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.

Hypoxia decreases hepatic hepcidin expression by a novel regulatory pathway exerted via PDGF-BB, leading to increased availability of circulating iron that can be used for erythropoiesis.

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...

Key Points• Genome-wide RNAi screen provides the first comprehensive list of putative hepatic hepcidin regulators.• Hepcidin suppression is linked to the control of mitogen stimulation and nutrient status via components of Ras/RAF MAPK and mTOR signaling.The hepatic hormone hepcidin is a key regulator of systemic iron metabolism. Its expression is largely regulated by 2 signaling pathways: the "iron-regulated" bone morphogenetic protein (BMP) and the inflammatory JAK-STAT pathways. To obtain broader insights into cellular processes that modulate hepcidin transcription and to provide a resource to identify novel genetic modifiers of systemic iron homeostasis, we designed an RNA interference (RNAi) screen that monitors hepcidin promoter activity after the knockdown of 19 599 genes in hepatocarcinoma cells. Interestingly, many of the putative hepcidin activators play roles in signal transduction, inflammation, or transcription, and affect hepcidin transcription through BMP-responsive elements. Furthermore, our work sheds light on new components of the transcriptional machinery that maintain steady-state levels of hepcidin expression and its responses to the BMP-and interleukin-6-triggered signals. Notably, we discover hepcidin suppression mediated via components of Ras/ RAF MAPK and mTOR signaling, linking hepcidin transcriptional control to the pathways that respond to mitogen stimulation and nutrient status. Thus using a combination of RNAi screening, reverse phase protein arrays, and small molecules testing, we identify links between the control of systemic iron homeostasis and critical liver processes such as regeneration, response to injury, carcinogenesis, and nutrient metabolism. (Blood. 2014;123(10):1574-1585

Hepcidin regulates systemic iron homeostasis. Suppression of hepcidin expression occurs physiologically in iron deficiency and increased erythropoiesis but is pathologic in thalassemia and hemochromatosis. Here we show that epigenetic events govern hepcidin expression. Erythropoiesis and iron deficiency suppress hepcidin via erythroferrone-dependent and -independent mechanisms, respectively, in vivo, but both involve reversible loss of H3K9ac and H3K4me3 at the hepcidin locus. In vitro, pan-histone deacetylase inhibition elevates hepcidin expression, and in vivo maintains H3K9ac at hepcidin-associated chromatin and abrogates hepcidin suppression by erythropoietin, iron deficiency, thalassemia, and hemochromatosis. Histone deacetylase 3 and its cofactor NCOR1 regulate hepcidin; histone deacetylase 3 binds chromatin at the hepcidin locus, and histone deacetylase 3 knockdown counteracts hepcidin suppression induced either by erythroferrone or by inhibiting bone morphogenetic protein signaling. In iron deficient mice, the histone deacetylase 3 inhibitor RGFP966 increases hepcidin, and RNA sequencing confirms hepcidin is one of the genes most differentially regulated by this drug in vivo. We conclude that suppression of hepcidin expression involves epigenetic regulation by histone deacetylase 3.

The inhibitory Smad7 acts as a critical suppressor of hepcidin, the major regulator of systemic iron homeostasis. In this study we define the mRNA expression of the two functionally related Smad proteins, Smad6 and Smad7, within pathways known to regulate hepcidin levels. Using mouse models for hereditary hemochromatosis (Hfe-, TfR2-, Hfe/TfR2-, Hjv- and hepcidin1-deficient mice) we show that hepcidin, Smad6 and Smad7 mRNA expression is coordinated in such a way that it correlates with the activity of the Bmp/Smad signaling pathway rather than with liver iron levels. This regulatory circuitry is disconnected by iron treatment of Hfe-/- and Hfe/TfR2 mice that significantly increases hepatic iron levels as well as hepcidin, Smad6 and Smad7 mRNA expression but fails to augment pSmad1/5/8 levels. This suggests that additional pathways contribute to the regulation of hepcidin, Smad6 and Smad7 under these conditions which do not require Hfe.

Podoplanin (PDPN), an O-glycosylated, transmembrane, mucin-type glycoprotein, is expressed by cancer associated fibroblasts (CAFs). In malignant transformation, PDPN is subjected to changes and its role is yet to be established. Here we show that it is involved in modulating the activity of the CCL21/CCR7 chemokine/receptor axis in a hypoxia-dependent manner. In the present model, breast cancer MDA-MB-231 cells and NKL3 cells express the surface CCR7 receptor for CCL21 chemokine which is a potent chemoattractant able to bind to PDPN. The impact of the CCL21/CCR7 axis in the molecular mechanism of the adhesion of NKL3 cells and of MDA-MB-231 breast cancer cells was reduced in a hypoxic tumor environment. In addition to its known effect on migration, CCL21/CCR7 interaction was shown to allow NK cell adhesion to endothelial cells (ECs) and its reduction by hypoxia. A PDPN expressing model of CAFs made it possible to demonstrate the same CCL21/CCR7 axis involvement in the tumor cells to CAFs recognition mechanism through PDPN binding of CCL21. PDPN was induced by hypoxia and its overexpression undergoes a reduction of adhesion, making it an anti-adhesion molecule in the absence of CCL21, in the tumor. CCL21/CCR7 modulated NK cells/ECs and MDA-MB-231 cells/CAF PDPN-dependent interactions were further shown to be linked to hypoxia-dependent microRNAs as miRs: miR-210 and specifically miR-21, miR-29b which influence PDPN expression.

Disorders of iron metabolism are largely attributed to an excessive or insufficient expression of hepcidin, the master regulator of systemic iron homeostasis. Here, we investigated whether drugs targeting genetic regulators of hepcidin can affect iron homeostasis. We focused our efforts on drugs approved for clinical use to enable repositioning strategies and/or to reveal iron-related side effects of widely prescribed therapeutics. To identify hepcidin-modulating therapeutics, we re-evaluated data generated by a genome-wide RNAi screen for hepcidin regulators. We identified ‘druggable’ screening hits and validated those by applying RNAi of potential drug targets and small-molecule testing in a hepatocytic cell line, in primary murine and human hepatocytes and in mice. We initially identified spironolactone, diclofenac, imatinib and Suberoylanilide hydroxamic acid (SAHA) as hepcidin modulating drugs in cellular assays. Among these, imatinib and spironolactone further suppressed liver hepcidin expression in mice. Our results demonstrate that a commonly used anti-hypertensive drug, spironolactone, which is prescribed for the treatment of heart failure, acne and female hirsutism, as well as imatinib, a first-line, lifelong therapeutic option for some frequent cancer types suppress hepcidin expression in cultured cells and in mice. We expect these results to be of relevance for patient management, which needs to be addressed in prospective clinical studies.