Hepcidin and Atoh8 mRNA Expression Are Decreased in Murine Models of Hemochromatosis However Hepcidin Expression Is Unaffected By Atoh8 Knockout

Atoh8 is a member of the bHLH transcription factor family, and hepatic expression of atoh8 mRNA is up-regulated by dietary iron and down-regulated by stimulation of erythropoiesis in mice, in parallel with changes in hepcidin mRNA (hamp1). It is a candidate transcription factor in the regulation of Hamp1 by iron status. Here we performed studies evaluating the contribution of genes regulating hepcidin to the regulation of Atoh8, as well as the role of Atoh8 in the regulation of hepcidin. In the former studies, wild type FVB and three hereditary hemochromatosis (HH) mouse models, Hfe^-/-, TfR2^Y245X/Y245X and Hfe^-/-TfR2^Y245X/Y245X mice, were placed on diets containing 60 ppm or 25,000 ppm iron at weaning for two weeks before euthanasia and tissue collection. Liver expression of Hamp1 and Atoh8 were evaluated. Additionally, the WT and HH mice were intraperitoneally injected with 30 mg/kg phenylhydrazine (PHZ) on days 49 and 50, and then euthanized on day 53. Liver and spleen non-heme iron concentrations, and serum hematologic and iron parameters were measured. The liver mRNAs for hamp1, atoh8, and bmp6 were measured by quantitative RT-PCR. Hfe^-/- and Tfr2^Y245X/Y245X mice each demonstrated lower Hamp1 and Atoh8 expression than did WT mice, despite hepatic iron loading. WT mice demonstrated the expected increases in Hamp1 and Atoh8 expression in response to dietary iron loading. Hamp1 increased in the HH mice with iron loading; however Atoh8 expression was not statistically different. The addition of Hfe knockout to the TfR2^Y245X/Y245X mutation suppressed Hamp1 expression further, but had no additive effect on Atoh8. PHZ treatment at 30 mg/kg mouse significantly decreased red blood cells and hematocrit, caused severe splenomegaly, and down-regulated Hamp1 and Atoh8 in WT mice, as well as in HH mice. These results are consistent with a role for Hfe and TfR2 in normal signaling to Hamp1 and Atoh8, but also suggest some regulation of Hamp1 in response to dietary iron independent of a change in Atoh8 expression. To evaluate the role of Atoh8 in the regulation of hepcidin, Atoh8 Δexon1 homozygous, heterozygous and WT mice were placed on diets containing standard (250 ppm) and euthanized at 5-6 weeks of age. The Atoh8 Δexon1 homozygous mice were significantly smaller in size than the WT or heterozygous littermates, but did not differ in any measured iron parameter (serum iron, hematocrit, liver iron, liver hamp1 mRNA, liver bmp6 mRNA). The Atoh8 Δexon1 homozygous mice also did not differ from heterozygous mice in PHZ response. These observations indicate that HFE and TFR2 are necessary for normal Atoh8 as well as hepcidin expression; however they do not support an essential role for Atoh8 in hepcidin regulation by iron demand.