RED CELLS, IRON, AND ERYTHROPOIESIS SMAD7 controls iron metabolism as a potent inhibitor of hepcidin expression Katarzyna Mleczko-Sanecka, 1-3 Guillem Casanovas, 1-3 Anan Ragab, 4 Katja Breitkopf, 5 Alexandra Mu ¨ ller, 5 Michael Boutros, 4 Steven Dooley, 5 Matthias W. Hentze, 2,3 and Martina U. Muckenthaler 1,2 1 Department of Pediatric Oncology, Hematology and Immunology, University of Heidelberg, Heidelberg; 2 Molecular Medicine Partnership Unit, Heidelberg; 3 European Molecular Biology Laboratory, Heidelberg; 4 Division of Signaling and Functional Genomics, German Cancer Research Center and Department of Cell and Molecular Biology, University of Heidelberg, Heidelberg; and 5 Department of Medicine II, Gastroenterology and Hepatology, University Hospital, Mannheim, Germany Hepcidin is the master regulatory hormone of systemic iron metabolism. Hepcidin defi- ciency causes common iron overload syn- dromes whereas its overexpression is re- sponsible for microcytic anemias. Hepcidin transcription is activated by the bone mor- phogenetic protein (BMP) and the inflamma- tory JAK-STAT pathways, whereas compara- tively little is known about how hepcidin expression is inhibited. By using high- throughput siRNA screening we identified SMAD7 as a potent hepcidin suppressor. SMAD7 is an inhibitory SMAD protein that mediates a negative feedback loop to both transforming growth factor- and BMP sig- naling and that recently was shown to be coregulated with hepcidin via SMAD4 in response to altered iron availability in vivo. We show that SMAD7 is coregulated with hepcidin by BMPs in primary murine hepato- cytes and that SMAD7 overexpression com- pletely abolishes hepcidin activation by BMPs and transforming growth factor-. We identify a distinct SMAD regulatory motif (GTCAAGAC) within the hepcidin promoter involved in SMAD7-dependent hepcidin sup- pression, demonstrating that SMAD7 does not simply antagonize the previously re- ported hemojuvelin/BMP-responsive ele- ments. This work identifies a potent inhibi- tory factor for hepcidin expression and uncovers a negative feedback pathway for hepcidin regulation, providing insight into a mechanism how hepcidin expression may be limited to avoid iron deficiency. (Blood. 2010;115(13):2657-2665) Introduction Hepcidin is an iron-regulated hepatic peptide hormone that controls systemic iron homeostasis. Iron excess or inflammatory cytokines stimulate hepcidin expression, leading to reduced plasma iron levels as the result of iron retention in macrophages and reduced intestinal iron absorption. Hypoxia, high erythropoietic activity, and iron deficiency inhibit hepcidin expression by largely unknown mechanisms to mobilize iron stores and increase iron absorption. 1 Hepcidin exerts its function by binding to the iron efflux channel ferroportin, which is predominantly expressed on macrophages, intestinal enterocytes, and hepatocytes, causing ferroportin internal- ization and degradation. 2 Hepcidin levels are inappropriately low in hereditary hemochromatosis, a disease caused by mutations in HFE, 3 transferrin receptor 2, 4 hemojuvelin (HJV, HFE2), 5 or hepcidin itself. 6 By contrast, constant induction of hepcidin by inflammatory cytokines is implicated in the pathogenesis of the anemia of inflammation, a disease commonly observed in hospital- ized patients. 7 Two major signaling pathways communicate systemic stimuli to activate hepcidin mRNA expression in hepatocytes. One is induced by bone morphogenetic proteins (BMPs), a group of cytokines of the transforming growth factor- (TGF-) family. 8 BMP-mediated hepcidin activation involves BMP receptors at the cell surface, as well as the BMP coreceptor HJV. 9,10 BMP-receptor interaction induces phosphorylation of receptor activated (R)- SMAD proteins and subsequent formation of active transcriptional complexes involving the co-SMAD factor, SMAD4. Two sequence motifs (the proximal BMP-RE1 and the distal BMP-RE2) within the human and murine hepcidin promoters are critical for the stimulation of hepcidin via HJV, BMP, and SMAD4. 11,12 The BMP signaling pathway communicates systemic iron levels, 13-15 main- tains steady-state hepcidin expression, and contributes to the activation of hepcidin by inflammatory stimuli at the level of SMAD4. 12,16,17 In addition, proinflammatory cytokines stimulate hepcidin transcription via the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway and a STAT binding motif proximal to the transcription start site. 18,19 Although the hepcidin activating pathways are beginning to be understood, comparatively little is known about how hepcidin expression is suppressed by hypoxia and active erythropoiesis to allow adequate iron uptake. Growth differentiation factor-15, twisted gastrulation 1, and erythropoietin have been implicated in mediating hepcidin suppression in response to augmented hematopoietic activity, 20-23 but their mode of involvement remains to be defined. Sensing of iron deficiency recently was linked to TMPRSS6, a protease shown to cleave HJV, 24 and to the von Hippel-Lindau–hypoxia-inducible factor pathway. 25 A further study by Braliou et al 26 suggested that hypoxia-mediated hepcidin suppression requires 2-oxoglutarate–dependent oxygenases but is independent of hypoxia-inducible factor-1. For all of the impli- cated negative regulators, it is unclear how repressive signals reach the hepcidin promoter. In this work, we identify SMAD7 as a potent repressor of hepcidin transcription and define its mechanism of action. In addition, our data assign functional importance to the previously reported observation that SMAD7 and hepcidin are coregulated in Submitted September 9, 2009; accepted December 1, 2009. Prepublished online as Blood First edition paper, December 29, 2009. DOI: 10.1182/blood- 2009-09-238105. The online version of this article contains a data supplement. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ‘‘advertisement’’ in accordance with 18 USC section 1734. © 2010 by The American Society of Hematology 2657 BLOOD, 1 APRIL 2010  VOLUME 115, NUMBER 13 For personal use only. on February 19, 2016. by guest www.bloodjournal.org From