Prolyl hydroxylase domain protein 2 (PHD2, also known as Egg Laying Defective Nine homolog 1) is a key oxygen-sensing protein
in metazoans. In an oxygen-dependent manner, PHD2 site-specifically prolyl hydroxylates the master transcription factor of
the hypoxic response, hypoxia-inducible factor-α (HIF-α), thereby targeting HIF-α for degradation. In this report we show
that the heat shock protein 90 (HSP90) co-chaperones p23 and FKBP38 interact via a conserved Pro-Xaa-Leu-Glu motif (where
Xaa = any amino acid) in these proteins with the N-terminal Myeloid Nervy and DEAF-1 (MYND)-type zinc finger of PHD2. Knockdown
of p23 augments hypoxia-induced HIF-1α protein levels and HIF target genes. We propose that p23 recruits PHD2 to the HSP90
machinery to facilitate HIF-1α hydroxylation. These findings identify a link between two ancient pathways, the PHD:HIF and
the HSP90 pathways, and suggest that this link was established concurrent with the emergence of the PHD:HIF pathway in evolution.
[Show abstract][Hide abstract] ABSTRACT: The central pathway for controlling red cell mass is the PHD (prolyl hydroxylase domain protein):hypoxia-inducible factor
(HIF) pathway. HIF, which is negatively regulated by PHD, activates numerous genes, including ones involved in erythropoiesis,
such as the ERYTHROPOIETIN (EPO) gene. Recent studies have implicated PHD2 as the key PHD isoform regulating red cell mass. Studies of humans have identified
erythrocytosis-associated, heterozygous point mutations in the PHD2 gene. A key question concerns the mechanism by which human mutations lead to phenotypes. In the present report, we generated
and characterized a mouse line in which a P294R knock-in mutation has been introduced into the mouse Phd2 locus to model the first reported human PHD2 mutation (P317R). Phd2P294R/+ mice display a degree of erythrocytosis equivalent to that seen in Phd2+/− mice. The Phd2P294R/+-associated erythrocytosis is reversed in a Hif2a+/−, but not a Hif1a+/− background. Additional studies using various conditional knock-outs of Phd2 reveal that erythrocytosis can be induced by homozygous and heterozygous knock-out of Phd2 in renal cortical interstitial cells using a Pax3-Cre transgene or by homozygous knock-out of Phd2 in hematopoietic progenitors driven by a Vav1-Cre transgene. These studies formally prove that a missense mutation in PHD2 is the cause of the erythrocytosis, show that
this occurs through haploinsufficiency, and point to multifactorial control of red cell mass by PHD2.
[Show abstract][Hide abstract] ABSTRACT: Hypoxia-mediated stress responses are important in tumor progression, especially when tumor growth causes the tumor to become deprived of its blood supply. The oxygen-labile transcription factor hypoxia-inducible factor-1 alpha (HIF-1α) plays a critical role in regulating hypoxia stress-related gene expression and is considered a novel therapeutic target. Lung adenocarcinoma cell lines were exposed to minocycline, followed by incubation at hypoxic condition for 3-6 h. Here, we show that minocycline, a second-generation tetracycline, can induce HIF-1α proteasomal degradation under hypoxia by increasing the expression prolyl hydroxylase-2 and HIF-1α/von Hippel-Lindau protein interaction, thereby overcoming hypoxia-induced HIF-1α stabilization. Neither repression of hypoxia-induced phosphatidylinositol-3 kinase/Akt/mammalian target of rapamycin pathway nor inhibition of Hsp90 was required for minocycline-induced HIF-1α degradation. The HIF-1α degradation-enhancing effect of minocycline was evident in both cancerous and primary cells. Minocycline-pretreated, hypoxia-conditioned cells showed a clear reduction in hypoxia response element reporter expression and amelioration of vascular endothelial growth factor C/D (VEGF-C/D), matrix metalloproteinase 2, and glucose transporter 1 expression. By decreasing VEGF secretion of cancerous cells, minocycline could suppress endothelial cell neovasculogenesis. These findings suggest a novel application of minocycline in the treatment of tumor angiogenesis as well as hypoxia-related diseases.
Archives of Toxicology 12/2013; 88(3). DOI:10.1007/s00204-013-1175-5 · 5.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The transcription of the erythropoietin (EPO) gene is tightly regulated by the hypoxia response pathway to maintain oxygen homeostasis. Elevations in serum EPO level may be reflected in an augmentation in the red cell mass, thereby causing erythrocytosis. Studies on erythrocytosis have provided insights into the function of the oxygen-sensing pathway and the critical proteins involved in the regulation of EPO transcription. The α subunits of the hypoxia-inducible transcription factor are hydroxylated by three prolyl hydroxylase domain (PHD) enzymes, which belong to the iron and 2-oxoglutarate-dependent oxygenase superfamily. Sequence analysis of the genes encoding the PHDs in patients with erythrocytosis has revealed heterozygous germline mutations only occurring in Egl nine homolog 1 (EGLN1, also known as PHD2), the gene that encodes PHD2. To date, 24 different EGLN1 mutations comprising missense, frameshift, and nonsense mutations have been described. The phenotypes associated with the patients carrying these mutations are fairly homogeneous and typically limited to erythrocytosis with normal to elevated EPO. However, exceptions exist; for example, there is one case with development of concurrent paraganglioma (PHD2-H374R). Analysis of the erythrocytosis-associated PHD2 missense mutations has shown heterogeneous results. Structural studies reveal that mutations can affect different domains of PHD2. Some are close to the hypoxia-inducible transcription factor α/2-oxoglutarate or the iron binding sites for PHD2. In silico studies demonstrate that the mutations do not always affect fully conserved residues. In vitro and in cellulo studies showed varying effects of the mutations, ranging from mild effects to severe loss of function. The exact mechanism of a potential tumor-suppressor role for PHD2 still needs to be elucidated. A knockin mouse model expressing the first reported PHD2-P317R mutation recapitulates the phenotype observed in humans (erythrocytosis with inappropriately normal serum EPO levels) and demonstrates that haploinsufficiency and partial deregulation of PHD2 is sufficient to cause erythrocytosis.
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