Hepcidin Bound to α2-Macroglobulin Reduces Ferroportin-1 Expression and Enhances Its Activity at Reducing Serum Iron Levels

Hepcidin regulates iron metabolism by down-regulating ferroportin-1 (Fpn1). We demonstrated that hepcidin is complexed to the blood transport protein, α 2 -macroglobulin (α 2 M) (Peslova, G., Petrak, J., Kuzelova, K., Hrdy, I., Halada, P., Kuchel, P. W., Soe-Lin, S., Ponka, P., Sutak, R., Becker, E., Huang, M. L., Suryo Rahmanto, Y., Richardson, D. R., and Vyoral, D. (2009) Blood 113, 6225–6236). However, nothing is known about the mechanism of hepcidin binding to α 2 M or the effects of the α 2 M·hepcidin complex in vivo . We show that decreased Fpn1 expression can be mediated by hepcidin bound to native α 2 M and also, for the first time, hepcidin bound to methylamine-activated α 2 M (α 2 M-MA). Passage of high molecular weight α 2 M·hepcidin or α 2 M-MA·hepcidin complexes (≈725 kDa) through a Sephadex G-25 size exclusion column retained their ability to decrease Fpn1 expression. Further studies using ultrafiltration indicated that hepcidin binding to α 2 M and α 2 M-MA was labile, resulting in some release from the protein, and this may explain its urinary excretion. To determine whether α 2 M-MA·hepcidin is delivered to cells via the α 2 M receptor (Lrp1), we assessed α 2 M uptake and Fpn1 expression in Lrp1 −/− and Lrp1 +/+ cells. Interestingly, α 2 M·hepcidin or α 2 M-MA·hepcidin demonstrated similar activities at decreasing Fpn1 expression in Lrp1 −/− and Lrp1 +/+ cells, indicating that Lrp1 is not essential for Fpn1 regulation. In vivo , hepcidin bound to α 2 M or α 2 M-MA did not affect plasma clearance of α 2 M/α 2 M-MA. However, serum iron levels were reduced to a significantly greater extent in mice treated with α 2 M·hepcidin or α 2 M-MA·hepcidin relative to unbound hepcidin. This effect could be mediated by the ability of α 2 M or α 2 M-MA to retard kidney filtration of bound hepcidin, increasing its half-life. A model is proposed that suggests that unlike proteases, which are irreversibly bound to activated α 2 M, hepcidin remains labile and available to down-regulate Fpn1. Background: Hepcidin is the hormone of iron metabolism that is bound by α 2 -macroglobulin (α 2 M) and its activated counterpart (α 2 M-MA). Results: Serum iron is reduced to a greater extent in mice treated with α 2 M·hepcidin or α 2 M-MA·hepcidin relative to unbound hepcidin. Conclusion: α 2 M retards hepcidin excretion by the kidney, increasing its efficacy. Significance: These results are important for understanding hepcidin transport and detection in blood.