SMAD7 controls iron metabolism as a potent inhibitor of hepcidin expression

Abstract

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...

The murine growth differentiation factor 15 is not essential for systemic iron homeostasis in phlebotomized mice

“…Smad6 mRNA levels are not further increased in phlebotomized Gdf15 -/-mice, which may be attributed to a saturation of the signaling pathways that activate Smad6 mRNA expression. By contrast, the mRNA levels of Smad7, a recently identified suppressor of hepcidin expression, 20 were reduced upon phlebotomy in wild-type mice, suggesting that it does not contribute to the reduction of hepcidin levels in response to acute anemia.…”

Regulation of TMPRSS6 by BMP6 and iron in human cells and mice

“…This time delay is unlike the time course of other BMP-SMAD targets, such as hepcidin and ID1, that can rapidly increase their expression within 1 hour of BMP-SMAD pathway activation. With promoter analysis software, we did not identify in the TMPRSS6 promoter any BMP responsive elements (BMP-RE) with sequences identical to the ones previously identified in the hepcidin, 51 ID1, 52 and SMAD7 45 promoters. Thus, it is unlikely that the BMP-SMAD pathway directly regulates TMPRSS6 mRNA through a BMP-RE in the TMPRSS6 promoter.…”

“…We hypothesized that SMAD7 could participate in TMPRSS6 regulation because it was demonstrated that SMAD7 is an inhibitory SMAD protein that is induced by BMP6-SMAD signaling in hepatocytes to antagonize TGF-␤/BMP signaling [42][43][44] and to suppress steady-state hepcidin expression as well as hepcidin induction by BMP stimulation. 45 To test whether SMAD7 is involved in TMPRSS6 mRNA regulation, we transfected Hep3B cells with a pool of siRNA directed against SMAD7 and a control siRNA pool. Treatment of Hep3B cells with BMP6 induced an increase in SMAD7 mRNA expression by 10-fold ( Figure 3A black bars), whereas silencing of SMAD7 with siRNA transfection in nontreated cells or cells treated with 25 ng/mL of BMP6 leads to a reduction of SMAD7 mRNA level by 70% ( Figure 3A gray bars).…”

Smad7 deficiency decreases iron and haemoglobin through hepcidin up‐regulation by multilayer compensatory mechanisms

“…Although Smad7 has been suggested to regulate hepcidin expression based on in vitro studies , 18, 19 this function has not been investigated in vivo. In cell lines, overexpressing Smad7 reduced hepcidin expression 18. Thus, Smad7 may serve as a negative feedback regulator of hepcidin expression.…”