HIF-1 and HIF-2 pathways in regulation of hepcidin Free

Hepcidin, the master regulator of iron metabolism, inhibits absorption and release of iron from macrophages through interaction with ferroportin (PMID:35905974). Hepcidin is upregulated 1) in response to higher intracellular iron stores and plasma iron concentrations as reflected by serum ferritin and elevated circulating transferrin-bound iron (PMID:20603012) and 2) inflammation through interleukin-6 and a JAK-STAT pathway (PMID:15124018). Hepcidin is downregulated by increased erythropoiesis via erythroferrone secreted by erythroblasts, which suppresses bone morphogenetic protein receptor complex signaling (PMID:20603012). The relationship of hepcidin to transferrin concentration in a model that includes ferritin, erythropoietin and upregulated hypoxia sensing has not previously been reported (PMIDs:21876117,9242677).

Hypoxia-inducible factors (HIFs) are dimers of a common HIF-b subunit and one of several HIF-a subunits that are regulated post-translationally; HIF-1 and HIF-2 are best studied. Prolyl hydroxylases (PHDs, Fe-dependent enzymes) are the principal negative regulators of HIF-a subunits. In the presence of O₂ and a-ketoglutarate, HIF-1a and HIF-2a subunits are hydroxylated by PHDs facilitating binding to von Hippel Lindau (VHL) protein, which leads to their ubiquitination and rapid proteosomal degradation (PMIDs:19364912,11292861,12639980) In hypoxia and iron-deficiency, degradation of HIF-a decreases leading to increased HIF-1 and HIF-2 heterodimers that augment hypoxia-inducible genes' transcription.

Homozygous germline loss-of-function VHL^R200W causes congenital Chuvash erythrocytosis (CE). VHL^R200W homozygotes have elevated HIF-1 and HIF-2 levels (PMID:37435906) with thrombosis as the major cause of morbidity and mortality. HIFs upregulate several genes that influence thrombosis (PMIDs:37435906,32203583). Erythropoietin (EPO), upregulated by hypoxia and iron deficiency via HIF-2, is increased in VHL^R200W homozygotes even in absence of hypoxia or iron deficiency and increases thrombosis (PMID:37435906). Transferrin, upregulated by HIF-1 during hypoxia and iron deficiency, is increased in VHL^R200W homozygotes even in absence of hypoxia or iron deficiency and provides protection from thrombosis (PMID:9242677).

We reported that in CE hepcidin and ferritin levels are low and EPO levels are high (PMID: 21876117). In multivariate analysis, hepcidin had an inverse relationship with VHL^R200W homozygosity and positive relationship with ferritin, but did not correlate with EPO after adjustment for VHL^R200W homozygosity. However, we did not examine the HIF-1 upregulated gene transferrin or perform a pathway analysis. We have now reexamined the relationship of CE homozygosity with hepcidin related to both HIF-2-mediated enhancement of EPO and HIF-1-augmentation of transferrin. The IRB approved research included 111 VHL^R200W homozygotes and 29 controls without mutated VHL. Hepcidin was measured by enzyme-linked immunosorbent assay (PMID:18689548); transferrin, EPO, and ferritin were analyzed as previously described (PMID:37435906).

In univariate analyses hepcidin (p<0.0001) and ferritin (p<0.0001) were lower in VHL^R200Whomozygotes, and EPO (p<0.0001) and transferrin (p<0.0001) were higher. In Spearman correlations ferritin had a positive relationship with hepcidin (p<0.0001) while age (p=0.006), VHL^R200W homozygosity(p<0.0001), EPO (p<0.0001), and transferrin (p<0.0001) had significant negative correlations. Female gender had no relationship.

We now show in a pathway analysis that the homozygous VHL^R200Wmutation strongly correlates with both increased EPO and increased transferrin concentrations, which in turn strongly correlate with lower hepcidin levels, in analyses that consider the influence of ferritin on hepcidin and on transferrin. Transferrin elevation is a classic feature of iron deficiency that may be induced by both hypoxia and iron deficiency via HIF-1. Many CE patients, as well as those with other causes of erythrocytosis, are phlebotomized leading to iron deficiency and impaired prolyl hydroxylase activity, which has additive effects in increasing HIF signaling. Our findings suggest a model of hepcidin regulation involving both HIF-1 and HIF-2 signaling with mediators transferrin and ferritin representing primarily iron effects on hepcidin, and with erythropoietin stimulating erythropoiesis to generate the hepcidin-suppressive erythrokine erythroferrone.