Regulation of erythropoiesis by hypoxia-inducible factors

Abstract

A classic physiologic response to systemic hypoxia is the increase in red blood cell production. Hypoxia-inducible factors (HIFs) orchestrate this response by inducing cell-type specific gene expression changes that result in increased erythropoietin (EPO) production in kidney and liver, in enhanced iron uptake and utilization and in adjustments of the bone marrow microenvironment that facilitate erythroid progenitor maturation and proliferation. In particular HIF-2 has emerged as the transcription factor that regulates EPO synthesis in the kidney and liver and plays a critical role in the regulation of intestinal iron uptake. Its key function in the hypoxic regulation of erythropoiesis is underscored by genetic studies in human populations that live at high-altitude and by mutational analysis of patients with familial erythrocytosis. This review provides a perspective on recent insights into HIF-controlled erythropoiesis and iron metabolism, and examines cell types that have EPO-producing capability. Furthermore, the review summarizes clinical syndromes associated with mutations in the O(2)-sensing pathway and the genetic changes that occur in high altitude natives. The therapeutic potential of pharmacologic HIF activation for the treatment of anemia is discussed.

Figure 1. Cellular sources of EPO

Shown is a schematic overview of cell types and tissues with EPO-producing capacity. In adults, the kidney and liver are the two major contributors to the serum EPO pool with the kidney being the main physiologic site of EPO synthesis. While EPO transcripts are not detectable at baseline, the liver produces EPO when stimulated with either moderate to severe hypoxia or pharmacologically. The contribution of other cell types to erythropoiesis under stress conditions is not clear. A list of cell types and tissues in which EPO transcripts have been detected under various experimental conditions is shown on the left. Renal EPO-producing cells (REPC) are peritubular interstitial fibroblasts (shown in orange). Tubular epithelial cells do not produce EPO and are shown in green. Under chronic injury conditions, REPC undergo trans-differentiation into collagen-producing myofibroblasts (blue) and loose their ability to produce EPO; injured renal tubuli are shown in grey color. In CKD kidneys, the number of REPC is reduced, which results in inadequate EPO production in response to hypoxic stimuli and leads to the development of anemia. Abb.: cv, central vein.

Figure 2. EPO is HIF-2-regulated

Shown is an overview of EPO gene regulation by HIF-2. The VHL-E3-ubiquitin ligase complex targets hydroxylated HIF-2α for proteasomal degradation (shown are key components of this complex). Hydroxylated HIF-α binds to the β-domain of VHL, which is contained within amino acid residues 64–154. The C-terminal α-domain links VHL to the E3-ligase via elongin C. HIF-2α hydroxylation is carried out by O₂- and iron-dependent HIF prolyl-4-hydroxylases (HIF-PHD). In the absence of molecular O₂, HIF-2α, which is constitutively synthesized, is no longer degraded and translocates to the nucleus where it forms a heterodimer with HIF-β also known as the aryl hydrocarbon receptor nuclear translocator (ARNT). HIF-2α/β heterodimers bind to the HIF consensus binding site 5′-RCGTG-3′ and increase EPO transcription in the presence of transcriptional coactivators, such as CREB-binding protein (CBP) and p300. Hypoxic induction of EPO in the liver is mediated by the liver-inducibility element located in the 3′-end of the EPO gene. The hypoxic induction of EPO in REPC requires the kidney inducibility element, which is located 6 – 14 kb upstream of its transcription start site. Nitric oxide, reactive O₂ species, succinate and fumarate, cobalt chloride and iron chelators such as desferrioxamine inhibit HIF-PHDs, which results in increased EPO transcription. Boxes depict EPO exons. EPO coding sequences are shown in red, non-translated regions are shown in blue. Numbers indicate distance from transcription start site in kilobases (kb; not drawn to scale). Also shown are 3′-HNF4 binding sites. Abb.: CoCl₂, cobalt chloride; Fe²⁺, ferrous iron; HNF4, hepatocyte nuclear factor 4; NO, nitric oxide; ROS, reactive oxygen species; ub, ubiquitin.

Figure 3. HIF coordinates EPO production with iron metabolism

Shown is a schematic overview of changes in iron metabolism as a consequence of HIF activation. HIF-regulated genes are indicated in red. HIF-2 activation (either by hypoxia, pharmacologic means or as a result of mutations) induces renal and hepatic EPO synthesis, which leads to an increase in serum EPO levels and stimulation of erythropoiesis. Iron metabolism is adjusted to match iron demand from increased erythropoiesis. In the duodenum, duodenal cytochrome b (DCYTB) reduces ferric iron (Fe³⁺) to its ferrous form (Fe²⁺), which is then transported into the cytosol of enterocytes by divalent metal transporter-1 (DMT1). DCYTB and DMT1 are both HIF-2-regulated. Absorbed iron is released into the circulation by ferroportin and is then transported in complex with transferrin (TF) to the liver, reticulo-endothelial cells (RES), bone marrow and other organs. TF is HIF-regulated and hypoxia increases its serum levels. EPO-induced erythropoiesis inhibits hepcidin synthesis in the liver and reduces serum hepcidin levels. As a result of low serum hepcidin ferroportin (FPN) cell surface expression is increased (hepcidin promotes ferroportin degradation). As a result, more iron is released from enterocytes, hepatocytes and RES cells. Growth differentiation factor 15 (GDF15) is made by erythroid precursor cells and has been shown to suppress hepcidin in hepatocytes.