University of Traditional Chinese Medicine, Hebei Province, China article info abstract

Article history: Both iron deficiency and excess are relatively common health concerns. Maintaining the body’s Received 18 October 2013 levels of iron within precise boundaries is critical for cell functions. However, the difference Accepted 15 December 2013 between iron deficiency and overload is often a question of a scant few milligrams of iron. The mammalian target of rapamycin (mTOR), an atypical Ser/Thr protein kinase, is attracting significant Keywords: amounts of interest due to its recently described role in iron homeostasis. Despite extensive study, Iron a complete understanding of mTOR function has remained elusive. mTOR can form two multi- Rapamycin protein complexes that consist of mTOR complex 1 (mTORC1) and mTOR complex 2. Recent Iron-sulfur clusters Tristetraprolin advances clearly demonstrate that mTORC1 can phosphorylate iron-sulfur cluster assembly Transferrin receptor enzyme ISCU and affect iron-sulfur clusters assembly. Moreover, mTOR is reported to control iron metabolism through modulation of tristetraprolin expression. It is now well appreciated that the hormonal hepcidin-ferroportin system and the cellular iron-responsive element/iron-regulatory protein regulatory network play important regulatory roles for systemic iron metabolism. Sus- tained ISCU protein levels enhanced by mTORC1 can inhibit iron-responsive element and iron- regulatory protein binding activities. In this study, hepcidin gene and protein expression in the livers of tristetraprolin knockout mice were dramatically reduced. Here, we highlight and sum- marize the current understanding of how mTOR pathways serve to modulate iron metabolism and homeostasis as the third iron-regulatory system. Ó 2014 Elsevier Inc. All rights reserved.

Introduction least two mechanisms, iron-regulatory proteins (IRPs) and RNA stem-loop iron-responsive elements (IREs) [1] and the tran- Iron is an essential cofactor in diverse biological processes scriptional regulation of hepcidin [2]. such as oxygen transport, cellular respiration, and DNA synthe- IRPs are central regulators of cellular iron homeostasis due to sis. The average adult human body contains 2 to 4 g of iron. Iron their regulation of specific mRNAs encoding proteins of iron deficiency can cause cellular growth arrest and death. However, uptake, export, storage, and utilization. IRP1 can assume two iron is extremely toxic when present in excess: Ferrous iron re- different functions in the cell, depending on conditions [3] acts with hydrogen peroxides or lipid peroxides to generate (Fig. 1). During iron deficiency, IRP1 binds to IREs to modulate hydroxyl or lipid radicals, respectively. So the levels of iron must the translation of iron metabolism genes. In iron-rich conditions, be tightly controlled inside individual organelles, cells, and tis- IRP1 binds an iron-sulfur cluster (ISC) to function as a cytosolic sues. However, the molecular circuits that achieve iron balance aconitase. IRP2 has w60% sequence identity to IRP1, but IRP2 under most conditions have only recently begun to be charac- does not display aconitase activity [4]. In the case of IRP2, iron terized. The iron content of the body is tightly regulated by at controls its RNA-binding activity by stimulating its ubiquitina- tion and rapid proteasomal degradation [5]. IRP2 exhibits a different pattern of affinities to the IRE family than IRP1, having This work was supported by National Natural Science Foundation of China in general weaker binding to the non-ferritin types [6]. IRPs (grant number 31200863), Science and Technology Research Youth Fund Project function to restore iron levels through stabilization of transferrin of Hebei Colleges and Universities (grant number QN20131009), and Hebei receptor 1 (TfR1) messenger RNA (mRNA) and increased iron Normal University Fund (grant number L2010 Q06). * Corresponding author. Tel.: þ86 311 80787579; fax: þ86 311 83930675. uptake, mobilization of cellular iron stores, and reduction in E-mail address: [email protected] (N. Wang). cellular iron export through the suppression of ferroportin [7].

Fig. 1. Regulation of iron homeostasis by IRP/IRE in mammals. Cellular iron loading switches IRP1 from its IRE-binding form to an Fe-S cluster containing cytoplasmic aconitase and triggers proteasomal degradation of IRP2. Low iron levels promote accumulation of active IRP1 in its apo form and stabilize IRP2. IRP1 and IRP2 interact with IREs to coordinate the expression of proteins involved in iron uptake, export, and storage. DMT, divalent metal transporter; Fe, iron; IRE, iron-responsive element; IRP, iron- regulatory protein; RBC, red blood cell.

Hepcidin binds to the iron-transport protein ferroportin, factor 1 (IGF-1) receptor via substrates that include Akt, serum/ resulting in its destruction and thereby inhibiting absorption of glucocorticoid-regulated kinase (SGK), and protein kinase C a iron from the gastrointestinal tract and release of iron for mac- (PKC a) [11]. It is clear that mTOR signaling regulates iron ho- rophages [8] (Fig. 2). Additionally, an iron-conservation response meostasis, as treatment of mouse embryonic fibroblasts (MEFs) has been recently described [9]. The study found that tandem or H9 C2 cardiomyocyte with rapamycin leads to coordinated zinc finger protein tristetraprolin (TTP) causes destabilization of reduction in the expression of TfR1 and ferroportin 1, resulting in mRNAs of nonessential iron-containing proteins and liberation a net accumulation of cellular iron, whereas mTOR activation has of iron for use in vital processes by modulating TfR1 stability and the opposite effect [9]. Induction of TTP by rapamycin is unal- altering cellular iron flux. TTP has already been demonstrated as tered in MEFs with defective IRP 1/2 signaling, suggesting that the downstream target of mammalian target of rapamycin IRP/IRE is not involved in mTOR-dependent regulation of TTP [9]. (mTOR) [9]. mTOR is a phosphatidylinositol 3-kinase (PI3 K)-like However, silencing of ISCU not only inappropriately activated the serine/threonine protein kinase that is evolutionarily conserved IRP1 but also resulted in marked activation of the IRE-binding in all eukaryotes. The past several years have seen an explosion activity of IRP2 [12], suggesting that mTOR act as a “brake” on of interest in the mTOR signaling pathway, spurred in large part the IRP1/2 system. In addition to its effects on IRP1/2 system, by the finding that inhibition of mTORC1 signaling can signifi- mTOR inhibition leads to coordinated reduction in the expres- cantly increase life span and protect from age-related diseases in sion of ferroportin, whereas mTOR inhibition by rapamycin in mouse models [10]. mTOR can form two separate protein com- mouse liver has no effect on the expression of systemic iron- plexes (mTORCs). mTOR complex 1 (mTORC1), which is acutely regulatory hormone hepcidin and two of its upstream regula- sensitive to rapamycin, regulates processes such as ribosomal tors, bone morphogenic protein 6 and hemojuvel in [9]. biogenesis, cap-dependent translation, lysosomal biogenesis, Moreover, hepcidin and its upstream activator bone morpho- and autophagy via substrates that include S6 kinase (S6 K), 4 E- genic protein 6 gene expression in TTP-deficient mouse livers binding protein 1 (4 E-BP1), TFEB1, and Ulk1. mTOR complex 2 were dramatically reduced. Thus, the function of mTOR/TTP/TfR (mTORC2), which is resistant to acute rapamycin treatment but in regulation of hepcidin appears to be complex and more can be disrupted by chronic rapamycin treatment in tissue cul- research is needed. This review focuses on the molecular control ture as well as in vivo, is sensitive to growth factor signaling and of cellular iron homeostasis by mTOR, which is a parallel iron regulates targets downstream of the insulin/insulin-like growth regulatory network. 970 P. Guan, N. Wang / Nutrition 30 (2014) 968–974

Fig. 2. Regulation of iron homeostasis by hepcidin in mammals. Dietary iron is absorbed by duodenal enterocytes. It circulates bound to transferrin in the plasma and is mainly used for the hemoglobinization of newly synthesized red blood cells. Systemic iron is recycled by macrophages that also export iron via ferroportin into bloodstream. Hepcidin, secreted by liver in response to hepatic iron accumulation, controls iron uptake and recycling by inducing proteosomal degradation of ferroportin. DMT, divalent metal transporter; Fe, iron; HAMP, hepcidin antimicrobial peptide; RBC, red blood cell.

Iron-regulatory mTOR signaling system Scientists discovered iron deficiency decreased mTOR activity in vivo and in vitro. One study found for the first time that 3-wk- Mammalian target of rapamycin old male Wistar-strain rats fed an iron-deficient diet for 4 wk showed reduced mTOR activity, meanwhile COS-1 cells cultured Target of rapamycin (TOR) was initially identified as a target with the iron chelator deferoxamine also responded similarly, for the antifungal properties of rapamycin, which leads to showing significantly decreased phosphorylation of mTOR at Ser growth inhibition in Saccharomyces cerevisiae. Later, it was 2448 [17]. Another study examined mTOR activity in iron shown that TOR is also involved in the regulation of autophagy chelation-treated human myeloid leukemia cell line K562 cells and that rapamycin is able to induce autophagy in yeast even [18]. The study found phosphorylated mTOR was decreased in a under nutrient-rich conditions [13]. mTOR is central to nutrient- dose-dependent manner after deferasirox treatment. Further- and energy-sensing networks, it regulates many fundamental more, S6 ribosomal protein as well as phosphorylated S6, which metabolic and physiological processes, including cell size and is known to be a target of mTOR, was significantly repressed in mass, proliferation, survival [14], and lipid metabolism [15].Itis deferasirox-treated K562 cells. The specific role of iron in regu- activated under energetically favorable, low-stress states, lation of mTOR signaling also was shown in the nervous system whereas adverse conditions, such as starvation, cytotoxic insults, by examining two hippocampal, pyramidal cell-specific, non- and DNA damage act to inhibit mTOR signaling and promote anemic, genetic mouse models of iron deficiency in 2013: A survival through conservation of cellular resources [16]. mTOR CAMKIIa cre-loxP permanent knockout of divalent metal can interact with a number of protein partners, including pro- transporter-1 (DMT-1 CKO) and a CAMKIIa-tTA-driven revers- teins that probably act as adaptors or scaffolds and others that ible, overexpression of nonfunctional, dominant negative TfR-1. regulate mTOR to form at least two functionally distinct com- In both models, mTORC1 and mTORC2 activity, assessed by plexes. The one sensitive to rapamycin, known as mTORC1, phosphorylation levels of mTOR (Ser2448) and Akt (Ser473) promotes protein synthesis in response to amino acids, stress, respectively, was up-regulated during development by iron oxygen, energy, and growth factors via the phosphorylation of deficiency [19]. p70 S6 K and 4 EBP1; whereas the other, known as mTORC2, Recent studies identified mTOR as a key regulator of cellular promotes cell migration and survival via the activation of Rho iron metabolism; the regulatory pathway is a parallel iron- GTPases and the phosphorylation of Akt, respectively. regulatory network. In vitro, cellular iron uptake via TfR-1 is P. Guan, N. Wang / Nutrition 30 (2014) 968–974 971 modulated by mTOR activity-induced TfR-1 surface expression in required for iron delivery from transferrin to cells [26]. Each HeLa cells [20]. Further mechanistic studies identify TTP, a pro- transferrin molecule can bind two atoms of ferric iron, forming a tein involved in anti-inflammatory response, as the downstream molecule of apotransferrin. Interaction of transferrin-iron with a target of mTOR that binds to and enhances degradation of TfR1 high-affinity TfR1 expressed on cell surface triggers its inter- mRNA [9]. Moreover, mTORC1 associates with and phosphory- nalization through the formation of clarithin-coated pits and lates ISCU protein, consequently stabilizing ISCU and enhancing endocytosis. Following endocytosis of TfR1-apotransferrin com- ISC assembly [21]. Therefore, unrestrained mTORC1-mediated plex, iron is released from transferrin by acidification of the stabilization of ISCU protein sensitizes cells to iron deprivation, vesicle by a proton pumping ATPase and transferrin molecules due to constitutive ISC biogenesis-triggered iron demand that are returned to the bloodstream by exocytosis. Computational outstrips supply. analysis of the 3’-UTR of TfR1 revealed multiple putative AREs, some of which were found in a close proximity to or overlapped ISCU with IREs. It has been found that TfR1 mRNA levels are reduced because TTP interacts with TfR1 mRNA and leads to its degra- The scaffold protein ISCU acts as a scaffold on which the ISCs dation [9]. An RNA co-immunoprecipitation experiment with are assembled and from which the clusters are delivered to four designed primer sets for TfR1 to target the regions near its various apoproteins. It is synthesized as a precursor in the 3’-UTR was performed in human HEK293 cells to assess the cytosol and migrates to the mitochondria where it becomes the physical interaction between TTP and TfR1 mRNA. TfR1 mRNA mature form after a two-step mitochondria target sequence levels were enriched in the TTP group compared with the cleavage. In humans, mutations of ISCU decrease its expression immunoglobulin G control, indicative of interaction between TTP and ultimately the activities of muscle aconitase and succinate and 3’-UTR of TfR1 [9]. dehydrogenase, for which ISCs serve as essential cofactors. It is reported that phosphorylation of ISCU by mTORC1 prevents its The function of mTOR regulatory system degradation and increases ISC [21]. on iron metabolism

Tristetraprolin mTOR is an important sensor of the cellular energy state and a major hub for integration of environmental cues [27]. Many of TTP is recognized as an early response gene that was induced the processes governed by mTOR are dependent on iron as a in response to growth factors, inflammatory stimuli, and phorbol cofactor and would require a steady supply of this metal. The esters [22]. In yeast, a tandem zinc finger protein Cth2 was found essential role of mTOR in iron metabolism was established to conserve cellular iron in states of deficiency by preferentially because clinical use of rapamycin is associated with the devel- degrading mRNA of non-essential iron-containing proteins, thus opment of microcytosis and polyglobulia, which is mechanisti- reducing iron requirements and liberating iron for vital func- cally distinct from the anemia of chronic disease [28,29]. tions. TTP was found to be the functional homolog of Cth2 in According to the World Health Organization definition, func- mammalian, which is the mediator of iron-regulatory effects of tional iron deficiency was present in 80% of mTOR-treated mTOR and provides a novel link between energy metabolism, orthotopic heart transplant patients [30]. There are two mech- inflammation, and iron-regulatory pathways. Iron chelation with anisms that can be used to analyze how the mTOR pathway desferoxamine resulted in a significant and dose-dependent up- serves to modulate iron metabolism and homeostasis: Through regulation of TTP mRNA and protein expression, whereas iron phosphorylation stabilizing ISCU protein and inhibiting TTP overload with ferric ammonium citrate suppressed TTP expres- expression separately. sion [9]. These results suggest that TTP is regulated by cellular First, mTORC1 plays an important role in ISCU gene expres- iron status in mammals. TTP is known to mediate its functions by sion and function, and thus enhances ISC assembly. ISCU is an altering the stability of mRNAs of several transiently expressed mTOR kinase target, and phosphorylation of S14 is essential for inflammatory genes. TTP binds directly to the AU-rich element mTORC1-mediated preservation of ISCU protein [21]. It is re- (ARE) in the 3’ untranslated region (UTR) of tumor necrosis factor ported that ISCU can form a protein complex with cytosolic ISCs (TNF)-a transcript leading to its destabilization and rapid and that these two proteins together facilitate assembly of the degradation [23]. In a knockout mouse model, the lack of TTP has ISC of IRP1 and yeast mitochondrial aconitase [31]. Moreover, been shown to be normal at birth, but within a few months RNA interference studies have confirmed that silencing of ISCU develops a characteristic phenotype that includes loss of body not only disrupted ISC biogenesis but also inappropriately acti- weight and body fat, a severe syndrome of growth retardation, vated the IRP1 and disrupted intracellular iron homeostasis [12]. cachexia, arthritis, inflammation and autoimmunity [24]. Phos- IRP1 registers cytosolic iron and oxidative stress through its phorylation of TTP can lead to its inactivation because the labile ISC. In tissue culture cells, when iron is abundant, IRP1 unphosphorylated or de-phosphorylated form of TTP recruits contains a [4 Fe-4 S] cluster, exhibits aconitase activity, and binds more efficiently the deadenylase and mRNA decapping com- IREs with low affinity. When iron is scarce, IRP1 loses its ISC and plexes to the AREs containing TNF-a transcript to cause rapid aconitase activity and binds IREs with high affinity. Interestingly, degradation [25]. Recent studies in MEFs found TTP to regulate silencing of ISCU also resulted in marked activation of the IRE- cellular iron homeostasis by binding to AREs and destabilizing binding activity of IRP2, although IRP2 is not known to depend the mRNAs of iron-requiring proteins, thus potentially opti- on an ISC for its regulation [12]. Therefore, Insufficiency of ISCU mizing iron utilization in low-iron states [9]. triggered by mTORC1 inhibition can increase the binding activity between IRP and IRE. The endogenous ISCU knockdown showed Transferrin receptor 1 that knockdown of ISCU partially prevented the iron starvation- triggered cell death. With the discovery that transferrin serves as the iron source Moreover, mTOR activity was shown to inhibit the tran- for hemoglobin-synthesizing immature red blood cells came the scription and/or prevents destabilization of TTP mRNA, which in demonstration that a cell surface receptor, now known as TfR 1, is turn regulates the levels and stability of TfR 1 mRNA and 972 P. Guan, N. Wang / Nutrition 30 (2014) 968–974 subsequently iron metabolism in hearts of mice [9]. As the discovered [9]. It was found that TfR1 mRNA levels were signif- downstream target of mTOR, TTP functions similar to iron- icantly enriched in the TTP group in the pull-down experiments regulating yeast cth1 p/cth2 p proteins to regulate iron homeo- performed in human HEK293 cells. In summary, taken together, stasis. The budding yeast Saccharomyces cerevisiae has provided TTP is induced by rapamycin and negatively regulates the significant insight into iron homeostasis in eukaryotes, including expression of TfR1, leading to reduced iron import and net iron humans [32]. Yeast can down-regulate iron-dependent pro- loss from the cell. Iron deficiency would activate IRP1/2 to pre- cesses to preserve intracellular iron by a posttranscriptional vent further iron loss. IRE/IRP complexes formed within the mechanism via the RNA-binding proteins cth1 p/cth2 p, which 5’UTR of an mRNA (e.g., H-ferritin, L-ferritin, ferroportin 1) inhibit are up-regulated in iron deficiency [33]. Cth1 p/cth2 p proteins translation, whereas IRP binding to IREs in the 3’UTR of DMT1 preferentially bind to AREs and then destabilize the mRNAs of and TFR1 mRNA prevents its degradation. Moreover, TTP con- iron-requiring proteins, thus salvaging iron for use in essential serves cellular iron by repressing non-essential iron-requiring functions [34]. TTP was also shown to perform a similar function proteins and thus sparing iron for vital processes. As levels of of cth1 p/cth2 p to suppress the expression of ARE-containing iron-containing proteins decrease, more iron becomes available. iron-requiring proteins potentially optimizing iron utilization in low-iron states [9]. ATP-binding cassette E1 (ABCE1) is an ISC Regulation of mTOR regulatory system protein that is essential for ribosome function and for cellular viability in eukaryotic systems [35]. In TTP overexpression, MEFs, mTOR is placed downstream of the PI3 K/Akt pathway, thus mRNA levels of ABCE1 were dramatically reduced. Moreover, its activity can be regulated by multiple upstream signaling treatment of TTP knockout MEFs with the iron-chelating agents, pathways. Rapamycin, when bound to its intracellular receptor deferoxamine, not only failed to decrease, but instead led to a FKBP12, weakens the interaction of mTORC1 with its binding sharp increase in the expression of ABCE1. The results suggest partners, which is eventually followed by disassociation of the mammalian TTP could serve a similar function as yeast cth1 p/ complex. Recent data suggest that PI3 K and its downstream cth2 p. effector, the protein kinase Akt, control mTOR through the TSC1- Rapamycin, an mTOR inhibitor, can lead to increased TTP TSC2 tumor suppressor complex [36]. Growth factor activation of expression and specifically to a global suppression of many iron- PI3 K/Akt results in the phosphorylation and inhibition of TSC1- regulatory genes such as TfR1 and ferroportin 1 which is consis- TSC2, leading to derepression of mTOR. It has been reported tent with a reduction in cellular iron flux, whereas the opposite that the a1 A adrenergic receptor activated a pathway that 2þ was observed with activation of mTOR [9] (Fig. 3). The effects of required an increase in [Ca ]i, activation of phospholipase D, rapamycin on the iron-regulatory network are specific because and accumulation of phosphatidic acid to induce mTOR activa- there is no down-regulation of the genes not involved in regu- tion [37]. Amino acids may also signal to mTOR via the lation of cellular iron upon rapamycin treatment of wildtype Ca(2þ)/CaM-dependent activation of the lysosomal membrane MEFs. An interaction between TTP and TfR1 mRNA has been protein Vps34. Negative regulation of mTOR is imposed by

Fig. 3. Regulation of iron homeostasis by mTOR. mTORC1 can phosphorylate iron-sulfur cluster (ISC) assembly enzyme ISCU and thus increase ISC assembly. Stabilization of mTORC1 can inhibit IRP1 and IRP2 inactivate IRE-binding activities. Induction of TTP by rapamycin through mTOR results in destabilization of TfR1 mRNA and reduction in cellular iron import. Moreover, TTP conserves cellular iron by repressing non-essential iron-requiring proteins and thus sparing iron for vital processes. apo-Tf, apotransferrin; DMT, divalent metal transporter; Fe, iron; IRP, iron-regulatory protein; mTORC, mammalian target of rapamycin complex; TfR, transferrin receptor; TTP, tristetraprolin. P. Guan, N. Wang / Nutrition 30 (2014) 968–974 973 lipopolysaccharides and inflammatory cytokines, that may generally accepted to be a master regulator of cell growth, cell impinge on upstream amino acid signaling and mTOR itself to proliferation, cell motility, cell survival, protein synthesis, and prevent the phosphorylation of mTOR substrates and translation transcription. The impressive progress that has been achieved in initiation [38]. The small G protein Rheb is known to promote recent years in unraveling the regulation of mTOR pathway on mTOR signaling. mTOR has been identified as a common iron metabolism and its function may well translate into new downstream target of Rheb [39]. When Rheb contains bound drugs to treat patients with iron metabolism disorders. GTP, mTOR is activated. Conversely, in the presence of GDP, Rheb prevents phosphorylation of mTOR substrates. Pathways down- References stream of mTOR may play a role in the regulation of TTP and cellular iron homeostasis. 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