Total iron quantification in liver cells from wild type and <i>Hjv</i> KO mice.

<p>HCs: hepatocytes. KCs: kupffer cells. LSECs: liver sinusoidal endothelial cells.</p><p>*: <i>Hjv</i> KO vs wild type <i>P</i> = 0.036.</p><p>^ns: <i>Hjv</i> KO vs wild type non significant.</p><p>Total iron quantification in liver cells from wild type and <i>Hjv</i> KO mice.</p

<i>Bmp6</i> Expression in Murine Liver Non Parenchymal Cells: A Mechanism to Control their High Iron Exporter Activity and Protect Hepatocytes from Iron Overload?

<div><p><i>Bmp6</i> is the main activator of hepcidin, the liver hormone that negatively regulates plasma iron influx by degrading the sole iron exporter ferroportin in enterocytes and macrophages. <i>Bmp6</i> expression is modulated by iron but the molecular mechanisms are unknown. Although hepcidin is expressed almost exclusively by hepatocytes (HCs), <i>Bmp6</i> is produced also by non-parenchymal cells (NPCs), mainly sinusoidal endothelial cells (LSECs). To investigate the regulation of <i>Bmp6</i> in HCs and NPCs, liver cells were isolated from adult wild type mice whose diet was modified in iron content in acute or chronic manner and in disease models of iron deficiency (<i>Tmprss6</i> KO mouse) and overload (<i>Hjv</i> KO mouse). With manipulation of dietary iron in wild-type mice, <i>Bmp6</i> and <i>Tfr1</i> expression in both HCs and NPCs was inversely related, as expected. When hepcidin expression is abnormal in murine models of iron overload (<i>Hjv</i> KO mice) and deficiency (<i>Tmprss6</i> KO mice), <i>Bmp6</i> expression in NPCs was not related to <i>Tfr1</i>. Despite the low <i>Bmp6</i> in NPCs from <i>Tmprss6</i> KO mice, <i>Tfr1</i> mRNA was also low. Conversely, despite body iron overload and high expression of <i>Bmp6</i> in NPCs from <i>Hjv</i> KO mice, <i>Tfr1</i> mRNA and protein were increased. However, in the same cells ferritin L was only slightly increased, but the iron content was not, suggesting that <i>Bmp6</i> in these cells reflects the high intracellular iron import and export. We propose that NPCs, sensing the iron flux, not only increase hepcidin through <i>Bmp6</i> with a paracrine mechanism to control systemic iron homeostasis but, controlling hepcidin, they regulate their own ferroportin, inducing iron retention or release and further modulating <i>Bmp6</i> production in an autocrine manner. This mechanism, that contributes to protect HC from iron loading or deficiency, is lost in disease models of hepcidin production.</p></div

<i>Bmp6</i> and <i>Tfr1</i> expression levels in acute dietary iron changes.

<p><i>Bmp6</i> (<b>A, C, E</b>) and <i>Tfr1</i> (<b>B, D, F</b>) mRNA expression was evaluated by qRT-PCR in HCs (<b>A, B</b>), KCs (<b>C, D</b>) and LSECs (<b>E, F)</b> isolated from 4–7 mice/group. <i>Hprt1</i> was used as housekeeping gene. mRNA expression ratio was normalized to control (ID-1 day) mean value set to 1. Error bars indicate SE. *: P<. 05; **: P<. 01; ***: P<. 001; ns: not significant.</p

<i>Bmp6</i> and <i>Tfr1</i> expression in chronic dietary iron changes.

<p><i>Bmp6</i> (<b>A, C, E</b>) and <i>Tfr1</i> (<b>B, D, F</b>) mRNA expression was evaluated by qRT-PCR in isolated HCs (<b>A, B</b>), KCs (<b>C, D</b>) and LSECs (<b>E, F</b>) from 4–12 mice/group. mRNA expression ratio was normGalized relative to housekeeping <i>Hprt1</i>. Mean control value of IB-treated mice was set to 1. Error bars indicate SE. *: P<. 05; **: P<. 01; ***: P<. 001; ns: not significant.</p

<i>Bmp6</i> and <i>Tfr1</i> expression in <i>Hjv</i> KO mice.

<p><i>Bmp6</i> (<b>A, C, E</b>) and <i>Tfr1</i> (<b>B, D, F</b>) mRNA expression was evaluated in liver cells isolated from 6–10 mice by qRT-PCR, using <i>Hprt1</i> as the referred housekeeping gene. mRNA expression ratio was normalized to control wild type (wt) mean value set to 1. Error bars indicate SE. **: P<. 01; ***: P<. 001; ****: P<. 0001.</p

Isolated liver cells characterization.

<p>Liver cells were isolated from 4–6 mice using the protocol reported in Experimental Procedures. Hepcidin (<i>Hamp</i>, <b>A</b>), Bone Morphogenetic Protein 6 (<i>Bmp6</i>, <b>B</b>), Transferrin Receptor 1 (<i>Tfr1</i>, <b>D</b>) and ferroportin (<i>Fpn</i>, <b>E</b>) mRNA expression was quantified by qRT-PCR relative to housekeeping <i>Hprt1</i> gene. (<b>C</b>) <b>Upper panel</b>: IRE/IRP-binding activity of HCs, KCs and LSECs, expressed as percentage of total activity. The plot refers to three independent experiments. <b>Lower panel</b>: IRE-IRPs electro mobility shift assay (EMSA). β-ME (beta-mercaptoethanol) was used to evaluate the total binding activity. A representative experiment was shown. Error bars indicate SE. *: P<. 05; **: P<. 01; ***: P<. 001.</p

Iron parameters in acute dietary iron changes.

<p>Transferrin saturation (TS, <b>A</b>) and non-heme liver (LIC, <b>B</b>) iron content were measured in mice (6–8/group) treated by an iron deficient diet for two weeks and then challenged with 1 day ID (ID-1day), iron balanced (IB-1day) and iron loaded (IL-1 day) diets. <i>Hamp</i> (<b>C</b>) expression was evaluated in HCs isolated from 4–7 mice/group. <i>Hprt1</i> was used as housekeeping gene. mRNA expression ratio was normalized to control (ID-1 day) mean value set to 1. Error bars indicate SE. *: P<. 05; **: P<. 01; ***: P<. 001; ns: not significant.</p

Proposed model of the crosstalk between <i>Bmp6</i>-producing NPCs and <i>hepcidin</i>-producing HCs in different conditions.

<p><b>(1)</b> NPC iron entry; <b>(2)</b> Bmp6 production; <b>(3)</b> Bmp6-mediated activation of the BMPR-Hjv complex and hepcidin production; (<b>4</b>) hepcidin-mediated degradation of ferroportin; <b>(5)</b> HC iron entry. The thickness of solid lines and arrows is proportional to the amount of iron, Bmp6, ferroportin and hepcidin. The dotted line indicates inhibition of the pathway. HC: hepatocyte; NPCs: non parenchymal cells (Kupffer cells and sinusoidal endothelial cells); Hamp: hepcidin; BMPR: Bone Morphogenetic Protein Receptor; Hjv: hemojuvelin; Fpn: ferroportin.</p

<i>Bmp6</i> and <i>Tfr1</i> expression in <i>Tmprss6</i> KO mice.

<p><i>Bmp6</i> (<b>A, C, E</b>) and <i>Tfr1</i> (<b>B, D, F</b>) expression was measured in liver cells isolated from wild type (wt) and <i>Tmprss6</i> KO mice (4–6 mice/group) by qRT-PCR, using <i>Hprt1</i> as housekeeping gene. mRNA expression ratio was normalized to wt mean value set to 1. Error bars indicate SE. *: P<. 05; **: P<. 01; ns: not significant.</p

A Strong Anti-Inflammatory Signature Revealed by Liver Transcription Profiling of <i>Tmprss6</i>−/− Mice

<div><p>Control of systemic iron homeostasis is interconnected with the inflammatory response through the key iron regulator, the antimicrobial peptide hepcidin. We have previously shown that mice with iron deficiency anemia (IDA)-low hepcidin show a pro-inflammatory response that is blunted in iron deficient-high hepcidin <i>Tmprss6</i> KO mice. The transcriptional response associated with chronic hepcidin overexpression due to genetic inactivation of <i>Tmprss6</i> is unknown. By using whole genome transcription profiling of the liver and analysis of spleen immune-related genes we identified several functional pathways differentially expressed in <i>Tmprss6</i> KO mice, compared to IDA animals and thus irrespective of the iron status. In the effort of defining genes potentially targets of Tmprss6 we analyzed liver gene expression changes according to the genotype and independently of treatment. <i>Tmprss6</i> inactivation causes down-regulation of liver pathways connected to immune and inflammatory response as well as spleen genes related to macrophage activation and inflammatory cytokines production. The anti-inflammatory status of <i>Tmprss6 KO</i> animals was confirmed by the down-regulation of pathways related to immunity, stress response and intracellular signaling in both liver and spleen after LPS treatment. Opposite to <i>Tmprss6</i> KO mice, <i>Hfe^−/−</i> mice are characterized by iron overload with inappropriately low hepcidin levels. Liver expression profiling of <i>Hfe^−/−</i> deficient <i>versus</i> iron loaded mice show the opposite expression of some of the genes modulated by the loss of <i>Tmprss6</i>. Altogether our results confirm the anti-inflammatory status of <i>Tmprss6</i> KO mice and identify new potential target pathways/genes of Tmprss6.</p></div