A quantitative analysis of hepcidin promoter regulation using mathematical modelling techniques to reveal principles underlying systemic iron homeostasis

Since the development of advanced mathematical modelling techniques in biology, thermodynamics (and therefore equilibrium statistical mechanics) has played a key role in mathematically quantifying biological activities. We use this underlying notion of thermodynamic ‘micro-states’ to attempt to uncover how the hormone hepcidin under the influence of two major signalling pathways maintains systemic iron homeostasis. Systemic iron homeostasis involves a negative feedback circuit in which the expression level of the peptide hormone hepcidin depends on and controls the iron blood levels. Hepcidin expression is regulated by the BMP6/SMAD and IL6/STAT signalling cascades. Deregulation of either pathway causes iron storage diseases such as hemochromatosis or anaemia of inflammation (AI). We quantitatively analyzed how BMP and IL6 control hepcidin expression in human hepatoma (HuH7) cells. We used data from our experimental collaborators who measured transcription factor phosphorylation and reporter gene expression under co-stimulation conditions and perturbed the promoter by mutagenesis. We applied statistical data analysis and mathematical modelling to reveal possible biological mechanisms that control hepcidin expression at the promoter level. Specifically we develop a thermodynamic modelling framework that is able to simulate and predict possible molecular mechanisms that might underlie iron homeostasis by hepcidin. Our results reveal that hepcidin cross- regulation primarily occurs by combinatorial transcription factor binding to the promoter, whereas signalling crosstalk is insignificant. We find that the presence of two BMP-responsive elements in the promoter ensures high sensitivity towards the iron-sensing BMP signalling axis, which promotes iron homeostasis in vivo. IL6 stimulation reduces the promoter sensitivity to the BMP signal that may explain the disturbance of iron homeostasis in AI. We get to understand why the iron homeostasis circuit is sensitive to certain perturbations implicated in disease. Taken together, our work reveals how mathematical quantification and modelling can aid in understanding biological phenomenon that underlies gene expression

Iron, hepcidin, and microcytosis in canine hepatocellular carcinoma

2021 Summer.Includes bibliographical references.Hepatocellular carcinoma (HCC) is the most common primary liver tumor found in dogs. There is evidence that iron dysregulation is associated with HCC pathogenesis in both humans and dogs. Anemia and thrombocytosis were common hematologic abnormalities detected in about half of dogs with massive HCC, and microcytosis was present in approximately 31% of dogs in one study. Additionally, humans with hereditary hemochromatosis have an increased risk of HCC. The liver is the major organ site for iron storage and metabolism containing numerous iron regulatory proteins which may play an important role in canine HCC. Since microcytosis is associated with iron restricted erythropoiesis, our first objective was to determine whether neoplastic hepatocytes exhibit differential expression of iron regulatory genes as well as hepatic iron stores in normocytic versus microcytic HCC cases in an initial pilot study. Next, we aimed to quantify and compare expressions of a larger set of iron regulatory and human HCC-related genes among canine HCC tumor tissue, adjacent peritumoral liver tissue, non-specific reactive hepatitis liver tissue from non-HCC dogs, and normal liver tissue, as well as to quantify and compare estimated hepatic iron stores. We hypothesized that canine HCC tumor tissue exhibits iron overloading and higher expression of hepcidin and its upstream regulators (IL-6 and BMP6), which would promote intracellular iron availability for neoplastic hepatocyte proliferation. We also hypothesized that microcytic HCC cases would exhibit higher expression levels of hepcidin in tumor tissue compared to tumors from normocytic dogs. Additionally, we explored associations between clinical parameters and RNA levels of iron regulatory genes as well as estimated hepatocellular iron stores in both HCC tumor and the adjacent, peritumoral tissues. We expected to find gene expression patterns in canine HCC tumor tissue related to abnormal regulation of iron metabolism and other pathways similar to what has been described in human malignancies. Cases were selected from a database search for canine HCC and included if complete pre-operative blood work was available and there was adequate formalin-fixed paraffin-embedded (FFPE) tissue for RNA isolation for all cases. Hematologic and clinical parameters were recorded and used for correlation studies. All liver sections were reviewed by a board-certified veterinary anatomic pathologist. RNA was isolated from FFPE blocks and NanoString nCounter platform was used to quantify RNA counts for selected genes. Sections were stained with Perls Prussian Blue stain and hepatocytic iron stores were estimated using NIS-Elements software. Contrary to our hypotheses, all canine HCC tumors had markedly decreased expression of hepcidin (HAMP) and depletion of hepatocellular iron stores. Other iron-related genes down-regulated in canine HCC tumor tissue included TfR2 (an upstream regulator of hepcidin), STEAP2, LTF, HMOX1, CYBRD1and SFXN5. Tumor tissue overexpressed TfR1, STEAP3, and LCN2. No significant differences in RNA levels or iron stores were found between tumors of microcytic and normocytic HCC cases, but the adjacent peritumoral tissue was markedly iron loaded and exhibited negative correlation between hepcidin RNA levels and mean cell volume (MCV) as well as serum iron. Microcytic HCC cases were associated with noteworthy clinical findings such as increased ALT, lower HCT and serum iron, and histologically more poorly differentiated tumors. Differential expression of genes involved in Wnt signaling and ferroptosis was observed in canine HCC tumor versus the adjacent peritumoral liver tissue

Transcriptional and Post-transcriptional Regulation of Hepcidin and Iron Metabolism by Lipid Signaling in the Liver

Although iron is required for essential biological processes, excess iron is detrimental due to oxidative damage induced by iron-mediated Fenton reactions, which promote tissue injury. Cellular iron uptake, transport and storage must therefore be tightly regulated. This task is accomplished mainly through hepcidin, the key iron-regulatory hormone. Hepcidin is synthesized primarily in hepatocytes as a circulatory antimicrobial peptide. It controls iron metabolism by inhibiting iron absorption from the duodenum and iron release from reticuloendothelial macrophages. Besides synthesizing hepcidin, the liver plays an important role in maintaining iron homeostasis by serving as the main storage organ for excess iron. Patients with liver diseases frequently display disturbances of iron metabolism but the underlying mechanisms are unclear. Due to obesity epidemic worldwide, the incidence of nonalcoholic fatty liver disease (NAFLD) is on the rise. This study therefore focuses on the regulation of hepcidin in NAFLD. NAFLD is the hepatic manifestation of metabolic syndrome, which is characterized by visceral adiposity, dyslipidemia and insulin resistance. Both the level and distribution of iron in the livers of NAFLD patients have been shown to correlate with disease severity. NAFLD patients have also been reported to display changes in hepcidin expression. The significance and relevance of these alterations regarding NAFLD pathogenesis are unclear. Although impaired fatty acid metabolism and lipid accumulation in the liver are major contributors to the pathogenesis of NAFLD, the role of lipids or lipid derivatives in hepcidin regulation have not been investigated. The studies presented in this dissertation identified new and unique mechanisms of hepcidin gene regulation by saturated fatty acids and the biologically active lipid derivative, ceramide in human hepatoma cells. The post-transcriptional regulation of hepcidin expression by palmitic acid was mediated through AU-rich element binding protein, Human Antigen R (HuR) and novel class of protein kinase C isoforms. Ceramide, on the other hand, induced hepcidin transcription via inflammatory JAK/STAT3 signaling. Furthermore, by using high fat-fed hepcidin knockout mice as an in vivo model, I have implicated a role for hepcidin in the regulation of hepatic lipid metabolism, and characterized these mice as a potential experimental model to study liver injury in NAFLD

Doctor of Philosophy

dissertationIron overload has been proposed to be a component of the insulin-resistance syndrome. On the other hand, dietary iron supplementation is associated with growth and increased appetite in children. We found the concentration of circulating ferritin is associated with low serum adiponectin, the insulin-sensitizing adipokine, and leptin, the adipokine that is responsible for regulating food intake. Similarly, mice fed a high iron diet and 3T3-L1 adipocytes treated with iron exhibited decreased adiponectin and leptin mRNA and protein. Iron negatively regulates transcription of adiponectin promoter-driven luciferase activity in a FOXO1-dependent manner. However, iron decreases the inactivated form of FOXO1, acetyl-FOXO1, while leaving phosphorylated FOXO1 and total FOXO1 unaffected. The mechanism of iron's effect on adiponectin and promotion of metabolic syndrome is demonstrated via the binding affinity of multiple transcription factors in the adiponectin promoter using ChIP studies. The higher activation of FOXO1 in iron-treated cells contributes to more inactivation of PPARγ through direct interaction, leading to decreased adiponectin transcription and expression. These findings directly demonstrate a causal role for iron as a risk factor for metabolic syndrome and a role for adipocytes in modulating metabolism through adiponectin in response to iron stores. We found iron negatively regulated leptin transcription via cAMP response element binding protein (CREB) activation. Two potential CREB-binding sites were identified in the mouse leptin promoter region. Mutation of both sites completely blocked the effect of iron on promoter activity. We also found enrichment of phospho-CREB iv binding to those two sites by ChIP in 3T3-L1 adipocytes treated with iron. CREB is modified and destabilized by O-GlcNAc modification. Iron also negatively regulates O-GlcNAc modification both in 3T3-L1 and epididymal fat, and mice heterozygous for deletion of the gene encoding the enzyme that removes O-GlcNAc, O-GlcNAcase, showed significantly increased serum leptin compared to wild-type mice. Glucosamine treatment rescued high iron-induced decrease of leptin promoter activity. These results suggest that the effects of iron on leptin may be via crosstalk of O-GlcNAcylation and phosphorylation of CREB. These findings indicate that levels of dietary iron play an important role in regulation of appetite and fat metabolism through CREB-dependent modulation of leptin expression

Molecular coordination of iron homeostasis by microRNA

Iron is an essential nutrient critical for oxygen transport, DNA synthesis, ATP generation, and cellular proliferation. At the molecular level, insufficient iron elicits a cascade of cellular events aimed at conserving iron for the maintenance of these life-preserving functions, but tissue-specific responses and metabolic adaptations to iron deficiency (ID) are not fully understood. Recently, small regulatory RNA molecules called microRNA (or miRNA) have been identified as an important mechanism for regulating various cellular processes. Therefore we sought to determine if the expression pattern of miRNA changes in response to dietary ID and to examine the potential regulatory capacity of miRNA in the adaptive response to ID. To do this, we first characterized the expression of miRNA in the livers of iron-sufficient and iron-deficient animals using next-generation sequencing technology. Results compiled from three different bioinformatics approaches indicate that ~10 miRNA are differentially expressed in the livers of ID rats. Further bioinformatics analyses suggested that at least two of these miRNA, miR-210 and miR-181d, had predicted targets directly involved in either the maintenance of iron homeostasis or the metabolic adaptation to iron deficiency. We then used reporter assays to validate the putative miRNA targets including the miR-210 target, cytoglobin, and the miR-181d targets, carnitine palmitoyltransferase 1B and mitoferrin 1. These findings have provided insight into the metabolic adaptation to ID and have demonstrated how miRNA contribute to the molecular coordination of iron homeostasis in a physiologic model of dietary ID

An RNAi screen identifies TLR2/6 as mediators of a novel inflammatory pathway for rapid hepcidin-independent hypoferremia

Systemic iron homeostasis is essential for human health. Its maintenance critically depends on the interaction between the hepatic hormone hepcidin and the sole known iron exporter ferroportin (FPN) predominantly expressed in hepatocytes, duodenal enterocytes and macrophages. Hepcidin binding leads to FPN internalization and degradation resulting in cellular iron retention. Iron is an essential nutrient also for pathogens and plays a central role in host-pathogen interactions. The innate immune system fights infections by sequestration of iron in macrophages of the reticuloendothelial system. The resulting hypoferremia represents a major host defence strategy. A current model posits that hepcidin is the crucial effector of this response, as its release from macrophages and hepatocytes provokes FPN protein decrease and, consequently, tissue iron retention. The aim of my PhD project was to identify novel cellular regulators of hepcidin-mediated ferroportin (FPN) degradation, a fundamental process that controls systemic iron homeostasis. To reach this aim I generated a HeLa cell line expressing a hFPN-renilla fusion protein, which was used for a focused high-throughput RNAi screen targeting kinases and related proteins. Out of 779 genes tested, the screen identified 71 putative regulators of FPN protein stability. Validation experiments confirmed the phenotype of 24 genes. Interestingly, most validated regulators of FPN expression conferred hepcidin-independent FPN regulation. From these I selected 14 genes associated with immune processes for further characterization in murine bone marrow-derived macrophages (BMDMs). Finally, my studies focused on Toll-like receptor 6 (TLR6) as an effective regulator of FPN expression in BMDMs and I investigated how the TLR6 activation pathway modulates iron regulation in the inflammatory context. TLR2/6 ligation by the synthetic lipoprotein derived from Mycoplasma: FSL1 triggered a profound decrease in FPN mRNA and protein expression in BMDMs as well as in the liver and the spleen of mice. Unexpectedly hepcidin expression remained unchanged. Hepcidin-independent FPN down regulation was a conserved response to different microbial lipopeptides and elicited a fast, hepcidin-independent hypoferremia pathway. These findings were further confirmed in C326S FPN knock-in mice with a disrupted hepcidin/FPN regulatory circuitry. This work challenges the prevailing role of hepcidin in inflammatory hypoferremia and suggests that rapid hepcidin-independent FPN down regulation may represent the first line response to restrict iron access to pathogens

Novel loci affecting iron homeostasis and their effects in individuals at risk for hemochromatosis

Variation in body iron is associated with or causes diseases, including anaemia and iron overload. Here, we analyse genetic association data on biochemical markers of iron status from 11 European-population studies, with replication in eight additional cohorts (total up to 48,972 subjects). We find 11 genome-wide-significant (

Novel loci affecting iron homeostasis and their effects in individuals at risk for hemochromatosis.

Variation in body iron is associated with or causes diseases, including anaemia and iron overload. Here, we analyse genetic association data on biochemical markers of iron status from 11 European-population studies, with replication in eight additional cohorts (total up to 48,972 subjects). We find 11 genome-wide-significant (P<5 × 10(-8)) loci, some including known iron-related genes (HFE, SLC40A1, TF, TFR2, TFRC, TMPRSS6) and others novel (ABO, ARNTL, FADS2, NAT2, TEX14). SNPs at ARNTL, TF, and TFR2 affect iron markers in HFE C282Y homozygotes at risk for hemochromatosis. There is substantial overlap between our iron loci and loci affecting erythrocyte and lipid phenotypes. These results will facilitate investigation of the roles of iron in disease

The evolution, modifications and interactions of proteins and RNAs

Proteins and RNAs are two of the most versatile macromolecules that carry out almost all functions within living organisms. In this thesis I have explored evolutionary and regulatory aspects of proteins and RNAs by studying their structures, modifications and interactions. In the first chapter of my thesis I investigate domain atrophy, a term I coined to describe large-scale deletions of core structural elements within protein domains. By looking into truncated domain boundaries across several domain families using Pfam, I was able to identify rare cases of domains that showed atrophy. Given that even point mutations can be deleterious, it is surprising that proteins can tolerate such large-scale deletions. Some of the structures of atrophied domains show novel protein-protein interaction interfaces that appear to compensate and stabilise their folds. Protein-protein interactions are largely influenced by the surface and charge complementarity, while RNA-RNA interactions are governed by base-pair complementarity; both interaction types are inherently different and these differences might be observed in their interaction networks. Based on this hypothesis I have explored the protein-protein, RNA-protein and the RNA-RNA interaction networks of yeast in the second chapter. By analysing the three networks I found no major differences in their network properties, which indicates an underlying uniformity in their interactomes despite their individual differences. In the third chapter I focus on RNA-protein interactions by investigating post-translational modifications (PTMs) in RNA-binding proteins (RBPs). By comparing occurrences of PTMs, I observe that RBPs significantly undergo more PTMs than non-RBPs. I also found that within RBPs, PTMs are more frequently targeted at regions that directly interact with RNA compared to regions that do not. Moreover disorderedness and amino acid composition were not observed to significantly influence the differential PTMs observed between RBPs and nonRBPs. The results point to a direct regulatory role of PTMs in RNA-protein interactions of RBPs. In the last chapter, I explore regulatory RNA-RNA interactions. Using differential expression data of mRNAs and lncRNAs from mouse models of hereditary hemochromatosis, I investigated competing regulatory interactions between mRNA, lncRNA and miRNA. A mutual interaction network was created from the predicted miRNA interaction sites on mRNAs and lncRNAs to identify regulatory RNAs in the disease. I also observed interesting relations between the sense-antisense mRNA-lncRNA pairs that indicate mutual regulation of expression levels through a yet unknown mechanism

Transferrin receptor 2 is emerging as a major player in the control of iron metabolism

Abstract Our knowledge of mammalian iron metabolism has advanced dramatically over recent years. Iron is an essential element for virtually all living organisms. Its intestinal absorption and accurate cellular regulation is strictly required to ensure the coordinated synthesis of the numerous iron-containing proteins involved in key metabolic processes, while avoiding the uptake of excess iron that can lead to organ damage. A range of different proteins exist to ensure this fine control within the various tissues of the body. Among these proteins, transferrin receptor (TFR2) seems to play a key role in the regulation of iron homeostasis. Disabling mutations in TFR2 are responsible for type 3 hereditary hemochromatosis (Type 3 HH). This review describes the biological properties of this membrane receptor, with a particular emphasis paid to the structure, function and cellular localization. Although much information has been garnered on TFR2, further efforts are needed to elucidate its function in the context of the iron regulatory network