Interplay Between Iron Overload and Osteoarthritis: Clinical Significance and Cellular Mechanisms

There are multiple diseases or conditions such as hereditary hemochromatosis, hemophilia, thalassemia, sickle cell disease, aging, and estrogen deficiency that can cause iron overload in the human body. These diseases or conditions are frequently associated with osteoarthritic phenotypes, such as progressive cartilage degradation, alterations in the microarchitecture and biomechanics of the subchondral bone, persistent joint inflammation, proliferative synovitis, and synovial pannus. Growing evidences suggest that the conditions of pathological iron overload are associated with these osteoarthritic phenotypes. Osteoarthritis (OA) is an important complication in patients suffering from iron overload-related diseases and conditions. This review aims to summarize the findings and observations made in the field of iron overload-related OA while conducting clinical and basic research works. OA is a whole-joint disease that affects the articular cartilage lining surfaces of bones, subchondral bones, and synovial tissues in the joint cavity. Chondrocytes, osteoclasts, osteoblasts, and synovial-derived cells are involved in the disease. In this review, we will elucidate the cellular and molecular mechanisms associated with iron overload and the negative influence that iron overload has on joint homeostasis. The promising value of interrupting the pathologic effects of iron overload is also well discussed for the development of improved therapeutics that can be used in the field of OA.

New Mutations in HFE2 and TFR2 Genes Causing Non HFE-Related Hereditary Hemochromatosis

Hereditary hemochromatosis (HH) is an iron metabolism disease clinically characterized by excessive iron deposition in parenchymal organs such as liver, heart, pancreas, and joints. It is caused by mutations in at least five different genes. HFE hemochromatosis is the most common type of hemochromatosis, while non-HFE related hemochromatosis are rare cases. Here, we describe six new patients of non-HFE related HH from five different families. Two families (Family 1 and 2) have novel nonsense mutations in the HFE2 gene have novel nonsense mutations (p.Arg63Ter and Asp36ThrfsTer96). Three families have mutations in the TFR2 gene, one case has one previously unreported mutation (Family A—p.Asp680Tyr) and two cases have known pathogenic mutations (Family B and D—p.Trp781Ter and p.Gln672Ter respectively). Clinical, biochemical, and genetic data are discussed in all these cases. These rare cases of non-HFE related hereditary hemochromatosis highlight the importance of an earlier molecular diagnosis in a specialized center to prevent serious clinical complications.

Dissecting the Mechanisms of Hepcidin and BMP-SMAD Pathway Regulation By FKBP12

The BMP-SMAD pathway is activated when a dimeric ligand (BMP) interacts with a dimeric serine threonine kinase receptor (BMPRII) and triggers the activation of a dimeric BMP type I receptor (BMPRI). Catalytically active BMPRIs phosphorylate SMAD1/5/8 that, upon SMAD4 binding, translocate to the nucleus to regulate the expression of BMP target genes, including hepcidin. Hepcidin is the main regulator of iron homeostasis that controls body iron levels by binding and blocking the sole iron exporter ferroportin. In agreement, hepcidin expression is homeostatically activated by serum and liver iron, and its deficiency is a common hallmark of Hereditary Hemochromatosis (HH) and the major cause of iron overload in beta thalassemia. The components of the BMP-SMAD pathway relevant for hepcidin regulation are ALK2 and ALK3 (BMPRI); BMPR2 and ACVR2A (BMPRII), BMP2 and BMP6 (BMP ligands). Recently, we have identified the immunophilin FKBP12 as an inhibitor of hepcidin and demonstrated that FKBP12 binds ALK2 to avoid ligand-independent activation of the BMP-SMAD pathway. To investigate the mechanism of BMP-SMAD pathway and hepcidin regulation by FKBP12, we performed in vitro, ex vivo and in vivo studies. We found that FKBP12 sequestration by the immunosuppressive drug Tacrolimus (TAC) stabilizes ALK2-ALK2 homodimers and ALK2-ALK3 heterodimers in a transfected human hepatoma cell line. In addition, it increases the interaction of ALK2 with ACVR2A and BMPR2. To investigate the role of FKBP12 on BMP-SMAD signaling, BMPRI and II were silenced in murine primary hepatocytes. Despite FKBP12 co-immunoprecipitates only with ALK2, silencing of Alk2 and Alk3 completely blunts TAC-mediated BMP-SMAD pathway activation, suggesting that FKBP12 functionally interacts also with ALK3. Acvr2a silencing impairs TAC-dependent hepcidin upregulation, whereas Bmpr2 silencing does not. As expected, Fkbp12 silencing abrogates hepcidin upregulation by TAC, confirming the main role of this immunophilin in hepcidin regulation. In vivo, TAC treatment upregulates hepcidin in wild type and HH mouse models, but surprisingly, Fkbp12 mRNA downregulation by ASOs does not. Interestingly, Fkbp 2, 4 and 8 are highly expressed in murine hepatocytes and, according to literature data, are able to bind to TAC. Of note, Fkbp12 is the least expressed immunophilin in murine primary hepatocytes. To further investigate the FKBPs involved in TAC-dependent hepcidin regulation, Fkbp2, 4 and 8 were knockdown in murine primary HCs that were then treated with TAC. The TAC effect is preserved in siFkbp2- and siFkbp4-derived HCs, but abolished when Fkbp8 was downregulated. Overall these data suggest that: 1) FKBP12 regulates BMP-SMAD signaling by favoring ALK2-ALK3 homo and heterodimerization, and interaction with BMPRII in the absence of ligands; 2) TAC-mediated hepcidin upregulation is dependent upon ALK2, ALK3, ACVR2A, FKBP12 and FKBP8. 3) In vivo, TAC treatment upregulates hepcidin whereas Fkbp12 silencing does not, suggesting the existence of redundancy between the different FKBPs. Further studies are needed to dissect the role of FKBP8 in BMP-SMAD pathway and hepcidin regulation. Disclosures Aghajan: Ionis Pharmaceuticals, Inc.: Current Employment. Muckenthaler: Silence Therapeutics: Research Funding. Guo: Ionis Pharmaceuticals, Inc.: Current Employment.

Rusfertide (PTG-300), a Hepcidin Mimetic, Maintains Liver Iron Concentration in the Absence of Phlebotomies in Patients with Hereditary Hemochromatosis

Introduction: Patients with hereditary hemochromatosis (HH) require continued phlebotomies to limit end-organ damage. Approximately 25% of patients in maintenance felt receiving phlebotomies was "inconvenient" or "very inconvenient" (Brisott et al, 2011). Patient compliance with phlebotomies generally declines with time (Hicken et al, 2003), and therapeutic phlebotomies may not be medically suitable for some HH patients. Rusfertide, a peptide mimetic of hepcidin, is an effective regulator of iron distribution and utilization that has demonstrated control of iron in an animal model of HH. Methods: We conducted an open-label, dose-finding efficacy study that investigated subcutaneous rusfertide in HH patients on a stable phlebotomy regimen of 0.25 to 1 phlebotomy per month for at least 6 months. Patients with clinical laboratory abnormalities and those receiving iron chelation therapy or erythrocytapheresis were excluded. Subjects received individually titrated rusfertide doses once or twice a week to maintain transferrin saturation (TSAT) below 45% and were followed for 6 months. Study measures included TSAT, serum iron, transferrin and ferritin, liver iron concentration (LIC) measured by MRI, and adverse events (AEs). Results: Sixteen subjects (10 male/6 female) were enrolled. Mean age and weight were 62.5 years and 88.1 kg, respectively. LIC values were maintained at pre-study levels, with minimal use of phlebotomies during the duration of the study (Figure 1A). Average pre-study phlebotomy rate was 0.27 phlebotomies/month compared to 0.03 phlebotomies/month during the study (p<0.0001; Figure 1B). There was a dose- and concentration-dependent decrease in serum iron and TSAT (Figure 2A and 2B). Transferrin levels were relatively constant over the course of the study. There were no notable changes in hematological parameters such as hematocrit, erythrocytes, leucocytes, or platelets. Rusfertide was generally well tolerated. Adverse events reported in 2 or more subjects included diarrhea, fatigue, injection site reactions (erythema, induration, pain, pruritis), dizziness, and headache. Conclusions: Rusfertide demonstrated a pharmacodynamic effect in reducing TSAT and serum iron, with corresponding significant reduction in the number of phlebotomies, and with LIC maintained at pre-study levels with minimal use of phlebotomies. These data indicate rusfertide was well tolerated in patients with HH and controls LIC, supporting development of rusfertide as a potential treatment for HH. Figure 1 Figure 1. Disclosures Kowdley: PTG: Consultancy, Research Funding. Modi: Protagonist Therapeutics: Current Employment. Valone: Protagonist Therapeutics: Current Employment, Current equity holder in publicly-traded company. Gupta: Protagonist Therapeutics: Current Employment.