P1: Role of iron in bone metabolism of Hfe-hemochromatosis

PI: Maja Vujic Spasic (Ulm)

Team: Peter Steele-Perkins (PhD student)

Objective: unravel the iron vs. endocrine effects of Hfe-hemochromatosis on bone

One of the most common forms of genetic iron overload disorders (hereditary hemochromatosis, HH) in Caucasians is caused by mutations in the HFE gene. Most of the HFE-HH cases are explained by attenuated BMP/SMAD signaling activity in hepatocytes and subsequently low expression of the liver hormone hepcidin. Given that there is no active mechanism to excrete the excess of iron, progressive iron accumulation in various tissues occurs, causing multiple organ dysfunction and eventually failure, including liver cirrhosis, hepatocellular carcinoma, cardiomyopathies, diabetes, and hypogonadism. Clinical observations have raised attention to the possible correlation between iron overload and the development of osteoporosis in HH. However, some inconsistencies between the data exist as to whether iron per se affects bone integrity in HFE-HH patients. We hypothesize that osteoporosis in HFE-HH does not primarily arise from iron overload, as our most recent work showed that systemic iron overload, at the degree present in Hfe-/- mice, does not associate with microarchitectural impairment of bones, thus excluding a negative effect of iron overload on bone integrity. We suggest that potential triggers throughout the course of disease progression could induce malfunction in bones. A more simple explanation would be gonadal deficiency, a commonly known cause of bone loss, due to its co-occurrence with iron overload in HFE-HH. Whether iron overload in Hfe-/- mice may act as additional culprit when other osteoporosis triggers are present, such as liver cirrhosis and endocrine defects, is currently unknown and will be investigated here.

P2: TMPRSS6-mediated regulation of iron via cleavage of BMP pathway components in liver and bone

PI: Andrea Steinbicker (Frankfurt) & Guiscard Seebohm (Münster)

Team: Isabelle Hornung (PhD student); Suma Choorapoikayil (PostDoc); Lisa Schrader (PostDoc); Anne Humberg (technician); Nathalie Strutz-Seebohm (Group leader)

Objective: elucidate effects of TMPRSS6 modulation on iron sensing and bone

The regulation of iron is crucial for liver and bone homeostasis. The transmembrane serine protease 6 (TMPRSS6) interacts with multiple components of the BMP signaling pathway including the BMP type I receptors ALK2 and ALK3, and thereby reduces the expression of the iron regulatory hormone hepcidin. Physiologically, hepcidin binds to and inactivates the iron exporter ferroportin if serum iron levels have reached sufficient levels. The loss of function mutation in TMPRSS6 causes iron-resistant-iron deficient anemia due to inappropriately high hepcidin levels. Even though aspects of TMPRSS6 binding and function in systemic iron homeostasis and liver regulation have been investigated, a detailed understanding of this regulatory system is elusive. The overall aim of this project is to investigate in depth signaling mechanisms and binding of TMPRSS6. Therefore, we will focus on the three main goals: 1) in silico-modeling and biochemistry of TMPRSS6 interactions with BMP receptors and the iron regulation proteins Hfe, Hjv, Tfr1, Tfr2, Kir2.1, Kv7.1, CaV2.1, 2) investigation of TMPRSS6 interactions with components of the BMP signaling pathway and other iron regulation proteins in vivo in mouse models, and 3) assessing the importance of TMPRSS6 in bone cells. In addition, we will investigate whether changes of systemic iron homeostasis impair the signaling mechanisms, which contributing factors occur, and how activation or blockage affects binding and receptor formation. This proposal will unravel mechanisms of TMPRSS6 actions in liver, iron sensing, and bone.

P3: Mechanisms of Tfr1-mediated iron uptake in bone and effects of iron on bone cell bioenergetics

PI: Martina Rauner (Dresden)

Team: Vanessa Passin (PhD student), Maria Ledesma-Colunga (associated PostDoc)

 Objective: unravel the role of iron on osteoblast bioenergetics and the importance of Tfr1 for iron uptake in bone

Osteoblasts rely on a delicate balance of iron for proper differentiation and function. While iron is necessary for various cellular processes including DNA replication, proliferation, and cellular respiration, high levels of iron suppress osteoblast function. Despite this knowledge, our basic understanding on the impact of iron on osteoblasts remains very limited. To date, it is unclear how iron affects cellular functions in osteoblasts and what metabolic pathways are involved in regulating cell function. It is even unknown how iron enters into osteoblasts. In this project, we will decipher these unresolved aspects. We hypothesize that transferrin receptor 1 (Tfr1) is the main iron-uptake receptor in osteoblasts. Moreover, our preliminary data lead us to hypothesize that high concentrations of iron promote osteoblast proliferation at the expense of differentiation, thereby limiting bone formation. These hypotheses will be tested by investigating the bone phenotype of osteoblast-specific Tfr1 knockout mice. As osteoclasts play a major role in bone resorption and the role of Tfr1 has also not been addressed in vivo, we will also characterize osteoclast-specific Tfr1 knock-out mice. To address how iron is utilized by specific osteoblast subsets, we will use single-cell RNA sequencing, untargeted metabolomics, and functional tests in vitro to link metabolic profiles to specific osteoblast subsets and their function (e.g. proliferation vs. matrix production vs. matrix mineralization). This project will unravel the importance of Tfr1 for iron uptake in the skeleton and will provide detailed insights into the role of iron in osteoblast function and bioenergetics.

P4: Defining the role of the iron-sensor Tfr2 in ossification processes

PI: Ulrike Baschant (Dresden)

Team: Sven Spangenberg (PhD student)

Objective: dissect the role of Tfr2 in ossification

Heterotopic ossification (HO) is a process of ossification at abnormal anatomical sites. It is a common severe medical complication in patients after total hip replacement and it is a hallmark of the rare disease, fibrodysplasia ossificans progressiva. HO develops through a process of endochondral ossification involving stages of inflammation, chondrogenesis and osteogenesis. Mechanistically, BMP signaling has been implicated in the pathogenesis of HO. The BMP pathway is also an important regulator of iron homeostasis and we have recently shown that the iron regulator transferrin receptor 2 (Tfr2) regulates bone formation via the BMP-p38-MAPK pathway. Based on our discovery that the extracellular domain of Tfr2 (Tfr2-ECD) can bind BMP ligands and can be used as a ligand-trap to reduce HO, we hypothesize that Tfr2 plays a crucial role in the development of HO. Therefore, we aim to define the regulation of Tfr2 and its source in the various phases of HO by analyzing the inflammatory as well as the chondrogenic and osteogenic stages of HO in WT and Tfr2-/–mice. Furthermore, we will determine the role of iron and other iron regulators in HO in mice. To unravel the underlying mechanisms of Tfr2 and BMP downstream signaling in HO, we will subject conditional Tfr2-deficient mice as well as conditional mice specific for BMP signal transducers to HO. Moreover, we will define the BMP binding sites within the Tfr2-ECD and test the ability of shorter BMP-binding Tfr2-ECD fragments to block BMP-2-induced ossification in vivo. The project will provide novel insights into the impact of iron regulators on HO. Refining the ligand trap may provide a novel therapeutic approach to abrogate HO.

P5: Identification of iron-related signals controlling BMP expression in liver non-parenchymal cells

PI: Martina Muckenthaler (Heidelberg)

Team: Ruiyue Qiu (PostDoc), Stefania Cucinelli (PhD student), Anja Schuh (Clinician Scientist)

Objective: understanding liver cell crosstalk during iron sensing

Control of systemic iron homeostasis evolved to maintain a plasma iron concentration that secures adequate supplies to tissues and cells while preventing organ iron overload. The small hepatocyte-derived peptide hormone hepcidin synchronizes systemic iron fluxes to control the amount of iron available in the circulation for cellular iron uptake (e.g. in bone cells). Hepcidin targets the iron export protein ferroportin to trigger its degradation, thus preventing dietary iron absorption and macrophage iron release.  A fundamental question in iron biology still remains unanswered: how are iron levels sensed by the liver? Hepcidin mRNA expression in hepatocytes is controlled by the BMP/Smad signaling pathway, whereby BMPs are expressed in liver non-parenchymal cells in response to changes in systemic iron levels. We hypothesize that a cross-talk of several liver cell types (hepatocytes, LSECs, stellate cells and Kupffer cells) will play a role for the hepcidin response to iron. By analyzing genetic mouse models of systemic iron overload and deficiency we will determine gene response patterns in all liver cell types and identify and validate pathways involved in the responses of BMPs to iron in primary cell (co) cultures. Additionally, we will test whether signals and pathways identified in the liver will also be operational in the bone for regulation of BMP levels.

P6: Dissecting the bone cell-specific functions of ferroportin

PI: Sandro Altamura (Heidelberg) & Lorenz Hofbauer (Dresden)

Team: Tiago Carvalho Oliveira (PhD student), Claudia Krause (technician)

Objective: dissect the role of Fpn in liver and bone

Iron balance is critical for bone health, and osteoporosis represents a frequent complication of iron overload disorders. Systemic iron homeostasis is maintained via the hepcidin/ferroportin (Fpn) system. Fpn mediates the release of iron from specialized iron-exporting cells such as duodenal enterocytes and splenic macrophages. Fpn levels, and thus the amount of iron exported, are post-translationally regulated by hepcidin, a hepatic hormone produced upon increased body iron levels that triggers Fpn internalization and degradation. Gain of function mutations of Fpn conferring resistance to hepcidin binding (FpnC326S) have been identified in patients with hereditary hemochromatosis type 4, characterized by an unrestricted systemic iron export that results in massive body iron overload. We have generated the FpnC326S knock-in mouse model of hereditary hemochromatosis type 4. As in the human disease, this model displays systemic iron overload and massive iron deposition in parenchymal tissues, including the liver. By contrast, cell types expressing Fpn are iron depleted due to hepcidin resistance. The central aim of this project is to identify the mechanisms whereby cell autonomous alteration in osteoblast and osteoclast iron content may affect bone remodeling and impair bone quality. Using customized mouse models of Fpn ablation (Fpn-ko) or stabilization (FpnC326S) specifically in osteoblasts and osteoclasts, we will address the following aims: (I) How do cell-specific alterations in bone iron content affect bone remodeling under normal and estrogen-deficient conditions? (II) How does Fpn prevent iron-related toxicity of osteoclasts/osteoblasts and impaired bone quality? (III) Which molecular patterns link imbalanced osteoblast/osteoclast iron content to altered bone remodeling and what are the translational consequences? This approach will allow us dissecting the component of the hormonal impairment from the cell-autonomous bone iron status in the pathogenesis of bone disorders.

P7: Unraveling mechanisms regulating the dynamics of BMP signal transduction and its impact on hepcidin expression with a systems biology approach

PI: Ursula Klingmüller (Heidelberg)

Team: Philipp Kastl (PostDoc)

Objective: disentangle the interactions between signaling pathways of iron sensing and their effects on bone

Hepatocytes are the major cells for iron storage and secrete the iron regulator hepcidin. Expression of hepcidin is induced by the BMP/Smad signaling pathway. Transferrin receptor 2 (Tfr2) binds BMP ligands and BMP receptors, and activates Smad signaling, whereas TMPRSS6 cleaves BMP receptors and decreases BMP signal transduction. Additionally, hepcidin is an acute phase protein that is induced by IL-6 via the JAK1-STAT3 pathway. It was suggested that the IL-6/STAT and BMP/Smad axes interact at the transcription factor level, but the impact of this cross-talk on the dynamics of BMP signaling and on hepcidin expression in hepatocytes remains to be mechanistically resolved. To disentangle such complex interrelations, we will use a systems biology approach. With this approach we identified the three most relevant Smad complexes and predicted their impact on target gene expression. Based on a mathematical model of the IL-6/STAT3 signaling pathway, we predicted optimal inhibitor dosing to modulate the acute phase response including hepcidin expression. In our project we will address the communication between bone cells and hepatocytes ensuring iron homeostasis. By a mass spectrometric approach we will quantify the dynamics of plasma factors in response to iron overload in mouse models. Utilizing our integrative mathematical model of IL-6/BMP signal transduction and a targeted mass spectrometry approach, we will elucidate the impact of Tfr2 and of TMPRSS6 on the dynamics of hepcidin expression and will identify mechanisms regulating hepcidin expression that are altered in chronic liver diseases. The integrative approach that quantitatively dissects the communication between bone and liver and the regulation of iron homeostasis promises to identify novel strategies to prevent iron overload.

P8: Impact of iron overload and anemia on fractures

PI: Andrea Burden (Zürich), Andrea Steinbicker (Frankfurt) & Lorenz Hofbauer (Dresden)

Team: Marcel Rauer (Clinician Scientist)

Objective: provide insights into the occurrence of fractures with iron disorders and their associated co-morbidities and co-medications

Anemia and osteoporosis are both frequent diseases and often coincide, especially among the aging population where low-grade inflammation is common. It has become evident that disturbances in iron metabolism are associated with bone disease. In addition, inflammation is a major cause of anemia and bone loss. Considering the clinical relevance of these diseases, the overall goal of this project is to investigate the mutual interactions between inflammation, iron homeostasis and bone metabolism in mice and men. To that end we will focus on determining the association of anemia/hemochromatosis and fractures in humans using the UK IQVIA Medical Research Database (IMRD). In preliminary analyses, we have shown that the cohort data is representative to the German population and that cohort analyses are feasible. A particular interest will be the analysis of co-morbidities, co-medication and fracture outcomes. Thus, this project will provide novel outcomes and insights into the occurrence of fractures with iron disorders and their associated co-morbidities and co-medications.