Characterization of Hepcidin-Inducing Cytokines in Multiple Myeloma.

Hepcidin, the principal iron-regulatory hormone, plays an important role in the development of anemia of inflammation and other iron-restricted anemias. Patients with multiple myeloma (MM) frequently present with anemia not attributable to a known mechanism. We previously found that hepcidin is increased in MM and thus could cause or contribute to the anemia of MM. The BMP and IL-6 pathways are the two known major transcriptional regulators of hepcidin.

To identify cytokines that increase hepcidin in MM patients, we screened patient sera with an in vitro cellular reporter system, consisting of human hepatoma 7 cell line (HuH7) and the hepcidin promoter-firefly luciferase reporter. Using site-directed mutagenesis, the promoter was mutated at the STAT3-binding site (STAT3-BS) and/or two BMP responsive elements (BREs), sequences known to be involved in the regulation of hepcidin expression by IL-6 and BMPs, respectively.

As expected, recombinant IL-6 and BMP-4, -6 and -9 activated wild-type hepcidin promoter activity several fold. Of note, IL-6 and BMP-9 interacted synergistically at low doses, at the level of the promoter. Mutations in STAT3-BS abrogated the response to IL-6. Mutations in either BRE site by itself did not abolish the response to BMPs, but concurrent mutagenesis of both sites resulted in a complete loss of hepcidin response. Importantly, STAT3-BS and BREs affected hepcidin promoter response independently from each other. Using the in vitro system, we compared sera from six MM patients with previously measured serum hepcidin levels to sera from healthy controls. Sera of four patients with high hepcidin and one with low hepcidin significantly induced hepcidin promoter activity, while serum of another patient with low hepcidin did not. Mutations in STAT3-BS only abrogated the response to two patient sera, both of which had high hepcidin. Mutations in two BREs abrogated the response in all six sera as did the triple mutation involving STAT3-BS and both BREs.

We could separately interrogate the signaling pathways by which IL-6 and BMPs induce hepcidin transcription, allowing us to discriminate between the effects of IL-6-like or BMP-like cytokines in each patient serum. BMP-like cytokines were involved in the upregulation of hepcidin in all tested MM patients, and in some patients IL-6 or related cytokines contributed as well, either independently or through a synergistic interaction. No residual activation of hepcidin promoter by other factors was noted. Antibody neutralization may identify the specific myeloma-associated cytokines that stimulate hepcidin production through the two canonical pathways.

Roodman:Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Research Funding, Speakers Bureau; Celgene: Consultancy; Acceleron: Consultancy. Nemeth:Intrinsic LifeSciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Ganz:Intrinsic LifeSciences: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Xenon Pharmaceuticals: Consultancy, Equity Ownership.