Iron overload as a high risk factor for microgravity-induced bone loss

Iron overload as a high risk factor for microgravity-induced bone loss

Acta Astronautica 164 (2019) 407–414 Contents lists available at ScienceDirect Acta Astronautica journal homepage: www.elsevier.com/locate/actaastro...

796KB Sizes 0 Downloads 12 Views

Acta Astronautica 164 (2019) 407–414

Contents lists available at ScienceDirect

Acta Astronautica journal homepage: www.elsevier.com/locate/actaastro

Review article

Iron overload as a high risk factor for microgravity-induced bone loss a,c

a,c

a,c

a,c

a,c

a,c

Xin Chen , Jiancheng Yang , Dandan Dong , Huanhuan Lv , Bin Zhao , Yanru Xue , Peng Shangb,c,∗ a b c

T

School of Life Sciences, Northwestern Polytechnical University, Xi'an 710072, China Research & Development Institute in Shenzhen, Northwestern Polytechnical University, Shenzhen 518057, China Key Laboratory for Space Bioscience and Biotechnology, Institute of Special Environmental Biophysics, Northwestern Polytechnical University, Xi'an 710072, China

ARTICLE INFO

ABSTRACT

Keywords: Iron overload Bone loss Microgravity Spaceflight

Exposure to microgravity during long-term habitation in space results in numerous physiological alterations in the human body. Among them, reduction in bone mineral density is well known as one of the most important changes, which limits further space exploration by humans. Although numerous studies have been conducted on microgravity-induced bone loss, the explicit mechanism behind its occurrence has not been fully elucidated. Even though mechanical unloading has been generally accepted as a dominant cause of microgravity-induced bone loss, the risks of iron-loading and oxidative damage due to increased iron stores and dietary iron intake during spaceflight have attracted much attention, especially in bone. Indeed, excessive iron accumulation has been found in astronauts exposed to microgravity during spaceflight. Moreover, a great deal of evidence from clinical, animal, and cellular studies indicates that iron-loading has direct adverse effects on bone metabolism. This review summarizes the latest findings on bone loss and iron status in microgravity conditions, as well as the association between iron-loading and bone abnormalities. We discuss the possible mechanisms of iron overloadinduced skeletal involution. Finally, we hypothesize that, in addition to mechanical unloading, iron overload due to long-term spaceflight missions is a high risk factor for microgravity-induced bone loss. The underlying mechanism by which it occurs is iron-loading that leads to an imbalance in bone remodeling, involving bone formation and bone resorption, mediated by oxidative stress via the Fenton reaction.

1. Introduction Five million years since the origin of humankind, we have now entered the space age. In the coming decades, public and private entities have set a goal of manned spaceflight missions to the Moon, asteroids, and Mars, which sounds fascinating [1,2]. In this context, it is necessary to better understand the effects of microgravity on human physiology during long-term spaceflights. Exposure to microgravity during long-term habitation in space results in a number of remarkable physiological alterations, which represent serious threats to the health of astronauts [1,3]. In addition to muscle atrophy [4], cardiovascular deconditioning [5], and immune dysfunction [6], bone loss is also an established serious physiological change that occurs in microgravity [7,8]. Bone loss primarily manifests in the lower limbs and spine, and requires extended periods of time to recover upon returning to Earth [2,9,10]. Due to the lack of effective countermeasures, continuous bone loss in microgravity may increase fracture incidence in the skeletal framework of astronauts during and after spaceflight [11,12]. This scenario represents a considerable barrier to humans in long-term space ∗

exploration [3,13]. Although there have been a number of experimental studies under both actual and simulated microgravity conditions, the mechanisms of microgravity-induced bone loss have yet to be completely defined [14,15]. Iron, an essential element in mammals, plays a crucial role in numerous fundamental biological processes, ranging from oxygen transport to DNA synthesis and oxidative phosphorylation [16]. However, despite the beneficial role it plays in normal conditions, an excessive accumulation of iron in the human body has recently been suggested to be connected with bone lesions such as osteopenia and osteoporosis [17,18]. Osteoporosis, osteopenia, and pathological bone fractures are complications in disorders that manifest with iron overload, including hereditary hemochromatosis, thalassemia, sicklemia, and the cessation of menstruation [19–23]. Moreover, results from animal studies have shown that iron overload decreases bone formation and increases bone resorption [24,25]. Also, data from in vitro studies have indicated that increased levels of iron inhibit osteoblastogenesis and facilitate osteoclastogenesis [26,27]. Reactive oxygen species (ROS) are short-lived molecules that are

Corresponding author. Research & Development Institute in Shenzhen, Northwestern Polytechnical University, Shenzhen 518057, China. E-mail address: [email protected] (P. Shang).

https://doi.org/10.1016/j.actaastro.2019.07.034 Received 24 December 2018; Received in revised form 30 July 2019; Accepted 31 July 2019 Available online 20 August 2019 0094-5765/ © 2019 IAA. Published by Elsevier Ltd. All rights reserved.

Acta Astronautica 164 (2019) 407–414

X. Chen, et al.

generated during mitochondrial respiration and cellular enzymatic reactions as the normal by-products of oxygen metabolism [28,29]. ROS consist of a variety of chemical species, mainly including superoxide and hydrogen peroxide [30]. They can be neutralized by cellular antioxidants such as superoxide dismutase (SOD) and reduced glutathione (GSH) [27]. Recent studies have suggested that increased levels of ROS are involved in both osteoporosis and the aging process [31]. It has been proven that increased ROS have opposing effects on osteoblasts and osteoclasts. ROS have an inhibitory effect on osteoblast differentiation [32], while simultaneously promoting osteoclast differentiation and bone-resorption activity [33]. Indeed, it has been confirmed that increased iron is associated with elevated ROS levels. Excessive iron accumulation catalyzes the formation of ROS mediated by the Fenton reaction, in which Fe2+ reacts with hydrogen peroxide to generate hydroxyl radicals, resulting in oxidative stress, which is an important factor in age-associated pathological conditions such as general aging and postmenopausal osteoporosis [22,34–36]. Evidence from spaceflight missions indicates that iron homeostasis is impaired in the bodies of astronauts after entering space [37]. Astronauts experience changes in iron metabolism as a result of adaptations to microgravity and an iron-rich food system in space. Serum ferritin, the main iron storage protein in humans, increases after shortand long-term spaceflight missions [37,38]. Evidence from the Spacelab 1 mission indicated that ferritin levels increased by 53% after 7 days in microgravity [38]. Evaluation of biomarkers related to iron metabolism in astronauts after long-term spaceflight aboard the International Space Station (ISS) revealed that their serum ferritin levels were elevated after spaceflight [39]. Data from a long-term spaceflight mission revealed that there is a positive correlation between serum ferritin levels and the markers of oxidative damage, whereas a negative correlation was found between serum ferritin levels and bone mineral density (BMD) [37]. Data pertaining to the impact of microgravity on the human body during long-term spaceflight missions have been extremely limited to date. Accordingly, in vivo and in vitro models have been developed to reproduce the bone loss in microgravity on the ground [40,41]. The hindlimb unloading (HLU) rodent model is a well-established classic microgravity model widely used to study various aspects of skeletal loading on the ground [42]. Recently, a study from our group showed that iron deposition contributes to unloading-induced bone loss in mice [43]. HLU in rats caused an increase of iron storage in the spleen and a decrease of circulating iron [44]. In this review, it is proposed that, in addition to mechanical unloading, iron overload due to long-term spaceflight missions is a high risk factor for microgravity-induced bone loss. The underlying mechanism is postulated to involve the disturbance of the balance between bone formation and bone resorption via oxidative stress resulting from the iron-mediated Fenton reaction.

during long-term spaceflight missions may increase fracture and renal calculus risks [48]. Data from the ISS missions showed that 7 of 8 cosmonauts exhibited a decrease in BMD in the range of 2.5%–10.6% in the lumbar vertebrae, 4 had a reduction in BMD in the range of 1.7%–10% in the femoral neck, and all 8 showed a decrease in BMD ranging from 3% to 10% in the femur [49]. Recently, it has been shown that the microstructure of weight-bearing bones did not recover during the year following 4- to 6-month stays in the ISS [50]. At present, due to the absence of effective countermeasures, bone loss has only been partially alleviated. Additionally, postflight rehabilitation of the skeleton in astronauts is also a health concern and is suspected to take 2 to 3 times longer than the mission length, increasing the risks for premature osteoporosis and fractures after their return to Earth [50,51]. In short, these results revealed trends towards pronounced bone loss at weight-bearing sites, especially in the lower body, and minimal changes at non-weight-bearing sites. 2.2. Bone loss in response to ground-based models Since spaceflight experiments are limited by high costs and few opportunities, much of the known pathology of bone loss in microgravity is derived from ground-based studies. Bed rest and the HLU rodent model are the two main ground-based models extensively used to study the effects of microgravity on bone metabolism. These models both induce skeletal system alterations that are similar to those that occur during spaceflight [7,41]. Combined and analyzed data from five bed-rest studies (n = 74; 50 men and 24 women) indicated that there was no pronounced difference between men and women in the response to bed rest. Men, however, exhibited elevated biomarkers of bone resorption as well as calcium in urinary excretion, leading to a higher risk of renal stones [52]. Cervinka et al. showed that the highest mean bed rest-induced bone loss occurs in the cortical bone, but only during the first 60 days. As with longer duration, pronounced loss was observed in trabecular bone [53]. Fourteen days of HLU resulted in 25.9% and 29.2% decreases in total bone mineral content and trabecular BMD, respectively. In addition, the trabecular microarchitecture was significantly changed in the proximal tibia [54]. Saxena et al. found a decrease in BMD in femurs, and a loss of cortical and trabecular microarchitecture in HLU mice [55]. These data are consistent with those from spaceflight missions. 2.3. Main mechanism associated with microgravity-induced bone loss The Wnt/β-catenin signaling pathway plays a vital role in microgravity-induced bone loss. Canonical Wnt signaling stimulates osteoblast differentiation and activity, while inhibiting bone resorption [56–58]. Sclerostin and Dickkopf-related protein 1 (Dkk-1), antagonists of the Wnt signaling pathway, suppress the canonical Wnt signaling pathway through binding to the two co-receptors, low-density lipoprotein receptor-related proteins 5 and 6 (LRP5, 6) [59]. Osteocytes are capable of sensing mechanical stress and secreting sclerostin and Dkk-1 [60]. Osteocytes forcibly inhibit osteoblasts by secreting sclerostin and Dkk-1 [60]. It has been indicated that microgravity upregulates the levels of sclerostin expression [61]. Bed-rest studies showed increased sclerostin levels, indicating that the Wnt signaling pathway contributes to disuse-induced bone loss [59]. In vitro studies found that mechanical unloading promotes the expression of sclerostin, suggesting that mechanical loading is involved in the regulation of intrinsic osteocyte responses [62]. In brief, these results showed that the Wnt/β-catenin signaling pathway plays a key role in the maintenance of bone homeostasis under microgravity conditions.

2. Microgravity-induced bone loss The health and safety of astronauts have been widely supervised before, during, and after spaceflight since the first man was sent up into space in 1961, revealing several health concerns for humans, including bone loss and muscle atrophy [7,45]. Bone loss has been regarded as the most serious health problem, and microgravity in space is its major cause [43,46]. Prolonged exposure to microgravity in space results in 1%–2% bone loss per month in astronauts, which is equivalent to the annual bone loss in postmenopausal women [47]. Bone loss has also been reproduced on the ground using in vivo and in vitro models. 2.1. Spaceflight-induced bone loss in astronauts It is well known that the skeletal system provides mechanical support for humans, while exposure to microgravity during spaceflight leads to mechanical unloading, resulting in loss of BMD, primarily in the lumbar spine and lower extremities [10]. Bone loss in astronauts

3. Iron status in microgravity conditions Iron is an essential trace element for virtually all living organisms [63]. Healthy human adults contain approximately 3–5 g of iron, nearly 408

Acta Astronautica 164 (2019) 407–414

X. Chen, et al.

80% of which is bound to hemoglobin in red blood cells (RBCs) or myoglobin in muscle cells in the form of heme. The remainder is mainly stored in the liver within ferritin, with a very small portion located in the intracellular labile iron pool (LIP) [64]. The human body has no positive pathway through which to excrete redundant iron, with the loss of iron from the body occurring only through the shedding of skin and mucosal cells, as well as bleeding [65]. Therefore, iron homeostasis must be tightly controlled. Several mechanisms are involved in the regulation of iron homeostasis, ranging from absorption by the intestine to release by macrophages and storage in hepatocytes [66]. The absorption of iron from iron-containing foods occurs in duodenal enterocytes, with several iron-regulatory proteins involved in this process [67]. Ingested iron reaches the duodenum in its ferric (FeIII or Fe3+) form and must be reduced to ferrous (FeII or Fe2+) form prior to gut uptake [63]. This reduction is mediated by the ferrireductase duodenal cytochrome B (DcytB or cybrd1), which is located in the intestinal cell apical membrane [63]. Fe2+ is then transported into enterocytes from the duodenal lumen by divalent metal transporter 1 (DMT1; gene name SLC11A2) via the apical membrane [65]. Ferroportin (FPN; gene name SLC40A1), the only known iron exporter, exports iron from enterocytes into the plasma via the basolateral membrane [68]. Prior to being transported into the plasma, Fe2+ is oxidized to Fe3+ by hephaestin and ceruloplasmin [69]. In the plasma, Fe3+ rapidly bounds to transferrin (TF), a monomeric glycoprotein that has 2 binding sites for Fe3+ and delivers it to tissues [69]. Hepcidin, a liver-produced small peptide hormone encoded by HAMP in humans (Hamp in mice), is a principal molecule in the modulation of systemic iron homeostasis [70,71]. Hepcidin binds to FPN and triggers its internalization and degradation in lysosomes in order to inhibit iron uptake from the duodenum and iron release from macrophages and hepatocytes [72,73]. Hepcidin levels are modulated by hepatic iron stores, inflammation, and hypoxia [74–76]. Higher levels of hepcidin inhibit iron absorption from duodenum enterocytes and vice versa. Bone morphogenetic protein 6 (BMP6) is regarded as a main regulator of hepcidin, which is elevated in response to hepatic iron stores. Hepatic iron stores transcriptionally regulate hepcidin expression through the activation of BMP6-ALK2/3-SMAD1/5/9 signaling [77]. Additionally, inflammatory stimuli also regulate hepatic hepcidin synthesis. Interleukin-6 (IL-6) participates in inflammation-related hepcidin transcription by activating STAT3, which can bind to specific sequences in the HAMP promoter [78]. Hypoxia is associated with the transcriptional suppression of hepcidin. It has been proposed that hypoxia downregulates hepcidin levels by upregulating hypoxia-inducible factors (HIFs) [79]. Several studies have clearly confirmed that iron homeostasis and hematology are altered soon after entering microgravity [37,39]. One of the most significant alterations in hematology is the reduction of circulating RBCs [80]. Upon returning to Earth, a delay in replacing RBCs causes decreases in hemoglobin, hematocrit, and mean corpuscular volume [81]. The decrease in circulating RBC mass leads to anemia, a condition that is referred to as “spaceflight anemia” [82]. In addition, serum ferritin is significantly increased, the amount of ferritin iron is slightly greater than preflight values, and the amount of transferrin is decreased [83]. Iron accumulation in tissues can impair bone and muscle metabolism, weaken immune function, increase cardiovascular disease and cancer risk, and enhance sensitivity to radiation injury [63,84–86]. In brief, iron overload and iron deficiency are both harmful to astronauts' health and limit the further exploration of space. Therefore, maintenance of iron homeostasis in the human body during long-term spaceflight is crucial, and the explicit mechanism whereby microgravity regulates iron metabolism must be completely elucidated.

decrease in RBC mass during spaceflight results from neocytolysis, the selective destruction of the youngest circulating RBCs [87,88]. The mechanism by which spaceflight induces neocytolysis consists of a decrease in plasma volume (PV), resulting in an increase in the concentration of hemoglobin that reduces erythropoietin (EPO) [89]. The reduced EPO level triggers neocytolysis by influencing surface-adhesion molecules [88]. One consequence of the reduced RBC mass is the subsequent transfer of iron from the destroyed erythrocytes into iron storage proteins and processes, such as ferritin [37]. Serum ferritin, an index of iron storage, is elevated after short- and long-term spaceflights. Biochemical data from blood samples in astronauts before and after 28 flights (average duration = 6 days) revealed an increase in serum ferritin [38]. Biomedical data related to iron metabolism from astronauts after long-term (duration 128–195 days) spaceflights showed increased serum ferritin [39]. Zwart et al. reported that serum ferritin and body iron stores increase early in spaceflight, while transferrin and transferrin receptors decrease later during long-term (duration 50–247 days) spaceflight, suggesting that the increased iron stores result from the mobilization of iron into storage tissues. Moreover, there were no changes in hepcidin level and acute phase proteins during spaceflight, indicating that long-term spaceflight-induced increased iron stores is not a result of an inflammatory response [37]. During spaceflight, the transferrin index, an estimate of transferrin saturation, significantly increases, suggesting that transferrin is more saturated during spaceflight [37]. Moreover, astronauts aboard the ISS exhibited an increase in tissue iron storage, which represented a risk factor for oxidative damage. Remarkably, the increase in iron stores after spaceflight continues to accumulate [80]. These changes in iron metabolism during microgravity exposure may contribute to an increase in oxidative damage markers, including increased urinary 8-hydroxy-2′-deoxyguanosine (8OHdG) and decreased superoxide dismutase (SOD) activity, upon landing relative to their preflight levels [37]. 3.2. Iron status in ground-based studies The prominent phenomena associated with the changes in iron metabolism during spaceflight have also been reproduced in groundbased models [44,90]. Cavey et al. found that 7 days of HLU in rats increases spleen iron storage and decreases circulating iron concentration through an inflammatory process that upregulates hepcidin expression [44]. IL-6, a pleotropic cytokine, plays an important role in the regulation of inflammatory response [91]. It is further confirmed that IL-6 facilitates hepcidin synthesis through the activation of signal transducer and activator of transcription 3 (STAT3) [44]. Xu et al. showed that 28 days of HLU induces the increase of iron content in hindlimb bone and the liver, and is connected with the regulation of hepcidin by the liver [90]. Recently, Yang et al. showed that 28 days of HLU leads to iron deposition in the liver, spleen, bone, and serum [43]. In addition, long-term (60-day) bed rest was found to induce increased iron stores [83]. In short, these data from ground-based studies demonstrate that microgravity affects the iron status, leading to increased iron stores. The results from Cavey et al. and Yang et al. are contradictory [43,44]. Hepcidin plays a central role in the modulation of serum iron concentrations by inhibiting iron uptake by duodenum and sequestrating iron in organs [92]. In acute inflammatory conditions, shortterm microgravity exposure induces elevated hepcidin level, which results in a decrease in serum iron concentrations. Long-term spaceflight decreases RBC mass resulting from neocytolysis. One consequence of the reduced RBC mass is the subsequent transfer of iron from the destroyed erythrocytes into iron storage proteins such as serum ferritin, which can exacerbate tissue iron storage [37]. Moreover, it has been suggested that iron overload orchestrates unloading-induced bone loss [90]. The higher hepcidin level as a feedback of iron overload can alleviate iron accumulation in tissues and serum. However, it is still inadequate in response to iron overload. Taken together, the data from

3.1. Iron status in spaceflight studies Spaceflight-induced hematological changes have been observed since the initial days of space exploration [80]. Of note, a 10%–15% 409

Acta Astronautica 164 (2019) 407–414

X. Chen, et al.

the different studies indicate that the short-term spaceflight-induced increase in iron stores may be due to inflammation-induced hepcidin expression upregulation. However, the long-term spaceflight-induced increase in iron stores may be due to the decrease in RBC mass, which is resulted from neocytolysis. Microgravity leads to iron overload in the human body which upregulates hepcidin level, thereby alleviating iron accumulation in tissues.

excess iron by reducing TfR1 and increasing ferritin expression [104]. In addition, excess iron has inhibitory effects on osteoblast activity, which is mediated by ferritin due to its ferroxidase activity [105]. Cellular iron levels are tightly controlled by the modulation of iron import, storage and export which are mainly regulated by iron-regulatory proteins (IRPs) [106]. During iron deprivation, IRPs bind to 5′ iron-responsive elements (IREs) in ferritin and FPN mRNAs to inhibit their translation, while binding to 3′ IREs in TfR1 and DMT1 mRNAs to suppress their degradation [107,108]. Iron surplus inactivates the IRP-

4. Iron overload and bone loss (Table 1)

Table 1 Major observations of the relationship between iron overload and bone remodeling. Experimental model

Type of iron preparation

Major observation

Reference

Wistar female rats C57BL/6 male mice C57BL/6 female mice ICR male mice ICR female mice C57BL/6 male mice C57BL/6 male mice Hfe-KO mice

iron lactate iron dextran ferric sulfate ferric ammonium citrate ferric ammonium citrate iron dextran iron dextran iron carbonyl

decreased bone formation increased bone resorption decreased bone formation promoted osteoclast differentiation and bone resorption inhibited osteoblast activity and promoted osteoclast differentiation inhibited osteoblast activity and promoted osteoclast activity inhibited osteogenic differentiation of mesenchymal stem cells decreased bone formation and promoted osteoclast activity

[31] [84] [118] [27] [17] [119] [116] [19]

Hfe-KO: hemochromatosis protein deficient.

Iron is essential for cell survival since it serves as a protein cofactor for hemoglobin and DNA synthesis [93]. Excess iron, however, catalyzes the formation of ROS and facilitates oxidative stress, phenomena that damage cellular macromolecules [67]. Hence, iron import, utilization, storage, and export must be tightly controlled in order to maintain cellular iron homeostasis [94]. Several specialized proteins are involved in this process. Plasma transferrin (TF) has the capacity to bind to ferric iron very tightly, but reversibly. The primary function of TF is to transport iron from circulation into cells upon binding to transferrin receptor 1 (TfR1), which is presented on cell membranes [95]. TfR1 is probably expressed in most cell types, including osteoblasts and osteoclasts. The complex of Fe2Tf-TfR1 is internalized within endocytic vesicles by clathrin-mediated endocytosis, and ferric iron is released from the TF by a reduction in endosome pH. The released Fe3+ is then reduced to Fe2+ by the sixtransmembrane epithelial antigen of prostate 3 (STEAP3) and subsequently delivered into the cytosolic LIP by DMT1 [96]. The apoTf-TfR1 complex is then recycled back to the cell membrane, where it liberates apoTf to circulation [95]. It has been suggested that ZIP14 mediates non-Tf-bound iron (NTBI) absorption into cells. The iron that is not needed immediately for cellular metabolism is stored in cytoplasmic ferritin. Excessive iron is exported out of cells by FPN, a Fe2+ transporter, which is expressed in most cell types [97]. Bone is a metabolically active tissue that undergoes continuous remodeling, wherein osteoclasts resorb old or damaged matrix and osteoblasts form new bone [98]. Osteoclasts are derived from the monocyte/macrophage hematopoietic lineage, while osteoblasts originate from multipotent mesenchymal stem cells (MSCs) [98]. Mounting evidence suggests that iron overload leads to an imbalance in bone remodeling by influencing the biological activities of osteoclasts and osteoblasts [99]. The OPG/RANKL/RANK system plays a critical role in osteoclast differentiation and bone-resorption activity [100]. It has been found that ferric iron facilitates RANKL-induced osteoclast formation in both RAW264.7 cells and bone marrow-derived macrophages [27]. Osteoclast differentiation is related to dramatic alterations in cellular iron homeostasis, and several specialized proteins—associated with iron uptake (TfR1, DMT1), utilization (Steap4), storage (ferritin), and export (FPN)—are involved in this process [99]. During osteoclast differentiation, the levels of TfR1 and DMT1 are upregulated, as are those of Steap4, a metalloreductase that is crucial to cellular iron utilization. The levels of FPN, however, are downregulated at the initial stages of osteoclast differentiation [101–103]. Osteoblasts respond to

IRE interaction, increasing ferritin and FPN proteins expression, while facilitating TfR1 and DMT1 mRNAs degradation [107,109]. The following sections briefly summarize the effects of iron overload on bone metabolism and discuss the underlying mechanisms. 4.1. Effects of iron overload on bone metabolism Osteoporosis is a systemic bone metabolic disease that afflicts millions of people worldwide. Osteoporosis is characterized by progressive bone loss and degeneration of bone microstructure, resulting in increased bone fragility and susceptibility to fracture [110–112]. As a medical problem, osteoporosis impacts patients' quality of life and health, and leads to heavy economic burdens to their family and society [113,114]. Therefore, it is of the utmost importance to uncover any additional risk factors underlying the pathogenesis of osteoporosis [115]. Recently, an increasing number of clinical observations have shown that osteoporosis and fractures are frequent complications in disorders characterized by iron overload, including hereditary hemochromatosis, thalassemia, sicklemia, and the cessation of menstruation, which suggests that iron overload is a new pathogenic factor for osteoporosis [19–23]. Numerous studies have described the detrimental impact of iron overload on bone metabolism [116]. Data from clinical studies revealed that there is a positive correlation between serum ferritin levels and BMD [117]. Additionally, evidence from an animal study showed that iron overload increases bone iron storage and bone resorption, resulting in changes in bone microarchitecture [84]. Overall, increased iron levels are independent risk factors for osteoporosis. Reduced iron accumulation can improve bone in vivo and is beneficial to bone cell metabolism in vitro. 4.2. Mechanism of the relationship between iron overload and bone loss Iron overload inhibits osteoblastogenesis and promotes osteoclastogenesis [26,27]. The predominant mechanism by which iron overload causes an imbalance between osteoclast and osteoblast activity leading to bone loss appears to be oxidative stress. Tsay et al. revealed that ironoverloaded mice exhibit increases in bone iron content, as well as alterations in bone microarchitecture and material properties, accompanied by increased bone resorption and elevated ROS levels. Treatment with N-acetyl-L-cysteine (NAC) was found to largely prevent the development of bone abnormalities [84]. Evidence from an iron-overload zebrafish model demonstrated that iron overload significantly 410

Acta Astronautica 164 (2019) 407–414

X. Chen, et al.

Fig. 1. Effects of iron overload on microgravity-induced bone loss and the possible mechanism involved in this process. O2-: superoxide anions; H2O2: hydrogen peroxide; OH·: hydroxyl radicals.

inhibits osteoblast activity and bone formation due to the production of ROS [115]. This finding was later confirmed by He et al. who demonstrated that iron overload inhibits osteoblast function through higher oxidative stress as a result of increased intracellular iron concentrations [120]. ROS elicit a range of responses, including proliferation, growth, differentiation arrest, and cell death through the activation of various signaling pathways. In fact, mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinases (ERK1/2), cJun-N terminal kinase (JNK), and p38 MAPK are involved in osteoblast apoptosis [30]. MSCs are multipotent cells and precursors to osteoblasts, and play an important role in the development of osteoporosis. Xu et al. found that iron accumulation induces ROS-mediated damage to MSC proliferation and inhibits MSC ability [121]. TfR1-mediated iron uptake facilitates osteoclast differentiation and bone-resorption activity through the induction of mitochondrial respiration and the formation of ROS, whereas iron chelation inhibits osteoclastic bone resorption [103]. In addition, Jia et al. found that ferric iron promotes RANKL-induced osteoclast formation in both RAW264.7 cells and bone marrow-derived macrophages through the production of ROS [27]. Nuclear factor-kB (NF-kB) is a pleiotropic transcription factor that regulates osteoclast formation, function, and survival [122]. The role of NF-kB in osteoclast formation was discovered serendipitously when NF-

kB1/p50 and NF-kB2/p52 double-knockout (dKO) mice were established and found to be accompanied by osteopetrosis because NF-kB p50/p52 dKO mice cannot form osteoclasts [122]. The NF-kB signaling pathway is activated by virtually all stimuli that affect NF-kB, including the receptor activator of the NF-kB ligand (RANKL, encoded by TNFSF11), the master osteoclastogenic cytokines, and ROS. Synthesizing these data, we propose that excess iron induces the production of ROS, which inhibit osteoblast function while stimulating osteoclast differentiation (Fig. 2). 5. Iron overload associated with microgravity-induced bone loss The relationship between iron overload and bone loss in microgravity has been reported in both actual and simulated microgravity studies. Data from ISS missions reported that higher serum ferritin concentrations are related to higher concentrations of biomarkers associated with oxidative injury, and related to greater decreases in BMD in the hip trochanter, hip neck, and pelvis after long-term microgravity exposure [37]. Data from an HLU study showed that iron accumulation in bone clearly increased, and is accompanied by increased osteoclast activity and reduced osteoblast activity [90]. Moreover, decreased BMD and damaged microstructure were found in the tibia of unloaded mice.

Fig. 2. Schematic diagram of the iron-mediated signaling pathways in osteoclast differentiation and activation. RANKL binding to its receptor RANK induces the recruitment and activation of TRAF6. Following this, TRAF6 activates IKK and MAPKs, including ERK, p38, and JNK. The increment of ROS due to the iron-mediated Fenton reaction regulates IKK activation, thereby activating the NF-kB signaling pathway. Finally, this signaling pathway leads to an increase in the expression of osteoclast-specific genes and bone resorption. DMT1: divalent metal transporter 1; STEAP4: six-transmembrane epithelial antigen of prostate 4; RANK: receptor activator of NF-kB; TRAF6: tumor necrosis factor receptor-associated factor 6; NFATc1: nuclear factor of activated T-cells cytoplasmic 1; IKK: IkB kinase; TRAP: tartrate-resistant acid phosphatase; CSK: cathepsin K; MMP-9: matrix metallopeptidase 9. 411

Acta Astronautica 164 (2019) 407–414

X. Chen, et al.

Remarkably, after treatment with deferoxamine (DFO), iron content in bone and the liver was reduced, concomitant with a notable alleviation of the decreased BMD and improvement of the damaged microstructure [90]. Recently, a study from our group found that iron accumulation is involved in unloading-induced bone loss in mice [43]. In short, all of these findings suggest that microgravity induces excessive iron accumulation in bone tissue, which is involved in microgravity-induced bone loss.

Osteoporos. Rep. 11 (2013) 92–98. [3] J.P. He, X. Feng, J.F. Wang, W.G. Shi, H. Li, S. Danilchenko, et al., Icariin prevents bone loss by inhibiting bone resorption and stabilizing bone biological apatite in a hindlimb suspension rodent model, Acta Pharmacol. Sin. 39 (2018) 1760–1767. [4] R.H. Fitts, S.W. Trappe, D.L. Costill, P.M. Gallagher, A.C. Creer, P.A. Colloton, et al., Prolonged space flight-induced alterations in the structure and function of human skeletal muscle fibres, J. Physiol. 588 (2010) 3567–3592. [5] G.C. Demontis, M.M. Germani, E.G. Caiani, I. Barravecchia, C. Passino, D. Angeloni, Human pathophysiological adaptations to the space environment, Front. Physiol. 8 (2017) 547. [6] H. Luo, C. Wang, M. Feng, Y. Zhao, Microgravity inhibits resting T cell immunity in an exposure time-dependent manner, Int. J. Med. Sci. 11 (2013) 87–96. [7] D. Grimm, J. Grosse, M. Wehland, V. Mann, J.E. Reseland, A. Sundaresan, et al., The impact of microgravity on bone in humans, Bone 87 (2016) 44–56. [8] Y.X. Qin, W. Lin, E. Mittra, Y. Xia, J. Cheng, S. Judex, et al., Prediction of trabecular bone qualitative properties using scanning quantitative ultrasound, Acta Astronaut. 92 (2013) 79–88. [9] R.D. Carpenter, A.D. LeBlanc, H. Evans, J.D. Sibonga, T.F. Lang, Long-term changes in the density and structure of the human hip and spine after longduration spaceflight, Acta Astronaut. 67 (2010) 71–81. [10] E.R. Spector, S.M. Smith, J.D. Sibonga, Skeletal effects of long-duration headdown bed rest, Aviat. Space Environ. Med. 80 (2009) A23–A28. [11] T. Lang, J. Van Loon, S. Bloomfield, L. Vico, A. Chopard, J. Rittweger, et al., Towards human exploration of space: the THESEUS review series on muscle and bone research priorities, NPJ Microgravity 3 (2017) 8. [12] Y.N. Zhang, W.G. Shi, H. Li, J.R. Hua, X. Feng, W.J. Wei, et al., Bone Loss induced by simulated microgravity, ionizing radiation and/or ultradian rhythms in the hindlimbs of rats, Biomed. Environ. Sci. 31 (2018) 126–135. [13] A.I. Grigoriev, A.S. Kaplansky, G.N. Durnova, I.A. Popova, Biochemical and morphological stress-reactions in humans and animals in microgravity, Acta Astronaut. 40 (1997) 51–56. [14] Y. Arfat, W.Z. Xiao, S. Iftikhar, F. Zhao, D.J. Li, Y.L. Sun, et al., Physiological effects of microgravity on bone cells, Calcif. Tissue Int. 94 (2014) 569–579. [15] P. Ethiraj, J.R. Link, J.M. Sinkway, G.D. Brown, W.A. Parler, S.V. Reddy, Microgravity modulation of syncytin-A expression enhance osteoclast formation, J. Cell. Biochem. 119 (2018) 5696–5703. [16] A. Pietrangelo, Iron and the liver, Liver Int. 36 (2016) 116–123. [17] X. Wang, B.B. Fei, G.S. Shen, Y. Jiang, W. Zhang, X. Huang, et al., Iron overload increases osteoclastogenesis and aggravates the effects of ovariectomy on bone mass, J. Endocrinol. 226 (2015) 121–134. [18] Y. Li, B. Bai, Y. Zhang, Expression of iron-regulators in the bone tissue of rats with and without iron overload, Biometals 31 (2018) 749–757. [19] M. Simão, A. Camacho, A. Ostertag, M. Cohen-Solal, I.J. Pinto, G. Porto, et al., Iron-enriched diet contributes to early onset of osteoporotic phenotype in a mouse model of hereditary hemochromatosis, PLoS One 13 (2018) e0207441. [20] A. Piga, Impact of bone disease and pain in thalassemia, Hematol. Am. Soc. Hematol. Educ. Progr. 2017 (2017) 272–277. [21] M. Sadat-Ali, O. Sultan, H. Al-Turki, A. Alelq, Does high serum iron level induce low bone mass in sickle cell anemia? Biometals 24 (2011) 19–22. [22] G.F. Li, Y.Z. Pan, P. Sirois, K. Li, Y.J. Xu, Iron homeostasis in osteoporosis and its clinical implications, Osteoporos. Int. 23 (2012) 2403–2408. [23] G. Liu, P. Men, G.H. Kenner, S.C. Miller, Age-associated iron accumulation in bone: implications for postmenopausal osteoporosis and a new target for prevention and treatment by chelation, Biometals 19 (2006) 245–251. [24] L. Wang, B. Fang, T. Fujiwara, K. Krager, A. Gorantla, C. Li, et al., Deletion of ferroportin in murine myeloid cells increases iron accumulation and stimulates osteoclastogenesis in vitro and in vivo, J. Biol. Chem. 293 (2018) 9248–9264. [25] Y. Jiang, B. Chen, Y. Yan, G.X. Zhu, Hepcidin protects against iron overload-induced inhibition of bone formation in zebrafish, Fish Physiol. Biochem. 45 (2019) 365–374. [26] K. Yamasaki, H. Hagiwara, Excess iron inhibits osteoblast metabolism, Toxicol. Lett. 191 (2009) 211–215. [27] P. Jia, Y.J. Xu, Z.L. Zhang, K. Li, B. Li, W. Zhang, et al., Ferric ion could facilitate osteoclast differentiation and bone resorption through the production of reactive oxygen species, J. Orthop. Res. 30 (2012) 1843–1852. [28] D. Morikawa, H. Nojiri, Y. Saita, K. Kobayashi, K. Watanabe, Y. Ozawa, et al., Cytoplasmic reactive oxygen species and SOD1 regulate bone mass during mechanical unloading, J. Bone Miner. Res. 28 (2013) 2368–2380. [29] X. Wang, B. Chen, J. Sun, Y. Jiang, H. Zhang, P. Zhang, et al., Iron-induced oxidative stress stimulates osteoclast differentiation via NF-κB signaling pathway in mouse model, Metabolism 83 (2018) 167–176. [30] V. Domazetovic, G. Marcucci, T. Iantomasi, M.L. Brandi, M.T. Vincenzini, Oxidative stress in bone remodeling: role of antioxidants, Clin. Cases Miner. Bone Metab. 14 (2017) 209–216. [31] H. Isomura, K. Fujie, K. Shibata, N. Inoue, T. Iizuka, G. Takebe, et al., Bone metabolism and oxidative stress in postmenopausal rats with iron overload, Toxicology 197 (2004) 93–100. [32] X.C. Bai, D. Lu, J. Bai, H. Zheng, Z.Y. Ke, X.M. Li, et al., Oxidative stress inhibits osteoblastic differentiation of bone cells by ERK and NF-kappaB, Biochem. Biophys. Res. Commun. 314 (2004) 197–207. [33] S. Hyeon, H. Lee, Y. Yang, W. Jeong, Nrf2 deficiency induces oxidative stress and promotes RANKL-induced osteoclast differentiation, Free Radic. Biol. Med. 65 (2013) 789–799. [34] K. Jomova, M. Valko, Advances in metal-induced oxidative stress and human disease, Toxicology 283 (2011) 65–87. [35] R.R. Crichton, S. Wilmet, R. Legssyer, R.J. Ward, Molecular and cellular

6. Conclusions and perspectives Exposure to microgravity in space represents a major threat to the health of astronauts. Bone loss is a well-documented phenomenon occurring in astronauts during long-term spaceflight. Microgravity-induced bone loss has adverse effects on the safety and performance of crew members during long-term habitation in space and puts them at increased risk for fracture and renal calculus. Although some progress has been made, the explicit mechanism by which microgravity induces bone loss has not yet been fully elucidated. Evidence from short- and long-term spaceflight missions has revealed that astronauts have increased iron stores [38,39]. Serum ferritin, the most effective index of iron stores, is used in clinical and public health settings [123]. Data from a long-term spaceflight aboard the ISS revealed that the levels of serum ferritin had increased by approximately 220% in women and 70% in men by the 15th day of spaceflight [37]. Additionally, higher serum ferritin concentrations were associated with greater reductions in BMD of the hip, trochanter, hip neck, and pelvis after long-term spaceflight [37]. Several animal studies have demonstrated that oxidative stress is involved in iron overload-induced bone loss [84,115]. Moreover, in vitro studies revealed that iron overload inhibits osteoblast function via elevated ROS levels following the increase of intracellular iron content [120]. In addition, iron overload promotes RANKL-induced osteoclast differentiation and bone resorption through the increased production of ROS [27]. Accordingly, it is proposed that exposure to microgravity may disturb iron metabolism, leading to increased iron content in the skeleton. Excessive iron elevates the production of ROS and causes oxidative stress, resulting in significant alterations in skeletal function and contributing to microgravity-induced bone loss (Fig. 1). Future research is required in order to gain better insight into the potential mechanisms of iron overload caused by microgravity and to find possible therapies targeting the iron metabolism pathway, thereby preventing bone loss in microgravity and helping to protect the health and safety of astronauts. Moreover, developing appropriate rodent models to simulate bone loss in microgravity is extremely important, given that spaceflight experiments are limited by high costs and few opportunities. Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. Acknowledgements This work was supported by the National Natural Science Foundation of China (51777171, 11502213, 81741127, 81803032) and the Northwestern Polytechnical University Foundation for Fundamental Research(3102018JGC012). References [1] S.A. Lloyd, S.E. Morony, V.L. Ferguson, S.J. Simske, L.S. Stodieck, K.S. Warmington, et al., Osteoprotegerin is an effective countermeasure for spaceflight-induced bone loss in mice, Bone 81 (2015) 562–572. [2] J.D. Sibonga, Spaceflight-induced bone loss: is there an osteoporosis risk? Curr.

412

Acta Astronautica 164 (2019) 407–414

X. Chen, et al.

[36] [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] [50]

[51] [52] [53] [54] [55] [56] [57] [58] [59] [60] [61] [62] [63] [64] [65] [66]

mechanisms of iron homeostasis and toxicity in mammalian cells, J. Inorg. Biochem. 91 (2002) 9–18. O.F. Sendur, Y. Turan, E. Tastaban, M. Serter, Antioxidant status in patients with osteoporosis: a controlled study, Jt. Bone Spine 76 (2009) 514–518. S.R. Zwart, J.L. Morgan, S.M. Smith, Iron status and its relations with oxidative damage and bone loss during long-duration space flight on the International Space Station, Am. J. Clin. Nutr. 98 (2013) 217–223. C.S. Leach, Biochemical and hematologic changes after short-term space flight, Microgravity Q. 2 (1992) 69–75. S.M. Smith, S.R. Zwart, G. Block, B.L. Rice, J.E. Davis-Street, The nutritional status of astronauts is altered after long-term space flight aboard the International Space Station, J. Nutr. 135 (2005) 437–443. A. Cazzaniga, J.A.M. Maier, S. Castiglioni, Impact of simulated microgravity on human bone stem cells: new hints for space medicine, Biochem. Biophys. Res. Commun. 473 (2016) 181–186. A.R. Hargens, L. Vico, Long duration bed rest as an analog to microgravity, J. Appl. Physiol. 120 (1985) 891–903 2016. E.R. Morey-Holton, R.K. Globus, Hindlimb unloading rodent model: technical aspects, J. Appl. Physiol. 92 (1985) 1367–1377 2002. J. Yang, X. Meng, D. Dong, Y. Xue, X. Chen, S. Wang, et al., Iron overload involved in the enhancement of unloading-induced bone loss by hypomagnetic field, Bone 114 (2018) 235–245. T. Cavey, N. Pierre, K. Nay, C. Allain, M. Ropert, O. Loréal, et al., Simulated microgravity decreases circulating iron in rats: role of inflammation-induced hepcidin upregulation, Exp. Physiol. 102 (2017) 291–298. N. Guo, X. Fan, Y. Wu, Z. Li, S. Liu, L. Wang, et al., Effect of constraint loading on the lower limb muscle forces in weightless treadmill exercise, J. Healthc. Eng. 2018 (2018) 8487308. M.P. Nagaraja, D. Risin, The current state of bone loss research: data from spaceflight and microgravity simulators, J. Cell. Biochem. 114 (2013) 1001–1008. J.H. Siamwala, S. Rajendran, S. Chatterjee, Strategies of manipulating BMP signaling in microgravity to prevent bone loss, Vitam. Horm. 99 (2015) 249–272. S.M. Smith, M. Heer, L.C. Shackelford, J.D. Sibonga, J. Spatz, R.A. Pietrzyk, et al., Bone metabolism and renal stone risk during International Space Station missions, Bone 81 (2015) 712–720. I.B. Kozlovskaya, A.I. Grigoriev, Russian system of countermeasures on board of the International Space Station (ISS): the first results, Acta Astronaut. 55 (2004) 233–237. L. Vico, B. van Rietbergen, N. Vilayphiou, M.T. Linossier, H. Locrelle, M. Normand, et al., Cortical and trabecular bone microstructure did not recover at weightbearing skeletal sites and progressively deteriorated at non-weight-bearing sites during the year following International Space Station missions, J. Bone Miner. Res. 32 (2017) 2010–2021. T.F. Lang, A.D. Leblanc, H.J. Evans, Y. Lu, Adaptation of the proximal femur to skeletal reloading after long-duration spaceflight, J. Bone Miner. Res. 21 (2006) 1224–1230. J.L. Morgan, M. Heer, A.R. Hargens, B.R. Macias, E.K. Hudson, L.C. Shackelford, et al., Sex-specific responses of bone metabolism and renal stone risk during bed rest, Physiol. Rep. 2 (2014). T. Cervinka, H. Sievänen, J. Hyttinen, J. Rittweger, Bone loss patterns in cortical, subcortical, and trabecular compartments during simulated microgravity, J. Appl. Physiol. 117 (1985) 80–88 2014. Y.C. Lau, X. Qian, K.T. Po, L.M. Li, X. Guo, Electrical stimulation at the dorsal root ganglion preserves trabecular bone mass and microarchitecture of the tibia in hindlimb-unloaded rats, Osteoporos. Int. 26 (2015) 481–488. R. Saxena, G. Pan, E.D. Dohm, J.M. McDonald, Modeled microgravity and hindlimb unloading sensitize osteoclast precursors to RANKL-mediated osteoclastogenesis, J. Bone Miner. Metab. 29 (2011) 111–122. A. Jackson, B. Vayssiere, T. Garcia, W. Newell, R. Baron, S. Roman-Roman, et al., Gene array analysis of Wnt-regulated genes in C3H10T1/2 cells, Bone 36 (2005) 585–598. M.M. Weivoda, M. Ruan, C.M. Hachfeld, L. Pederson, A. Howe, R.A. Davey, et al., Wnt signaling inhibits osteoclast differentiation by activating canonical and noncanonical cAMP/PKA pathways, J. Bone Miner. Res. 31 (2016) 65–75. J.B. Regard, Z. Zhong, B.O. Williams, Y. Yang, Wnt signaling in bone development and disease: making stronger bone with Wnts, Cold Spring Harb. Perspect. Biol. 4 (2012). P. Frings-Meuthen, G. Boehme, A.M. Liphardt, N. Baecker, M. Heer, J. Rittweger, Sclerostin and DKK1 levels during 14 and 21 days of bed rest in healthy young men, J. Musculoskelet. Neuronal Interact. 13 (2013) 45–52. T.A. Burgers, B.O. Williams, Regulation of Wnt/beta-catenin signaling within and from osteocytes, Bone 54 (2013) 244–249. X. Yang, L.W. Sun, M. Liang, X.N. Wang, Y.B. Fan, The response of Wnt/β-catenin signaling pathway in osteocytes under simulated microgravity, Microgravity Sci. Technol. 27 (2015) 473–483. J.M. Spatz, M.N. Wein, J.H. Gooi, Y. Qu, J.L. Garr, S. Liu, et al., The wnt inhibitor sclerostin is up-regulated by mechanical unloading in osteocytes in vitro, J. Biol. Chem. 290 (2015) 16744–16758. S. Dev, J.L. Babitt, Overview of iron metabolism in health and disease, Hemodial. Int. 21 (2017) S6–S20. K. Pantopoulos, S.K. Porwal, A. Tartakoff, L. Devireddy, Mechanisms of mammalian iron homeostasis, Biochemistry 51 (2012) 5705–5724. H. Gunshin, Y. Fujiwara, A.O. Custodio, C. Direnzo, S. Robine, N.C. Andrews, Slc11a2 is required for intestinal iron absorption and erythropoiesis but dispensable in placenta and liver, J. Clin. Investig. 115 (2005) 1258–1266. M.D. Knutson, Iron transport proteins: gateways of cellular and systemic iron

homeostasis, J. Biol. Chem. 292 (2017) 12735–12743. [67] K. Gkouvatsos, G. Papanikolaou, K. Pantopoulos, Regulation of iron transport and the role of transferrin, Biochim. Biophys. Acta 1820 (2012) 188–202. [68] H. Drakesmith, E. Nemeth, T. Ganz, Ironing out ferroportin, Cell Metabol. 22 (2015) 777–787. [69] J. Chifman, R. Laubenbacher, S.V. Torti, A systems biology approach to iron metabolism, Adv. Exp. Med. Biol. 844 (2014) 201–225. [70] S.R. Pasricha, P.J. Lim, T.L. Duarte, C. Casu, D. Oosterhuis, K. Mleczko-Sanecka, et al., Hepcidin is regulated by promoter-associated histone acetylation and HDAC3, Nat. Commun. 8 (2017) 403. [71] P.J. Schmidt, Regulation of iron metabolism by hepcidin under conditions of inflammation, J. Biol. Chem. 290 (2015) 18975–18983. [72] B. Andriopoulos Jr., E. Corradini, Y. Xia, S.A. Faasse, S. Chen, L. Grgurevic, BMP6 is a key endogenous regulator of hepcidin expression and iron metabolism, Nat. Genet. 41 (2009) 482–487. [73] E. Nemeth, M.S. Tuttle, J. Powelson, M.B. Vaughn, A. Donovan, D.M. Ward, et al., Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization, Science 306 (2004) 2090–2093. [74] A.U. Steinbicker, M.U. Muckenthaler, Out of balance-systemic iron homeostasis in iron-related disorders, Nutrients 5 (2013) 3034–3061. [75] T. Ganz, E. Nemeth, Regulation of iron acquisition and iron distribution in mammals, Biochim. Biophys. Acta 1763 (2006) 690–699. [76] L. Détivaud, E. Nemeth, K. Boudjema, B. Turlin, M.B. Troadec, P. Leroyer, et al., Hepcidin levels in humans are correlated with hepatic iron stores, hemoglobin levels, and hepatic function, Blood 106 (2005) 746–748. [77] J. Frydlova, D.W. Rogalsky, J. Truksa, E. Nečas, M. Vokurka, J. Krijt, Effect of stimulated erythropoiesis on liver SMAD signaling pathway in iron-overloaded and iron-deficient mice, PLoS One 14 (2019) e0215028. [78] M.V. Verga Falzacappa, M. Vujic Spasic, R. Kessler, J. Stolte, M.W. Hentze, M.U. Muckenthaler, STAT3 mediates hepatic hepcidin expression and its inflammatory stimulation, Blood 109 (2007) 353–358. [79] N.P. Talbot, T.G. Smith, S. Lakhal-Littleton, C. Gülsever, M. Rivera-Ch, K.L. Dorrington, et al., Suppression of plasma hepcidin by venesection during steady-state hypoxia, Blood 127 (2016) 1206–1207. [80] S.M. Smith, Red blood cell and iron metabolism during space flight, Nutrition 18 (2002) 864–866. [81] S.M. Smith, J.E. Davis-Street, B.L. Rice, J.L. Nillen, P.L. Gillman, G. Block, Nutritional status assessment in semiclosed environments: ground-based and space flight studies in humans, J. Nutr. 131 (2001) 2053–2061. [82] M. Tavassoli, Anemia of spaceflight, Blood 60 (1982) 1059–1067. [83] S.R. Zwart, S.A. Oliver, J.V. Fesperman, G. Kala, J. Krauhs, K. Ericson, et al., Nutritional status assessment before, during, and after long-duration head-down bed rest, Aviat. Space Environ. Med. 80 (2009) A15–A22. [84] J. Tsay, Z. Yang, F.P. Ross, S. Cunningham-Rundles, H. Lin, R. Coleman, et al., Bone loss caused by iron overload in a murine model: importance of oxidative stress, Blood 116 (2010) 2582–2589. [85] T.F. Reardon, D.G. Allen, Iron injections in mice increase skeletal muscle iron content, induce oxidative stress and reduce exercise performance, Exp. Physiol. 94 (2009) 720–730. [86] J. Shaw, A. Chakraborty, A. Nag, A. Chattopadyay, A.K. Dasgupta, M. Bhattacharyya, Intracellular iron overload leading to DNA damage of lymphocytes and immune dysfunction in thalassemia major patients, Eur. J. Haematol. 99 (2017) 399–408. [87] H.W. Lane, C.P. Alfrey, T.B. Driscoll, S.M. Smith, L.E. Nyquist, Control of red blood cell mass during spaceflight, J. Gravitational Physiol. 3 (1996) 87–88. [88] C.P. Alfrey, L. Rice, M.M. Udden, T.B. Driscoll, Neocytolysis: physiological downregulator of red-cell mass, Lancet 349 (1997) 1389–1390. [89] C.P. Alfrey, M.M. Udden, C. Leach-Huntoon, T. Driscoll, M.H. Pickett, Control of red blood cell mass in spaceflight, J. Appl. Physiol. 81 (1985) 98–104 1996. [90] Z. Xu, W. Sun, Y. Li, S. Ling, C. Zhao, G. Zhong, et al., The regulation of iron metabolism by hepcidin contributes to unloading-induced bone loss, Bone 94 (2017) 152–161. [91] S. Askar, S.N. Deveboynu, H. Er, T.K. Askar, A.A. Hismiogullari, Changes in proinflammatory cytokines and antimicrobial proteins in elderly women with iron deficiency anemia, Pak. J. Med. Sci. 35 (2019) 298–301. [92] V. Sangkhae, E. Nemeth, Regulation of the iron homeostatic hormone hepcidin, Adv. Nutr. 8 (2017) 126–136. [93] L. Bo, Z. Liu, Y. Zhong, J. Huang, B. Chen, H. Wang, Y. Xu, Iron deficiency anemia's effect on bone formation in zebrafish mutant, Biochem. Biophys. Res. Commun. 475 (2016) 271–276. [94] L. Zhou, B. Zhao, L. Zhang, S. Wang, D. Dong, H. Lv, et al., Alterations in cellular iron metabolism provide more therapeutic opportunities for cancer, Int. J. Mol. Sci. 19 (2018). [95] E. Gammella, P. Buratti, G. Cairo, S. Recalcati, The transferrin receptor: the cellular iron gate, Metallomics 9 (2017) 1367–1375. [96] C. Chen, D. Garcia-Santos, Y. Ishikawa, A. Seguin, L. Li, K.H. Fegan, et al., Snx3 regulates recycling of the transferrin receptor and iron assimilation, Cell Metabol. 17 (2013) 343–352. [97] I. De Domenico, D.M. Ward, C. Langelier, M.B. Vaughn, E. Nemeth, W.I. Sundquist, et al., The molecular mechanism of hepcidin-mediated ferroportin down-regulation, Mol. Biol. Cell 18 (2007) 2569–2578. [98] V. Jeney, Clinical impact and cellular mechanisms of iron overload-associated bone loss, Front. Pharmacol. 8 (2017) 77. [99] E. Balogh, G. Paragh, V. Jeney, Influence of iron on bone homeostasis, Pharmaceuticals (Basel) 11 (2018). [100] L.C. Hofbauer, C.A. Kühne, V. Viereck, The OPG/RANKL/RANK system in

413

Acta Astronautica 164 (2019) 407–414

X. Chen, et al. metabolic bone diseases, J. Musculoskelet. Neuronal Interact. 4 (2004) 268–275. [101] Z. Gu, H. Wang, J. Xia, Y. Yang, Z. Jin, H. Xu, et al., Decreased ferroportin promotes myeloma cell growth and osteoclast differentiation, Cancer Res. 75 (2015) 2211–2221. [102] J. Zhou, S. Ye, T. Fujiwara, S.C. Manolagas, H. Zhao, Steap4 plays a critical role in osteoclastogenesis in vitro by regulating cellular iron/reactive oxygen species (ROS) levels and cAMP response element-binding protein (CREB) activation, J. Biol. Chem. 288 (2013) 30064–30074. [103] K.A. Ishii, T. Fumoto, K. Iwai, S. Takeshita, M. Ito, N. Shimohata, et al., Coordination of PGC-1beta and iron uptake in mitochondrial biogenesis and osteoclast activation, Nat. Med. 15 (2009) 259–266. [104] J.G. Messer, A.K. Kilbarger, K.M. Erikson, D.E. Kipp, Iron overload alters ironregulatory genes and proteins, down-regulates osteoblastic phenotype, and is associated with apoptosis in fetal rat calvaria cultures, Bone 45 (2009) 972–979. [105] A. Zarjou, V. Jeney, P. Arosio, M. Poli, E. Zavaczki, G. Balla, et al., Ferritin ferroxidase activity: a potent inhibitor of osteogenesis, J. Bone Miner. Res. 25 (2010) 164–172. [106] M. Miyazawa, A.R. Bogdan, K. Hashimoto, Y. Tsuji, Regulation of transferrin receptor-1 mRNA by the interplay between IRE-binding proteins and miR-7/miR141 in the 3'-IRE stem-loops, RNA 24 (2018) 468–479. [107] C.P. Anderson, M. Shen, R.S. Eisenstein, E.A. Leibold, Mammalian iron metabolism and its control by iron regulatory proteins, Biochim. Biophys. Acta 1823 (2012) 1468–1483. [108] L.C. Kühn, Iron regulatory proteins and their role in controlling iron metabolism, Metallomics 7 (2015) 232–243. [109] N. Hubert, M.W. Hentze, Previously uncharacterized isoforms of divalent metal transporter (DMT)-1: implications for regulation and cellular function, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 12345–12350. [110] B. Chen, G.F. Li, Y. Shen, X.I. Huang, Y.J. Xu, Reducing iron accumulation: a potential approach for the prevention and treatment of postmenopausal osteoporosis, Exp. Ther. Med. 10 (2015) 7–11. [111] J.E. Compston, M.R. McClung, W.D. Leslie, Osteoporosis, Lancet 393 (2019) 364–376. [112] J.C. Yang, Z.Q. Yang, W.B. Li, Y.R. Xue, H.Y. Xu, J.B. Li, et al., Glucocorticoid: a

[113] [114] [115] [116] [117]

[118] [119] [120] [121] [122] [123]

414

potential role in microgravity-induced bone loss, Acta Astronaut. 140 (2017) 206–212. Y. Ma, J. Chu, J. Ma, L. Ning, K. Zhou, X. Fang, Sanguinarine protects against ovariectomy-induced osteoporosis in mice, Mol. Med. Rep. 16 (2017) 288–294. W.L. Zhang, H.Z. Meng, M.W. Yang, Regulation of DMT1 on bone microstructure in type 2 diabetes, Int. J. Med. Sci. 12 (2015) 441–449. B. Chen, Y.L. Yan, C. Liu, L. Bo, G.F. Li, H. Wang, et al., Therapeutic effect of deferoxamine on iron overload-induced inhibition of osteogenesis in a zebrafish model, Calcif. Tissue Int. 94 (2014) 353–360. E. Balogh, E. Tolnai, B. Nagy Jr., B. Nagy, G. Balla, J. Balla, et al., Iron overload inhibits osteogenic commitment and differentiation of mesenchymal stem cells via the induction of ferritin, Biochim. Biophys. Acta 1862 (2016) 1640–1649. K.S. Lee, J.S. Jang, D.R. Lee, Y.H. Kim, G.E. Nam, B.D. Han, et al., Serum ferritin levels are positively associated with bone mineral density in elderly Korean men: the 2008-2010 Korea National Health and Nutrition Examination Surveys, J. Bone Miner. Metab. 32 (2014) 683–690. Q. Yang, J. Jian, S.B. Abramson, X. Huang, Inhibitory effects of iron on bone morphogenetic protein 2-induced osteoblastogenesis, J. Bone Miner. Res. 26 (2011) 1188–1196. L. Zhao, Y. Wang, Z. Wang, Z. Xu, Q. Zhang, M. Yin, Effects of dietary resveratrol on excess-iron-induced bone loss via antioxidative character, J. Nutr. Biochem. 26 (2015) 1174–1182. Y.F. He, Y. Ma, C. Gao, G.Y. Zhao, L.L. Zhang, G.F. Li, et al., Iron overload inhibits osteoblast biological activity through oxidative stress, Biol. Trace Elem. Res. 152 (2013) 292–296. Y. Yuan, F. Xu, Y. Cao, L. Xu, C. Yu, F. Yang, et al., Iron accumulation leads to bone loss by inducing mesenchymal stem cell apoptosis through the activation of caspase3, Biol. Trace Elem. Res. 187 (2019) 434–441. N.S. Soysa, N. Alles, NF-kappaB functions in osteoclasts, Biochem. Biophys. Res. Commun. 378 (2009) 1–5. J. Daru, K. Colman, S.J. Stanworth, B. De La Salle, E.M. Wood, S.R. Pasricha, Serum ferritin as an indicator of iron status: what do we need to know? Am. J. Clin. Nutr. 106 (2017) 1634S–1639S.