Hemojuvelin-mediated hepcidin induction requires both bone morphogenetic protein type I receptors ALK2 and ALK3.

ABSTRACT: Hemojuvelin (HJV) is a glycosylphosphatidylinositol-anchored protein of the repulsive guidance molecule family acting as a bone morphogenetic protein (BMP) coreceptor to induce the hepatic iron regulatory protein hepcidin. Hepcidin causes ubiquitination and degradation of the sole known iron exporter ferroportin, thereby limiting iron availability. The detailed signaling mechanism of HJV in vivo has yet to be investigated. In the current manuscript, we used an established model of adeno-associated virus (AAV)-mediated liver-specific overexpression of HJV in murine models of hepatocyte-specific deficiency of the BMP type I receptors Alk2 or Alk3. In control mice, HJV overexpression increased hepatic Hamp messenger RNA (mRNA) levels, soluble HJV (sHJV), splenic iron content (SIC), as well as phosphorylated small mothers against decapentaplegic protein (pSMAD1/5/8) levels. In contrast, in Alk2fl/fl;Alb-Cre and Alk3fl/fl;Alb-Cre mice, which present with moderate and severe iron overload, respectively, the administration of AAV-HJV induced HJV and sHJV. However, it did not rescue the iron overload phenotypes of those mice. Serum iron levels were induced in Alk2fl/fl;Alb-Cre mice after HJV overexpression. In phosphate-buffered saline-injected Alk3fl/fl;Alb-Cre mice, serum iron levels and the expression of duodenal ferroportin remained high, whereas Hamp mRNA levels were decreased to 1% to 5% of the levels detected in controls. This was reduced even further by AAV-HJV overexpression. SIC remained low in mice with hepatocyte-specific Alk2 or Alk3 deficiency, reflecting disturbed iron homeostasis with high serum iron levels and transferrin saturation and an inability to induce hepcidin by HJV overexpression. The data indicate that ALK2 and ALK3 are both required in vivo for the HJV-mediated induction of hepcidin.

FKBP12 inhibits hepcidin expression by modulating BMP receptors interaction and ligand responsiveness in hepatocytes.

The expression of the iron regulatory hormone hepcidin in hepatocytes is regulated by the BMP-SMAD pathway through the type I receptors ALK2 and ALK3, the type II receptors ACVR2A and BMPR2, and the ligands BMP2 and BMP6. We previously identified the immunophilin FKBP12 as a new hepcidin inhibitor that acts by blocking ALK2. Both the physiologic ALK2 ligand BMP6 and the immunosuppressive drug Tacrolimus (TAC) displace FKBP12 from ALK2 and activate the signaling. However, the molecular mechanism whereby FKBP12 regulates BMP-SMAD pathway activity and thus hepcidin expression remains unclear. Here, we show that FKBP12 acts by modulating BMP receptor interactions and ligand responsiveness. We first demonstrate that in primary murine hepatocytes TAC regulates hepcidin expression exclusively via FKBP12. Downregulation of the BMP receptors reveals that ALK2, to a lesser extent ALK3, and ACVR2A are required for hepcidin upregulation in response to both BMP6 and TAC. Mechanistically, TAC and BMP6 increase ALK2 homo-oligomerization and ALK2-ALK3 hetero-oligomerization and the interaction between ALK2 and the type II receptors. By acting on the same receptors, TAC and BMP6 cooperate in BMP pathway activation and hepcidin expression both in vitro and in vivo. Interestingly, the activation state of ALK3 modulates its interaction with FKBP12, which may explain the cell-specific activity of FKBP12. Overall, our results identify the mechanism whereby FKBP12 regulates the BMP-SMAD pathway and hepcidin expression in hepatocytes, and suggest that FKBP12-ALK2 interaction is a potential pharmacologic target in disorders caused by defective BMP-SMAD signaling and characterized by low hepcidin and high BMP6 expression.