Final Report Summary - EUROIRON1 (Genetic control of the pathogenesis of diseases based on iron accumulation)
The occurrence of diseases of iron accumulation such as Genetic iron overload (GIO) and Anaemia of chronic diseases (ACD), and their phenotypic variability, involve genetic abnormalities that affect the expression of proteins involved in iron metabolism. The project investigated the genetic factors involved, directly or indirectly, with expression of the key genes of iron metabolism associated with pathological iron accumulation. It assessed the functional consequences of their abnormal expression in cellular and animal models. It also explored how this knowledge can be used for introducing diagnostic and prognostic markers for defining the particular type of GIO or ACD concerned and its optimal therapeutic management by established and experimental procedures. This contributes to the improved knowledge of the genetic control of the pathogenesis of other diseases based on iron metabolism disorders. The technological objectives consisted in the development of innovative methodological approaches and tools, based on animal and cellular models, and resorting to analytical methods for in situ tracing of cellular labile iron pool and gene expression studies.
Summary of work performed, achievements, end results
Genetic control of cellular iron uptake:
- Focusing on intestinal heme iron uptake, so far poorly characterised in contrast of non heme iron uptake, this research has shown that:
i) in knock-out mouse models mimicking the genetic iron overload situations of type 1 (HFE-related) and type 2 (hepcidin-related) haemochromatosis, heme iron uptake is also enhanced, therefore contributing to the development of iron overload;
ii) competition exists between substrates heme and folate for the transport protein HCP1.
- Using the differentiated HepaRG cell model, an increased expression of L-ferritin gene by spermine and spermidine was found, reflecting at least partly an increased cellular iron storage.
- A significant reducing effect of the calcium channel blocker verapamil on tissue iron loading in HPX (hypotransferrinemic) mice, in contrast of hepcidin knock-out mice, suggesting that genetic factors may modulate the verapamil effect.
Genetic control of cellular iron egress:
Two main pathological situations, characterised by decreased cellular iron egress, have been studied: the ferroportin disease and the anaemia of chronic disease (ACD).
Studying ferroportin expression in macrophages from patients with ferroportin disease revealed that ferroportin expression for all tested mutations was increased in ferroportin patients' macrophages, predominantly located intracellularly and not inhibited by hepcidin. These data indicate that ferroportin mutations impair ferroportin trafficking to the plasma membrane and induce iron retention in human monocytes-macrophages.
Studying ACD and ACD + IDA (iron deficiency anaemia) revealed low duodenal ferroportin expression and high serum hepcidin levels, in ACD whereas an opposite situation was observed in ACD + IDA. This has important clinical applications since hepcidin levels, which are more responsive to the erythropoietic demand for iron than to inflammation, may help to differentiate ACD and ACD + IDA and to select appropriate iron therapy in these patients. Using a coculture model, a stimulation of ferroportin expression upon certain pathogens was found. This enhanced ferroportin expression promoted iron export, therefore limiting iron availability for pathogens.
Genetic control of systemic iron regulation
Determining the role of hepcidin in iron regulation has revealed that:
- A human monoclonal antibody has been patented. It is of high affinity for hepcidin and speficic to human hepcidin. It recognises serum human hepcidin and possesses a neutralising activity on hepcidin in vitro as well as in vivo. Therefore, this antibody should allow developing an ELISA assay for clinical applications.
- In contrast to macrophagic cell types, hepatocytes had been poorly studied in terms of the presence of the iron exporter ferroportin. The present data, obtained in a hepatocyte culture system and in hepcidin-deficient mice, showed that hepatocytic ferroportin is a target of hepcidin. The fact that hepcidin can trigger ferroportin degradation in hepatocytes should be taken into account when considering hepcidin therapeutics.
- The inhibition, through specific SiRNAs, of hepcidin expression in the coculture model associating mouse hepatocytes and rat liver epithelial cells, has provided a list of promising genes that are regulated by hepcidin, independently of iron overload.
- Transcriptomic and proteomic analysis of hepcidin knock-out mice showed a decrease in liver and serum haptoglobin due to failure to fold and assembly proteins in the endoplasmic reticulum affected by the iron related oxidative stress. This result is important knowing the many clinical situations which can induce endoplasmic reticulum stress.
Characterisation of genes involved in the control of systemic iron regulation revealed:
- A new coculture model, combining mouse hepatocytes and rat liver epithelial cells, has been set up. It allows a very high level of hepcidin mRNA expression and therefore represents a valuable tool for studying hepcidin regulation. Natural (curcumin) and synthetic STAT3 inhibitors have been shown, in this model, to reduce hepcidin expression in differentiated mouse hepatocytes expressing the active phosphorylated STAT3 form.
- Accumulation of iron within periportal hepatocytes is a feature of iron overload of digestive origin. No metabolic lobular zonation was found for most genes, including in particular hepcidin and ferroportin. However, a preferential periportal expression of ceruloplasmin was observed, raising the issue of its special role in iron overload disorders involving a defect in cellular iron export.
- Hemojuvelin is known as a key protein of iron metabolism, leading, when mutated, to juvenile haemochromatosis. Characterisation of hemojuvelin showed that defective targeting to the plasma membrane accounts for the inability of most hemojuvelin mutants to activate hepcidin. Investigating the origin of soluble hemojuvelin also showed that it is produced by a furin cleavage.
- SMAD7 controls iron metabolism as a potent inhibitor of hepcidin expression.
- BMP6 has been demonstrated as a major gene involved in the control of hepcidin expression as demonstrated by the strong haemochromatosis phenotype of BMP6 -/- mice. BMP6 may constitute a strong candidate to modulate endogenously the production of hepcidin.
- Despite the key implication of BMP6 in hepcidin expression, Hfe -/- mice exhibit an increased BMP6 expression (related to hepatic iron overload) contrasting with decreased hepcidin expression. These data suggest that Hfe contributes to the BMP / SMAD cascade in a way which is critical for the regulation of hepcidin expression.
- Focusing on the novel major hepcidin suppressor, TMPRSS6 (matriptase-2), identified by others in 2008, we demonstrated that matriptase-2 inhibits hepcidin only in the presence of hemojuvelin and by cleaving the hemojuvelin isoform localised on the plasma membrane.
- CREBH involved in the endoplasmic stress response, has been identified as a critical factor in the response to hepcidin promoter in stress situations. This implies that endoplasmic reticulum stress controls iron metabolism through induction of hepcidin.
- Cross-talk between the mitogen activated protein kinase and bone morphogenetic protein / hemojuvelin pathways is required for the induction of hepcidin by holotransferrin in primary mouse hepatocytes.
- HIF-2 alpha, but not HIF-1 alpha, promotes iron absorption in mice. This finding may support new strategies to improve iron homeostasis in patients with iron disorders.
- Secondary iron overload in mice leads to decreased plasma iron bioavailability related to hepcidin increase with subsequent limitation of splenic iron release. These data support the importance of managing hepcidin levels before starting venesection therapy in patients with secondary iron overload and eligible for phlebotomies.
Validation studies of the targeted genes and pathophysiological mechanisms in humans revealed:
- A new mutation in the hepcidin promoter impairs its BMP response (proximal BMP-RE) and contributes to a severe phenotype in Hfe-related haemochromatosis. This helps diagnose unexplained iron overload situations.
- Daily regulation of serum hepcidin is not influenced by submaximal muscle exercise in healthy volunteers, suggesting that muscles may not be physiologically a major modulator of iron homeostasis, and having potential implications for hepcidin sampling in medical practice.
- Studying serum levels of GDF-15, a growth differentiation factor associated to iron metabolism through modulation of hepcidin expression, in patients with ACD, ACD + IDA, and IDA implied that other ACD-related factors may overcome the regulatory effects of GDF-15 on hepcidin expression during inflammation.
- A novel form of secondary iron overload has been identified. It corresponds to acquired aceruloplasminemia related to copper deficiency, itself due at least partly to excessive zinc intake. This underlines the strong interactions between zinc, copper and iron metabolisms.
Animal and cellular models and novel technologies
- Generating conditional HFE knock-out mice
- Using fluorescent probes, demonstration that the iron chelator deferiprone can act as a siderophore
- Using frataxin-deficient cells, demonstration that deferiprone-mediated relocation of iron restores cell functions impaired by frataxin deficiency
- Development of animal models involving Iron regulatory proteins (IRP)
- Development of animal models involving HFE
- Development of cell models involved in the pathogenesis of disorders of iron metabolism
- Development of novel technologies for the functional assessment of genes related to iron metabolism.
A systematic approach to identify new genes controlling body iron stores
Genotyping of 120 HFE -/- mice, completed for almost 400 SNP markers, has identified 8 clusters of genes exhibiting a summary expression trait significantly correlated with iron.
The phenotypic and genotypic study of a genetic Italian isolate consisting of 1800 subjects has confirmed that the widely reported effect of HFE and TMPRSS6 on haemoglobin and erythrocyte trait variations is mediated by their effect on iron homeostasis. They also suggest that TMPRSS6 variants, opposite to HFE C282Y, are good candidate modifiers of the phenotype of the C282Y / C282Y patients and might contribute to the non-penetrance of HFE hemochromatosis.
Almost 200 patients with either severe or mild / asymptomatic C282Y homozygotes have been genotyped for 250K SNPs. Preliminary analyses have shown associations between SNPs on different chromosomes and disease severity. Some of them are located in genes that could be functionally relevant for iron metabolism.
These complementary approaches have therefore led to the identification of a manageable set of genes to test for functional causality. In conclusion, identifying new physiological actors intervening in iron metabolism, elucidating the mechanisms whereby mutated genes generate iron disorders, and discovering genes which account for the variable phenotypic expression, have constituted the main scientific goals of the present consortium.
The improved understanding of the genetic mechanisms underlying widespread diseases related to iron accumulation, which has been permitted by EuroIron, should lead in the near future to novel diagnostic, prognostic and therapeutic approaches which will ameliorate the overall curative and preventive management of these disorders.
Publishable results for using and disseminating the knowledge
The present project has provided new tools for exploring iron metabolism and its disorders, as well as new models in vivo and in vitro providing interesting alternative to in vivo models, such as cocultures. It provided new methods for tracking intracellular labile iron with the design of agents for attaining iron distribution and led to the production of a patented human monoclonal hepcidin antibody. A major result was the identification of new genes of iron metabolism and / or characterisation of new regulatory processes for iron metabolism. The project also revealed improved mechanistic knowledge in iron-related disorders. During the whole duration of the contract, the knowledge dissemination among the medicoscientific community has been the following: 58 original articles published in peer-reviewed Journals (among them: Nature Genetics, Science, Gastroenterology, Cell Metabolism, Blood), 36 general reviews, 63 invited conferences, 31 oral presentations, and 24 posters.
Summary of work performed, achievements, end results
Genetic control of cellular iron uptake:
- Focusing on intestinal heme iron uptake, so far poorly characterised in contrast of non heme iron uptake, this research has shown that:
i) in knock-out mouse models mimicking the genetic iron overload situations of type 1 (HFE-related) and type 2 (hepcidin-related) haemochromatosis, heme iron uptake is also enhanced, therefore contributing to the development of iron overload;
ii) competition exists between substrates heme and folate for the transport protein HCP1.
- Using the differentiated HepaRG cell model, an increased expression of L-ferritin gene by spermine and spermidine was found, reflecting at least partly an increased cellular iron storage.
- A significant reducing effect of the calcium channel blocker verapamil on tissue iron loading in HPX (hypotransferrinemic) mice, in contrast of hepcidin knock-out mice, suggesting that genetic factors may modulate the verapamil effect.
Genetic control of cellular iron egress:
Two main pathological situations, characterised by decreased cellular iron egress, have been studied: the ferroportin disease and the anaemia of chronic disease (ACD).
Studying ferroportin expression in macrophages from patients with ferroportin disease revealed that ferroportin expression for all tested mutations was increased in ferroportin patients' macrophages, predominantly located intracellularly and not inhibited by hepcidin. These data indicate that ferroportin mutations impair ferroportin trafficking to the plasma membrane and induce iron retention in human monocytes-macrophages.
Studying ACD and ACD + IDA (iron deficiency anaemia) revealed low duodenal ferroportin expression and high serum hepcidin levels, in ACD whereas an opposite situation was observed in ACD + IDA. This has important clinical applications since hepcidin levels, which are more responsive to the erythropoietic demand for iron than to inflammation, may help to differentiate ACD and ACD + IDA and to select appropriate iron therapy in these patients. Using a coculture model, a stimulation of ferroportin expression upon certain pathogens was found. This enhanced ferroportin expression promoted iron export, therefore limiting iron availability for pathogens.
Genetic control of systemic iron regulation
Determining the role of hepcidin in iron regulation has revealed that:
- A human monoclonal antibody has been patented. It is of high affinity for hepcidin and speficic to human hepcidin. It recognises serum human hepcidin and possesses a neutralising activity on hepcidin in vitro as well as in vivo. Therefore, this antibody should allow developing an ELISA assay for clinical applications.
- In contrast to macrophagic cell types, hepatocytes had been poorly studied in terms of the presence of the iron exporter ferroportin. The present data, obtained in a hepatocyte culture system and in hepcidin-deficient mice, showed that hepatocytic ferroportin is a target of hepcidin. The fact that hepcidin can trigger ferroportin degradation in hepatocytes should be taken into account when considering hepcidin therapeutics.
- The inhibition, through specific SiRNAs, of hepcidin expression in the coculture model associating mouse hepatocytes and rat liver epithelial cells, has provided a list of promising genes that are regulated by hepcidin, independently of iron overload.
- Transcriptomic and proteomic analysis of hepcidin knock-out mice showed a decrease in liver and serum haptoglobin due to failure to fold and assembly proteins in the endoplasmic reticulum affected by the iron related oxidative stress. This result is important knowing the many clinical situations which can induce endoplasmic reticulum stress.
Characterisation of genes involved in the control of systemic iron regulation revealed:
- A new coculture model, combining mouse hepatocytes and rat liver epithelial cells, has been set up. It allows a very high level of hepcidin mRNA expression and therefore represents a valuable tool for studying hepcidin regulation. Natural (curcumin) and synthetic STAT3 inhibitors have been shown, in this model, to reduce hepcidin expression in differentiated mouse hepatocytes expressing the active phosphorylated STAT3 form.
- Accumulation of iron within periportal hepatocytes is a feature of iron overload of digestive origin. No metabolic lobular zonation was found for most genes, including in particular hepcidin and ferroportin. However, a preferential periportal expression of ceruloplasmin was observed, raising the issue of its special role in iron overload disorders involving a defect in cellular iron export.
- Hemojuvelin is known as a key protein of iron metabolism, leading, when mutated, to juvenile haemochromatosis. Characterisation of hemojuvelin showed that defective targeting to the plasma membrane accounts for the inability of most hemojuvelin mutants to activate hepcidin. Investigating the origin of soluble hemojuvelin also showed that it is produced by a furin cleavage.
- SMAD7 controls iron metabolism as a potent inhibitor of hepcidin expression.
- BMP6 has been demonstrated as a major gene involved in the control of hepcidin expression as demonstrated by the strong haemochromatosis phenotype of BMP6 -/- mice. BMP6 may constitute a strong candidate to modulate endogenously the production of hepcidin.
- Despite the key implication of BMP6 in hepcidin expression, Hfe -/- mice exhibit an increased BMP6 expression (related to hepatic iron overload) contrasting with decreased hepcidin expression. These data suggest that Hfe contributes to the BMP / SMAD cascade in a way which is critical for the regulation of hepcidin expression.
- Focusing on the novel major hepcidin suppressor, TMPRSS6 (matriptase-2), identified by others in 2008, we demonstrated that matriptase-2 inhibits hepcidin only in the presence of hemojuvelin and by cleaving the hemojuvelin isoform localised on the plasma membrane.
- CREBH involved in the endoplasmic stress response, has been identified as a critical factor in the response to hepcidin promoter in stress situations. This implies that endoplasmic reticulum stress controls iron metabolism through induction of hepcidin.
- Cross-talk between the mitogen activated protein kinase and bone morphogenetic protein / hemojuvelin pathways is required for the induction of hepcidin by holotransferrin in primary mouse hepatocytes.
- HIF-2 alpha, but not HIF-1 alpha, promotes iron absorption in mice. This finding may support new strategies to improve iron homeostasis in patients with iron disorders.
- Secondary iron overload in mice leads to decreased plasma iron bioavailability related to hepcidin increase with subsequent limitation of splenic iron release. These data support the importance of managing hepcidin levels before starting venesection therapy in patients with secondary iron overload and eligible for phlebotomies.
Validation studies of the targeted genes and pathophysiological mechanisms in humans revealed:
- A new mutation in the hepcidin promoter impairs its BMP response (proximal BMP-RE) and contributes to a severe phenotype in Hfe-related haemochromatosis. This helps diagnose unexplained iron overload situations.
- Daily regulation of serum hepcidin is not influenced by submaximal muscle exercise in healthy volunteers, suggesting that muscles may not be physiologically a major modulator of iron homeostasis, and having potential implications for hepcidin sampling in medical practice.
- Studying serum levels of GDF-15, a growth differentiation factor associated to iron metabolism through modulation of hepcidin expression, in patients with ACD, ACD + IDA, and IDA implied that other ACD-related factors may overcome the regulatory effects of GDF-15 on hepcidin expression during inflammation.
- A novel form of secondary iron overload has been identified. It corresponds to acquired aceruloplasminemia related to copper deficiency, itself due at least partly to excessive zinc intake. This underlines the strong interactions between zinc, copper and iron metabolisms.
Animal and cellular models and novel technologies
- Generating conditional HFE knock-out mice
- Using fluorescent probes, demonstration that the iron chelator deferiprone can act as a siderophore
- Using frataxin-deficient cells, demonstration that deferiprone-mediated relocation of iron restores cell functions impaired by frataxin deficiency
- Development of animal models involving Iron regulatory proteins (IRP)
- Development of animal models involving HFE
- Development of cell models involved in the pathogenesis of disorders of iron metabolism
- Development of novel technologies for the functional assessment of genes related to iron metabolism.
A systematic approach to identify new genes controlling body iron stores
Genotyping of 120 HFE -/- mice, completed for almost 400 SNP markers, has identified 8 clusters of genes exhibiting a summary expression trait significantly correlated with iron.
The phenotypic and genotypic study of a genetic Italian isolate consisting of 1800 subjects has confirmed that the widely reported effect of HFE and TMPRSS6 on haemoglobin and erythrocyte trait variations is mediated by their effect on iron homeostasis. They also suggest that TMPRSS6 variants, opposite to HFE C282Y, are good candidate modifiers of the phenotype of the C282Y / C282Y patients and might contribute to the non-penetrance of HFE hemochromatosis.
Almost 200 patients with either severe or mild / asymptomatic C282Y homozygotes have been genotyped for 250K SNPs. Preliminary analyses have shown associations between SNPs on different chromosomes and disease severity. Some of them are located in genes that could be functionally relevant for iron metabolism.
These complementary approaches have therefore led to the identification of a manageable set of genes to test for functional causality. In conclusion, identifying new physiological actors intervening in iron metabolism, elucidating the mechanisms whereby mutated genes generate iron disorders, and discovering genes which account for the variable phenotypic expression, have constituted the main scientific goals of the present consortium.
The improved understanding of the genetic mechanisms underlying widespread diseases related to iron accumulation, which has been permitted by EuroIron, should lead in the near future to novel diagnostic, prognostic and therapeutic approaches which will ameliorate the overall curative and preventive management of these disorders.
Publishable results for using and disseminating the knowledge
The present project has provided new tools for exploring iron metabolism and its disorders, as well as new models in vivo and in vitro providing interesting alternative to in vivo models, such as cocultures. It provided new methods for tracking intracellular labile iron with the design of agents for attaining iron distribution and led to the production of a patented human monoclonal hepcidin antibody. A major result was the identification of new genes of iron metabolism and / or characterisation of new regulatory processes for iron metabolism. The project also revealed improved mechanistic knowledge in iron-related disorders. During the whole duration of the contract, the knowledge dissemination among the medicoscientific community has been the following: 58 original articles published in peer-reviewed Journals (among them: Nature Genetics, Science, Gastroenterology, Cell Metabolism, Blood), 36 general reviews, 63 invited conferences, 31 oral presentations, and 24 posters.
