VHL, transferrin, and erythropoietin in the regulation of hepcidin

Hepcidin, the master regulator of iron metabolism, is a hepatic peptide hormone that inhibits the absorption of iron by enterocytes and the release of iron from macrophages through interaction with ferroportin in the cellular membrane.1 Hepcidin binds to ferroportin, occluding it and causing it to move to the interior of cells, thereby preventing the release of cellular iron to plasma, and also causing a conformational change that leads to ferroportin ubiquitination and lysosomal degradation.2 Hepcidin is upregulated in response to (1) higher intracellular iron stores and plasma iron concentrations as reflected by serum ferritin and elevated circulating transferrin-bound iron3 via a bone morphogenetic protein receptor complex4 and (2) inflammation through interleukin-6 and a JAK-STAT pathway.5 Hepcidin is downregulated by increased erythropoiesis via erythroferrone secreted by erythroblasts, which suppresses bone morphogenetic protein receptor complex signaling.6 Hypoxia downregulates hepcidin mostly indirectly, via erythropoietin and erythroferrone.7 Upregulation of hypoxia sensing as seen in Chuvash erythrocytosis also leads to increased erythropoietin and erythroferrone and decreased hepcidin.8-10 The relationship of hepcidin to transferrin concentration in a model that includes ferritin, erythropoietin, and upregulated hypoxia sensing has not previously been reported. Hypoxia-inducible factors (HIFs) are dimers of a common HIF-β subunit and one of several HIF-α subunits that are regulated posttranslationally; HIF-1 and HIF-2 are best studied.11 Prolyl hydroxylases (PHDs, Fe-dependent enzymes) are the principal negative regulators of HIF-α subunits. In the presence of O2 and α-ketoglutarate, HIF-1α and HIF-2α subunits are hydroxylated by PHDs, facilitating binding to von Hippel–Lindau (VHL) protein, which leads to their ubiquitination and rapid proteosomal degradation.12, 13 In hypoxia and iron deficiency, degradation of HIF-α decreases, leading to increased HIF-1 and HIF-2 heterodimers that augment transcription of hypoxia-inducible genes.11 Homozygous germline loss-of-function VHLR200W causes congenital Chuvash erythrocytosis with thrombosis as the major cause of morbidity and mortality. VHLR200W homozygosity leads to decreased ubiquitination of HIF-1α and HIF-2α,8 which is necessary for their degradation. In turn, HIFs upregulate several genes that influence oxygen homeostasis, erythropoiesis, and iron metabolism.8, 14, 15 Erythropoietin, upregulated by hypoxia and iron deficiency via HIF-2,16 is increased in VHLR200W homozygotes even in the absence of hypoxia or iron deficiency,8, 17 and erythroferrone expression is increased in VHLR200W homozygote erythroid progenitors.10 Transferrin, upregulated at least in part by HIF-1 during hypoxia and iron deficiency,14 is also increased in VHLR200W homozygotes even in the absence of hypoxia or iron deficiency.18 We reported that in Chuvash erythrocytosis, hepcidin and ferritin levels are low, and erythropoietin levels are high.9 In multivariable analysis, hepcidin was decreased by VHLR200W homozygosity and correlated positively with ferritin, but did not correlate with erythropoietin after adjustment for VHLR200W homozygosity. However, we did not examine the HIF upregulated gene, transferrin, or perform a pathway analysis. We have now reexamined the relationship of VHLR200W homozygosity with hepcidin related to both HIF-2-mediated enhancement of erythropoietin and HIF-mediated augmentation of transferrin, which is at least in part mediated by HIF-1. The research was Institutional Review Board (IRB) approved. Participants lived in the Chuvash Autonomous Republic of the Russian Federation, located about 650 km southeast of Moscow along the Volga River. We studied 111 VHLR200W homozygotes and 29 controls without mutated VHL. We previously reported hepcidin, ferritin, and erythropoietin levels but not transferrin concentrations in the controls and 90 of the VHLR200W homozygotes in the present study.9 Participants were recruited from the community in their usual state of health, and all provided written informed consent. They were characterized by medical history, physical examination, and laboratory tests of the peripheral blood. Serum samples were stored at −70°C. Hepcidin was measured by enzyme-linked immunosorbent assay19; transferrin, erythropoietin, and ferritin were analyzed as previously described.18 About half of the patients with Chuvash erythrocytosis had been treated with phlebotomy within the year before the date of the study. The other half had never undergone phlebotomy or had received phlebotomy more than one year before the date of the study. The primary study comparison was between VHLR200W homozygotes and genotypically normal participants with regard to serum hepcidin concentration using multiple linear regression and pathway analysis. Skewed continuous variables were log-transformed to better approximate a normal distribution. Generalized linear models were also applied to validate the linear regression. The clinical characteristics of the VHLR200W homozygotes and controls were assessed with the Student's t-test or the Pearson chi-square. Bivariate relationships of various measurements with hepcidin were performed with Spearman correlation. Analyses were performed by Stata 18.0 (StataCorp, College Station, TX). Pathway analysis was performed to explore VHLR200W homozygosity as a predictor of hepcidin concentration, with a specific interest in considering erythropoietin and transferrin as mediators of the increase in HIF-2 and HIF-1 pathways, respectively, in predicting serum hepcidin concentration. As shown in Table S1 and consistent with our previous report, in univariate analyses, serum concentrations of hepcidin and ferritin were significantly lower in VHLR200W homozygotes, and serum erythropoietin concentration, a marker of the HIF-2 pathway, was significantly higher. Serum transferrin concentration, at least in part a marker of the HIF-1 pathway, was also significantly higher in the VHLR200W homozygotes. As shown in Table S2, in univariate analyses, serum ferritin had a significant positive Spearman correlation with serum hepcidin, while age, VHLR200W homozygosity, serum erythropoietin, and serum transferrin had significant negative correlations with hepcidin. Multivariable analyses of associations with serum hepcidin concentration are shown in Table 1. We first analyzed the relationship of hepcidin with serum ferritin and VHLR200W homozygosity (Table 1A). Serum ferritin had a strong independent positive relationship with hepcidin (P = 0.0001) while VHLR200W homozygosity had a strong negative relationship (P = 0.0001) with an r2 of 0.367 for the model. We next performed a multivariable analysis of serum hepcidin that included serum ferritin, VHLR200W homozygosity, serum erythropoietin to represent increased HIF-2 signaling, and serum transferrin to represent increased HIF-1 signaling (Table 1B). Serum ferritin continued to have a strong positive relationship with hepcidin (P = 0.004), but VHLR200W homozygosity no longer had a significant negative relationship (P = 0.085). Rather, serum transferrin had a significant negative relationship (P = 0.015) and serum erythropoietin a borderline negative association (P = 0.094), suggesting that the negative association of VHLR200W homozygosity with hepcidin is represented by transferrin and erythropoietin. The r2 of 0.404 for this model was higher than the r2 for the model that included only ferritin and VHLR200W homozygosity. A multivariable analysis of serum hepcidin that removed VHLR200W homozygosity as a predictor of hepcidin level but replaced it with both erythropoietin and transferrin (Table 1C) confirmed that the effect of VHLR200W homozygosity on hepcidin is represented by erythropoietin and transferrin. Serum ferritin continued to have a strong positive relationship with hepcidin (P = 0.005) while erythropoietin (P = 0.004) and transferrin (P = 0.0004) had significant negative relationships with an r2 for the model of 0.391 that is higher than the r2 for the model with ferritin and VHLR200W homozygosity. As shown in Figure 1, pathway analysis suggests that VHLR200W homozygosity has strong relationships with increased HIF-2-regulated serum erythropoietin and with increased serum transferrin, at least in part regulated by HIF-1. In turn, increased erythropoietin and transferrin concentrations are strongly associated with reduced hepcidin levels. At the same time, increased ferritin concentration has a strong relationship with increased hepcidin level. Concurrently and as expected, higher serum ferritin concentration is associated with lower serum transferrin level. To summarize, we now show in multivariable and pathway analyses that the homozygous VHLR200W mutation strongly correlates with increases of both serum erythropoietin and transferrin concentrations, which in turn strongly correlate with lower serum hepcidin levels in analyses that consider the influence of ferritin on hepcidin and on transferrin. The present study included 90 VHLR200W homozygotes for whom we previously reported the relationship between erythropoietin and hepcidin9 and an additional 21 VHLR200W homozygotes to provide a more robust statistical analysis. Transferrin elevation is a classic feature of iron deficiency20 that may be induced by both hypoxia and iron deficiency, at least in part via HIF-1.14 Many Chuvash erythrocytosis patients, as well as those with other causes of erythrocytosis, are phlebotomized leading to iron deficiency and impaired PHD activity, which has additive effects in increasing HIF signaling.8, 17 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. In this model, increased transferrin is associated with lower plasma hepcidin levels after accounting for the relationship of higher hepcidin levels with greater amounts of storage iron as reflected in higher ferritin concentrations. Ivan Sergeev: Conceptualization (supporting); writing—review and editing (equal). Josef T. Prchal: Conceptualization (supporting); writing—review and editing (equal). Seyed Mehdi Nouraie: Formal analysis (lead); software (lead). Binal N. Shah: Conceptualization. Xu Zhang: Conceptualization; formal analysis. Adelina Sergueeva: Conceptualization (supporting). Galina Miasnikova: Conceptualization (supporting). Tomas Ganz: Conceptualization (lead); writing—review and editing (equal). Victor R. Gordeuk: Writing—original draft (lead); conceptualization (supporting); formal analysis (contributing); writing—review and editing (equal). The authors declare no conflicts of interest. The research was IRB approved. This work was supported in part by grant no. UH1-HL03679-05 from the National Heart, Lung, and Blood Institute (to V.R.G.) and the Office of Research on Minority Health; by Howard University General Clinical Research Center grant MO1-RR10284; and by National Institutes of Health grants R01HL079912-01 (to V.R.G.) and R01HL50077-14 (to J.T.P.). Participants were recruited from the community in their usual state of health, and all provided written informed consent. The data that support the findings of this study are available from the corresponding author upon reasonable request. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.