Prolyl Hydroxylase Domain Isoforms Research Articles

Whole-Body Prolyl Hydroxylase Domain (PHD) 3 Deficiency Increased Plasma Lipids and Hematocrit Without Impacting Plaque Size in Low-Density Lipoprotein Receptor Knockout Mice.

Background and aims: Atherosclerosis is an important cause of clinical cardiovascular events. Atherosclerotic plaques are hypoxic, and reoxygenation improves plaque phenotype. Central players in hypoxia are hypoxia inducible factors (HIF) and their regulators, HIF-prolyl hydroxylase (PHD) isoforms 1, 2, and 3. PHD inhibitors, targeting all three isoforms, are used to alleviate anemia in chronic kidney disease. Likewise, whole-body PHD1 and PHD2ko ameliorate hypercholesterolemia and atherogenesis. As the effect of whole-body PHD3 is unknown, we investigated the effects of germline whole-body PHD3ko on atherosclerosis.Approach and Results: To initiate hypercholesterolemia and atherosclerosis low-density lipoprotein receptor knockout (LDLrko) and PHD3/LDLr double knockout (PHD3dko), mice were fed a high-cholesterol diet. Atherosclerosis and hypoxia marker pimonidazole were analyzed in aortic roots and brachiocephalic arteries. In contrast to earlier reports on PHD1- and PHD2-deficient mice, a small elevation in the body weight and an increase in the plasma cholesterol and triglyceride levels were observed after 10 weeks of diet. Dyslipidemia might be explained by an increase in hepatic mRNA expression of Cyp7a1 and fatty acid synthase, while lipid efflux of PHD3dko macrophages was comparable to controls. Despite dyslipidemia, plaque size, hypoxia, and phenotype were not altered in the aortic root or in the brachiocephalic artery of PHD3dko mice. Additionally, PHD3dko mice showed enhanced blood hematocrit levels, but no changes in circulating, splenic or lymphoid immune cell subsets.Conclusion: Here, we report that whole-body PHD3dko instigated an unfavorable lipid profile and increased hematocrit, in contrast to other PHD isoforms, yet without altering atherosclerotic plaque development.

Are all erythropoiesis-stimulating agents created equal?

Erythropoiesis-stimulating agents (ESAs) are effective drugs to correct and maintain haemoglobin (Hb) levels, however, their use at doses to reach high Hb targets has been associated with an increased risk of cardiovascular adverse events, mortality and cancer. Presently used ESAs have a common mechanism of action but different pharmacokinetic and pharmacodynamic characteristics. Accordingly, the mode of activation of the erythropoietin (EPO) receptor can exert marked differences in downstream events. It is unknown whether the various ESA molecules have different efficacy/safety profiles. The relative mortality and morbidity risks associated with the use of different types of ESAs remains poorly evaluated. Recently an observational study and a randomized clinical trial provided conflicting results regarding this matter. However, these two studies displayed several differences in patient characteristics and ESA molecules used. More importantly, by definition, randomized clinical trials avoid bias by indication and suffer less from confounding factors. Therefore they bring a higher degree of evidence. The scenario becomes even more complex when considering the new class of ESAs, called prolyl-hydroxylase domain (PHD) inhibitors. They are oral drugs that mimic exposure to hypoxia and stabilize hypoxia-inducible factor α. They profoundly differ from presently used ESAs, as they have multiple targets of action, including the stimulation of endogenous EPO synthesis, direct mobilization/absorption of iron and a higher reduction of hepcidin. Accordingly, they have the potential to be more effective in inflamed patients with functional iron deficiency, i.e. the setting of patients who are at higher risk of cardiovascular events and mortality in response to present ESA use. As for ESAs, individual PHD inhibitors differ in molecular structure and degree of selectivity for the three main PHD isoforms; their efficacy and safety profiles may therefore be different from that of presently available ESAs.

Inhibition of prolyl hydroxylase domain proteins selectively enhances venous thrombus neovascularisation

BackgroundHypoxia within acute venous thrombi is thought to drive resolution through stabilisation of hypoxia inducible factor 1 alpha (HIF1α). Prolyl hydroxylase domain (PHD) isoforms are critical regulators of HIF1α stability. Non-selective inhibition of PHD isoforms with l-mimosine has been shown to increase HIF1α stabilisation and promote thrombus resolution. ObjectiveThe aim of this study was to investigate the therapeutic potential of PHD inhibition in venous thrombus resolution. MethodsThrombosis was induced in the inferior vena cava of mice using a combination of flow restriction and endothelial activation. Gene and protein expression of PHD isoforms in the resolving thrombus was measured by RT-PCR and immunohistochemistry. Thrombus resolution was quantified in mice treated with pan PHD inhibitors AKB-4924 and JNJ-42041935 or inducible all-cell Phd2 knockouts by micro-computed tomography, 3D high frequency ultrasound or endpoint histology. ResultsResolving venous thrombi demonstrated significant temporal gene expression profiles for PHD2 and PHD3 (P < 0.05), but not for PHD1. PHD isoform protein expression was localised to early and late inflammatory cell infiltrates. Treatment with selective pan PHD inhibitors, AKB-4924 and JNJ-42041935, enhanced thrombus neovascularisation (P < 0.05), but had no significant effect on overall thrombus resolution. Thrombus resolution or its markers, macrophage accumulation and neovascularisation, did not differ significantly in inducible all-cell homozygous Phd2 knockouts compared with littermate controls (P > 0.05). ConclusionsThis data suggests that PHD-mediated thrombus neovascularisation has a limited role in the resolution of venous thrombi. Directly targeting angiogenesis alone may not be a viable therapeutic strategy to enhance venous thrombus resolution.

Structure-Function Studies of the Hypoxia-Inducible Proyly Hydroxylases

Prolyl-hydroxylase domain (PHD)-containing proteins are the primary oxygen sensors in the cell and have been identified as important potential targets for treatment of ischemic events including heart attack and stroke. There are three known PHD isoforms that differ significantly in size and activity. We are performing structure-function studies of the PHD-3 (27.3 kDa) and the catalytic domain of PHD-2 (27.5 kDa) using fluorescence-based assays and nuclear magnetic resonance (NMR). These two isoforms are the most active in maintaining cellular oxygen levels. A protocol for refolding from inclusion bodies and purification for both proteins will be presented. Because the known structure of trPHD2 shows that the catalytic center is buried, an important role for protein dynamics in enzymatic turnover has been proposed. Molecular dynamics simulations of the trPHD2 protein in various ligand-bound forms are being used to investigate the functional relevance of internal motions. NMR experiments investigating the slow motions of these proteins provide experimental evidence for the locations of motions with potential functional relevance, and new inhibitory molecules are being developed as potential new tools for understanding this important pathway and as possible new therapeutic leads. This project was funded by the New Jersey Health Foundation.

Identification of Small-Molecule PHD2 Zinc Finger Inhibitors that Activate Hypoxia Inducible Factor.

The prolyl hydroxylase domain (PHD) protein:hypoxia inducible factor (HIF) pathway is the main pathway by which changes in oxygen concentration are transduced to changes in gene expression. In mammals, there are three PHD paralogues, and PHD2 has emerged as a particularly critical one for regulating HIF target genes such as erythropoietin (EPO), which controls red cell mass and hematocrit. PHD2 is distinctive among the three PHDs in that it contains an N-terminal MYND-type zinc finger. We have proposed that this zinc finger binds a Pro-Xaa-Leu-Glu (PXLE) motif found in proteins of the HSP90 pathway to facilitate HIF-α hydroxylation. Targeting this motif could provide a means of specifically inhibiting this PHD isoform. Here, we screened a library of chemical compounds for their capacity to inhibit the zinc finger of PHD2. We identified compounds that, in vitro, can inhibit PHD2 binding to a PXLE-containing peptide and induce activation of HIF. Injection of one of these compounds into mice induces an increase in hematocrit. This study offers proof of principle that inhibition of the zinc finger of PHD2 can provide a means of selectively targeting PHD2 to activate the HIF pathway.

A Knock-in Mouse Model of Human PHD2 Gene-associated Erythrocytosis Establishes a Haploinsufficiency Mechanism

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). Phd2(P294R/+) mice display a degree of erythrocytosis equivalent to that seen in Phd2(+/-) mice. The Phd2(P294R/+)-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.

You Can't Run From Inflammation

### Macrophage Skewing by Phd2 Haplodeficiency Prevents Ischaemia by Inducing Arteriogenesis Takeda et al Nature . 2011;479:122–126. Prolyl hydroxylase domain (PHD) molecules sense oxygen availability in mammalian cells and can serve as drug targets. Recent work from Takeda et al suggest a previously unappreciated role for PHD2—the main HIF prolyl hydroxylase—in arteriogenesis via macrophage skewing. Deletion of PHD2 in macrophages associated with activation of the canonical NF-κB pathway and arteriogenesis. Work done by many investigators over the last decade has shed light onto the mechanisms of oxygen sensing in cells and in organisms.1 The prolyl hydroxylase domain (PHD; also called EglN) proteins belong to a family of Fe(II) and 2-oxoglutarate–dependent dioxygenases, whose activity depends on oxygen bioavailability.2 One well-described substrate of PHDs is the master transcription factor hypoxia-inducible factor (HIF). HIF hydroxylation by PHDs targets HIF for degradation; hence, PHDs inhibit HIF activity. Whereas mammalian cells contain 3 PHD isoforms, PHD2 appears to be the primary HIF prolyl hydroxylase, although other PHD family members (PHD1 and PHD3) can contribute to HIF regulation in certain conditions.2 Multiple lines of evidence suggest that PHD molecules (particularly PHD2) act as “oxygen sensors” in mammalian cells. For example, genetic knockdown of PHD2 in mice recapitulates many of the hallmarks of hypoxia in vivo.3 In a recent paper in Nature , Mazzone and colleagues4 describe a novel, and previously unappreciated, …

Biochemical characterization of human HIF hydroxylases using HIF protein substrates that contain all three hydroxylation sites

The HIF (hypoxia-inducible factor) plays a central regulatory role in oxygen homoeostasis. HIF proteins are regulated by three Fe(II)- and α-KG (α-ketoglutarate)-dependent prolyl hydroxylase enzymes [PHD (prolyl hydroxylase domain) isoenzymes 1-3 or PHD1, PHD2 and PHD3] and one asparaginyl hydroxylase [FIH (factor inhibiting HIF)]. The prolyl hydroxylases control the abundance of HIF through oxygen-dependent hydroxylation of specific proline residues in HIF proteins, triggering subsequent ubiquitination and proteasomal degradation. FIH inhibits the HIF transcription activation through asparagine hydroxylation. Understanding the precise roles and regulation of these four Fe(II)- and α-KG-dependent hydroxylases is of great importance. In the present paper, we report the biochemical characterization of the first HIF protein substrates that contain the CODDD (C-terminal oxygen-dependent degradation domain), the NODDD (N-terminal oxygen-dependent degradation domain) and the CAD (C-terminal transactivation domain). Using LC-MS/MS (liquid chromatography-tandem MS) detection, we show that all three PHD isoenzymes have a strong preference for hydroxylation of the CODDD proline residue over the NODDD proline residue and the preference is observed for both HIF1α and HIF2α protein substrates. In addition, steady-state kinetic analyses show differential substrate selectivity for HIF and α-KG in reference to the three PHD isoforms and FIH.

Inhibition of the histone demethylase JMJD2E by 3-substituted pyridine 2,4-dicarboxylates.

Based on structural analysis of the human 2-oxoglutarate (2OG) dependent JMJD2 histone N(ε)-methyl lysyl demethylase family, 3-substituted pyridine 2,4-dicarboxylic acids were identified as potential inhibitors with possible selectivity over other human 2OG oxygenases. Microwave-assisted palladium-catalysed cross coupling methodology was developed to install a diverse set of substituents on the sterically demanding C-3 position of a pyridine 2,4-dicarboxylate scaffold. The subsequently prepared di-acids were tested for in vitro inhibition of the histone demethylase JMJD2E and another human 2OG oxygenase, prolyl-hydroxylase domain isoform 2 (PHD2, EGLN1). A subset of substitution patterns yielded inhibitors with selectivity for JMJD2E over PHD2, demonstrating that structure-based inhibitor design can enable selective inhibition of histone demethylases over related human 2OG oxygenases.

Prolyl hydroxylase in human corpora lutea during menstrual cycle and early pregnancy

Immunohistochemical staining for human prolyl hydroxylase revealed intense staining of the human corpora lutea (CL) parencyma during early pregnancy compared with those in the menstrual cycle. These results suggest that human prolyl hydroxylase might play an important role in determining the physiology and structure of the CL during the menstrual cycle and early pregnancy.

Regulation of adult erythropoiesis by prolyl hydroxylase domain proteins

AbstractPolycythemia is often associated with erythropoietin (EPO) overexpression and defective oxygen sensing. In normal cells, intracellular oxygen concentrations are directly sensed by prolyl hydroxylase domain (PHD)–containing proteins, which tag hypoxia-inducible factor (HIF) α subunits for polyubiquitination and proteasomal degradation by oxygen-dependent prolyl hydroxylation. Here we show that different PHD isoforms differentially regulate HIF-α stability in the adult liver and kidney and suppress Epo expression and erythropoiesis through distinct mechanisms. Although Phd1−/− or Phd3−/− mice had no apparent defects, double knockout of Phd1 and Phd3 led to moderate erythrocytosis. HIF-2α, which is known to activate Epo expression, accumulated in the liver. In adult mice deficient for PHD2, the prototypic Epo transcriptional activator HIF-1α accumulated in both the kidney and liver. Elevated HIF-1α levels were associated with dramatically increased concentrations of both Epo mRNA in the kidney and Epo protein in the serum, which led to severe erythrocytosis. In contrast, heterozygous mutation of Phd2 had no detectable effects on blood homeostasis. These findings suggest that PHD1/3 double deficiency leads to erythrocytosis partly by activating the hepatic HIF-2α/Epo pathway, whereas PHD2 deficiency leads to erythrocytosis by activating the renal Epo pathway.

Role and regulation of prolyl hydroxylase domain proteins

Oxygen-dependent hydroxylation of hypoxia-inducible factor (HIF)-alpha subunits by prolyl hydroxylase domain (PHD) proteins signals their polyubiquitination and proteasomal degradation, and plays a critical role in regulating HIF abundance and oxygen homeostasis. While oxygen concentration plays a major role in determining the efficiency of PHD-catalyzed hydroxylation reactions, many other environmental and intracellular factors also significantly modulate PHD activities. In addition, PHDs may also employ hydroxylase-independent mechanisms to modify HIF activity. Interestingly, while PHDs regulate HIF-alpha protein stability, PHD2 and PHD3 themselves are subject to feedback upregulation by HIFs. Functionally, different PHD isoforms may differentially contribute to specific pathophysiological processes, including angiogenesis, erythropoiesis, tumorigenesis, and cell growth, differentiation and survival. Because of diverse roles of PHDs in many different processes, loss of PHD expression or function triggers multi-faceted pathophysiological changes as has been shown in mice lacking different PHD isoforms. Future investigations are needed to explore in vivo specificity of PHDs over different HIF-alpha subunits and differential roles of PHD isoforms in different biological processes.

Essential Role for Prolyl Hydroxylase Domain Protein 2 in Oxygen Homeostasis of the Adult Vascular System

Prolyl hydroxylase domain (PHD) proteins, including PHD1, PHD2, and PHD3, mediate oxygen-dependent degradation of hypoxia-inducible factor (HIF)-alpha subunits. Although angiogenic roles of hypoxia-inducible factors are well known, the roles of PHDs in the vascular system remain to be established. We evaluated angiogenic phenotypes in mice carrying targeted disruptions in genes encoding different PHD isoforms. Although Phd1-/- and Phd3-/- mice did not display apparent angiogenic defects, broad-spectrum conditional knockout of Phd2 led to hyperactive angiogenesis and angiectasia. Blood vessels in PHD2-deficient mice were highly perfusable. Furthermore, examination of medium-sized vessels in subendocardial layer in the heart demonstrated successful recruitment of vascular smooth muscle cells. Surprisingly, increased vascular growth was independent of local efficiency of Phd2 disruption. Mice carrying significant Phd2 disruption in multiple organs, including the liver, heart, kidney, and lung, displayed excessive vascular growth not only in these organs but also in the brain, where Phd2 disruption was very inefficient. More surprisingly, increased accumulation of hypoxia-inducible factor-1alpha and angiectasia in the liver were not accompanied by corresponding increases in hepatic expression of Vegfa or angiopoietin-1. However, the serum vascular endothelial growth factor-A level was significantly increased in PHD2-deficient mice. PHD2, but not PHD1 and PHD3, is a major negative regulator for vascular growth in adult mice. Increased angiogenesis in PHD2-deficient mice may be mediated by a novel systemic mechanism.