Iron overload accelerates bone loss in mice lacking the bone morphogenetic protein 6 (Bmp6) gene, which is the key endogenous regulator of hepcidin, iron homeostasis gene. We investigated involvement of other BMPs in preventing haemochromatosis and subsequent osteopenia in Bmp6-/- mice.
Iron-treated wild-type (WT) and Bmp6-/- mice were analysed for hepcidin messenger RNA (mRNA) and tissue and blood BMP levels by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR), immunohistochemistry, Western blot, enzyme-linked immunosorbent assay (ELISA) and proximity extension assay. BMPs labeled with technetium-99m were used in pharmacokinetic studies.
In WT mice, 4 h following iron challenge, liver Bmp6 and hepcidin expression were increased, while expression of other Bmps was not affected. In parallel, we provided the first evidence that BMP6 circulates in WT mice and that iron increased the BMP6 serum level and the specific liver uptake of 99mTc-BMP6. In Bmp6-/- mice, iron challenge led to blunted activation of liver Smad signaling and hepcidin expression with a delay of 24 h, associated with increased Bmp5 and Bmp7 expression and increased Bmp2, 4, 5 and 9 expression in the duodenum. Liver Bmp7 expression and increased circulating BMP9 eventually contributed to the late hepcidin response. This was further supported by exogenous BMP7 therapy resulting in an effective hepcidin expression followed by a rapid normalisation of plasma iron values and restored osteopenia in Bmp6-/- mice.
In Bmp6-/- mice, iron activated endogenous compensatory mechanisms of other BMPs that were not sufficient for preventing hemochromatosis and bone loss. Administration of exogenous BMP7 was effective in correcting the plasma iron level and bone loss, indicating that BMP6 is an essential but not exclusive in vivo regulator of iron homeostasis.
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Bone morphogenetic protein
- pSmad 1/5/8:
Phosphorylated Smad1, Smad5 and Smad8 protein
Guggenbuhl P, Deugnier Y, Boisdet JF, Rolland Y, Perdriger A, Pawlotsky Y, Chalès G (2005) Bone mineral density in men with genetic hemochromatosis and HFE gene mutation. Osteoporos Int 16:1809–1814
Kim BJ, Ahn SH, Bae SJ, Kim EH, Lee SH, Kim HK, Choe JW, Koh JM, Kim GS (2012) Iron overload accelerates bone loss in healthy postmenopausal women and middle-aged men: a 3-year retrospective longitudinal study. J Bone Miner Res 27:2279–22790
Tsay J, Yang Z, Ross FP, Cunningham-Rundles S, Lin H, Coleman R, Mayer-Kuckuk P, Doty SB, Grady RW, Giardina PJ, Boskey AL, Vogiatzi MG (2010) Bone loss caused by iron overload in a murine model: importance of oxidative stress. Blood 116:2582–2589
Vukicevic S, Oppermann H, Verbanac D et al (2014) The clinical use of bone morphogenetic proteins (BMPs) revisited: A novel BMP6 biocompatible carrier device OSTEOGROW for bone healing. Int Orthop 38:635–664
Pigeon C, Ilyin G, Courselaud B, Leroyer P, Turlin B, Brissot B, Loréal O (2001) A new mouse liver-specific gene, encoding a protein homologous to human antimicrobial peptide hepcidin, is overexpressed during iron overload. J Biol Chem 276:7811–7819
Nemeth E, Tuttle MS, Powelson J, Vaughn MB, Donovan A, Ward DM, Ganz T, Kaplan J (2004) Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization. Sci 306:2090–2093
Nemeth E, Rivera S, Gabayan V, Keller C, Taudorf S, Pedersen BK, Ganz T (2004) IL-6 mediates hypoferremia of inflammation by inducing the synthesis of the iron regulatory hormone hepcidin. J Clin Invest 113:1271–1276
Nicolas G, Chauvet C, Viatte L, Danan JL, Bigard X, Devaux I, Beaumont C, Kahn A, Vaulont S (2002) The gene encoding the iron regulatory peptide hepcidin is regulated by anemia, hypoxia, and inflammation. J Clin Invest 110:1037–1044
Babitt JL, Huang FW, Wrighting DM, Xia Y, Sidis Y, Samad TA, Campagna JA, Chung RT, Schneyer AL, Woolf CJ, Andrews NC, Lin HY (2006) Bone morphogenetic protein signaling by hemojuvelin regulates hepcidin expression. Nat Genet 38:531–539
Babitt JL, Huang FW, Xia Y, Sidis Y, Andrews NC, Lin HY (2007) Modulation of bone morphogenetic protein signaling in vivo regulates systemic iron balance. J Clin Invest 117:1933–1939
Shi Y, Massague J (2003) Mechanisms of TGF-beta signaling from cell membrane to the nucleus. Cell 113:685–700
Vukicevic S, Stavljenic A, Pecina M (1995) Discovery and clinical applications of bone morphogenetic proteins. Eur J Clin Chem Clin Biochem 33:661–671
Wang RH, Li C, Xu X, Zheng Y, Xiao C, Zerfas P, Cooperman S, Eckhaus M, Rouault T, Mishra L, Deng CX (2005) A role of SMAD4 in iron metabolism through the positive regulation of hepcidin expression. Cell Metab 2:399–409
Andriopoulos BJ, Corradini E, Xia Y, Faasse SA, Chen S, Grgurevic L, Knutson MD, Pietrangelo A, Vukicevic S, Lin HY, Babitt JL (2009) BMP6 is a key endogenous regulator of hepcidin expression and iron metabolism. Nat Genet 41:482–487
Meynard D, Kautz L, Darnaud V, Canonne-Hergaux F, Coppin H, Roth MP (2009) Lack of the bone morphogenetic protein BMP6 induces massive iron overload. Nat Genet 41:478–481
Corradini E, Schmidt PJ, Meynard D, Garuti C, Montosi G, Chen S, Vukicevic S, Pietrangelo A, Lin HY, Babitt JL (2010) BMP6 treatment compensates for the molecular defect and ameliorates hemochromatosis in Hfe knockout mice. Gastroenterol 139:1721–1729
Yu PB, Hong CC, Sachidanandan C, Babitt JL, Deng DY, Hoyng SA, Lin HY, Bloch KD, Peterson RT (2008) Dorsomorphin inhibits BMP signals required for embryogenesis and iron metabolism. Nat Chem Biol 4:33–41
Truksa J, Peng H, Lee P, Beutler E (2006) Bone morphogenetic proteins 2, 4, and 9 stimulate murine hepcidin 1 expression independently of Hfe, transferrin receptor 2 (Tfr2), and IL-6. Proc Natl Acad Sci U S A 103:10289–10293
Ramos E, Kautz L, Rodriguez R, Hansen M, Gabayan V, Ginzburg Y, Roth MP, Nemeth E, Ganz T (2011) Evidence for distinct pathways of hepcidin regulation by acute and chronic iron loading in mice. Hepatol 53:1333–1341
Vukicevic S, Kopp JB, Luyten FP, Sampath TK (1996) Induction of nephrogenic mesenchyme by osteogenic protein 1 (bone morphogenetic protein 7). Proc Natl Acad Sci U S A 93:9021–9026
Vukicevic S, Grgurevic L (2009) BMP-6 and mesenchymal stem cell differentiation. Cytokine Growth Factor Rev 20:441–448
Kautz L, Meynard D, Monnier A, Darnaud V, Bouvet R, Wang RH, Deng C, Vaulont S, Mosser J, Coppin H, Roth MP (2008) Iron regulates phosphorylation of Smad1/5/8 and gene expression of Bmp6, Smad7, Id1, and Atoh8 in the mouse liver. Blood 112:1503–1509
Arndt S, Maegdefrau U, Dorn C, Schardt K, Hellerbrand C, Bosserhoff AK (2010) Iron induced expression of bone morphogenic protein 6 in intestinal cells is the main regulator of hepatic hepcidin expression in vivo. Gastroenterol 138:372–382
Kautz L, Besson-Fournier C, Meynard D, Latour C, Roth MP, Coppin H (2011) Iron overload induces Bmp6 expression in the liver but not in the duodenum. Haematol 96:199–203
Corradini E, Meynard D, Wu Q, Chen S, Ventura P, Pietrangelo A, Babitt JL (2011) Serum and liver iron differently regulate the bone morphogenetic protein 6 (BMP6)-SMAD signaling pathway in mice. Hepatol 54:273–284
Zhang AS, Gao J, Koeberl DD, Enns CA (2010) The role of hepatocyte hemojuvelin in the regulation of bone morphogenic protein-6 and hepcidin expression in vivo. J Biol Chem 285:16416–16423
Solloway MJ, Dudley AT, Bikoff EK, Lyons KM, Hogan BL, Robertson EJ (1998) Mice lacking Bmp6 function. Dev Genet 22:321–339
Grgurevic L, Macek B, Healy DR, Brault AL, Erjavec I, Cipcic A, Grgurevic I, Rogic D, Galesic K, Brkljacic J, Stern-Padovan R, Paralkar VM, Vukicevic S (2011) Circulating bone morphogenetic protein 1-3 isoform increases renal fibrosis. J Am Soc Nephrol 22:681–692
Brkljacic J, Pauk M, Erjavec I, Cipcic A, Grgurevic L, Zadro R, Inman GJ, Vukicevic S (2013) Exogenous heparin binds and inhibits bone morphogenetic protein 6 biological activity. Int Orthop 37:529–541
Grgurevic L, Macek B, Durdevic D, Vukicevic S (2007) Detection of bone and cartilage-related proteins in plasma of patients with a bone fracture using liquid chromatography-mass spectrometry. Int Orthop 31:743–751
Tacke F, Gäbele E, Bataille F, Schwabe RF, Hellerbrand C, Klebl F, Straub RH, Luedde T, Manns MP, Trautwein C, Brenner DA, Schölmerich J, Schnabl B (2007) Bone morphogenetic protein 7 is elevated in patients with chronic liver disease and exerts fibrogenic effects on human hepatic stellate cells. Dig Dis Sci 52:3404–3415
David L, Mallet C, Keramidas M, Lamandé N, Gasc JM, Dupuis-Girod S, Plauchu H, Feige JJ, Bailly S (2008) Bone morphogenetic protein-9 is a circulating vascular quiescence factor. Circ Res 102:914–922
Knittel T, Fellmer P, Müller L, Ramadori G (1997) Bone morphogenetic protein-6 is expressed in nonparenchymal liver cells and upregulated by transforming growth factor-beta 1. Exp Cell Res 232:263–269
Ganz T, Olbina G, Girelli D, Nemeth E, Westerman M (2008) Immunoassay for human serum hepcidin. Blood 112:4292–4297
Wu Q, Sun CC, Lin HY, Babitt JL (2012) Repulsive guidance molecule (RGM) family proteins exhibit differential binding kinetics for bone morphogenetic proteins (BMPs). PLoS One 7:e46307
Ozkaynak E, Schnegelsberg PN, Oppermann H (1991) Murine osteogenic protein (OP-1): high levels of mRNA in kidney. Biochem Biophys Res Commun 179:116–123
Theurl I, Schroll A, Nairz M, Seifert M, Theurl M, Sonnweber T, Kulaksiz H, Weiss G (2011) Pathways for the regulation of hepcidin expression in anemia of chronic disease and iron deficiency anemia in vivo. Haematol 96:1761–1769
We thank Djurdja Car and Mirjana M. Renic for expert animal care.
Conflict of Interest
JLB and HYL have ownership interest in a start-up company Ferrumax Pharmaceuticals, which has licensed technology from the Massachusetts General Hospital based on their work.
This study was supported by grants from the Ministry of Science and Technology of the Republic of Croatia. No conflict of interest for any authors. JLB was supported in part by NIH grant RO1 DK087727.
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Pauk, M., Grgurevic, L., Brkljacic, J. et al. Exogenous BMP7 corrects plasma iron overload and bone loss in Bmp6-/- mice. International Orthopaedics (SICOT) 39, 161–172 (2015). https://doi.org/10.1007/s00264-014-2550-4
- Bone morphogenetic protein