BMPER Protein Is a Negative Regulator of Hepcidin and Is Up-regulated in Hypotransferrinemic Mice
2011; Elsevier BV; Volume: 287; Issue: 6 Linguagem: Inglês
10.1074/jbc.m111.310789
ISSN1083-351X
AutoresNeeta Patel, Patarabutr Masaratana, Javier Díaz‐Castro, Gladys O. Latunde‐Dada, Aakafa Qureshi, Pamela Lockyer, Molly Jacob, Matthew Arno, Pavle Matak, Ragai R. Mitry, Robin D. Hughes, Anil Dhawan, Cam Patterson, Robert J. Simpson, Andrew T. McKie,
Tópico(s)Trace Elements in Health
ResumoThe BMP/SMAD4 pathway has major effects on liver hepcidin levels. Bone morphogenetic protein-binding endothelial cell precursor-derived regulator (Bmper), a known regulator of BMP signaling, was found to be overexpressed at the mRNA and protein levels in liver of genetically hypotransferrinemic mice (Trfhpx/hpx). Soluble BMPER peptide inhibited BMP2- and BMP6-dependent hepcidin promoter activity in both HepG2 and HuH7 cells. These effects correlated with reduced cellular levels of pSMAD1/5/8. Addition of BMPER peptide to primary human hepatocytes abolished the BMP2-dependent increase in hepcidin mRNA, whereas injection of Bmper peptide into mice resulted in reduced liver hepcidin and increased serum iron levels. Thus Bmper may play an important role in suppressing hepcidin production in hypotransferrinemic mice. The BMP/SMAD4 pathway has major effects on liver hepcidin levels. Bone morphogenetic protein-binding endothelial cell precursor-derived regulator (Bmper), a known regulator of BMP signaling, was found to be overexpressed at the mRNA and protein levels in liver of genetically hypotransferrinemic mice (Trfhpx/hpx). Soluble BMPER peptide inhibited BMP2- and BMP6-dependent hepcidin promoter activity in both HepG2 and HuH7 cells. These effects correlated with reduced cellular levels of pSMAD1/5/8. Addition of BMPER peptide to primary human hepatocytes abolished the BMP2-dependent increase in hepcidin mRNA, whereas injection of Bmper peptide into mice resulted in reduced liver hepcidin and increased serum iron levels. Thus Bmper may play an important role in suppressing hepcidin production in hypotransferrinemic mice. IntroductionHepcidin is secreted into the circulation by hepatocytes and plays a central role in the regulation of iron metabolism (1Pigeon C. Ilyin G. Courselaud B. Leroyer P. Turlin B. Brissot P. Loréal O. A new mouse liver-specific gene, encoding a protein homologous to human antimicrobial peptide hepcidin, is overexpressed during iron overload.J. Biol. Chem. 2001; 276: 7811-7819Abstract Full Text Full Text PDF PubMed Scopus (1403) Google Scholar, 2Park C.H. Valore E.V. Waring A.J. Ganz T. Hepcidin, a urinary antimicrobial peptide synthesized in the liver.J. Biol. Chem. 2001; 276: 7806-7810Abstract Full Text Full Text PDF PubMed Scopus (1722) Google Scholar, 3Nicolas G. Bennoun M. Devaux I. Beaumont C. Grandchamp B. Kahn A. Vaulont S. Lack of hepcidin gene expression and severe tissue iron overload in upstream stimulatory factor 2 (USF2) knockout mice.Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 8780-8785Crossref PubMed Scopus (1075) Google Scholar). Hepatic hepcidin levels are regulated by iron stores (1Pigeon C. Ilyin G. Courselaud B. Leroyer P. Turlin B. Brissot P. Loréal O. A new mouse liver-specific gene, encoding a protein homologous to human antimicrobial peptide hepcidin, is overexpressed during iron overload.J. Biol. Chem. 2001; 276: 7811-7819Abstract Full Text Full Text PDF PubMed Scopus (1403) Google Scholar), hypoxia, erythropoietic rate, and inflammatory status (4Nicolas G. Chauvet C. Viatte L. Danan J.L. Bigard X. Devaux I. Beaumont C. Kahn A. Vaulont S. The gene encoding the iron regulatory peptide hepcidin is regulated by anemia, hypoxia, and inflammation.J. Clin. Invest. 2002; 110: 1037-1044Crossref PubMed Scopus (1331) Google Scholar, 5Nemeth E. Valore E.V. Territo M. Schiller G. Lichtenstein A. Ganz T. Hepcidin, a putative mediator of anemia of inflammation, is a type II acute-phase protein.Blood. 2003; 101: 2461-2463Crossref PubMed Scopus (1165) Google Scholar). Hepcidin works by blocking the entry of iron into the circulation from the gut and the macrophages by binding to the iron transporter Ferroportin resulting in its ubiquitylation and degradation (6Nemeth E. Tuttle M.S. Powelson J. Vaughn M.B. Donovan A. Ward D.M. Ganz T. Kaplan J. Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization.Science. 2004; 306: 2090-2093Crossref PubMed Scopus (3533) Google Scholar, 7De Domenico I. Ward D.M. Langelier C. Vaughn M.B. Nemeth E. Sundquist W.I. Ganz T. Musci G. Kaplan J. The molecular mechanism of hepcidin-mediated ferroportin down-regulation.Mol. Biol. Cell. 2007; 18: 2569-2578Crossref PubMed Scopus (354) Google Scholar). Low levels of circulating hepcidin are associated with many forms of genetic iron overload (8Nemeth E. Ganz T. Regulation of iron metabolism by hepcidin.Annu. Rev. Nutr. 2006; 26: 323-342Crossref PubMed Scopus (559) Google Scholar, 9Nemeth E. Ganz T. Hepcidin and iron-loading anemias.Haematologica. 2006; 91: 727-732PubMed Google Scholar). Various signaling pathways have been shown to regulate hepatic hepcidin levels with bone morphogenetic proteins, Bmps 2The abbreviations used are: Bmpbone morphogenetic proteinBmperBmp-binding endothelial cell precursor-derived regulatorCV-2crossveinless-2Twsg1Twisted gastrulationhjvhemojuvelinANOVAanalysis of variance. (especially Bmp2, Bmp4, Bmp6, and Bmp9), which signal via SMADs, emerging as key players (10Babitt 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. Andrews N.C. Lin H.Y. Bone morphogenetic protein signaling by hemojuvelin regulates hepcidin expression.Nat. Genet. 2006; 38: 531-539Crossref PubMed Scopus (824) Google Scholar, 11Babitt J.L. Huang F.W. Xia Y. Sidis Y. Andrews N.C. Lin H.Y. Modulation of bone morphogenetic protein signaling in vivo regulates systemic iron balance.J. Clin. Invest. 2007; 117: 1933-1939Crossref PubMed Scopus (359) Google Scholar, 12Andriopoulos Jr., B. Corradini E. Xia Y. Faasse S.A. Chen S. Grgurevic L. Knutson M.D. Pietrangelo A. Vukicevic S. Lin H.Y. Babitt J.L. BMP6 is a key endogenous regulator of hepcidin expression and iron metabolism.Nat. Genet. 2009; 41: 482-487Crossref PubMed Scopus (588) Google Scholar, 13Meynard D. Kautz L. Darnaud V. Canonne-Hergaux F. Coppin H. Roth M.P. Lack of the bone morphogenetic protein BMP6 induces massive iron overload.Nat. Genet. 2009; 41: 478-481Crossref PubMed Scopus (466) Google Scholar). Bmp signaling is extraordinarily complex with a number of extracellular Bmp ligands, including crossveinless-2 (CV-2), Twisted gastrulation (Twsg1), chordin, noggin, and hemojuvelin (hjv), which can act as either pro- and anti-Bmp effectors depending on the context and concentration (14Bier E. Intriguing extracellular regulation of BMP signaling.Dev. Cell. 2008; 15: 176-177Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). TWSG1 was recently shown to be elevated in patients with thalassemia and to negatively regulate hepcidin by inhibiting the effects of BMP2 and 4 (15Tanno T. Porayette P. Sripichai O. Noh S.J. Byrnes C. Bhupatiraju A. Lee Y.T. Goodnough J.B. Harandi O. Ganz T. Paulson R.F. Miller J.L. Identification of TWSG1 as a second novel erythroid regulator of hepcidin expression in murine and human cells.Blood. 2009; 114: 181-186Crossref PubMed Scopus (281) Google Scholar).Crossveinless-2 (Cv-2) was identified in Drosophila where it was implicated in Bmp signaling and shown to be essential for the formation of small blood vessels or "crossveins" in the wing (16Conley C.A. Silburn R. Singer M.A. Ralston A. Rohwer-Nutter D. Olson D.J. Gelbart W. Blair S.S. Crossveinless 2 contains cysteine-rich domains and is required for high levels of BMP-like activity during the formation of the cross veins in Drosophila.Development. 2000; 127: 3947-3959Crossref PubMed Google Scholar). The orthologous mouse protein named bone morphogenetic protein (Bmp)-binding endothelial cell precursor-derived regulator (Bmper) is highly expressed in developing endothelial cells and binds to Bmp2, -4, and -6 inhibiting BMP signaling (17Moser M. Binder O. Wu Y. Aitsebaomo J. Ren R. Bode C. Bautch V.L. Conlon F.L. Patterson C. BMPER, a novel endothelial cell precursor-derived protein, antagonizes bone morphogenetic protein signaling and endothelial cell differentiation.Mol. Cell. Biol. 2003; 23: 5664-5679Crossref PubMed Scopus (181) Google Scholar). Further evidence in zebrafish Bmper mutants supports a role in Bmp signaling during vascular development (18Moser M. Yu Q. Bode C. Xiong J.W. Patterson C. BMPER is a conserved regulator of hematopoietic and vascular development in zebrafish.J. Mol. Cell. Cardiol. 2007; 43: 243-253Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Bmper has been shown to have both pro- and anti-Bmp activity (16Conley C.A. Silburn R. Singer M.A. Ralston A. Rohwer-Nutter D. Olson D.J. Gelbart W. Blair S.S. Crossveinless 2 contains cysteine-rich domains and is required for high levels of BMP-like activity during the formation of the cross veins in Drosophila.Development. 2000; 127: 3947-3959Crossref PubMed Google Scholar, 17Moser M. Binder O. Wu Y. Aitsebaomo J. Ren R. Bode C. Bautch V.L. Conlon F.L. Patterson C. BMPER, a novel endothelial cell precursor-derived protein, antagonizes bone morphogenetic protein signaling and endothelial cell differentiation.Mol. Cell. Biol. 2003; 23: 5664-5679Crossref PubMed Scopus (181) Google Scholar, 19Rentzsch F. Zhang J. Kramer C. Sebald W. Hammerschmidt M. Crossveinless 2 is an essential positive feedback regulator of Bmp signaling during zebrafish gastrulation.Development. 2006; 133: 801-811Crossref PubMed Scopus (82) Google Scholar, 20Binnerts M.E. Wen X. Canté-Barrett K. Bright J. Chen H.T. Asundi V. Sattari P. Tang T. Boyle B. Funk W. Rupp F. Human Crossveinless-2 is a novel inhibitor of bone morphogenetic proteins.Biochem. Biophys. Res. Commun. 2004; 315: 272-280Crossref PubMed Scopus (62) Google Scholar). The protein exists in two forms, a full-length membrane-associated form and a soluble form consisting of a heterodimer of C- and N-terminal cleavage fragments connected via disulfide bonding (19Rentzsch F. Zhang J. Kramer C. Sebald W. Hammerschmidt M. Crossveinless 2 is an essential positive feedback regulator of Bmp signaling during zebrafish gastrulation.Development. 2006; 133: 801-811Crossref PubMed Scopus (82) Google Scholar). The cleavage is autocatalytic via a conserved acid-sensitive cleavage site (FGDPH) and is suggested to account for the ability of the protein to act as a pro- or anti-Bmp in vivo (19Rentzsch F. Zhang J. Kramer C. Sebald W. Hammerschmidt M. Crossveinless 2 is an essential positive feedback regulator of Bmp signaling during zebrafish gastrulation.Development. 2006; 133: 801-811Crossref PubMed Scopus (82) Google Scholar). The N terminus of Bmper contains five cysteine-rich von Willebrand type C-like domains (named CR1–5) responsible for ligand binding that are conserved in other BMP-binding proteins such as chordin. Bmper binds Bmps with high affinity similar to that of Bmp type I and type II receptors themselves (19Rentzsch F. Zhang J. Kramer C. Sebald W. Hammerschmidt M. Crossveinless 2 is an essential positive feedback regulator of Bmp signaling during zebrafish gastrulation.Development. 2006; 133: 801-811Crossref PubMed Scopus (82) Google Scholar). The C-terminal of Bmper contains a furin-like, a von Willebrand type D, and a trypsin inhibitor domain (16Conley C.A. Silburn R. Singer M.A. Ralston A. Rohwer-Nutter D. Olson D.J. Gelbart W. Blair S.S. Crossveinless 2 contains cysteine-rich domains and is required for high levels of BMP-like activity during the formation of the cross veins in Drosophila.Development. 2000; 127: 3947-3959Crossref PubMed Google Scholar).Hypotransferrinemic mice (Trfhpx/hpx) have defective transferrin gene expression leading to low levels of plasma transferrin (<1% of wild type) and, despite massive iron loading, have very low levels of hepcidin in hepatocytes (21Weinstein D.A. Roy C.N. Fleming M.D. Loda M.F. Wolfsdorf J.I. Andrews N.C. Inappropriate expression of hepcidin is associated with iron refractory anemia. Implications for the anemia of chronic disease.Blood. 2002; 100: 3776-3781Crossref PubMed Scopus (542) Google Scholar). The precise mechanism of hepcidin suppression in Trfhpx/hpx mice, as in other conditions of enhanced erythropoiesis, anemia, and hypoxia, remains unclear and may involve a number of signaling pathways. We sought to identify potential hepcidin regulators produced locally in liver of Trfhpx/hpx mice. We identified a known Bmp regulator Bmper as being highly up-regulated in liver of Trfhpx/hpx mice. In this report we show that Bmper can suppress hepcidin promoter activity and reduce hepcidin levels in liver cells both in vitro and in vivo via effects on the BMP pathway.DISCUSSIONAnemia has been shown to strongly down-regulate liver hepcidin expression, even when iron stores are greatly increased as found in Trfhpx/hpx mice (4Nicolas G. Chauvet C. Viatte L. Danan J.L. Bigard X. Devaux I. Beaumont C. Kahn A. Vaulont S. The gene encoding the iron regulatory peptide hepcidin is regulated by anemia, hypoxia, and inflammation.J. Clin. Invest. 2002; 110: 1037-1044Crossref PubMed Scopus (1331) Google Scholar, 21Weinstein D.A. Roy C.N. Fleming M.D. Loda M.F. Wolfsdorf J.I. Andrews N.C. Inappropriate expression of hepcidin is associated with iron refractory anemia. Implications for the anemia of chronic disease.Blood. 2002; 100: 3776-3781Crossref PubMed Scopus (542) Google Scholar). This has led to the notion that one or more secreted humoral factors are released from the marrow under increased erythropoietic drive that can influence hepatic hepcidin levels. Levels of GDF15 and TWSG1 in the marrow are increased in patients with β-thalassemia, and both have been shown to inhibit hepcidin production in vitro, the latter by inhibiting the action of BMP4 and BMP2 (15Tanno T. Porayette P. Sripichai O. Noh S.J. Byrnes C. Bhupatiraju A. Lee Y.T. Goodnough J.B. Harandi O. Ganz T. Paulson R.F. Miller J.L. Identification of TWSG1 as a second novel erythroid regulator of hepcidin expression in murine and human cells.Blood. 2009; 114: 181-186Crossref PubMed Scopus (281) Google Scholar). Bartnikas et al. found that both Gdf15 and Twsg1 levels were raised in spleen of Trfhpx/hpx mice; however, when erythropoiesis was inhibited with doxorubicin, levels of both decreased without significant change in liver hepcidin levels. This suggests Gdf15 and Twsg1 may be markers for increased erythropoiesis but do not directly regulate hepcidin in vivo (26Bartnikas T.B. Andrews N.C. Fleming M.D. Transferrin is a major determinant of hepcidin expression in hypotransferrinemic mice.Blood. 2011; 117: 630-637Crossref PubMed Scopus (66) Google Scholar). We found that Gdf15 levels were increased in liver of Trfhpx/hpx mice, which may be reflective of some extra medullary or stress erythropoiesis occurring in liver of Trfhpx/hpx mice. However, no changes in Twsg1, Hjv, or other known BMP antagonists Noggin and Chordin were observed. We also found no changes in Hjv, Noggin, or Chordin in skeletal muscle of Trfhpx/hpx. Hence, of the BMP antagonists tested, only Bmper was highly up-regulated in Trfhpx/hpx mice.Our results revealed that excess soluble BMPER peptide inhibited BMP-dependent hepcidin transcription as well as endogenous hepcidin production in hepatocytes by acting as an anti-Bmp and inhibiting the SMAD pathway. The mechanism is likely based on the ability of BMPER to bind and sequester BMP molecules with high affinity and thereby prevent BMPs from binding to BMP type I and II receptors (28Zhang J.L. Huang Y. Qiu L.Y. Nickel J. Sebald W. von Willebrand factor type C domain-containing proteins regulate bone morphogenetic protein signaling through different recognition mechanisms.J. Biol. Chem. 2007; 282: 20002-20014Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 29Zhang J.L. Qiu L.Y. Kotzsch A. Weidauer S. Patterson L. Hammerschmidt M. Sebald W. Mueller T.D. Crystal structure analysis reveals how the Chordin family member crossveinless 2 blocks BMP-2 receptor binding.Dev. Cell. 2008; 14: 739-750Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). It is noteworthy that in some situations Bmper has been shown to elicit pro-BMP responses, a function that may be dependent on whether the protein is membrane-associated or soluble (28Zhang J.L. Huang Y. Qiu L.Y. Nickel J. Sebald W. von Willebrand factor type C domain-containing proteins regulate bone morphogenetic protein signaling through different recognition mechanisms.J. Biol. Chem. 2007; 282: 20002-20014Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). In this respect, BMPER may function in a similar manner to HJV where the soluble form is anti-BMP and membrane form is pro-BMP. Both HJV and BMPER share several structural features such as a conserved autocatalytic cleavage site and C-terminal furin cleavage sites. In our study we did not test the effect of full-length membrane-associated BMPER; however, we did not observe pro-BMP effects using the soluble BMPER peptide.BMPER has been shown to bind to BMP2, -4, and -6 (17Moser M. Binder O. Wu Y. Aitsebaomo J. Ren R. Bode C. Bautch V.L. Conlon F.L. Patterson C. BMPER, a novel endothelial cell precursor-derived protein, antagonizes bone morphogenetic protein signaling and endothelial cell differentiation.Mol. Cell. Biol. 2003; 23: 5664-5679Crossref PubMed Scopus (181) Google Scholar). In agreement our results showed that BMPER inhibited the effects of both BMP2 and BMP6 on hepcidin production, although the effect on BMP6 was less pronounced than that for BMP2. The reasons for this are not clear as yet; however, BMPER is likely to bind to the various BMPs with different affinities.TWSG1 has been shown to inhibit hepcidin (15Tanno T. Porayette P. Sripichai O. Noh S.J. Byrnes C. Bhupatiraju A. Lee Y.T. Goodnough J.B. Harandi O. Ganz T. Paulson R.F. Miller J.L. Identification of TWSG1 as a second novel erythroid regulator of hepcidin expression in murine and human cells.Blood. 2009; 114: 181-186Crossref PubMed Scopus (281) Google Scholar) production induced by BMP2 and -4 in hepatocytes, whereas other reports suggest TWSG1 forms a ternary complex with Bmper and Bmp4 (27Ambrosio A.L. Taelman V.F. Lee H.X. Metzinger C.A. Coffinier C. De Robertis E.M. Crossveinless-2 Is a BMP feedback inhibitor that binds Chordin/BMP to regulate Xenopus embryonic patterning.Dev. Cell. 2008; 15: 248-260Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar) thereby increasing the binding of Bmp4 to Bmper. In our experiment comparing relative effects of BMPER and TWSG1 we used 25 ng of BMP2 and 1000 ng of TWSG1 and BMPER. This is equivalent to a 1:23 molar ratio of BMP2 to TWSG1 and a 1:6 molar ratio of BMP2 to BMPER. Hence using four times less BMPER than TWSG1 resulted in an 80% inhibition of BMP2-dependent activity, compared with ∼40% for TWSG1. We therefore conclude that BMPER is more potent than TWSG1 at inhibiting BMP-induced promoter activity. This is consistent with data of others showing that the zebrafish orthologue of BMPER, Cv-2, had a binding affinity for BMP2 some 20-fold higher than TWSG1 and was considerably more potent at inhibiting the binding of BMP2 to both BMP type I and type II receptors (28Zhang J.L. Huang Y. Qiu L.Y. Nickel J. Sebald W. von Willebrand factor type C domain-containing proteins regulate bone morphogenetic protein signaling through different recognition mechanisms.J. Biol. Chem. 2007; 282: 20002-20014Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). In summary we found that BMPER was more potent at inhibiting BMP effects compared with TWSG1, and there was no evidence of a significant additive anti-BMP effect of BMPER and TWSG1 together.Trfhpx/hpx mice have very low circulating levels of diferric transferrin, and in common with human patients with β- thalassemia, very low hepatic hepcidin levels associated with increased erythropoiesis. However, unlike β-thalassemic patients, Trfhpx/hpx mice do not have ineffective erythropoiesis. Instead they have expanded marrow due to iron-restricted erythropoiesis and exhibit life-long anemia and tissue hypoxia due to the lack of transferrin. Reduced levels of Tfr2 have also been reported in Trfhpx/hpx mouse liver, which may also contribute to reduced hepcidin transcription (30Robb A. Wessling-Resnick M. Regulation of transferrin receptor 2 protein levels by transferrin.Blood. 2004; 104: 4294-4299Crossref PubMed Scopus (166) Google Scholar). Although it is not known whether one or a combination of these factors contributes to the lowered hepcidin in Trfhpx/hpx mice, evidence suggests that both diferric transferrin levels and the anemia are important (26Bartnikas T.B. Andrews N.C. Fleming M.D. Transferrin is a major determinant of hepcidin expression in hypotransferrinemic mice.Blood. 2011; 117: 630-637Crossref PubMed Scopus (66) Google Scholar, 31Raja K.B. Pountney D.J. Simpson R.J. Peters T.J. Importance of anemia and transferrin levels in the regulation of intestinal iron absorption in hypotransferrinemic mice.Blood. 1999; 94: 3185-3192Crossref PubMed Google Scholar). Iron absorption is much higher and hepcidin levels lower in Trfhpx/hpx mice compared with b-thalassemic mice, and as a result iron accumulation in the liver is some 6-fold higher as well (32Latunde-Dada G.O. McKie A.T. Simpson R.J. Animal models with enhanced erythropoiesis and iron absorption.Biochim. Biophys. Acta. 2006; 1762: 414-423Crossref PubMed Scopus (37) Google Scholar). This implies other factors are more important than the anemia alone for hepcidin down-regulation in Trfhpx/hpx mice. Bmp6 levels are increased in Trfhpx/hpx mouse liver indicating that iron sensing is intact (26Bartnikas T.B. Andrews N.C. Fleming M.D. Transferrin is a major determinant of hepcidin expression in hypotransferrinemic mice.Blood. 2011; 117: 630-637Crossref PubMed Scopus (66) Google Scholar). Our finding that the BMP ligand BMPER is significantly increased in Trfhpx/hpx liver could explain why Trfhpx/hpx mice fail to elicit a hepcidin response despite increased hepatic iron and BMP6 levels.It has been suggested that BMPER plays a role in blood vessel sprouting and remodeling, because mutations in the Bmper gene (Cv-2) adversely affect the formation of small blood vessels in Drosophila wing (16Conley C.A. Silburn R. Singer M.A. Ralston A. Rohwer-Nutter D. Olson D.J. Gelbart W. Blair S.S. Crossveinless 2 contains cysteine-rich domains and is required for high levels of BMP-like activity during the formation of the cross veins in Drosophila.Development. 2000; 127: 3947-3959Crossref PubMed Google Scholar), whereas in zebrafish mutations affect vasculogenesis and blood vessel remodeling (18Moser M. Yu Q. Bode C. Xiong J.W. Patterson C. BMPER is a conserved regulator of hematopoietic and vascular development in zebrafish.J. Mol. Cell. Cardiol. 2007; 43: 243-253Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). We hypothesize that, under severe chronic anemia as found in Trfhpx/hpx mice, BMPER levels are raised to support blood vessel development and remodeling, to counteract the anemia, leading to increased levels of soluble BMPER, sequestration of BMPs, and the down-regulation of hepatic hepcidin levels through inhibition of the SMAD signaling pathway (Fig. 8).In summary, hepcidin down-regulation in Trfhpx/hpx mice is likely to be a complex interplay of a number of factors exerting downward repression on hepcidin. BMPER adds to the list of factors that can suppress hepcidin levels and therefore affect iron metabolism. Increased BMPER levels in Trfhpx/hpx mice would also explain why increased BMP6 fails to induce an increase in hepcidin levels. BMPER may play a role in suppressing hepcidin in other forms of severe chronic anemia with iron loading or in diseases where there is a significant amount of angiogenesis. IntroductionHepcidin is secreted into the circulation by hepatocytes and plays a central role in the regulation of iron metabolism (1Pigeon C. Ilyin G. Courselaud B. Leroyer P. Turlin B. Brissot P. Loréal O. A new mouse liver-specific gene, encoding a protein homologous to human antimicrobial peptide hepcidin, is overexpressed during iron overload.J. Biol. Chem. 2001; 276: 7811-7819Abstract Full Text Full Text PDF PubMed Scopus (1403) Google Scholar, 2Park C.H. Valore E.V. Waring A.J. Ganz T. Hepcidin, a urinary antimicrobial peptide synthesized in the liver.J. Biol. Chem. 2001; 276: 7806-7810Abstract Full Text Full Text PDF PubMed Scopus (1722) Google Scholar, 3Nicolas G. Bennoun M. Devaux I. Beaumont C. Grandchamp B. Kahn A. Vaulont S. Lack of hepcidin gene expression and severe tissue iron overload in upstream stimulatory factor 2 (USF2) knockout mice.Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 8780-8785Crossref PubMed Scopus (1075) Google Scholar). Hepatic hepcidin levels are regulated by iron stores (1Pigeon C. Ilyin G. Courselaud B. Leroyer P. Turlin B. Brissot P. Loréal O. A new mouse liver-specific gene, encoding a protein homologous to human antimicrobial peptide hepcidin, is overexpressed during iron overload.J. Biol. Chem. 2001; 276: 7811-7819Abstract Full Text Full Text PDF PubMed Scopus (1403) Google Scholar), hypoxia, erythropoietic rate, and inflammatory status (4Nicolas G. Chauvet C. Viatte L. Danan J.L. Bigard X. Devaux I. Beaumont C. Kahn A. Vaulont S. The gene encoding the iron regulatory peptide hepcidin is regulated by anemia, hypoxia, and inflammation.J. Clin. Invest. 2002; 110: 1037-1044Crossref PubMed Scopus (1331) Google Scholar, 5Nemeth E. Valore E.V. Territo M. Schiller G. Lichtenstein A. Ganz T. Hepcidin, a putative mediator of anemia of inflammation, is a type II acute-phase protein.Blood. 2003; 101: 2461-2463Crossref PubMed Scopus (1165) Google Scholar). Hepcidin works by blocking the entry of iron into the circulation from the gut and the macrophages by binding to the iron transporter Ferroportin resulting in its ubiquitylation and degradation (6Nemeth E. Tuttle M.S. Powelson J. Vaughn M.B. Donovan A. Ward D.M. Ganz T. Kaplan J. Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization.Science. 2004; 306: 2090-2093Crossref PubMed Scopus (3533) Google Scholar, 7De Domenico I. Ward D.M. Langelier C. Vaughn M.B. Nemeth E. Sundquist W.I. Ganz T. Musci G. Kaplan J. The molecular mechanism of hepcidin-mediated ferroportin down-regulation.Mol. Biol. Cell. 2007; 18: 2569-2578Crossref PubMed Scopus (354) Google Scholar). Low levels of circulating hepcidin are associated with many forms of genetic iron overload (8Nemeth E. Ganz T. Regulation of iron metabolism by hepcidin.Annu. Rev. Nutr. 2006; 26: 323-342Crossref PubMed Scopus (559) Google Scholar, 9Nemeth E. Ganz T. Hepcidin and iron-loading anemias.Haematologica. 2006; 91: 727-732PubMed Google Scholar). Various signaling pathways have been shown to regulate hepatic hepcidin levels with bone morphogenetic proteins, Bmps 2The abbreviations used are: Bmpbone morphogenetic proteinBmperBmp-binding endothelial cell precursor-derived regulatorCV-2crossveinless-2Twsg1Twisted gastrulationhjvhemojuvelinANOVAanalysis of variance. (especially Bmp2, Bmp4, Bmp6, and Bmp9), which signal via SMADs, emerging as key players (10Babitt 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. Andrews N.C. Lin H.Y. Bone morphogenetic protein signaling by hemojuvelin regulates hepcidin expression.Nat. Genet. 2006; 38: 531-539Crossref PubMed Scopus (824) Google Scholar, 11Babitt J.L. Huang F.W. Xia Y. Sidis Y. Andrews N.C. Lin H.Y. Modulation of bone morphogenetic protein signaling in vivo regulates systemic iron balance.J. Clin. Invest. 2007; 117: 1933-1939Crossref PubMed Scopus (359) Google Scholar, 12Andriopoulos Jr., B. Corradini E. Xia Y. Faasse S.A. Chen S. Grgurevic L. Knutson M.D. Pietrangelo A. Vukicevic S. Lin H.Y. Babitt J.L. BMP6 is a key endogenous regulator of hepcidin expression and iron metabolism.Nat. Genet. 2009; 41: 482-487Crossref PubMed Scopus (588) Google Scholar, 13Meynard D. Kautz L. Darnaud V. Canonne-Hergaux F. Coppin H. Roth M.P. Lack of the bone morphogenetic protein BMP6 induces massive iron overload.Nat. Genet. 2009; 41: 478-481Crossref PubMed Scopus (466) Google Scholar). Bmp signaling is extraordinarily complex with a number of extracellular Bmp ligands, including crossveinless-2 (CV-2), Twisted gastrulation (Twsg1), chordin, noggin, and hemojuvelin (hjv), which can act as either pro- and anti-Bmp effectors depending on the context and concentration (14Bier E. Intriguing extracellular regulation of BMP signaling.Dev. Cell. 2008; 15: 176-177Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). TWSG1 was recently shown to be elevated in patients with thalassemia and to negatively regulate hepcidin by inhibiting the effects of BMP2 and 4 (15Tanno T. Porayette P. Sripichai O. Noh S.J. Byrnes C. Bhupatiraju A. Lee Y.T. Goodnough J.B. Harandi O. Ganz T. Paulson R.F. Miller J.L. Identification of TWSG1 as a second novel erythroid regulator of hepcidin expression in murine and human cells.Blood. 2009; 114: 181-186Crossref PubMed Scopus (281) Google Scholar).Crossveinless-2 (Cv-2) was identified in Drosophila where it was implicated in Bmp signaling and shown to be essential for the formation of small blood vessels or "crossveins" in the wing (16Conley C.A. Silburn R. Singer M.A. Ralston A. Rohwer-Nutter D. Olson D.J. Gelbart W. Blair S.S. Crossveinless 2 contains cysteine-rich domains and is required for high levels of BMP-like activity during the formation of the cross veins in Drosophila.Development. 2000; 127: 3947-3959Crossref PubMed Google Scholar). The orthologous mouse protein named bone morphogenetic protein (Bmp)-binding endothelial cell precursor-derived regulator (Bmper) is highly expressed in developing endothelial cells and binds to Bmp2, -4, and -6 inhibiting BMP signaling (17Moser M. Binder O. Wu Y. Aitsebaomo J. Ren R. Bode C. Bautch V.L. Conlon F.L. Patterson C. BMPER, a novel endothelial cell precursor-derived protein, antagonizes bone morphogenetic protein signaling and endothelial cell differentiation.Mol. Cell. Biol. 2003; 23: 5664-5679Crossref PubMed Scopus (181) Google Scholar). Further evidence in zebrafish Bmper mutants supports a role in Bmp signaling during vascular development (18Moser M. Yu Q. Bode C. Xiong J.W. Patterson C. BMPER is a conserved regulator of hematopoietic and vascular development in zebrafish.J. Mol. Cell. Cardiol. 2007; 43: 243-253Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Bmper has been shown to have both pro- and anti-Bmp activity (16Conley C.A. Silburn R. Singer M.A. Ralston A. Rohwer-Nutter D. Olson D.J. Gelbart W. Blair S.S. Crossveinless 2 contains cysteine-rich domains and is required for high levels of BMP-like activity during the formation of the cross veins in Drosophila.Development. 2000; 127: 3947-3959Crossref PubMed Google Scholar, 17Moser M. Binder O. Wu Y. Aitsebaomo J. Ren R. Bode C. Bautch V.L. Conlon F.L. Patterson C. BMPER, a novel endothelial cell precursor-derived protein, antagonizes bone morphogenetic protein signaling and endothelial cell differentiation.Mol. Cell. Biol. 2003; 23: 5664-5679Crossref PubMed Scopus (181) Google Scholar, 19Rentzsch F. Zhang J. Kramer C. Sebald W. Hammerschmidt M. Crossveinless 2 is an essential positive feedback regulator of Bmp signaling during zebrafish gastrulation.Development. 2006; 133: 801-811Crossref PubMed Scopus (82) Google Scholar, 20Binnerts M.E. Wen X. Canté-Barrett K. Bright J. Chen H.T. Asundi V. Sattari P. Tang T. Boyle B. Funk W. Rupp F. Human Crossveinless-2 is a novel inhibitor of bone morphogenetic proteins.Biochem. Biophys. Res. Commun. 2004; 315: 272-280Crossref PubMed Scopus (62) Google Scholar). The protein exists in two forms, a full-length membrane-associated form and a soluble form consisting of a heterodimer of C- and N-terminal cleavage fragments connected via disulfide bonding (19Rentzsch F. Zhang J. Kramer C. Sebald W. Hammerschmidt M. Crossveinless 2 is an essential positive feedback regulator of Bmp signaling during zebrafish gastrulation.Development. 2006; 133: 801-811Crossref PubMed Scopus (82) Google Scholar). The cleavage is autocatalytic via a conserved acid-sensitive cleavage site (FGDPH) and is suggested to account for the ability of the protein to act as a pro- or anti-Bmp in vivo (19Rentzsch F. Zhang J. Kramer C. Sebald W. Hammerschmidt M. Crossveinless 2 is an essential positive feedback regulator of Bmp signaling during zebrafish gastrulation.Development. 2006; 133: 801-811Crossref PubMed Scopus (82) Google Scholar). The N terminus of Bmper contains five cysteine-rich von Willebrand type C-like domains (named CR1–5) responsible for ligand binding that are conserved in other BMP-binding proteins such as chordin. Bmper binds Bmps with high affinity similar to that of Bmp type I and type II receptors themselves (19Rentzsch F. Zhang J. Kramer C. Sebald W. Hammerschmidt M. Crossveinless 2 is an essential positive feedback regulator of Bmp signaling during zebrafish gastrulation.Development. 2006; 133: 801-811Crossref PubMed Scopus (82) Google Scholar). The C-terminal of Bmper contains a furin-like, a von Willebrand type D, and a trypsin inhibitor domain (16Conley C.A. Silburn R. Singer M.A. Ralston A. Rohwer-Nutter D. Olson D.J. Gelbart W. Blair S.S. Crossveinless 2 contains cysteine-rich domains and is required for high levels of BMP-like activity during the formation of the cross veins in Drosophila.Development. 2000; 127: 3947-3959Crossref PubMed Google Scholar).Hypotransferrinemic mice (Trfhpx/hpx) have defective transferrin gene expression leading to low levels of plasma transferrin (<1% of wild type) and, despite massive iron loading, have very low levels of hepcidin in hepatocytes (21Weinstein D.A. Roy C.N. Fleming M.D. Loda M.F. Wolfsdorf J.I. Andrews N.C. Inappropriate expression of hepcidin is associated with iron refractory anemia. Implications for the anemia of chronic disease.Blood. 2002; 100: 3776-3781Crossref PubMed Scopus (542) Google Scholar). The precise mechanism of hepcidin suppression in Trfhpx/hpx mice, as in other conditions of enhanced erythropoiesis, anemia, and hypoxia, remains unclear and may involve a number of signaling pathways. We sought to identify potential hepcidin regulators produced locally in liver of Trfhpx/hpx mice. We identified a known Bmp regulator Bmper as being highly up-regulated in liver of Trfhpx/hpx mice. In this report we show that Bmper can suppress hepcidin promoter activity and reduce hepcidin levels in liver cells both in vitro and in vivo via effects on the BMP pathway.
Referência(s)