MOLECULAR MECHANISMS OF IRON UPTAKE BY HEPG2 HEPATOMA CELLS

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1 MOLECULAR MECHANISMS OF IRON UPTAKE BY HEPG2 HEPATOMA CELLS By LIN ZHANG A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA

2 2012 LIN ZHANG 2

3 To my beloved wife: without your endless love and support, none of these would have meanings 3

4 ACKNOWLEDGMENTS I would like to express my deepest appreciation to my super advisor, Dr. Mitchell Knutson, who gave me the chance to study in the department of Food Science and Human Nutrition. I was very impressed by not only his enthusiasm toward research, but his excellent personality as well. I have been learning from him what I need to know to be a good scientist and a good advisor. Without his persistent help and guidance, this thesis would not have been successful. Additionally, I would like to thank each of my committee members: Dr. James Collins, and Dr. Michael King. They were abundantly helpful and offered invaluable advice, and guidance. Their efforts for my thesis were highly valued and appreciated. I would like also thank all my lab members: Chia-yu Wang, Supak Jenkitkasemwong, Wei Zhang, and Richard Coffey for help. I had a wonderful time with them both in and out of the lab. Their help makes this thesis much easier to accomplish. Finally, I would like to thank my wife--yan Ren, my parents, and all the family members, for their support and encouragement for me to pursue this degree. Thank you all. 4

5 TABLE OF CONTENTS page ACKNOWLEDGMENTS... 4 LIST OF FIGURES... 7 LIST OF ABBREVIATIONS... 8 ABSTRACT CHAPTER 1 INTRODUCTION LITERATURE REVIEW Introduction to Iron Iron Metabolism Biological Functions of Iron Whole Body Iron Distribution Iron Recycling Iron Storage Iron Excretion Iron-Related Diseases Liver Iron Metabolism Function of Liver in Iron Metabolism Hepcidin HFE TF TFR Hepatic Iron Uptake TBI Uptake TFR1-mediated TBI uptake TFR2-mediated TBI uptake TFR1/2-independent TBI uptake NTBI Uptake in the Liver The Role of Endocytosis in TBI Uptake into Hepatocytes Endocytosis-Dependent TBI Uptake TFR1/2-mediated endocytosis Fluid-phase endocytosis (pinocytosis) Endocytosis-Independent Pathway Reductive iron release at the cell surface Iron Transporters in Hepatocytes DMT ZIP14 and Iron Metabolism Identification and characterization of ZIP

6 ZIP14 and cellular iron uptake Specific Aims Aim 1. To Test the Hypothesis that Endocytosis Is not Required for TBI Uptake in HepG2 Cells Aim 2. To Test the Hypothesis that TF Interacts with ZIP MATERIALS AND METHODS Knockdown of Clathrin and TFR1/2 Using RNAi Measurement of TF Uptake and Assimilation of Iron from TF DuoLink In Situ Proximity Ligation Assay Far-Western Blotting Analyses Co-Immunoprecipitation Assay Statistical Analysis RESULTS Inhibiting Clathrin-Mediated Endocytosis in HepG2 cells Does not Affect Iron Uptake from TF The Effects of Knocking down TFR1/2 on TF Uptake Are Different at Low and High TF Levels ZIP14 Can Interact with TF both In Vitro and In Vivo DISCUSSION LIST OF REFERENCES BIOGRAPHICAL SKETCH

7 LIST OF FIGURES Figure page 1-1 Assimilation of iron through TFR-dependent pathway in developing erythroid cells Clathrin heavy chain (CHC) knockdown with sirna decreases uptake of TF, but does not affect uptake of iron from TF TFR1 and TFR2 knockdown with sirna decreases uptake of TF at 50 nm level TFR1 and TFR2 knockdown with sirna increases uptake of TF at 5 µm level ZIP14 can interact with TF in vivo in FLAG-tagged HepG2 cells ZIP14 can bind TF directly in vitro ZIP14 interact with endogenous TF

8 LIST OF ABBREVIATIONS AAV BSA CO-IP DAPI DMEM Adeno-associated virus Bovine serum albumin co-immunoprecipitation 4', 6-diamidino-2-phenylindole Dulbecco s modified Eagle s medium DMT1 Divalent metal transporter 1 FENTA Iron-nitrilotriacetate FPN1 Ferroportin 1 HFE HH HRP IRT IRP IRE KDA LPS NTBI PFA PLA RES RNAI ROS SFM SIRNA High Fe gene (Gene mutated in the most common form of hemochromatosis) Hereditary hemochromatosis Horseradish peroxidase Iron-regulated transporter Iron-regulatory protein Iron-responsive element Kilodalton Lipopolysaccharide Non-transferrin-bound iron Paraformaldehyde Proximity Ligation Assay Reticuloendothelial system RNA interference Reactive oxygen species Serum-free medium Small interfering RNA 8

9 SLC STEAP TBI TBS TBST TF Solute carrier Six-transmembrane epithelial antigen of the prostate Transferrin-bound iron Tris-buffered saline Tris-buffered saline Tween-20 Transferrin TFR1 Transferrin receptor 1 TFR2 Transferrin receptor 2 ORF PBS WT ZIP ZRT Open reading frame Phosphate-buffered saline Wild-type ZRT/IRT-like proteins Zinc-regulated transporter 9

10 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science MOLECULAR MECHANISMS OF IRON UPTAKE BY HEPG2 HEPATOMA CELLS Chair: Mitchell Knutson Major: Food Science and Human Nutrition By Lin Zhang August 2012 After erythroid cells of the bone marrow, hepatocytes of the liver represent the second largest consumer of transferrin-bound iron (TBI), accounting for 10-20% of iron exchange with the plasma. Although it is well known that transferrin (TF) is taken up by cells via endocytosis, a previous study in hepatocytes found that chemical inhibitors of endocytosis did not inhibit the assimilation of iron from TF. The first aim of the present study was to investigate the role of clathrin-mediated endocytosis in the uptake of TBI by HepG2 cells, a human hepatoma cell line. I used sirna to suppress the expression of clathrin heavy chain (CHC) and then measured the iron uptake level after supplying the cells with 59 Fe-labeled TF to see whether endocytosis is required for the uptake of iron from TF. The second aim of this study was to test the hypothesis that ZIP14 (SLC39A14) interacts with TF and may play a role in the iron uptake from TF by hepatocytes by utilizing the novel PLA assay, co-ip and far-western blotting. 10

11 CHAPTER 1 INTRODUCTION Iron is necessary to almost all known organisms. It plays an essential role in the functioning of many biochemical processes including erythropoiesis, electron transfer reactions, oxygen-carrying as well as growth and differentiation of cells [1,2]. Iron deficiency can cause serious health problems such as growth retardation, anemia, and impaired cognitive function [3]. On the other side, excessive free iron due to iron overload accelerate the formation of reactive oxygen species (ROS), which when in excess can damage DNA, proteins, and lipids [4]. Therefore, iron status must be tightly controlled to ensure our body has sufficient, but not excessive, iron levels. TF is an abundant plasma iron transport protein. Approximately 0.1% of the 3 grams body iron circulates in the plasma as an exchangeable pool by binding to TF [5]. This chelation serves three purposes: it renders iron soluble under physiologic conditions, it prevents free radical iron toxicity, and it facilitates iron transport to cells [6]. TF receptor (TFR) is a receptor for TF at the cell surface. TFR-mediated iron uptake is the best described model of iron uptake in developing erythroid cells (Figure 1-1). It imports iron by internalizing the TF-iron complex through receptor-mediated endocytosis. Briefly, at the neutral ph (7.2) in plasma, TF Fe(III) complex binds to the TFR1 on the cell surface from where it is internalized by receptor-mediated endocytosis through clathrin-coated pits. The internalized vesicle, or endosome, becomes acidified by the action of H + ATPase, which results in a conformational change in TF TFR complex and the subsequent detachment of the metal. Fe(III) is then converted to Fe(II) by the endosomal reductase six-transmembrane epithelial antigen of the prostate-3 (STEAP3), which is finally transported out of the endosome into the cytosol by divalent 11

12 metal transporter-1 (DMT1). Fe(II) can be stored in ferritin in non-erythroid cells or incorporated into hemoglobin in erythroid cells. The TF TFR complex is recycled back to the cell surface and apo-tf is released. Figure 1-1. Assimilation of iron through TFR-dependent pathway in developing erythroid cells The liver functions as one of the most important iron storage organ of the body by exchanging iron with plasma TF. The liver takes up TBI almost exclusively into hepatocytes [7]. Hepatocytes can take up TBI through the TFR1-dependent endocytic pathway [7,8], or the recently reported transferrin receptor 2 (TFR2)-dependent endocytic pathway [9]. TFR2 is a homologue of TFR1and can also bind Fe(III)-TF, but with a significantly lower affinity than TFR1. Unlike the wide expression pattern and ironregulated expression of TFR1, TFR2 is mainly detected in hepatocytes of liver and its expression is not regulated by the intracellular iron level [10]. It appears that TFR2 functions not primarily in cellular iron uptake and delivery, but more as an iron status 12

13 sensor for systemic homeostasis [11]. Besides the TFR-mediated endocytic TBI uptake, hepatocytes were reported to also be able to take up iron from TF without the involvement of endocytosis [12,13,14]. Chua et al. [14] recently found that liver could still take up iron in TFR2 knockout mice. Given TFR1 levels are very low in these iron overload mice, there probably exists another pathway for TBI uptake, which is independent of both TFR1 and TFR2. The consequence that knockdown of both TFR1 and TFR2 with sirna in hepatoma cells had little effect on iron uptake at high TF concentration further provides support for this hypothesis [15]. These findings suggested the existence of a new type of TFR other than TFR1 or TFR2 is required for TBI uptake by hepatocytes. The most possible way for hepatocytes taking up iron during this process is through a TF-hepatocyte plasma membrane interaction in which iron is removed from the TF and passed through membrane. ZIP14 is a transmembrane protein that belongs to the solute carrier 39 (SLC39A) family. The word ZIP is derived from ZRT1 (zinc-regulated transporter 1) and IRT1 (iron-regulated transporter 1), the first two members identified in this family [16]. ZIP14 was first characterized in 2005 and was reported to stimulate the uptake of zinc into the cytosol of Chinese hamster ovary (CHO) cells when overexpressed [17]. One year later, Liuzzi et al. [18] reported that overexpression of ZIP14 in HEK 293 cells and Sf9 insect cells enhanced not only the uptake of zinc, but also iron. In addition, a previous study in our lab found that ZIP14 may take up iron from TF [19]. Specifically, it was found that overexpression of ZIP14 in HEK 293T cells increased the assimilation of iron from TF without increasing levels of TFR1 or the uptake of TF. It was also found that suppressing endogenous ZIP14 by sirna in HepG2 cells decreased iron uptake from 13

14 TF by 50% compared with controls. This result was consistent with previous finding that a downregulation of ZIP14 was associated with reduced uptake and assimilation of iron from TF [20]. The interesting finding that ZIP14 partially colocalizes with TF using immunofluorescence assay suggested these two proteins could potentially interact with each other. 14

15 CHAPTER 2 LITERATURE REVIEW Introduction to Iron Iron, atomic number 26, is a transit metal with the symbol Fe. It is the fourth most common element in the Earth's crust, constituting much of the planet s outer and inner core. Iron is an important micronutrient for living organisms. As far as we know, almost all living organisms from Archaea to man need iron for maintaining their normal metabolic activities. It is essential for numerous biological and biochemical processes including erythropoiesis, DNA biosynthesis, gene regulation, oxygen transport, photosynthesis, electron transfer reaction and other key metabolic reactions [2]. The role that iron plays in the body s homeostasis is so important and delicate that both iron deficiency and iron overload have serious or even fatal consequences. Therefore, the cellular and whole body homeostatic mechanisms which regulate the absorption, transport, storage and mobilization of iron appear to be very crucial in iron metabolism [1]. Biological Functions of Iron Iron Metabolism Iron can exist in various oxidation states because it has unfilled d orbitals. The capacity of easily interconverting between ferric (Fe 2+ ) and ferrous (Fe 3+ ) forms makes it a useful component of oxygen-binding molecules, cytochromes, and many enzymes. However, the same high redox potential that enables iron to readily switch between the ferrous and ferric states also makes the iron potentially damaging to fatty acids, proteins, and nucleic acids in the cell through catalyzing the production of dangerous free radicals [21]. 15

16 Most of total body iron (about 60-70%) is present in hemoglobin in red blood cells, with a smaller amount (5-10%) in myoglobin in muscle tissue. Both hemoglobin and myoglobin belong to the family of heme-containing proteins called hemoproteins [22]. Heme is a prosthetic group composed of a porphyrin ring structure with a central iron atom. The major role of heme iron in hemoglobin and myoglobin is to help transport and store oxygen. In mitochondria, the important hemoproteins cytochromes play a role in electron transport and related metabolic pathways which are essential to cellular energy production as part of the electron transport chain [23]. Heme iron in these hemoproteins serves as electron carriers during the synthesis of ATP in cells. Compared to the predominance of heme iron, only very small amount (20-30%) of iron exists in non-heme iron-containing proteins, but they still have key functions in the body. Several iron-containing enzymes, like succinate dehydrogenase and cytochrome c reductase, are involved primarily in energy metabolism; iron storage and transport proteins (TF, lactoferrin, ferritin and hemosiderin, all bind to iron) participating in iron uptake, transport and storage in the body, play important roles in maintaining cellular iron homeostasis, whose gene expressions are also regulated by the iron levels [24]. Iron also plays important roles in cellular processes such as the synthesis of DNA, RNA and proteins; cell proliferation and differentiation. Most importantly, in the brain, iron is required for the formation of myelin and neuronal dendritic tree development [25]. As a result, iron homeostasis is very important for normal brain function, such as learning and memory [26]. Iron is essential for all eukaryotes but can also be toxic; easily gaining and losing electrons by iron can result in the donation its electrons to oxygen, increasing the 16

17 generation of the hydroxyl radical and superoxide anions. These oxygen metabolites react readily with biological molecules, including proteins, lipids and DNA [21]. Under normal physiological conditions, only trace amount of free iron is present in the body. Extra free iron will be quickly bound by TF in the plasma or by ferritin and other ironbinding proteins in the cytoplasm so that the toxicity of free iron is kept at a minimum. Whole Body Iron Distribution Under normal conditions, adult men have 35 to 45 mg of iron per kg of body weight, while women have lower iron stores (~ 23 mg of iron per kg of body weight) as a result of their recurrent blood loss through menstruation [27]. At the cellular level, greater than two thirds of the body s iron is incorporated into hemoglobin in developing erythroid precursors and mature red cells. The remaining body iron is mostly found in hepatocytes and reticuloendothelial macrophages, which are known as the iron storage depots [2]. However, there s still approximately 0.1% of the total body iron existing in blood plasma is bound to TF, which is responsible for delivering iron to developing erythroid precursors, as well as to other tissues of the body [5]. Iron Recycling In the average adult, about 2 million red blood cells are being turned over every second. During this process, nearly 25 mg of iron will be recycled each day and used by the hemoglobin of developing erythrocytes, for heme reproduction and erythropoiesis [28]. This recycling process is a necessity because the erythron require about 20 mg of iron one day, but only less than 2 mg of iron normally absorbed by the body each day through the intestine. Reticuloendothelial macrophages mainly found in the liver, spleen, and bone marrow are critical in the process of iron recycling [29]. Nearly 80% of body iron found in hemoglobin within decaying erythrocytes is phagocytosed by macrophages 17

18 of the reticuloendothelial system. Each day, these macrophages quickly and efficiently recycle those 25 mg of erythrocyte-derived iron and return it to the plasma [30]. The recycled iron is loaded on TF and incorporated into the bone marrow for reuse in erythropoiesis [31]. Disturbances in this tightly regulated and highly efficient process will result in anemia [32], iron overload [33], and other pathologies [24,34]. Iron Storage In normal human subjects approximately one-third of the iron in the body is present as iron stores [35] and the liver (mainly hepatocytes) serves as the primary depots for iron storage which accounts for approximately 50% of the storage iron [36]. In rats, most iron (~ 98%) stored in the liver is found in parenchymal cells. Kupffer cells are the second largest depot for iron; only very small amounts iron is present in stellate cells, endothelial cells and bile duct cells [37]. Most of the iron stored in the hepatocytes is present in the form of ferritin the primary physiologic source of reserve iron in the body, which can be mobilized when needed elsewhere in the body. Ferritin has a protein component apoferritin, consisting of 24 subunits arranged as a hollow sphere and can store up to 4500 atoms of iron [38]. Macrophages of the reticuloendothelial system also serve as iron store during the process of breaking down hemoglobin from phagocytosed red blood cells, especially among patients with iron overload who have frequent blood cell destruction and transfusions happen in their bodies. Under normal condition, hemosiderin in macrophages only represents a small portion of normal body iron stores. However, in iron overload conditions, it is dramatically increased a hundred fold in comparison to that of ferritin, which only increases tenfold [39]. 18

19 Both ferritin and hemosiderin iron can be mobilized to compensate for iron lost when iron deficiency occurs. Studies in normal rats indicated that around 6% of storage iron is released from the hepatocytes per day [40]. In addition to hepatic iron, each day a considerable amount of iron from phagocytosed senescent red blood cells are also released from Kupffer cells and incorporated into plasma TF [36,41]. Iron Excretion The body s iron level is mainly controlled during the process of intestinal absorption, the efficiency of which can be up-regulated about 15-fold in inverse proportion to body iron content [42]. Unlike other metal nutrients, there is no specific iron excretion pathway in the human body [43]. Basal iron excretion (about 1 mg), mainly through feces and cellular exfoliation from GI mucosa, skin, hair, and nails, is limited, and is the primary factor determining human s nutritional requirements for absorbed iron. As a result, body iron is highly conserved except for iron losses from menstruation, other bleeding, and pregnancy [44]. Iron-Related Diseases Maintenance of normal iron levels, proper iron distribution and timely delivery of iron are critical for normal iron metabolism in all tissues. Failure to keep iron homeostasis balanced will eventually lead to iron deficiency or iron overload, both of which are harmful to human body. Disorders of iron homeostasis are among the most common diseases of humans with diverse clinical manifestations. Diseases such as anemia, cancer and neurodegenerative disorders are all related to disturbed iron metabolism [45]. More than half a billion people worldwide have adverse effects as a result of iron deficiency [27], whereas iron overload disorders (hereditary 19

20 hemochromatosis) are among the most frequent single gene disorders in humans [46,47]. Liver Iron Metabolism Function of Liver in Iron Metabolism The liver plays a critical role in iron metabolism. It is responsible for approximately 8% of plasma iron turnover in humans [48]. When the body is starved of iron, the liver releases iron into the circulation to meet tissue iron needs. In addition, the liver is the major storage organ for iron, because it has first-pass access to dietary iron entering the portal circulation from the gut [49]. The liver also clears the plasma of iron that exceeds the binding capacity of plasma TF during iron overload conditions [50] and stores it in ferritin and hemosiderin. Most importantly, the liver regulates iron traffic into and around the body through its production and release of the master peptide hepcidin. The liver is made up of several different types of cells: parenchymal cells (hepatocytes), endothelial cells, Kupffer cells and stellate cells. Of all these types of cells in liver, hepatocytes occupy 72% of the total liver volume and play a key role in maintaining the proper function of liver [51]. Hepatocytes function as the body's primary iron store, and exhibit iron uptake rates 10-20% of that observed for bone marrow [52]. Besides TF and ceruloplasmin, hepatocytes also produce and secrete other proteins participating in iron homeostasis including hepcidin, TFR2, and hemojuvelin [53]. Functional loss of any of these genes results in HH. In addition, hepatocytes synthesize ~ 15% of the total body heme [54], as well as haptoglobin and hemopexin which recover heme after intravascular hemolysis [55]. Kupffer cells are resident macrophages in the liver. The main function of Kupffer cells in iron metabolism appears to be ingesting senescent or damaged red blood cells, 20

21 catabolizing the hemoglobin and releasing the iron into the circulation [56]. The quantity of iron recycled through the macrophage on a daily basis is 10 ~ 20 times more than that taken up through the intestine. Hepcidin Hepcidin (liver-expressed antimicrobial peptide, LEAP-1) is a 25 amino acids peptide synthesized and secreted predominantly by hepatocytes [57], which has been identified as an important iron-regulatory hormone by two independent groups in 2001 [58]. Hepcidin negatively regulates the main iron flows that enter the plasma: the absorption of dietary iron in the duodenum, the release of recycled iron from macrophages, and the release of stored iron from hepatocytes. In turn, the production of hepcidin is regulated by iron, so that more hepcidin is produced by hepatocytes when iron is abundant, limiting further iron absorption and release from stores. When iron is deficient, hepatocytes produce little or no hepcidin, allowing more iron to enter bloodstream. Both diferric plasma TF and stored iron in hepatocytes increase the synthesis of hepcidin [59]. The regulatory role of hepcidin in iron was supported by the observation that disruption of hepcidin synthesis by Usf2 (upstream stimulatory factor2) knockout developed a severe iron overload in various tissues, such as liver, pancreas and heart in mice [60]. In contrast, transgenic mice over-expressing hepcidin in the liver developed severe iron-deficiency anemia [61,62]. The mechanism by which hepcidin regulates iron levels in the body is through its binding to the only known cellular iron exporter, FPN, which is predominantly expressed in macrophages and basolateral membrane of enterocytes, where it regulates iron entry into blood. Hepcidin binding to FPN triggers its internalization and subsequent degradation, thus reducing iron export into the circulating system [63]. 21

22 HFE HFE, a product of the hereditary hemochromatosis gene, is expressed ubiquitously at low levels, but at high levels in hepatocytes [64]. HFE requires the protein β2-microglobulin for its correct localization to the cell surface. Mutation of a single base pair in the HFE gene results in a substitution of tyrosine for cysteine (C282Y) in the protein, which disrupts a disulfide bond required for proper folding, preventing it from binding to β2-microglobulin and trafficking to the cell surface [20]. This dysfunction of HFE protein can lead to iron overload in the liver, heart, pancreas, and parathyroid and pituitary glands, resulting in multi-organ dysfunction [65]. The C282Y mutation in the HFE genes is responsible for the vast majority of patients with hereditary hemochromatosis. Several mechanisms have been proposed by which HFE regulates iron metabolism. HFE competes with diferric TF for binding to the similar motif of TFR1 lowering iron uptake into cells [66]. Alternately, the binding of diferric TF to TFR1 releases HFE. Consequently, free HFE binds to TFR2 in hepatocytes to increase hepcidin secretion by the liver, which negatively regulates dietary iron uptake by the intestine and iron release from macrophages. Recently, HFE was also found to inhibit iron uptake via down-regulation of ZIP14 protein, a member of the SLC39A metal ion transporter family, which was reported to be abundantly expressed in hepatocytes and involved in iron uptake [18,20]. TF TF, a major plasma protein of biological interest, is primarily synthesized in hepatocytes [67]. In vertebrates, TF plays a significant physiological role in the transport of iron among the sites of absorption, storage, and utilization [68]. Alterations in the 22

23 level of TF cause changes in iron distribution, delivery and storage and could be involved in diseases of iron metabolism [69]. For instance, hypotransferrinemia (blood TF level < 10 mg/dl ), a rare recessive disorder, is characterized by severe iron deficiency anemia, and iron overloading of the liver and other parenchymal organs [70]. TF functions by binding to and transporting ferric iron into cells through receptormediated endocytosis process, during which TF and its receptor are reutilized repeatedly for iron delivery while ferrous iron is released into cytoplasm. The storage and synthesis of TF are regulated by iron and estrogen levels, and also influenced by nutritional status [71]. In human, the serum TF concentrations decrease during iron overload and increase during chronic iron deficiency. TFR2 In 1999, Kawabata [72] reported the cloning of TFR2, a type of transmembrane protein which shared significant sequence similarity to TFR1. TFR2 displays a restricted expression pattern, with highest levels in hepatocytes. Unlike TFR1, TFR2 has no IRE elements and its protein level is not dependent on cellular iron content, but on diferric TF level. TFR2 also binds diferric TF as TFR1 does, specifically in a ph-dependent manner but with an affinity times lower than TFR1 [73,74]. The role of TFR2 in TF-mediated iron-uptake by the liver seems not so important. In fact, more occurring evidences indicate that TFR2 binds diferric TF and HFE to regulate hepcidin expression [75,76] and is more likely considered a sensor of TF saturation of the blood [11]. Although how TFR2 functions in the iron uptake is still not very clear, the critical role it plays in iron homeostasis is obvious: mutation of TFR2 causes a rare form of hereditary hemochromatosis [77] and mice with either germline or liver conditional inactivation of TFR2 also develop iron overload [78,79]. 23

24 Overall, the liver plays a central role in iron metabolism. Hepatocytes express a number of genes participating in iron homeostasis; functional loss of any of these genes results in the hereditary hemochromatosis. Through producing and secreting hepcidin, the master in regulating the iron uptake from enterocytes and iron release from macrophages, liver regulates the whole body iron homeostasis. The liver is also among the first organs damaged in iron-overload disorders such as hemochromatosis and transfusional hemosiderosis [35]. Thus, it is imperative to study the mechanism of liver function in iron mechanism, especially, how hepatocytes absorb iron from the environment and its regulation. Hepatic Iron Uptake Iron assimilation by liver hepatocytes is a highly versatile process, and pathways of iron transport through the hepatocytes membrane are more complex and diverse than in any other cell type. Hepatocytes can acquire non-heme iron from the circulation mainly in two forms: TBI at physiologic iron concentrations and non-transferrin-bound iron (NTBI, mainly in the form of ferric citrate) in iron overload conditions. TBI Uptake Since under physiological conditions the vast majority of iron circulating in the blood is bound to TF, diferric TF is the dominant source of iron for the liver. TFR1-mediated TBI uptake TFR1, also known as TFRC (Transferrin Receptor) or CD71 (Cluster of Differentiation 71), was identified as a specific receptor for circulating TF and responsible for cellular TBI uptake [80]. TFR1 is a transmembrane glycoprotein consisting of two identical subunits (~ 90 kda each) conjugated by disulfide bonds [81]. Each subunit contains an N-terminal 24

25 cytoplasmic domain required for inducing the receptor-mediated endocytosis, followed by a 28-residue transmembrane domain which serves as an anchor tethering the receptor to the plasma membrane. The C-terminal extracellular domain of the protein is important for TF binding and receptor dimerization [82]. TFR1 is capable of binding diferric TF at 2:2 ratios, with each subunit binding one molecule of TF and delivering 2 iron atoms to cells [83]. This TFR1-TF interaction is reversible depending on environmental ph and the iron occupation state of TF [84,85]. The affinity constant of TFR1 for diferric TF is 10 9, about 100 times higher than that for monoferric TF, and even higher than that for apo-tf [86]. The affinity of TFR1 for apo- TF increases at the ph of 5.5 (as in the endosome), while decreases at the ph of 7.4 (as in extracellular surface). For diferric TF, its affinity with TFR1 changes in reverse way. TFR1-mediated TBI uptake is regulated in several ways. First, TFR1 expression level is regulated both at a posttranscriptional level by intracellular iron and at a transcriptional level by iron and some other small molecules. Low intracellular iron levels increase while high iron levels decrease TFR1 mrna stability, hence regulating protein translation [87,88]. For example, in iron-deficient hepatocytes, TFR1 expression and subsequent TBI uptake is up-regulated [89]. In contrast, in iron overload disorders, such as HH, hepatic TFR1 was not detectable [90]. Small molecules, like nitric oxide and hydrogen peroxide generated by oxidative stress, cytokines, such as interleukin-2, mitogens and growth factors [91,92,93], and transcription factors, such as the hypoxiainducible factor and transforming-specific protein-1, all of which alter TFR1 transcription and influence TBI uptake by cells [53,94,95]. Second, the TBI uptake by TFR1 is 25

26 affected by the interaction between TFR1 and HFE. HFE associates with TFR1 at cell surface [66] and decreases iron uptake from TF probably through competing with TF for binding TFR1 at the same motif [96]. Third, TBI uptake by TFR1 is influenced by inflammation. Under chronic inflammation condition, serum iron decreases and hepatic iron accumulates because of reticuloendothelial system iron blockade [97]. In vivo stimulation of IL-6, an inflammation promoter, increases a rapid hepatic uptake of serum iron through TFR1 [98]. TFR2-mediated TBI uptake After TFR1, a second TFR has been identified and was named TFR2, which is highly homologous to TFR1 [72]. Like TFR1, TFR2 also localizes to both plasma membrane and endosome, and mediates TBI uptake through a receptor-mediated endocytic process [9]. Unlike the low expression level of TFR1, TFR2 is predominantly expressed in hepatocytes [72]. Its affinity to circulating TF is significantly lower (up to 30-fold) compared to TFR1, but its binding capacity is higher than TFR1 because of approximately five times higher expression level than TFR1 in liver [99,100]. Hepatocytes may acquire TBI via both TFR1-dependent and TFR1-independent pathways. Iron and TF uptake by the TFR1-independent pathway was approximately 100-fold greater than that by the TFR1 pathway. This high capacity TFR1-independent pathway is believed to be a low-affinity pathway, for which TFR2-mediated TBI uptake seems to fit well. During iron overload, TF saturation and diferric TF levels are increased, resulting in an up-regulation of TFR2 protein expression [10,101], suggesting that hepatocytes iron loading may involve TFR2 [102]. Although TFR2 may also mediate iron uptake from TF by hepatocytes, it may not be indispensible. Recent studies in hepatocytes and liver indicated that TBI uptake 26

27 correlated less with TFR2. TFR1 knockdown reduced iron uptake by 80% in human hepatoma cells whereas TFR2 knockdown did not affect uptake [15]. In mice fed irondeficient or iron-loaded diets, hepatic TFR2 mrna expression did not change [103]. Similarly, in HepG2 cells treated with an iron chelator iron-nitrilotriacetate (FeNTA), TFR2 mrna and protein levels did not show any difference [73]. Diferric TF upregulates hepatocyte TFR2, resulting in a small increase in TF but not iron uptake [102]. These data suggest that TFR2 may have a limited role in the TFR1-independent pathway. Instead of playing a vital role in iron transport, TFR2 appears to more likely function as a diferric TF sensor in the regulation of cellular and whole body iron metabolism through hepcidin. Hepcidin is synthesized by hepatocytes and is the key regulator of iron homeostasis. TFR2 is also predominantly expressed in hepatocytes and has recently been shown to interact with HFE, another highly expressed protein by hepatocytes. It has been proposed that the competing binding of diferric TF with HFE to the TFR1 or TFR2 regulates hepcidin synthesis [104]. Mutation in either TFR2 or HFE results in low hepcidin levels [105]. TFR1/2-independent TBI uptake Since the high-affinity TFR1-dependent TF-bound iron uptake becomes saturated at relatively low extracellular TF concentrations (50 to 100 nm) [106], and TFR2- dependent pathway has a limited role in the TBI uptake, there must be some other TFR1/TFR2-independent high-capacity pathway exists in the hepatocytes and predominates at normal physiological level of plasma TF (25 to 50 μm) [107]. The most compelling evidence is that in TFR2 knockout mice, the iron-loaded liver can still avidly take up iron from TF when TFR1 is markedly downregulated [14]. One possible explanation for the TFR1/2-independent TBI uptake is the existence of another receptor 27

28 for TF at the cell membrane. In macrophage cells and CHO-TRVb cells, a new TFR but an old protein GAPDH was identified to have the ability bind diferric TF and mediate iron uptake into cells [108,109]. It is possible that GAPDH or another protein may function similarly in hepatocytes. A second explanation for the TFR1/2-independent TBI uptake is via fluid-phase endocytosis, a low efficiency process taking up iron from the fluid phase of the endocytic vesicles in a non-specific manner [110,111,112]. However, the rate of fluidphase endocytosis reported for hepatocytes is only sufficient to account for less than 20% of observed uptake of TBI [113]. Another possible way is that iron is released from TF at the cell surface and then transported across the cell membrane by an iron transporter. Thorstensen and Romslo [114] proposed a reductive release model for the uptake of iron from TF at the cell surface of hepatocytes based on their and other s experimental data. According to this model, TF still binds to its receptor as in the receptor-mediated endocytosis model. But the concerted action of protons and reducing equivalents furnished by the NADH: ferricyanide oxidoreductase in close proximity to the TFR destabilizes the TF-iron bond and reduces ferric iron to ferrous iron. The ferrous iron is then bound by a membrane carrier and translocated across the membrane to the cytosolic side where it is picked up by cytosolic iron acceptors (ferritin or heme) [115]. NTBI Uptake in the Liver Since free iron is quite toxic to cells, under normal conditions the vast majority of iron in the plasma is bound to TF. However, there is still a very small proportion of iron present as NTBI [116]. In various iron-loading syndromes, such as HFE-associated hemochromatosis, the amount of free iron in the plasma can increase to even higher 28

29 since the capacity of TF to sequester it is exceeded [117]. Specifically, TF is only 20-50% saturated with iron under normal conditions, but in iron overload conditions plasma TF can become fully saturated, giving rise to NTBI [118,119]. NTBI can exist at least in two forms. The major fraction of NTBI in the blood is in the form of ferric citrate [120], but smaller amounts could be bound loosely to proteins such as albumin. NTBI can be taken up very efficiently by the liver, mainly by hepatocytes through first-pass extraction [121]. This is well demonstrated in humans with congenital TF deficiency (atransferrinemia). Despite lacking TF, those patients can still absorb iron from their diet very efficiently and large amounts are deposited in the liver [122]. In mice, after given sufficient intravenous iron to saturate their circulating TF, and a subsequent dose of radioactive NTBI orally or intravenously, over 70% of the radioactive iron finally ended up in the liver [123]. Studies in isolated perfused livers have also shown very efficient extraction of NTBI [50]. The molecular basis of NTBI uptake has not been fully resolved. DMT1, the only known iron transporter is reported to be involved in NTBI uptake into cells. However, Dmt1 knockout mice can still accumulate iron in the liver even under the anemia condition, indicating that DMT1 is not the major iron transporter in the liver [124]. ZIP14 (SLC39A14, solute carrier 39 family, member 14) is another candidate. How NTBI is taken up by the liver hepatocytes remains unclear. The liver can also take up iron from circulating heme, hemoglobin, ferritin and other iron-binding molecules [53]. (1) Iron-bound ferritin binds to the specific receptor of ferritin at the cell surface and is endocytosed. Iron is delivered to the transit cytosol pool or transferred to endogenous ferritin. (2) The iron-heme-hemopexin binds to a specific 29

30 receptor, CD91, at the cell surface and is endocytosed. (3) The hemoglobin-haptoglobin complex binds to the receptor of haptoglobin at the cell surface and delivers the iron through endocytosis, a process during which the complex is targeted to the lysosomes for degradation or released into bile. (4) Lactoferrin-bound iron binds to the cellular membrane receptor of lactoferrin and is internalized into the cell. The amount of iron taken up by these other mechanisms appears to be very low in normal conditions. But when hemolysis occurs or ferritin releases from damaged tissues, the liver can play an important role in removing these molecules from the circulation. The Role of Endocytosis in TBI Uptake into Hepatocytes Although hepatocytes receive their iron primarily from TF, most aspects of the interaction of TF with hepatocytes, the subsequent release of iron from TF and the assimilation of iron into hepatocytes remain obscure. The role of endocytosis in TBI uptake by hepatocytes will be discussed here. Endocytosis-Dependent TBI Uptake TBI may be taken up by different mechanisms in hepatocytes, but receptormediated endocytosis is the most commonly described model in hepatocytes until now. Both TFR1 and TFR2-dependent TF-bound iron uptake mentioned above belong to this model. TFR1/2-mediated endocytosis Hepatocytes obtain iron from TF via interacting with its cell surface receptor, TFR1/2, which induce the subsequent formation of membrane invagination and initiate the internalization (endocytosis) of the receptor-tf-iron complex, followed by release and translocation of iron into cytoplasm. 30

31 As for this model, the process of TFR-mediated endocytosis can be distinguished into 6 main steps: binding, internalization (endocytosis), acidification, dissociation and reduction, translocation, and cytosolic transfer of iron into intracellular compounds such as ferritin or heme [125]. The binding of TF to the extracellular domain of TFR occurs at the cell surface, which initiates the uptake of TF-iron process [126]. After the binding, a signal is sent through the membrane, leading to membrane coating, and formation of a membrane invagination. The receptor and TF-iron complex are then clustered together and opsonized in clathrin-coated vesicles, which finally form endocytic vesicles termed endosomes. Through the action of an ATP-dependent proton pump in the endosomal membrane, an acid environment is generated at the luminal side of the endosome [127,128,129,130,131]. The low ph facilitates iron release from TF and the ferric iron is reduced to ferrous iron by Steap3 as in erythroid cells [132]. After that, ferrous iron is exported from the endosomal vesicle by DMT1 [124] and enters the cytosol labile iron pool. Under the low ph value of endosome, the apo-tf binds tightly to the TFR and is sorted together into exocytic vesicles followed by recycling to the plasma membrane. Under the extracellular ph, apo-tf dissociates with TFR and will be utilized for another cycle of TF-iron uptake by cells [133]. Knockdown of Steap3 or DMT1 in liver does not inhibit TBI uptake by hepatocytes, indicating hepatocytes use different reductase and transporter for TBI assimilation compared to erythroid cells [134,135]. As discussed above, although TFR1/2-dependent endocytosis may play a role to some extent in mediating hepatocytes TBI uptake, it is probably not the primary pathway under physiological conditions. 31

32 Fluid-phase endocytosis (pinocytosis) Fluid-phase endocytosis is also believed to be involved in the nonspecific uptake of iron by hepatocytes, probably as a minor mechanism. Fluid-phase endocytosis is a form of endocytosis in which small particles are brought into the cell suspended within small vesicles which subsequently fuse with lysosomes to break down the particles. This process requires a lot of energy. Unlike receptor-mediated endocytosis, fluid-phase endocytosis is nonspecific in the substances that it transports. The cell takes in surrounding fluids, including all solutes present [136]. As a result, it is possible that hepatocytes can use this pathway for nonspecific, high capacity TBI uptake. In fact, a computer simulation performed by Bakøy et al. [137], based on models of receptormediated endocytosis and fluid-phase endocytosis, showed that the differences in iron uptake processes observed between reticulocytes and hepatocytes may be explained by fluid-phase endocytosis of iron-tf complex in hepatocytes, corroborating the existence of fluid-phase endocytosis of iron-tf indirectly. However, Page et al [110] determined the rate of fluid-phase endocytosis of adult rat hepatocytes in culture from the cellular uptake of 125 I-polyvinylpyrrolidone during the h period after commencement of culture. TF uptake at this rate could account for only about 5% of the measured rate of iron uptake by cells cultured in the presence of diferric TF at this concentration. Other studies also showed that fluid-phase endocytosis accounts for less than 20% of the observed TBI uptake in hepatocytes [138,139]. Whether fluid-phase endocytosis play a key role in the TF-bound iron uptake by hepatocytes remains obscure. 32

33 Endocytosis-Independent Pathway After its introduction in 1963, the hypothesis that diferric TF binds to the TFRs on the cell membrane and is endocytosed into the cell was widely accepted. However, a variety of observations led us to suspect that the TF molecule may actually enter the cell during the process of iron donation and many studies have even shown directly that endocytosis is not required during TBI uptake by hepatocytes or the liver. This endocytosis-independent mechanism probably contributes most of the iron taken up by hepatocytes since plasma TF concentration is several orders of magnitude greater than that needed to saturate TFR1 [95,106,138]. TFR1 and TFR2 are the only two TFRs known to exist in hepatocytes. The TBI uptake processes mediated by both of them are endocytosis-involved. Experiment done by Herbison et al. [107] showed that knockdown of both TFR1 and TFR2 with sirna in hepatoma cells inhibited iron uptake from TF at low diferric TF concentration (50 nm), but had little effect on iron uptake at higher diferric-tf concentration (5 µm), suggesting that TFR1/2-dependent endocytosis is not required for TF-bound iron uptake. In vivo study in iron-loaded TFR2 mutant mice, with TFR1 level in hepatocytes being very low due to the overload condition in liver, liver hepatocytes can still take up iron from TF avidly. This result also suggests the presence of another route of TBI uptake in hepatocytes that is independent of TFR1 and TFR2-mediated endocytosis processes [14]. Isolated hepatocyte membranes from the rat liver was reported to being able to release iron from TF, and Fe-TF seems to not use internalization as a major way during iron uptake by hepatocytes [140]. 33

34 Hepatic iron uptake was also studied by subcellular fractionation of rat liver homogenates after injection of rats with either native or denatured rat TF labeled with l25 I and 59 Fe. Although labeled iron from denatured TF could be detected in the lysosomal fraction, iron from native TF did not appear in either the mitochondrial or lysosomal fractions. The study suggests that hepatic iron uptake from native TF does not involve endocytosis [13,141]. The most compelling evidence for the cell surface uptake of iron from TF by hepatocytes came from a study done by Thorstensen [12]. In this study, the uptake of iron from TF by isolated rat hepatocytes and rat reticulocytes were compared. The perturbation of endocytosis and endosomal acidification had no inhibitory effect on hepatocyte iron uptake, but inhibited reticulocyte iron uptake from TF, the cell in which TFR-dependent endocytosis is known to be actively involved. Reductive iron release at the cell surface Several models proposed that iron released from TF at the cell surface was reduced by some oxidoreductase (iron reduced from ferric to ferrous state) before its uptake by the cells. Crane et al [142] proposed that a plasma membrane electron transport enzyme that is found on nearly all cells could act as such a diferric TF complex reductase. The existence of a NADH diferric transferrin reductase on the plasma membrane was reported by the further work [143]. It was proposed that plasma membrane NADH: ferricyanide oxidoreductase activity is approximately 60-fold higher in the hepatocyte than in the reticulocyte. This reductive process was also believed to be accompanied by proton release from the cell and this accompanying proton flux could assist in the removal of iron from TF, allowing reduction to occur at a lower negative potential [144]. 34

35 Evidence for this reductive iron release at the cell surface was also provided by determining the effect of iron chelators on iron uptake. Ferrous iron chelators that cannot pass through the cell membrane have been reported to inhibit the uptake of iron from TF by hepatocytes [12], suggesting that the iron is reduced to its ferrous form outside the cell. Second, the released iron from TF is transported across the cell membrane through an iron transporter which seems to be shared by NTBI. Specifically, the TBI uptake by hepatocyte and hepatoma cell is inhibited by the uptake of NTBI [145]. Conversely, the uptake of NTBI in livers is reduced by the TBI [146]. This indicates that iron from TBI and NTBI is taken up by a common pathway. However, which iron transporter are involved remains unknown. In summary, these studies above proved the involvement of an endocytosisindependent, cell surface iron release and reduction mechanism during hepatocyte uptake of iron from TF and described the importance of its contribution to the iron uptake under physiological condition, comparing with the predominance of receptormediated endocytosis in reticulocyte. Iron Transporters in Hepatocytes DMT1 DMT1, also known as DCT1 (divalent cation transporter 1) and Nramp2 (natural resistance-associated macrophage protein 2), is a transmembrane protein encoded by the SLC11A2 (solute carrier family 11, member 2) gene, which is widely distributed in body tissues [147]. As its name suggests, DMT1 transports divalent metals, such as cadmium (Cd 2+ ), manganese (Mn 2+ ) and copper (Cu 2+ ), and is best known as ferrous iron (Fe 2+ ) transporter. It is the first found and best-described ferrous iron 35

36 transporter in the body. It majorly functions to transport iron across the brush border membrane of intestinal epithelial cells, but also actively participates in the delivery of TF-derived iron from the endosome to the cytosol in erythroid cells [148,149]. The role of DMT1 in the iron uptake by liver is unclear. Unlike its high expression in duodenum, it is expressed at low to moderate amounts in liver [150]. Also, in the duodenum, DMT1 expression is increased significantly under iron-deficient conditions, but surprisingly the opposite effect is seen in the liver and hepatoma cells where DMT1 levels are enhanced by iron loading [151,152]. A recent study by our lab [135] showed that hepatocyte-specific Dmt1 knockout mice (Dmt1liv/liv) had normal hepatic non-heme iron concentrations and took up NTBI very efficiently. These data indicate that DMT1 is not required for hepatic iron accumulation under normal or iron overload conditions. Dmt1-/- globally-knockout mice exhibit growth retardation and are notably pale at birth. Their blood smear shows anisocytosis and poikilocytosis, both of which are the signs of severe iron deficiency anemia. However, the livers of these mice are iron overloaded. The fact that iron continues to accumulate in their Dmt1-/- liver after iron dextran injection suggests there is other iron transporter present in the liver [124]. A similar result was found in human patients affected with DMT1 mutation [153]. Three patients with this recessively inherited condition displayed severe microcytic anemia at birth. Serum markers (high serum iron, normal total iron-binding capacity (TIBC), increased saturation of TF, slightly elevated ferritin, and increased soluble transferrin receptor (stfr)) all indicated the iron-deficient anemia. However, liver iron 36

37 overload was present in all of the affected patients, indicating that hepatocytes have an alternative DMT1-independent iron uptake pathway. ZIP14 and Iron Metabolism Identification and characterization of ZIP14 ZIP14, or ZRT/IRT (zinc regulated transporter/ iron regulated transporter) -like Protein 14 is one of the members of ZIP metal-ion transporter superfamily (SLC39A, solute carrier 39 family) [154]. It is the second identified iron-import protein to date and was reported to transport both zinc and iron [18], but other metals, like manganese and cadmium as well [155,156]. ZIP14 has a ubiquitously tissue expression pattern, with highest expression in the liver, moderate expression in the pancreas and heart [17], and lowest expression in the spleen, thymus, and peripheral blood leukocytes. Human ZIP14 protein has a predicted molecular weight of around 53 kda with 8 putative transmembrane domains and may function as a dimer or trimer [17,157]. The metal transport activity of ZIP14 was first characterized in Originally, Taylor et al. [17] showed that ZIP14 overexpression in CHO cells stimulated the uptake of zinc into the cytosol. Liuzzi et al. [158] followed by finding that transfection of mouse ZIP14 cdna into HEK 293 cells could increase zinc uptake and that ZIP14 was the most upregulated zinc transporter in response to lipopolysaccharide (LPS) treatment or turpentine-induced inflammation in the mouse. Immunohistochemical analysis showed that, in hepatocytes, plasma membrane expression of ZIP14 increased in response to both LPS and turpentine, suggesting ZIP14 plays a major role in the hypozincemia accompanying the acute-phase response to inflammation and infection. Mice lacking ZIP14 (SLC39A14 KO mice) exhibit growth retardation and impaired gluconeogenesis, 37

38 indicating that ZIP14 plays an important role in body development and glucose metabolism [159]. Recent studies by Pinilla-Tenas et al. [160] and Fujishiro et al. [155] showed that ZIP14 not only transports iron and zinc, but also mediates the uptake of Cd 2+ and Mn 2+, revealing that ZIP14 is a complex, broad-scope metal-ion transporter. ZIP14 and cellular iron uptake In 2006, Liuzzi et al. [18] reported that overexpression of ZIP14 enhanced not only the uptake of zinc, but also iron in the form of ferric citrate, the major form of NTBI present in plasma during iron overload. In addition, suppression of endogenous ZIP14 expression with sirna in AML12 cells resulted in reduced uptake of NTBI from ferric citrate. Gao et al. [20] showed that ZIP14 overexpression stimulated NTBI uptake in HeLa cells, and that suppression of endogenous ZIP14 in the human hepatoma cell line, HepG2, decreased NTBI uptake. In iron-loading disorders, such as hemochromatosis or dietary iron overload, excess iron is mainly found in the liver, pancreas and heart [27]. Notably, ZIP14 is abundantly expressed in the liver, heart, and pancreas [17,161], organs where excess iron is stored, suggesting that ZIP14 may play a role in NTBI uptake [160]. ZIP14 localizes to the plasma membrane and can optimally transport iron at ph 7.4 [18], the ph at the plasma membrane surface of hepatocytes, whereas DMT1 efficiently transports iron at ph 5.5 [162] and is located to endosomes but not on the plasma membrane of hepatocytes [163]. These observations suggest that ZIP14, rather than DMT1, may mediate NTBI uptake into hepatocytes, probably at the cell surface. HFE-associated HH is the most prevalent form of hemochromatosis and is one of the most common inherited human disorders [164]. In this disease, HFE is known to be 38

39 involved in iron metabolism and important for regulating iron homeostasis probably through switching its binding between TFR1 and TFR2 and controlling hepcidin synthesis by hepatocytes. Mutation of a single amino acid (C282Y) in the HFE gene causes iron overload [165]. A recent study showed that overexpression of HFE in HepG2 cells by destabilizing ZIP14 at protein level [20]. The reduced ZIP14 levels were associated with decreased uptake of not only NTBI but also iron from TF, suggesting that ZIP14 may participate in both NTBI and TBI uptake by the hepatocytes. The role of ZIP14 in iron uptake from TF in hepatocytes has other evidences to support it. Isolated rat hepatocytes have been shown to be able to take up more TBI after stimulation of LPS [166]. Similarly in HepG2 cells, stimulation with the inflammatory cytokine, IL-6, enhanced the uptake of TBI [167]. Both LPS and IL-6 stimulation have been shown to potentially increase levels of ZIP14 in mouse liver and in isolated hepatocytes [158], which suggest that ZIP14 may mediate TBI uptake under this physiological condition. A recent study by our lab also suggested that ZIP14 may take up iron from TF in hepatocytes [19]. Specifically, overexpression of ZIP14 in HEK 293T cells increased the assimilation of iron from TF without increasing levels of TFR1 or the uptake of TF. Suppressing endogenous ZIP14 by sirna in HepG2 cells decreased iron uptake from TF by 50%compared with control cells. This result is consistent with the previous finding that downregulation of ZIP14 was associated with reduced uptake and assimilation of iron from TF [20]. The interesting finding that ZIP14 partially colocalizes with TF shown by immunofluorescence suggested these two proteins could potentially interact with each other, and ZIP14 could play a role in TF-bound iron uptake by hepatocytes [19]. 39

40 Specific Aims Aim 1. To Test the Hypothesis that Endocytosis Is not Required for TBI Uptake in HepG2 Cells. I hypothesize that hepatocytes can take up iron from TF without the involvement of TFR mediated endocytosis. By transfecting specific sirna targeting clathrin heavy chain (CHC), I will abolish the TFR mediated endocytosis and detect its effect on iron uptake from TF by HepG2 cells. Aim 2. To Test the Hypothesis that TF Interacts with ZIP14. I postulate that TF interacts with ZIP14 in HepG2 cells. To test this hypothesis, here we will take advantage of two novel techniques: DuoLink in situ assay, which is used for detecting protein-protein interactions and far-western blotting, which is a technology used to detect direct protein-protein binding. 40

41 CHAPTER 3 MATERIALS AND METHODS Knockdown of Clathrin and TFR1/2 Using RNAi Small interfering RNA template: 5 -AATGAACCTGCGCTCTGGAGT-3 specific for human clathrin heavy chain mrna (GenBank accession number NM_004859), was purchased from Eurofins MWG Operon. Validated sirna specifically targeting human TFR1 and TFR2 mrna (Catalog #: and ) and negative control sirna with a sequence that does not target any known gene product (Catalog #: AM4611) were purchased from Ambion. Lipofectamine RNAiMAX transfection reagent (Invitrogen) was used to transfect sirna into HepG2 cells. Briefly, 6 µl of Lipofectamine RNAiMAX and 60 pmol of RNAi duplex were mixed in 600 µl of Opti-MEM (Invitrogen) and added into each well of a 6-well plate. After incubating at room temperature for 15 min, 2 x10 5 cells were added to each well containing 1.8 ml of DMEM supplemented with 4.5 g/liter glucose, 4 mm L-glutamine, 1 mm sodium pyruvate, 1x minimum Eagle s medium nonessential amino acids and 10% FBS, without antibiotics. Cells were incubated with the sirna for 72 h. To confirm knockdown, clathrin heavy chain levels were determined by western blotting. Measurement of TF Uptake and Assimilation of Iron from TF 59 Fe-TF was prepared by saturating human apo-tf (Sigma) with 59 Fe-ferric nitrilotriacetic acid. After incubating for 1 h in PBS containing 10 mm NaHCO 3, unbound iron was removed by repeated washing through a Centricon centrifugal filter (MWCO 30,000; Millipore). Cells were washed twice with serum-free DMEM medium (SFM) and incubated at 37 C for 60 min to deplete TF from cells. For TF uptake experiment, cells were then incubated in SFM containing 10 nm biotin-labeled holo-tf (Sigma) for 10 min 41

42 (CHC knockdown experiment) or 50 nm and 5 µm biotin-conjugated TF for 1 h (TFR1/2 knockdown experiment). For iron assimilation experiment, cells were incubated with SFM containing 10 nm 59 Fe-TF, 20 mm Hepes (ph 7.4) and 2 mg/ml ovalbumin. To stop uptake, cells were placed on ice, and externally bound TF was stripped with an acidic buffer (0.2 N acetic acid, 500 mm NaCl, 1 mm FeCl 3 ) for 3 min. After washing suspended cells twice in cold SFM, cells were lysed in a solution containing 0.2% SDS and 0.2 M NaOH. Cell-associated radioactivity was determined by γ-counting. Protein concentrations of the cell extracts were measured by using the RC-DC Protein Assay (Bio-Rad). TF uptake levels were determined by western blotting and probing for TF. Assimilation of 59 Fe was expressed cpm to mg protein. DuoLink In Situ Proximity Ligation Assay To detect a possible interaction between ZIP14 and TF, I used the DuoLink in situ Proximity Ligation Assay (PLA) (Olink Bioscience, Uppsala, Sweden) according to the manufacturer s protocol. HepG2 cells, grown in culture dishes (MatTek), were incubated with 30 µg/ml holo-tf (Sigma) for 30 min at 37 C and washed three times with PBS before fixation with 2% paraformaldehyde (PFA) freshly prepared in PBS. After 15 min incubation, fixation buffer was aspirated and 50 mm NH 4 Cl was then added to cells to quench remnant PFA followed by 2 times PBS washing. After 10 min incubation with 1% Triton X-100 solution in PBS and further 30 min blocking in the blocking buffer (supplied in the kit) at room temperature, cells were immunolabeled with primary antibodies (M2 against FLAG tag was bought from Invitrogen and TF antibody from Stressgen) for 2 h at 37 C. The secondary antibodies conjugated with oligonucleotides (PLA probe MINUS and PLA probe PLUS) were added and a Ligation solution was used to hybridize the two PLA probes. The Amplification buffer was then added to 42

43 fluorescently label the PLA probes and amplify the signals. The glass coverslip with cells on it was then mounted to slide with mounting medium with DAPI. Red fluorescence signal was then observed by Olympus DSU-IX81 Spinning Disc Confocal microscopy at 60x magnification, with excitation and emission peaks at 598 nm and 634 nm. A fluorescence signal indicates that two proteins within cells are located within 40 nm to each other ( Far-Western Blotting Analyses To purify ZIP14 proteins, both wild-type and FLAG tag knock-in HepG2 cells were lysed with immunoprecipitation (IP) lysis buffer (Pierce). Lysates were precleared with mouse IgG agarose and the supernatants were incubated with 50 µl M2-conjugated agarose (Sigma) to bind ZIP14-FLAG. Proteins were eluted with 100 µg/ml FLAG Peptide (Sigma) and separated with 7% SDS-PAGE. After protein transfer, membranes were blocked with 5% non-fat milk blocking buffer and incubated with 30 µg/ml of TFbiotin or BSA-biotin overnight at 4 C. Membrane was then washed completely with TBST and probed with streptavidin for 2 h at room temperature. The membranes were rewashed three times with TBST, followed by incubation with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific). Signals were detected using FluoChem E System (Cell Bioscience). Co-Immunoprecipitation Assay FLAG-tagged HepG2 cell extracts were prepared by lysing approximately cells in IP lysis buffer (Peirce) on ice for 15 min and centrifuging at 13,000 g for 30 min at 4 C. The supernatant was collected and precleared with protein A- conjugated agarose (for pulldown of endogenous TF) or empty agarose (for the pulldown of endogenous ZIP14-FLAG). For the pulldown of endogenous TF, 15 µg 43

44 rabbit derived against human TF or nonspecific IgG antibody were incubated with 100 µl protein A-conjugated beads together with 500 µl NETT buffer (protease inhibitor included) for 3 h and centrifuged to pellet the beads. After 3 washes with NETT buffer, beads were collected and incubated with each half of the precleared supernatant overnight at 4 C. For the pulldown of endogenous ZIP14-FLAG, 50 µl mouse M2- conjugated agarose or non-specific IgG-conjugated agarose were incubated with each half of the precleared supernatant for 30 min at 4 C. After sufficient washes with IP lysis buffer, beads were collected and incubated (for pulldown of ZIP14-FLAG, proteins were eluted with FLAG Peptides first) with equivalent amount of 2x protein sample buffer at 37 C and followed by gel electrophoresis analysis. TF and ZIP14 were detected by probing the membrane with anti-tf or M2 antibody. Statistical Analysis Data represent mean ± SE and were analyzed by unpaired Student s t test (GraphPad Prism, GraphPad Software). A probability level of p < 0.05 was defined as a significant difference. 44

45 CHAPTER 4 RESULTS Inhibiting Clathrin-Mediated Endocytosis in HepG2 cells Does not Affect Iron Uptake from TF Clathrin-mediated endocytosis is the major pathway for uptake of Fe(III)-TF-TFR complex by reticulocytes [168]. To test whether this is the case in hepatocytes, I inhibited endocytosis by transfecting cells with clathrin heavy chain-specific sirna, which was previously shown to be effective in knocking down clathrin and abolishing endocytosis in HepG2 cells [169]. Here, TF uptake level decreased by nearly 70% after clathrin knockdown in my experiment (Figure 4-1 A). To detect the effect of clathrin knockdown on TBI uptake, HepG2 cells were incubated for 1 h with 59 Fe-TF. Iron uptake level was determined by measuring cell-associated radioactivity. TBI uptake Figure 4-1. Clathrin heavy chain (CHC) knockdown with sirna decreases uptake of TF, but does not affect uptake of iron from TF. (A) To inhibit endocytosis, HepG2 cells were incubated with CHC-specific sirna for 72 h. CHC protein levels and TF uptake after knockdown were detected by western blot and normalized to tubulin. For negative control, nonspecific sirna was used. (B) HepG2 cells transfected with negative control or CHC sirna were incubated with 10 nm 59 Fe for 1 h. Cells were harvested, and cell-associated radioactivity was determined by γ-counting. The amount of 59 Fe assimilated by the cells is expressed as cpm/mg protein. Data represent the mean ±S.E. (error bars) of three independent experiments. 45

46 level did not change significantly after clathrin heavy chain knockdown compared with negative control, indicating that clathrin-mediated endocytosis is dispensable for TBI uptake by hepatocytes (Figure 4-1 B). The Effects of Knocking down TFR1/2 on TF Uptake Are Different at Low and High TF Levels It is known that the binding of diferric TF to TFR1 or TFR2 at the cell surface induces the subsequent formation of membrane invagination and initiate the endocytosis of the receptor-tf-iron complex, followed by release and translocation of iron into cytoplasm [72,170]. According to this model, TFR1 and TFR2 may play a role in this endocytosis process through which uptake of TF and assimilation of iron from TF by cells are accomplished. To investigate this in hepatocytes, I inhibited TFR1 and TFR2 expression by transfecting cells with sirnas specific to TFR1 and TFR2. To Figure 4-2 TFR1 and TFR2 knockdown with sirna decreases uptake of TF at 50 nm level. HepG2 cells were incubated with control sirna or TFR1 and TFR2- specific sirna for 72 h. Cells were then incubated for 1 h with 50 nm of biotinylated TF. After stopping uptake, cells were washed to remove unbound TF, and cell lysates were obtained. (A) Western blot analysis was used to determined levels of TFR1, TFR2, TF, and actin as lane-loading control. (B) Densitometric analysis of TF levels of cells treated with control or TFRs sirna. Data (mean ± SE, n=3) are representative of two independent experiments with similar results. 46

47 detect the effect of knocking down TFRs on TF uptake, cells were then incubated with 50 nm or 5 µm biotin-labeled holo-tf for 1 h and TF uptake levels were determined with western blotting. As shown in Figure 4-2, TF uptake by cells incubated with 50 nm TF decreased by ~ 80% after knocking down TFRs compared to negative control, which indicated that TFR1/2 are necessary for TF uptake at this TF concentration. By contrast, results obtained from using 5 µm TF (Figure 4-3) were opposite from those with 5 nm. After knockdown of TFRs, TF uptake by cells increased by ~ 60% compared to negative control. This difference in TF uptake may reflect the versatile mechanisms of TF uptake, probably also iron uptake from TF, by hepatocytes under different conditions. TFR1/2-mediated endocytosis may not be necessary for TF uptake at least at high TF concentration. Since iron is bound to TF in this case, iron assimilation by hepatocytes is probably also independent of TFR1/2-mediated endocytosis. Figure 4-3 TFR1 and TFR2 knockdown with sirna increases uptake of TF at 5 µm level. HepG2 cells were incubated with control sirna or TFR1 and TFR2- specific sirna for 72 h. Cells were then incubated for 1 h with 5 µm of biotinylated TF. After stopping uptake, cells were washed to remove unbound TF, and cell lysates were obtained. (A) Western blot analysis was used to determined levels of TFR1, TFR2, TF, and actin as lane-loading control. (B) Densitometric analysis of TF levels of cells treated with control or TFRs sirna. Data (mean ± SE, n=3) are representative of two independent experiments with similar results. 47

48 ZIP14 Can Interact with TF both In Vitro and In Vivo Since endocytosis is not necessary for TBI uptake by hepatocytes, iron may be taken up directly at the cell surface, perhaps using a receptor other than TFR1/TFR2. Interestingly, alternate receptor proteins for TF have been described both for erythrocyte precursors [55] and macrophage cells [108]. As a cell surface transmembrane protein, it is possible that ZIP14 could serve as a receptor for TBI in hepatocytes. To investigate the interaction between ZIP14 and TF, I first used a DuoLink in situ proximity ligation assay to detect in vivo fluorescence signal that indicates that two proteins are localized within 40 nm of each other. The Duolink assay detects endogenous protein interactions, e.g. ligand binding of membrane proteins and Figure 4-4. ZIP14 can interact with TF in vivo in FLAG-tagged HepG2 cells. To confirm the interaction between ZIP14 and TF, both ZIP14 FLAG-tagged HepG2 cells (A) and wild-type HepG2 cells (B) were depleted in serum-free DMEM for 1 h and incubated with 30 µg/ml holo-tf for 30 min before being probed with FLAG and TF antibodies. DuoLink in situ proximity ligation assay was carried out subsequently to detect the signals which indicate interaction between two proteins. intracellular proteins, through a pair of antibodies that bind to proteins in close proximity. Individual protein interactions are made visible through DNA amplification. The level of 48