The hemochromatosis proteins HFE, transferrin receptor 2 (TfR2) and hemojuvelin (HJV, HFE2) positively control expression of the major iron regulatory hormone hepcidin. HJV is a bone morphogenetic protein (BMP) co-receptor that enhances the cellular response to BMP cytokines via the phosphorylation of SMAD proteins. In this study, we show that two highly conserved and sequence-identical BMP-responsive elements located at positions -84/-79 (BMP-RE1) and -2,255/-2,250 (BMP-RE2) of the human hepcidin promoter are critical for both the basal hepcidin mRNA expression and the hepcidin response to BMP-2 and BMP-6. While BMP-RE1 and BMP-RE2 show additive effects in responding to HJV-mediated BMP signals, only BMP-RE1 that is located in close proximity to a previously identified STAT-binding site is important for the hepcidin response to IL-6. These data identify a missing link between the HJV/BMP signaling pathways and hepcidin transcription, and further define the connection between inflammation and BMP-dependent hepcidin promoter activation. As such, they provide important new information furthering our understanding of disorders of iron metabolism and the anemia of inflammation.

The precise regulation of the iron-regulatory hormone hepcidin is essential to maintain body iron homeostasis: Hepcidin deficiency induces iron overload, and hepcidin excess results in anaemia. Mutations in the gene HFE2 cause severe iron overload and are associated with low hepcidin expression. Recent data suggest that HFE2 is a bone morphogenetic protein (BMP) co-receptor, and that the decreased hepcidin mRNA expression because of HFE2 dysfunction is a result of impaired BMP signalling ability. In this study, we identify a critical BMP-responsive element (BMP-RE) at position -84/-79 of the hepcidin promoter. We show that this element mediates HFE2-dependent basal hepcidin mRNA expression under control conditions. Unexpectedly, the mutation of the same BMP-RE element also severely impairs hepcidin activation in response to IL-6. These data uncover a missing link in the HFE2-mediated control of hepcidin expression and suggest that the BMP-RE controls hepcidin promoter activity mediated by HFE2 and inflammatory stimuli.

Hepcidin is the master regulatory hormone of systemic iron metabolism. Hepcidin deficiency causes common iron overload syndromes whereas its overexpression is responsible for microcytic anemias. Hepcidin transcription is activated by the bone morphogenetic protein (BMP) and the inflammatory JAK-STAT pathways, whereas comparatively little is known about how hepcidin expression is inhibited. By using highthroughput 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 signaling 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 hepatocytes and that SMAD7 overexpression completely 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 suppression, demonstrating that SMAD7 does not simply antagonize the previously reported hemojuvelin/BMP-responsive elements. This work identifies a potent inhibitory 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) IntroductionHepcidin 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 internalization 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 hospitalized 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, SM...