Hypoxia-Dependent Hepcidin Down-Regulation: In Vitro and In Vivo Studies

Hepcidin is the main regulator of systemic iron homeostasis, acting by inhibiting macrophages iron release and intestinal iron absorption. During hypoxia hepcidin expression is inhibited to allow iron requirement to bone marrow to sustain erythropoietic expansion, but mechanisms of hypoxia-induced hepcidin down-regulation are partially unknown. Our aim was to improve our knowledge on hypoxia modulation of hepatic hepcidin, investigating the role of erythroid and iron-related pathways using in vitro and in vivo studies. We firstly studied the effect of hypoxia on healthy volunteers living at high altitude for 24h and 72h, confirming that hypoxia is a strong inhibitor of hepcidin production and that this inhibition followed EPO induction. Indeed s-hepcidin was strictly down-regulated at 72h of hypoxia while EPO levels were already markedly increased at 24h slightly decreasing thereafter. We then analyzed the effect of sera of these subjects on hepcidin expression in HuH-7 cells using both luciferase assay and RT-PCR. HAMP wild-type promoter activity decreased after exposure to hypoxic sera, suggesting that circulating factors present in sera may modulate hepcidin expression. However, analysis of the transcripts gave less homogenous results. In fact only a subgroup of hypoxic sera significantly inhibited hepcidin. These sera also induced a significant ID1 down-regulation indicating that inhibitory factor(s) seems to act through the SMADs pathway. Our results also suggest that a cellular model cannot reproduce what really happens in vivo that might depend to complex events involving both circulating and tissue factors. Therefore, we took advantages from a mouse model. For hypoxia challenge, C57BL/6 mice were housed in a hypoxic chamber at 10% O2 for 6 and 15h. mRNA levels of erythroid genes were assessed in liver, spleen and bone marrow by RT-PCR. Hamp1 levels were slightly increased after 6h of hypoxia and strongly down-regulated after 15h. After 6h we already observed a marked activation of transcription of all the erythroid genes analyzed (Pdgf-b, Fam132b, Gdf-15 and Twsg-1) in bone marrow (but not in spleen) that return to baseline levels after 15h. To better analyze the role of PDGF-BB, normoxic and hypoxic mice were treated with PDGF-BB neutralizing antibody or PDGF-BB receptor inhibitor immediately prior to hypoxic challenge. Inactivation of PDGF-BB or its receptor suppressed the hypoxia-induced hepcidin inhibition confirming the importance of PDGF-BB in hepcidin regulation. However, no changes in CREB/H protein were observed suggesting that this molecule is not involved in hepcidin regulation in this setting. In conclusion, our studies demonstrated that hypoxia-induced hepcidin regulation is a quick event secondary to erythropoietic activation. Indeed hepcidin decreased after up-regulation of transcription of several erythroid genes that return to baseline when hepcidin reached the maximum inhibition. Our studies indicate that PDGF-BB is a good candidate as hepcidin erythroid regulators but also suggest that hypoxia induced-hepcidin suppression is the result of cooperation of multiple signals that need to be dissected.