Examination of the role of hemojuvelin and matriptase-2 in the modulation of hepcidin, a key iron regulator

Elizabeth Leddy (2010). Examination of the role of hemojuvelin and matriptase-2 in the modulation of hepcidin, a key iron regulator MPhil Thesis, School of Medicine, The University of Queensland.

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Author Elizabeth Leddy
Thesis Title Examination of the role of hemojuvelin and matriptase-2 in the modulation of hepcidin, a key iron regulator
School, Centre or Institute School of Medicine
Institution The University of Queensland
Publication date 2010-05
Thesis type MPhil Thesis
Supervisor Nathan Subramaniam
Daniel Wallace
Total pages 157
Total colour pages 16
Total black and white pages 141
Subjects 11 Medical and Health Sciences
Abstract/Summary Iron plays important functional roles in mammalian cells, including oxygen transport and redox reactions. The liver expressed peptide hepcidin plays a central role in the regulation of iron homeostasis. Hepcidin levels are inversely correlated with iron levels in the body, and are regulated by a number of iron-related genes. Hemojuvelin is a liver-expressed protein, mutations in which lead to severe iron-overload early in life (known as juvenile haemochromatosis). Complications of iron accumulation in the liver, pancreas, heart and pituitary gland can lead to diabetes mellitus, cardiomyopathy, cirrhosis and hypogonadism. Hemojuvelin is known to be a positive regulator of hepcidin. Matriptase-2 is a serine protease expressed in the liver. Patients with mutations in matriptase-2 manifest iron refractory iron deficiency anaemia, indicating that matriptase-2 is a hepcidin suppressor. The mechanism of suppression of hepcidin expression by matriptase-2 is therefore a new area of interest in iron metabolism. The overall aims of this thesis were to define the relationship between hemojuvelin and matriptase-2 and examine their role in regulating hepcidin and therefore iron levels in the body. In order to further understand how hemojuvelin regulates hepcidin, disease-causing mutations in hemojuvelin were replicated in a liver cell line model to examine the dysregulation in hepcidin modulation upon mutation as compared to wild-type. It was hypothesised that mutations in hemojuvelin would lead to changes in the secretion and trafficking of hemojuvelin in addition to dysregulation of hepcidin. An antibody to hemojuvelin was produced in order to detect hemojuvelin in lieu of adequate commercial antibodies. In order to elucidate the relationship between the liver expressed proteins hemojuvelin and matriptase-2, an in vitro model was used to determine changes in mRNA and protein of each respective gene when co-expressed. It was hypothesised that hemojuvelin may be a substrate for matriptase-2 in the regulation of the key iron regulator, hepcidin. This thesis (Chapter 3) examines the effect of two juvenile haemochromatosis disease-causing mutations of hemojuvelin (G320V and R326X) on the secretion of hemojuvelin and response of hepcidin to induction with bone morphogenetic protein (BMP)-9 in a human liver cell line (Huh7). The functional regulation of hepcidin in a HepG2 liver cell line in response to exposure to secreted mutant hemojuvelin was also investigated. It was observed that mutations in hemojuvelin have significant effects including dysregulation of hepcidin, leading to altered iron homeostasis. Rabbit and sheep anti-hemojuvelin antibodies were generated and characterised in their detection of hemojuvelin levels in cell lysate and human liver (Chapter 4). The antibodies were generated in host animals via injection of a tagged recombinant human hemojuvelin peptide, located within the first half of the protein. The immunological reaction was monitored by Western blot analysis and subsequent test bleeds indicated low antibody titre. A total of eight injections over seven months produced an antibody that was of high concentration and purity. However, specificity was too low to be of further use and the antibody was therefore not used for subsequent experiments. The final Chapter (5) investigates the proposal that hemojuvelin is processed by matriptase-2 in the regulation of hepcidin. It was observed that Huh7 cells co-expressing hemojuvelin and matriptase-2 had a significant down-regulation of hepcidin when stimulated with BMP 9 but not BMP 2. At the same time, the hetero-dimeric membrane-bound form of hemojuvelin was observed to be reduced alongside the release of the serine protease domain of matriptase-2, indicating activity of this protease. This is a major finding of this thesis and one that was confirmed within months of discovery by the publication of similar findings. Additionally, it was observed that soluble hemojuvelin, known to bind BMP 2, did so in cell culture significantly, thus inhibiting the induction of hepcidin in this system. While BMP 9 is not known to bind hemojuvelin, it served as an alternative model to BMP 2 in this system as it was found that hemojuvelin significantly enhanced BMP 9 induced mRNA expression of hepcidin. Overall this thesis examines the relationship between hemojuvelin, matriptase-2 and hepcidin. The study of the molecular and cellular consequences of juvenile haemochromatosis causing mutations in hemojuvelin has provided further elucidation of the significant role of hemojuvelin in regulating hepcidin in healthy and disease states. The production of anti-hemojuvelin antibodies has provided an insight into the complexity and difficulty of producing antibodies against this protein. Finally, the finding that hemojuvelin acts as a substrate for matriptase-2 is a significant finding of this thesis, providing important new insights into the iron-regulatory pathway. The discovery has been confirmed by a similar in vitro study and is compatible with recent findings in an in vivo system comprising of hemojuvelin and matriptase-2 double mutant mice. The significant role played by matriptase-2 in regulating hepcidin through hemojuvelin is only just beginning to be fully understood and further clarification has broad implications for the understanding of iron homeostasis and the potential for development of new treatments for both iron deficiency and overload.
Keyword Haemochromatosis
Additional Notes Colour pages: 22, 25, 29, 32, 33, 35, 74, 77, 83, 98, 130, 132, 133, 134, 140, 142 Landscape pages: 90, 91, 138, 140, 142, 144

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Created: Wed, 10 Nov 2010, 14:04:14 EST by Ms Elizabeth Leddy on behalf of Library - Information Access Service