A classic physiologic response to hypoxia in humans is the up-regulation of the ERYTHROPOIETIN (EPO) gene, which is the central regulator of red blood cell mass. The EPO gene, in turn, is activated by hypoxia inducible factor (HIF). HIF is a transcription factor consisting of an ␣ subunit (HIF-␣) and a ␤ subunit (HIF-␤). Under normoxic conditions, prolyl hydroxylase domain protein (PHD, also known as HIF prolyl hydroxylase and egg laying-defective nine protein) site specifically hydroxylates HIF-␣ in a conserved LXXLAP motif (where underlining indicates the hydroxylacceptor proline). This provides a recognition motif for the von Hippel Lindau protein, a component of an E3 ubiquitin ligase complex that targets hydroxylated HIF-␣ for degradation. Under hypoxic conditions, this inherently oxygen-dependent modification is arrested, thereby stabilizing HIF-␣ and allowing it to activate the EPO gene. We previously identified and characterized an erythrocytosis-associated HIF2A mutation, G537W. More recently, we reported two additional erythrocytosis-associated HIF2A mutations, G537R and M535V. Here, we describe the functional characterization of these two mutants as well as a third novel erythrocytosisassociated mutation, P534L. These mutations affect residues C-terminal to the LXXLAP motif. We find that all result in impaired degradation and thus aberrant stabilization of HIF-2␣. However, each exhibits a distinct profile with respect to their effects on PHD2 binding and von Hippel Lindau interaction. These findings reinforce the importance of HIF-2␣ in human EPO regulation, demonstrate heterogeneity of functional defects arising from these mutations, and point to a critical role for residues C-terminal to the LXXLAP motif in HIF-␣.

No abstract

The central transcriptional response to hypoxia is mediated by the Prolyl Hydroxylase Domain protein (PHD):Hypoxia Inducible Factor (HIF) pathway. In this pathway, PHD prolyl hydroxylates and thereby negatively regulates the α-subunit of the transcription factor HIF (HIF-α). An important HIF target gene is that for Erythropoietin (EPO), which controls red cell mass. Recent studies have identified PHD2 as the critical PHD isoform regulating the EPO gene. Other studies have shown that the inducibility of the HIF pathway diminishes as a function of age. Thus, an important question is whether the PHD2:EPO pathway is altered in the aging. Here, we employed a mouse line with a globally-inducible Phd2 conditional knockout allele to examine the integrity of the Phd2:Epo axis in young (six to eight month old) and aging (sixteen to twenty month old) mice. We find that acute global deletion of Phd2 results in a robust erythrocytosis in both young and aging mice, with both age groups showing marked extramedullary hematopoiesis in the spleen. Epo mRNA is dramatically upregulated in the kidney, but not in the liver, in both age groups. Conversely, other Hif targets, including Vegf, Pgk1, and Phd3 are upregulated in the liver but not in the kidney in both age groups. These findings have implications for targeting this pathway in the aging.

Transcriptionally erythropoietin (Epo) synthesis is tightly regulated by the hypoxia inducible factor (HIF), which is composed of one alpha and one beta subunit that are constitutively expressed. The beta subunit is non-variable, but three different alpha subunits give rise to three isoforms of HIF. The alpha subunit is proteasomally regulated in the presence of oxygen by hydroxylation of the proline in the LXXLAP motif of the oxygen dependent degradation (ODD) domain of HIFalpha, catalysed by members of the prolyl hydroxylase domain (PHD) family of enzymes. This allows the von Hippel Lindau (VHL) protein to associate with the alpha subunit, which is subsequently tagged with ubiquitin and degraded by the proteasome. Any defect in the oxygen sensing pathway that allows the alpha subunit to escape proteasomal regulation leads to elevated expression of HIF target genes. Recently mutations in both VHL and PHD2 have been identified in a cohort of patients with erythrocytosis, but no mutations were found in the ODD domain of HIF1alpha. Instead, investigation of the homologous region in HIF-2alpha revealed four different mutations, Pro534Leu, Met535Val, Gly537Arg and Gly537Trp in seven individuals/families. Affected individuals presented at a young age with elevated serum Epo. Several individuals have a clinical history of thrombosis, but no evidence of a von Hippel Lindau-like syndrome. To define how the four mutations relate to the erythrocytosis phenotype functional assays were performed in vitro. Binding of PHD2 to the four HIF-2alpha mutants was impaired to varying degrees, with both the Gly537 mutants showing the greatest reduction. The association of VHL with the hydroxylated Met535Val mutant peptide was similar to wild type HIF- 2alpha, but was decreased in the other three HIF-2alpha mutants. Expression of three HIF- 2alpha target genes, adrenomedullin, NDRG1 and VEGF, was significantly up-regulated in cells stably transfected with the mutants under normoxia compared to wild type HIF-2alpha. Mutations in the ODD domain of HIF-2alpha disrupt proteasomal regulation by reducing the association with PHD2 and hence hydroxylation. Furthermore the binding of VHL is also impaired, even when HIF-2alpha is hydroxylated. Examination of the three-dimensional structure of hydroxylated HIF-1alpha bound to VHL confirms that amino acids close to site of hydroxylation (Pro-531 in isoform 2) are important for this association. These observations, together with recent studies utilising murine models of erythrocytosis, support the PHD2-HIF-2alpha-VHL axis as the major regulator of erythropoietin.