Publications by authors named "Frank S Lee"

72 Publications

gain-of-function mutation modulates the stiffness of smooth muscle cells and compromises vascular mechanics.

iScience 2021 Apr 2;24(4):102246. Epub 2021 Mar 2.

Department of Chemical and Biomolecular Engineering and Institute for NanoBioTechnology, Johns Hopkins University, Baltimore, MD 21218, USA.

Heterozygous gain-of-function (GOF) mutations of hypoxia-inducible factor 2α (HIF2A), a key hypoxia-sensing regulator, are associated with erythrocytosis, thrombosis, and vascular complications that account for morbidity and mortality of patients. We demonstrated that the vascular pathology of HIF2A GOF mutations is independent of erythrocytosis. We generated HIF2A GOF-induced pluripotent stem cells (iPSCs) and differentiated them into endothelial cells (ECs) and smooth muscle cells (SMCs). Unexpectedly, HIF2A-SMCs, but not HIF2A-ECs, were phenotypically aberrant, more contractile, stiffer, and overexpressed endothelin 1 (EDN1), myosin heavy chain, elastin, and fibrillin. EDN1 inhibition and knockdown of -receptors both reduced HIF2-SMC stiffness. Hif2A GOF heterozygous mice displayed pulmonary hypertension, had SMCs with more disorganized stress fibers and higher stiffness in their pulmonary arterial smooth muscle cells, and had more deformable pulmonary arteries compared with wild-type mice. Our findings suggest that targeting these vascular aberrations could benefit patients with HIF2A GOF and conditions of augmented hypoxia signaling.
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http://dx.doi.org/10.1016/j.isci.2021.102246DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7995528PMC
April 2021

High-altitude deer mouse hypoxia-inducible factor-2α shows defective interaction with CREB-binding protein.

J Biol Chem 2021 Jan-Jun;296:100461. Epub 2021 Feb 25.

Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA. Electronic address:

Numerous mammalian species have adapted to the chronic hypoxia of high altitude. Recent genomic studies have identified evidence for natural selection of genes and associated genetic changes in these species. A major gap in our knowledge is an understanding of the functional significance, if any, of these changes. Deer mice (Peromyscus maniculatus) live at both low and high altitudes in North America, providing an opportunity to identify functionally important genetic changes. High-altitude deer mice show evidence of natural selection on the Epas1 gene, which encodes for hypoxia-inducible factor-2α (Hif-2α), a central transcription factor of the hypoxia-inducible factor pathway. An SNP encoding for a T755M change in the Hif-2α protein is highly enriched in high-altitude deer mice, but its functional significance is unknown. Here, using coimmunoprecipitation and transcriptional activity assays, we show that the T755M mutation produces a defect in the interaction of Hif-2α with the transcriptional coactivator CREB-binding protein. This results in a loss of function because of decreased transcriptional activity. Intriguingly, the effect of this mutation depends on the amino acid context. Interchanges between methionine and threonine at the corresponding position in house mouse (Mus musculus) Hif-2α are without effects on CREB-binding protein binding. Furthermore, transfer of a set of deer mouse-specific Hif-2α amino acids to house mouse Hif-2α is sufficient to confer sensitivity of house mouse Hif-2α to the T755M substitution. These findings provide insight into high-altitude adaptation in deer mice and evolution at the Epas1 locus.
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http://dx.doi.org/10.1016/j.jbc.2021.100461DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8024697PMC
February 2021

Tibetan , an allele with loss-of-function properties.

Proc Natl Acad Sci U S A 2020 06 15;117(22):12230-12238. Epub 2020 May 15.

Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104;

Tibetans have adapted to the chronic hypoxia of high altitude and display a distinctive suite of physiologic adaptations, including augmented hypoxic ventilatory response and resistance to pulmonary hypertension. Genome-wide studies have consistently identified compelling genetic signatures of natural selection in two genes of the Hypoxia Inducible Factor pathway, and The product of the former induces the degradation of the product of the latter. Key issues regarding Tibetan are whether it is a gain-of-function or loss-of-function allele, and how it might contribute to high-altitude adaptation. Tibetan PHD2 possesses two amino acid changes, D4E and C127S. We previously showed that in vitro, Tibetan PHD2 is defective in its interaction with p23, a cochaperone of the HSP90 pathway, and we proposed that Tibetan is a loss-of-function allele. Here, we report that additional PHD2 mutations at or near Asp-4 or Cys-127 impair interaction with p23 in vitro. We find that mice with the Tibetan allele display augmented hypoxic ventilatory response, supporting this loss-of-function proposal. This is phenocopied by mice with a mutation in that abrogates the PHD2:p23 interaction. haploinsufficiency, but not the Tibetan allele, ameliorates hypoxia-induced increases in right ventricular systolic pressure. The Tibetan allele is not associated with hemoglobin levels in mice. We propose that Tibetans possess genetic alterations that both activate and inhibit selective outputs of the HIF pathway to facilitate successful adaptation to the chronic hypoxia of high altitude.
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http://dx.doi.org/10.1073/pnas.1920546117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7275716PMC
June 2020

An Erythrocytosis-Associated Mutation in the Zinc Finger of PHD2 Provides Insights into Its Binding of p23.

Hypoxia (Auckl) 2019 13;7:81-86. Epub 2019 Dec 13.

Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Background: Loss of function mutations in the gene are a cause of erythrocytosis. encodes for prolyl hydroxylase domain protein 2 (PHD2). PHD2 hydroxylates and downregulates hypoxia-inducible factor-2α (HIF-2α), a transcription factor that regulates erythropoiesis. While the large majority of erythrocytosis-associated mutations occur within its catalytic domain, rare mutations reside in its zinc finger. This zinc finger binds a Pro-Xaa-Leu-Glu motif in p23, an HSP90 cochaperone that facilitates hydroxylation of HIF-α, an HSP90 client. Essentially nothing is known about the specific interactions between the PHD2 zinc finger and p23.

Results: Here, we characterize an erythrocytosis-associated mutation in the zinc finger, K55N, that abolishes interaction with p23. We provide evidence that the affected residue, Lys-55, interacts with Asp-152 of p23. We also present results that indicate that PHD2 Arg-32 interacts with p23 Glu-160.

Conclusion: These studies not only reinforce the importance of the PHD2 zinc finger in the control of erythropoiesis, but also lead to a model in which a peptide motif in p23 binds in a specific orientation to a predicted groove in the zinc finger of PHD2.
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http://dx.doi.org/10.2147/HP.S230502DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6916684PMC
December 2019

Association of gene with high aerobic capacity of Peruvian Quechua at high altitude.

Proc Natl Acad Sci U S A 2019 11 11;116(48):24006-24011. Epub 2019 Nov 11.

Department of Anthropology, University of Michigan, Ann Arbor, MI 48109-1107.

Highland native Andeans have resided at altitude for millennia. They display high aerobic capacity (VOmax) at altitude, which may be a reflection of genetic adaptation to hypoxia. Previous genomewide (GW) scans for natural selection have nominated gene () as a candidate gene. The encoded protein, EGLN1/PHD2, is an O sensor that controls levels of the Hypoxia Inducible Factor-α (HIF-α), which regulates the cellular response to hypoxia. From GW association and analysis of covariance performed on a total sample of 429 Peruvian Quechua and 94 US lowland referents, we identified 5 SNPs associated with higher VOmax (L⋅min and mL⋅min⋅kg) in hypoxia (rs1769793, rs2064766, rs2437150, rs2491403, rs479200). For 4 of these SNPs, Quechua had the highest frequency of the advantageous (high VOmax) allele compared with 25 diverse lowland comparison populations from the 1000 Genomes Project. Genotype effects were substantial, with high versus low VOmax genotype categories differing by ∼11% (e.g., for rs1769793 SNP genotype TT = 34.2 mL⋅min⋅kg vs. CC = 30.5 mL⋅min⋅kg). To guard against spurious association, we controlled for population stratification. Findings were replicated for SNP rs1769793 in an independent Andean sample collected in 2002. These findings contextualize previous reports of natural selection at in Andeans, and support the hypothesis that natural selection has increased the frequency of an causal variant that enhances O delivery or use during exercise at altitude in Peruvian Quechua.
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http://dx.doi.org/10.1073/pnas.1906171116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6883775PMC
November 2019

Substrates of PHD.

Authors:
Frank S Lee

Cell Metab 2019 10;30(4):626-627

Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

The discovery of prolyl hydroxylase domain proteins (PHDs) as key enzymes in the hypoxia inducible factor (HIF) pathway has been followed by reports of a multitude of non-HIF substrates of PHD. Reporting in eLife, Cockman et al. (2019) find a surprising lack of detectable PHD activity toward any of them.
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http://dx.doi.org/10.1016/j.cmet.2019.08.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7454164PMC
October 2019

Update on mutations in the HIF: EPO pathway and their role in erythrocytosis.

Blood Rev 2019 09 16;37:100590. Epub 2019 Jul 16.

Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.

Identification of the underlying defects in congenital erythrocytosis has provided mechanistic insights into the regulation of erythropoiesis and oxygen homeostasis. The Hypoxia Inducible Factor (HIF) pathway plays a key role in this regard. In this pathway, an enzyme, Prolyl Hydroxylase Domain protein 2 (PHD2), constitutively prolyl hydroxylates HIF-2α, thereby targeting HIF-2α for degradation by the von Hippel Lindau (VHL) tumor suppressor protein. Under hypoxia, this modification is attenuated, resulting in the stabilization of HIF-2α and transcriptional activation of the erythropoietin (EPO) gene. Circulating EPO then binds to the EPO receptor (EPOR) on red cell progenitors in the bone marrow, leading to expansion of red cell mass. Loss of function mutations in PHD2 and VHL, as well as gain of function mutations in HIF-2α and EPOR, are well established causes of erythrocytosis. Here, we highlight recent developments that show that the study of this condition is still evolving. Specifically, novel mutations have been identified that either change amino acids in the zinc finger domain of PHD2 or alter splicing of the VHL gene. In addition, continued study of HIF-2α mutations has revealed a distinctive genotype-phenotype correlation. Finally, novel mutations have recently been identified in the EPO gene itself. Thus, the cascade of genes that at a molecular level leads to EPO action, namely PHD2 - > HIF2A - > VHL - > EPO - > EPOR, are all mutational targets in congenital erythrocytosis.
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http://dx.doi.org/10.1016/j.blre.2019.100590DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7484688PMC
September 2019

Author Correction: Loss of Phd2 cooperates with BRAF to drive melanomagenesis.

Nat Commun 2019 03 11;10(1):1211. Epub 2019 Mar 11.

Center for Research on Reproduction & Women's Health, University of Pennsylvania, Philadelphia, PA, 19104, USA.

The original version of this Article contained an error in the spelling of the author Brett L. Ecker, which was incorrectly given as Brett Ecker. This has now been corrected in both the PDF and HTML versions of the Article.
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http://dx.doi.org/10.1038/s41467-019-09195-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6411891PMC
March 2019

Loss of Phd2 cooperates with BRAF to drive melanomagenesis.

Nat Commun 2018 12 21;9(1):5426. Epub 2018 Dec 21.

Center for Research on Reproduction & Women's Health, University of Pennsylvania, Philadelphia, PA, 19104, USA.

Prolyl hydroxylase domain protein 2 (PHD2) is a well-known master oxygen sensor. However, the role of PHD2 in tumor initiation remains controversial. We find that during the transition of human nevi to melanoma, the expression of PHD2 protein is significantly decreased and lower expression PHD2 in melanoma is associated with worse clinical outcome. Knockdown of PHD2 leads to elevated Akt phosphorylation in human melanocytes. Mice with conditional melanocyte-specific expression of Phd2 (Tyr::CreER;Phd2) fail to develop pigmented lesions. However, deletion of Phd2 in combination with expression of BRaf in melanocytes (Tyr::CreER;Phd2;BRaf) leads to the development of melanoma with 100% penetrance and frequent lymph node metastasis. Analysis of tumor tissues using reverse phase protein arrays demonstrates that Phd2 deletion activates the AKT-mTOR-S6 signaling axis in the recovered tumors. These data indicate that PHD2 is capable of suppressing tumor initiation largely mediated through inhibiting of the Akt-mTOR signaling pathway in the melanocyte lineage.
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http://dx.doi.org/10.1038/s41467-018-07126-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6303344PMC
December 2018

At the crossroads of oxygen and iron sensing: hepcidin control of HIF-2α.

Authors:
Frank S Lee

J Clin Invest 2019 01 10;129(1):72-74. Epub 2018 Dec 10.

Hepcidin is the master regulator of iron metabolism. It plays a key role in the regulation of iron transport across the duodenal epithelium, which in turn is dependent on the oxygen-regulated transcription factor hypoxia-inducible factor 2α (HIF-2α). In this issue of the JCI, Schwartz and colleagues show that duodenal HIF-2α is itself regulated by hepcidin, thereby indicating that this transcription factor is not only regulated by oxygen, but also by iron. This work indicates that the crosstalk between liver hepcidin and intestinal HIF-2α plays an important role during iron overload, systemic iron deficiency, and anemia.
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http://dx.doi.org/10.1172/JCI125509DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6307931PMC
January 2019

Loss-of-function zinc finger mutation in the gene associated with erythrocytosis.

Blood 2018 09 15;132(13):1455-1458. Epub 2018 Aug 15.

Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA.

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http://dx.doi.org/10.1182/blood-2018-06-854711DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6161772PMC
September 2018

Functional Assays to Screen and Dissect Genomic Hits: Doubling Down on the National Investment in Genomic Research.

Circ Genom Precis Med 2018 04;11(4):e002178

Cardiovascular Institute, Department of Medicine (K.M.), Department of Genetics (K.M.), and Department of Pathology and Laboratory Medicine (F.S.L.), Perelman School of Medicine at the University of Pennsylvania, Philadelphia. Department of Pediatrics (D.B.), Cardiovascular Institute (D.B., T.Q.), and Department of Medicine (T.Q.), Stanford University, CA. Edward Mallinckrodt Department of Pediatrics, Washington University School of Medicine, St. Louis, MO (F.S.C.). St. Louis Children's Hospital, MO (F.S.C.). Pediatric Genomics Discovery Program, Department of Pediatrics and Genetics, Yale University School of Medicine, New Haven, CT (M.K.K.). Division of Cardiology, Department of Medicine, University of Washington, Seattle (S.L.). Department of Cardiovascular Sciences, University of South Florida Morsani College of Medicine, Tampa, FL (T.V.M.). Department of Pediatrics (I.P.M.), Department of Pathology (I.P.M.), and Department of Human Genetics (I.P.M.), The University of Chicago, IL. Division of Hematology/ Oncology, Boston Children's Hospital, MA (V.G.S.). Department of Pediatric Oncology, Dana-Farber Cancer Institute (V.G.S.) and Channing Division of Network Medicine, Brigham and Women's Hospital (E.K.S., X.Z.), Harvard Medical School, Boston. Broad Institute of MIT and Harvard, Cambridge, MA (V.G.S.). University of Colorado, Aurora (D.A.S.). Division of Cardiovascular Sciences, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD (A.A.K.H., X.-z.J.L.).

The National Institutes of Health have made substantial investments in genomic studies and technologies to identify DNA sequence variants associated with human disease phenotypes. The National Heart, Lung, and Blood Institute has been at the forefront of these commitments to ascertain genetic variation associated with heart, lung, blood, and sleep diseases and related clinical traits. Genome-wide association studies, exome- and genome-sequencing studies, and exome-genotyping studies of the National Heart, Lung, and Blood Institute-funded epidemiological and clinical case-control studies are identifying large numbers of genetic variants associated with heart, lung, blood, and sleep phenotypes. However, investigators face challenges in identification of genomic variants that are functionally disruptive among the myriad of computationally implicated variants. Studies to define mechanisms of genetic disruption encoded by computationally identified genomic variants require reproducible, adaptable, and inexpensive methods to screen candidate variant and gene function. High-throughput strategies will permit a tiered variant discovery and genetic mechanism approach that begins with rapid functional screening of a large number of computationally implicated variants and genes for discovery of those that merit mechanistic investigation. As such, improved variant-to-gene and gene-to-function screens-and adequate support for such studies-are critical to accelerating the translation of genomic findings. In this White Paper, we outline the variety of novel technologies, assays, and model systems that are making such screens faster, cheaper, and more accurate, referencing published work and ongoing work supported by the National Heart, Lung, and Blood Institute's R21/R33 Functional Assays to Screen Genomic Hits program. We discuss priorities that can accelerate the impressive but incomplete progress represented by big data genomic research.
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http://dx.doi.org/10.1161/CIRCGEN.118.002178DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5901889PMC
April 2018

Characterization and health risk assessment of PM-bound organics inside and outside of Chinese smoking lounges.

Chemosphere 2017 Nov 4;186:438-445. Epub 2017 Aug 4.

Key Lab of Aerosol Chemistry & Physics, Institute of Earth Environment, Chinese Academy of Sciences, Xi'an, China; State Key Lab of Loess and Quaternary Geology (SKLLQG), Institute of Earth Environment, Chinese Academy of Sciences, Xi'an, China. Electronic address:

PM samples were collected at six indoor public places that contained dedicated smoking lounges. Samples were taken in the smoking lounges, at two indoor locations outside of the lounges, and in outdoor air near the venues. Organic carbon (OC), elemental carbon (EC), and non-polar organic compounds including polycyclic aromatic hydrocarbons (PAHs), n-alkanes (n-C to n-C), iso/anteiso-alkanes (C to C), hopanes and phthalate esters (PAEs) were quantified. Average PM levels of 170.2 ± 85.9 μg/m in the lounges exceeded limits of 25 μg/m set by World Health Organization (WHO); these levels were 5.4 and 3.9 times higher than those indoors and outdoors, respectively. High ratios of OC to PM, OC to EC, and PAHs diagnostic ratios in the lounges indicated contributions from environmental tobacco smoke (ETS). The maximum carbon number (C) and carbon preference indices (CPI) for n-alkanes showed ETS transport from the enclosed lounges to nearby indoor non-smoking areas. Iso/anteiso-alkanes in the lounges were 876.5 ng/m, ∼80 times higher than outdoor levels. 17α(H)-21β(H),30-norhopane and 17α(H)-21β(H),(22R)-homohopane were much higher in the lounges than outdoor air, but they cannot be directly attributed to ETS. Estimated carcinogenic risks of PAHs in the lounges exceeded the acceptable level of 10.
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http://dx.doi.org/10.1016/j.chemosphere.2017.08.006DOI Listing
November 2017

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

Chembiochem 2016 Dec 11;17(24):2316-2323. Epub 2016 Nov 11.

Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, 605 Stellar Chance Labs, 422 Curie Blvd, Philadelphia, PA, 19104, USA.

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.
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http://dx.doi.org/10.1002/cbic.201600493DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5163474PMC
December 2016

The Zinc Finger of Prolyl Hydroxylase Domain Protein 2 Is Essential for Efficient Hydroxylation of Hypoxia-Inducible Factor α.

Mol Cell Biol 2016 09 26;36(18):2328-43. Epub 2016 Aug 26.

Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA

Prolyl hydroxylase domain protein 2 (PHD2) (also known as EGLN1) is a key oxygen sensor in mammals that posttranslationally modifies hypoxia-inducible factor α (HIF-α) and targets it for degradation. In addition to its catalytic domain, PHD2 contains an evolutionarily conserved zinc finger domain, which we have previously proposed recruits PHD2 to the HSP90 pathway to promote HIF-α hydroxylation. Here, we provide evidence that this recruitment is critical both in vitro and in vivo We show that in vitro, the zinc finger can function as an autonomous recruitment domain to facilitate interaction with HIF-α. In vivo, ablation of zinc finger function by a C36S/C42S Egln1 knock-in mutation results in upregulation of the erythropoietin gene, erythrocytosis, and augmented hypoxic ventilatory response, all hallmarks of Egln1 loss of function and HIF stabilization. Hence, the zinc finger ordinarily performs a critical positive regulatory function. Intriguingly, the function of this zinc finger is impaired in high-altitude-adapted Tibetans, suggesting that their adaptation to high altitude may, in part, be due to a loss-of-function EGLN1 allele. Thus, these findings have important implications for understanding both the molecular mechanism of the hypoxic response and human adaptation to high altitude.
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http://dx.doi.org/10.1128/MCB.00090-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5007793PMC
September 2016

Case 221: Hereditary Leiomyomatosis and Renal Cell Cancer Syndrome.

Radiology 2015 Sep;276(3):922-7

From the Department of Diagnostic Imaging, The Ottawa Hospital, 1053 Carling Ave, Ottawa, ON, Canada K1Y 4E9.

History A 24-year-old woman presented to the emergency department with a history of acute urinary retention, gross hematuria, and left flank pain for 2 days. Past history was unrevealing. Her last menstrual period occurred 2 weeks prior to presentation. At physical examination, she had a temperature of 38.4°C. A palpable mass was noted in the suprapubic region, and a second mass was palpated in the left upper quadrant. Blood work revealed a hemoglobin level of 4.7 g/dL (normal range, 11.5-15.5 g/dL). Her coagulation profile and white blood cell count were within normal limits. Ultrasonography (US) of the abdomen and pelvis was performed and was followed by contrast material-enhanced (80 mL of iopamidol) computed tomography (CT) of the chest, abdomen, and pelvis. Magnetic resonance (MR) imaging of the abdomen and pelvis also was performed.
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http://dx.doi.org/10.1148/radiol.2015132798DOI Listing
September 2015

Novel Homozygous Mutation of the Internal Translation Initiation Start Site of VHL is Exclusively Associated with Erythrocytosis: Indications for Distinct Functional Roles of von Hippel-Lindau Tumor Suppressor Isoforms.

Hum Mutat 2015 Nov 17;36(11):1039-42. Epub 2015 Aug 17.

Department of Clinical Chemistry and Haematology, University Medical Center Utrecht, Utrecht, The Netherlands.

Congenital secondary erythrocytosis is a rare disorder characterized by increased red blood cell production. An important cause involves defects in the oxygen sensing pathway, in particular the PHD2-VHL-HIF axis. Mutations in VHL are also associated with the von Hippel-Lindau tumor predisposition syndrome. The differences in phenotypic expression of VHL mutations are poorly understood. We report on three patients with erythrocytosis, from two unrelated families. All patients show exceptionally high erythropoietin (EPO) levels, and are homozygous for a novel missense mutation in VHL: c.162G>C p.(Met54Ile). The c.162G>C mutation is the most upstream homozygous VHL mutation described so far in patients with erythrocytosis. It abolishes the internal translational start codon, which directs expression of VHLp19, resulting in the production of only VHLp30. The exceptionally high EPO levels and the absence of VHL-associated tumors in the patients suggest that VHLp19 has a role for regulating EPO levels that VHLp30 does not have, whereas VHLp30 is really the tumor suppressor isoform.
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http://dx.doi.org/10.1002/humu.22846DOI Listing
November 2015

Identification of prolyl hydroxylation modifications in mammalian cell proteins.

Proteomics 2015 Apr 19;15(7):1259-67. Epub 2015 Jan 19.

Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, PA, USA.

Prolyl hydroxylation is a PTM that plays an important role in the formation of collagen fibrils and in the oxygen-dependent regulation of hypoxia inducible factor-α (HIF-α). While this modification has been well characterized in the context of these proteins, it remains unclear to what extent it occurs in the remaining mammalian proteome. We explored this question using MS to analyze cellular extracts subjected to various fractionation strategies. In one strategy, we employed the von Hippel Lindau tumor suppressor protein, which recognizes prolyl hydroxylated HIF-α, as a scaffold for generating hydroxyproline capture reagents. We report novel sites of prolyl hydroxylation within five proteins: FK506-binding protein 10, myosin heavy chain 10, hexokinase 2, pyruvate kinase, and C-1 Tetrahydrofolate synthase. Furthermore, we show that identification of prolyl hydroxylation presents a significant technical challenge owing to widespread isobaric methionine oxidation, and that manual inspection of spectra of modified peptides in this context is critical for validation.
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http://dx.doi.org/10.1002/pmic.201400398DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4438755PMC
April 2015

Human high-altitude adaptation: forward genetics meets the HIF pathway.

Genes Dev 2014 Oct;28(20):2189-204

Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA

Humans have adapted to the chronic hypoxia of high altitude in several locations, and recent genome-wide studies have indicated a genetic basis. In some populations, genetic signatures have been identified in the hypoxia-inducible factor (HIF) pathway, which orchestrates the transcriptional response to hypoxia. In Tibetans, they have been found in the HIF2A (EPAS1) gene, which encodes for HIF-2α, and the prolyl hydroxylase domain protein 2 (PHD2, also known as EGLN1) gene, which encodes for one of its key regulators, PHD2. High-altitude adaptation may be due to multiple genes that act in concert with one another. Unraveling their mechanism of action can offer new therapeutic approaches toward treating common human diseases characterized by chronic hypoxia.
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http://dx.doi.org/10.1101/gad.250167.114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4201282PMC
October 2014

The role of PHD2 mutations in the pathogenesis of erythrocytosis.

Hypoxia (Auckl) 2014 1;2:71-90. Epub 2014 Jul 1.

Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.

The transcription of the erythropoietin () 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 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 (, also known as ), the gene that encodes PHD2. To date, 24 different 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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http://dx.doi.org/10.2147/HP.S54455DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5045058PMC
July 2014

Defective Tibetan PHD2 binding to p23 links high altitude adaption to altered oxygen sensing.

J Biol Chem 2014 May 7;289(21):14656-65. Epub 2014 Apr 7.

From the Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104 and

The Tibetan population has adapted to the chronic hypoxia of high altitude. Tibetans bear a genetic signature in the prolyl hydroxylase domain protein 2 (PHD2/EGLN1) gene, which encodes for the central oxygen sensor of the hypoxia-inducible factor (HIF) pathway. Recent studies have focused attention on two nonsynonymous coding region substitutions, D4E and C127S, both of which are markedly enriched in the Tibetan population. These amino acids reside in a region of PHD2 that harbors a zinc finger, which we have previously discovered binds to a Pro-Xaa-Leu-Glu (PXLE) motif in the HSP90 cochaperone p23, thereby recruiting PHD2 to the HSP90 pathway to facilitate HIF-α hydroxylation. We herein report that the Tibetan PHD2 haplotype (D4E/C127S) strikingly diminishes the interaction of PHD2 with p23, resulting in impaired PHD2 down-regulation of the HIF pathway. The defective binding to p23 depends on both the D4E and C127S substitutions. We also identify a PXLE motif in HSP90 itself that can mediate binding to PHD2 but find that this interaction is maintained with the D4E/C127S PHD2 haplotype. We propose that the Tibetan PHD2 variant is a loss of function (hypomorphic) allele, leading to augmented HIF activation to facilitate adaptation to high altitude.
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http://dx.doi.org/10.1074/jbc.M113.541227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4031521PMC
May 2014

A knock-in mouse model of human PHD2 gene-associated erythrocytosis establishes a haploinsufficiency mechanism.

J Biol Chem 2013 Nov 11;288(47):33571-33584. Epub 2013 Oct 11.

Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104. Electronic address:

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.
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http://dx.doi.org/10.1074/jbc.M113.482364DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3837105PMC
November 2013

Erythrocytosis and pulmonary hypertension in a mouse model of human HIF2A gain of function mutation.

J Biol Chem 2013 Jun 2;288(24):17134-44. Epub 2013 May 2.

Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

The central pathway for oxygen-dependent control of red cell mass is the prolyl hydroxylase domain protein (PHD):hypoxia inducible factor (HIF) pathway. PHD site specifically prolyl hydroxylates the transcription factor HIF-α, thereby targeting the latter for degradation. Under hypoxia, this modification is attenuated, allowing stabilized HIF-α to activate target genes, including that for erythropoietin (EPO). Studies employing genetically modified mice point to Hif-2α, one of two main Hif-α isoforms, as being the critical regulator of Epo in the adult mouse. More recently, erythrocytosis patients with heterozygous point mutations in the HIF2A gene have been identified; whether these mutations were polymorphisms unrelated to the phenotype could not be ruled out. In the present report, we characterize a mouse line bearing a G536W missense mutation in the Hif2a gene that corresponds to the first such human mutation identified (G537W). We obtained mice bearing both heterozygous and homozygous mutations at this locus. We find that these mice display, in a mutation dose-dependent manner, erythrocytosis and pulmonary hypertension with a high degree of penetrance. These findings firmly establish missense mutations in HIF-2α as a cause of erythrocytosis, highlight the importance of this HIF-α isoform in erythropoiesis, and point to physiologic consequences of HIF-2α dysregulation.
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http://dx.doi.org/10.1074/jbc.M112.444059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3682519PMC
June 2013

Prolyl hydroxylase domain protein 2 (PHD2) binds a Pro-Xaa-Leu-Glu motif, linking it to the heat shock protein 90 pathway.

J Biol Chem 2013 Apr 14;288(14):9662-9674. Epub 2013 Feb 14.

Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104. Electronic address:

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.
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http://dx.doi.org/10.1074/jbc.M112.440552DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3617269PMC
April 2013

[Application of synchronous fluorescence in identification of spilled oil at sea].

Guang Pu Xue Yu Guang Pu Fen Xi 2011 Jan;31(1):154-7

Research Center of Marine Ecology, The First Institute of Oceanography, State Ocean Administration , and Qingdao Key Lab of Analytical Technology Development and Standardization of Chinese Medicine, Qingdao 266061, China.

In order to screen and identify the source of spilled oils at sea, synchronous fluorescence scans combined with clustering analysis are proposed and applied to different crude oil and weathering crude oil. SFS data of deltal = 25 nm were recorded and dealt with clustering analysis. The cluster results of SFS data in the range of 300 - 500 nm show that the crude oil and the weathering oil could separate completely. And the crude oils from different sea areas, also collected at different time, clustered into different groups, respectively. The results indicate that this method could preliminarily selected, and maybe serves as an assistant method in oil spill identification.
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January 2011

Mouse knock-out of IOP1 protein reveals its essential role in mammalian cytosolic iron-sulfur protein biogenesis.

J Biol Chem 2011 May 2;286(18):15797-805. Epub 2011 Mar 2.

Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.

Iron-sulfur proteins play an essential role in a variety of biologic processes and exist in multiple cellular compartments. The biogenesis of these proteins has been the subject of extensive investigation, and particular focus has been placed on the pathways that assemble iron-sulfur clusters in the different cellular compartments. Iron-only hydrogenase-like protein 1 (IOP1; also known as nuclear prelamin A recognition factor like protein, or NARFL) is a human protein that is homologous to Nar1, a protein in Saccharomyces cerevisiae that, in turn, is an essential component of the cytosolic iron-sulfur protein assembly pathway in yeast. Previous siRNA-induced knockdown studies using mammalian cells point to a similar role for IOP1 in mammals. In the present studies, we pursued this further by knocking out Iop1 in Mus musculus. We find that Iop1 knock-out results in embryonic lethality before embryonic day 10.5. Acute, inducible global knock-out of Iop1 in adult mice results in lethality and significantly diminished activity of cytosolic aconitase, an iron-sulfur protein, in liver extracts. Inducible knock-out of Iop1 in mouse embryonic fibroblasts results in diminished activity of cytosolic but not mitochondrial aconitase and loss of cell viability. Therefore, just as with knock-out of Nar1 in yeast, we find that knock-out of Iop1/Narfl in mice results in lethality and defective cytosolic iron-sulfur cluster assembly. The findings demonstrate an essential role for IOP1 in this pathway.
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http://dx.doi.org/10.1074/jbc.M110.201731DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3091189PMC
May 2011

Respiratory health and lung function in Chinese restaurant kitchen workers.

Occup Environ Med 2011 Oct 5;68(10):746-52. Epub 2011 Feb 5.

School of Public Health and Primary Care, The Chinese University of Hong Kong, Hong Kong, China.

Objectives: To measure air pollutant concentrations in Chinese restaurant kitchens using different stove types and assess their influence on workers' respiratory health.

Methods: 393 kitchen workers from 53 Chinese restaurants were surveyed over 16 months: 115 workers from 21 restaurants using only electric stoves and 278 workers from 32 restaurants using only gas stoves. Workers were interviewed about their respiratory symptoms and had their lung function tested. Concentrations of nitric oxide (NO), nitrogen dioxide (NO(2)), carbon monoxide (CO), carbon dioxide (CO(2)), methane (CH(4)), non-methane hydrocarbons (NMHC), total volatile organic compounds (TVOC) and fine particulate matter (PM(2.5)) were measured using portable monitors and air-bag sampling. Temperature and noise levels were assessed.

Results: Median concentrations of NO, NO(2) and CO were 7.4, 1.5 and 1.6 times higher in gas-fuelled kitchens than in electric ones and average concentrations of PM(2.5) and TVOC were 81% and 78% higher, respectively. Differences were smaller for CH(4) and NMHC. Electricity-run kitchens were 4.5°C cooler and 9 dBA less noisy than gas-fuelled ones. Workers using electric cookers had significantly better lung function than their gas-using counterparts and their mean FEV(1) and FVC values were 5.4% and 3.8% higher, respectively, after adjustment for confounders. Wheeze, phlegm, cough and sore throat were more prevalent in workers using gas. The adjusted OR for having phlegm regularly was significantly higher.

Conclusions: The poorer lung function and higher prevalence of respiratory symptoms among workers in gas-fuelled kitchens compared to those in electricity-powered kitchens may be associated with exposure to higher concentrations of toxic air pollutants generated during gas cooking.
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http://dx.doi.org/10.1136/oem.2010.059378DOI Listing
October 2011

The HIF pathway and erythrocytosis.

Annu Rev Pathol 2011 ;6:165-92

Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, 19104, USA.

Because of the central role that red blood cells play in the delivery of oxygen to tissues of the body, red blood cell mass must be controlled at precise levels. The glycoprotein hormone erythropoietin (EPO) regulates red blood cell mass. EPO transcription, in turn, is regulated by a distinctive oxygen-sensing mechanism. In this pathway, prolyl hydroxylase domain protein (PHD) site-specifically hydroxylates the α-subunit of the transcription factor hypoxia-inducible factor α (HIF-α), thereby targeting the latter for degradation by the von Hippel-Lindau tumor-suppressor protein (VHL). Under hypoxic conditions, this posttranslational modification of HIF-α is inhibited, which stabilizes it and promotes the transcriptional activation of genes, including that for EPO. Rare patients with erythrocytosis have mutations in the genes encoding for PHD2, HIF-2α, and VHL, which implicates these proteins as critical to the proper control of red blood cell mass in humans.
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http://dx.doi.org/10.1146/annurev-pathol-011110-130321DOI Listing
April 2011
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