Notch Downregulation andExtramedullary Erythrocytosis inHypoxia-Inducible Factor Prolyl4-Hydroxylase 2-Deficient Mice

Mikko N. M. Myllymäki,a Jenni Määttä,a Elitsa Y. Dimova,a Valerio Izzi,a

Timo Väisänen,b Johanna Myllyharju,a Peppi Koivunen,a Raisa Serpia

Biocenter Oulu, Faculty of Biochemistry and Molecular Medicine, Oulu Center for Cell-Matrix Research,University of Oulu, Oulu, Finlanda; Department of Pathology, Oulu University Hospital, and Department ofPathology, Cancer and Translational Medicine Research Unit, University of Oulu, Oulu, Finlandb

ABSTRACT Erythrocytosis is driven mainly by erythropoietin, which is regulated byhypoxia-inducible factor (HIF). Mutations in HIF prolyl 4-hydroxylase 2 (HIF-P4H-2)(PHD2/EGLN1), the major downregulator of HIF� subunits, are found in familiar erythro-cytosis, and large-spectrum conditional inactivation of HIF-P4H-2 in mice leads to severeerythrocytosis. Although bone marrow is the primary site for erythropoiesis, spleen re-mains capable of extramedullary erythropoiesis. We studied HIF-P4H-2-deficient (Hif-p4h-2gt/gt) mice, which show slightly induced erythropoiesis upon aging despite non-increased erythropoietin levels, and identified spleen as the site of extramedullaryerythropoiesis. Splenic hematopoietic stem cells (HSCs) of these mice exhibited in-creased erythroid burst-forming unit (BFU-E) growth, and the mice were pro-tected against anemia. HIF-1� and HIF-2� were stabilized in the spleens, whilethe Notch ligand genes Jag1, Jag2, and Dll1 and target Hes1 became downregu-lated upon aging HIF-2� dependently. Inhibition of Notch signaling in wild-typespleen HSCs phenocopied the increased BFU-E growth. HIF� stabilization can thus me-diate non-erythropoietin-driven splenic erythropoiesis via altered Notch signaling.

KEYWORDS erythrocytosis, hypoxia, hypoxia-inducible factor prolyl-4-hydroxylase 2,spleen

Erythrocytosis, defined as an absolute increase in red cell mass, is associated withincreased hematocrit and hemoglobin values. Erythropoietin (EPO) stimulates

erythropoiesis by binding to the EPO receptor (EPOR) on hematopoietic progenitors topromote erythroid differentiation, survival, and proliferation (1, 2). Classically, erythro-cytosis is classified as either primary, caused by intrinsic defects in erythroid progenitorcells in the presence of normal or low serum EPO levels, or secondary. The causes ofprimary erythrocytosis include mutations in the Janus kinase 2 (JAK2) and EPOR genes,which can lead to EPO-independent proliferation of erythroid precursors or hypersen-sitivity to EPO (3). Secondary erythrocytosis is due to defects in the oxygen-sensingpathway, including mutations in the genes for hypoxia-inducible factor (HIF) prolyl4-hydroxylase 2 (HIF-P4H-2/PHD2/EGLN1), HIF-2�, and von Hippel Lindau (VHL) protein,and impaired oxygen delivery or tissue hypoxia, all associated with the activation of theEPO pathway and elevated serum EPO levels (3–5).

During human development, the bone marrow becomes a functional site forhematopoiesis in the fetus at 4 to 5 months, whereas in mice, bone marrow hemato-poiesis becomes active after birth (6). To supply oxygen for the needs of the developingfetus, nonmarrow tissues, such as the spleen and liver, serve as sites of extramedullary

Received 27 September 2016 Returned formodification 18 October 2016 Accepted 27October 2016

Accepted manuscript posted online 7November 2016

Citation Myllymäki MNM, Määttä J, Dimova EY,Izzi V, Väisänen T, Myllyharju J, Koivunen P,Serpi R. 2017. Notch downregulation andextramedullary erythrocytosis in hypoxia-inducible factor prolyl 4-hydroxylase 2-deficient mice. Mol Cell Biol 37:e00529-16.https://doi.org/10.1128/MCB.00529-16.

Copyright © 2017 American Society forMicrobiology. All Rights Reserved.

Address correspondence to Raisa Serpi,raisa.serpi@oulu.fi.

P.K. and R.S. contributed equally to this work.

RESEARCH ARTICLE

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hematopoiesis (6). In the case of mice, the spleen remains a hematopoietic organthroughout their lives albeit at low levels (7).

HIF regulates the expression of a large number of genes, many of which are involvedin erythropoiesis (8–10). HIF-P4H-1 to -3 are central regulators of the hypoxia response(8–10). They hydroxylate one or two prolyl residues in the HIF� subunit in the presenceof oxygen, leading to the subsequent binding of the VHL protein, polyubiquitinylation,and rapid proteasomal degradation of HIF�. Under hypoxia, they are inactivated so thatHIF� accumulates and binds to HIF�, and the HIF�� dimer then transcriptionallyactivates HIF target genes (8).

HIF-P4H-2 is the major regulator of the stability of HIF� subunits. Knockout of Hif-p4h-2results in embryonic lethality (11), while large-spectrum conditional inactivation ofHif-p4h-2 after birth leads to severe erythrocytosis associated with splenomegaly (12,13). Hif-p4h-1 or Hif-p4h-3 knockout mice do not display erythrocytosis (3). A mouse linewith a conditional Hif-p4h-2 deficiency in CD68� macrophages and in the hematopoi-etic system displays excessive erythrocytosis, driven exclusively by HIF-2� (14). Usingthe gene trap (gt) strategy, we have generated a mouse line with partial Hif-p4h-2inactivation (Hif-p4h-2gt/gt) that expresses decreased amounts of wild-type Hif-p4h-2mRNA in tissues and shows stabilization of the HIF� subunits (15). These mice areprotected against cardiac and skeletal muscle ischemia (15, 16), metabolic syndrome,and atherosclerosis (17, 18) due to the activation of the hypoxia response pathway.Hif-p4h-2gt/gt mice did not develop erythrocytosis when studied up to 5 months age(15).

Clinical trials with small-molecule compounds that inhibit HIF-P4Hs, stabilize HIF�,and induce EPO for the treatment of anemias of chronic kidney diseases are ongoing(10, 19, 20). We have shown previously that administration of a small-molecule HIF-P4Hinhibitor to Hif-p4h-2gt/gt mice increased their hemoglobin and hematocrit values moremarkedly than in their wild-type littermates (21), and we show here that splenic HIF�

stabilization due to partial genetic Hif-p4h-2 inactivation is associated with the inhibi-tion of Notch signaling and age-dependent erythropoiesis in the spleen and protectionagainst inflammation-induced anemia.

RESULTSErythrocytosis in aged Hif-p4h-2 hypomorphic mice. Where our previous studies

with young adult (2- to 5-month-old) Hif-p4h-2 hypomorphic (Hif-p4h-2gt/gt) micerevealed no erythrocytosis (15, 21), in the present study, we found �20% higher levelsof red blood cells (RBCs) and hemoglobin and hematocrit values in 1-year-old femaleHif-p4h-2gt/gt mice than in their wild-type littermates (Fig. 1A). There were no differ-ences in the levels of white blood cells, platelets, or plasma total iron (Fig. 1B). In viewof the observed differences in erythrocytosis between young (15, 21) and old Hif-p4h-2gt/gt mice, we then monitored the hematocrit values for a group of male mice frombirth to 1 year of age and observed that Hif-p4h-2gt/gt mice had a mild increase inhematocrit values relative to those for wild-type mice from 6 months onwards (Fig. 1C).Both genders thus expressed age-dependent erythrocytosis (Fig. 1A and C), the mostobvious mechanism of which would be elevated EPO levels. However, no significantdifference in renal Epo mRNA levels was seen between genotypes (100% � 39% inwild-type versus 214% � 61% in Hif-p4h-2gt/gt mice [n � 8 for both genotypes]; P �

0.14), and no detectable levels of Epo mRNA were found in the spleens or livers of eitherHif-p4h-2gt/gt or wild-type mice. Serum EPO levels in 5-month-old and 12-month-oldanimals showed a small but statistically nonsignificant, and apparently not physiolog-ically relevant, upregulation in Hif-p4h-2gt/gt mice (Fig. 1D). The logarithmic transformsof serum EPO levels in Hif-p4h-2gt/gt mice, when plotted against their hematocrit values,lay above the trend line for the wild type in the majority of cases (Fig. 1E). We thereforecannot exclude the possibility that the Hif-p4h-2gt/gt mice were slightly hypersensitiveto EPO.

Induced erythroid and megakaryocytic hyperplasia in the spleen but not in thebone marrow of Hif-p4h-2 hypomorphic mice. To study the origin of erythrocytosis

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in Hif-p4h-2gt/gt mice, we first performed a histological analysis of bone marrow samplesbut saw no differences in either morphology or cell composition between Hif-p4h-2gt/gt

and wild-type mice (Fig. 2A) or the numbers of proerythroblasts and erythrocytes orgranulocytes when analyzed by fluorescence-activated cell sorter (FACS) analysis (Fig.2B). Hif-p4h-2gt/gt mice of both genders had increased spleen weights compared tothose of wild-type mice at 12 months, but no difference in liver weight was observed(Fig. 2C). Also, the architecture of Hif-p4h-2gt/gt mouse spleens was distorted, withincreased erythroid hyperplasia (Fig. 2D) and higher numbers of megakaryocytes (Fig.2E) and CD41� cells (Fig. 2F). FACS analyses indicated that the numbers of Ter119�

proerythroblasts and erythrocytes were significantly increased in the spleens of Hif-p4h-2gt/gt mice (Fig. 2G) (P � 0.001), whereas the numbers of granulocytes weredownregulated (Fig. 2G) (P � 0.001), indicating a shift toward higher levels of eryth-ropoiesis and lower levels of myelopoiesis. Further FACS analyses were performed toevaluate the proportion of proerythroid, immature erythroid, or mature erythroid cellsin the spleen. Hif-p4h-2gt/gt mice had a higher number of splenic CD71� Ter119�

immature erythroid cells (P � 0.01), and the number of mature CD71� Ter119�

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FIG 1 Hif-p4h-2gt/gt mice show age-dependent erythrocytosis. (A and B) Hematologic parameters for1-year-old female wild-type (wt) and Hif-p4h-2gt/gt (gt/gt) mice. Red blood cell (RBC), hemoglobin (Hb),hematocrit (Hct), white blood cell (WBC), platelet, and plasma iron (p-total iron) levels were measured(n � 7 [wild type] and n � 13 [gt/gt], except for plasma iron [n � 8 {wild type} and n � 5 {gt/gt}]). (C)Hematocrit levels for a group of male mice measured for a 1-year follow-up period (n � 12 [wild type]and n � 5 [gt/gt]). (D) Serum EPO levels of 5- and 12-month-old Hif-p4h-2gt/gt mice and wild-typelittermates (n � 8 [wild type] and n � 7 [gt/gt] at 5 months and n � 13 [wild type] and n � 12 [gt/gt]at 12 months). (E) Correlation between log serum EPO levels and hematocrit values in 5- and 12-month-old Hif-p4h-2gt/gt and wild-type mice (n � 8 [wild type] and n � 7 [gt/gt] at 5 months and n � 7 [wildtype] and n � 7 [gt/gt] at 12 months). Student’s t test was used for comparisons between two groups.*, P � 0.05; **, P � 0.01; ***, P � 0.001. Error bars represent SEM.

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FIG 2 Extramedullary erythropoiesis in spleens of Hif-p4h-2gt/gt mice. (A) Bone marrow histology of Hif-p4h-2gt/gt (gt/gt) and wild-type (wt) mice studied by using toluidine blue (2-month-old mice) (left) andLeder (6-month-old mice) (right) staining. Representative figures are shown. Bars, 200 �m (left) and 50�m (right). (B) Representative flow cytometric dot plots of red blood cells and their precursors (Precs)(Ter119) versus granulocytes (Gr-1) in bone marrow and quantification of the number of these cells in4-month-old wild-type and Hif-p4h-2gt/gt mice. (C) Spleen and liver weights for 12-month-old females (f)and males (m) and representative pictures of spleens from 10-month-old wild-type and Hif-p4h-2gt/gt

male mice (for spleen weights, n � 17 [wild type] and n � 23 [gt/gt] for females and n � 6 [wild type]and n � 7 [gt/gt] for males); (for liver weights, n � 9 [wild type] and n � 14 [gt/gt] for females and n �7 [wild type] and n � 8 [gt/gt] for males). (D) Hematoxylin-eosin staining of 12-month-old wild-type andHif-p4h-2gt/gt spleens. Bars, 500 �m (left) and 200 �m (right). (E) Numbers of megakaryocytes in spleensof 12-month-old mice per high-power field (HPF) (n � 6 [wild type] and n � 10 [gt/gt]). (F) Percentageof CD41-positive cells in 12-month-old wild-type and Hif-p4h-2gt/gt spleens (n � 16 [wild type] and n �22 [gt/gt]). (G) Representative flow cytometric dot plots of red blood cells and their precursors (Precs)(Ter119) versus granulocytes (Gr-1) in spleen and quantification of the number of these cells in spleens

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erythrocytes was significantly increased relative to the numbers in wild-type mice (Fig.3A) (P � 0.05). This observation was further supported by immunohistological stainingwith an anti-CD71 antibody, which pointed to an increased number of CD71-positivecells in Hif-p4h-2gt/gt spleens (Fig. 3B).

To ascertain the reason for the increased numbers of erythroid precursors inHif-p4h-2gt/gt mouse spleens, we evaluated the rates of cell death and proliferation. Thenumbers of terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end label-ing (TUNEL)-positive apoptotic cells in whole spleens of Hif-p4h-2gt/gt mice revealed aslight, although statistically nonsignificant, decrease relative to the numbers in thespleens of wild-type littermates (Fig. 3C). FACS analysis of the death of Ter119�

proerythroblasts by means of 7-aminoactinomycin D (7-AAD) dye showed this to bereduced in Hif-p4h-2gt/gt spleens (Fig. 3D) (P � 0.01), but there was no indication ofincreased EPOR-mediated antiapoptotic signaling, as evidenced by the absence ofa difference in the mRNA levels of the EpoR, Suppressor of cytokine signaling 3(Socs3), B-cell lymphoma 2 (Bcl-2), B-cell lymphoma-extra large (Bcl-xl), Fas receptor(Fas), Fas ligand (FasL), or Serine protease inhibitor 2A (Spi2A) gene and the phos-phorylation of the JAK2 protein (Fig. 3E). The extent of splenic cell proliferation, asanalyzed in terms of the Ki67 immunohistochemistry of the whole organ, did notdiffer between genotypes (Fig. 3F).

Hif-p4h-2gt/gt spleens show stabilization of HIF-1� and HIF-2� and alteredNotch signaling. Western blotting indicated normoxic stabilization of the HIF-1� andHIF-2� proteins in the spleens of Hif-p4h-2gt/gt mice but not in wild-type mice (Fig. 4A).Hif-p4h-2gt/gt mice express reduced levels of wild-type Hif-p4h-2 mRNA in their tissues.This level was 29% of that in the spleens of wild-type mice (P � 0.001) at 5 months and34% of that (P � 0.001) at 12 months (Fig. 4B). There were no significant changes in theexpression levels of Hif-p4h-1 or Hif-p4h-3 mRNAs. Of the established glycolytic HIF1target genes analyzed, only Glyceraldehyde phosphate dehydrogenase (Gapdh) mRNAwas slightly but nonsignificantly upregulated (120%) in the spleens of Hif-p4h-2gt/gt

mice at both ages compared to the levels in wild-type mice (Fig. 4B), whereas the mRNAlevels of the Glucose transporter 1 (Glut1), Phosphofructokinase, liver type (Pfkl), Phos-phoglycerate kinase 1 (Pgk1), Lactate dehydrogenase A (Ldha), and Pyruvate dehydroge-nase kinase 4 (Pdk4) genes were not altered. The expression of mRNA for C-X-C motifchemokine 12 (Cxcl12) was downregulated in the spleens of Hif-p4h-2gt/gt mice, being78% of the level in wild-type mice in the 5-month-old mice and 54% of that in the12-month-old mice (Fig. 4B). Also, Cd34 mRNA was downregulated to 76% in the5-month-old Hif-p4h-2gt/gt mice and to 40% in the 12-month-old ones (P � 0.01) (Fig.4B). Interestingly, the expression levels of the mRNAs for the Notch ligands Jagged 1(Jag1) (58%; P � 0.01), Jagged 2 (Jag2) (48%; P � 0.24), and Delta-like ligand 1 (Dll1)(67%; P � 0.08) were downregulated in 12-month-old Hif-p4h-2gt/gt mice but not in5-month-old mice relative to wild-type mice (Fig. 4B). Immunohistological staining ofsplenic Jag1 supported the observation of its downregulation in Hif-p4h-2gt/gt mice (Fig.4C). No differences in Notch1 and Notch4 mRNA levels were seen, but a downregulationof Notch2 mRNA was found in 5-month-old Hif-p4h-2gt/gt mice (Fig. 4B). Of the estab-lished Notch target genes, the expression levels of the mRNA for Hairy and enhancer ofsplit 1 (Hes1) were downregulated to 74% in the 5-month-old mice (P � 0.05) and to72% in the 12-month-old ones (P � 0.05) (Fig. 4B). This downregulation was also seenfor the protein levels in 12-month-old mice (Fig. 4D).

To find out whether these changes in mRNA levels were specific for the spleen, weanalyzed the levels of mRNAs for Hif-p4hs, established HIF1 target genes, Notch ligands,and Hes1 in the bone marrow of 12-month-old Hif-p4h-2gt/gt mice relative to wild-type

FIG 2 Legend (Continued)of 4-month-old wild-type and Hif-p4h-2gt/gt mice. Analysis of variance was used for flow cytometrycomparisons between groups, Student’s t test was used for comparisons between the two genotypes. Inpanels A and D, a Leica DM LB2 microscope and a Leica DFC 320 camera were used. *, P � 0.05; **, P �0.01; ***, P � 0.001. Error bars represent SEM.

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FIG 3 Hif-p4h-2gt/gt mice have increased numbers of erythroid precursors in their spleens. (A) Represen-tative flow cytometric dot plots of erythroid precursors (CD71) versus proerythroblasts and erythrocytes(Ter119) and quantification of the numbers of immature erythroid precursors (Imm-E) (CD71� Ter119�)and mature erythrocytes (Mat-E) (CD71� Ter119�) in spleens of wild-type (wt) and Hif-p4h-2gt/gt (gt/gt)mice (n � 5 for both genotypes). (B) Immunofluorescence staining for CD71 in spleen. Bar, 50 �m. (C)Representative images of TUNEL staining in spleen and analyses of numbers of TUNEL-positive cells perhigh-power field (HPF) (n � 6 [wild type] and n � 7 [gt/gt]). Bar, 50 �m. Student’s t test was used forcomparisons between two groups. (D) Percentages of cell death measured in terms of 7-aminoactinomycin

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mice. Hif-p4h-2 mRNA was downregulated in Hif-p4h-2gt/gt bone marrow to 33% of thatin wild-type mice (Fig. 4E), and the HIF1 target genes Phosphofructokinase, liver type(Pfkl), Pyruvate dehydrogenase kinase 1 (Pdk1), and Glut1 showed a tendency forupregulation, indicating putative HIF1 stabilization in the bone marrow. The mRNAlevels of the Notch ligands Jag1, Jag2, and Dll1 as well as a Notch target gene, Hes1,were not altered in Hif-p4h-2gt/gt bone marrow (Fig. 4E).

Since there was no induction of the established HIF1 target genes in the spleens ofHif-p4h-2gt/gt mice despite HIF-1� stabilization, we silenced HIF-2� in primary spleno-cytes extracted from Hif-p4h-2gt/gt mice with small interfering RNA (siRNA). This led toa 13-fold induction of Jag1 mRNA compared to that in scrambled siRNA-transfectedcells (P � 0.01) (Fig. 5A). Also, the expression of Jag2 was slightly upregulated (P �

0.05), and that of Dll1 was upregulated 6-fold (P � 0.01), suggesting that the down-regulation of the Notch ligands in Hif-p4h-2gt/gt spleens was HIF-2� dependent.

When an erythroid burst-forming unit (BFU-E) colony assay was performed, hema-topoietic stem cells (HSCs) of Hif-p4h-2gt/gt spleens formed significantly more BFU-Ewithout EPO relative to the wild type, and this difference persisted, but did not increase,following supplementation with EPO (Fig. 5B). There was also induced growth of CFUgranulocyte-erythroid-macrophage-megakaryocyte (CFU-GEMM) colonies in the HSCsof Hif-p4h-2gt/gt spleens in the presence and absence of exogenous EPO, whereas nosignificant difference was seen in the numbers of CFU granulocyte-macrophage (CFU-GM) colonies (Fig. 5B), supporting the notion of a shift toward the erythroid lineage.When Notch signaling was inhibited in wild-type spleen HSCs with a gamma-secretaseinhibitor (GSI), in the presence of EPO, wild-type HSCs formed BFU-Es to an extentsimilar to that for Hif-p4h-2gt/gt spleen HSCs (Fig. 5C) (P � 0.05 for wild-type BFU-Es withGSI and EPO versus the control and EPO), indicating that, indeed, diminished Notchsignaling is an important regulator of induced splenic erythropoiesis. No differences inthe numbers of BFU-Es from bone marrow were detected between genotypes eitherwithout or with EPO (Fig. 5D).

Hif-p4h-2 hypomorphic mice are protected against inflammation-induced ane-mia. To determine whether splenic extramedullary hematopoiesis was protectiveagainst anemia, we subjected Hif-p4h-2gt/gt and wild-type mice to two inflammation-induced anemia models. First, 3-month-old mice, which had no difference in erythroidcell parameters between the genotypes, were treated with lipopolysaccharide (LPS) andzymosan A to cause acute anemia, whereupon the levels of RBCs and hemoglobin andhematocrit values declined significantly from the baseline in wild-type mice after 1week, whereas they remained steady in Hif-p4h-2gt/gt mice (Fig. 6A). After 2 weeks, thewild-type mice developed more severe anemia, as manifested by a greater decrease inthe RBC levels (P � 0.01) and hemoglobin (P � 0.05) and hematocrit (P � 0.01) valuesthan for Hif-p4h-2gt/gt mice (Fig. 6A). Supporting the hypothesis that the protectiveeffect against the development of anemia may originate from the spleen, the numbersof spleen megakaryocytes were significantly higher in Hif-p4h-2gt/gt mice than inwild-type mice after the 2-week follow-up (Fig. 6B) (P � 0.05). No difference was seenbetween genotypes in the levels of serum EPO (Fig. 6C) or plasma total iron (Fig. 6D).

In the second model, 3- to 4-month-old mice were injected with peptidoglycan-polysaccharide polymer (PGPS), which causes a more chronic type of anemia. Thedifference in erythroid cell parameters between genotypes became significant after 3

FIG 3 Legend (Continued)(7-AAD) positivity in Ter119� cells by flow cytometry (n � 6 for both genotypes). Analysis of variance wasused for comparisons between groups. (E) Expression of Erythropoietin receptor (EpoR), Suppressor ofcytokine signaling 3 (Socs3), B-cell lymphoma 2 (Bcl-2), B-cell lymphoma-extra large (Bcl-xl), Fas receptor(Fas), Fas ligand (FasL), and Serine protease inhibitor 2A (Spi2A) mRNAs in spleens of the Hif-p4h-2gt/gt micepresented as percentages of values for the wild type (n � 6 [wild type] and n � 7 [gt/gt]). Student’s ttest was used for comparison between the genotypes. Representative Western blots for phosphorylatedJAK2. �-Actin was used as a loading control. (F) Spleens were stained with Ki67 to determine theproliferation rate and graded as either Ki67 positive or negative. Error bars represent SEM. *, P � 0.05;**, P � 0.01.

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FIG 4 Stabilization of HIF-1� and HIF-2� and altered expression of Notch ligands in spleens of Hif-p4h-2gt/gt mice. (A) Representative Western blots for HIF-1� and HIF-2� in spleens of Hif-p4h-2gt/gt

(gt/gt) and wild-type (wt) mice. (B) Expression of Hypoxia-inducible factor prolyl 4-hydroxylase 1(Hif-p4h-1), Hif-p4h-2, Hif-p4h-3, Glyceraldehyde phosphate dehydrogenase (Gapdh), C-X-C motifchemokine 12 (Cxcl12), Cd34, Jagged 1 (Jag1), Jag2, Delta-like ligand 1 (Dll1), Notch1, Notch2, Notch4,and Hairy and enhancer of split 1 (Hes1) mRNAs in spleens of Hif-p4h-2gt/gt mice presented aspercentages of values for the wild type (n � 5 to 8 [wild type] and n � 6 to 8 [gt/gt]). (C)Immunofluorescence staining for Jag1. Bar, 50 �m. (D) Representative Western blot for Hes1 inspleens of 1-year-old Hif-p4h-2gt/gt (gt/gt) and wild-type mice. Vinculin was used as a loading control.

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weeks of the 6-week follow-up period, when the levels of RBCs and hemoglobin andhematocrit values were all lower in wild-type than in Hif-p4h-2gt/gt mice (Fig. 6E) (P �

0.01, P � 0.001, and P � 0.05, respectively). FACS analysis of the spleens after 6 weeksshowed significantly higher numbers of CD71� proerythroblasts and erythrocytes anda lower rate of cell death among Hif-p4h-2gt/gt mice than among wild-type mice (Fig. 6F)(P � 0.01), suggesting protection against anemia by splenic extramedullary erythro-poiesis in the former. Again, no difference in the plasma iron level was seen (Fig. 6G).

DISCUSSION

Members of the Notch family play critical roles in cell fate determination and in themaintenance of progenitor cells in many developmental systems (22–24). Notch pro-teins function both as cell surface receptors and as transcriptional regulators (23,25–27). Activation of Notch signaling requires cell-cell contact between Notch ligand-and receptor-presenting cells, leading to a receptor-ligand interaction that results in therelease of the Notch intracellular domain (NICD) (28). Four receptors (Notch1 to -4) (29)and five ligands (Dll1, -3, and -4 [30–32] and Jag1 and -2 [33–35]) have been describedto date. The Notch receptors are found on primitive hematopoietic precursors, whileligand expression has been found on the surface of stromal cells, suggesting a role forNotch signaling in mammalian hematopoietic development (36). Notch signaling en-hances the survival of primitive multipotent precursors (37–39), and a role in earlyerythropoiesis has been suggested (36).

Given that HSCs and multipotent progenitors must continuously undergo lineagecommitment, differentiation, and proliferation while also maintaining a pool of uncom-mitted progenitors, Notch, as a regulator of cell fate determination, limits the numberof committed cells. Induced Jag1-Notch1 signaling promotes the maintenance andexpansion of hematopoietic progenitors (37, 38), whereas inhibition of Notch signalingleads to a higher rate of differentiation and thus the inhibition of HSC maintenance ina native bone marrow microenvironment (40). Also, the chemokine CXCL12 plays a rolein the maintenance of HSCs in the bone marrow and in the splenic perisinusoidal nichefor extramedullary hematopoiesis (41). We show here that downregulation of Jag1 andCxcl12 mRNAs in the spleens of Hif-p4h-2gt/gt mice was associated with an increasednumber of CD71� Ter119� immature erythroid cells and reduced death of Ter119�

cells, leading to increased extramedullary erythropoiesis, possibly via induced HSCdifferentiation. Both the downregulation of Notch signaling and the induction oferythropoiesis in Hif-p4h-2gt/gt mice were age dependent, suggesting a potential causallink between these two phenomena.

Recent studies have demonstrated molecular connections between HIF and theNotch signaling pathways. HIF-1� has been shown to interact with the NICD, leading toincreased expression of Notch target genes under hypoxia (42), whereas HIF-2� candirectly bind the RBP-J-associated module (RAM) domain of the NICD (43) and repressNotch signaling (44). Results obtained by using glioma stem cells suggest that HIF-1�

and HIF-2� bind competitively to the NICD in the same domain (44). Those authorsthen proposed a model involving the differential regulation of NICD transactivationactivity depending on oxygen levels, where HIF-2� stabilization represses Notch sig-naling in mild hypoxia, whereas HIF-1� competes with HIF-2� for NICD binding insevere hypoxia, thus leading to the upregulation of Notch signaling (44). Moreover,acute global deletion of Hif-p4h-2 led to increased spleen weight and extramedullaryhematopoiesis, which were restored by concurrent HIF-2� deletion (45). These data (44,45) are in line with our in vivo results with regard to the stabilization of splenic HIF-2�

regulating extramedullary hematopoiesis and the concomitant downregulation of

FIG 4 Legend (Continued)(E) Expression of Hif-p4h-1, Hif-p4h-2, Hif-p4h-3, Phosphofructokinase, liver type (Pfkl), Gapdh, Lactatedehydrogenase A (Ldha), Pyruvate dehydrogenase kinase 1 (Pdk1), Glucose transporter 1 (Glut1), Eporeceptor (EpoR), Jag1, Jag2, Dll1, and Hes1 mRNAs in bone marrow of Hif-p4h-2gt/gt mice presentedas percentages of values for the wild type (n � 8 [wild type] and n � 8 [gt/gt]).

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Notch signaling. Also, when we silenced Hif2a in primary splenocytes of Hif-p4h-2gt/gt

mice by means of siRNA, we observed increased expression levels of the Jag1, Jag2, andDll1 mRNAs in comparison with the levels in scramble-treated control cells, suggestingthat HIF-2� is indeed responsible for the downregulation of the expression of theseNotch ligands in Hif-p4h-2gt/gt mouse spleens.

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FIG 5 Increased number of BFU-E colonies from spleen HSCs of Hif-p4h-2gt/gt mice and from spleen HSCsof wild-type mice treated with GSI. (A) Expression of Hif1a (hypoxia-inducible factor 1�), Hif2a (hypoxia-inducible factor 2�), Jagged 1 (Jag1), Jag2, and Delta-like ligand 1 (Dll1) mRNAs in primary splenocytes ofHif-p4h-2gt/gt mice treated with siRNA against Hif-2�, presented as percentages of expression levels inscrambled siRNA-treated cells (n � 2 for both siRNAs). (B) Average numbers of erythroid burst-formingunit (BFU-E), CFU granulocyte-erythroid-macrophage-megakaryocyte (CFU-GEMM), and CFU granulocyte-macrophage (CFU-GM) colonies grown from spleen HSCs of Hif-p4h-2gt/gt and wild-type mice in thepresence or absence of 3 U/ml EPO (n � 3 [wild type] and n � 5 [gt/gt]). Student’s t test was used forcomparisons between two groups. (C) Average numbers of BFU-E colonies grown from spleens ofHif-p4h-2gt/gt and wild-type mice treated with either DMSO (control) or 8 �M gamma-secretase inhibitor(GSI) in the presence or absence of 3 U/ml EPO (n � 3 [wild type] and n � 4 [gt/gt]). Student’s t test wasused for comparisons between two groups. (D) Numbers of BFU-E colonies grown from bone marrowHSCs with no EPO and 30 and 300 mU/ml of EPO (n � 3 for both genotypes). Mice were 6 months ofage. *, P � 0.05; **, P � 0.01. Error bars represent SEM.

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HIF-P4Hs are known to control red cell mass, with HIF-P4H-2 being implicated as thekey isoform in this respect (45). Traditionally, erythrocytosis driven by the HIF-P4H-2inhibition-induced accumulation of HIF�s has been attributed to increased expressionof EPO from kidney interstitial cells (13), but also, wider, multifactorial control of red cellmass by HIF-P4H-2 has been proposed (45). In our present data, no physiologicallyrelevant induction of the serum EPO level was seen in Hif-p4h-2gt/gt mice despiteage-dependent erythrocytosis. Also, no significant differences were seen in the expres-sion levels of EpoR, Socs3, Bcl-2, Bcl-xl, Fas, FasL, or Spi2A mRNAs; the phosphorylationof the JAK2 protein; or the overall rate of apoptosis, suggesting that there was noinduction of the EPO-signaling pathway in the spleens of Hif-p4h-2gt/gt mice, whichcould have explained the erythrocytosis observed in these mice.

The development of agents that stimulate erythropoiesis, such as recombinanthuman EPO, has resulted in substantial health benefits for patients with end-stage renalfailure (46). A considerable proportion of these patients, however, exhibit a suboptimalresponse to treatment, with inflammation being recognized with increasing frequencyas a cause of the hyporesponsiveness to EPO (46). The unresponsiveness in thesepatients is evidenced by the persistence of anemia, creating a considerable challengefor their treatment. The induction of erythropoiesis in Hif-p4h-2gt/gt mice without anelevation of the serum EPO level could therefore offer an intriguing new possibility fortreating EPO-resistant anemia and help in improving the clinical outcome for thesepatients.

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FIG 6 Hif-p4h-2gt/gt mice are protected against inflammation-induced anemia. (A) Hematologic param-eters at baseline (BL) and after 1 and 2 weeks of follow-up after lipopolysaccharide and zymosan Ainjection. (B) Numbers of spleen megakaryocytes per high-power field (HPF). (C and D) Serum EPO (C) andplasma iron (D) levels in wild-type (wt) and Hif-p4h-2gt/gt (gt/gt) mice after a 2-week follow-up. (For panelsA to D, n � 7 [wild type] and n � 5 [gt/gt].) (E) Hematologic parameters at baseline (BL) and after 3- and6-week follow-ups after peptidoglycan-polysaccharide-polymer (PGPS) injection (n � 9 [wild type] andn � 11 [gt/gt]). (F) Percentages of CD71-positive cells and of 7-aminoactinomycin (7-AAD)-positive cellsin spleens of the mice 6 weeks after PGPS injection (n � 6 [wild type] and n � 4 [gt/gt]). Analysis ofvariance was used for comparisons between groups. (G) Plasma iron levels 6 weeks after PGPS injection(n � 11 for both genotypes). Student’s t test was used for comparisons between two groups. *, P � 0.05;**, P � 0.01; ***, P � 0.001. Error bars represent SEM.

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When we cultured HSCs from bone marrow with different concentrations of EPO, nodifferences in the numbers of BFU-Es were seen between genotypes. Surprisingly,however, BFU-Es from the spleens of Hif-p4h-2gt/gt mice grew to a significant extenteven without EPO, whereas BFU-Es from their wild-type counterparts did not, indicatingthat they had an intrinsic growth advantage, putatively due to altered Notch signaling.Inhibition of Notch signaling with a GSI in wild-type splenic HSCs mimicked thisphenotype with induced growth of BFU-Es. This supports the hypothesis that Notchsignaling plays an important role in induced extramedullary erythropoiesis in Hif-p4h-2gt/gt mice. Activation of HIF-1� by the pan-prolyl hydroxylase inhibitor dimethylox-alylglycine (DMOG) has been reported to synergize with glucocorticoids to enhance theproduction of CFU-E, suggesting that prolyl hydroxylase inhibitors stimulate erythro-poiesis not only through enhanced EPO production but also by intrinsically stimulatingBFU-E self-renewal (47).

Since both aging and inflammatory diseases are said to impose an increased anemiaburden, we set out to test whether Hif-p4h-2gt/gt mice were protected against the devel-opment of inflammation-induced anemia by virtue of their Notch-dependent erythrocyto-sis. Using two models, performed on young adult mice with no apparent erythro-cytosis at the start of the challenge, we saw that Hif-p4h-2gt/gt mice were protectedagainst the development of inflammation-induced anemia and that this protectionwas non-EPO driven but associated with increased splenic erythropoiesis. It is thuslikely that protection against the development of inflammation-induced anemia isdue to a mechanism similar to that for the induced erythropoiesis seen upon aging.In a broader sense, inflammation and aging can be considered stress reactions, andthese data suggest that the HIF-2�-dependent downregulation of Jag1-Notchsignaling could be a common phenomenon in the regulation of extramedullaryerythropoiesis linked to such challenges.

In conclusion, we show here that HIF-2� stabilization-mediated downregulation ofthe Notch ligands Jag1 and -2 and Dll1 and a Notch target gene, Hes1, in Hif-p4h-2hypomorphic mice leads to induced extramedullary erythropoiesis in the spleen (Fig. 7)but not in the bone marrow and that this is manifested in increased hematocrit valuesupon aging and protection against inflammation-induced anemia.

MATERIALS AND METHODSHif-p4h-2gt/gt mice. The method for generating the Hif-p4h-2gt/gt mouse line was described previ-

ously (15). In short, a GeneTrap targeting vector was introduced into intron 1 of the Hif-p4h-2 gene. Inaddition to the truncated trapped Hif-p4h-2 mRNA, the targeted alleles yield some wild-type Hif-p4h-2mRNA because of partial skipping of the targeting vector. The knockdown level of Hif-p4h-2 variesbetween tissues in these mice, being, for example, �35% in the kidney, which appears not to be enoughto induce Epo mRNA and cause massive erythrocytosis (15). Gender-matched wild-type littermates wereused as controls in all experiments. The mice were sedated with fentanyl and midazolam when indicated.

FIG 7 Schematic model of the molecular-level effects of HIF-P4H-2 deficiency leading to splenicextramedullary erythropoiesis. HIF-P4H-2, hypoxia-inducible factor prolyl 4-hydroxylase 2; HIF-1�,hypoxia-inducible factor 1�; Jag1, Jagged 1; Dll1, Delta-like ligand 1; Hes1, Hairy and enhancer ofsplit 1.

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All experiments were performed according to protocols approved by the National Animal ExperimentBoard of Finland.

Analysis of hematologic parameters. For analysis of hematologic parameters, 20 �l of hind-limbvenous blood was collected into a capillary. The samples were analyzed by using the Exigo veterinaryhematology system (Boule Diagnostics).

Plasma iron and serum EPO. For determination of plasma iron and serum EPO levels, an iron assaykit (catalog number ab83366; Abcam) and the Quantikine Mouse EPO immunoassay kit (R&D Systems),respectively, were used.

Histological analyses. Five-micrometer sections from formaldehyde-fixed paraffin-embedded spleensamples were stained with hematoxylin-eosin (HE) and viewed and photographed with a Leica DM LB2microscope and a Leica DFC 320 camera. Representative pictures (5 to 8 pictures/mouse) were taken, and themegakaryocytes were counted in a blind manner. CD41 immunohistochemistry (with anti-CD41 antibodyab63983; Abcam) was used to count the CD41� spleen cells. Ki67 immunohistochemistry (catalog numberM7248; Dako) was used to score the rate of proliferation in spleens.

To analyze the level of apoptosis, spleen samples were stained with the In Situ Cell Death Detectionkit, fluorescein (Roche), and the number of apoptotic cells or bodies was calculated from 5 to 8 picturesper mouse by using an Olympus BX21 microscope and an Olympus XC50 camera. To visualize theexpression of CD71 and Jag1, immunofluorescence staining was performed with anti-transferrin receptor(CD71) antibody (catalog number ab84036; Abcam) and donkey anti-rabbit antibody–Alexa Fluor488 (Jackson ImmunoResearch) and with anti-Jag1 antibody (catalog number ab109536; Abcam) anddonkey anti-rabbit antibody–Cy3 (Jackson ImmunoResearch). Diamidinophenylindole dihydrochloride(DAPI) was used to stain nuclei. Stainings were photographed with a Zeiss AxioScope A1 microscope andan AxioCam MRm camera.

Femurs of 2-month-old mice were decalcified; fixed for 3 weeks in 10% EDTA, 1% glutaraldehyde, and4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS); and embedded in Epon LX112 (LaddResearch Industries). Semithin blocks were cut and stained with toluidine blue. For Leder staining,formalin-EDTA-decalcified femurs were embedded in paraffin. Tissue sections were stained by using thestandard Leder protocol.

FACS analysis. Spleen and liver samples were rapidly filtered with a Falcon nylon cell strainer(Thermo Fischer Scientific) and flushed with 1� PBS. Femurs were cut at both ends, and bone marrowtissues were collected by flushing them with 1� PBS filtered through a Falcon nylon cell strainer. Thenumbers of erythroid precursors/immature erythrocytes/proerythroblasts and erythrocytes were evalu-ated with anti-Ter119 (catalog number 557915; BD Biosciences) and anti-CD71 (catalog number 563504;BD Biosciences) antibodies, respectively, and the number of granulocytes was evaluated with phyco-erythrin (PE)-Cy7 (catalog number 552985; BD Biosciences) and allophycocyanin (APC) (catalog number553129; BD Biosciences) antibodies. The extent of cell death was evaluated with 7-AAD dye. Experimentswere carried out by using a FACSCalibur instrument running CellQuest software, and data were analyzedby using FlowJo.

Western blotting. Dissected spleens were snap-frozen in liquid nitrogen and crushed to a powderby using beads. Nuclear and cytosolic fractions for the detection of HIF-1� and HIF-2� proteins wereextracted with the NE-PER kit (Pierce). To detect phosphorylated JAK2 protein, the total protein fractionswere extracted with a solution containing 25 mM Tris (pH 8.0), 60 mM NaCl, 0.25% NP-40, and 3 M ureasupplemented with Pierce protease and phosphatase inhibitor minitablets (Thermo Fisher Scientific).Protein concentrations were determined with the Bio-Rad protein assay, and the samples were resolvedby SDS-PAGE and blotted onto Immobilon-P membranes (Millipore). The following primary antibodieswere used: anti-HIF-1� (catalog number NB100-479; Novus Biologicals), anti-HIF-2� (catalog numberab199; Abcam), anti-phosphorylated JAK2 (catalog number ab195055; Abcam), and anti-�-actin (catalognumber NB600-501; Novus Biologicals).

Quantitative real-time reverse transcription-PCR. Total RNA from tissues was isolated with EZNAtotal RNA kit II (Omega Bio-Tek) or TriPure isolation reagent (Roche) and reverse transcribed with aniScript cDNA synthesis kit (Bio-Rad). Quantitative PCR (qPCR) was performed by using iTaq SYBR greensupermix and ROX (Bio-Rad) with a Touch thermal cycler and a CFX96 real-time system (Bio-Rad). Theprimers used are presented in Table 1.

Colony-forming assays. Bone marrow from femurs and spleen cells were isolated from 6- to10-month-old mice. Nucleated bone marrow cells (3 � 104 cells/ml) and nucleated splenocytes (1 � 105

cells/ml) were plated in triplicate in methylcellulose medium (catalog number M3534; StemCellTechnologies) in the absence or presence of EPO (30 mU/ml and 300 mU/ml EPO for bone marrowcells and 3,000 mU/ml for spleen cells). Bone marrow and spleen BFU-E, CFU-GEMM, and CFU-GMcolonies were scored after 7 days of incubation at 37°C in a humidified atmosphere containing 5%CO2. In another set of experiments, spleen BFU-E, CFU-GEMM, and CFU-GM colonies were scoredafter treatment with GSI XII (Calbiochem) or dimethyl sulfoxide (DMSO) in the presence or absenceof 3,000 mU/ml EPO on day 7.

siRNA transfection. The spleens of 4- to 5-month-old male Hif-p4h-2gt/gt mice were isolated andmeshed through a Falcon nylon cell strainer (Thermo Fischer Scientific). After several washes, theresulting cell suspension was dispersed in Iscove’s modified Dulbecco’s medium (catalog number12440-046; Thermo Fischer Scientific) supplemented with 10% fetal calf serum, 1% penicillin-streptomycin, and 100 �M 2-mercaptoethanol. About 2 � 106 cells were transfected according to a“reverse-transfection” protocol with 150 pmol of X-tremeGENE siRNA transfection reagent (Roche,Sigma-Aldrich) per well in six-well plates with an siRNA targeting HIF-2� (Sigma-Aldrich) or a scrambleduniversal negative control. The cells were collected for total RNA isolation 24 to 48 h after transfection.

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Anemia models. In one model, 3-month-old mice were injected intraperitoneally with LPS (2 �g/g)and zymosan A (800 �g/g) 6 days later in order to cause inflammation. Blood samples were collected atthe beginning of the experiment, after 7 days, and upon sacrifice 14 days after the zymosan A injection.Other 3- to 4-month-old mice were injected intraperitoneally with PGPS (15 �g/g), and blood sampleswere collected at the beginning of the experiment, 3 weeks after the PGPS injection, and at sacrifice 6weeks after the PGPS injection. Tissues were collected at sacrifice.

Statistical analyses. Student’s two-tailed t test was used for comparisons between two groups. Sincethe BFU-E values in the GSI experiment were not distributed normally, statistical analysis in this case wasperformed after logarithmic transformation of the values. Differences between multiple groups wereevaluated by using one-way analysis of variance (ANOVA) followed by the Newman-Keuls post hoc test.A P value of �0.05 was considered statistically significant. All data are presented as means � standarderrors of the means (SEM).

ACKNOWLEDGMENTSWe thank K. I. Kivirikko for valuable comments on the manuscript; T. Aatsinki, E.

Lehtimäki, R. Salmu, and M. Siurua for excellent technical assistance; S. Kauppila forevaluating the Leder-stained bone marrow samples; and the Biocenter Oulu corefacilities for electron microscopy and transgenic animals.

Elitsa Y. Dimova, Peppi Koivunen, and Raisa Serpi designed the experiments;Mikko N. M. Myllymäki, Jenni Määttä, Elitsa Y. Dimova, Valerio Izzi, Timo Väisänen,Peppi Koivunen, and Raisa Serpi performed experiments and analyzed the data;Johanna Myllyharju contributed to generating the Hif-p4h-2gt/gt mouse line; PeppiKoivunen supervised the work; and Peppi Koivunen and Raisa Serpi wrote themanuscript.

Johanna Myllyharju owns equity in FibroGen Inc., which develops HIF-P4H inhibitorsas potential therapeutics, and the company currently supports research headed by her.We have no additional financial interests.

This study was supported by Academy of Finland grants 218129 (Peppi Koivunen)and Center of Excellence 2012-2017 grant 251314 (Johanna Myllyharju) and by the S.

TABLE 1 Sequences of qPCR primers

GeneForward primer or trade name(manufacturer) Reverse primer

�-Actin AGAGGGAAATCGTGCGTGAC CAATAGTGATGACCTGGCCGTEpoR Quantitect primer assays (Qiagen)Socs3 ATGGTCACCCACAGCAAGTTT TCCAGTAGAATCCGCTCTCCTBcl-2 CTCGTCGCTACCGTCGTGACTTCG CAGATGCCGGTTCAGGTACTCAGTCBcl-xl TGGAGTAAACTGGGGGTCGCATCG AGCCACCGTCATGCCCGTCAGGFas GCGGGTTCGTGAAACTGATAA GCAAAATGGGCCTCCTTGATAFasL TCCGTGAGTTCACCAACCAAA GGGGGTTCCCTGTTAAATGGGSpi2a CTTCCCAACGGCTGGAATCTA ACTGTCCAATCAGGCATAGCGHif-p4h-1 GGCAACTACGTCATCAATG ACCTTAACATCCCAGTTCTGAHif-p4h-2 CTGGGCAACTACAGGATAAAC GCGTCCCAGTCTTTATTTAGATAHif-p4h-3 AGGCCCAACGTACCCCACCT GTGTGTTGGCTGGGAGCCCAGapdh TGTGTCCGTCGTGGATCTGA TTGCTGTTGAAGTCGCAGGAGCxcl12 TGCATCAGTGACGGTAAACCA TTCTTCAGCCGTGCAACAATCCd34 GGTAGCTCTCTGCCTGATGAG TGGTAGGAACTGATGGGGATATTJag1 CCTCGGGTCAGTTTGAGCTG CCTTGAGGCACACTTTGAAGTAJag2 TGGCTGTCACCGAGGTCAA ACGTTCTTTCCTGCGCTTTCDll1 CAGGACCTTCTTTCGCGTATG AAGGGGAATCGGATGGGGTTNotch1 RT2 Profiler PCR array mouse

hematopoiesis (Qiagen)Notch2 RT2 Profiler PCR array mouse

hematopoiesis (Qiagen)Notch4 RT2 Profiler PCR array mouse

hematopoiesis (Qiagen)Hes1 CGGCATTCCAAGCTAGAGAAGG GGTAGGTCATGGCGTTGATCTGGlut1 Quantitect primer assays (Qiagen)Pfkl Quantitect primer assays (Qiagen)Pgk1 GGAGCGGGTCGTGATGA GCCTTGATCCTTTGGTTGTTTGLdha GGATGAGCTTGCCCTTGTTGA GACCAGCTTGGAGTTCGCAGTTAPdk1 Quantitect primer assays (Qiagen)Pdk4 Quantitect primer assays (Qiagen)

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Jusélius Foundation (Peppi Koivunen and Johanna Myllyharju), the Emil AaltonenFoundation (Peppi Koivunen), the Jane and Aatos Erkko Foundation (Peppi Koivunenand Johanna Myllyharju), and FibroGen Inc. (Johanna Myllyharju).

REFERENCES1. Palis J. 2014. Primitive and definitive erythropoiesis in mammals. Front

Physiol 5:3. https://doi.org/10.3389/fphys.2014.00003.2. Jelkmann W. 2013. Physiology and pharmacology of erythropoietin.

Transfus Med Hemother 40:302–309. https://doi.org/10.1159/000356193.

3. Lee FS, Percy MJ. 2011. The HIF pathway and erythrocytosis. Annu RevPathol Mech Dis 6:165–192. https://doi.org/10.1146/annurev-pathol-011110-130321.

4. Prchal JT, Sokol L. 1996. Benign erythrocytosis and other familial andcongenital polycythemias. Eur J Haematol 57:263–268.

5. Patnaik MM, Tefferi A. 2009. The complete evaluation of erythrocytosis:congenital and acquired. Leukemia 23:834–844. https://doi.org/10.1038/leu.2009.54.

6. Kim CH. 2010. Homeostatic and pathogenic extramedullary hematopoi-esis. J Blood Med 1:13–19. https://doi.org/10.2147/JBM.S7224.

7. Wolber FM, Leonard E, Michael S, Orschell-Traycoff CM, Yoder MC, SrourEF. 2002. Roles of spleen and liver in development of the murinehematopoietic system. Exp Hematol 30:1010 –1019. https://doi.org/10.1016/S0301-472X(02)00881-0.

8. Kaelin WG, Jr, Ratcliffe PJ. 2008. Oxygen sensing by metazoans: thecentral role of the HIF hydroxylase pathway. Mol Cell 30:393– 402.https://doi.org/10.1016/j.molcel.2008.04.009.

9. Semenza GL. 2012. Hypoxia-inducible factors in physiology and medi-cine. Cell 148:399 – 408. https://doi.org/10.1016/j.cell.2012.01.021.

10. Myllyharju J, Koivunen P. 2013. Hypoxia-inducible factor prolyl4-hydroxylases: common and specific roles. Biol Chem 394:435– 448.https://doi.org/10.1515/hsz-2012-0328.

11. Takeda K, Ho VC, Takeda H, Duan LJ, Nagy A, Fong G-H. 2006. Placentalbut not heart defects are associated with elevated hypoxia-induciblefactor � levels in mice lacking prolyl hydroxylase domain protein 2. MolCell Biol 26:8336 – 8346. https://doi.org/10.1128/MCB.00425-06.

12. Minamishima YA, Moslehi J, Bardeesy Y, Cullen D, Bronson RT, KaelinWG, Jr. 2008. Somatic inactivation of the PHD2 prolyl hydroxylase causespolycythemia and congestive heart failure. Blood 111:3236 –3244.https://doi.org/10.1182/blood-2007-10-117812.

13. Takeda K, Aguila HL, Parikh NS, Li X, Lamothe K, Duan LJ, Takeda H, LeeFS, Fong GH. 2008. Regulation of adult erythropoiesis by prolyl hydrox-ylase domain proteins. Blood 111:3229 –3235. https://doi.org/10.1182/blood-2007-09-114561.

14. Franke K, Kalucka J, Mamlouk S, Singh RP, Muschter A, Weidemann A,Iyengar V, Jahn S, Wieczorek K, Geiger K, Muders M, Sykes AM, Poitz DM,Ripich T, Otto T, Bergmann S, Breier G, Baretton G, Fong GH, Greaves DR,Bornstein S, Chavakis T, Fandrey J, Gassmann M, Wielockx B. 2013.HIF-1� is a protective factor in conditional PHD2-deficient mice sufferingfrom severe HIF-2�-induced excessive erythropoiesis. Blood 121:1436 –1445. https://doi.org/10.1182/blood-2012-08-449181.

15. Hyvärinen J, Hassinen IE, Sormunen R, Mäki JM, Kivirikko KI, KoivunenP, Myllyharju J. 2010. Hearts of hypoxia-inducible factor prolyl4-hydroxylase-2 hypomorphic mice show protection against acuteischemia-reperfusion injury. J Biol Chem 285:13646 –13657. https://doi.org/10.1074/jbc.M109.084855.

16. Kerkelä R, Karsikas S, Szabo Z, Serpi R, Magga J, Gao E, Alitalo K, AnisimovA, Sormunen R, Pietilä I, Vainio L, Koch WJ, Kivirikko KI, Myllyharju J,Koivunen P. 2013. Activation of hypoxia response in endothelial cellscontributes to ischemic cardioprotection. Mol Cell Biol 33:3321–3329.https://doi.org/10.1128/MCB.00432-13.

17. Rahtu-Korpela L, Karsikas S, Hörkkö S, Blanco Sequeiros R, Lammen-tausta E, Mäkelä KA, Herzig KH, Walkinshaw G, Kivirikko KI, Myllyharju J,Serpi R, Koivunen P. 2014. HIF prolyl-4-hydroxylase-2 inhibition protectsmice against obesity and metabolic syndrome. Diabetes 63:3324 –3333.https://doi.org/10.2337/db14-0472.

18. Rahtu-Korpela L, Määttä J, Dimova EY, Hörkkö S, Gylling H, WalkinshawG, Hakkola J, Kivirikko KI, Myllyharju J, Serpi R, Koivunen P. 2016.Hypoxia-inducible factor-prolyl 4-hydroxylase-2 inhibition protectsagainst development of atherosclerosis. Arterioscler Thromb Vasc Biol36:608 – 617. https://doi.org/10.1161/ATVBAHA.115.307136.

19. Besarab A, Chernyavskaya E, Motylev I, Shutov E, Kumbar LM, Gur-evich K, Chan DT, Leong R, Poole L, Zhong M, Saikali KG, Franco M,Hemmerich S, Yu KH, Neff TB. 2016. Roxadustat (FG-4592): correctionof anemia in incident dialysis patients. J Am Soc Nephrol 27:1225–1233. https://doi.org/10.1681/ASN.2015030241.

20. Besarab A, Provenzano R, Hertel J, Zabaneh R, Klaus SJ, Lee T, Leong R,Hemmerich S, Yu KH, Neff TB. 2015. Randomized placebo-controlleddose-ranging and pharmacodynamics study of roxadustat (FG-4592) totreat anemia in nondialysis-dependent chronic kidney disease (NDD-CKD) patients. Nephrol Dial Transplant 30:1665–1673. https://doi.org/10.1093/ndt/gfv302.

21. Laitala A, Aro E, Walkinshaw G, Mäki JM, Rossi M, Heikkilä M, Savolainen ER,Arend M, Kivirikko KI, Koivunen P, Myllyharju J. 2012. Transmembrane prolyl4-hydroxylase is a fourth prolyl 4-hydroxylase regulating EPO productionand erythropoiesis. Blood 120:3336–3344. https://doi.org/10.1182/blood-2012-07-441824.

22. Fortini ME, Artavanis-Tsakonas S. 1993. Notch: neurogenesis is only partof the picture. Cell 75:1245–1247. https://doi.org/10.1016/0092-8674(93)90611-S.

23. Artavanis-Tsakonas S, Matsuno K, Fortini ME. 1995. Notch signaling.Science 268:225–232. https://doi.org/10.1126/science.7716513.

24. Egan SE, St Pierre B, Leow CC. 1998. Notch receptors, partners andregulators: from conserved domains to powerful functions. Curr TopMicrobiol Immunol 228:273–324.

25. Kopan R, Turner DL. 1996. The Notch pathway: democracy and aristoc-racy in the selection of cell fate. Curr Opin Neurobiol 6:594 – 601. https://doi.org/10.1016/S0959-4388(96)80090-0.

26. Honjo T. 1996. The shortest path from the surface to the nucleus:RBP-J�/Su(H) transcription factor. Genes Cells 1:1–9. https://doi.org/10.1046/j.1365-2443.1996.10010.x.

27. Lewis J. 1998. Notch signalling. A short cut to the nucleus. Nature393:304 –305. https://doi.org/10.1038/30597.

28. Borgrrefe T, Lauth M, Zwijsen A, Huylebroeck D, Oswald F, Giaimo BD.2016. The notch intracellular domain integrates signals from Wnt,Hedgehog, TGF�/BMP and hypoxia pathways. Biochim Biophys Acta1863:303–313. https://doi.org/10.1016/j.bbamcr.2015.11.020.

29. Weinmaster G. 1997. The ins and outs of Notch signaling. Mol CellNeurosci 9:91–102. https://doi.org/10.1006/mcne.1997.0612.

30. Bettenhausen B, Hrabe de Angelis M, Simon D, Guénet J-L. 1995. Tran-sient and restricted expression during mouse embryogenesis of Dll1, amurine gene closely related to Drosophila Delta. Development 121:2407–2418.

31. Dunwoodie SL, Henrique D, Harrison SM, Beddington SP. 1997. MouseDll3: a novel divergent Delta gene which may complement the functionof other Delta homologues during early pattern formation in the mouseembryo. Development 124:3065–3076.

32. Rao PK, Dorsch M, Chickering T, Zheng G, Jiang C, Goodearl A, Kadesch T,McCarthy S. 2000. Isolation and characterization of the Notch ligand Delta4.Exp Cell Res 260:379 –386. https://doi.org/10.1006/excr.2000.5034.

33. Lindsell CE, Shawber CJ, Boulter J, Weinmaster G. 1995. Jagged: amammalian ligand that activates Notch1. Cell 80:909 –917. https://doi.org/10.1016/0092-8674(95)90294-5.

34. Shawber C, Boulter J, Lindsell CE, Weinmaster G. 1996. Jagged2: a serrate-like gene expressed during rat embryogenesis. Dev Biol 180:370 –376.https://doi.org/10.1006/dbio.1996.0310.

35. Luo B, Aster JC, Hasserjian RP, Kuo F, Sklar J. 1997. Isolation andfunctional analysis of a cDNA for human Jagged2, a gene encoding aligand for the Notch1 receptor. Mol Cell Biol 17:6057– 6067. https://doi.org/10.1128/MCB.17.10.6057.

36. Walker L, Carlson A, Tan-Pertel HT, Weinmaster G, Gasson J. 2001. Thenotch receptor and its ligands are selectively expressed during hema-topoietic development in the mouse. Stem Cells 19:543–552. https://doi.org/10.1634/stemcells.19-6-543.

37. Jones P, May G, Healy L, Brown J, Hoyne G, Delassus S, Enver T. 1998.Stromal expression of Jagged 1 promotes colony formation by fetalhematopoietic progenitor cells. Blood 92:1505–1511.

HIF-P4H-2 Inhibition and Splenic Erythrocytosis Molecular and Cellular Biology

January 2017 Volume 37 Issue 2 e00529-16 mcb.asm.org 15

on March 27, 2018 by U

NIV

OF

OU

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cb.asm.org/

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nloaded from

38. Varnum-Finney B, Purton LE, Yu M, Brashem-Stein C, Flowers D, Staats S,Moore KA, Le Roux I, Mann R, Gray G, Artavanis-Tsakonas S, Bernstein ID.1998. The Notch ligand, Jagged-1, influences the development of prim-itive hematopoietic precursor cells. Blood 91:4084 – 4091.

39. Walker L, Lynch M, Silverman S, Fraser J, Boulter J, Weinmaster G, GassonJC. 1999. The Notch/Jagged pathway inhibits proliferation of humanhematopoietic progenitors in vitro. Stem Cells 17:162–171. https://doi.org/10.1002/stem.170162.

40. Duncan AW, Rattis FM, DiMascio LN, Congdon KL, Pazianos G, Zhao C,Yoon K, Cook JM, Willert K, Gaiano N, Reya T. 2005. Integration of Notchand Wnt signaling in hematopoietic stem cell maintenance. Nat Immu-nol 6:314 –322. https://doi.org/10.1038/ni1164.

41. Inra CN, Zhou BO, Acar M, Murphy MM, Richardson J, Zhao Z, MorrisonSJ. 2015. A perisinusoidal niche for extramedullary haematopoiesis inthe spleen. Nature 527:466 – 471. https://doi.org/10.1038/nature15530.

42. Gustafsson MV, Zheng X, Pereira T, Gradin K, Jin S, Lundkvist J, Ruas JL,Poellinger L, Lendahl U, Bondesson M. 2005. Hypoxia requires notchsignaling to maintain the undifferentiated cell state. Dev Cell 9:617– 628.https://doi.org/10.1016/j.devcel.2005.09.010.

43. Chen H, Houshmand G, Mishra S, Fong G-H, Gittes GK. 2010. Impairedpancreatic development in Hif2-alpha deficient mice. Biochem BiophysRes Commun 399:440 – 445. https://doi.org/10.1016/j.bbrc.2010.07.111.

44. Hu YY, Fu LA, Li SZ, Chen Y, Li JC, Han J, Liang L, Li L, Ji CC, Zheng MH,Han H. 2014. Hif-1� and Hif-2� differentially regulate Notch signalingthrough competitive interaction with the intracellular domain of Notchreceptors in glioma stem cells. Cancer Lett 349:67–76. https://doi.org/10.1016/j.canlet.2014.03.035.

45. Arsenault PR, Pei F, Lee R, Kerestes H, Percy MJ, Keith B, Simon MC,Lappin TR, Khurana TS, Lee FS. 2013. A knock-in mouse model ofhuman PHD2 gene-associated erythrocytosis establishes a haploin-sufficiency mechanism. J Biol Chem 288:33571–33584. https://doi.org/10.1074/jbc.M113.482364.

46. Johnson DW, Pollock CA, MacDougall I. 2007. Erythropoiesis-stimulatingagent hyporesponsiveness. Nephrology 12:321–330. https://doi.org/10.1111/j.1440-1797.2007.00810.x.

47. Flygare J, Estrada VR, Shin C, Gupta S, Lodish HF. 2011. HIF1� synergizeswith glucocorticoids to promote BFU-E progenitor self-renewal. Blood117:3435–3444. https://doi.org/10.1182/blood-2010-07-295550.

Myllymäki et al. Molecular and Cellular Biology

January 2017 Volume 37 Issue 2 e00529-16 mcb.asm.org 16

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nloaded from