Activin B Induces Noncanonical SMAD1/5/8 Signaling via BMP Type I Receptors in Hepatocytes: Evidence for a Role in Hepcidin Induction by Inflammation in Male Mice

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• Endothelial Bmp6 conditional knockout mice exhibit hemochromatosis, whereas hepatocyte and macrophage Bmp6 conditional knockout mice do not.• Our data support a model in which EC Bmp6 has paracrine actions on hepatocyte hemojuvelin to regulate hepcidin production.Bone morphogenetic protein 6 (BMP6) signaling in hepatocytes is a central transcriptional regulator of the iron hormone hepcidin that controls systemic iron balance. How iron levels are sensed to regulate hepcidin production is not known, but local induction of liver BMP6 expression by iron is proposed to have a critical role. To identify the cellular source of BMP6 responsible for hepcidin and iron homeostasis regulation, we generated mice with tissue-specific ablation of Bmp6 in different liver cell populations and evaluated their iron phenotype. Efficiency and specificity of Cre-mediated recombination was assessed by using Cre-reporter mice, polymerase chain reaction of genomic DNA, and quantitation of Bmp6 messenger RNA expression from isolated liver cell populations. Localization of the BMP co-receptor hemojuvelin was visualized by immunofluorescence microscopy. Analysis of the Bmp6 conditional knockout mice revealed that liver endothelial cells (ECs) expressed Bmp6, whereas resident liver macrophages (Kupffer cells) and hepatocytes did not. Loss of Bmp6 in ECs recapitulated the hemochromatosis phenotype of global Bmp6 knockout mice, whereas hepatocyte and macrophage Bmp6 conditional knockout mice exhibited no iron phenotype. Hemojuvelin was localized on the hepatocyte sinusoidal membrane immediately adjacent to Bmp6-producing sinusoidal ECs. Together, these data demonstrate that ECs are the predominant source of BMP6 in the liver and support a model in which EC BMP6 has paracrine actions on hepatocyte hemojuvelin to regulate hepcidin transcription and maintain systemic iron homeostasis. (Blood. 2017;129(4):405-414) IntroductionIron homeostasis is tightly controlled to ensure sufficient iron for erythropoiesis and other fundamental metabolic processes and to prevent the toxicity of excess iron.1 The peptide hormone hepcidin is a master regulator of systemic iron balance by mediating the degradation of the iron export protein ferroportin to limit dietary iron absorption and macrophage iron recycling.2 Hepcidin production in the liver is carefully titrated in response to changes in organismal iron content and iron utilization requirements.1 Failure to appropriately induce hepcidin in response to iron is a central feature of hereditary hemochromatosis, a genetic disorder characterized by excess iron deposits in the liver, heart, and endocrine glands, resulting in organ dysfunction. 3 The most severe juvenile-onset form of this disease is caused by mutations in the genes encoding hepcidin itself (HAMP) or hemojuvelin (HFE2, also known as HJV), which regulates hepcidin transcription in hepatocytes. 4,5 Mice with global or hepatocyte-specific deletion of these genes have a similar iron overload phenotype, 6-11 confirming the essential role of hepat...

Section: Discussionmentioning

confidence: 99%

Endothelial cells produce bone morphogenetic protein 6 required for iron homeostasis in mice

Canali

Zumbrennen-Bullough

Core

et al. 2017

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“…Interestingly, in mouse models of acute (lipopolysaccharide [LPS]) and chronic (Brucella abortus) inflammation, treatment with follistatin, an activin inhibitor that binds at high efficiency both Activin A and Activin B, reverts hepcidin activation. 39 Activin B, a member of the TGF-b superfamily released in inflammation, initially proposed as a potential ligand, 40 was recently dismissed as hepcidin regulator. 41 Activin A, a critical component of the inflammatory response, secreted in the circulation, was not previously reported to take part in hepcidin activation.…”

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confidence: 99%

“…41 Activin A, a critical component of the inflammatory response, secreted in the circulation, was not previously reported to take part in hepcidin activation. 39,42 Activin A binds BMPR-II, ACVR2A and ACVR2B, and the BMPR-I ALK4. 25,43 The complex signals through phosphorylation and nuclear translocation of the transcription factors SMAD2 and SMAD3 (supplemental Figure 6A).…”

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confidence: 99%

The immunophilin FKBP12 inhibits hepcidin expression by binding the BMP type I receptor ALK2 in hepatocytes

Colucci

Pagani

Pettinato

et al. 2017

Key Points• FKBP12 suppresses hepcidin by interaction with the BMP receptor ALK2.• Disruption of FKBP12-ALK2 interaction increases hepcidin and renders the receptor responsive to the inflammatory ligand Activin A.The expression of the key regulator of iron homeostasis hepcidin is activated by the BMP-SMAD pathway in response to iron and inflammation and among drugs, by rapamycin, which inhibits mTOR in complex with the immunophilin FKBP12. FKBP12 interacts with BMP type I receptors to avoid uncontrolled signaling. By pharmacologic and genetic studies, we identify FKBP12 as a novel hepcidin regulator. Sequestration of FKBP12 by rapamycin or tacrolimus activates hepcidin both in vitro and in murine hepatocytes. Acute tacrolimus treatment transiently increases hepcidin in wild-type mice. FKBP12 preferentially targets the BMP receptor ALK2. ALK2 mutants defective in binding FKBP12 increase hepcidin expression in a ligand-independent manner, through BMP-SMAD signaling. ALK2 free of FKBP12 becomes responsive to the noncanonical inflammatory ligand Activin A. Our results identify a novel hepcidin regulator and a potential therapeutic target to increase defective BMP signaling in

“…1,2 To confirm the cause and effect relationships among activin B, Smad1/ 5/8 phosphorylation, and hepcidin in vivo, we challenged Inhbb 2/2 mice 3 (deficient in activin B) with lipopolysaccharide (LPS) or infected them with an Escherichia coli septicemic strain, as indicated in supplemental Methods (available on the Blood Web site).To examine whether, as observed in human hepatoma cell lines and in mouse primary hepatocytes, activin B also stimulates canonical SMAD2/3 and noncanonical SMAD1/5/8 signaling in vivo, we compared by western blot analysis Smad2 and Smad5 phosphorylation levels in the liver of wild-type and Inhbb 2/2 mice 4 hours after LPS stimulation ( Figure 1A) or E coli infection ( Figure 1B). Whereas these inflammatory stimuli strongly induce Smad2 and Smad5 phosphorylation in wild-type mice, none of these Smad effectors are activated in mice deficient in activin B.…”

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confidence: 99%

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confidence: 99%

Hepcidin upregulation by inflammation is independent of Smad1/5/8 signaling by activin B

Besson-Fournier

Gineste

Latour

et al. 2017

Activin B, which is strongly induced by inflammatory stimuli in the mouse liver, has recently appeared as a potent inductor of hepcidin in vitro, via the crossactivation of noncanonical SMAD1/5/8 signaling. 1,2 To confirm the cause and effect relationships among activin B, Smad1/ 5/8 phosphorylation, and hepcidin in vivo, we challenged Inhbb 2/2 mice 3 (deficient in activin B) with lipopolysaccharide (LPS) or infected them with an Escherichia coli septicemic strain, as indicated in supplemental Methods (available on the Blood Web site).To examine whether, as observed in human hepatoma cell lines and in mouse primary hepatocytes, activin B also stimulates canonical SMAD2/3 and noncanonical SMAD1/5/8 signaling in vivo, we compared by western blot analysis Smad2 and Smad5 phosphorylation levels in the liver of wild-type and Inhbb 2/2 mice 4 hours after LPS stimulation ( Figure 1A) or E coli infection ( Figure 1B). Whereas these inflammatory stimuli strongly induce Smad2 and Smad5 phosphorylation in wild-type mice, none of these Smad effectors are activated in mice deficient in activin B. These data demonstrate that activin B, whose messenger RNA (mRNA) expression is strongly induced after LPS administration ( Figure 1C) or E coli infection ( Figure 1D), is required for activation of Smad1/5/8 signaling in these mice. In parallel to activating SMAD1/5/8 phosphorylation in vitro, activin B was also shown to induce hepcidin expression.1,2 Remarkably, pretreatment of hepatocytes with the BMP type I receptor inhibitor LDN-193189 prevented both the induction of SMAD/5/8 phosphorylation and the upregulation of the hepcidin (HAMP) gene expression by activin B, 1 suggesting that, at least in vitro, the effect of activin B on hepcidin expression was entirely attributable to the activation of noncanonical SMAD signaling. Since activin B also activates noncanonical Smad1/5/8 signaling in vivo, we expected that induction of hepcidin expression in response to LPS administration or E coli infection would be impaired in Inhbb 2/2 mice. However, the magnitude of hepcidin mRNA induction and its evolution over time was unexpectedly similar in wild-type and in Inhbb 2/2 mice challenged with LPS ( Figure 1E) or infected with E coli (Figure 1F), pointing out the limitations of in vitro studies. To confirm the data at the protein level, serum hepcidin was quantified by competitive enzyme-linked immunosorbent assay (ELISA) in wild-type and in Inhbb 2/2 mice before and 4 hours after a LPS challenge. As suggested by the quantitative polymerase chain reaction (PCR) data, and similarly to wild-type mice, Inhbb 2/2 mice produce on average 3 times more hepcidin after endotoxin administration ( Figure 1G). These results show that neither activation of Smad1/5/8 signaling nor activin B induction is necessary for upregulation of hepcidin production by inflammatory stimuli such as LPS administration or E coli infection.