The transcription factor ATOH8 is regulated by erythropoietic activity and regulates HAMP transcription and cellular pSMAD1,5,8 levels

Neeta Patel,Patarabutr Masaratana,Robert J Simpson,Molly Jacob,Gladys O Latunde‐Dada,Joe Varghese,Andrew T Mckie

doi:10.1111/bjh.12649

Neeta Patel, Patarabutr Masaratana + Show 5 more

Open Access

https://doi.org/10.1111/bjh.12649

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Abstract

SummaryATOH8 has previously been shown to be an iron-regulated transcription factor, however its role in iron metabolism is not known. ATOH8 expression in HEK293 cells resulted in increased endogenous HAMP mRNA levels as well as HAMP promoter activity. Mutation of the E-box or SMAD response elements within the HAMP promoter significantly reduced the effects of ATOH8, indicating that ATOH8 activates HAMP transcription directly as well as through bone morphogenic protein (BMP) signalling. In support of the former, Chromatin immunoprecipitation assays provided evidence that ATOH8 binds to E-box regions within the HAMP promoter while the latter was supported by the finding that ATOH8 expression in HEK293 cells led to increased phosphorylated SMAD1,5,8 levels. Liver Atoh8 levels were reduced in mice under conditions associated with increased erythropoietic activity such as hypoxia, haemolytic anaemia, hypotransferrinaemia and erythropoietin treatment and increased by inhibitors of erythropoiesis. Hepatic Atoh8mRNA levels increased in mice treated with holo transferrin, suggesting that Atoh8 responds to changes in plasma iron. ATOH8 is therefore a novel transcriptional regulator of HAMP, which is responsive to changes in plasma iron and erythroid activity and could explain how changes in erythroid activity lead to regulation of HAMP.

Highlights

MethodsHypotransferrinaemic mice (HPX or Trfhpx/hpx) were bred and maintained as previously described (Simpson et al, 1991)
Mutation of the E-box or SMAD response elements within the HAMP promoter significantly reduced the effects of ATOH8, indicating that ATOH8 activates HAMP transcription directly as well as through bone morphogenic protein (BMP) signalling
In support of the former, Chromatin immunoprecipitation assays provided evidence that ATOH8 binds to E-box regions within the HAMP promoter while the latter was supported by the finding that ATOH8 expression in HEK293 cells led to increased phosphorylated SMAD1,5,8 levels

Summary

Methods

Hypotransferrinaemic mice (HPX or Trfhpx/hpx) were bred and maintained as previously described (Simpson et al, 1991). Hypoxia was induced by placing 7-week-old male CD1 mice in a hypobaric chamber for 24–72 h, as previously described (Raja et al, 1988); controls of the same gender and age were maintained under normoxic conditions. Hamp1À/À mice and wild type (WT) littermates (all female C57BL/6/129 mixed background, aged 5–7 weeks old) were injected intraperitoneally with 60 mg/kg body weight of neutralized phenylhydrazine (PHZ) or saline solution twice on consecutive days as previously described (Masaratana et al, 2011) and sacrificed 3 d after the last injection. Apo and Holo transferrin (10 mg) dissolved in 100 ll of saline was injected i.p (control mice received saline alone). All animal experiments were performed under the authority of a UK Home Office license

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