Regulation Of Hepcidin Expression Research Articles

Role of Ferroptosis in ALD: Focusing on Hepcidin and Besides Hepcidin.

Ascending incidence and poor outcomes make Alcoholic Liver Disease (ALD) a considerable public health concern. This review concluded the iron metabolism under physiology conditions and alcohol disturbance (leading to ferroptosis in ALD) and summarized the novel treatment, diagnosis, and prognosis of ferroptosis for ALD. ALD is characterized by alcohol-induced chronic metabolism disorder, peroxidation damage, and dysfunction of the anti-oxidant system. Current animal experiments and clinical studies identified ferroptosis as a new form of regulated cell death involved in ALD. One strong evidence is that the key iron regulatory hormone, hepcidin, is downgraded in ALD through NF-κB/IL-6/STAT3, BMP/SMAD, and Jak/STAT3 pathways, which would impair iron hemostasis and induce ferroptosis in ALD. Also, imbalance metabolism and other pathological responses in ALD induce and regulate ferroptosis, which proves ferroptosis participates in the pathophysiology of ALD via oxidative stress, steatosis, and fibrosis. Inhibition of ferroptosis via regulating hepcidin expression and metabolism impairment may provide new therapies for ALD.

RGM Family Involved in the Regulation of Hepcidin Expression in Anemia of Chronic Disease

The persistent production of inflammatory cytokines causes anemia of chronic disease (ACD). Playing a central role in the pathophysiology of ACD is hepcidin, a key regulator of iron metabolism. The regulation of hepcidin expression is a complex process intricately controlled by multiple pathways. These include the BMP/SMAD, the HFE–TFR2, and the IL-6/STAT3 pathway, each playing a significant role in this regulation. We detail the critical role of the repulsive guidance molecule (RGM) family, especially hemojuvelin (HJV/RGMc), in regulating hepcidin expression in ACD. HJV functions as a co-receptor for BMPs and positively regulates hepcidin expression. RGMa and RGMb may also regulate hepcidin expression and inflammatory responses. RGM family proteins play essential roles in the interplay between inflammation, iron metabolism, and the immune system, and elucidating them could lead to a better understanding of the pathophysiology of ACD and the development of new therapeutic strategies.

A functional interplay between the two BMP-SMAD pathway inhibitors TMPRSS6 and FKBP12 regulates hepcidin expression in vivo.

The Activin A Receptor type I (ALK2) is a critical component of BMP-SMAD signaling that, in the presence of ligands, phosphorylates cytosolic SMAD1/5/8 and modulates important biological processes, including bone formation and iron metabolism. In hepatocytes, the BMP-SMAD pathway controls the expression of hepcidin, the liver peptide hormone that regulates body iron homeostasis via the BMP receptors ALK2 and ALK3, and the hemochromatosis proteins. The main negative regulator of the pathway in the liver is transmembrane serine protease 6 (TMPRSS6), which downregulates hepcidin by cleaving the BMP coreceptor hemojuvelin. ALK2 function is inhibited also by the immunophilin FKBP12, which maintains the receptor in an inactive conformation. FKBP12 sequestration by tacrolimus or its silencing upregulates hepcidin in primary hepatocytes and in vivo in acute but not chronic settings. Interestingly, gain-of-function mutations in ALK2 that impair FKBP12 binding to the receptor and activate the pathway cause a bone phenotype in patients affected by Fibrodysplasia Ossificans Progressiva but not hepcidin and iron metabolism dysfunction. This observation suggests that additional mechanisms are active in the liver to compensate for the increased BMP-SMAD signaling. Here we demonstrate that Fkbp12 downregulation in hepatocytes by antisense oligonucleotide treatment upregulates the expression of the main hepcidin inhibitor Tmprss6, thus counteracting the ALK2-mediated activation of the pathway. Combined downregulation of both Fkbp12 and Tmprss6 blocks this compensatory mechanism. Our findings reveal a previously unrecognized functional cross talk between FKBP12 and TMPRSS6, the main BMP-SMAD pathway inhibitors, in the control of hepcidin transcription.NEW & NOTEWORTHY This study uncovers a previously unrecognized mechanism of hepcidin and BMP-SMAD pathway regulation in hepatocytes mediated by the immunophilin FKBP12 and the transmembrane serine protease TMPRSS6.

Circulating microRNAs and hepcidin as predictors of iron homeostasis and anemia among school children: a biochemical and cross-sectional survey analysis

BackgroundMicroRNAs (miRNAs) can control several biological processes. Thus, the existence of these molecules plays a significant role in regulating human iron metabolism or homeostasis.PurposeThe study aimed to determine the role of circulating microRNAs and hepcidin in controlling iron homeostasis and evaluating possible anemia among school children.MethodsThe study was based on a biochemical and cross-sectional survey study that included three hundred fifty school children aged 12–18 years old. RT–PCR and immunoassay analysis were accomplished to estimate iron concentration, Hgb, serum ferritin (SF), soluble transferrin receptor (sTfR), total body iron stores (TIBs), total oxidative stress (TOS), total antioxidant capacity (TAC), α-1-acid glycoprotein (AGP), high sensitive C-reactive protein (hs-CRP), and miRNAs; miR-146a, miR-129b, and miR-122 in 350 school adolescents.ResultsIron disorders were cross-sectionally predicted in 28.54% of the study population; they were classified into 14.26% with ID, 5.7% with IDA, and 8.6% with iron overload. The overall proportion of iron depletion was significantly higher in girls (20.0%) than in boys (8.6%). MicroRNAs; miR-146a, miR-125b, and miR-122 were significantly upregulated with lower hepcidin expression in adolescence with ID and IDA compared to iron-overloaded subjects, whereas downregulation of these miRNAs was linked with higher hepcidin. Also, a significant correlation was recorded between miRNAs, hepcidin levels, AGP, hs-CRP, TAC, and other iron-related indicators.ConclusionMolecular microRNAs such as miR-146a, miR-125b, and miR-122 were shown to provide an additional means of controlling or regulating cellular iron uptake or metabolism either via the oxidative stress pathway or regulation of hepcidin expression via activating genes encoding Hfe and Hjv activators, which promote iron regulation. Thus, circulating miRNAs as molecular markers and serum hepcidin could provide an additional means of controlling or regulating cellular iron and be associated as valuable markers in diagnosing and treating cases with different iron deficiencies.

Differential Effects of Monoferric Transferrin Forms on Erythropoiesis Are Mediated By TFR2

Transferrin (TF) is a bilobed 80kD glycoprotein with N- and C-lobe iron binding sites. TF circulates as four forms: unbound to iron (apo-TF), monoferric iron bound to the N-lobe or C-lobe (mono-TF) or to both lobes (holo-TF). All TF forms interact with TF receptor-1 (TFR1), which is ubiquitously expressed and serves as the main mechanism for cellular iron delivery. TF also interacts with TFR2 which is mainly expressed by hepatocytes and erythroid precursors to modulate cellular signaling events regulating hepcidin expression and erythropoiesis. We previously generated N-lobe blocked ( Tf N-bl) or C-lobe blocked ( Tf C-bl) TF mutant mice, and observed marked differences between them in iron parameters, RBC count and EPO sensitivity (Parrow et al., 2019). In this current work, we investigate the effects of the mutated TFs on ineffective erythropoiesis in murine β-thalassemia intermedia. We moreover examine the consequences of erythroid knockout of TFR2 in the TF N-blocked and C-blocked mice. Based on observations in the TF mutant mice, we hypothesized that Hbb th3/+ mice crossed with Tf C-bl would demonstrate improved erythropoietic and iron parameters compared with Hbb th3/+Tf N-bl. Hbb th3/+Tf C-bl mice demonstrated significantly increased RBC counts, elevated hemoglobin, improved erythrocyte morphology, decreased splenomegaly, fewer bone marrow erythroblasts, and improvement of IE. Additionally, serum erythroferrone (ERFE) was significantly reduced and hepcidin levels were increased in Hbb th3/+Tf C-bl relative to Hbb th3/+Tf +/+controls. By contrast, similar improvements in RBC counts and hemoglobin were not observed in Hbb th3/+Tf N-bl mice. Nonetheless, the Hbb th3/+Tf N-blmice displayed lower reticulocytes, increased platelets, decreased IE, decreased splenomegaly, and decreased serum ERFE compared to Hbb th3/+Tf +/+controls and thus displayed a phenotype intermediate between Hbb th3/+Tf C-bl and Hbb th3/+Tf +/+controls. Serum EPO was elevated in Hbb th3/+Tf N-blcompared to either Hbb th3/+Tf C-blor Hbb th3/+Tf +/+mice. Moreover, the increased hepcidin observed in Hbb th3/+Tf C-bl compared with Hbb th3/+Tf +/+was not observed in Hbb th3/+Tf N-bl mice. To examine the contribution of erythroid TFR2 to the different phenotypes of the N-blocked and C-blocked TF mice, we generated a mouse line expressing TFR2-3xFLAG that is flanked by loxp sites ( TFR2-3xFLAG fl/fl). These mice were used to generate mice with erythroid knockout of TFR2 in the TF mutant mice; i.e., EPOR Cre-tdtomTf N-blTFR2 cko and EPOR Cre-tdtomTf C-blTFR2 cko. N-blocked mice with conditional knockout of TFR2 demonstrated increases in RBC counts and Hb levels compared with the TF N-blocked mice ( EPOR Cre-tdtom Tf N-blTFR2 cko vs Tf N-bl/Tf N-blTFR2-3xFLAG fl/fl), and had values similar to the C-blocked mice ( Tf C-bl/Tf C-blTFR2-3xFLAG fl/fl or EPOR Cre-tdtomTf N-blTFR2 cko). Likewise, conditional KO of TFR2 in the N-blocked mice resulted in lower EPO levels, which were similar to those in the C-blocked mice. These data support the hypothesis that differences observed between the mono-TF forms at the erythroid level are mediated via TFR2. These data support a model by which lobe-specific iron occupancy of TF influences hematopoiesis via erythroid TFR2.

A Phase 1b Double-Blind, Placebo-Controlled Study of DISC-0974, an Anti-Hemojuvelin Antibody, in Patients with Non-Dialysis Dependent Chronic Kidney Disease and Anemia

Hepcidin, a central regulator of iron homeostasis, is pathologically elevated in patients with non-dialysis-dependent chronic kidney disease (NDD-CKD) and anemia. Elevations in hepcidin contribute to the onset, maintenance, and severity of anemia. DISC-0974 is an investigational, first-in-class, monoclonal antibody that blocks hemojuvelin (HJV), a co-receptor in the bone morphogenetic protein-signaling pathway regulating hepcidin expression. In a healthy volunteer study, DISC-0974 demonstrated dose-dependent reductions in serum hepcidin, increases in serum iron with doses up to 56 mg administered subcutaneously (SC), and increasing trends in reticulocyte count, reticulocyte hemoglobin, mean corpuscular hemoglobin, total hemoglobin (Hgb), and red blood cell count. In a rat model of anemia in CKD, DISC-0974 decreased hepcidin, increased serum iron, and normalized hemoglobin (Wu et al., ASH 2022). DISC-0974-103 is a Phase 1b, double-blind, placebo-controlled, single-ascending dose study (NCT05745883) to assess the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of DISC-0974 in patients with NDD-CKD and anemia. Eligible participants include patients over 18 years of age with NDD-CKD, stages 2-5, hemoglobin (Hgb) <10.5 g/dL in women and <11.0 g/dL in men, serum ferritin ≥100 μg/L, and transferrin saturation (TSAT) ≤30%. Major exclusionary criteria include: concomitant treatment with therapeutic oral iron, intravenous iron, erythropoietin-stimulating agents, or blood transfusions; splenectomy; and nutritional, genetic, infectious, or autoimmune causes of anemia. Approximately 32 participants will be enrolled. On Day 1, participants will receive a single dose of DISC-0974 SC with evaluations for safety, PK, and PD through 57 days of follow-up. Dose escalation is planned across 4 cohorts consisting of 8 participants each, randomized 3:1 active treatment to placebo, treated at single doses of 28, 40, 60, and 90 mg SC DISC-0974. Safety Review Committee assessments are planned to gate escalation decisions. Primary endpoints include adverse events (AEs), clinical laboratory assessments, vital signs, physical examinations, and electrocardiograms. Secondary endpoints include changes in serum hepcidin-25, iron, TSAT, ferritin, Hgb, and other hematology biomarkers. Enrollment is currently ongoing, and available study results will be presented at the meeting.

Weak response of bovine hepcidin induction to iron through decreased expression of Smad4.

Hepcidin negatively regulates systemic iron levels by inhibiting iron entry into the circulation. Hepcidin production is increased in response to an increase in systemic iron via the activation of the bone morphogenetic protein (BMP) pathway. Regulation of hepcidin expression by iron status has been proposed on the basis of evidence mainly from rodents and humans. We evaluated the effect of iron administration on plasma hepcidin concentrations in calves and the expression of bovine hepcidin by the BMP pathway in a cell culture study. Hematocrit as well as levels of blood hemoglobin and plasma iron were lower than the reference level in calves aged 1-4 weeks. Although intramuscular administration of iron increased iron-related parameters, plasma hepcidin concentrations were unaffected. Treatment with BMP6 increased hepcidin expression in human liver-derived cells but not in bovine liver-derived cells. A luciferase-based reporter assay revealed that Smad4 was required for hepcidin reporter transcription induced by Smad1. The reporter activity of hepcidin was lower in the cells transfected with bovine Smad4 than in those transfected with murine Smad4. The lower expression levels of bovine Smad4 were responsible for the lower activity of the hepcidin reporter, which might be due to the instability of bovine Smad4 mRNA. In fact, the endogenous Smad4 protein levels were lower in bovine cells than in human and murine cells. Smad4 also confers TGF-β/activin-mediated signaling. Induction of TGF-β-responsive genes was also lower after treatment with TGF-β1 in bovine hepatocytes than in human hepatoma cells. We revealed the unique regulation of bovine hepcidin expression and the characteristic TGF-β family signaling mediated by bovine Smad4. The present study suggests that knowledge of the regulatory expression of hepcidin as well as TGF-β family signaling obtained in murine and human cells is not always applicable to bovine cells.

A Decrease in Maternal Iron Levels Is the Predominant Factor Suppressing Hepcidin during Pregnancy in Mice.

In order to supply adequate iron during pregnancy, the levels of the iron regulatory hormone hepcidin in the maternal circulation are suppressed, thereby increasing dietary iron absorption and storage iron release. Whether this decrease in maternal hepcidin is caused by changes in factors known to regulate hepcidin expression, or by other unidentified pregnancy factors, is not known. To investigate this, we examined iron parameters during pregnancy in mice. We observed that hepatic iron stores and transferrin saturation, both established regulators of hepcidin production, were decreased in mid and late pregnancy in normal and iron loaded dams, indicating an increase in iron utilization. This can be explained by a significant increase in maternal erythropoiesis, a known suppressor of hepcidin production, by mid-pregnancy, as indicated by an elevation in circulating erythropoietin and an increase in spleen size and splenic iron uptake. Iron utilization increased further in late pregnancy due to elevated fetal iron demand. By increasing maternal iron levels in late gestation, we were able to stimulate the expression of the gene encoding hepcidin, suggesting that the iron status of the mother is the predominant factor influencing hepcidin levels during pregnancy. Our data indicate that pregnancy-induced hepcidin suppression likely occurs because of reductions in maternal iron reserves due to increased iron requirements, which predominantly reflect stimulated erythropoiesis in mid-gestation and increased fetal iron requirements in late gestation, and that there is no need to invoke other factors, including novel pregnancy factor(s), to explain these changes.

Lactate modulates iron metabolism by binding soluble adenylyl cyclase

Overproduction of lactate (LA) can occur during exercise and in many diseases such as cancers. Individuals with hyperlactatemia often display anemia, decreased serum iron, and elevated hepcidin, a key regulator of iron metabolism. However, it is unknown whether and how LA regulates hepcidin expression. Here, we show LA binds to soluble adenylyl cyclase (sAC) in normal hepatocytes and affects systemic iron homeostasis in mice by increasing hepcidin expression. Comprehensive invitro, invivo, and in silico experiments show that the LA-sAC interaction raises cyclic adenosine monophosphate (cAMP) levels, which activates the PKA-Smad1/5/8 signaling pathway to increase hepcidin transcription. We verified this regulatory axis in wild-type mice and in mice with disordered iron homeostasis. LA also regulates hepcidin in humans at rest and subjected to extensive exercise that produce elevated LA. Our study links hyperlactatemia to iron deficiency, offering a mechanistic explanation for anemias seen in athletes and patients with lactic acidosis.

FKBP12 inhibits hepcidin expression by modulating BMP receptors interaction and ligand responsiveness in hepatocytes.

The expression of the iron regulatory hormone hepcidin in hepatocytes is regulated by the BMP-SMAD pathway through the type I receptors ALK2 and ALK3, the type II receptors ACVR2A and BMPR2, and the ligands BMP2 and BMP6. We previously identified the immunophilin FKBP12 as a new hepcidin inhibitor that acts by blocking ALK2. Both the physiologic ALK2 ligand BMP6 and the immunosuppressive drug Tacrolimus (TAC) displace FKBP12 from ALK2 and activate the signaling. However, the molecular mechanism whereby FKBP12 regulates BMP-SMAD pathway activity and thus hepcidin expression remains unclear. Here, we show that FKBP12 acts by modulating BMP receptor interactions and ligand responsiveness. We first demonstrate that in primary murine hepatocytes TAC regulates hepcidin expression exclusively via FKBP12. Downregulation of the BMP receptors reveals that ALK2, to a lesser extent ALK3, and ACVR2A are required for hepcidin upregulation in response to both BMP6 and TAC. Mechanistically, TAC and BMP6 increase ALK2 homo-oligomerization and ALK2-ALK3 hetero-oligomerization and the interaction between ALK2 and the type II receptors. By acting on the same receptors, TAC and BMP6 cooperate in BMP pathway activation and hepcidin expression both in vitro and in vivo. Interestingly, the activation state of ALK3 modulates its interaction with FKBP12, which may explain the cell-specific activity of FKBP12. Overall, our results identify the mechanism whereby FKBP12 regulates the BMP-SMAD pathway and hepcidin expression in hepatocytes, and suggest that FKBP12-ALK2 interaction is a potential pharmacologic target in disorders caused by defective BMP-SMAD signaling and characterized by low hepcidin and high BMP6 expression.

How Histone Deacetylase 3 Controls Hepcidin Expression and Hepatitis C Virus Replication

The key role of histone deacetylases (HDACs) in the regulation of the cellular response to infection with the hepatitis C virus (HCV) was first demonstrated in 2008. Studying the metabolism of iron in the liver tissues of patients with chronic hepatitis C, the authors found that the expression of the hepcidin gene (HAMP), a hormone regulator of iron export, is markedly reduced in hepatocytes under conditions of oxidative stress caused by viral infection. HDACs were involved in the regulation of hepcidin expression through the control of acetylation level of histones and transcription factors, primarily STAT3, associated with the HAMP promoter. The purpose of this review is to summarize current data on the functioning of the HCV-HDAC3-STAT3-HAMP regulatory circuit as an example of a well-characterized interaction between the virus and the epigenetic apparatus of the host cell.

Distinctive modulation of hepcidin in cancer and its therapeutic relevance.

Hepcidin, a short peptide synthesized primarily by hepatocytes in response to increased body iron and inflammation, is a crucial iron-regulating factor. Hepcidin regulates intestinal iron absorption and releases iron from macrophages into plasma through a negative iron feedback mechanism. The discovery of hepcidin inspired a torrent of research into iron metabolism and related problems, which have radically altered our understanding of human diseases caused by an excess of iron, an iron deficiency, or an iron disparity. It is critical to decipher how tumor cells manage hepcidin expression for their metabolic requirements because iron is necessary for cell survival, particularly for highly active cells like tumor cells. Studies show that tumor and non-tumor cells express and control hepcidin differently. These variations should be explored to produce potential novel cancer treatments. The ability to regulate hepcidin expression to deprive cancer cells of iron may be a new weapon against cancer cells.

DISC-0974, an Anti-Hemojuvelin (HJV) Monoclonal Antibody, Reduced Hepcidin and Improved Anemia in a Rat Model of Chronic Kidney Disease

Introduction DISC-0974 is a humanized monoclonal antibody against hemojuvelin (HJV) and is currently in Phase I clinical studies. HJV is a pathway-specific coreceptor for bone morphogenetic protein (BMP) signaling that regulates hepcidin expression and iron metabolism. Loss of function mutations of the HFE2 gene encoding HJV in humans cause profound reductions in hepcidin synthesis and severe iron overload. Consequently, it is hypothesized that pharmacological reduction of HJV activity will lead to reduction of hepcidin synthesis and increased iron availability. This hypothesis has been confirmed in studies of DISC-0974 in rodents and non-human primates and in healthy human volunteers. We have conducted studies to evaluate the effect of DISC-0974 in an animal model of CKD anemia. Anemia is a common complication in patients with CKD and has been associated with multiple adverse outcomes in this population. In CKD hepcidin is increased because of both reduced renal clearance and increased synthesis. This increase is believed to be a central contributor to the development of anemia by reducing the availability of iron from systemic iron stores and by reducing dietary iron absorption. Supporting this hypothesis, treatment with hepcidin lowering drugs has been demonstrated to increase serum iron and to increase hemoglobin (Sheetz et al, Br J Clin Pharmacol. 2019;85:935-948). We hypothesize that by downregulating hepcidin, DISC-0974 will have beneficial effects in treating anemia in the CKD patients because it will make iron available for improved erythropoiesis and increased hemoglobin synthesis as well as reducing erythropoietin resistance. Material and Methods The effect of DISC-0974 on improving anemia was evaluated in an adenine-induced rat model of CKD. In this study, renal impairment was induced by giving Sprague Dawley rats a diet containing 0.75% adenine for 3 weeks to induce kidney injury and then switched to a control diet for the remainder of the study (i.e., 3 more weeks). DISC-0974 (20 mg/kg) or vehicle (n-5/group) was administered intravenously once per week starting on Day 7 and continuing to Day 35. An additional group of rats (n=5/group) was fed with the control diet for the entire study and dosed intravenously with vehicle. Rats were euthanized on Day 42. Blood was collected on Days 0, 7, 21, 35, and 42 for evaluation of hematology parameters, as well as serum iron and hepcidin. Livers were also collected at study end for evaluation of hepcidin gene (HAMP) mRNA expression level. Results Consistent with a previous report (Sun et al, Nephrol Dial Transplant. 2013;28:1733-1743), the 0.75% adenine diet induced kidney dysfunction, as evidenced by marked increase in both serum creatinine and blood urea nitrogen. Rats on adenine diet developed reduced serum iron, reduced red cell production, and lowered hemoglobin with a hypochromic microcytic profile reflecting reduced iron availability. CKD rats appeared to tolerate DISC-0974 treatment well, as DISC-0974 @ 20mg/kg did not affect body weight. Consistent with the mechanism of action of blocking the formation of the BMP/BMPR/HJV complex, DISC-0974 reduced HAMP gene expression in the liver as measured at the end the study, which led to reduced serum hepcidin and increased serum iron concentration. Cellular hemoglobinization measured as reticulocyte Hemoglobin was increased. The maximum improvement in hemoglobin by DISC-0974 compared to the vehicle group was 17g/L in the rat CKD model. Conclusions We conclude that treatment with DISC-0974 at a dose that reduces hepcidin gene expression is able to increase iron availability and substantially prevents the reduction in hemoglobin that is seen in animals that develop adenine-induced renal impairment. This study supports the development of DISC-0974 for the treatment of patients with CKD anemia. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Differential Effects of Monoferric Forms of Transferrin on Mouse Models of Anemia of Inflammation and Congenital Anemia

Transferrin (TF) is a bilobed 80kD glycoprotein with N- and C-lobe iron binding sites. TF circulates as four forms: unbound to iron (apo-TF), iron bound to the N-lobe, the C-lobe or to both lobes (diferric-TF). Most circulating TF under physiological conditions is monoferric (Dirusso et al., 1985). The TF forms interact with TF receptor-1 (TFR1), which is ubiquitously expressed and serves as the main mechanism for cellular iron delivery. TF also interacts with TF receptor-2 (TFR2) which is expressed on hepatocytes, erythroblasts, and other cells of the bone marrow and is thought to primarily influence cellular signaling events regulating hepcidin expression and erythropoiesis. In erythrocytes, loss of TFR2 results in abnormally high red blood cell production under conditions of iron-deficiency (Artuso et al., 2018), suggesting it functions as an iron sensor. Additional evidence suggests TFR2 modulates erythropoiesis via the erythropoietin (EPO) receptor (EPOR) (Forejtnikovà et al., 2010). To understand the role of monoferric TF forms in vivo, we previously generated N-lobe blocked (TfN-bl) or C-lobe blocked (TfC-bl) TF mutant mice. Characterization of these mice demonstrated dramatic differences in RBC levels, hepcidin expression relative to iron status, and EPO sensitivity (Parrow et al., 2019). More recently, we have become interested in characterizing the immune compartment of these mice. Recent studies point to iron being an important regulator of immune responses, which has important implications worldwide in understanding how to treat iron-deficient patients without exacerbating latent infections. Cells belonging to the immune branch have also been shown to express TFR2 and EPOR (Cantarelli et al., 2019; Roetto et al., 2011; Kawabata et al., 2001), however their function is largely unexplored. Therefore, in this work, we investigate the role of the two monoferric forms of TF on ineffective erythropoiesis (IE) using a mouse model of β-thalassemia (BT) intermedia (Hbbth3/+) and chronic anemia of inflammation using heat-killed Brucella Abortus (HKBA). Based on observations in BT mice treated with exogenous TF (Li et al., 2010), which led to reduced erythroid iron intake, reduced EPO requirement (as in TfC-bl mutant mice) and improved anemia, we hypothesized that Hbbth3/+ mice crossed with TfC-bl would demonstrate improved erythropoietic and iron parameters compared with Hbbth3/+TfN-bl. We measured effects on hematologic parameters, IE, and expression of relevant iron regulatory serum proteins. Hbbth3/+TfC-bl mice demonstrated significantly increased RBC counts, elevated hemoglobin, improved erythrocyte morphology, decreased splenomegaly, fewer bone marrow erythroblasts, and improvement of IE. Additionally, serum erythroferrone (ERFE) was significantly reduced and hepcidin levels were increased in Hbbth3/+TfC-bl relative to Hbbth3/+Tf+/+ controls. However, hematological parameters from Hbbth3/+TfN-bl mice did not show these improvements. In our studies examining the immune compartments of TfC-bl and TfN-bl mice we found striking differences in their expression of TFR1 and response to immunological challenge by HKBA. Our preliminary data shows that expression levels of TFR1 were elevated in both TF mutant mice relative to control at baseline on lymphocyte, monocyte and granulocyte populations of the bone marrow. When challenged with a single intraperitoneal injection of HKBA, TfC-bl-HKBA mice suffered a more severe anemic phenotype with a 30% probability of survival following HKBA treatment. On the contrary, TfN-bl-HKBA mice displayed a blunted response to the immunological challenge, with faster recovery and no death. Taken together, our work provides additional evidence that TF is not only delivering iron cargo to cells, but also functioning as a signaling molecule via the lobe that is bound to iron. Despite each mouse only possessing a TF capable of binding one atom of iron, the two mouse models show profound differences in the pathophysiology of BT and AI. We hypothesize that the monoferric TF forms exert their influence via TFR2. We are currently characterizing a new loxp flanked TFR2-FLAG mouse model generated by our lab, which has been crossed to the TF monoferric mutant mice to test this hypothesis. Additionally, we are also exploring the translation potential of the two TF forms to treat anemia in BT and AI.

Engineering Peptide Inhibitors of the HFE-Transferrin Receptor 1 Complex.

The protein HFE (homeostatic iron regulator) is a key regulator of iron metabolism, and mutations in HFE underlie the most frequent form of hereditary haemochromatosis (HH-type I). Studies have shown that HFE interacts with transferrin receptor 1 (TFR1), a homodimeric type II transmembrane glycoprotein that is responsible for the cellular uptake of iron via iron-loaded transferrin (holo-transferrin) binding. It has been hypothesised that the HFE/TFR1 interaction serves as a sensor to the level of iron-loaded transferrin in circulation by means of a competition mechanism between HFE and iron-loaded transferrin association with TFR1. To investigate this, a series of peptides based on the helical binding interface between HFE and TFR1 were generated and shown to significantly interfere with the HFE/TFR1 interaction in an in vitro proximity ligation assay. The helical conformation of one of these peptides, corresponding to the α1 and α2 helices of HFE, was stabilised by the introduction of sidechain lactam “staples”, but this did not result in an increase in the ability of the peptide to disrupt the HFE/TFR1 interaction. These peptides inhibitors of the protein–protein interaction between HFE and TFR1 are potentially useful tools for the analysis of the functional role of HFE in the regulation of hepcidin expression.

Red Blood Cell Function and Disorders of Iron Metabolism

The red blood cell, or erythrocyte, is an anucleate biconcave disk, approximately 7 µm in diameter. The principal function of the red blood cell is to exchange carbon dioxide for oxygen while in the pulmonary circulation, exchange that oxygen for carbon dioxide in the peripheral tissue, and carry that carbon dioxide back to the lungs. The physicochemical composition of the red cell is aligned to optimize that function. The state of tissue oxygenation, in turn, regulates the production of red cells. This review covers red blood cell function, iron metabolism, iron deficiency, iron overload, and primary iron overload. Figures show a model of the hemoglobin molecule showing the relative alignment of the α chains and β chains; the normal oxygen-hemoglobin dissociation curve shifted by changes in temperature, pH, and the intracellular concentration of 2,3-diphosphoglycerate; body iron supply and storage; regulation of hepcidin expression and its role in disease; blood smear from a patient with iron deficiency; and mechanisms contributing to iron overload in iron-loading anemias. Tables list laboratory results associated with decreased iron balance, causes of iron deficiency, oral iron replacement therapy, parenteral iron replacement therapy, primary iron overload syndromes, secondary iron overload syndromes, laboratory results associated with increased iron stores, and other rare disorders of iron overload. This review contains 6 figures, 20 tables, and 111 references Keywords: Red blood cell, iron deficiency anemia, iron overload, hemochromatosis, hemoglobin, iron balance

Red Blood Cell Function and Disorders of Iron Metabolism

The red blood cell, or erythrocyte, is an anucleate biconcave disk, approximately 7 µm in diameter. The principal function of the red blood cell is to exchange carbon dioxide for oxygen while in the pulmonary circulation, exchange that oxygen for carbon dioxide in the peripheral tissue, and carry that carbon dioxide back to the lungs. The physicochemical composition of the red cell is aligned to optimize that function. The state of tissue oxygenation, in turn, regulates the production of red cells. This review covers red blood cell function, iron metabolism, iron deficiency, iron overload, and primary iron overload. Figures show a model of the hemoglobin molecule showing the relative alignment of the α chains and β chains; the normal oxygen-hemoglobin dissociation curve shifted by changes in temperature, pH, and the intracellular concentration of 2,3-diphosphoglycerate; body iron supply and storage; regulation of hepcidin expression and its role in disease; blood smear from a patient with iron deficiency; and mechanisms contributing to iron overload in iron-loading anemias. Tables list laboratory results associated with decreased iron balance, causes of iron deficiency, oral iron replacement therapy, parenteral iron replacement therapy, primary iron overload syndromes, secondary iron overload syndromes, laboratory results associated with increased iron stores, and other rare disorders of iron overload. This review contains 6 figures, 20 tables, and 111 references Keywords: Red blood cell, iron deficiency anemia, iron overload, hemochromatosis, hemoglobin, iron balance

Interrelations between Iron and Vitamin A-Studied Using Systems Approach.

A deficiency of vitamin A (VAD) and iron is the most common nutritional problem affecting people worldwide. Given the scale of the problem, the interactions between vitamin A and iron levels are widely studied. However, the exact mechanism of the impact of vitamin A on the regulation of iron metabolism remains unclear. An extremely significant issue becomes a better understanding of the nature of the studied biological phenomenon, which is possible by using a systems approach through developing and analyzing a mathematical model based on a Petri net. To study the considered system, the t-cluster analysis, the significance analysis, and the analysis of the average number of transition firings were performed. The used analyses have allowed distinguishing the most important mechanisms (both subprocesses and elementary processes) positively and negatively regulating an expression of hepcidin and allowed to distinguish elementary processes with a higher frequency of occurrence compared to others. The analysis also allowed to resolve doubts about the discrepancy in literature reports, where VAD leads to positive regulation of hepcidin expression or to negative regulation of hepcidin expression. The more detailed analyses have shown that VAD more frequently positively stimulates hepcidin expression and this mechanism is more significant than the mechanism inhibiting hepcidin expression indirectly by VAD.

Pentosan polysulfate regulates hepcidin expression in native Mongolian horses.

The aim of this study was to investigate the anti-hepcidin effect of pentosan polysulfate (PPS) in Mongolian horses. Twenty-six healthy horses were randomly allocated in to two-groups; one group was treated with a PPS once a week for 4-weeks while another group keeping as placebo. Blood samples at day 0 (D0), before race (BR; day 28) and after race (AR; day 28) were analyzed for serum biochemistry, hepcidin and iron concentrations. Significant reduction of hepcidin was observed at AR in PPS group when compared with BR placebo (P<0.05) and AR placebo (P<0.01). Mean hepcidin concentration difference of D0-BR and BR-AR in PPS was greater than the placebo whereas the iron concentration difference is reduced compared to placebo. Results indicate a novel therapeutic application of PPS as an anti-hepcidin compound to control hepcidin in horses while emphasizing further molecular studies.

Obligate N-Terminal but Not C-Terminal Monoferric Transferrin Ameliorates Anemia in β-Thalassemic Mice

Transferrin (TF) is a bilobed 80kD glycoprotein with N- and C-lobe iron binding sites. TF circulates as four forms: unbound to iron (apo-TF), iron bound to the N-lobe (monoferric N-TF), the C-lobe (monoferric-C), or to both lobes (diferric-TF). Most circulating TF under physiological conditions is monoferric. The iron-bound TF forms interact with TF receptor-1 (TFR1), which is ubiquitously expressed and serves as the main mechanism for cellular iron delivery. Iron-bound TF also interacts with TF receptor-2 (TFR2) which is expressed on hepatocytes, erythroblasts, and bone cells. Whereas TFR1 serves primarily as a cargo receptor, TFR2 serves primarily to influence cellular signaling events regulating hepcidin expression, erythropoiesis, and bone formation. We proposed that different transferrin forms provide differential signaling properties in this regulation. We thus generated TF mutant mice in which all iron-containing TF was either monoferric N (Tf monoN) or monoferric C (Tf monoC). Compared with Tf monoC mice, the Tf monoN mice demonstrated increased RBC production and increased hepcidin expression relative to iron status (Parrow et al. Blood). Based on observations in β-thalassemic mice treated with exogenous TF (Li et al. Nat Med), we hypothesized that β-thalassemic mice obligate for monoN TF would demonstrate improved erythropoietic and iron parameters compared with β-thalassemic mice obligate for monoC TF.To address this hypothesis, we crossed Hbb th3/+ mice (a mouse model of β-thalassemia intermedia) with Tf monoN and Tf monoC mice. Compared with Hbb th3Tf +/+mice, in Hbb th3/+Tf monoN mice demonstrated significantly increased RBC counts, elevated hemoglobin, improved erythrocyte morphology (Figure 1A-B), decreased splenomegaly, fewer bone marrow erythroblasts, and improvement of ineffective erythropoiesis (as measured by the ratio of progenitors to RBC in the bone marrow). Additionally, serum ERFE was significantly reduced and hepcidin levels were increased in Hbb th3/+Tf monoN relative to Hbb th3/+Tf +/+ controls. Conversely, hematological parameters from Hbb th3/+Tf monoC mice were comparable to Hbb th3/+Tf +/+ mice. Similarly, Hbb th3/+Tf monoC mice had no improvements in markers of ineffective erythropoiesis in the bone marrow compared with Hbb th3/+Tf +/+ mice.In summary, we demonstrate that the differential regulatory effects of monoN and monoC TF on erythropoiesis are relevant not only in steady-state, but also in the ineffective erythropoiesis that is characteristic of β-thalassemia. Because both monoN and monoC TF forms can deliver only one iron atom per TF-TFR1 binding event, our findings suggest that the improvements observed only in the Hbb th3/+Tf monoN mice were not due to iron restriction alone. We are now elucidating the mechanisms by which the two TF lobes exert their differential effects on ineffective erythropoiesis and exploring the translational potential of obligate monoN TF in the treatment of β-thalassemia. [Display omitted] DisclosuresRivella: Ionis Pharmaceuticals: Consultancy; Meira GTx: Consultancy.