Iron-Dependent Regulation of Hepcidin in Hjv−/− Mice: Evidence That Hemojuvelin Is Dispensable for Sensing Body Iron Levels

<div><p>Hemojuvelin (Hjv) is a bone morphogenetic protein (BMP) co-receptor involved in the control of systemic iron homeostasis. Functional inactivation of Hjv leads to severe iron overload in humans and mice due to marked suppression of the iron-regulatory hormone hepcidin. To investigate the role of Hjv in body iron sensing, Hjv−/− mice and isogenic wild type controls were placed on a moderately low, a standard or a high iron diet for four weeks. Hjv−/− mice developed systemic iron overload under all regimens. Transferrin (Tf) was highly saturated regardless of the dietary iron content, while liver iron deposition was proportional to it. Hepcidin mRNA expression responded to fluctuations in dietary iron intake, despite the absence of Hjv. Nevertheless, iron-dependent upregulation of hepcidin was more than an order of magnitude lower compared to that seen in wild type controls. Likewise, iron signaling via the BMP/Smad pathway was preserved but substantially attenuated. These findings suggest that Hjv is not required for sensing of body iron levels and merely functions as an enhancer for iron signaling to hepcidin.</p></div

Residual iron-dependent regulation of hepatic hepcidin mRNA expression in Hjv−/− mice.

<p>RNA was extracted from tissues of the Hjv−/− and wild type mice described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085530#pone-0085530-g001" target="_blank">Fig. 1</a> and used for qPCR analysis. (A) Expression of hepatic hepcidin mRNA. (B) Expression of splenic hepcidin mRNA. Note that absolute hepcidin mRNA levels in the spleen are >100 times lower than in the liver. Data are presented as the mean ± SEM. The p values were calculated by using one-way ANOVA with Bonferroni post-test correction. Detailed statistical analysis is provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085530#pone.0085530.s006" target="_blank">Table S1</a>.</p

Effects of dietary iron manipulations on hepatic and splenic iron content.

<p>Livers and spleens from the Hjv−/− and wild type mice described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085530#pone-0085530-g001" target="_blank">Fig. 1</a> were used for histological detection of iron by staining with Perls’ Prussian blue, and for tissue iron quantification by the ferrozine assay. (A) Visualization of ferric deposits in representative liver sections (magnification: 10×). (B) Quantification of non-heme hepatic iron. (C) Visualization of ferric deposits in representative spleen sections (original magnification: 10×). (D) Quantification of non-heme splenic iron. Data in (B) and (D) are presented as the mean ± SEM. The p values were calculated by using one-way ANOVA with Bonferroni post-test correction. Detailed statistical analysis is provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085530#pone.0085530.s006" target="_blank">Table S1</a>.</p

List of primers used for qPCR or genotyping.

<p>List of primers used for qPCR or genotyping.</p

Hjv−/− mice exhibit elevated serum iron indices independently of dietary iron intake.

<p>Ten-week old male Hjv−/− and wild type mice (n = 10 for each group) in C57BL/6 background were placed on diets with variable iron content (low: 75–100 ppm; normal: 225 ppm; high: 225 ppm plus 2% carbonyl iron). After four weeks the animals were sacrificed and sera were analyzed for iron (A), transferrin saturation (B), and ferritin (C). Data are presented as the mean ± SEM. Statistical analysis is provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085530#pone.0085530.s006" target="_blank">Table S1</a>.</p

Grouping of states from binding trajectories into a coarse-grained metastable state model.

<p>(A) Unbound states. One hundred randomly selected conformations of pyruvate from unbound states S2 (yellow) and S6 (green) are presented against a cartoon representation of DHDPS. Collectively, poses within these states lack any conserved interactions with the protein surface. (B) The DHDPS-pyruvate bound complex (S13). Conformations of pyruvate and active site residues from this state (green) are contrasted with a crystallographic reference structure of pyruvate bound to DHDPS (PDB ID 3DI1; silver). Key active site residues Thr46, Tyr109, Tyr135, and Lys163 are indicated. Conformations of pyruvate within this state deviate from the reference structure by as little as 1.85 Å. Note that Tyr109 is shown from the opposing DHDPS subunit. (C) Individual metastable states, labelled accordingly, are shown as nodes within rounded boxes. Edges depict bidirectional interstate transitions, where edge shade reflects the transition probability (darker arrows indicate higher probabilities). States classified as unbound (S2, S6) are shaded in red, whereas the DHDPS-pyruvate bound state (S13) is shaded in green. For clarity, only highly-populated states (equilibrium populations greater than 4%) are shown.</p

Serum and liver iron indices in wild type and Hjv−/− mice of 129S6/SvEvTac or C57BL/6 genetic background (n = 10 male C57BL/6 mice for each genotype; n = 5 male 129S6/SvEvTac mice for each genotype).

<p>p<0.05;</p><p>p<0.01;</p><p>p<0.001 vs 129S6/SvEvTac mice of the same genotype (Student’s t test).</p><p>All differences among wild type and Hjv−/− mice of the same strain are statistically significant (p values not shown).</p

Kinetic mechanism and structure.

<p>(A) The DHDPS-catalyzed reaction follows a classic bi-bi substrate model, requiring the first substrate (PYR; pyruvate) bind the enzyme for the second substrate (ASA) to be recruited to the active site and ultimately liberate the reaction product (HTPA). The initial pyruvate-binding portion of the reaction scheme is highlighted in cyan. (B) Quaternary structure of the DHDPS dimer. (C) Licorice representation of key active site residues. Protein chains A and B are shown in yellow and green, respectively.</p

Umbrella sampling PMF.

<p>PMF curve calculated using 28 windows of umbrella sampling along an arbitrary <i>Z</i>-coordinate. Each curve, colored from red to blue, represents successive truncation of the data in 5% increments from the beginning of each simulation window until the final 50% of data (2.5 ns) remained. Several bound states identified are labelled according to their average <i>Z</i>-coordinate. State S13, which was bimodal with respect to the <i>Z</i>-coordinate, is highlighted as a gray box.</p

The major pyruvate-binding pathway is multi-step.

<p>(A) Pyruvate, indicated using green carbon atoms, must successively pass through several binding intermediates to reach the crystallographic bound pose. From bulk solvent, pyruvate forms a transient interaction with an arginine residue at the entryway to the active site (T1), moves into the active site cavity (T2), and in the penultimate step penetrates deeper into the active site to assume a ‘pre-bound’ pose (T3). Finally, from the ‘pre-bound’ pose pyruvate undergoes a twisting motion (T4) and achieves the crystallographic DHDPS-pyruvate complex. (B) Multiple sequence alignment of bacterial DHDPS enzymes. Several interacting residues from the binding pathway depicted in (A) are absolutely conserved across species. Sequence alignment was performed using CLUSTALO [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004811#pcbi.1004811.ref037" target="_blank">37</a>].</p