Knockdown of Matriptase-2 Increases Hepcidin Expression in a BMP-Dependent Manner in Zebrafish Embryos.

Abstract 4050

Poster Board III-985

Hepatocytes produce hepcidin, a transcriptionally regulated peptide hormone, in response to iron overload, resulting in decreased intestinal iron absorption. Hepcidin levels are inappropriately low in patients with thalassemia major and hemochromatosis. We have been developing the zebrafish embryo as a model to identify hepcidin regulators, which may be exploited to prevent and treat iron overload syndromes. Recently, it has been shown in mammalian models that the membrane-bound serine protease, matriptase-2 (also known as TMPRSS-6), inhibits hepcidin expression by cleaving membrane-bound hemojuvelin (hjv), a bone morphogenic protein (BMP) co-receptor, which is mutated in patients with juvenile hemochromatosis. Previously, we demonstrated that hepcidin expression increases in response to iron loading or activation of the BMP signaling pathway, but that hjv is not required for hepcidin expression in zebrafish embryos. We hypothesized that hepcidin expression in zebrafish embryos is normally hjv-independent, either because of low levels of hjv expression in the liver or because of the presence of matriptase-2. We used whole mount in situ hybridization, quantitative realtime RT-PCR, morpholino knockdowns and overexpression experiments to address these questions. As hjv was undetectable in the zebrafish embryonic liver by in situ hybridization, we used flow cytometry to sort zebrafish embryonic hepatocytes and assess hjv expression by RT-PCR. We found that hjv was weakly expressed in the embryonic liver, but strongly expressed in zebrafish adult liver. We overexpressed zebrafish hjv in human hepatocytes and found that it potentiated the effects of BMP6 to activate a human hepcidin promoter and a BMP response element. In contrast, overexpression of zebrafish hjv in zebrafish embryos failed to increase hepcidin expression. Knockdown of matriptase-2 in zebrafish embryos caused developmental delay and a smaller liver, but increased hepcidin expression, relative to liver size. Furthermore, treatment with the BMP inhibitor dorsomorphin abrogated the increased hepcidin expression associated with matriptase-2 knockdown. Experiments are underway to determine whether zebrafish hjv has little effect on hepcidin expression during embryonic development because of the activity of matriptase-2.