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Fine Tuning of Hepcidin Expression by Positive and Negative Regulators

2008; Cell Press; Volume: 8; Issue: 1 Linguagem: Inglês

10.1016/j.cmet.2008.06.009

1932-7420

Martina U. Muckenthaler,

Erythropoietin and Anemia Treatment

Hepcidin, the systemic regulator of iron homeostasis is activated by proteins responsible for hereditary hemochromatosis, bone morphogenetic proteins (BMPs), and inflammatory cytokines. Three recent publications now identify a novel hepcidin suppressor, the transmembrane serine protease TMPRSS6 (also known as matriptase-2), which is required to sense iron deficiency. Hepcidin, the systemic regulator of iron homeostasis is activated by proteins responsible for hereditary hemochromatosis, bone morphogenetic proteins (BMPs), and inflammatory cytokines. Three recent publications now identify a novel hepcidin suppressor, the transmembrane serine protease TMPRSS6 (also known as matriptase-2), which is required to sense iron deficiency. Balancing systemic iron levels within narrow limits is critical for human health: diseases of iron deficiency and overload belong to the most frequent disorders worldwide. Regulation of systemic iron homeostasis evolved to maintain a plasma iron concentration that secures adequate supplies while preventing organ iron overload. The regulatory system responds to signals from pathways that consume iron (e.g., erythropoiesis) and transmits signals to cells that supply iron (e.g., duodenal enterocytes that absorb dietary iron, macrophages that recycle iron from effete erythrocytes, and hepatocytes that store iron). The small hepatic peptide hormone hepcidin synchronizes these systemic iron fluxes by binding to the iron export channel ferroportin located on the surface of macrophages, hepatocytes, and intestinal enterocytes to cause its internalization and proteolysis. In accordance with this mechanism chronically elevated hepcidin levels (e.g., in the anemia of chronic diseases; ACD) cause systemic iron deficiency and inappropriately low hepcidin levels (e.g., in hereditary hemochromatosis; HH) result in iron overload (Nemeth and Ganz, 2006Nemeth E. Ganz T. Annu. Rev. Nutr. 2006; 26: 323-342Crossref PubMed Scopus (525) Google Scholar). Here, we review recent advances that identify the transmembrane serine protease TMPRSS6 as a suppressor of hepcidin expression. TMPRSS6 is mutated in individuals with iron-refractory iron deficiency anemia (IRIDA) and is critical for adequate dietary iron uptake to prevent iron deficiency (Du et al., 2008Du X. She E. Gelbart T. Truksa J. Lee P. Xia Y. Khovananth K. Mudd S. Mann N. Moresco E.M. et al.Science. 2008; 320: 1088-1092Crossref PubMed Scopus (442) Google Scholar, Folgueras et al., 2008Folgueras A.R. Martin de Lara F. Pendas A.M. Garabaya C. Rodriguez F. Astudillo A. Bernal T. Cabanillas R. Lopez-Otin C. Velasco G. Blood Press. 2008; (Published online June 3, 2008)https://doi.org/10.1182/blood-2008-04-149773Crossref Scopus (239) Google Scholar, Finberg et al., 2008Finberg K.E. Heeney M.M. Campagna D.R. Aydinok Y. Pearson H.A. Hartman K.R. Mayo M.M. Samuel S.M. Strouse J.J. Markianos K. et al.Nat. Genet. 2008; 40: 569-571Crossref PubMed Scopus (491) Google Scholar). Key research within the field aims to identify activators and suppressors of hepcidin synthesis. Hepcidin activators include proteins responsible for hereditary hemochromatosis (HFE, TfR2, and hemojuvelin [HFE2]) and the bone morphogenetic proteins (BMPs) that induce hepcidin transcription via the SMAD signaling pathway (e.g., Babitt et al., 2006Babitt J.L. Huang F.W. Wrighting D.M. Xia Y. Sidis Y. Samad T.A. Campagna J.A. Chung R.T. Schneyer A.L. Woolf C.J. et al.Nat. Genet. 2006; 38: 531-539Crossref PubMed Scopus (792) Google Scholar, Wang et al., 2005Wang R.H. Li C. Xu X. Zheng Y. Xiao C. Zerfas P. Cooperman S. Eckhaus M. Rouault T. Mishra L. Deng C.X. Cell Metab. 2005; 2: 399-409Abstract Full Text Full Text PDF PubMed Scopus (474) Google Scholar) (Figure 1). Additionally, inflammatory cytokines increase hepcidin expression via the JAK/STAT pathway (Wrighting and Andrews, 2006Wrighting D.M. Andrews N.C. Blood. 2006; 108: 3204-3209Crossref PubMed Scopus (642) Google Scholar). Comparatively little is known about mechanisms that suppress hepcidin synthesis during iron deficiency. In recent work by Du et al., 2008Du X. She E. Gelbart T. Truksa J. Lee P. Xia Y. Khovananth K. Mudd S. Mann N. Moresco E.M. et al.Science. 2008; 320: 1088-1092Crossref PubMed Scopus (442) Google Scholar, such a negative regulator of hepcidin expression, the putative serine protease TMPRSS6, was discovered. A role for TMPRSS6 in maintaining iron homeostasis was identified when the chemically induced mutant mouse phenotype “mask” was analyzed that is characterized by progressive loss of body hair, microcytic anemia, low plasma iron levels, and low body iron stores. The mask mutation maps to chromosome 15 and generates abnormal splice products that lack the proteolytic domain of TMPRSS6. TMPRSS6 mutant mice show impaired dietary iron uptake and fail to suppress hepcidin mRNA expression in response to dietary iron deficiency. More recently, analysis of a mouse line with a TMPRSS6 null allele yielded highly similar data and extended the observations from mask mutant mice in that high hepatic hepcidin mRNA levels correlate with low ferroportin protein expression and iron accumulation in duodenal enterocytes (Folgueras et al., 2008Folgueras A.R. Martin de Lara F. Pendas A.M. Garabaya C. Rodriguez F. Astudillo A. Bernal T. Cabanillas R. Lopez-Otin C. Velasco G. Blood Press. 2008; (Published online June 3, 2008)https://doi.org/10.1182/blood-2008-04-149773Crossref Scopus (239) Google Scholar). TMPRSS6 thus is required to render mice sensitive to low iron stores and to suppress hepcidin expression. Consistent with the data in mice, TMPRSS6 overexpression in a hepatoma cell line decreases hepcidin promoter activity and counteracts stimuli known to activate hepcidin expression. How TMPRSS6 regulates the expression of hepcidin is still unknown but the crosstalk to the hepcidin promoter involves the proximal 200 bp adjacent to the start of transcription (Du et al., 2008Du X. She E. Gelbart T. Truksa J. Lee P. Xia Y. Khovananth K. Mudd S. Mann N. Moresco E.M. et al.Science. 2008; 320: 1088-1092Crossref PubMed Scopus (442) Google Scholar) where the STAT-3 binding site that controls IL6-mediated hepcidin transcription (Verga Falzacappa et al., 2006Verga Falzacappa M.V. Vujic Spasic M. Kessler R. Stolte J. Hentze M.W. Muckenthaler M.U. Blood. 2006; 109: 353-358Crossref PubMed Scopus (423) Google Scholar) and the multifunctional BMP-responsive element important for both HJV/BMP and IL6-mediated hepcidin activation (Verga Falzacappa et al., 2008Verga Falzacappa M.V. Casanovas G. Hentze M.W. Muckenthaler M.U. J. Mol. Med. 2008; 86: 531-540Crossref PubMed Scopus (109) Google Scholar) are located. In an independent study, Finberg et al., 2008Finberg K.E. Heeney M.M. Campagna D.R. Aydinok Y. Pearson H.A. Hartman K.R. Mayo M.M. Samuel S.M. Strouse J.J. Markianos K. et al.Nat. Genet. 2008; 40: 569-571Crossref PubMed Scopus (491) Google Scholar investigated gene defects in five families characterized by congenital hypochromic, microcytic anemia, low mean corpuscular erythrocyte volume, and low transferrin saturation. Because in these individuals anemia does not resolve following treatment with oral iron and the response to parenteral iron administration is incomplete, this disease entity is termed iron-refractory iron deficiency anemia (IRIDA). Genetic analysis of these families indicates linkage of IRIDA to chromosome 22q12-13. Interestingly, the TMPRSS6 gene is located within this critical interval, and mutations in the TMPRSS6 gene are detected in all five families studied in addition to two individuals with sporadic IRIDA. All identified mutations lie distal to exon 8 in the highly conserved region of the conserved trypsin-like serine protease domain that was shown by Du et al., 2008Du X. She E. Gelbart T. Truksa J. Lee P. Xia Y. Khovananth K. Mudd S. Mann N. Moresco E.M. et al.Science. 2008; 320: 1088-1092Crossref PubMed Scopus (442) Google Scholar to be required for TMPRSS6-mediated hepcidin suppression in cultured cells. Consistent with the findings in mask mutant (Du et al., 2008Du X. She E. Gelbart T. Truksa J. Lee P. Xia Y. Khovananth K. Mudd S. Mann N. Moresco E.M. et al.Science. 2008; 320: 1088-1092Crossref PubMed Scopus (442) Google Scholar) and TMPRSS6 knockout (Folgueras et al., 2008Folgueras A.R. Martin de Lara F. Pendas A.M. Garabaya C. Rodriguez F. Astudillo A. Bernal T. Cabanillas R. Lopez-Otin C. Velasco G. Blood Press. 2008; (Published online June 3, 2008)https://doi.org/10.1182/blood-2008-04-149773Crossref Scopus (239) Google Scholar) mice, urinary hepcidin levels in individuals with IRIDA are inappropriately high, although hepcidin is typically undetectable in patients with iron deficiency. Chronically elevated hepcidin expression prevents iron release from duodenal enterocytes and macrophages explaining why both orally applied and parenteral injected iron cannot be efficiently utilized by IRIDA patients. While in many aspects the phenotype of IRIDA patients resembles the one of TMPRSS6-deficient mice (Table 1), it is currently unclear why oral and parenteral iron application is unable to normalize iron deficiency in individuals with IRIDA while anemia and hair loss is resolved upon iron treatment in both murine TMPRSS6-deficiency models (Du et al., 2008Du X. She E. Gelbart T. Truksa J. Lee P. Xia Y. Khovananth K. Mudd S. Mann N. Moresco E.M. et al.Science. 2008; 320: 1088-1092Crossref PubMed Scopus (442) Google Scholar, Folgueras et al., 2008Folgueras A.R. Martin de Lara F. Pendas A.M. Garabaya C. Rodriguez F. Astudillo A. Bernal T. Cabanillas R. Lopez-Otin C. Velasco G. Blood Press. 2008; (Published online June 3, 2008)https://doi.org/10.1182/blood-2008-04-149773Crossref Scopus (239) Google Scholar). Analysis of hepcidin expression in TMPRSS6-deficient mice following iron application may yield further insight into this question.Table 1Clinical and Biochemical Parameters in IRIDA Patients Mask Mice and Mice with a TMPRSS6 Null AlleleIRIDA Patients (Finberg et al., 2008Finberg K.E. Heeney M.M. Campagna D.R. Aydinok Y. Pearson H.A. Hartman K.R. Mayo M.M. Samuel S.M. Strouse J.J. Markianos K. et al.Nat. Genet. 2008; 40: 569-571Crossref PubMed Scopus (491) Google Scholar)Mask Mutant Mice (Du et al., 2008Du X. She E. Gelbart T. Truksa J. Lee P. Xia Y. Khovananth K. Mudd S. Mann N. Moresco E.M. et al.Science. 2008; 320: 1088-1092Crossref PubMed Scopus (442) Google Scholar)TMPRSS6−/− Mice (Folgueras et al., 2008Folgueras A.R. Martin de Lara F. Pendas A.M. Garabaya C. Rodriguez F. Astudillo A. Bernal T. Cabanillas R. Lopez-Otin C. Velasco G. Blood Press. 2008; (Published online June 3, 2008)https://doi.org/10.1182/blood-2008-04-149773Crossref Scopus (239) Google Scholar)Red blood cell counts (RBC)Normal rangeNDNDHemoglobin (Hb)LowLowLowMean corpuscular volume (MCV)LowLowLowPlasma iron levelsNDLowLowTransferrin saturationLowNDLowIron absorptionAbnormalAbnormalAbnormalHepcidin levelsHighHighHighIron stores (spleenic iron)NDLowNDAbnormal iron absorption in IRIDIA patients was concluded by a lack of hematological improvement following treatment with oral iron. In mask mutant mice, iron absorption was reduced, while in TMPRSS6−/− mice low ferroportin expression and iron accumulation within duodenal enterocytes was indicative of anomalies in dietary iron uptake. Low in IRIDA patients indicates that values lie below the respective reference range. "Low" or "High" in TMPRSS6-deficient mice indicates that values are significantly lower or higher, respectively, compared to wild-type mice. ND, not determined. Open table in a new tab Abnormal iron absorption in IRIDIA patients was concluded by a lack of hematological improvement following treatment with oral iron. In mask mutant mice, iron absorption was reduced, while in TMPRSS6−/− mice low ferroportin expression and iron accumulation within duodenal enterocytes was indicative of anomalies in dietary iron uptake. Low in IRIDA patients indicates that values lie below the respective reference range. "Low" or "High" in TMPRSS6-deficient mice indicates that values are significantly lower or higher, respectively, compared to wild-type mice. ND, not determined. Three recent studies suggest that TMPRSS6 suppresses hepcidin production to permit adequate dietary iron uptake. If active hepcidin suppression fails due to TMPRSS6 inactivation, severe iron deficiency prevails. TMPRSS6 is predominantly expressed in the liver and consists of a C-terminal, extracellular trypsin-like serine protease domain, three low density lipoprotein receptor (LDLR) tandem repeats, two CUB (complement factor C1s/C1r, urchin embryonic growth factor, bone morphogenetic protein) domains, and a membrane-proximal SEA (sea urchin sperm protein, enteropeptidase, agrin) domain that contains a potential cleavage motif that may release the enzyme from the cell surface. TMPRSS6 cleaves extracellular matrix proteins in vitro (Velasco et al., 2002Velasco G. Cal S. Quesada V. Sánchez L.M. López-Otín C. J. Biol. Chem. 2002; 277: 37637-37646Crossref PubMed Scopus (150) Google Scholar), but it is not understood whether this function is required to maintain iron homeostasis or whether additional phenotypes exist in TMPRSS6-deficient mice. Future studies also need to address how TMPRSS6-mediated hepcidin suppression is fine tuned to maintain systemic iron levels within narrow limits. Possibilities include binding of a putative ligand to the TMPRSS6 extracellular domain whose expression is regulated by systemic iron availability. Alternatively, TMPRSS6 expression levels may be controlled directly by intracellular iron levels. Further, it will be interesting to unravel how TMPRSS6 suppresses hepcidin transcription. In principal, TMPRSS6 protease cleavage and/or TMPRSS6-mediated signaling may function to inactivate a protein involved in a hepcidin activating pathway as those controlled by IL6, the HH-associated proteins, and BMPs (Figure 1). Alternatively, TMPRSS6 may activate a protein involved in a hepcidin suppressing pathway. Hypoxia inducible factor (HIF)-1α, growth differentiation factor (GDF) 15, and a cleavage product of hemojuvelin that is detectable in the plasma were previously implicated as hepcidin suppressors. The finding that TMPRSS6 regulates hepcidin transcription via a hepcidin promoter region that contains two activating elements (Wrighting and Andrews, 2006Wrighting D.M. Andrews N.C. Blood. 2006; 108: 3204-3209Crossref PubMed Scopus (642) Google Scholar, Verga Falzacappa et al., 2006Verga Falzacappa M.V. Vujic Spasic M. Kessler R. Stolte J. Hentze M.W. Muckenthaler M.U. Blood. 2006; 109: 353-358Crossref PubMed Scopus (423) Google Scholar, Verga Falzacappa et al., 2008Verga Falzacappa M.V. Casanovas G. Hentze M.W. Muckenthaler M.U. J. Mol. Med. 2008; 86: 531-540Crossref PubMed Scopus (109) Google Scholar) may give first clues regarding such a mechanism. The identification of TMPRSS6 as a novel hepcidin regulator is an important advancement in our understanding of hepcidin regulation that may have clinical implications for the treatment of frequent iron disorders: inhibition of the putative protease function may be applied in disorders such as HH or iron-loading anemias, in which hepcidin levels are inappropriately low. Similarly, strategies stimulating TMPRSS6 may be useful in ACD that is hallmarked by high hepcidin levels. The requirement for both activating and repressing pathways to maintain precise hepcidin levels underscores the importance for tight control of hepcidin expression. Mutations in HFE, TfR2, hemojuvelin, and hepcidin explain most known cases of hereditary hemochromatosis. Therefore, the discovery of novel hepcidin regulators in the future is likely to arise from the subgroup of hepcidin suppressors that control the response to iron deficiency and may be mutated in so-far-uncharacterized cases of anemias in patients and mice. M.M. is generously supported by the Deutsche Forschungsgemeinschaft (MU 1108/4-1), EEC Framework 6 (LSHM-CT-2006037296 EuroIron1), Landesstiftung Baden Württemberg, and the BMBF (HepatoSy-Iron_liver).