The regulation of the hepcidin by modulators of the bone morphogenetic protein (BMP) pathway
Hepcidin, the iron regulatory peptide, has emerged as the master regulator of systemic iron homeostasis. Under normal circumstances, hepcidin expression is upregulated by excess iron and inflammation; and downregulated by iron deficiency, anaemia and hypoxia. Insights gained into the pathogenesis of iron-storage disorders such as hereditary haemochromatosis (HH) and hypotransferrinaemia (HPX), have contributed to the identification of the molecular mechanisms governing hepcidin expression. In these disorders, hepcidin expression is inappropriately low, causing increased absorption of iron by the gut thus leading to iron overload. The precise mechanism by which hepcidin is suppressed in these disorders is unclear. The regulation of hepcidin is principally transcriptional, where the Bone Morphogenetic Protein (BMP) pathway has been shown to be a major network governing hepcidin expression. The current study utilised the HPX mouse model to identify potential regulators of hepcidin that are produced locally in liver with a focus on the regulation of hepcidin by the BMP signalling pathway. A known regulator of the BMP pathway, bone morphogenetic protein [BMP]-binding endothelial cell precursor-derived regulator (BMPER), was found to be overexpressed in the HPX mouse liver. Soluble BMPER peptide in excess strongly inhibited BMP-dependent hepcidin promoter activity in both HepG2 and Huh7, cells abolishing the effects of BMP2 and attenuating the effects of BMP6. These effects correlated with reduced cellular pSMAD levels. Addition of recombinant BMPER peptide to primary human hepatocytes strongly downregulated hepcidin mRNA levels and abolished the effects of BMP2. These effects were reflected in vivo where the injection of mice with recombinant BMPER peptide significantly reduced hepcidin mRNA expression which correlated with increased serum iron levels. Thus, the protein may play an important role in suppressing hepcidin production to increase iron availability under conditions of chronic anaemia. Using the same HPX model, several other genes related to the BMP pathway also demonstrated differential gene expression. Atonal Homologue 8 (ATOH8), a basic helix-loop helix (bHLH) transcription factor, was previously shown to be regulated by iron loading however its precise role in iron metabolism was unknown. The studies presented in this thesis identified hepatic ATOH8 mRNA and protein expression to be robustly downregulated in various mouse models of altered iron metabolism where increased erythropoietic activity was shown to suppress hepcidin. Further investigations demonstrated that ATOH8 expression in HEK-293 cells directly regulated the hepcidin promoter and increased cellular pSMAD levels, thereby establishing a hitherto missing link between the regulation of hepcidin, erythropoietic activity and the BMP/SMAD pathway. Finally, the little known BMP, BMP8b, and the clotting factor, von Willebrand Factor which were also found to be significantly increased in the liver of the HPX mouse, were investigated as potential regulators of hepcidin. BMPSb has the potential to form heterodimeric complexes with other BMP members and therefore could be important in modulating BMP signalling and thus in turn hepcidin promoter activity. Whereas von Willebrand factor contains BMP binding sites and therefore may sequester BMPs, thus inhibiting BMP signalling. However the present study was unable to show consistent effects of either molecule on hepcidin promoter activity and so the roles of BMPSb and von Willebrand Factor in iron metabolism, if any, remains unclear. In conclusion, the analyses presented in this thesis demonstrate the novel molecular interactions governing hepcidin regulation by the BMP pathway. The new knowledge generated will be useful in the development of therapeutic strategies to diagnose, prevent, or mitigate disorders of iron homeostasis.
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