Volume 138, Issue 1, Pages 372-382 (January 2010) Iron-Induced Expression of Bone Morphogenic Protein 6 in Intestinal Cells Is the Main Regulator of Hepatic Hepcidin Expression In Vivo  Stephanie Arndt, Ulrike Maegdefrau, Christoph Dorn, Katharina Schardt, Claus Hellerbrand, Anja–Katrin Bosserhoff  Gastroenterology  Volume 138, Issue 1, Pages 372-382 (January 2010) DOI: 10.1053/j.gastro.2009.09.048 Copyright © 2010 AGA Institute Terms and Conditions

Figure 1 Hepatic iron accumulation and hepcidin expression in BMP6−/− mice on 129Sv/Ev background. (A) Iron accumulation in liver sections of BMP6−/− mice with increasing age. BMP6−/− mice older than 5 months develop a massive granular iron accumulation across all zones of the liver. In BMP6−/− mice older than 8 months, a Scheuer grade29 4 iron overload with focal deposition in portal macrophages was observed. BMP6+/+ mice did not show pathologic changes. (B) Hepatic iron accumulation in sections of BMP6−/− mice fed diets with different iron content or a methionine-choline-deficient (MCD) diet. Two- to 3-month-old mice were fed for 3 weeks with an iron-supplemented diet. An iron gradient was identified with maximum iron in periportal (zone 1) hepatocytes in BMP6−/− animals, whereas no apparent accumulation was observed in BMP6+/+ mice. MCD-fed BMP6−/− mice developed a massive and uniform distributed iron accumulation throughout the liver, whereas no iron accumulation was visible in the liver of BMP6+/+ mice. Prussian Blue staining does not reveal major differences regarding iron accumulation in BMP6−/− and BMP6+/+ mice fed iron-balanced (standard) or iron-deficient diet for 3 weeks (n = 4–11 mice per group in analogy to Table 2). (C) Expression of hepcidin in the liver after intravenous iron injection. mRNA analysis by quantitative RT-PCR revealed no induction of hepcidin in the liver of BMP6−/− mice after intravenous iron-dextran injection, whereas this treatment resulted in significantly increased hepcidin expression in BMP6+/+ mice (*P < .05 compared with control). (D) BMP6 serum levels after intravenous iron injection or feeding a diet with elevated iron content. Parenteral application of iron-dextran or feeding an iron-supplemented diet induced BMP6 protein in the serum of BMP6+/+ mice as assessed by Western blot analysis. The 23-kilodalton band of mature BMP6 migrates in serum probes as a subtle doublet, which was most apparent in lower percentage polyacrylamide gels. This may have been caused by variations in glycosylation as described.28 Gastroenterology 2010 138, 372-382DOI: (10.1053/j.gastro.2009.09.048) Copyright © 2010 AGA Institute Terms and Conditions

Figure 2 BMP6 and hepcidin expression after sensing iron in vitro in macrophages and hepatic cells. BMP6 mRNA expression was analyzed in macrophages (A) and hepatic cells (B) of BMP6+/+ mice after treatment with FeSO4 or holo-transferrin (50 μmol/L for 1 hour) by quantitative RT-PCR. Neither macrophages nor hepatocytes (HEP), hepatic stellate cells (HSC), or Kupffer cells (KC) revealed a significant induction of BMP6 expression after sensing iron. (C) Furthermore, treatment with FeSO4 or holo-transferrin did not induce hepcidin mRNA expression of hepatic cells in vitro. Gastroenterology 2010 138, 372-382DOI: (10.1053/j.gastro.2009.09.048) Copyright © 2010 AGA Institute Terms and Conditions

Figure 3 BMP6 and hepcidin expression in hepatic and small intestinal tissue after sensing iron ex vivo. Ex vivo assessment of BMP6 mRNA (A) and protein (B) expression in tissue slices of the liver and the small intestine after treatment with FeSO4 or holo-transferrin (50 μmol/L; 1 hour for RNA and 4 hours for protein analysis). Only intestinal tissue revealed a marked increase of BMP6 expression after sensing iron (*P < .05 compared with PBS control). (C) Hepatic hepcidin mRNA expression was not altered by treatment with FeSO4 or holo-transferrin. Gastroenterology 2010 138, 372-382DOI: (10.1053/j.gastro.2009.09.048) Copyright © 2010 AGA Institute Terms and Conditions

Figure 4 BMP6 expression in the liver and the small intestine after sensing iron in vivo. BMP6 mRNA (A and C) and protein (B and D) expression were analyzed in the liver and the small intestine after (a single) parenteral application of iron-dextran or dextran as control (n = 5 mice/group). Furthermore, mice were fed an iron-balanced (control; 5 or 6 mice/group), an iron-supplemented (11 mice/group), or an iron-deficient (8 mice/group) diet for 3 weeks. Iron sensing resulted in a significant induction of BMP6 expression in the small intestine. Immunohistologic analysis of the liver (E) and the small intestine (F) after iron exposure. (E) Liver sections of 2- to 3-month-old BMP6+/+ mice revealed no specific BMP6 staining after parenteral application of iron-dextran or feeding iron-supplemented and iron-deficient diets. The visible staining around the hepatocytes is nonspecific and because of iron overload as determined in the BMP6−/− mice. (F) Immunohistochemical analysis of the small intestine revealed specific BMP6 staining in enterocytes of 2- to 3-month-old BMP6+/+ mice injected with iron-dextran or fed with an iron-supplemented diet. Gastroenterology 2010 138, 372-382DOI: (10.1053/j.gastro.2009.09.048) Copyright © 2010 AGA Institute Terms and Conditions

Figure 5 Intestinal BMP expression after sensing iron in vivo, effect of different BMPs on hepatocellular hepcidin expression, and analysis of hepatocellular BMP-receptor expression in vitro. (A) Feeding an iron-supplemented diet increased expression levels of BMP2, BMP4, and BMP9 in the small intestine of BMP6−/− but not BMP6+/+ mice as determined by quantitative RT-PCR (*P < .05 compared with BMP6+/+ mice). (B) In accordance, treatment of primary hepatocytes isolated from BMP6+/+ mice with different recombinant BMPs (100 ng/mL BMP2, 4, 6, or 9 for 12 hours) revealed BMP6 as the most potent stimulator of hepcidin mRNA expression (*P < .05 compared with control). (C) Ex vivo coculture experiments with tissue samples from the small intestine and the liver of BMP6−/− and BMP6+/+ mice. Incubation of cocultures of small intestine from BMP6+/+ and liver from BMP6−/− mice with FeSO4 (5 hours) resulted in a strong induction of hepatic hepcidin mRNA expression (P < .05 compared with PBS). (D) The expression of different BMP-receptor subunits in primary hepatocytes isolated from BMP6−/− and BMP6+/+ mice was analyzed by quantitative RT-PCR. BMPRIA (Alk-3), BMPR2, and ActRIIA are strongly expressed in hepatocytes. BMPRIB (Alk-6), ActRIIB, AMHR2, and ActRIA (Alk-2) are only weakly expressed in hepatocytes. Gastroenterology 2010 138, 372-382DOI: (10.1053/j.gastro.2009.09.048) Copyright © 2010 AGA Institute Terms and Conditions

Figure 6 Working model for hepcidin regulation after iron exposure. Communication between the small intestine and the liver regulates hepcidin expression. Iron absorption in enterocytes leads to the activation of BMP6 expression and, subsequently, to the delivery of BMP6 to the liver via portal circulation. BMP6 binds to type I and II BMP receptors BMPRIA and ActRIIA and to the coreceptor hemojuvelin (HJV) on hepatocytes and activates the Smad signaling pathway through phosphorylation of Smad1/5/8 and complex formation with Smad4. This complex translocates into the nucleus and stimulates the expression of hepcidin.8,10 Gastroenterology 2010 138, 372-382DOI: (10.1053/j.gastro.2009.09.048) Copyright © 2010 AGA Institute Terms and Conditions