ABSTRACT
Hemojuvelin (Hjv), a member of the repulsive-guidance molecule (RGM) family, upregulates transcription of the iron regulatory hormone hepcidin by activating the bone morphogenetic protein (BMP) signaling pathway in mammalian cells. Mammalian models have identified furin, neogenin, and matriptase-2 as modifiers of Hjv's function. Using the zebrafish model, we evaluated the effects of hjv and its interacting proteins on hepcidin expression during embryonic development. We found that hjv is strongly expressed in the notochord and somites of the zebrafish embryo and that morpholino knockdown of hjv impaired the development of these structures. Knockdown of hjv or other hjv-related genes, including zebrafish orthologs of furin or neogenin, however, failed to decrease hepcidin expression relative to liver size. In contrast, overexpression of bmp2b or knockdown of matriptase-2 enhanced the intensity and extent of hepcidin expression in zebrafish embryos, but this occurred in an hjv-independent manner. Furthermore, we demonstrated that zebrafish hjv can activate the human hepcidin promoter and enhance BMP responsive gene expression in vitro, but is expressed at low levels in the zebrafish embryonic liver. Taken together, these data support an alternative mechanism for hepcidin regulation during zebrafish embryonic development, which is independent of hjv.
Subject(s)
Antimicrobial Cationic Peptides/analysis , Bone Morphogenetic Proteins/metabolism , Signal Transduction , Trans-Activators/physiology , Zebrafish Proteins/physiology , Zebrafish/metabolism , Animals , Anti-Bacterial Agents , Antimicrobial Cationic Peptides/biosynthesis , Antimicrobial Cationic Peptides/genetics , Bone Morphogenetic Protein 2 , Embryo, Nonmammalian , GPI-Linked Proteins , Hemochromatosis/congenital , Hemochromatosis Protein , Hepcidins , Humans , Liver/chemistry , Liver/metabolism , Notochord/chemistry , Promoter Regions, Genetic , Serine Endopeptidases , Somites/chemistry , Zebrafish/geneticsABSTRACT
Acetaminophen (APAP) toxicity is the most common drug-induced cause of acute liver failure in the United States. The only available treatment, N-acetylcysteine (NAC), has a limited time window of efficacy, indicating a need for additional therapeutic options. Zebrafish have emerged as a powerful tool for drug discovery. Here, we developed a clinically relevant zebrafish model of APAP toxicity. APAP depleted glutathione stores, elevated aminotransferase levels, increased apoptosis, and caused dose-dependent hepatocyte necrosis. These outcomes were limited by NAC and conserved in zebrafish embryos. In a targeted embryonic chemical screen, prostaglandin E2 (PGE2) was identified as a potential therapeutic agent; in the adult, PGE2 similarly decreased APAP-associated toxicity. Significantly, when combined with NAC, PGE2 extended the time window for a successful intervention, synergistically reducing apoptosis, improving liver enzymes, and preventing death. Use of a wnt reporter zebrafish line and chemical genetic epistasis showed that the effects of PGE2 are mediated through the wnt signaling pathway. Zebrafish can be used as a clinically relevant toxicological model amenable to the identification of additional therapeutics and biomarkers of APAP injury; our data suggest combinatorial PGE2 and NAC treatment would be beneficial for patients with APAP-induced liver damage.
