Matriptase-2 regulates iron homeostasis primarily by setting the basal levels of hepatic hepcidin expression through a nonproteolytic mechanism.

Matriptase-2 (MT2), encoded by TMPRSS6, is a membrane-anchored serine protease. It plays a key role in iron homeostasis by suppressing the iron-regulatory hormone, hepcidin. Lack of functional MT2 results in an inappropriately high hepcidin and iron-refractory iron-deficiency anemia. Mt2 cleaves multiple components of the hepcidin-induction pathway in vitro. It is inhibited by the membrane-anchored serine protease inhibitor, Hai-2. Earlier in vivo studies show that Mt2 can suppress hepcidin expression independently of its proteolytic activity. In this study, our data indicate that hepatic Mt2 was a limiting factor in suppressing hepcidin. Studies in Tmprss6-/- mice revealed that increases in dietary iron to ∼0.5% were sufficient to overcome the high hepcidin barrier and to correct iron-deficiency anemia. Interestingly, the increased iron in Tmprss6-/- mice was able to further upregulate hepcidin expression to a similar magnitude as in wild-type mice. These results suggest that a lack of Mt2 does not impact the iron induction of hepcidin. Additional studies of wild-type Mt2 and the proteolytic-dead form, fMt2S762A, indicated that the function of Mt2 is to lower the basal levels of hepcidin expression in a manner that primarily relies on its nonproteolytic role. This idea is supported by the studies in mice with the hepatocyte-specific ablation of Hai-2, which showed a marginal impact on iron homeostasis and no significant effects on iron regulation of hepcidin. Together, these observations suggest that the function of Mt2 is to set the basal levels of hepcidin expression and that this process is primarily accomplished through a nonproteolytic mechanism.

Hepatocyte neogenin is required for hemojuvelin-mediated hepcidin expression and iron homeostasis in mice.

Neogenin (NEO1) is a ubiquitously expressed multifunctional transmembrane protein. It interacts with hemojuvelin (HJV), a BMP coreceptor that plays a pivotal role in hepatic hepcidin expression. Earlier studies suggest that the function of HJV relies on its interaction with NEO1. However, the role of NEO1 in iron homeostasis remains controversial because of the lack of an appropriate animal model. Here, we generated a hepatocyte-specific Neo1 knockout (Neo1fl/fl;Alb-Cre+) mouse model that circumvented the developmental and lethality issues of the global Neo1 mutant. Results show that ablation of hepatocyte Neo1 decreased hepcidin expression and caused iron overload. This iron overload did not result from altered iron utilization by erythropoiesis. Replacement studies revealed that expression of the Neo1L1046E mutant that does not interact with Hjv, was unable to correct the decreased hepcidin expression and high serum iron in Neo1fl/fl;Alb-Cre+ mice. In Hjv-/- mice, expression of HjvA183R mutant that has reduced interaction with Neo1, also displayed a blunted induction of hepcidin expression. These observations indicate that Neo1-Hjv interaction is essential for hepcidin expression. Further analyses suggest that the Hjv binding triggered the cleavage of the Neo1 cytoplasmic domain by a protease, which resulted in accumulation of truncated Neo1 on the plasma membrane. Additional studies did not support that Neo1 functions by inhibiting Hjv shedding as previously proposed. Together, our data favor a model in which Neo1 interaction with Hjv leads to accumulation of cleaved Neo1 on the plasma membrane, where Neo1 acts as a scaffold to induce the Bmp signaling and hepcidin expression.

The ectodomain of matriptase-2 plays an important nonproteolytic role in suppressing hepcidin expression in mice.

Matriptase-2 (MT2), encoded by TMPRSS6, is a membrane-anchored serine protease that plays a key role in suppressing hepatic hepcidin expression. MT2 is synthesized as a zymogen and undergoes autocleavage for activation. Previous studies suggest that MT2 suppresses hepcidin by cleaving hemojuvelin and other components of the bone morphogenetic protein-signaling pathway. However, the underlying mechanism is still debatable. Here we dissected the contributions of the nonproteolytic and proteolytic activities of Mt2 by taking advantage of Mt2 mutants and Tmprss6-/- mice. Studies of the protease-dead full-length Mt2 (Mt2S762A) and the truncated Mt2 that lacks the catalytic domain (Mt2mask) indicate that the catalytic domain, but not its proteolytic activity, was required for Mt2 to suppress hepcidin expression. This process was likely accomplished by the binding of Mt2 ectodomain to Hjv and Hfe. We found that Mt2 specifically cleaved the key components of the hepcidin-induction pathway, including Hjv, Alk3, ActRIIA, and Hfe, when overexpressed in hepatoma cells. Nevertheless, studies of a murine iron-refractory iron-deficiency anemia-causing mutant (Mt2I286F) in the complement protein subcomponents C1r/C1s, urchin embryonic growth factor, and bone morphogenetic protein 1 domain indicate that Mt2I286F can be activated, but it exhibited a largely compromised ability to suppress hepcidin expression. Coimmunoprecipitation analysis revealed that Mt2I286F, but not Mt2S762A, had reduced interactions with Hjv, ActRIIA, and Hfe. In addition, increased expression of a serine protease inhibitor, the hepatocyte growth factor activator inhibitor-2, in the liver failed to alter hepcidin. Together, these observations support the idea that the substrate interaction with Mt2 plays a determinant role and suggest that the proteolytic activity is not an appropriate target to modulate the function of MT2 for clinical applications.

Extrahepatic deficiency of transferrin receptor 2 is associated with increased erythropoiesis independent of iron overload.

Transferrin receptor 2 (TFR2) is a transmembrane protein expressed mainly in hepatocytes and in developing erythroid cells and is an important focal point in systemic iron regulation. Loss of TFR2 function results in a rare form of the iron-overload disease hereditary hemochromatosis. Although TFR2 in the liver has been shown to be important for regulating iron homeostasis in the body, TFR2's function in erythroid progenitors remains controversial. In this report, we analyzed TFR2-deficient mice in the presence or absence of iron overload to distinguish between the effects caused by a high iron load and those caused by loss of TFR2 function. Analysis of bone marrow from TFR2-deficient mice revealed a reduction in the early burst-forming unit-erythroid and an expansion of late-stage erythroblasts that was independent of iron overload. Spleens of TFR2-deficient mice displayed an increase in colony-forming unit-erythroid progenitors and in all erythroblast populations regardless of iron overload. This expansion of the erythroid compartment coincided with increased erythroferrone (ERFE) expression and serum erythropoietin (EPO) levels. Rescue of hepatic TFR2 expression normalized hepcidin expression and the total cell count of the bone marrow and spleen, but it had no effect on erythroid progenitor frequency. On the basis of these results, we propose a model of TFR2's function in murine erythropoiesis, indicating that deficiency in this receptor is associated with increased erythroid development and expression of EPO and ERFE in extrahepatic tissues independent of TFR's role in the liver.

VIPER is a genetically encoded peptide tag for fluorescence and electron microscopy.

Many discoveries in cell biology rely on making specific proteins visible within their native cellular environment. There are various genetically encoded tags, such as fluorescent proteins, developed for fluorescence microscopy (FM). However, there are almost no genetically encoded tags that enable cellular proteins to be observed by both FM and electron microscopy (EM). Herein, we describe a technology for labeling proteins with diverse chemical reporters, including bright organic fluorophores for FM and electron-dense nanoparticles for EM. Our technology uses versatile interacting peptide (VIP) tags, a class of genetically encoded tag. We present VIPER, which consists of a coiled-coil heterodimer formed between the genetic tag, CoilE, and a probe-labeled peptide, CoilR. Using confocal FM, we demonstrate that VIPER can be used to highlight subcellular structures or to image receptor-mediated iron uptake. Additionally, we used VIPER to image the iron uptake machinery by correlative light and EM (CLEM). VIPER compared favorably with immunolabeling for imaging proteins by CLEM, and is an enabling technology for protein targets that cannot be immunolabeled. VIPER is a versatile peptide tag that can be used to label and track proteins with diverse chemical reporters observable by both FM and EM instrumentation.

The catalytic, stem, and transmembrane portions of matriptase-2 are required for suppressing the expression of the iron-regulatory hormone hepcidin.

Matriptase-2 (MT2) is a type-II transmembrane, trypsin-like serine protease that is predominantly expressed in the liver. It is a key suppressor for the expression of hepatic hepcidin, an iron-regulatory hormone that is induced via the bone morphogenetic protein signaling pathway. A current model predicts that MT2 suppresses hepcidin expression by cleaving multiple components of the induction pathway. MT2 is synthesized as a zymogen that undergoes autocleavage for activation and shedding. However, the biologically active form of MT2 and, importantly, the contributions of different MT2 domains to its function are largely unknown. Here we examined the activities of truncated MT2 that were generated by site-directed mutagenesis or Gibson assembly master mix, and found that the stem region of MT2 determines the specificity and efficacy for substrate cleavage. The transmembrane domain allowed MT2 activation after reaching the plasma membrane, and the cytoplasmic domain facilitated these processes. Further in vivo rescue studies indicated that the entire extracellular and transmembrane domains of MT2 are required to correct the low-hemoglobin, low-serum iron, and high-hepcidin status in MT2-/- mice. Unlike in cell lines, no autocleavage of MT2 was detected in vivo in the liver, implying that MT2 may also function independently of its proteolytic activity. In conjunction with our previous studies implicating the cytoplasmic domain as an intracellular iron sensor, these observations reveal the importance of each MT2 domain for MT2-mediated substrate cleavage and for its biological function.

Insights into basic science: what basic science can teach us about iron homeostasis in trauma patients.

PURPOSE OF REVIEW: This review summarizes recent basic science studies on homeostasis of iron, an essential dietary nutrient and potentially toxic metal, and explores the relevance of these studies to our understanding of trauma and related severe, acute events. RECENT FINDINGS: Recent studies in experimental models of iron homeostasis have added to our understanding of how iron levels are regulated in the body and how iron levels and iron-dependent biological processes contribute to trauma and related events. Iron deficiency, a common nutritional disorder, can impair critical organ function and wound and injury repair. Iron excess, typically because of genetic defects, can cause toxicity to tissues and, like iron deficiency, impair wound and injury repair. Finally, pharmacologic inhibition of ferroptosis, a novel form of iron-dependent cell death, is beneficial in animal models of cardiac, hepatic, and intestinal injury and intracerebral hemorrhage, suggesting that ferroptosis inhibitors could serve as novel therapeutic agents for trauma and related events. SUMMARY: Perturbations in iron homeostasis can contribute significantly to an individual's predisposition to trauma and their ability to recover posttrauma, whereas pharmacologic targeting of ferroptosis may attenuate severity of trauma-induced organ dysfunction.

Transferrin Receptors TfR1 and TfR2 Bind Transferrin through Differing Mechanisms.

Hereditary hemochromatosis (HH), a disease marked by chronic iron overload from insufficient expression of the hormone hepcidin, is one of the most common genetic diseases. One form of HH (type III) results from mutations in transferrin receptor-2 (TfR2). TfR2 is postulated to be a part of signaling system that is capable of modulating hepcidin expression. However, the molecular details of TfR2's role in this system remain unclear. TfR2 is predicted to bind the iron carrier transferrin (Tf) when the iron saturation of Tf is high. To better understand the nature of these TfR-Tf interactions, a binding study with the full-length receptors was conducted. In agreement with previous studies with truncated forms of these receptors, holo-Tf binds to the TfR1 homologue significantly stronger than to TfR2. However, the binding constant for Tf-TfR2 is still far above that of physiological holo-Tf levels, inconsistent with the hypothetical model, suggesting that other factors mediate the interaction. One possible factor, apo-Tf, only weakly binds TfR2 at serum pH and thus will not be able to effectively compete with holo-Tf. Tf binding to a TfR2 chimera containing the TfR1 helical domain indicates that the differences in the helical domain account for differences in the on rate of Tf, and nonconserved inter-receptor interactions are necessary for the stabilization of the complex. Conserved residues at one possible site of stabilization, the apical arm junction, are not important for TfR1-Tf binding but are critical for the TfR2-Tf interaction. Our results highlight the differences in Tf interactions with the two TfRs.

Matriptase-2 suppresses hepcidin expression by cleaving multiple components of the hepcidin induction pathway.

Systemic iron homeostasis is maintained by regulation of iron absorption in the duodenum, iron recycling from erythrocytes, and iron mobilization from the liver and is controlled by the hepatic hormone hepcidin. Hepcidin expression is induced via the bone morphogenetic protein (BMP) signaling pathway that preferentially uses two type I (ALK2 and ALK3) and two type II (ActRIIA and BMPR2) BMP receptors. Hemojuvelin (HJV), HFE, and transferrin receptor-2 (TfR2) facilitate this process presumably by forming a plasma membrane complex with BMP receptors. Matriptase-2 (MT2) is a protease and key suppressor of hepatic hepcidin expression and cleaves HJV. Previous studies have therefore suggested that MT2 exerts its inhibitory effect by inactivating HJV. Here, we report that MT2 suppresses hepcidin expression independently of HJV. In Hjv-/- mice, increased expression of exogenous MT2 in the liver significantly reduced hepcidin expression similarly as observed in wild-type mice. Exogenous MT2 could fully correct abnormally high hepcidin expression and iron deficiency in MT2-/- mice. In contrast to MT2, increased Hjv expression caused no significant changes in wild-type mice, suggesting that Hjv is not a limiting factor for hepcidin expression. Further studies revealed that MT2 cleaves ALK2, ALK3, ActRIIA, Bmpr2, Hfe, and, to a lesser extent, Hjv and Tfr2. MT2-mediated Tfr2 cleavage was also observed in HepG2 cells endogenously expressing MT2 and TfR2. Moreover, iron-loaded transferrin blocked MT2-mediated Tfr2 cleavage, providing further insights into the mechanism of Tfr2's regulation by transferrin. Together, these observations indicate that MT2 suppresses hepcidin expression by cleaving multiple components of the hepcidin induction pathway.

Neogenin Facilitates the Induction of Hepcidin Expression by Hemojuvelin in the Liver.

Hemojuvelin (HJV) regulates iron homeostasis by direct interaction with bone morphogenetic protein (BMP) ligands to induce hepcidin expression through the BMP signaling pathway in the liver. Crystallography studies indicate that HJV can simultaneously bind to both BMP2 and the ubiquitously expressed cell surface receptor neogenin. However, the role of the neogenin-HJV interaction in the function of HJV is unknown. Here we identify a mutation in HJV that specifically lowers its interaction with neogenin. Expression of this mutant Hjv in the liver of Hjv(-/-) mice dramatically attenuated its induction of BMP signaling and hepcidin mRNA, suggesting that interaction with neogenin is critical for the iron regulatory function of HJV. Further studies revealed that neogenin co-immunoprecipitated with ALK3, an essential type-I BMP receptor for hepatic hepcidin expression. Neogenin has also been shown to facilitate the cleavage of HJV by furin in transfected cells. Surprisingly, although cleavage of HJV by furin has been implicated in the regulation of HJV function in cell culture models and furin-cleaved soluble Hjv is detectable in the serum of mice, mutating the furin cleavage site did not alter the stimulation of hepcidin expression by Hjv in mice. In vivo studies validated the important role of HJV-BMP interaction for Hjv stimulation of BMP signaling and hepcidin expression. Together these data support a model in which neogenin acts as a scaffold to facilitate assembly of the HJV·BMP·BMP receptor complex to induce hepcidin expression.

Versatile Interacting Peptide (VIP) Tags for Labeling Proteins with Bright Chemical Reporters.

Fluorescence microscopy is an essential tool for the biosciences, enabling the direct observation of proteins in their cellular environment. New methods that facilitate attachment of photostable synthetic fluorophores with genetic specificity are needed to advance the frontiers of biological imaging. Here, we describe a new set of small, selective, genetically encoded tags for proteins based on a heterodimeric coiled-coil interaction between two peptides: CoilY and CoilZ. Proteins expressed as a fusion to CoilZ were selectively labeled with the complementary CoilY fluorescent probe peptide. Fluorophore-labeled target proteins were readily detected in cell lysates with high specificity and sensitivity. We found that these versatile interacting peptide (VIP) tags allowed rapid and specific delivery of bright organic dyes or quantum dots to proteins displayed on living cells. Additionally, we validated that either CoilY or CoilZ could serve as the VIP tag, which enabled us to observe two distinct cell-surface protein targets with this one heterodimeric pair.

An iron-regulated and glycosylation-dependent proteasomal degradation pathway for the plasma membrane metal transporter ZIP14.

Protein degradation is instrumental in regulating cellular function. Plasma membrane proteins targeted for degradation are internalized and sorted to multivesicular bodies, which fuse with lysosomes, where they are degraded. ZIP14 is a newly identified iron transporter with multitransmembrane domains. In an attempt to dissect the molecular mechanisms by which iron regulates ZIP14 levels, we found that ZIP14 is endocytosed, extracted from membranes, deglycosylated, and degraded by proteasomes. This pathway did not depend on the retrograde trafficking to the endoplasmic reticulum and thus did not involve the well-defined endoplasmic reticulum-associated protein degradation pathway. Iron inhibited membrane extraction of internalized ZIP14, resulting in higher steady-state levels of ZIP14. Asparagine-linked (N-linked) glycosylation of ZIP14, particularly the glycosylation at N102, was required for efficient membrane extraction of ZIP14 and therefore is necessary for its iron sensitivity. These findings highlight the importance of proteasomes in the degradation of endocytosed plasma membrane proteins.

CD81 promotes both the degradation of transferrin receptor 2 (TfR2) and the Tfr2-mediated maintenance of hepcidin expression.

Mutations in transferrin receptor 2 (TfR2) cause a rare form of the hereditary hemochromatosis, resulting in iron overload predominantly in the liver. TfR2 is primarily expressed in hepatocytes and is hypothesized to sense iron levels in the blood to positively regulate the expression of hepcidin through activation of the BMP signaling pathway. Hepcidin is a peptide hormone that negatively regulates iron egress from cells and thus limits intestinal iron uptake. In this study, a yeast two-hybrid approach using the cytoplasmic domain of TfR2 identified CD81 as an interacting protein. CD81 is an abundant tetraspanin in the liver. Co-precipitations of CD81 with different TfR2 constructs demonstrated that both the cytoplasmic and ecto-transmembrane domains of TfR2 interact with CD81. Knockdown of CD81 using siRNA significantly increased TfR2 levels by increasing the half-life of TfR2, indicating that CD81 promotes degradation of TfR2. Previous studies showed that CD81 is targeted for degradation by GRAIL, an ubiquitin E3 ligase. Knockdown of GRAIL in Hep3B-TfR2 cells increased TfR2 levels, consistent with inhibition of CD81 ubiquitination. These results suggest that down-regulation of CD81 by GRAIL targets TfR2 for degradation. Surprisingly, knockdown of CD81 decreased hepcidin expression, implying that the TfR2/CD81 complex is involved in the maintenance of hepcidin mRNA. Moreover, knockdown of CD81 did not affect the stimulation of hepcidin expression by BMP6 but increased both the expression of ID1 and SMAD7, direct targets of BMP signaling pathway, and the phosphorylation of ERK1/2, indicating that the CD81 regulates hepcidin expression differently from the BMP and ERK1/2 signaling pathways.

Low intracellular iron increases the stability of matriptase-2.

Matriptase-2 (MT2) is a type II transmembrane serine protease that is predominantly expressed in hepatocytes. It suppresses the expression of hepatic hepcidin, an iron regulatory hormone, by cleaving membrane hemojuvelin into an inactive form. Hemojuvelin is a bone morphogenetic protein (BMP) co-receptor. Here, we report that MT2 is up-regulated under iron deprivation. In HepG2 cells stably expressing the coding sequence of the MT2 gene, TMPRSS6, incubation with apo-transferrin or the membrane-impermeable iron chelator, deferoxamine mesylate salt, was able to increase MT2 levels. This increase did not result from the inhibition of MT2 shedding from the cells. Rather, studies using a membrane-permeable iron chelator, salicylaldehyde isonicotinoyl hydrazone, revealed that depletion of cellular iron was able to decrease the degradation of MT2 independently of internalization. We found that lack of the putative endocytosis motif in its cytoplasmic domain largely abolished the sensitivity of MT2 to iron depletion. Neither acute nor chronic iron deficiency was able to alter the association of Tmprss6 mRNA with polyribosomes in the liver of rats indicating a lack of translational regulation by low iron levels. Studies in mice showed that Tmprss6 mRNA was not regulated by iron nor the BMP-mediated signaling with no evident correlation with either Bmp6 mRNA or Id1 mRNA, a target of BMP signaling. These results suggest that regulation of MT2 occurs at the level of protein degradation rather than by changes in the rate of internalization and translational or transcriptional mechanisms and that the cytoplasmic domain of MT2 is necessary for its regulation.

N-linked glycosylation is required for transferrin-induced stabilization of transferrin receptor 2, but not for transferrin binding or trafficking to the cell surface.

Transferrin receptor 2 (TfR2) is a member of the transferrin receptor-like family of proteins. Mutations in TfR2 can lead to a rare form of the iron overload disease, hereditary hemochromatosis. TfR2 is proposed to sense body iron levels and increase the level of expression of the iron regulatory hormone, hepcidin. Human TfR2 (hTfR2) contains four potential Asn-linked (N-linked) glycosylation sites on its ectodomain. The importance of glycosylation in TfR2 function has not been elucidated. In this study, by employing site-directed mutagenesis to remove glycosylation sites of hTfR2 individually or in combination, we found that hTfR2 was glycosylated at Asn 240, 339, and 754, while the consensus sequence for N-linked glycosylation at Asn 540 was not utilized. Cell surface protein biotinylation and biotin-labeled Tf indicated that in the absence of N-linked oligosaccharides, hTfR2 still moved to the plasma membrane and bound its ligand, holo-Tf. However, without N-linked glycosylation, hTfR2 did not form the intersubunit disulfide bonds as efficiently as the wild type (WT). Moreover, the unglycosylated form of hTfR2 could not be stabilized by holo-Tf. We further provide evidence that the unglycosylated hTfR2 behaved in manner different from that of the WT in response to holo-Tf treatment. Thus, the putative iron-sensing function of TfR2 could not be achieved in the absence of N-linked oligosaccharides. On the basis of our analyses, we conclude that unlike TfR1, N-linked glycosylation is dispensable for the cell surface expression and holo-Tf binding, but it is required for efficient intersubunit disulfide bond formation and holo-Tf-induced stabilization of TfR2.

Neogenin interacts with matriptase-2 to facilitate hemojuvelin cleavage.

Hemojuvelin (HJV) and matriptase-2 (MT2) are co-expressed in hepatocytes, and both are essential for systemic iron homeostasis. HJV is a glycosylphosphatidylinositol-linked membrane protein that acts as a co-receptor for bone morphogenetic proteins to induce hepcidin expression. MT2 regulates the levels of membrane-bound HJV in hepatocytes by binding to and cleaving HJV into an inactive soluble form that is released from cells. HJV also interacts with neogenin, a ubiquitously expressed transmembrane protein with multiple functions. In this study, we showed that neogenin interacted with MT2 as well as with HJV and facilitated the cleavage of HJV by MT2. In contrast, neogenin was not cleaved by MT2, indicating some degree of specificity by MT2. Down-regulation of neogenin with siRNA increased the amount of MT2 and HJV on the plasma membrane, suggesting a lack of neogenin involvement in their trafficking to the cell surface. The increase in MT2 and HJV upon neogenin knockdown was likely due to the inhibition of cell surface MT2 and HJV internalization. Analysis of the Asn-linked oligosaccharides showed that MT2 cleavage of cell surface HJV was coupled to a transition from high mannose oligosaccharides to complex oligosaccharides on HJV. These results suggest that neogenin forms a ternary complex with both MT2 and HJV at the plasma membrane. The complex facilitates HJV cleavage by MT2, and release of the cleaved HJV from the cell occurs after a retrograde trafficking through the TGN/Golgi compartments.

Hereditary hemochromatosis and transferrin receptor 2.

BACKGROUND: Multicellular organisms regulate the uptake of calories, trace elements, and other nutrients by complex feedback mechanisms. In the case of iron, the body senses internal iron stores, iron requirements for hematopoiesis, and inflammatory status, and regulates iron uptake by modulating the uptake of dietary iron from the intestine. Both the liver and the intestine participate in the coordination of iron uptake and distribution in the body. The liver senses inflammatory signals and iron status of the organism and secretes a peptide hormone, hepcidin. Under high iron or inflammatory conditions hepcidin levels increase. Hepcidin binds to the iron transport protein, ferroportin (FPN), promoting FPN internalization and degradation. Decreased FPN levels reduce iron efflux out of intestinal epithelial cells and macrophages into the circulation. Derangements in iron metabolism result in either the abnormal accumulation of iron in the body, or in anemias. The identification of the mutations that cause the iron overload disease, hereditary hemochromatosis (HH), or iron-refractory iron-deficiency anemia has revealed many of the proteins used to regulate iron uptake. SCOPE OF THE REVIEW: In this review we discuss recent data concerning the regulation of iron homeostasis in the body by the liver and how transferrin receptor 2 (TfR2) affects this process. MAJOR CONCLUSIONS: TfR2 plays a key role in regulating iron homeostasis in the body. GENERAL SIGNIFICANCE: The regulation of iron homeostasis is important. One third of the people in the world are anemic. HH is the most common inherited disease in people of Northern European origin and can lead to severe health complications if left untreated. This article is part of a Special Issue entitled Transferrins: Molecular mechanisms of iron transport and disorders.

Suppression of hepatic hepcidin expression in response to acute iron deprivation is associated with an increase of matriptase-2 protein.

Recent studies demonstrate a pivotal role for bone morphogenic protein-6 (BMP6) and matriptase-2, a protein encoded by the TMPRSS6 gene, in the induction and suppression of hepatic hepcidin expression, respectively. We examined their expression profiles in the liver and showed a predominant localization of BMP6 mRNA in nonparenchymal cells and exclusive expression of TMPRSS6 mRNA in hepatocytes. In rats fed an iron-deficient (ID) diet for 24 hours, the rapid decrease of transferrin saturation from 71% to 24% (control vs ID diet) was associated with a 100-fold decrease in hepcidin mRNA compared with the corresponding controls. These results indicated a close correlation of low transferrin saturation with decreased hepcidin mRNA. The lower phosphorylated Smad1/5/8 detected in the ID rat livers suggests that the suppressed hepcidin expression results from the inhibition of BMP signaling. Quantitative real-time reverse transcription polymerase chain reaction analysis revealed no significant change in either BMP6 or TMPRSS6 mRNA in the liver. However, an increase in matriptase-2 protein in the liver from ID rats was detected, suggesting that suppression of hepcidin expression in response to acute iron deprivation is mediated by an increase in matriptase-2 protein levels.