Accepted Manuscript The Mammalian Target of Rapamycin Coordinates Iron Metabolism with Iron-sulfur Cluster Assembly Enzyme and Tristetraprolin Guan Peng, Ph.D Wang Na, M.D PII:
S0899-9007(14)00034-3
DOI:
10.1016/j.nut.2013.12.016
Reference:
NUT 9193
To appear in:
Nutrition
Received Date: 18 October 2013 Revised Date:
13 December 2013
Accepted Date: 15 December 2013
Please cite this article as: Peng G, Na W, The Mammalian Target of Rapamycin Coordinates Iron Metabolism with Iron-sulfur Cluster Assembly Enzyme and Tristetraprolin, Nutrition (2014), doi: 10.1016/ j.nut.2013.12.016. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Title: The Mammalian Target of Rapamycin Coordinates Iron Metabolism with Iron-sulfur Cluster
Assembly Enzyme and Tristetraprolin
1
Key Laboratory of Animal Physiology, Biochemistry and Molecular Biology of Hebei Province,
Hebei Normal University, Shijiazhuang 050024, Hebei Province, China;
School of Basic Medical Sciences, Hebei University of Traditional Chinese Medicine, Shijiazhuang
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2
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Guan Peng Ph.D. 1, Wang Na M.D. 1, 2*
* Correspondence:
Tel.: +86 311 80787579
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050091, Hebei Province, China
Email address: [email protected] (N. Wang).
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Key Words: iron, rapamycin, iron-sulfur clusters, tristetraprolin, transferrin receptor 1
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running head: A link between mTOR and iron
ACCEPTED MANUSCRIPT Abstract Both iron deficiency and excess are relatively common health concerns. Maintaining the body’s levels of iron within precise boundaries is critical for cell functions. However, the difference between iron
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deficiency or 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 amounts of interest due to its recently described role in iron homeostasis. Despite extensive study, a complete understanding of
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mTOR function has remained elusive. mTOR can form two multi-protein complexes that consist of
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mTOR Complex 1 (mTORC1) and mTOR Complex 2 (mTORC2). Recent advances clearly demonstrate that mTORC1 can phosphorylate iron-sulfur cluster assembly enzyme ISCU and affect iron-sulfur clusters (ISCs) assembly. Moreover, mTOR is reported to control iron metabolism through modulation of tristetraprolin expression. It is now well appreciated that the hormonal
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hepcidin/ferroportin system and the cellular iron-responsive element/iron-regulatory protein (IRE/IRP) regulatory network play important regulatory roles for systemic iron metabolism. Sustained ISCU protein levels enhanced by mTORC1 can inhibit IRP and IRE binding activities, while hepcidin gene
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and protein expression in the livers of TTP KO mice were dramatically reduced. Here, we highlight and
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summarize the current understanding of how mTOR pathways serve to modulate iron metabolism and homeostasis as the third iron regulatory system.
ACCEPTED MANUSCRIPT Iron is an essential cofactor in diverse biological processes such as oxygen transport, cellular respiration, and DNA synthesis. The average adult human contains 2~4 g of iron. Iron deficiency can cause cellular growth arrest and death. However, iron is extremely toxic when present in excess:
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ferrous iron reacts with hydrogen peroxides or lipid peroxides to generate hydroxyl or lipid radicals, respectively. So the levels of iron must be tightly controlled inside individual organelles, cells and tissues. However, the molecular circuits that achieve iron balance under most conditions have only
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recently begun to be characterized. The iron content of the body is tightly regulated by at least two
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mechanisms, iron regulatory proteins (IRPs) and RNA stem-loop iron regulatory elements (IREs) mechanism[1] and the transcriptional regulation of hepcidin[2].
IRPs are central regulators of cellular iron homeostasis due to their regulation of specific mRNAs encoding proteins of iron uptake, export, storage, and utilization. IRP1 can assume two different
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functions in the cell, depending on conditions[3] (Figure 1). During iron deficiency, IRP1 binds to IREs to modulate the translation of iron metabolism genes. In iron-rich conditions, IRP1 binds an ISC to function as a cytosolic aconitase. IRP2 has ∼60% sequence identity to IRP1, but IRP2 does not display
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aconitase activity[4]. In the case of IRP2, iron controls its RNA binding activity by stimulating its
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ubiquitination and rapid proteasomal degradation[5]. IRP2 exhibits a different pattern of affinities to the IRE family than IRP1, having in general weaker binding to the non-ferritin types[6]. IRPs function to restore iron levels through stabilization of transferrin receptor 1 (TfR1) messenger RNA (mRNA) and increased iron uptake, mobilization of cellular iron stores, and reduction in cellular iron export through the suppression of ferroportin[7]. Hepcidin binds to the iron-transport protein ferroportin, resulting in its destruction and thereby inhibiting absorption of iron from the gastrointestinal tract and release of iron for macrophages[8]
ACCEPTED MANUSCRIPT (Figure 2). Additionally, an iron conservation response has been recently described by Bayeva et al.[9], their study found that tandem zinc finger (TZF) protein tristetraprolin (TTP) causes destabilization of mRNAs of nonessential iron-containing proteins and liberation of iron for use in vital processes by
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modulating transferrin receptor 1 (TfR1) stability and altering cellular iron flux. TTP has already been demonstrated as the downstream target of mammalian target of rapamycin (mTOR)[9]. mTOR is a phosphatidylinositol 3-kinase (PI3K)-like serine/threonine protein kinase that is evolutionarily
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conserved in all eukaryotes. The past several years have seen an explosion of interest in the mTOR
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signaling pathway, spurred in large part by the finding that inhibition of mTORC1 signaling can significantly increase lifespan and protect from age-related diseases in mouse models[10]. The mTOR can form two separate protein complexes (mTORCs). mTOR complex 1 (mTORC1), which is acutely sensitive to rapamycin, regulates processes such as ribosomal biogenesis, cap-dependent translation,
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lysosomal biogenesis, and autophagy via substrates that include S6 kinase (S6K), 4E-binding protein 1 (4E-BP1), TFEB1, and Ulk1. mTOR complex 2 (mTORC2), which is resistant to acute rapamycin treatment but can be disrupted by chronic rapamycin treatment in tissue culture as well as in vivo, is
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sensitive to growth factor signaling and regulates targets downstream of the insulin/insulin-like growth
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factor 1 (IGF-1) receptor via substrates that include Akt, serum/glucocorticoid-regulated kinase (SGK), and protein kinase C α (PKC α) [11]. It is clear that mTOR signaling regulates iron homeostasis, as treatment of MEFs or H9c2 with rapamycin leads to coordinated reduction in the expression of TfR1 and ferroportin 1, resulting in a net accumulation of cellular iron, while mTOR activation has the opposite effect[9]. Induction of TTP by rapamycin is unaltered in MEFs with defective IRP 1/2 signaling, suggesting that IRP/IRE is not involved in mTOR-dependent regulation of TTP [9]. However, silencing of ISCU not only inappropriately activated the IRP1 but also resulted in marked activation of
ACCEPTED MANUSCRIPT the IRE binding activity of IRP2 [12], suggesting that mTOR act as a ‘‘brake’’ on the IRP1/2 system. In addition to its effects on IRP1/2 system, mTOR inhibition leads to coordinated reduction in the expression of ferroportin, while mTOR inhibition by rapamycin in mouse liver has no effect on the
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expression of systemic iron-regulatory hormone hepcidin and two of its upstream regulators, bone morphogenic protein 6 (BMP6) and hemojuvel in (HJV) [9]. Moreover, hepcidin and its upstream activator BMP6 gene expression in TTP-deficient mouse livers were dramatically reduced. Thus, the
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function of mTOR/TTP/TfR in regulation of hepcidin appears to be complex, more research is needed.
iron regulatory network.
1 The consist of mTOR regulatory system
1.1 The mammalian target of rapamycin
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This review focuses on the molecular control of cellular iron homeostasis by mTOR, which is a parallel
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Target of rapamycin (TOR) was initially identified as a target for the antifungal properties of rapamycin, which leads to growth inhibition in Saccharomyces cerevisiae. Later, it was shown that TOR is also
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involved in the regulation of autophagy and that rapamycin is able to induce autophagy in yeast even under nutrient-rich conditions[13]. The mTOR is central to nutrient and energy-sensing networks, it
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regulates many fundamental metabolic and physiological processes, including cell size and mass, proliferation, survival[14] and lipid metabolism[15]. It will be activated under energetically favorable, low-stress states, while adverse conditions, such as starvation, cytotoxic insults and DNA damage, act to inhibit mTOR signaling and promote survival through conservation of cellular resources[16]. The mTOR can interact with a number of protein partners, including proteins that probably act as adaptors or scaffolds and others that regulate mTOR to form at least two functionally distinct complexes. The one sensitive to rapamycin, known as mTORC1, promotes protein synthesis in response to amino acids,
ACCEPTED MANUSCRIPT stress, oxygen, energy, and growth factors via the phosphorylation of p70S6K and 4EBP1; While the other, known as mTORC2, promotes cell migration and survival via the activation of Rho GTPases and the phosphorylation of Akt, respectively.
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Scientists discovered iron-deficiency decreased mTOR activity in vivo and vitro. In a study conducted by Ndong et al. in 2009[17], it was found for the first time that three-week-old male Wistar-strain rats fed with iron-deficient diet for 4 weeks showed reduced mTOR activity,
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meanwhile, COS-1 cells cultured with the iron chelator deferoxamine also responded similarly,
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showing significantly decreased phosphorylation of mTOR at Ser 2448. In 2009, Ohyashiki et al.[18] studied the mTOR activity in iron chelation-treated in human myeloid leukemia cell line K562 cells. They found phosphorylated mTOR was decreased in a dose-dependent manner after deferasirox treated. Furthermore, S6 ribosomal protein as well as phosphorylated S6, which is
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known to be a target of mTOR, was significantly repressed in deferasirox-treated K562 cells. The specific role of iron in regulation of mTOR signaling was also shown in nervous system by examining two hippocampal, pyramidal cell-specific, nonanemic, genetic mouse models of iron
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deficiency in 2013: a CAMKIIα cre-loxP permanent knockout of divalent metal transporter-1
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(DMT-1 CKO) and a CAMKIIα-tTA-driven reversible, overexpression of nonfunctional, dominant negative transferrin receptor-1 (DN TfR-1). In both models, mTORC1 and mTORC2 activity, assessed by phosphorylation levels of mTOR (Ser2448) and Akt (Ser473) respectively, was upregulated during development by iron deficiency[19]. Recently studies identify mTOR as a key regulator of cellular iron metabolism, the regulatory pathway is a parallel iron regulatory network. In vitro, cellular iron uptake via TfR-1 is modulated by mTOR activity-induced TfR-1 surface expression in HeLa cells[20]. Further mechanistic studies identify
ACCEPTED MANUSCRIPT triste-traprolin (TTP), a protein involved in anti-inflammatory response, as the downstream target of mTOR that binds to and enhances degradation of TfR1 mRNA[9]. Moreover, mTORC1 can associates with and phosphorylates ISCU protein, consequently stabilizing ISCU and enhancing ISC
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assembly[21]. Therefore, unrestrained mTORC1-mediated stabilization of ISCU protein sensitizes cells to iron deprivation, due to constitutive ISC biogenesis-triggered iron demand that outstrips supply. 1.2 ISCU
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The scaffold protein ISCU acts as a scaffold on which the ISCs are assembled and from which the
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clusters are delivered to various apoproteins. It is synthesized as a precursor in the cytosol and migrates to the mitochondria where it becomes the mature form after a two-step mitochondria target sequence cleavage. In humans, mutations of ISCU decrease its expression and ultimately the activities of muscle aconitase and succinate dehydrogenase, for which ISCs serve as essential cofactors. It is reported that
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phosphorylation of ISCU by mTORC1 prevents its degradation and increases ISC [21]. 1.3 Tristetraprolin
Tristetraprolin (TTP) is recognized as an early response gene which was induced in response to growth
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factors, inflammatory stimuli and phorbol esters[22]. In yeast, a tandem zinc finger (TZF) protein
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Cth2 was found to conserve cellular iron in states of deficiency by preferentially degrading mRNA of non-essential iron-containing proteins thus reducing iron requirements and liberating iron for vital functions. TTP was found to be the functional homolog of Cth2 in mammalian, which is the mediator of iron-regulatory effects of mTOR and provide a novel link between energy metabolism, inflammation and iron regulatory pathways. Iron chelation with DFO resulted in a significant and dose-dependent upregulation of TTP mRNA and protein expression, while iron overload with ferric ammonium citrate (FAC) suppressed TTP expression [9]. These results suggest that TTP is regulated by
ACCEPTED MANUSCRIPT cellular iron status in mammals. TTP is known to mediate its functions by altering the stability of mRNAs of several transiently expressed inflammatory genes. TTP binds directly to the AU-rich element in the 3′-UTR of TNF-α transcript leading to its destabilization and rapid degradation [23]. In a
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knock-out mouse model, the lack of TTP has been shown to be normal at birth, but within a few months develop a characteristic phenotype that includes loss of body weight and body fat, a severe syndrome of growth retardation, cachexia, arthritis,
inflammation and autoimmunity[24].
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Phosphorylation of TTP can lead to its inactivation because the unphosphorylated or de-phosphorylated
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form of TTP recruits more efficiently the deadenylase and mRNA decapping complexes to the AU-rich element (AREs) containing TNF-α transcript to cause rapid degradation[25]. Recent studies in mouse embryonic fibroblasts (MEFs) found TTP to regulate cellular iron homeostasis by binding to AREs and destabilizing the mRNAs of iron-requiring proteins, thus potentially optimizing iron utilization in
1.4 Transferrin receptor 1
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low-iron states[9].
With the discovery that transferrin serves as the iron source for hemoglobin-synthesizing immature red
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blood cells came the demonstration that a cell surface receptor, now known as transferrin receptor 1
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(TfR 1), is required for iron delivery from transferrin to cells[26]. Each transferrin molecule can bind two atoms of ferric iron, forming a molecule of apotransferrin. Interaction of transferrin-iron with a high-affinity TfR1 expressed on cell surface triggers its internalization through the formation of a clarithin-coated pits and endocytosis. Following endocytosis of TfR1-apotransferrin complex, iron is released from transferrin by acidification of the vesicle by a proton pumping ATPase and transferrin molecules are returned to the bloodstream by exocytosis. Computational analysis of the 3’ untranslated region (UTR) of TfR1 revealed multiple putative AREs, some of which were found in a close
ACCEPTED MANUSCRIPT proximity to or overlapped with IREs. Bayeva et al. found TfR1 mRNA levels to be reduced because TTP interacts with TfR1 mRNA and leads to its degradation[9]. RNA co-immunoprecipitation experiment with four designed primer sets for TfR1 to target the regions near its 3’UTR was performed
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in human HEK293 cells to assess the physical interaction between TTP and TfR1 mRNA. TfR1 mRNA levels were enriched in the TTP group compared to the IgG control, indicative of interaction between
2 The function of mTOR regulatory system on iron metabolism
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TTP and 3’UTR of TfR1 [9].
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mTOR is an important sensor of cellular energy state and a major hub for integration of environmental cues[27], many of the processes governed by mTOR are dependent on iron as a cofactor and would require a steady supply of this metal. The essential role of mTOR in iron metabolism was established as clinical use of rapamycin is associated with the development of microcytosis and polyglobulia that is
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mechanistically distinct from the anemia of chronic disease[28, 29]. According to the World Health Organization definition, functional iron deficiency was present in 80% of mTOR-treated orthotopic
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heart transplant patients[30]. There are two mechanisms that can be used to analyze how mTOR pathway serves to modulate iron metabolism and homeostasis: through phosphorylation stabilizing
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ISCU protein and inhibiting TTP expression separately. First, mTORC1 plays an important role in ISCU gene expression and function, and thus enhance ISC assembly. ISCU is an mTOR kinase target, and phosphorylation of S14 is essential for mTORC1-mediated preservation of ISCU protein[21]. It is reported that ISCU can form a protein complex with cytosolic ISCS and that these two proteins together facilitate assembly of the ISC of IRP1 and yeast mitochondrial aconitase [31]. Moreover, RNA interference studies have confirmed that silencing of ISCU not only disrupted ISC biogenesis but also inappropriately activated the IRP1 and
ACCEPTED MANUSCRIPT disrupted intracellular iron homeostasis [12]. IRP1 registers cytosolic iron and oxidative stress
through its labile ISC. In tissue culture cells, when iron is abundant, IRP1 contains a [4Fe-4S] cluster, exhibits aconitase activity, and binds IREs with low affinity. When iron is scarce, IRP1
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loses its ISC and aconitase activity and binds IREs with high affinity. Interestingly, silencing of ISCU also resulted in marked activation of the IRE binding activity of IRP2, although IRP2 is not known to depend on an ISC for its regulation[12]. Therefore, Insufficiency of ISCU triggered by
knockdown
showed
that
starvation-triggered cell death.
knockdown
of ISCU
partially prevented
the
iron
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ISCU
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mTORC1 inhibition can increase the binding activity between IRP and IRE. The endogenous
Moreover, mTOR activity was shown to inhibit the transcription and/or prevents destabilization of Tristetraprolin (TTP) mRNA, which in turn regulates the levels and stability of TfR 1 mRNA and
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subsequently iron metabolism in hearts of mice [9]. As the downstream target of mTOR, TTP functions similar to iron-regulating yeast cth1p/cth2p proteins to regulate iron homeostasis. The budding yeast Saccharomyces cerevisiae has provided significant insight into iron homeostasis in eukaryotes,
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including humans[32]. Yeast can down-regulate iron-dependent processes to preserve intracellular iron
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by a post-transcriptional mechanism via the RNA-binding proteins cth1p/cth2p, which are up-regulated in iron deficiency[33]. Cth1p/cth2p proteins preferentially bind to AU-rich elements (AREs) and then destabilize the mRNAs of iron-requiring proteins, thus salvaging iron for use in essential functions[34]. TTP was also shown a similar function of cth1p/cth2p to suppress the expression of ARE-containing iron-requiring proteins potentially optimizing iron utilization in low-iron states[9]. ATP binding cassette E1 (ABCE1) is an ISC protein which is essential for ribosome function and for cellular viability in eukaryotic systems[35]. In TTP overexpression MEFs, mRNA levels of ABCE1 were
ACCEPTED MANUSCRIPT dramatically reduced. Moreover, treatment of TTP KO MEFs with the iron chelating agents, deferoxamine, not only failed to decrease, but instead led to a sharp increase in the expression of ABCE1. The results suggest mammalian TTP could serve a similar function as yeast Cth1p/Cth2p.
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Rapamycin, an mTOR inhibitor, can lead to increased TTP expression and specifically lead to a global suppression of many iron-regulatory genes such as TfR1, Ferroportin1 which is consistent with a reduction in cellular iron flux, while the opposite was observed with activation of mTOR[9] (Figure 3).
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The effects of rapamycin on iron-regulatory network are specific because there is no down-regulation
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of several genes not involved in regulation of cellular iron upon rapamycin treatment of WT MEFs. Bayeva et al.[9] discovered an interaction between TTP and TfR1 mRNA, they found TfR1 mRNA levels were significantly enriched in the TTP group in the pull-down experiments performed in human HEK293 cells. In summary, Taken together, TTP is induced by rapamycin and negatively regulates the
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expression of TfR1, leading to reduced iron import and net iron loss from the cell. Iron deficiency would activate IRP1/2 to prevent further iron loss. IRE/IRP complexes formed within the 5’UTR of an mRNA (e.g., H-ferritin, L-ferritin, ferroportin 1) inhibit translation, whereas IRP binding to IREs in the
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3’UTR of DMT1 and TFR1 mRNA prevents its degradation. Moreover, TTP conserves cellular iron by
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repressing non-essential iron-requiring proteins and thus sparing iron for vital processes. As levels of iron-containing proteins decrease and more iron becomes available.
3 The regulation of mTOR regulatory system
The mTOR is placed downstream of the PI3K/Akt pathway, thus its activity can be regulated by multiple upstream signaling pathways. Rapamycin, when bound to its intracellular receptor FKBP12, weakens the interaction of mTORC1 with its binding partners, which is eventually followed by disassociation of the complex. Recent data suggest that PI3K and its downstream effector, the protein
ACCEPTED MANUSCRIPT kinase Akt, control mTOR through the TSC1-TSC2 tumor suppressor complex[36]. Growth factor activation of PI3K/Akt results in the phosphorylation and inhibition of TSC1-TSC2, leading to derepression of mTOR. Ballou and coworkers reported that the α1A adrenergic receptor activated a
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pathway that required an increase in [Ca2+]i, activation of phospholipase D, and accumulation of phosphatidic acid to induce mTOR activation[37]. Amino acids may also signal to mTOR via the Ca(2+)/CaM-dependent activation of the lysosomal membrane protein Vps34. Negative regulation of
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mTOR is imposed by lipopolysaccharides and inflammatory cytokines, that may impinge on upstream
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amino acid signaling and mTOR itself to prevent the phosphorylation of mTOR substrates and translation initiation[38]. The small G protein Rheb is known to promote mTOR signaling. Tee et al. identified mTOR as a common downstream target of Rheb[39]. When Rheb contains bound GTP, mTOR is activated. Conversely, in the presence of GDP, Rheb prevents phosphorylation of mTOR
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substrates. Pathways downstream of mTOR may play a role in the regulation of TTP and cellular iron homeostasis.
Understanding the functional interconnections between iron and the mTOR system represents an
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important challenge to fully comprehend iron homeostasis. Iron deficiency was also shown to inhibit
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mTOR signaling. Ndong et al.[17] found iron-deficient diet depressed Akt activity in rats and in COS-1 cells, leading to a decrease in mTOR activity. In addition, it was observed that down-regulation of mTOR following marked down-regulation of the phosphorylated S6 protein in deferasirox-treated leukemia cells[18]. Additional investigations showed that rapamycin and iron chelation with DFO have an additive effect on TTP induction[9]. Increasing TTP can promote downregulation of iron-requiring genes and modulates survival in low-iron states.
4 Perspective
ACCEPTED MANUSCRIPT Mammalian iron homeostasis is maintained through the concerted action of sensory and regulatory networks that modulate the expression of proteins of iron metabolism at the transcriptional and/or post-transcriptional levels. For 30 years or more we have known that maintenance of cellular iron
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homeostasis is dependent on RNA-binding iron-regulatory proteins 1 and 2 (IRP1/2), which are activated under iron-deficient conditions. IRP1/2 function to restore iron levels through stabilization of TfR1 mRNA and increased iron uptake through stabilization of DMT1 mRNA, mobilization of cellular
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iron stores, and reduction in cellular iron export through the suppression of ferroportin 1[40]. Since the
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beginning of this century, the understanding of systemic iron homeostasis and its disorders has rapidly progressed, and regulation by the hepcidin/ferroportin system can now explain key features of systemic iron balance[1]. mTOR/TTP/TfR1 system is a new pathway mediating the iron homeostasis in parallel with two well-established IRP1/2 and hepcidin systems. mTOR/TTP/TfR1 system may act as a
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‘‘brake’’ on the IRP1/2 system to prevent the unnecessary overactivation of iron uptake as the cell is reducing its iron use through TTP-dependent downregulation of iron-containing proteins. While the mTOR pathway plays a role in regulation of cellular iron availability and utilization, its main function
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is seen as a mechanism for cell survival. In instances where the cell is suffering from iron starvation, it
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reduces the levels of proteins that require the nutrient, conserving it for use by essential proteins only. This in turn keeps the cell viable and prevents cell death. On the other hand, considering mTOR is frequently activated in tumors, it would benefit patients to apply iron chelators to tumors. Tumor cells exhibit increased uptake and utilization of iron by virtue of possessing significantly higher levels of transferrin receptor 1 than healthy cells[41]. This has led to the suggestion that agents that deplete intracellular iron may be useful in cancer therapy. With the deeper understanding of iron metabolism and its regulation will offer avenues for new treatment options. The mTOR kinase is generally accepted
ACCEPTED MANUSCRIPT to be a master regulator of cell growth, cell proliferation, cell motility, cell survival, protein synthesis, and transcription, the impressive progress that has been achieved during the past years in unraveling the regulation of mTOR pathway on iron metabolism and its function may well translate into new drugs
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to treat patients with iron metabolism disorder. Funding
This work was supported by National Natural Science Foundation of China (grant number 31200863),
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Science and Technology Research Youth Fund Project of Hebei Colleges and Universities (grant
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number QN20131009) and Hebei Normal University Fund (grant number L2010Q06). References
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ACCEPTED MANUSCRIPT [29] Sofroniadou S, Kassimatis T, Goldsmith D. Anaemia, microcytosis and sirolimus--is iron the missing link? Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association. 2010; 25:1667-75. [30] Przybylowski P, Malyszko JS, Macdougall IC, Malyszko J. Iron metabolism, hepcidin, and anemia in orthotopic heart transplantation recipients treated with mammalian target of rapamycin. Transplantation proceedings. 2013; 45:387-90. [31] Li K, Tong WH, Hughes RM, Rouault TA. Roles of the mammalian cytosolic cysteine desulfurase, 2006; 281:12344-51.
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ISCS, and scaffold protein, ISCU, in iron-sulfur cluster assembly. The Journal of biological chemistry. [32] Kaplan J, McVey Ward D, Crisp RJ, Philpott CC. Iron-dependent metabolic remodeling in S. cerevisiae. Biochimica et biophysica acta. 2006; 1763:646-51.
[33] Puig S, Askeland E, Thiele DJ. Coordinated remodeling of cellular metabolism during iron
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deficiency through targeted mRNA degradation. Cell. 2005; 120:99-110.
[34] Puig S, Vergara SV, Thiele DJ. Cooperation of two mRNA-binding proteins drives metabolic adaptation to iron deficiency. Cell metabolism. 2008; 7:555-64.
[35] Sims LM, Igarashi RY. Regulation of the ATPase activity of ABCE1 from Pyrococcus abyssi by
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Fe-S cluster status and Mg(2)(+): implication for ribosomal function. Archives of biochemistry and biophysics. 2012; 524:114-22.
[36] Marygold SJ, Leevers SJ. Growth signaling: TSC takes its place. Current biology : CB. 2002; 12:R785-7.
[37] Ballou LM, Jiang YP, Du G, Frohman MA, Lin RZ. Ca(2+)- and phospholipase D-dependent and -independent pathways activate mTOR signaling. FEBS letters. 2003; 550:51-6. [38] Frost RA, Lang CH. mTor signaling in skeletal muscle during sepsis and inflammation: where
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does it all go wrong? Physiology (Bethesda). 2011; 26:83-96.
[39] Tee AR, Blenis J, Proud CG. Analysis of mTOR signaling by the small G-proteins, Rheb and RhebL1. FEBS letters. 2005; 579:4763-8.
[40] Eisenstein RS. Iron regulatory proteins and the molecular control of mammalian iron metabolism. Annual review of nutrition. 2000; 20:627-62.
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[41] Faulk WP, Hsi BL, Stevens PJ. Transferrin and transferrin receptors in carcinoma of the breast.
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Lancet. 1980; 2:390-2.
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Figure 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.
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Figure 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.
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Figure 3. Regulation of iron homeostasis by mTOR. mTORC1 can phosphorylate iron-sulfur cluster 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.
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S0899-9007(14)00034-3
DOI:
10.1016/j.nut.2013.12.016
Reference:
NUT 9193
To appear in:
Nutrition
Received Date: 18 October 2013 Revised Date:
13 December 2013
Accepted Date: 15 December 2013
Please cite this article as: Peng G, Na W, The Mammalian Target of Rapamycin Coordinates Iron Metabolism with Iron-sulfur Cluster Assembly Enzyme and Tristetraprolin, Nutrition (2014), doi: 10.1016/ j.nut.2013.12.016. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Title: The Mammalian Target of Rapamycin Coordinates Iron Metabolism with Iron-sulfur Cluster
Assembly Enzyme and Tristetraprolin
1
Key Laboratory of Animal Physiology, Biochemistry and Molecular Biology of Hebei Province,
Hebei Normal University, Shijiazhuang 050024, Hebei Province, China;
School of Basic Medical Sciences, Hebei University of Traditional Chinese Medicine, Shijiazhuang
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2
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Guan Peng Ph.D. 1, Wang Na M.D. 1, 2*
* Correspondence:
Tel.: +86 311 80787579
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050091, Hebei Province, China
Email address: [email protected] (N. Wang).
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Key Words: iron, rapamycin, iron-sulfur clusters, tristetraprolin, transferrin receptor 1
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running head: A link between mTOR and iron
ACCEPTED MANUSCRIPT Abstract Both iron deficiency and excess are relatively common health concerns. Maintaining the body’s levels of iron within precise boundaries is critical for cell functions. However, the difference between iron
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deficiency or 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 amounts of interest due to its recently described role in iron homeostasis. Despite extensive study, a complete understanding of
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mTOR function has remained elusive. mTOR can form two multi-protein complexes that consist of
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mTOR Complex 1 (mTORC1) and mTOR Complex 2 (mTORC2). Recent advances clearly demonstrate that mTORC1 can phosphorylate iron-sulfur cluster assembly enzyme ISCU and affect iron-sulfur clusters (ISCs) assembly. Moreover, mTOR is reported to control iron metabolism through modulation of tristetraprolin expression. It is now well appreciated that the hormonal
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hepcidin/ferroportin system and the cellular iron-responsive element/iron-regulatory protein (IRE/IRP) regulatory network play important regulatory roles for systemic iron metabolism. Sustained ISCU protein levels enhanced by mTORC1 can inhibit IRP and IRE binding activities, while hepcidin gene
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and protein expression in the livers of TTP KO mice were dramatically reduced. Here, we highlight and
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summarize the current understanding of how mTOR pathways serve to modulate iron metabolism and homeostasis as the third iron regulatory system.
ACCEPTED MANUSCRIPT Iron is an essential cofactor in diverse biological processes such as oxygen transport, cellular respiration, and DNA synthesis. The average adult human contains 2~4 g of iron. Iron deficiency can cause cellular growth arrest and death. However, iron is extremely toxic when present in excess:
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ferrous iron reacts with hydrogen peroxides or lipid peroxides to generate hydroxyl or lipid radicals, respectively. So the levels of iron must be tightly controlled inside individual organelles, cells and tissues. However, the molecular circuits that achieve iron balance under most conditions have only
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recently begun to be characterized. The iron content of the body is tightly regulated by at least two
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mechanisms, iron regulatory proteins (IRPs) and RNA stem-loop iron regulatory elements (IREs) mechanism[1] and the transcriptional regulation of hepcidin[2].
IRPs are central regulators of cellular iron homeostasis due to their regulation of specific mRNAs encoding proteins of iron uptake, export, storage, and utilization. IRP1 can assume two different
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functions in the cell, depending on conditions[3] (Figure 1). During iron deficiency, IRP1 binds to IREs to modulate the translation of iron metabolism genes. In iron-rich conditions, IRP1 binds an ISC to function as a cytosolic aconitase. IRP2 has ∼60% sequence identity to IRP1, but IRP2 does not display
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aconitase activity[4]. In the case of IRP2, iron controls its RNA binding activity by stimulating its
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ubiquitination and rapid proteasomal degradation[5]. IRP2 exhibits a different pattern of affinities to the IRE family than IRP1, having in general weaker binding to the non-ferritin types[6]. IRPs function to restore iron levels through stabilization of transferrin receptor 1 (TfR1) messenger RNA (mRNA) and increased iron uptake, mobilization of cellular iron stores, and reduction in cellular iron export through the suppression of ferroportin[7]. Hepcidin binds to the iron-transport protein ferroportin, resulting in its destruction and thereby inhibiting absorption of iron from the gastrointestinal tract and release of iron for macrophages[8]
ACCEPTED MANUSCRIPT (Figure 2). Additionally, an iron conservation response has been recently described by Bayeva et al.[9], their study found that tandem zinc finger (TZF) protein tristetraprolin (TTP) causes destabilization of mRNAs of nonessential iron-containing proteins and liberation of iron for use in vital processes by
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modulating transferrin receptor 1 (TfR1) stability and altering cellular iron flux. TTP has already been demonstrated as the downstream target of mammalian target of rapamycin (mTOR)[9]. mTOR is a phosphatidylinositol 3-kinase (PI3K)-like serine/threonine protein kinase that is evolutionarily
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conserved in all eukaryotes. The past several years have seen an explosion of interest in the mTOR
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signaling pathway, spurred in large part by the finding that inhibition of mTORC1 signaling can significantly increase lifespan and protect from age-related diseases in mouse models[10]. The mTOR can form two separate protein complexes (mTORCs). mTOR complex 1 (mTORC1), which is acutely sensitive to rapamycin, regulates processes such as ribosomal biogenesis, cap-dependent translation,
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lysosomal biogenesis, and autophagy via substrates that include S6 kinase (S6K), 4E-binding protein 1 (4E-BP1), TFEB1, and Ulk1. mTOR complex 2 (mTORC2), which is resistant to acute rapamycin treatment but can be disrupted by chronic rapamycin treatment in tissue culture as well as in vivo, is
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sensitive to growth factor signaling and regulates targets downstream of the insulin/insulin-like growth
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factor 1 (IGF-1) receptor via substrates that include Akt, serum/glucocorticoid-regulated kinase (SGK), and protein kinase C α (PKC α) [11]. It is clear that mTOR signaling regulates iron homeostasis, as treatment of MEFs or H9c2 with rapamycin leads to coordinated reduction in the expression of TfR1 and ferroportin 1, resulting in a net accumulation of cellular iron, while mTOR activation has the opposite effect[9]. Induction of TTP by rapamycin is unaltered in MEFs with defective IRP 1/2 signaling, suggesting that IRP/IRE is not involved in mTOR-dependent regulation of TTP [9]. However, silencing of ISCU not only inappropriately activated the IRP1 but also resulted in marked activation of
ACCEPTED MANUSCRIPT the IRE binding activity of IRP2 [12], suggesting that mTOR act as a ‘‘brake’’ on the IRP1/2 system. In addition to its effects on IRP1/2 system, mTOR inhibition leads to coordinated reduction in the expression of ferroportin, while mTOR inhibition by rapamycin in mouse liver has no effect on the
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expression of systemic iron-regulatory hormone hepcidin and two of its upstream regulators, bone morphogenic protein 6 (BMP6) and hemojuvel in (HJV) [9]. Moreover, hepcidin and its upstream activator BMP6 gene expression in TTP-deficient mouse livers were dramatically reduced. Thus, the
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function of mTOR/TTP/TfR in regulation of hepcidin appears to be complex, more research is needed.
iron regulatory network.
1 The consist of mTOR regulatory system
1.1 The mammalian target of rapamycin
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This review focuses on the molecular control of cellular iron homeostasis by mTOR, which is a parallel
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Target of rapamycin (TOR) was initially identified as a target for the antifungal properties of rapamycin, which leads to growth inhibition in Saccharomyces cerevisiae. Later, it was shown that TOR is also
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involved in the regulation of autophagy and that rapamycin is able to induce autophagy in yeast even under nutrient-rich conditions[13]. The mTOR is central to nutrient and energy-sensing networks, it
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regulates many fundamental metabolic and physiological processes, including cell size and mass, proliferation, survival[14] and lipid metabolism[15]. It will be activated under energetically favorable, low-stress states, while adverse conditions, such as starvation, cytotoxic insults and DNA damage, act to inhibit mTOR signaling and promote survival through conservation of cellular resources[16]. The mTOR can interact with a number of protein partners, including proteins that probably act as adaptors or scaffolds and others that regulate mTOR to form at least two functionally distinct complexes. The one sensitive to rapamycin, known as mTORC1, promotes protein synthesis in response to amino acids,
ACCEPTED MANUSCRIPT stress, oxygen, energy, and growth factors via the phosphorylation of p70S6K and 4EBP1; While the other, known as mTORC2, promotes cell migration and survival via the activation of Rho GTPases and the phosphorylation of Akt, respectively.
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Scientists discovered iron-deficiency decreased mTOR activity in vivo and vitro. In a study conducted by Ndong et al. in 2009[17], it was found for the first time that three-week-old male Wistar-strain rats fed with iron-deficient diet for 4 weeks showed reduced mTOR activity,
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meanwhile, COS-1 cells cultured with the iron chelator deferoxamine also responded similarly,
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showing significantly decreased phosphorylation of mTOR at Ser 2448. In 2009, Ohyashiki et al.[18] studied the mTOR activity in iron chelation-treated in human myeloid leukemia cell line K562 cells. They found phosphorylated mTOR was decreased in a dose-dependent manner after deferasirox treated. Furthermore, S6 ribosomal protein as well as phosphorylated S6, which is
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known to be a target of mTOR, was significantly repressed in deferasirox-treated K562 cells. The specific role of iron in regulation of mTOR signaling was also shown in nervous system by examining two hippocampal, pyramidal cell-specific, nonanemic, genetic mouse models of iron
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deficiency in 2013: a CAMKIIα cre-loxP permanent knockout of divalent metal transporter-1
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(DMT-1 CKO) and a CAMKIIα-tTA-driven reversible, overexpression of nonfunctional, dominant negative transferrin receptor-1 (DN TfR-1). In both models, mTORC1 and mTORC2 activity, assessed by phosphorylation levels of mTOR (Ser2448) and Akt (Ser473) respectively, was upregulated during development by iron deficiency[19]. Recently studies identify mTOR as a key regulator of cellular iron metabolism, the regulatory pathway is a parallel iron regulatory network. In vitro, cellular iron uptake via TfR-1 is modulated by mTOR activity-induced TfR-1 surface expression in HeLa cells[20]. Further mechanistic studies identify
ACCEPTED MANUSCRIPT triste-traprolin (TTP), a protein involved in anti-inflammatory response, as the downstream target of mTOR that binds to and enhances degradation of TfR1 mRNA[9]. Moreover, mTORC1 can associates with and phosphorylates ISCU protein, consequently stabilizing ISCU and enhancing ISC
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assembly[21]. Therefore, unrestrained mTORC1-mediated stabilization of ISCU protein sensitizes cells to iron deprivation, due to constitutive ISC biogenesis-triggered iron demand that outstrips supply. 1.2 ISCU
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The scaffold protein ISCU acts as a scaffold on which the ISCs are assembled and from which the
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clusters are delivered to various apoproteins. It is synthesized as a precursor in the cytosol and migrates to the mitochondria where it becomes the mature form after a two-step mitochondria target sequence cleavage. In humans, mutations of ISCU decrease its expression and ultimately the activities of muscle aconitase and succinate dehydrogenase, for which ISCs serve as essential cofactors. It is reported that
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phosphorylation of ISCU by mTORC1 prevents its degradation and increases ISC [21]. 1.3 Tristetraprolin
Tristetraprolin (TTP) is recognized as an early response gene which was induced in response to growth
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factors, inflammatory stimuli and phorbol esters[22]. In yeast, a tandem zinc finger (TZF) protein
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Cth2 was found to conserve cellular iron in states of deficiency by preferentially degrading mRNA of non-essential iron-containing proteins thus reducing iron requirements and liberating iron for vital functions. TTP was found to be the functional homolog of Cth2 in mammalian, which is the mediator of iron-regulatory effects of mTOR and provide a novel link between energy metabolism, inflammation and iron regulatory pathways. Iron chelation with DFO resulted in a significant and dose-dependent upregulation of TTP mRNA and protein expression, while iron overload with ferric ammonium citrate (FAC) suppressed TTP expression [9]. These results suggest that TTP is regulated by
ACCEPTED MANUSCRIPT cellular iron status in mammals. TTP is known to mediate its functions by altering the stability of mRNAs of several transiently expressed inflammatory genes. TTP binds directly to the AU-rich element in the 3′-UTR of TNF-α transcript leading to its destabilization and rapid degradation [23]. In a
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knock-out mouse model, the lack of TTP has been shown to be normal at birth, but within a few months develop a characteristic phenotype that includes loss of body weight and body fat, a severe syndrome of growth retardation, cachexia, arthritis,
inflammation and autoimmunity[24].
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Phosphorylation of TTP can lead to its inactivation because the unphosphorylated or de-phosphorylated
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form of TTP recruits more efficiently the deadenylase and mRNA decapping complexes to the AU-rich element (AREs) containing TNF-α transcript to cause rapid degradation[25]. Recent studies in mouse embryonic fibroblasts (MEFs) found TTP to regulate cellular iron homeostasis by binding to AREs and destabilizing the mRNAs of iron-requiring proteins, thus potentially optimizing iron utilization in
1.4 Transferrin receptor 1
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low-iron states[9].
With the discovery that transferrin serves as the iron source for hemoglobin-synthesizing immature red
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blood cells came the demonstration that a cell surface receptor, now known as transferrin receptor 1
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(TfR 1), is required for iron delivery from transferrin to cells[26]. Each transferrin molecule can bind two atoms of ferric iron, forming a molecule of apotransferrin. Interaction of transferrin-iron with a high-affinity TfR1 expressed on cell surface triggers its internalization through the formation of a clarithin-coated pits and endocytosis. Following endocytosis of TfR1-apotransferrin complex, iron is released from transferrin by acidification of the vesicle by a proton pumping ATPase and transferrin molecules are returned to the bloodstream by exocytosis. Computational analysis of the 3’ untranslated region (UTR) of TfR1 revealed multiple putative AREs, some of which were found in a close
ACCEPTED MANUSCRIPT proximity to or overlapped with IREs. Bayeva et al. found TfR1 mRNA levels to be reduced because TTP interacts with TfR1 mRNA and leads to its degradation[9]. RNA co-immunoprecipitation experiment with four designed primer sets for TfR1 to target the regions near its 3’UTR was performed
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in human HEK293 cells to assess the physical interaction between TTP and TfR1 mRNA. TfR1 mRNA levels were enriched in the TTP group compared to the IgG control, indicative of interaction between
2 The function of mTOR regulatory system on iron metabolism
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TTP and 3’UTR of TfR1 [9].
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mTOR is an important sensor of cellular energy state and a major hub for integration of environmental cues[27], many of the processes governed by mTOR are dependent on iron as a cofactor and would require a steady supply of this metal. The essential role of mTOR in iron metabolism was established as clinical use of rapamycin is associated with the development of microcytosis and polyglobulia that is
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mechanistically distinct from the anemia of chronic disease[28, 29]. According to the World Health Organization definition, functional iron deficiency was present in 80% of mTOR-treated orthotopic
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heart transplant patients[30]. There are two mechanisms that can be used to analyze how mTOR pathway serves to modulate iron metabolism and homeostasis: through phosphorylation stabilizing
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ISCU protein and inhibiting TTP expression separately. First, mTORC1 plays an important role in ISCU gene expression and function, and thus enhance ISC assembly. ISCU is an mTOR kinase target, and phosphorylation of S14 is essential for mTORC1-mediated preservation of ISCU protein[21]. It is reported that ISCU can form a protein complex with cytosolic ISCS and that these two proteins together facilitate assembly of the ISC of IRP1 and yeast mitochondrial aconitase [31]. Moreover, RNA interference studies have confirmed that silencing of ISCU not only disrupted ISC biogenesis but also inappropriately activated the IRP1 and
ACCEPTED MANUSCRIPT disrupted intracellular iron homeostasis [12]. IRP1 registers cytosolic iron and oxidative stress
through its labile ISC. In tissue culture cells, when iron is abundant, IRP1 contains a [4Fe-4S] cluster, exhibits aconitase activity, and binds IREs with low affinity. When iron is scarce, IRP1
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loses its ISC and aconitase activity and binds IREs with high affinity. Interestingly, silencing of ISCU also resulted in marked activation of the IRE binding activity of IRP2, although IRP2 is not known to depend on an ISC for its regulation[12]. Therefore, Insufficiency of ISCU triggered by
knockdown
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starvation-triggered cell death.
knockdown
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ISCU
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mTORC1 inhibition can increase the binding activity between IRP and IRE. The endogenous
Moreover, mTOR activity was shown to inhibit the transcription and/or prevents destabilization of Tristetraprolin (TTP) mRNA, which in turn regulates the levels and stability of TfR 1 mRNA and
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subsequently iron metabolism in hearts of mice [9]. As the downstream target of mTOR, TTP functions similar to iron-regulating yeast cth1p/cth2p proteins to regulate iron homeostasis. The budding yeast Saccharomyces cerevisiae has provided significant insight into iron homeostasis in eukaryotes,
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including humans[32]. Yeast can down-regulate iron-dependent processes to preserve intracellular iron
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by a post-transcriptional mechanism via the RNA-binding proteins cth1p/cth2p, which are up-regulated in iron deficiency[33]. Cth1p/cth2p proteins preferentially bind to AU-rich elements (AREs) and then destabilize the mRNAs of iron-requiring proteins, thus salvaging iron for use in essential functions[34]. TTP was also shown a similar function of cth1p/cth2p to suppress the expression of ARE-containing iron-requiring proteins potentially optimizing iron utilization in low-iron states[9]. ATP binding cassette E1 (ABCE1) is an ISC protein which is essential for ribosome function and for cellular viability in eukaryotic systems[35]. In TTP overexpression MEFs, mRNA levels of ABCE1 were
ACCEPTED MANUSCRIPT dramatically reduced. Moreover, treatment of TTP KO MEFs with the iron chelating agents, deferoxamine, not only failed to decrease, but instead led to a sharp increase in the expression of ABCE1. The results suggest mammalian TTP could serve a similar function as yeast Cth1p/Cth2p.
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Rapamycin, an mTOR inhibitor, can lead to increased TTP expression and specifically lead to a global suppression of many iron-regulatory genes such as TfR1, Ferroportin1 which is consistent with a reduction in cellular iron flux, while the opposite was observed with activation of mTOR[9] (Figure 3).
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The effects of rapamycin on iron-regulatory network are specific because there is no down-regulation
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of several genes not involved in regulation of cellular iron upon rapamycin treatment of WT MEFs. Bayeva et al.[9] discovered an interaction between TTP and TfR1 mRNA, they found TfR1 mRNA levels were significantly enriched in the TTP group in the pull-down experiments performed in human HEK293 cells. In summary, Taken together, TTP is induced by rapamycin and negatively regulates the
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expression of TfR1, leading to reduced iron import and net iron loss from the cell. Iron deficiency would activate IRP1/2 to prevent further iron loss. IRE/IRP complexes formed within the 5’UTR of an mRNA (e.g., H-ferritin, L-ferritin, ferroportin 1) inhibit translation, whereas IRP binding to IREs in the
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3’UTR of DMT1 and TFR1 mRNA prevents its degradation. Moreover, TTP conserves cellular iron by
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repressing non-essential iron-requiring proteins and thus sparing iron for vital processes. As levels of iron-containing proteins decrease and more iron becomes available.
3 The regulation of mTOR regulatory system
The mTOR is placed downstream of the PI3K/Akt pathway, thus its activity can be regulated by multiple upstream signaling pathways. Rapamycin, when bound to its intracellular receptor FKBP12, weakens the interaction of mTORC1 with its binding partners, which is eventually followed by disassociation of the complex. Recent data suggest that PI3K and its downstream effector, the protein
ACCEPTED MANUSCRIPT kinase Akt, control mTOR through the TSC1-TSC2 tumor suppressor complex[36]. Growth factor activation of PI3K/Akt results in the phosphorylation and inhibition of TSC1-TSC2, leading to derepression of mTOR. Ballou and coworkers reported that the α1A adrenergic receptor activated a
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pathway that required an increase in [Ca2+]i, activation of phospholipase D, and accumulation of phosphatidic acid to induce mTOR activation[37]. Amino acids may also signal to mTOR via the Ca(2+)/CaM-dependent activation of the lysosomal membrane protein Vps34. Negative regulation of
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mTOR is imposed by lipopolysaccharides and inflammatory cytokines, that may impinge on upstream
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amino acid signaling and mTOR itself to prevent the phosphorylation of mTOR substrates and translation initiation[38]. The small G protein Rheb is known to promote mTOR signaling. Tee et al. identified mTOR as a common downstream target of Rheb[39]. When Rheb contains bound GTP, mTOR is activated. Conversely, in the presence of GDP, Rheb prevents phosphorylation of mTOR
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substrates. Pathways downstream of mTOR may play a role in the regulation of TTP and cellular iron homeostasis.
Understanding the functional interconnections between iron and the mTOR system represents an
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important challenge to fully comprehend iron homeostasis. Iron deficiency was also shown to inhibit
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mTOR signaling. Ndong et al.[17] found iron-deficient diet depressed Akt activity in rats and in COS-1 cells, leading to a decrease in mTOR activity. In addition, it was observed that down-regulation of mTOR following marked down-regulation of the phosphorylated S6 protein in deferasirox-treated leukemia cells[18]. Additional investigations showed that rapamycin and iron chelation with DFO have an additive effect on TTP induction[9]. Increasing TTP can promote downregulation of iron-requiring genes and modulates survival in low-iron states.
4 Perspective
ACCEPTED MANUSCRIPT Mammalian iron homeostasis is maintained through the concerted action of sensory and regulatory networks that modulate the expression of proteins of iron metabolism at the transcriptional and/or post-transcriptional levels. For 30 years or more we have known that maintenance of cellular iron
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homeostasis is dependent on RNA-binding iron-regulatory proteins 1 and 2 (IRP1/2), which are activated under iron-deficient conditions. IRP1/2 function to restore iron levels through stabilization of TfR1 mRNA and increased iron uptake through stabilization of DMT1 mRNA, mobilization of cellular
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iron stores, and reduction in cellular iron export through the suppression of ferroportin 1[40]. Since the
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beginning of this century, the understanding of systemic iron homeostasis and its disorders has rapidly progressed, and regulation by the hepcidin/ferroportin system can now explain key features of systemic iron balance[1]. mTOR/TTP/TfR1 system is a new pathway mediating the iron homeostasis in parallel with two well-established IRP1/2 and hepcidin systems. mTOR/TTP/TfR1 system may act as a
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‘‘brake’’ on the IRP1/2 system to prevent the unnecessary overactivation of iron uptake as the cell is reducing its iron use through TTP-dependent downregulation of iron-containing proteins. While the mTOR pathway plays a role in regulation of cellular iron availability and utilization, its main function
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is seen as a mechanism for cell survival. In instances where the cell is suffering from iron starvation, it
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reduces the levels of proteins that require the nutrient, conserving it for use by essential proteins only. This in turn keeps the cell viable and prevents cell death. On the other hand, considering mTOR is frequently activated in tumors, it would benefit patients to apply iron chelators to tumors. Tumor cells exhibit increased uptake and utilization of iron by virtue of possessing significantly higher levels of transferrin receptor 1 than healthy cells[41]. This has led to the suggestion that agents that deplete intracellular iron may be useful in cancer therapy. With the deeper understanding of iron metabolism and its regulation will offer avenues for new treatment options. The mTOR kinase is generally accepted
ACCEPTED MANUSCRIPT to be a master regulator of cell growth, cell proliferation, cell motility, cell survival, protein synthesis, and transcription, the impressive progress that has been achieved during the past years in unraveling the regulation of mTOR pathway on iron metabolism and its function may well translate into new drugs
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to treat patients with iron metabolism disorder. Funding
This work was supported by National Natural Science Foundation of China (grant number 31200863),
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Science and Technology Research Youth Fund Project of Hebei Colleges and Universities (grant
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number QN20131009) and Hebei Normal University Fund (grant number L2010Q06). References
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[3] Volz K. The functional duality of iron regulatory protein 1. Current opinion in structural biology. 2008; 18:106-11.
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[5] Hanson ES, Rawlins ML, Leibold EA. Oxygen and iron regulation of iron regulatory protein 2. The Journal of biological chemistry. 2003; 278:40337-42. [6] Ke Y, Wu J, Leibold EA, Walden WE, Theil EC. Loops and bulge/loops in iron-responsive element
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Figure 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.
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Figure 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.
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Figure 3. Regulation of iron homeostasis by mTOR. mTORC1 can phosphorylate iron-sulfur cluster 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.
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