D 1277

OULU 2014

UNIV ER S IT Y OF OULU P. O. B R[ 00 FI-90014 UNIVERSITY OF OULU FINLAND

U N I V E R S I TAT I S

S E R I E S

SCIENTIAE RERUM NATURALIUM Professor Esa Hohtola

HUMANIORA University Lecturer Santeri Palviainen

TECHNICA Postdoctoral research fellow Sanna Taskila

MEDICA Professor Olli Vuolteenaho

SCIENTIAE RERUM SOCIALIUM University Lecturer Veli-Matti Ulvinen

SCRIPTA ACADEMICA

ACTA

U N I V E R S I T AT I S O U L U E N S I S

Anu Laitala

E D I T O R S

Anu Laitala

A B C D E F G

O U L U E N S I S

ACTA

A C TA

D 1277

HYPOXIA-INDUCIBLE FACTOR PROLYL 4-HYDROXYLASES REGULATING ERYTHROPOIESIS, AND HYPOXIA-INDUCIBLE LYSYL OXIDASE REGULATING SKELETAL MUSCLE DEVELOPMENT DURING EMBRYOGENESIS

Director Sinikka Eskelinen

OECONOMICA Professor Jari Juga

EDITOR IN CHIEF Professor Olli Vuolteenaho PUBLICATIONS EDITOR Publications Editor Kirsti Nurkkala ISBN 978-952-62-0693-6 (Paperback) ISBN 978-952-62-0694-3 (PDF) ISSN 0355-3221 (Print) ISSN 1796-2234 (Online)

UNIVERSITY OF OULU GRADUATE SCHOOL; UNIVERSITY OF OULU, FACULTY OF BIOCHEMISTRY AND MOLECULAR MEDICINE; BIOCENTER OULU; OULU CENTER FOR CELL-MATRIX RESEARCH

D

MEDICA

ACTA UNIVERSITATIS OULUENSIS

D Medica 1277

ANU LAITALA

HYPOXIA-INDUCIBLE FACTOR PROLYL 4-HYDROXYLASES REGULATING ERYTHROPOIESIS, AND HYPOXIAINDUCIBLE LYSYL OXIDASE REGULATING SKELETAL MUSCLE DEVELOPMENT DURING EMBRYOGENESIS Academic dissertation to be presented with the assent of the Doctoral Training Committee of Health and Biosciences of the University of Oulu for public defence in the Leena Palotie auditorium (101A) of the Faculty of Medicine (Aapistie 5 A), on 12 December 2014, at 9 a.m.

U N I VE R S I T Y O F O U L U , O U L U 2 0 1 4

Copyright © 2014 Acta Univ. Oul. D 1277, 2014

Supervised by Professor Johanna Myllyharju Doctor Joni Mäki

Reviewed by Professor Carine Michiels Professor David Hoogewijs

Opponent Associate Professor Janine Erler

ISBN 978-952-62-0693-6 (Paperback) ISBN 978-952-62-0694-3 (PDF) ISSN 0355-3221 (Printed) ISSN 1796-2234 (Online)

Cover Design Raimo Ahonen

JUVENES PRINT TAMPERE 2014

Laitala, Anu, Hypoxia-inducible factor prolyl 4-hydroxylases regulating erythropoiesis, and hypoxia-inducible lysyl oxidase regulating skeletal muscle development during embryogenesis. University of Oulu Graduate School; University of Oulu, Faculty of Biochemistry and Molecular Medicine; Biocenter Oulu; Oulu Center for Cell-Matrix Research Acta Univ. Oul. D 1277, 2014 University of Oulu, P.O. Box 8000, FI-90014 University of Oulu, Finland

Abstract Erythropoiesis is the process of red blood cell production. The main regulator is the erythropoietin (EPO) hormone, which is strongly upregulated in low oxygen concentration (hypoxia) in cells via the hypoxia-inducible transcription factor HIF. The stability of HIF is regulated in an oxygendependent manner by three HIF prolyl 4-hydroxylases, all of which are known to participate in the regulation of erythropoiesis. A role in erythropoiesis of a fourth prolyl 4-hydroxylase, P4H-TM, which possesses a transmembrane domain, is not known, but it is able to hydroxylate HIF at least in vitro and in cellulo. The role of P4H-TM in erythropoiesis was studied by administering a HIFP4H inhibitor, FG-4497, to P4h-tm null, Hif-p4h-3 null, and Hif-p4h-2 hypomorph mouse lines. The current study suggests that P4H-TM is involved in the regulation of EPO production, hepcidin expression and erythropoiesis. P4H-TM can thus be a new target for inhibition when designing novel pharmacological treatment strategies for anemia. LOX is required for crosslink formation between lysine residues in fibrillar collagens and elastin. These crosslinks enhance the tensile strength of collagen fibers and provide elasticity to elastic fibers and thus generate important structural support for tissues. LOX is required for normal embryonic development of the cardiovascular and pulmonary systems, and its depletion leads to a generalized elastinopathy and collagenolysis leading to perinatal death of Lox null mice. The development of muscles is a delicate process, which requires coordinated signaling and a homeostatic balance between the muscle and muscle connective tissue. Based on the drastic defects that were found in the present study in the skeletal muscle of Lox null mice, lack of LOX clearly disturbs this balance and increases transforming growth factor β (TGF-β) signaling, which leads to defects in the skeletal muscles. The impaired balance can cause muscle disorders, such as Duchenne Muscular Dystrophy (DMD). Despite the clinical significance, very little is known about the mechanisms controlling this homeostatic balance. The discovery of LOX as a regulating factor during skeletal muscle development will help to clarify the role of extracellular matrix (ECM) in muscle development and in muscle related congenital diseases.

Keywords: erythropoiesis, hypoxia, hypoxia-inducible factor, lysyl oxidase, prolyl 4hydroxylase, skeletal muscle

Laitala, Anu, Hypoksiaindusoituvaa tekijää säätelevät prolyyli-4-hydroksylaasit erytropoieesin säätelyssä ja hypoksiassa indusoituva lysyylioksidaasi luustolihaksen kehityksessä. Oulun yliopiston tutkijakoulu; Oulun yliopisto, Biokemian ja molekyylilääketieteen tiedekunta; Biocenter Oulu; Oulu Center for Cell-Matrix Research Acta Univ. Oul. D 1277, 2014 Oulun yliopisto, PL 8000, 90014 Oulun yliopisto

Tiivistelmä Erytropoieesi on fysiologinen prosessi, jossa tuotetaan veren punasoluja ja jonka pääsäätelijänä toimii erytropoietiini (EPO) hormoni. EPO:n geeni ilmentyy voimakkaasti alhaisessa happipitoisuudessa (hypoksia) hypoksia-indusoituvan transkriptiotekijän (HIF) toimesta. HIF-tekijän stabiilisuutta säätelee kolme HIF-prolyyli-4-hydroksylaasientsyymiä (HIF-P4H) hapesta riippuvaisesti, ja niiden tiedetään siten osallistuvan myös erytropoieesin säätelyyn, HIF-P4H-2:n toimiessa pääsäätelijänä. Neljännen transmembraanisen prolyyli-4-hydroksylaasin (P4H-TM) roolia erytropoieesissa ei vielä tiedetä, mutta sen tiedetään säätelevän HIF-tekijää. Työssä käytettiin Hif-p4h-2, Hif-p4h-3 ja P4h-tm muuntogeenisiä hiirilinjoja, joiden entsymaattinen aktiivisuus on alentunut tai poistettu. P4H-TM:n osallisuutta erytropoieesin säätelyyn tutkittiin antamalla hiirilinjoille HIF-P4H-entsyymejä inhiboivaa lääkettä. Tutkimuksen tulokset osoittavat ensimmäistä kertaa P4H-TM:n säätelevän EPO-geenin ilmentymistä ja siten erytropoieesia. Ennestään tiedettyjen HIF-P4H entsyymien inhiboinnin lisäksi P4H-TM:n inhibointia voidaan pitää uutena kohteena uusien farmakologisten hoitokeinojen kehityksessä. Lysyylioksidaasi (LOX) katalysoi säikeisten kollageenien välisten sekä elastisten säikeiden välisten poikkisidosten muodostumista. Pokkisidokset antavat vetolujuutta kollageeneille ja joustavuutta elastisille säikeille ja ovat siten tärkeitä kudoksen rakenteelle. LOX:ia tarvitaan sikiön kehityksen aikana mm. hengitys-, sydän- ja verisuonielimistöjen kehityksessä. LOX:in puutos hiirillä aiheuttaa viallisia elastisia- ja kollageenisäikeitä, johtaen poikasten kuolemaan synnytyksen yhteydessä. Lihasten kehitys on tarkoin säädelty prosessi, jossa lihas ja lihaksen sidekudos säätelevät toisiansa. LOX:n suhteen poistogeenisissä Lox-/- sikiöissä löydettiin selviä ongelmia luurankolihasten kehityksessä. LOX:n puutoksen osoitettiin lisäävän transformoivan kasvutekijä beetan (TGF-β) määrää, joka estää luustolihaksia kehittymästä normaalisti. LOX kykenee sitoutumaan TGF-β:aan ja inhiboimaan sen aktiivisuutta ja LOX:n puuttuessa inhibointia ei tapahdu. Tutkimus osoittaa LOX:n olevan keskeinen tekijä lihaksen kehityksessä ja siten auttaa ymmärtämään sidekudoksen merkitystä luurankolihasten kehityksessä ja siihen liittyvissä sairauksissa.

Asiasanat: erythropoieesi, hypokisa indusoituva tekijä, hypoksia, luurankolihas, lysyylioksidaasi, prolyyli-4-hydroksylaasi

Acknowledgements This research was carried out in the Faculty of Biochemistry and Molecular Medicine, formerly the Department of Medical Biochemistry and Molecular Biology, Faculty of Medicine, University of Oulu during the years 2009-2014. I wish to express my deepest gratitude to Professor Johanna Myllyharju for the opportunity to work in her outstanding research group and prepare my thesis under her guidance. She is an excellent example of a hardworking and successful scientist. I am also deeply grateful for my other supervisor Doctor Joni Mäki, who has guided me through the projects. He has taught me how to do science and look at things from an optimistic point of view. I want to thank Professor Peppi Karppinen for the collaboration and efficient guidance in the first project. I wish to acknowledge also Academy Professor Emeritus Kari Kivirikko, whose knowledge of science is extensive. I am also grateful for the personnel of the Faculty who have created an excellent environment to do science. I want to thank the key persons; Professor Emeritus Ilmo Hassinen, Professor Taina Pihlajaniemi, Professor Seppo Vainio, Docent Minna Männikkö, Docent Aki Manninen, Docent Lauri Eklund and other group leaders in the new Faculty. Also I want to thank Auli Kinnunen, Pertti Vuokila, Risto Helminen and Seppo Lähdesmäki for helping with the practical matters. I wish to thank reviewers Professor Carine Michiels and Professor David Hoogewijs for their valuable comments and feedback on the thesis manuscript. Also I would like to acknowledge Deborah Kaska for the language revision of the thesis. I want to thank Docent Eeva-Riitta Savolainen, Docent Raija Sormunen, Doctor Peleg Hasson and his research group and all the other collaborators and coauthors. I am especially thankful for Riitta Polojärvi for her valuable efforts with the everyday technical laboratory work, and Minna Siurua for her advice and help in the lab. I wish to thank all my current and former colleagues in the Faculty and in the JM group for the pleasant working atmosphere. I would like to thank Minna Komu for her advice with the laboratory work. I thank also Ellinoora Aro for cooperation with the first project and sharing the office with me. I want to thank also Ann-Helen Rosendahl for cooperation in the second project and for the friendship outside the office together with Fazeh Moafi, Kati Drushinin and Nadiya Byts. It is always nice to talk about science with you and especially about things not related to science (chocolate and other important stuff). I want to thank Mari Aikio and Johanna Korvala for their advice and help with all the practical

arrangements to become a Ph.D. I wish to thank Vanessa Harjunen for sharing the arrangements and this special day with me. I want to thank my family and friends for the never ending support I have received throughout my life. I thank my cousin Miia for the friendship and sharing a wild youth with me. Special thanks go to my big sister Mari and little brother Ville. Mari inspired me to study biochemistry and has helped me with my studies. I would like to thank my mother Tarja, who has always supported me no matter what and pushed me forward to achieve my ambitions. Kiitos äiti! Finally, I would like to thank Tuomas for the support and love. You have made my life more fun. I acknowledge the financial support received from Biocenter Oulu Doctoral Programme. Oulu, October 2014

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Anu Laitala

Abbreviations ATF4 ATP β2AR BAPN bHLH C-TAD CKD DMD DNA ECM EPO ER FAK FIH Hb HCLK Hct HIF HIF-P4H HRE HSP90 LOX LOX-PP LOXL LTQ MCT miRNA N-TAD 2OG TAD ODDD P4H-TM PKM2 PAS

activating transcription factor 4 Adenosine triphosphate β2-adrenergic receptor β-aminopropionitrile basic helix-loop-helix C-terminal transactivation domain chronic kidney disease Duchenne muscular dystrophy Deoxyribonucleic acid extracellular matrix erythropoietin endoplasmic reticulum focal adhesion kinase factor inhibiting HIF hemoglobin human homolog of the Caenorhabditis elegans biological clock protein CLK-2 hematocrit hypoxia-inducible factor HIF prolyl 4-hydroxylase hypoxia response element heat shock protein 90 lysyl oxidase LOX propeptide LOX-like proteins lysine tyrosylquinone muscle connective tissue microRNA N-terminal transactivation domain 2-oxoglutarate transactivation domain oxygen-dependent degradation domain P4H with a transmembrane domain pyruvate kinase M2 per-arnt-sim 9

PCR PDK1 PDH pVHL qPCR s-EPO SUMO TCF-4 TGF-β VEGF

10

polymerase chain reaction pyruvate dehydrogenase kinase 1 pyruvate dehydrogenase von Hippel-Lindau tumor suppressor protein real-time quantitative PCR serum EPO small ubiquitin-related modifier transcription factor 4 transforming growth factor  vascular endothelial growth factor

List of original papers This thesis is based on the following publications, which are referred to throughout the text by their Roman numerals: I

II

Anu Laitala, Ellinoora Aro, Gail Walkinshaw, Joni M. Mäki, Maarit Rossi, Minna Heikkilä, Eeva-Riitta Savolainen, Michael Arend, Kari I. Kivirikko, Peppi Koivunen*, and Johanna Myllyharju* (2012) Transmembrane prolyl 4-hydroxylase is a fourth prolyl 4-hydroxylase regulating EPO production and erythropoiesis. Blood 120(16):3336-3344. Liora Kutchuk*, Anu Laitala*, Sharon Soueid-Bomgarten, Pessia Shentzer, AnnHelen Rosendahl, Shelly Eilot, Moran Grossman, Irit Sagi, Raija Sormunen, Johanna Myllyharju, Joni M. Mäki and Peleg Hasson (2014) Muscle composition is regulated by a lysyl oxidase-transforming growth factor  feedback loop. Manuscript.

*Equal contributions

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Contents Abstract Tiivistelmä Acknowledgements 7  Abbreviations 9  List of original papers 11  Contents 13  1  Introduction 15  2  Review of the literature 17  2.1  Oxygen sensing and hypoxia .................................................................. 17  2.2  Hypoxia-inducible factor (HIF) .............................................................. 17  2.2.1  Structure and regulation ............................................................... 17  2.2.2  Functions of HIF .......................................................................... 19  2.3  HIF prolyl 4-hydroxylases (HIF-P4Hs) .................................................. 21  2.3.1  Molecular properties..................................................................... 21  2.3.2  Reaction mechanism, substrates and cosubstrates ........................ 23  2.3.3  Regulation and distribution .......................................................... 25  2.3.4  Transmembrane prolyl 4-hydroxylase (P4H-TM) ........................ 29  2.3.5  HIF-P4Hs in human diseases........................................................ 30  2.4  Erythropoiesis, the production of red blood cells.................................... 31  2.4.1  Role of HIF in the regulation of erythropoiesis ............................ 33  2.5  Lysyl oxidase (LOX), modifier of extracellular matrix .......................... 35  2.5.1  Biosynthesis and structure of LOX .............................................. 37  2.5.2  Reaction mechanism, cofactors and substrates ............................. 39  2.5.3  Regulation .................................................................................... 43  2.5.4  Lysyl oxidase like proteins LOXL1-4 .......................................... 44  2.5.5  LOX in mouse development and human diseases ........................ 45  2.6  Skeletal muscle development in mice ..................................................... 48  3  Aims of the present research 53  4  Materials and methods 55  5  Results 57  5.1  Role of P4H-TM in erythropoiesis (I) ..................................................... 57  5.1.1  Inhibition of P4H-TM and HIF-P4Hs by FG-4497 ...................... 57  5.1.2  Increased HIF-1α and HIF-2α stabilization in the kidneys of P4h-tm-/- relative to wild-type mice leads to increased EPO mRNA levels after FG-4497 treatment ................................ 58  13

5.1.3  Increased serum EPO level in the P4h-tm-/- relative to wild-type mice after FG-4497 treatment ...................................... 61  5.1.4  No difference in blood hemoglobin and hematocrit values between P4h-tm-/- and wild-type mice after FG-4497 treatment ....................................................................................... 61  5.1.5  Hepcidin mRNA levels are decreased more in P4h-tm-/than wild-type mice after FG-4497 treatment .............................. 62  5.1.6  Hif-p4h-2gt/gt/P4h-tm-/- double gene-modified mice have higher hemoglobin and hematocrit values .................................... 62  5.2  Role of LOX in skeletal muscle development (II) .................................. 63  5.2.1  Reduced myofiber content and disorganized connective tissue in Lox-/- limbs ..................................................................... 63  5.2.2  LOX is expressed in myofibers during embryonic myogenesis ................................................................................... 65  5.2.3  Increased TGF-β signaling in Lox-/- mice ..................................... 66  5.2.4  Inhibition of TGF-β signaling rescues the Lox-/- muscle phenotype ..................................................................................... 67  6  Discussion 69  6.1  P4H-TM contributes to regulation of erythropoiesis............................... 69  6.2  LOX participates in skeletal muscle development via TGF-β signaling .................................................................................................. 72  7  Conclusions and future prospects 77  References 79  List of original papers 103 

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1

Introduction

Decreased oxygen availability is associated with many diseases, such as anemia, myocardial infarction and ischemia, but it is also a normal condition in specific tissues particularly during development. Hypoxic signaling is mediated via the hypoxia-inducible transcription factor (HIF), which consists of two subunits, HIFα and HIF-β, and its stability is regulated by HIF prolyl 4-hydroxylases (HIFP4Hs). Three HIF-P4H isoenzymes hydroxylate the HIF-α subunit in normoxia leading to its degradation. In hypoxia the HIF-P4H mediated hydroxylation does not occur, HIF-α escapes degradation, forms a dimer with HIF-β and is able to mediate hypoxic signaling by regulating over 100 genes, including erythropoietin (EPO), whose expression is enhanced by hypoxia. EPO regulates the red blood cell production, erythropoiesis, which serves to restore the oxygen concentration in the body during hypoxia. Anemia is a disease where erythropoiesis is deficient and leads to reduced red blood cell number. When recombinant DNA technology enabled the production of recombinant EPO, it revolutionized the treatment of anemia patients. However, EPO treatment is not able to affect iron metabolism, which is an important factor in erythropoiesis. Later also a risk of adverse cardiac effects related to recombinant EPO treatment was noticed. HIF-P4H inhibition appears to be a promising therapeutic strategy to treat anemia, since stabilization of HIF results in both activation of erythropoiesis and iron metabolism. In addition to the three originally characterized HIF-P4H isoenzymes, a novel P4H, P4H-TM, which possesses a transmembrane domain, has been found and shown to be able to hydroxylate HIF-α in vitro and in cellulo, but nothing is known about its potential role in regulating erythropoiesis in vivo. In the first part of the thesis, the putative role of P4H-TM in erythropoiesis was studied. Three genetically modified mouse lines, a HIF-P4H-2 hypomorph mouse line (Hif-p4h-2gt/gt) and knockout mouse lines HIF-P4H-3 and P4H-TM were used in the experiments. Their responses to a HIF-P4H inhibitor (FG-4497) were studied and the results from the P4h-tm-/- mice were compared to those from wildtype, Hif-p4h-2gt/gt and Hif-p4h-3-/- mice. Our hypothesis was that if a particular P4H participates in the regulation of erythropoiesis, treatment of mice that lack or have reduced amounts of that enzyme should be more sensitive to FG-4497 mediated induction of erythropoiesis. In addition, the P4h-tm-/- mice were crossed with Hif-p4h-2gt/gt mice to study the effect of simultaneous deficiency of these two enzymes. 15

Skeletal muscle development is a carefully regulated process, where small interferences can cause drastic defects in the muscle phenotype. Many players are involved in the development process and all the muscle-composing parts as well as their surroundings in the developing limb have a role in the crosstalk. The roles of the muscle connective tissue ensheathing the muscles, muscle bundles and single muscle fibers have been observed to be more versatile than just providing a structural scaffold. However, little is still known about the regulatory mechanisms required for the development of a healthy functional skeletal muscle. Lysyl oxidase (LOX) is an enzyme that catalyzes the formation of crosslinks in fibrillar collagens and elastin to give tensile strength and elasticity to tissues. The importance of the crosslinking reactions is evident from the phenotype of Lox-/- mice. They die perinatally due to a weak arterial wall extracellular matrix (ECM), which leads to aneurysms. Defects have also been seen in other ECMs leading to immature lungs and ruptures in the diaphragm. In the present study we discovered that Lox-/- mice have defects in embryonic skeletal muscles. It was thus apparent that lack of LOX during the development of the skeletal muscles disturbs the balance between the muscle composing factors and its surroundings. In the second part of the thesis the role of LOX in skeletal muscle development was studied in detail.

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2

Review of the literature

2.1

Oxygen sensing and hypoxia

Oxygen is essential for multicellular organisms. It is required for the energy generating process, mitochondrial oxidative phosphorylation, to produce ATP. Global changes in oxygen concentrations in blood are sensed in the carotid body, more specifically in the glomus cells. Decreases in oxygen levels trigger physiological effects such as hyperventilation. Oxygen level sensing also occurs at the cellular level. Reduced oxygen availability (hypoxia) causes changes in the expression of genes that are required for cell survival in hypoxia. The key mediator in this hypoxia response is the hypoxia-inducible transcription factor (HIF). HIF is known to affect the expression of over 100 genes, which help cells to adapt to hypoxic conditions by decreasing oxygen consumption and increasing its supply. This includes shifting energy metabolism to the glycolytic pathway, which does not require oxygen, and reducing the amounts of harmful reactive oxygen species that are generated by oxidative phosphorylation at low oxygen concentrations. Upregulation of the erythropoietin (EPO) production in hypoxia produces more red blood cells, which deliver oxygen to the body. Stimulation of the vascular endothelial growth factor (VEGF) increases vascular formation to the hypoxic regions of the body (Palmer & Clegg 2014, Semenza 2014). Hypoxia can be caused by diseases, which affect blood circulation or its ability to carry oxygen, such as anemia, and myocardial and limb ischemia. Solid tumors can also be hypoxic inside. Hypoxia can also occur in normal physiological states, such as in embryos where it functions as a stimulus during development and growth, but also in adults in specific tissues such as the kidney medulla and bone marrow niches (Semenza 2014, Simon & Keith 2008). 2.2

Hypoxia-inducible factor (HIF)

2.2.1 Structure and regulation HIF is an αβ heterodimer the β subunit of which is constitutively expressed. The three α subunit isoforms are regulated in an oxygen-dependent manner. HIF-α protein is produced constitutively, but one or both proline residues in two -Leu-XX-Leu-Ala-Pro- sequences (Pro402, Pro564 in human HIF-1α; Pro405, Pro531 in 17

human HIF-2α) are hydroxylated by HIF-P4Hs under normoxic conditions. The 4-hydroxyproline residue is required for binding of the von Hippel–Lindau protein (pVHL) E3 ubiquitin ligase complex, which targets HIF-α to degradation in proteasomes (Bruick & McKnight 2001, Epstein et al. 2001, Ivan et al. 2001, Jaakkola et al. 2001, Maxwell et al. 1999, Yu et al. 2001). HIF-P4Hs cannot function in hypoxic conditions because oxygen is required in the hydroxylation reaction and consequently HIF-α escapes degradation in hypoxia. HIF-α forms a dimer with HIF-β, which is able to bind HRE-elements in the target genes. In addition to hydroxylation by HIF-P4Hs, HIF-1α and HIF-2α are also hydroxylated by factor inhibiting HIF (FIH) in normoxic conditions. The asparagine hydroxylated by FIH is located in the C-terminal transactivation domain (C-TAD) and inhibits the binding of the p300 and CBP coactivators that stabilize the transcription initiation complex, and thus inhibits the transcriptional activation of HIF (Fig. 1) (Loboda et al. 2012, Semenza 2010). In vertebrates there are three different HIF-α isoforms, HIF-1α, HIF-2α and HIF-3α, from which the first two are the most studied. HIF-1α and HIF-2α have a similar overall structure. They consist of basic helix-loop-helix (bHLH), PERARNT-SIM (PAS), oxygen-dependent degradation (ODDD) and N- and Cterminal transactivation (N-TAD and C-TAD) domains. The proline residues that can be hydroxylated, are located in the ODDD, in N- and C-terminal sites of the domain (Huang et al. 1998). bHLH and PAS domains are important for DNA binding and dimerization (Kewley et al. 2004). N-TAD is important for target gene specificity and C-TAD for expression of the target gene (Jiang et al. 1996). HIF-3α is unique in that it undergoes extensive alternative splicing, but all variants differ from HIF-1α and HIF-2α in that they lack the C-TAD domain (Hara et al. 2001, Maynard et al. 2003, Pasanen et al. 2010). In addition to the regulation of HIF-α stability and activity by the HIF-P4Hs and FIH in an oxygen-dependent manner, the stability and activity can be affected also by other factors. MicroRNAs (miRNAs) have been shown to decrease HIF-α mRNA stability and decrease HIF amounts (Bruning et al. 2011, Taguchi et al. 2008). HIF-α degradation is also regulated by SUMOylation. In hypoxia SUMOylation is increased and the small ubiquitin-related modifier protein (SUMO) is conjugated to HIF-α. SUMOylation can lead to VHL- and proteasome-dependent degradation of HIF-α. SUMOylation can be reversed with SENP1, which detaches the SUMO protein (Carbia-Nagashima et al. 2007, Cheng et al. 2007). 18

2.2.2 Functions of HIF HIF is a transcription factor that can regulate the expression of over 100 genes by binding to specific hypoxia response elements (HRE) that are located in the regulatory regions of HIF responsive genes (Wenger et al. 2005). Upregulation of most of the HIF target genes requires the binding of HIF to the HRE that contains the core sequence 5’-(A/G)CGTG-3’. However, certain HIF target genes are suppressed in hypoxia. Downregulation by HIF is thought to happen indirectly by some other transcriptional repressor (Schodel et al. 2011, Semenza 2010). From the three HIF-α isoforms HIF-1α is the most widely expressed and is found in essentially all tissues. HIF-2α (also known as endothelial PAS domain protein 1, EPAS1) expression is more restricted to certain tissues, such as the kidney, lung, heart and small intestine, and more specifically in the parenchyma and interstitial cells (Patel & Simon 2008). The function of HIF-3α (also known as the inhibitory PAS protein, IPAS) is less well known. It is subjected to extensive alternative splicing and has many splicing variants (Maynard et al. 2003, Pasanen et al. 2010). As all HIF-3α variants lack the C-TAD domain, their function as a transcription activator was considered very unlikely. However, HIF-3α has been shown to have both inductive and suppressive effects on HIF target genes in a variant specific manner (Heikkilä et al. 2011, Makino et al. 2001, Makino et al. 2007, Pasanen et al. 2010). From the HIF-α isoforms only HIF-3α is itself induced in hypoxia, by HIF-1 (Heidbreder et al. 2003, Pasanen et al. 2010, Tanaka et al. 2009). HIF-1α and HIF-2α have significant sequence homology and most of the differences between them are seen in the N-TAD domain, which confers different binding specificities for HIF-1α and HIF-2α. The C-TADs of HIF-1α and HIF-2α are homologous and contribute to the transcription of shared target genes (Hu et al. 2007). Even though HIF-1α and HIF-2α are highly similar, they have distinct tissue distribution and their inactivation in mouse leads to different phenotypes. Hif-1α deletion in the mouse causes embryonic lethality and death between embryonic days 8 and 11 (E8.5-E.11.5) due to defects in the heart and vascularity (Iyer et al. 1998, Kotch et al. 1999, Ryan et al. 1998). Mice with an Hif-2α deletion die at an embryonic or postnatal stage, depending on the mouse background. They suffer from vascular defects, bradycardia, impaired lung maturation and defective catecholamine synthesis (Compernolle et al. 2002, Peng et al. 2000, Tian et al. 1998).

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HIF has many biological roles. It has been linked, for example, to embryonic development, metabolism, angiogenesis, erythropoiesis, cancer, and inflammation. HIF-1 and HIF-2 have many common target genes, but also preferential induction of certain genes and processes by only one of the isoforms occurs. HIF-1α is thought to be the main regulator of genes involved in the glycolytic pathway, such as pyruvate dehydrogenase kinase 1 (PDK1), which inhibits pyruvate dehydrogenase (PDH). PDH connects glycolysis with the tricarboxylic acid cycle. HIF-2α has been shown to be the main regulator of erythropoiesis and iron metabolism, inducing EPO (Fig. 1) (Flamant et al. 2009, Hu et al. 2003, Kapitsinou et al. 2010, Mastrogiannaki et al. 2009, Morita et al. 2003). In addition to regulation of hypoxia responsive genes, HIF affects the activity of noncoding miRNAs. Hypoxia increases a set of miRNAs, which bind to target mRNAs leading to their degradation or translational inhibition and in this way miRNAs also contribute to the hypoxic cell response. Many of these miRNAs are overexpressed in human tumors. For example, miR-120 is increased in all ischemic diseases and tumors so far analyzed (Devlin et al. 2011, Kulshreshtha et al. 2007). HIF can also interact with other signaling pathways. Hypoxic conditions have been shown to maintain cells in an undifferentiated state. For example, HIF-1α interaction with the intracellular domain of Notch activates downstream targets of Notch in hypoxia (Gustafsson et al. 2005). HIF-1α can interfere with the β-catenin-T-cell factor-4 (TCF-4) complex and thus inhibits Wnt signaling and its target genes, such as the Myc oncogene. In addition, βcatenin enhances HIF-1α transcription activity (Kaidi et al. 2007). HIF-1α can also displace Myc from the p21cip1 promoter and suppress the cell cycle by upregulating Myc suppressed genes (Koshiji et al. 2004). On the other hand, HIF2α has been reported to have the opposite effect on Myc (Gordan et al. 2007). Recently hypoxia has been shown to have an effect on long noncoding RNAs (lncRNAs) via HIFs that also influence the transcriptional output of the certain genes (Choudhry et al. 2014, Ferdin et al. 2013). For example, hypoxia induces the accumulation of the Ephrin-A3, a cell surface protein that modulates cellular adhesion and repulsion (Gomez-Maldonado et al. 2014). In this study the mRNA level of the gene coding Ephrin-A3 was not increased, wherwas the amount of the novel group of lncRNAs form the same locus were increased leading to increased Ephrin-A3 protein amounts.

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Fig. 1. Degradation of HIF-α in an oxygen-dependent manner. In normoxia two prolines (Pro) in HIF-α are hydroxylated by HIF-P4Hs and an asparagine (Asn) by FIH. 4hydroxyprolines are required for the binding of pVHL, leading to ubiquitination and subsequent proteasomal degradation of HIF-α. Hydroxylation of the asparagine inhibits binding of the transcriptional coactivator CBP/p300. In hypoxia HIF-P4Hs and FIH are inhibited and HIF-α forms a dimer with HIF-β that is able to regulate genes that contain a hypoxia response element (HRE). HIF-1 and HIF-2 have distinct functions and show preference towards certain genes.

2.3

HIF prolyl 4-hydroxylases (HIF-P4Hs)

2.3.1 Molecular properties HIF-P4Hs catalyze the formation of 4-hydroxyproline in the HIF-α ODDD. They belong to the 2-oxoglutarate dioxygenase superfamily. Vertebrates have three HIF-P4H isoenzymes (HIF-P4Hs 1-3, also known as PHDs 1-3 and EGLNs 2, 1 and 3) that hydroxylate HIF-α (Bruick & McKnight 2001, Epstein et al. 2001, Ivan et al. 2002). They are composed of 407, 426 and 239 amino acids (Fig. 2), 21

respectively, and have 42-59% overall amino acid similarity to each other, the highest degree of homology being present in the C-terminal regions that contain the catalytic site. They have high Km values for oxygen, about 230-250 μM, when measured with a 19-20-residue peptide representing the C-terminal hydroxylation site of the ODDD (Hirsilä et al. 2003). When measured with longer synthetic peptides or the full-length ODDD, the Km values for oxygen range from 65 to 100 μM (Ehrismann et al. 2007, Koivunen et al. 2006). However, even these values are much higher than physiological O2 concentrations, meaning they can quickly respond to changes in O2 availability (Ehrismann et al. 2007, Koivunen et al. 2006). HIF-P4H-2 has a MYND domain in its N terminus, which has been found to decrease the enzymatic activity. The MYND domain is known to be involved in protein-protein interactions and may thus mediate cellular regulation of HIFP4H-2 activity (Choi et al. 2005). Variants of HIF-P4H isoforms are known to exist. HIF-P4H-1 has two forms, which arise from two alternative initiation sites. The shorter one has a shorter half-life but the enzyme activities of the two variants are very similar (Tian et al. 2006). HIF-P4H-2 and 3 are subjected to alternative splicing, both producing two shorter variants (Cervera et al. 2006, Hirsilä et al. 2003). The shorter HIF-P4H-2 transcripts both encode inactive polypeptides. Of the shorter HIF-P4H-3 variants, one did not have any enzyme activity, but the other had at least partial activity and was expressed mainly in primary cancer tissue.

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Fig. 2. Schematic representation of the human HIF-P4Hs and P4H-TM. HIF-P4H-1 is found in the nucleus, HIF-P4H-2 mainly in the

cytoplasm and HIF-P4H-3 in both

nucleus and cytoplasm. P4H-TM is located in the ER membrane so that the C-terminal catalytic site is inside the lumen. All three HIF-P4Hs are known to hydroxylate HIFs, but the substrate of P4H-TM in vivo is currently not identified.

2.3.2 Reaction mechanism, substrates and cosubstrates HIF-P4Hs belong to a dioxygenase family that requires 2-oxoglutarate (2OG), Fe2+, O2, and ascorbate in their catalytic activity (Myllyharju 2013). The active site is typically located in the C-terminal end, in a jelly-roll core formed by a double-stranded β-helix. Fe2+ binds to the active site by highly conserved residues, two histidines and one aspartate. Binding of 2OG requires a conserved positively charged residue, either arginine or lysine (Myllyharju 2013). In the hydroxylation reaction, HIF-P4H that contains the Fe2+ first binds 2OG, followed by binding of the HIF substrate and lastly the molecular oxygen. Molecular oxygen is split in two; one half is used in the hydroxylation of HIF and the other in the oxidative decarboxylation of 2OG to succinate and CO2. Ascorbate is needed for the full catalytic activity and is thought to reduce Fe3+ to Fe2+ in the event of uncoupled reaction cycles, that is, reactions in which decarboxylation of 2OG occurs without hydroxylation of the substrate (Kaelin & Ratcliffe 2008, Schofield & Ratcliffe 2005).

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HIF-P4Hs have also been suggested to have multiple non-HIF targets (Table 1). Hypoxia is known to activate NFκB and it was discovered that this occurs via HIF-P4Hs. HIF-P4Hs, especially HIF-P4H-1 and HIF-P4H-2, are reported to hydroxylate IKKβ, which leads to inactivation of NFκB signaling in normoxia. In hypoxia NFκB signaling is activated since HIF-P4Hs cannot catalyze the IKKβ hydroxylation (Cummins et al. 2006). HIF-P4H-2 has been shown to provide a link between the oxygen sensing and heat shock protein 90 (HSP90) pathways. It binds to p23, a co-chaperone of HSP90, via its N-terminal MYND-domain and is thus recruited to HSP90, which promotes efficient HIF-α hydroxylation by HIF-P4H-2 (Song et al. 2013). HIF-P4H-3 seems to be the isoenzyme with the highest number of non-HIF related functions. HIF-P4H-3 has been shown to bind activating transcription factor-4 (ATF4), which influences DNA repair and cell fate decisions (Koditz et al. 2007) and the β2-adrenergic receptor (β2AR), which is a G protein coupled receptor mediating cardiovascular and pulmonary functions (Xie et al. 2009). Interaction with HIF-P4H-3 regulates their stability in normoxia. Later, HIF-P4H2 was also shown to bind to β2AR and regulate the internalization of the receptors by concentrating them to clathrin-coated pits (Yan et al. 2011). Hydroxylation of the human homolog of the Caenorhabditis elegans biological clock protein CLK2 (HCLK2) by HIF-P4H-3 activates the pathway leading to apoptosis in response to DNA damage (Xie et al. 2012b). HIF-P4H-3 has also been shown to hydroxylate pyruvate kinase M2 (PKM2), which is an HIF-1α target gene (Luo et al. 2011). PKM2 interacts with HIF-1α and stimulates the transcriptional activity of HIF-1α on genes involved in glycolytic metabolism as well as VEGF in cancer cells. Hydroxylation of PKM2 by HIF-P4H-3 enhances its binding to HIF-1α. This presents a positive feedback loop that promotes HIF-1α action in cancer (Luo et al. 2011). A recent study has shown that HIF-P4H-3 hydroxylates also nonmuscle β and -actin and inhibits their polymerization, which negatively affects cell motility (Luo et al. 2014). HIF-P4H-3 also interacts with PDH-E1β, which is one of the four subunits forming PDH (Kikuchi et al. 2014). As stated above HIF-1α induces PDK1, which inhibits PDH and shifts the ATP production into the glycolytic pathway. The study revealed that HIF-P4H-3 maintains PDH activity in moderate hypoxia and thus works against HIF-1α (Kikuchi et al. 2014).

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Table 1. non-HIF-P4H targets HIF-P4H

Target

Effect

HIF-P4H-1

Cdr2 (cerebellar degeneration-related

attenuated hypoxic response in tumors

protein 2) HIF-P4H-1,

IKKβ (inhibitor of Nuclear Factor Kappa- inactivation of NFκB signaling in

HIF-P4H-2

B)

normoxia

HIF-P4H-2

FKBP38 (peptidyl prolyl cis/trans

decreases stability of HIF-P4H-2

isomerase FK506-binding protein 38) HIF-P4H-2

p23 (a co-chaperone of HSP90)

HIF-P4H-2

β2AR (β2-adrenergic receptor)

promotes efficient HIF-α hydroxylation internalization of the receptor

HIF-P4H-2

MAGE-11 (melanoma antigen-11)

Inhibition of HIF-P4H-2

HIF-P4H-3

ATF4 (activating transcription factor-4)

DNA repair and cell fate

HIF-P4H-3

β2AR (β2-adrenergic receptor)

cardiovascular and pulmonary functions

HIF-P4H-3

HCLK2 (Caenorhabditis elegans

apoptosis in response to DNA damage

biological clock protein CLK-2 HIF-P4H-3

Siah 2 (RING (really interesting new

decrease in the availability and activity

gene) finger E3 ligase)

of HIF-P4H-3

HIF-P4H-3

nonmuscle β and γ actin

cell motility

HIF-P4H-3

PHD-E1β (subunit of puruvate

maintains PDH activity

dehydrogenase, PDH) (Aprelikova et al. 2009, Balamurugan et al. 2009, Barth et al. 2007, Cummins et al. 2006, Kikuchi et al. 2014, Koditz et al. 2007, Luo et al. 2011, Luo et al. 2014, Nakayama et al. 2007, Song et al. 2013, Xie et al. 2009, Xie et al. 2012b, Yan et al. 2011)

2.3.3 Regulation and distribution HIF-P4Hs are widely expressed in different tissues, but in varying amounts (Li et al. 2007, Lieb et al. 2002, Oehme et al. 2002, Willam et al. 2006). HIF-P4H-2 is present in practically all tissues. The highest amount of HIF-P4H-3 is seen in the heart, whereas the highest amount of HIF-P4H-1 is found in the placenta and testis. All three isoenzymes have been located in the medullar part of the kidney. HIF-P4H-3 expression increases in the mouse heart as the animal gets older (Rohrbach et al. 2005). The same has been seen in human hearts. As the amount of HIF-P4H-3 increases the amount of HIF-1α decreases thus affecting expression of the hypoxia-inducible genes. The cellular localization of HIF-P4Hs differs between the isoenzymes. HIFP4H-1 is restricted to the nucleus and HIF-P4H-2 is found in the cytosol, while HIF-P4H-3 is distributed both in the nucleus and cytosol (Metzen et al. 2003). Later, HIF-P4H-2 was also shown to shuttle between the nucleus and cytosol 25

(Steinhoff et al. 2009). Increased levels of HIF-P4H-2 in the nucleus have been associated with with more aggressive tumor behavior (Jokilehto et al. 2006). The nuclear localization of enzymatically active HIF-P4H-2 is important for HIF-1α hydroxylation (Pientka et al. 2012), but the promotion of tumor growth is hypothesized to be at least partly an HIF-independent activity of HIF-P4H-2 (Jokilehto & Jaakkola 2010). HIF-P4Hs have different preferences towards HIF-1α and HIF-2α (Appelhoff et al. 2004). HIF-P4H-2 has more influence on HIF-1α, whereas HIF-P4H-3 has more affect on HIF-2α. Both HIF-P4H-2 and HIF-P4H-3 are themselves hypoxiainducible, HIF-1α having more effect on the upregulation of HIF-P4H-2 and HIF2α on HIF-P4H-3 (Aprelikova et al. 2004). As HIF-P4H-3 is upregulated after a longer period of hypoxia, it is thought to play an important role in reoxygenation (Minamishima et al. 2009). HIF-P4H-2 is the most abundant isoenzyme and it is the main HIF regulator. Silencing it alone in normoxic cells is enough to stabilize HIF, unlike silencing of HIF-P4H-1 or HIF-P4H-3 alone (Berra et al. 2003). Inactivation of Hif-p4h-2 in mice is lethal, causing death during embryogenesis due to placental defects (Takeda et al. 2006). Defects were also seen in heart development. Knockout mouse models of Hif-p4h-1 and Hif-p4h-3 are viable and have no obvious developmental defects (Takeda et al. 2006). HIF-P4H inhibitors Many compounds have been found to inhibit HIF-P4Hs with respect to their cosubstrates or substrates. Iron chelators such as DFO (desferrioxamine) stabilize HIF-α by inhibiting the activity of iron-dependent prolyl hydroxylases and lead to activation of its target genes (Ivan et al. 2001, Jaakkola et al. 2001). Bivalent cations such as Co2+ and Zn2+ also inhibit HIF-P4Hs by competing with Fe2+ for binding the active site (Bruick & McKnight 2001, Epstein et al. 2001, Hirsilä et al. 2005). Small molecular chemical compounds, that are structurally similar to 2oxoglutarate, such as DMOG (dimethyloxalylglycine) inhibit HIF-P4Hs by blocking the binding of the cosubstrate (Epstein et al. 2001, Jaakkola et al. 2001). These inhibitors inhibit not only HIF-P4Hs specifically, but also collagen prolyl 4-hydroxylases (c-P4Hs) and FIH, as well as other 2-OG enzyme family members (Bruick & McKnight 2001, Epstein et al. 2001, Hirsilä et al. 2005, Myllyharju 2003, Myllyharju 2008, Myllyharju 2009). 26

Distinct differences have been seen for example in many 2-oxoglutarate analogues between the inhibition of HIF-P4Hs and C-P4Hs, which enables development of HIF-P4H specific inhibitors (Hirsila et al. 2003). The HIF-P4H specific inhibitors can potentially be used in a variety of therapeutic applications (Table 2.) (Myllyharju 2013). Many publications have shown that pre-treatment with an inhibitor provides protection against cardiac, focal cerebral and renal ischemia in animal experiments, or even after onset of the injury in some cases (Bernhardt et al. 2006, Ockaili et al. 2005, Ogle et al. 2012, Rosenberger et al. 2008, Siddiq et al. 2005). Treating anemic patients with an HIF-P4H inhibitor is a very promising strategy to induce endogenous erythropoiesis (Haase 2010). In experiments done with macaques and human patients suffering from end-stage renal disease, the inhibition of HIF-P4Hs increased erythropoiesis, which prevents anemia (Bernhardt et al. 2010, Hsieh et al. 2007). Several inhibitors are currently being tested in clinical trials for use as an anemia treatment in patients suffering from kidney disease (Myllyharju 2013). Inhibition has also been shown to stimulate angiogenesis, for example in mice and rats, with subcutaneous sponge models (Nangaku et al. 2007, Warnecke et al. 2003). Hypoxia has been associated with inflammation and activates the inflammatory signaling by activating nuclear factor κB. Inhibiton of HIF-P4Hs can thus affect the inflammatory diseases (Fraisl et al. 2009). Table 2. List of selected HIF-P4H inhibitors Inhibitor

Principle of inhibition

Specificity

Physiological effect

Metal ions (e.g.

Replaces Fe2+

Less for HIF-P4Hs, more for

Promotes hypoxia tolerance

Co2+ and Cu2+)

(interferes with

FIH and C-P4Hs

cofactor) DFO

DMOG

Fe2+ chelator

2-OG analog

Less for HIF-P4Hs, more for

Ischemic preconditioning,

FIH and C-P4Hs

promotes hypoxia tolerance

HIF-P4Hs, FIH, C-P4Hs, many

Ischemic preconditioning,

2-OG dependent enzymes

promote angiogenesis,

HIF-P4Hs

Ischemic preconditioning,

supresses inflammation FG-4497

2-OG analog

promotes erythropoiesis, supresses inflammation ICA

2-OG analog

HIF-P4Hs

Ischemic preconditioning,

ICA; 2-(1-chloro-4-hydroxyisoquinoline-3-carboxamido) acetate, DFO; desferrioxamine, DMOG; dimethyloxalylglycine, 2-OG; 2-oxoglutarate. (Fraisl et al. 2009, Hirsilä et al. 2005, Myllyharju 2013)

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HIF-P4Hs in mouse models In later studies with HIF-P4H-1 deficient mice, they were discovered to have a shift to more anaerobic ATP production in skeletal muscles by activation of the Pparα pathway, which led to upregulation of pyruvate dehydrogenase kinase 4 (Pdk4), a known negative regulator of the PDH complex (Aragones et al. 2008). This was shown to be mainly HIF-2 mediated. The shift in metabolism protected the myofibers against ischemia, but lowered the oxidative muscle performance in normal conditions. A later study with HIF-P4H-3 deficient mice also showed, that these null mice have an increased cell number in the superior cervical ganglion, resulting from reduced apoptosis (Bishop et al. 2008). Increased cell numbers were also seen in the carotid body and adrenal medulla. These changes led to decreases in the systemic blood pressure, adrenal medullary secretion and sympathoadrenal responses. These are mediated at least partly via increased HIF-2α signaling, but the contribution of other HIF-P4H-3 substrates cannot be ruled out. Since HIF-P4H-2 deficient mice die during embryonic development, many conditional knockout mouse models have been produced. Wide inactivation of Hif-p4h-2 leads to hyperactive angiogenesis and angiectasia caused by increased VEGF-A (Takeda et al. 2007). Blood homeostasis in mice is also mainly regulated by HIF-P4H-2, since wide inactivation of its gene led to a drastic increase in red blood cell production or erythrocytosis via renal HIF-1α accumulation and increased serum EPO values (Minamishima et al. 2008, Takeda et al. 2008). This led to premature death probably due to high blood viscosity. The HIF-P4H-2 deficient mice also developed dilated cardiomyopathy (Minamishima et al. 2008). Hif-p4h-1 and Hif-p4h-3 null mice do not have any signs of erythrocytosis, but double knockout mice had increased hematocrit values via hepatic HIF-2α accumulation and EPO expression (Takeda et al. 2008). Inactivation of any of the Hif-p4h genes alone in the liver did not have an effect on erythropoiesis, but combinational inactivation of all three isoenzymes specifically in the liver increased the serum EPO and hematocrit values drastically, even above the levels resulting from Hif-p4h-2 inactivation in the kidney (Minamishima & Kaelin 2010). Both HIF-P4H-1 and HIF-P4H-2 deficient mice have shown protection against cardiac ischemia-reperfusion injury. Hif-p4h-1 null mice had increased HIF-1α expression in the heart, which led to a decrease in the infarct size via an increase of multiple factors, including β-catenin, eNOS and p65 (NF-κB subunit) 28

(Adluri et al. 2011). The HIF-P4H-1 deficient mice also had increased expression of the anti-apoptotic protein Bcl-2 leading to reduced cardiomyocyte apoptosis. Acute inhibition of Hif-p4h-2 with siRNA led to an increase in cardiac HIF-1α stabilization and smaller infarct size after myocardial ischemia (Eckle et al. 2008). This was achieved by induction of CD37, a regulator of extracellular adenosine generation and A2BR, an adenosine receptor. In an Hif-p4h-2 hypomorph mouse line, where 8% of the wild-type HIF-P4H-2 mRNA expression level is left in the heart, stabilization of HIF-1α and HIF-2α occurs, and these activate many genes involved in cardiac function, glucose metabolism and regulation of blood pressure. These changes protect against cardiac ischemiareperfusion and infarct injuries (Hyvärinen et al. 2010a, Kerkelä et al. 2013). Hif-p4h-2 hypomorphic mice also have improved glucose tolerance and insulin sensitivity, which protects against obesity and diabetes (Rahtu-Korpela et al. 2014). Similar results were also seen in mice with acute hepatic Hif-p4h-3 deletion (Taniguchi et al. 2013). 2.3.4 Transmembrane prolyl 4-hydroxylase (P4H-TM) A fourth P4H, a transmembrane P4H (P4H-TM), hydroxylating HIF has been found in the endoplasmic reticulum (ER) (Koivunen et al. 2007, Oehme et al. 2002). P4H-TM is a 502-amino-acid polypeptide that has a transmembrane domain in the N terminus and is located in the ER membranes so that the Cterminal catalytic site is inside the lumen (Fig. 2). The amino acid sequence of P4H-TM is in fact more closely related to the collagen P4Hs than HIF-P4Hs, the sequence similarities between the catalytically important C-terminal regions of P4H-TM and collagen P4Hs is 26-28%, while it is 13-15% between P4H-TM and HIF-P4Hs. However, P4H-TM does not have the peptide substrate-binding domain that is characteristic of collagen P4Hs and it is not able to hydroxylate collagen peptides in vitro. In contrast, P4H-TM has been shown to hydroxylate HIF-α in vitro, with a preference towards the proline in the C-terminal site of the ODDD. Overexpression and RNA interference studies also indicated hydroxylation of HIF in cellulo. The mechanism by which an ER bound enzyme, with the active site inside the lumen, can hydroxylate HIF, a nuclear or cytoplasmic protein, remains to be clarified. A truncated P4H-TM that is lacking the N-terminal transmembrane domain has been identified in human cell lines, but whether its subcellular location is changed is currently unknown. P4H-TM is induced in hypoxia, but its localization is not changed. 29

In zebrafish P4H-TM has its highest expression in the eye and brain and knockdown of the enzyme led to dysfunctions in the glomerular and lens capsule basement membranes (Hyvärinen et al. 2010b). Based on the subcellular location and the zebrafish phenotype, it was hypothesized that P4H-TM has additional substrates besides HIF, since even though HIF affects over 100 genes, the probability is small that the observed phenotype is only HIF-mediated. 2.3.5 HIF-P4Hs in human diseases Most of the known human HIF-P4H mutations are located in the HIF-P4H-2 gene and are associated with erythrocytosis (Percy et al. 2006, Percy et al. 2007). These mutations include a heterozygous 950C>G mutation that results in a change at codon 317 from proline to arginine and a heterozygous 1112G>A mutation leading to a change in amino acid 371 from arginine to histidine. Both mutations are located close to the conserved iron binding residues in the catalytic site. Further studies revealed that these mutations affect the HIF-P4H-2 activity towards HIF-1α and HIF-2α by decreasing the binding and hydroxylation of HIFα, which leads to secondary erythrocytosis. Surprisingly, the serum EPO levels were normal in these patients. Results from these patients do not exclude the possibility of HIF-P4H-2 interacting with proteins unrelated to HIF that could add to the phenotype Three additional mutations in the HIF-P4H-2 gene have been associated with erythrocytosis (Al-Sheikh et al. 2008). These were heterozygous frameshifts due to a deletion (606delG) or insertion (840_841insA) and a nonsense point mutation (1129C>T) in exon 1 that all led to a truncated HIF-P4H2 protein. In one case a patient with erythrocytosis due to an HIF-P4H-2 gene mutation was associated with a tumor (Ladroue et al. 2008). Recurrence of the paraganglioma with an extra-adrenal localization led to development of secondary erythrocytosis. The patient had a heterozygous missense mutation c.1121A>G, which led to impaired enzyme activity. Tumor analysis showed loss of heterozygosity, suggesting a potential tumor suppressor role for HIF-P4H-2. Recently more mutations of HIF-P4H-2 have been found and these distinct mutations have differential effects on HIF regulation (Albiero et al. 2012, Ladroue et al. 2012). A genetic adaptation to hypoxia has developed in populations living at high altitudes. The high-altitude Tibetan variant c.[12C>G; 380G>C] enhances HIF-P4H-2 activity and prevents the formation of polycythemia at high altitudes (Lorenzo et al. 2014). 30

HIF-P4H-1 is has been found to be estrogen-inducible in breast carcinoma cells and induces cyclin D1, which promotes cell proliferation (Zhang et al. 2009). This interaction is not mediated by HIF, but requires the HIF-P4H-1 catalytic activity. Inhibition of HIF-P4H-1 decreases cyclin D1 and suppresses proliferation, which could be a potential therapeutic strategy for hormone sensitive breast cancer. In renal cell carcinomas HIF-P4H-3 has been found to be frequently upregulated HIF-independently by the phosphatidylinositol-3 kinase/Akt/mTOR pathway and was found to reduce cell proliferation suggesting it to be a potential antitumor molecule (Tanaka et al. 2014). 2.4

Erythropoiesis, the production of red blood cells

Oxygen is carried to the organs by red blood cells, which are produced by a process known as erythropoiesis. The main regulator of erythropoiesis is the erythropoietin hormone (EPO). It is produced in the liver during embryonic development, but the primary producer in adults is the kidney, which is responsible for approximately 80% of the EPO production. The switch between the organs happens during birth and the time point varies in different species (Bunn 2013, Haase 2013, Wenger & Hoogewijs 2010). After the switch the liver is still able to contribute to systemic EPO amounts (Fried 1972, Kapitsinou et al. 2010, Minamishima & Kaelin 2010). Peritubular interstitial cells of kidney, that have neuronal and fibroblastic features, secrete the EPO hormone and are situated in the cortex and outer medulla of the kidney (Bachmann et al. 1993, Maxwell et al. 1993, Obara et al. 2008, Paliege et al. 2010, Pan et al. 2011, Souma et al. 2013, Yamazaki et al. 2013). In the liver the hepatocytes around the central vein are the EPO producing cells and EPO has also been detected in hepatic satellite cells (ITO cells) (Koury et al. 1991, Maxwell et al. 1994). EPO translocates via the blood circulation to the bone marrow where it binds to the EPO receptor (EPOr) on the cell membrane of erythroid progenitor cells (CFU-E, colony-forming unit-erythroid) and promotes their survival by inhibiting apoptosis and leading to differentiation to reticulocytes (Fig. 2) (Koury & Bondurant 1990). CFU-E cells differentiate from multipotent hematopoietic stem and progenitor cells (HSPCs), which give rise to all types of blood cells. They differentiate to reticulocytes in the bone marrow and enter the bloodstream where they mature into erythrocytes within 1-2 days and stay viable approximately 120 days. A basal rate of erythropoiesis is needed for replacing the senescent red blood cells (Yoon et al. 2011). 31

Other tissues have been found to produce EPO as well, such as the brain, heart, bone marrow, lung, spleen and bone osteoblasts (Bernaudin et al. 2000, Dame et al. 2000, Fandrey & Bunn 1993, Marti et al. 1996, Marti et al. 1997, Miro-Murillo et al. 2011, Rankin et al. 2012). Whether the EPO produced from these extra-renal sites has real systemic effects on erythropoiesis remains to be studied. They most probably participate in nonhematopoietic effects such as cellular protection and angiogenesis (Haase 2013). In brain EPO has affects locally by paracrine signaling and has been shown to work as a neuroprotective agent in brain injury (Brines et al. 2000, Sakanaka et al. 1998). In heart EPO has also been shown to protect against cardiac ischemia (Nishiya et al. 2006, Parsa et al. 2004). However, certain studies provide evidence that EPO produced in these extra-renal tissues can affect erythropoiesis. Hypoxic treatment of rat brain was enough to induce EPO production in the kidney. The signaling between the brain and kidney was hypothesized to occur via humoral factors (von Wussow et al. 2005). Inactivation of the Vhl gene in mouse epidermis led to dramatically increased serum EPO levels via nitric oxide, which mediates cutaneous vasodilation (Boutin et al. 2008). Astrocytes also produce EPO and deletion of Vhl in mouse astrocytes was shown to lead to erythrocytosis via HIF-2α revealing their contribution to the systemic erythropoietic response (Weidemann et al. 2009). Deletion of Vhl and subsequent HIF activation in osteoblasts increased their EPO production and increased the number of early progenitors and hematopoietic stem cells, which together increased red blood cell production (Rankin et al. 2012).

32

Fig. 3. Schematic figure of erythropoiesis.

2.4.1 Role of HIF in the regulation of erythropoiesis The role of HIF in erythropoiesis is probably the most studied HIF regulated process. HIF was in fact discovered during the investigation of transcriptional activation of the Epo gene in hypoxia (Semenza & Wang 1992). In normoxia Epo expression is supressed by a GATA motif bound by GATA-2 and GATA-3 transcription factors, leading to a low basal level of EPO (Imagawa et al. 1994, Imagawa et al. 1997, Obara et al. 2008). Under hypoxic conditions binding of HIF, or more specifically HIF-2, to the HRE-element of the Epo gene increases the expression. The Epo coding sequence has two additional HREs in both sides of it, from which the 5’ element is used in the kidney and the 3’ element is required for Epo induction in the liver (Semenza et al. 1990, Semenza & Wang 1992, Storti et al. 2014, Suzuki et al. 2011). Larger increases in EPO amounts are thought to result from increases in the number of EPO producing cells, at least in the kidney (Koury et al. 1989, Obara et al. 2008). Many studies have revealed 33

that HIF-2 is the main regulator of erythropoiesis and the resulting induction can be several hundred-fold higher than in normoxia. Postnatal broad-spectrum conditional inactivation of HIF-1α and HIF-2α in mice revealed the important role of HIF-2α in erythropoiesis (Gruber et al. 2007). HIF-2α loss decreased the hematocrit (Hct) value by 32%, whereas HIF-1α deficient mice had no change in Hct. Simultaneous inactivation of HIF-2α was enough to suppress the development of polycythemia by decreasing EPO mRNA amounts in Vhl deficient mice or wild-type mice that had induced anemia (Rankin et al. 2007). The same was not achieved with HIF-1α inactivation. Inactivation of HIF-2α just in the mouse kidney led to severe EPO-deficient anemia (Kapitsinou et al. 2010). Serum EPO levels were reduced by 60%-70%. Liver EPO production was increased, but was not enough to compensate for the loss of kidney EPO production. Histological findings also support the central role of HIF-2α in EPO regulation. EPO producing cells in the kidney, the renal interstitial fibroblast like cells, and HIF-2α expression correspond with each other (Paliege et al. 2010, Rosenberger et al. 2002). Erythropoiesis requires also iron and the key regulator of iron homeostasis is the liver produced hepcidin hormone (Haase 2013, Shah & Xie 2014). Hepcidin downregulates the iron exporter ferroportin by binding to it, which leads to degradation of the complex. This decreases iron uptake in the intestine and release from the internal stores. The expression of hepcidin is sensitive to iron and oxygen amounts; its expression is reduced in low levels of iron whereas high levels result in increased expression. Hepcidin expression is downregulated in hypoxia, but it is not a direct HIF target. HIF induces EPO production in the kidney and liver, which results in erythropoiesis in the bone marrow. These together produce a systemic signal from bone marrow, which can downregulate hepcidin via a still unknown mechanism (Liu et al. 2012, Mastrogiannaki et al. 2012, Volke et al. 2009). HIF-2 also directly regulates other factors required in iron uptake, such as DMT1 (iron transporter from the lumen of the gut into the cell), DCYTB (reduces Fe3+ to Fe2+), transferrin (transports iron in the serum) and its receptor (Lee et al. 1997, Mukhopadhyay et al. 2000, Rolfs et al. 1997). Mutations in the HIF pathway can lead to erythrocytosis where excess amounts of red blood cells are produced. Mutations have been found in genes coding for HIF-2α, HIF-P4H-2 and pVHL. No mutation in the gene for HIF-1α associated with erythrocytosis has yet been found, highlighting the role of HIF-2 in erythropoiesis (Haase 2013). For example in Chuvash polycythemia a mutation in pVHL impairs the degradation of hydroxylated HIF-1α and HIF-2α, which 34

leads to increased expression of HIF target genes, including those for VEGF, plasminogen activator (PAI-1) and EPO. Erythrocytosis also leads cerebral ischemic lesions in the patients (Yoon et al. 2011). Known human mutations in the gene encoding HIF-P4H-2 resulting in erythrocytosis are described above in 2.2.5. As described before, acute global inactivation of Hif-p4h-2 in mice leads to dramatic increases in red blood cell production and severe erythrocytosis (Minamishima et al. 2008, Takeda et al. 2008). This results from an increased EPO mRNA level in the kidney but not in the liver. Hif-p4h-1 and Hif-p4h-3 deficient mice did not have an erythrocytotic phenotype individually, but double inactivation of both genes led to moderate increases in the hematologic values. To achieve maximal renal EPO production mere inactivation of Hif-p4h-2 is enough, but to have maximal EPO production from the liver, all three Hif-p4h genes need to be inactivated together (Minamishima & Kaelin 2010). 2.5

Lysyl oxidase (LOX), modifier of extracellular matrix

Lysyl oxidase (LOX) is one of the enzymes involved in the formation and repair of the extracellular matrix (ECM). LOX catalyzes the crosslinking of fibrillar collagens and elastin. It belongs to the copper amine oxidase enzyme family and it has a tyrosine-derived quinone cofactor and Cu2+ in its structure. The enzyme was described first by Pinnell and Martin in 1968. They used chicken bone extracts and studied their ability to convert lysyl residues to aldehydes, which then spontaneously condense to crosslink with other aldehydes or peptidyl lysines (Finney et al. 2014, Kagan & Li 2003, Mäki 2009). All tissues have ECM, which is a complex structure consisting of many proteins and polysaccharides, and they differ in composition and structures that are formed during development. ECM is a dynamic structure that is constantly being remodeled. ECM not only provides structural strength to the tissues and organs, but it is also a major player in cell behavior, such as cell adhesion and migration. In addition, ECM participates in signaling by binding and storing growth factors or interacting with receptors on the cell surfaces. The two primary components of ECM are proteoglycans and fibrous proteins. Proteoglycans are glycoproteins that colocalize with collagen fibres. They are able to store water, which results in space-filling and lubricating abilities (Daley et al. 2008, Frantz et al. 2010). 35

Fibrous proteins of the ECM consist, for example, of collagens, laminins, fibronectin, fibrillins and elastin. Collagens, which function in the formation and maintenance of the structure of many tissues, are the most abundant proteins constituting up to one-third of the total protein mass of the human body. They provide the strength in the tissue, but participate also in the regulation of cell adhesion and migration. Biosynthesis of fibrillar collagens requires posttranslational modifications that take place both inside and outside the cell. First, the procollagen polypeptide chains are hydroxylated by three different collagen hydroxylases, (collagen P4Hs, lysyl hydroxylases and prolyl 3-hydroxylases) and glycosylated with two collagen glycosyltransferases (collagen gal-transferase and glc-transferases). After this the three procollagen chains fold into a triple helical procollagen molecule that is exported from the cell and the N and C-terminal propeptides are cleaved by procollagen proteinases. In the final step lysyl oxidase oxidizes specific lysine and hydroxylysine residues and the resulting aldehydes condense with peptidyl aldehydes or ε-amino groups of peptidyl lysine and form covalent crosslinks, which provide the high tensile strength for which the collagen fibrils are well known (Fig. 4) (Frantz et al. 2010, Myllyharju & Kivirikko 2004). Elastin is the major component of elastic fibers, comprising up to 90% of their structure. These fibers provide the elasticity and resilience to tissues that stretch. Tropoelastin is the soluble and uncrosslinked precursor of elastin, which self-aggregates in a process called coacervation, and elastin aggregates assemble upon a microfibril scaffold in the ECM, where the crosslinking domains align. Monomers bind to each other and the formation is stabilized by desmosine and isodesmosine crosslinking of the lysine derivatives, which is catalyzed by LOX. These elastic fibers start to form in mid-gestation and very little assembly is seen in adults. Apart from the tropoelastin and fibrillin, elastic fibers consist of many additional molecules, which are involved with the functions of the elastin fiber (Baldwin et al. 2013, Wagenseil & Mecham 2007, Yeo et al. 2011). Initially LOX was only known for its functions in modifying ECM structures, but later on many more diverse biological functions have been associated with LOX. All the proposed new functions do not require the enzyme activity of LOX and also the LOX propeptide has been shown to be a mediator in some of the functions (Csiszar 2001, Finney et al. 2014, Payne et al. 2007).

36

Fig. 4. Biosynthesis of fibrillar collagen. Polypeptide chains are synthesized into the rough

endoplasmic

reticulum

(ER),

where

certain

prolines

and

lysines

are

hydroxylated and some of the hydroxylysines are glycosylated. Three propeptides assembly and the triple helix is formed in a zipper-like manner, propagating from the C-terminal nucleus toward the N terminus. The procollagen molecules are transported from the ER to the ECM in secretory vesicles. In the ECM N and C propeptides are cleaved and the resulting collagen molecules spontaneously self-assemble into fibrils. Intramolecular

and

intermolecular

covalent

lysine-

and

hydroxylysine-derived

crosslinks are intiated by lysyl oxidase.

2.5.1 Biosynthesis and structure of LOX The cDNA of LOX was first identified from rat aorta. It produces a 47-kDa precursor protein (Trackman et al. 1990, Trackman et al. 1991), which is significantly larger than the purified mature LOX isolated from different tissues, for example the bovine and rat lung, and human placenta (Kagan et al. 1979, Kuivaniemi et al. 1984, Trackman et al. 1990, Trackman et al. 1991). LOX is synthesized as a preproenzyme (Fig. 5). The first 21 N-terminal amino acids form 37

the signal peptide required for secretion. The signal peptide is cleaved yielding a ~50-kDa N-glycosylated proenzyme, which is secreted to the ECM as an inactive protein (Trackman et al. 1992). In the ECM the proLOX binds to cellular fibronectin, which is required for the proteolytic cleavage to produce the 32-kDa catalytically active enzyme (LOX) and an 18-kDa propeptide (LOX-PP) (Fig. 3) (Fogelgren et al. 2005). The cleavage is produced by procollagen C-proteinase (Cronshaw et al. 1995, Panchenko et al. 1996) and this proteinase activity in mammals is provided by the Bmp-1 gene splicing variants bone morphogenetic protein 1 (BMP-1) and mammalian tolloid (mTLD) (Takahara et al. 1994). Other proteinases with procollagen C-proteinase activity can also cleave proLOX, such as mammalian tolloid-like-1 and 2 (mTLL-1 and 2), but at lower efficiency (Uzel et al. 2001). Another kind of proteolytic processing of the proLOX has also been suggested. In vitro cell culture experiments revealed a 25-kDa truncated LOX (tLOX), which had been cleaved at the C terminus by an unknown serine protease. The tLOX still had enzyme activity, but the biological significance of tLOX is not known (Atsawasuwan et al. 2011). In humans and mice the gene encoding LOX is located in chromosome 5 (Hämäläinen et al. 1991) and 18 (Mock et al. 1992), respectively, and both human and mouse LOX cDNAs were sequenced in the early 1990s (Contente et al. 1993, Hämäläinen et al. 1991, Mariani et al. 1992). They show both conserved and divergent sequences. Most of the differences are in the N-terminal region where the signal peptide is located, but nearly identical sequences can be found within the catalytic domain in the C-terminal part of the protein (Csiszar 2001, Kagan & Li 2003). Mature LOX includes a copper-binding domain (Krebs & Krawetz 1993), a cytokine receptor-like domain (CRL) (Bazan 1989, Bazan 1990) and conserved tyrosine and lysine residues for the binding of lysine tyrosylquinone (LTQ) (Dove et al. 1996, Wang et al. 1996). Copper and LTQ are needed for the enzyme activity, but the role of the CRL domain is not completely understood (Csiszar 2001). CRL may add stability to the protein interactions of LOX, for example the binding of LOX with cellular fibronectin is thought to occur via this domain (Fogelgren et al. 2005, Kagan & Li 2003).

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Fig. 5. Schematic structure and processing of preproLOX and the roles of mature LOX and LOX-PP.

2.5.2 Reaction mechanism, cofactors and substrates LOX contains one tightly bound Cu2+ cofactor, which is thought to be important to the structure and formation of lysine tyrosylquinone and therefore affects the catalytic activity (Gacheru et al. 1990). Cu2+ is incorporated to the copper binding domain of proLOX inside the cell (Kosonen et al. 1997) and failure of copper delivery to tissues due to mutation in copper transport in Menkes disease and occipital horn syndrome decreases LOX activity (Vulpe & Packman 1995). LOX also has a covalently bound lysine tyrosylquinone (LTQ) cofactor in its active site. LTQ is formed by a self-processing reaction and requires two conserved amino acid residues, a lysine and tyrosine. Cu2+ binds in the copper binding domain, which is in close proximity to the critical LTQ forming amino acids. This catalyzes the oxidation of the tyrosine residue, which then forms a covalent crosslink with the lysine (Bollinger et al. 2005). 39

LOX oxidizes peptidyl lysines in elastin and collagens and they form αamino-δ-semialdehydes. These aldehydes can then condense with other peptidyl aldehydes or ε-amino groups of peptidyl lysine to form covalent crosslinks. Early on, a lack of copper was found to inactivate the catalytic activity of LOX (Pinnell & Martin 1968, Williamson & Kagan 1986, Williamson & Kagan 1987) and later Cu2+ was defined as a requirement for the formation of LTQ, which has an important role in the catalytic mechanism of LOX as an electron receiver (Bollinger et al. 2005, Dove et al. 1996, Gacheru et al. 1990, Lucero & Kagan 2006, Wang et al. 1996). In the LOX catalyzed reaction, the amine substrate binds to one of the two carbonyls of LTQ forming a Schiff base (imine) and two electrons migrate from the substrate to the LTQ. Hydrolysis of the Schiff base linkage releases an aldehyde leaving behind a reduced enzyme bound with the amino group from the substrate. Molecular oxygen binds and oxidizes LTQ to its original state by producing and releasing hydrogen peroxide and ammonia (Fig. 6) (Lucero & Kagan 2006). Commonly known LOX substrates are the soluble precursors of elastin and fibrillar collagens, but the possibility for other substrates arose when in vitro assays demonstrated that LOX is able to oxidize globular proteins with an isoelectric point (pI) ≥ 8.0, such as histone H1 (Kagan et al. 1984) and nonpeptidyl amine substrates (Trackman et al. 1981). The amino acid sequences around the oxidized lysines are not conserved as can be seen from the sequence differences between fibrillar collagens and tropoelastin (Siegel 1974). Tropoelastin has a positive net charge and is thus readily oxidized by LOX (Nagan & Kagan 1994). Fibrillar collagens on the other hand are secreted into the ECM as procollagen precursors and have to be modified before LOX can oxidize them (Siegel 1974). The N and C-terminal procollagen propeptides are removed by N and C-procollagen proteinases, respectively, to produce mature collagen molecules. The mature collagen molecule still has a negative charge around the oxidized lysines and it is hypothesized that one mature molecule needs to form microfibril aggregations with two other molecules before oxidation. It is assumed that in this quarter-staggered formation the collagen molecules can neutralize each other’s negative charges thus enabling the oxidation reaction (Lucero & Kagan 2006, Nagan & Kagan 1994). Although LOX is commonly called an extracellular enzyme, it has also been found inside cells and further in the nucleus. The mechanism for internalization is not known, although LOX catalytic activity is not required for this internalization. 40

It is suggested that LOX is first secreted to produce the catalytically active mature LOX and then imported back inside the cell (Li et al. 1997, Nellaiappan et al. 2000). Results of a recent overexpression study suggest that LOX binds to the transcription repressor p66β by its catalytic domain and the complex is colocalized into the nucleus (Okkelman et al. 2014). The intracellular and nuclear localization of LOX strongly support the new biological roles of LOX that have been discovered. LOX has been found to bind and oxidize substrates other than collagen and elastin and alter the activity of the oxidized protein. LOX has also been shown to act as a transcriptional activator for specific genes (Kagan & Li 2003). Overexpression of LOX results in increased elastin and type III procollagen (COL3A1) transcription. In both cases LOX activity was required for the activation, but LOX itself did not bind to the promoters. In the case of elastin, TGF-β1 abolished the induction (Oleggini et al. 2007). The COL3A1 promoter activation is thought to be mediated via the Ku antigen, which is involved in a complex mediating V(J)D recombination and DNA repair (Giampuzzi et al. 2000). LOX is known to oxidize histone H1 and interact with H2 in vitro (Giampuzzi et al. 2003, Kagan et al. 1984) and recently the action on H1 has been demonstrated also in vivo (Mello et al. 2011, Oleggini & Di Donato 2011). H1 was found to have desmosine and isodesmosine crosslinks when LOX was overexpressed. Also the interaction with H1, which controls chromatin organization, loosened the H1-DNA complex and resulted in chromatin remodeling. LOX is also suspected to affect the mouse mammary tumor virus (MMTV) promoter via H1 interactions (Mello et al. 2011, Oleggini & Di Donato 2011). LOX has been shown to oxidize basic fibroblast growth factor in mouse 3T3 fibroblasts resulting in inactivation of mitogenic activity (Li et al. 2003). LOX also binds to mature TGF-β and inhibits its signaling in vitro. The inhibition occurs most probably via oxidation, since the use of the LOX inhibitor aminopropionitrile (BAPN) rescues the TGF-β signaling (Atsawasuwan et al. 2008). LOX can also oxidize plasma membrane proteins. One of these is the platelet-derived growth factor  receptor (PDGFR-β) on the cell membrane of vascular smooth muscle cells (VSMC). Oxidation of this receptor enables the binding of PDGF to its receptor and thus primes the cells for the chemotactic response (Lucero et al. 2011). 41

The by-product of the LOX catalytic reaction, hydrogen peroxide (H2O2), has also a role of its own. H2O2 can attract VSMC migration and has been associated with the progress of atherosclerosis (Li et al. 2000). Later the chemoattraction of H2O2 has been associated with focal adhesion kinase (FAK)/Src signaling. Even though LOX catalyzed oxidation occurs mainly in the ECM, H2O2 can easily diffuse through the cell membrane and can induce cellular responses and signaling. H2O2 induces FAK and paxillin activation. They are major components in the cell adhesion complex that can promote tumor cell invasion (Laczko et al. 2007, Payne et al. 2005, Webb et al. 2004). The enzymatically active mature LOX is not the only part of the LOX gene product that has a biological role. In addition to its requirement for LOX secretion (Grimsby et al. 2010) LOX-PP has a role of its own in cancer suppression (see part 2.5.5).

Fig. 6. Reaction mechanism of LOX. The oxidation reaction goes through a ping pong bi ter mechanism (modified from Lucero & Kagan 2006).

42

2.5.3 Regulation LOX expression at the protein and mRNA levels has been characterized in different species and has been localized in the human to the heart, placenta, lung, skeletal muscle, kidney, and pancreas, but only marginally to the brain and liver (Kim et al. 1995). Different studies have shown various transcriptional and posttranscriptional mechanisms that regulate the expression and activity of LOX in different physiological states including ECM remodeling, development, wound healing and ageing, copper deficiency and tumorigenesis (Csiszar 2001). LOX has many roles and is itself regulated by many factors. Decreased amounts or total loss of LOX mRNA have been discovered in cases where the Lox gene has been methylated. Methylation-related inhibition of LOX has been found in cancer cells and in pelvic organ prolapses (Contente et al. 1999, Kaneda et al. 2004, Klutke et al. 2010). Many cytokines, growth factors and transcription factors regulate LOX. For example, fibroblast growth factor (FGF)-2 and insulin-like growth factor (IGF)-1 increase LOX expression in inflamed rat oral tissues, but the mechanism is not known (Trackman et al. 1998). In contrast, LOX is not seen in the non-inflamed periapical tissue. Previous reports have shown an increase in LOX expression by TGF-β in murine osteoblastic cells (Feres-Filho et al. 1995), VSMCs (Gacheru et al. 1997, Shanley et al. 1997) and in chronic renal fibrosis in hereditary nephrotic mice (Goto et al. 2005). Many of the studies show that rather than activating the promoter, TGF-β affects the processing of the post-transcriptional level of LOX. The transcriptional activator IFN regulatory factor 1 (IRF-1) has been shown to upregulate LOX expression by binding to an IRF-E motif that is located in the LOX promoter (Tan et al. 1996). LOX has been shown to be strongly upregulated in hypoxia by HIF, which binds to a functional HRE in the LOX promoter sequence (Erler et al. 2006a). In a recent report LOX was upregulated in C3H10T1/2 pluripotent cells by Wnt3a via canonical Wnt signaling (Khosravi et al. 2014). In the same study three putative TCF/LEF sites were found in the LOX promoter of which one gave a significant response. Upregulation of LOX via Wnt3a was limited to pluripotent cells indicating its role in cell differentiation. It was hypothesized that the activity of LOX produces mesenchymal progenitor cells that can become osteoblasts, adipocytes or chondrocytes. Cytokine tumor necrosis factor α (TNF-α) inhibits LOX expression in endothelial cells, but the mechanism is not known (Rodriguez et al. 2008). Later TNF-α was found to inhibit Wnt3a induced LOX expression via miR203 that targets LOX mRNA 43

(Khosravi et al. 2014). A recent study has revealed an alternative promoter located in the LOX gene, which produces a novel variant of LOX, LOX transcription variant 2 (LOX-v2). It is smaller in size, only 22 kDa, which results from the lack of the exon 1 of LOX. It also has different tissue specificity and has been suggested to mediate the tumor progression activity assigned to LOX, since it lacks the propeptide region, which has a role as a tumor suppressor (Kim et al. 2014). In cell culture and animal experiments BAPN has been used to inhibit LOX activity. It is an irreversible LOX inhibitor, which was found to cause lathyrism in animals. It increased the amount of soluble collagen and led to defects in connective tissue and supporting structures. BAPN binds to the active site of LOX in a competitive manner and inhibits the enzyme activity (Tang et al. 1983). 2.5.4 Lysyl oxidase like proteins LOXL1-4 The lysyl oxidase gene family consists of LOX itself and four LOX-like proteins (LOXL1, LOXL2, LOXL3 and LOXL4) (Agra et al. 2013, Asuncion et al. 2001, Huang et al. 2001, Ito et al. 2001, Jourdan-Le Saux et al. 2001, Kenyon et al. 1993, Mäki & Kivirikko 2001, Mäki et al. 2001, Saito et al. 1997). They are all highly similar in the C-terminal region where the catalytic domain is located and are all catalytically active enzymes. The major differences between the sequences occur at the N-terminal part. They each have a signal peptide, but the following region is variable in sequence and size (Lucero & Kagan 2006). The most similar one to LOX is LOXL1. The LOXL1 cDNA was first characterized by Kenyon et al. (1993), and the protein has a proline-rich domain following the signal peptide. LOXL1 is expressed in ocular tissues, such as the iris, and in female reproductive tissues and can act as a tumor suppressor in bladder cancer (Finney et al. 2014). It is required in elastic fiber homeostasis, since depletion of LOXL1 leads to abnormal elastic fibers in mice and is associated with pelvic prolapses (Liu et al. 2004b). A mutation in LOXL1 has also been associated with exfoliative glaucoma (Ritch 2008), an age-related disorder characterized by accumulation of fibrillar extracellular material in many ocular tissues. LOXL2-4 have four cysteine-rich scavenger receptor-like (SRCR) domains following the signal peptide. SRCR domains are not found in LOX and LOXL1. LOXL2 (Saito et al. 1997) is highly expressed in reproductive tissues such as the placenta, uterus and prostate (Finney et al. 2014). It is suggested to affect metastasis of cancer cells by having a role in adhesion and it is also highly upregulated in invasive and metastatic breast cancer 44

cells (Mäki 2009). LOXL3 is the least studied of the LOXL proteins, although it is expressed in many tissues, the highest levels being found in the placenta, heart, ovary, testis, small intestine and spleen (Huang et al. 2001, Jourdan-Le Saux et al. 2001, Mäki & Kivirikko 2001). LOXL4 is also expressed in many tissues such as the skeletal muscle, testis, pancreas and cartilage (Asuncion et al. 2001, Ito et al. 2001, Mäki et al. 2001). It has been shown to be upregulated in invasive and metastatic cancer cells. In bladder cancer the LOXL4 gene is methylated and thus the expression is suppressed (Mäki 2009). The roles of SRCRs are not yet clear. They are known to be involved in cell adhesion or cell signaling via proteinprotein interactions (Finney et al. 2014). 2.5.5 LOX in mouse development and human diseases During embryogenesis, the clearest function of LOX seems to be to ensure the proper physical endurance of the ECM in the developing tissues, especially in the vascular walls (Mäki 2009). When the expression of LOX was studied during embryonic development, LOX expression was found from embryonic day 9.5 (E9.5) onwards in rats and mRNA levels increased until E15.5, but the protein activity remained relatively constant (Tchaparian et al. 2000). In mouse, LOX expression can be seen at E11.5 in the cardiovascular system (Tsuda et al. 2003). Knocking out the Lox gene causes drastic defects in mice that cannot be compensated by any of the other LOX isoforms and leads to perinatal death (Mäki et al. 2002). Lox-/- embryos have defects in the aortic walls, which can be seen as a detachment of the internal elastic lamina, leading to aneurysms and cardiovascular dysfunction. Also the development of the respiratory system is disturbed, since Lox-/- embryos have severe defects in the branching of the distal and proximal airways. Characteristic of these problems are abnormal structure and distribution of collagen fibers and elastinolysis in various tissues (Hornstra et al. 2003, Mäki et al. 2002, Mäki et al. 2005). In full-term Lox-/- mice the collagen crosslinking is reduced by 40% in the total body (Hornstra et al. 2003) and in E18.5 embryos the reduction of LOX activity in the skin and aortic smooth muscle cells is 80% (Mäki et al. 2005). Changes in LOX expression or activity have been associated with different human diseases. Decreased LOX activity has been linked to Menkes syndrome and its less severe variant, the occipital horn syndrome (Kaler 1998, Kemppainen et al. 1996, OMIM 309400). Characteristic of these recessively inherited disorders is abnormalities in copper metabolism. Mutations in the ATP7a gene 45

coding for a copper-transporting P-type ATPase causes low concentrations of serum copper and ceruloplasmin. The LOX mRNA level is also downregulated in fibroblasts isolated from Menkes patients. In Menkes disease the lack of copper leads also to inhibition of other copper requiring enzymes in addition to the LOX enzyme family and results in a wide spectrum of manifestations, such as cerebral and cerebellar neuronal cell loss, bladder diverticula, loose skin and loose joints. The condition leads to death usually by the age of 3. In occipital horn syndrome the neurological involvement is not very profound and the connective-tissue symptoms are more prominent. A decrease in LOX mRNA and activity has also been found in women with pelvic organ prolapse (Alarab et al. 2010, Klutke et al. 2010). Studies have shown a significantly higher rate of methylation in the LOX promoter region in women with pelvic organ prolapse leading to silencing of the LOX gene. The disease has been shown to have strong familial history. In Wilson’s disease, there is also a defect in copper metabolism, but the mutation is in the ATP7b gene, leading to copper accumulation and strong LOX upregulation in the patients’ livers (Vadasz et al. 2005). The patients suffer from hepatic damage caused by progressive hepatic cirrhosis and rapidly progressive liver failure accompanied by fibrosis. LOX is seen as a promoter of the fibrosis by oxidizing the accumulating collagen fibers in hepatocytes. Increased LOX activity has been associated also with other fibrotic disorders such as fibrotic liver disease, scleroderma and pulmonary fibrosis. LOX contributes significantly to the stabilization of collagen fibers leading to severe and dense fibrosis (Kagan 1994). LOX is also upregulated in Alzheimer’s disease, where active LOX molecules in the ECM are suggested to contribute to plaque formation in the hippocampus of the patient’s brain (Gilad et al. 2005). Upregulation of LOX mRNA has been found in various tumor tissues (Perryman & Erler 2014). In breast cancer the tumor progression has been correlated with LOX expression and a high level of LOX mRNA is a poor prognostic factor. Inhibition of LOX does not significantly affect the primary tumor size, but the metastatic nature of the breast cancer cells were inhibited both in early and late stages of metastasis (Erler et al. 2006, Kirschmann et al. 2002). In the process of metastasis, LOX mediates the migration of the breast cancer cells via H2O2, which is the byproduct of the LOX reaction. It affects FAK and Src kinase (FAK/Src), which are the main proteins of the adhesion complex (Payne et al. 2005). The secreted mature LOX from the primary tumor prepares the invasion sites in the lung for metastasis by crosslinking collagen in the 46

preparation of a pre-metastatic niche (Erler et al. 2009). In colorectal cancer LOX is also upregulated and causes increased tissue stiffness and activation of FAK/Src signaling by crosslinking the collagens, which promotes cancer progression (Baker et al. 2013a). LOX was also shown to induce VEGF via PDGFRβmediated activation of Akt (Baker et al. 2013b). In addition to the ability of LOX to promote cancer cell invasion and metastasis, it can also work against cancer. LOX is able to inhibit transformation of mouse NIH 3T3 cells by suppressing Ras activity. It has 92% sequence similarity with rrg (ras recission gene) at the cDNA level and is able to work as a tumor suppressor (Contente et al. 1990, Kenyon et al. 1991). LOX inhibits the Akt and ERK kinases leading to inhibition of signaling pathways that lead to the activation of the transcription factor nuclear factor-κB (NF-κB), which mediates the transformation by Ras (Jeay et al. 2003). Later it was discovered that this LOX mediated transformation inhibition does not require LOX enzyme activity, but instead requires LOXPP (Palamakumbura et al. 2004). Since the discovery of the LOX antitumor activity many studies have shown that LOX expression is indeed decreased in many cancer cell lines and their corresponding tumor tissues, for example in basal and squamous cell carcinoma, colon carcinoma, melanoma and pancreatic cancer (Payne et al. 2007). Several additional reports highlight the antitumor role of LOX-PP in different cancers. In the Her-2/neu-driven mouse breast tumor cell line, LOX-PP inhibits the transformation and the tumor formation in vivo, by inhibiting Her-2/neu induced signaling cascades that function via Ras (Min et al. 2007). In addition to breast cancer, LOX-PP has an inhibiting role in prostate (Palamakumbura et al. 2009) and lung cancer (Wu et al. 2007), Ewing sarcoma (Agra et al. 2013) and hepatocellular carcinoma (Zheng et al. 2014). LOX-PP has also been discovered to target DNA repair and sensitize prostate cancer to ionizing radiation (Bais et al. 2014).

47

2.6

Skeletal muscle development in mice

Fig. 7. Schematic of myogenesis and the specific markers of each stage. Satellite cells express transcription factor Pax3 during E10.5-E12.5. Pax7 is upregulated at E11.5 and continues to be expressed through myogenesis and in the adults. Myogenic regulatory factors (MRFs) are expressed in myoblasts, myocytes and myofibers. Myf5 (E10.5) is expressed in myoblasts. MyoD (10.5) is expressed in myoblasts and myofibers. Myogenin (E11.5) is expressed mainly in differentiated myocytes and myofibers. Mrf4 is the last MRF expressed (E13.5) and is expressed in myofibers. (Modified from Zammit et al. 2006)

Skeletal muscle is the most abundant tissue and constitutes nearly half of the human body mass (Grefte et al. 2007, Murphy & Kardon 2011). The muscle cell (also known as the myocyte or muscle fiber) is multinucleated and made up of many bundles of myofibrils, which are formed of repeating sarcomeres. Many muscle fibers bundle together and form fascicles and a complete muscle is made of many fascicles. All individual muscle fibers, fascicles as well as the whole muscle, are ensheathed with muscle connective tissue (MCT). Pax3 transcription factor positive progenitors originating in the somites produce the limb skeletal muscle. They migrate into the forelimbs by the 48

embryonic day 10.5 (E10.5) and a bit later to the hindlimbs. Once the migration is completed (E11.5) Pax7 is upregulated, followed by Pax3 downregulation during the fetal period (E12.5) (Relaix & Zammit 2012). Pax transcriptionfactors control the activation of the myogenic regulatory transcription factors (MRFs), which are inducers of myogenesis and are required in skeletal muscle differentiation. They are also markers for the cells that are differentiating into skeletal muscle and upregulation of Myf5, Mrf4 and MyoD lead to differentiation of progenitor cells to myoblasts and further the expression of myogenin leads to myocytes (Fig. 7) (Mok & Sweetman 2011, Relaix & Zammit 2012). In vertebrates myogenesis occurs in four subsequent phases and in the limb the muscle patterning is established during the first phase known as embryonic myogenesis (E10.5-E12.5). Muscle growth and maturation occur during fetal (E14.5-P0; P, postnatal day) and neonatal (P0-P21) myogenesis. This is followed by adult myogenesis (after P21), which includes postnatal growth and damage repair. During all the phases, progenitor cells divide and differentiate to myoblasts and then further to myocytes. Eventually the myocytes fuse forming multinucleated muscle fibers. Primary fibers are formed during embryonic myogenesis, and these form the scaffolding for secondary fibers. During fetal myogenesis, myocytes can either fuse with primary fibers or with one another to produce secondary muscle fibers. During fetal and early neonatal life, muscle fibers can grow by fusion of myocytes to the existing fibers. After this, muscle fibers increase in cross-sectional size by hypertrophy. After birth, muscle maturation progenitor cells enter quiescence as a form of Pax7 positive satellite cells, and are situated between the basal lamina and the muscle fiber membrane. Satellite cells have the potential to differentiate into a new fiber (Grefte et al. 2007, Mathew et al. 2011, Murphy & Kardon 2011). Extracellular signals have been also shown to direct muscle development, but they seem to regulate differentially embryonic and fetal myoblasts (Murphy & Kardon 2011). TGF-β and BMP signaling have no effect on embryonic myoblasts, whereas during fetal myogenesis as well as in adults, they both inhibit myoblast differentiation. Similarly wnt/β-catenin signaling was shown to affect only fetal myoblasts (Murphy & Kardon 2011). Muscle development requires delicate signaling and crosstalk between all the factors composing it. Increasingly the role of MCT has been emphasized not only participating in the muscle patterning but also contributing to the muscle development and differentiation (Hasson 2011). Early MCT contains plenty of fibroblasts, but lesser amounts of ECM components, such as type I collagen. 49

From the late stages of development, E15.5 onwards, the situation is reversed and MCT is rich in fibrillar collagen and proteoglycans and has fewer fibroblasts (Hasson 2011, Kardon et al. 2003). The development of muscle connective tissue is closely related to muscle development in a temporal and spatial manner and transcription factor Tcf4 positive mesodermal cells give rise to MCT fibroblasts (Kardon et al. 2003). Tcf4 is a member of the Tcf/Lef family and acts downstream of Wnt signaling. MCT fibroblasts regulate the muscle maturation and fiber type, which is both dependent and independent of Tcf4 (Anakwe et al. 2003, Kardon et al. 2003, Mathew et al. 2011). TGF-β is also expressed during myogenesis and has been shown to participate also in the regulation of the fiber-type composition of the myotubes. Myotubes formed without TGF-β differentiate into slow fibers, whereas the myotubes formed next to MCT expressing TGF-β will differentiate into fast fibers (McLennan 1993). TGF-β signaling can occur via a canonical or a noncanonical pathway. TGF-β binds to its receptor on the cell membrane and in the canonical pathway the binding phosphorylates the Smad2 and Smad3 signal transducers (Figure 4). The p-Smad2/p-Smad3 complex binds to Smad4 and the whole complex translocates into the nucleus and activates the transcription of TGF-β target genes (Fig. 8) (Rifkin 2005, Shi & Massague 2003).

50

Fig. 8. Canonical TGF-β signaling pathway. Binding of TGF-β to its receptors leads to phosphorylation of the downstream effectors Smad2 and Smad3, which form a complex and subsequently bind to Smad4 and translocate into the nucleus, where the complex acts as a transcription factor.

51

52

3

Aims of the present research

HIF-P4H enzymes regulate HIF-related pathways such as erythropoiesis, the production of red blood cells. HIF-P4H-2 is the main regulator of renal EPO production. HIF-P4H-1 and HIF-P4H-3 participate in the regulation, but to a much lesser extent. Inhibition of these enzymes has been considered as a potential pharmacological strategy to treat hypoxic diseases, such as anemia. A novel P4H-TM enzyme has been discovered and shown to be able to hydroxylate HIF in vitro and in cellulo. The mechanism for this is not clear, since the enzyme is bound to the ER and the catalytically active site is inside the lumen. The role of P4H-TM as a HIF-hydroxylating enzyme in vivo is not yet established. The first part of this study was designed to determine whether P4H-TM regulates HIF-α in vivo and whether it participates in the regulation of erythropoiesis in vivo. We hypothesized that if P4H-TM participates in the regulation of erythropoiesis, P4h-tm-/- mice should be more sensitive to an HIFP4H inhibitor (FG-4497) elicited erythropoietic response than wild-type mice. P4h-tm-/- mice, as well as hypomorphic Hif-p4h-2gt/gt, Hif-p4h-3-/- and wild-type mice were treated with FG-4497. FG-4497 is a 2OG analogue and has been shown to inhibit HIF-P4Hs in cell culture and in animals leading to HIF-1α and HIF-2α stabilization. We also crossed P4h-tm-/- mice with the Hif-p4h-2gt/gt mice to obtain a double gene-modified line. We studied the erythropoietic response of the mouse lines to the inhibitor and the hematological parameters of the doubledeficient Hif-p4h-2gt/gt/P4h-tm-/- mice. In earlier studies, LOX has been shown to be a crucial factor in cardiovascular and pulmonary development. The lack of LOX causes deficient crosslinking in collagens and elastin leading to poor arterial wall integrity, deficient development of the airways and diaphragmatic hernias. The impaired quality of ECM, due to the lack of LOX, has been seen also in certain other tissues. The development of muscle is a delicately regulated process. Muscles consist of many different components, such as the muscle fibers, satellite cells and fibroblasts. Individual muscle fibers, fasciculi of many fibers and the whole muscle itself are sheathed with MCT. Recent studies have shown that defects in MCT can unbalance muscle development, which is seen in many human muscular diseases. In the second part of the study, skeletal muscledevelopment was found 53

to be severely impaired in Lox-/- mice and the underlying mechanisms were investigated.

54

4

Materials and methods

The materials and methods used in this thesis are summarized in the tables below. Detailed descriptions with references can be found in the original articles I-II. Table 3. Materials. Knockout (KO) mice

Knockout strategy

P4h-tm-/-

Conventional KO

Used in I

Hif-p4h-2gt/gt*

Genetrap

I

Hif-p4h-3-/-

Conventional KO

I

Hif-p4h-2gt/gt/P4h-tm-/-

Genetrap/Conventional KO

I

Lox-/-

Conventional KO

II

*Various levels of wild-type Hif-p4h-2 mRNA expressed in the tissues: 8% in the heart; 15% in the skeletal muscle; 35% in the kidney and 85% in the liver (Hyvärinen et al. 2010).

Table 4. Methods. Level

Method

Used in

DNA

PCR

I, II

RNA

RNA isolation

I, II

RT-PCR

I, II

Quantitative real-time PCR

I, II

shRNA Protein

Cells and tissues

Animals Analysis

SDS-PAGE and Western Blotting

I, II

ELISA

I

Expression and purification of recombinant P4Hs

I

P4H activity assay using 2-oxo-[1-14C]glutarate

I

Analysis of hematological parameters

I

Cell culture

II

Preparation and staining of paraffin sections

II

Immunohistochemical staining

II

in situ hybridization

II

Electron microscopy and immunoelectron microscopy

II

Two-photon microscopy

II

Second harmony imaging microscopy

II

Administration of HIF-P4H inhibitor

I

Harvesting of tissues and collection of blood samples

I, II

Statistics

I, II

55

56

5

Results

5.1

Role of P4H-TM in erythropoiesis (I)

5.1.1 Inhibition of P4H-TM and HIF-P4Hs by FG-4497 FG-4497 is a small molecule HIF-P4H inhibitor (2-oxoglutarate analogue) provided by FibroGen. The efficiency of FG-4497 inhibition of P4H-TM was defined by studying the uncoupled decarboxylation of 2-oxo-[1-14C]glutarate without any peptide substrate, since no synthetic substrate is available for P4HTM. The concentration of FG-4497 required for 50% inhibition (IC50) of P4H-TM was 40 μM. The IC50 values for HIF-P4H-1, HIF-P4H-2 and HIF-P4H-3 were determined by measuring the hydroxylation-coupled release of 14CO2 from 2-oxo[1-14C]glutarate with a synthetic peptide (DLDLEMLAPYIPMDDDFQL (Innovagen)), which corresponds to the C-terminal hydroxylation site of the human HIF-1α ODDD. The corresponding IC50 values for HIF-P4Hs were 0.2-0.3 μM. To rule out the possibility that the less effective inhibition of P4H-TM resulted from the difference in the analysis, i.e. uncoupled decarboxylation vs. hydroxylation-coupled decarboxylation, the inhibition of HIF-P4H-2 by FG-4497 was defined also in a reaction without the substrate, which gave an identical IC50 value of 0.2 μM. The results thus showed that FG-4497 inhibits HIF-P4Hs more efficiently than P4H-TM. In a pilot study, the minimal effective dose of FG-4497 required to stabilize HIF-1α and HIF-2α in the liver and kidney in wild-type mice was defined. Different concentrations (6, 20, 60 and 100 mg/kg) of FG-4497 were administered to mice on days 1, 3, 6 and 8 and samples were collected 6 h after the last dose. The smallest dosage (6 mg/kg) was enough to stabilize HIF-1α in the kidney, but the stabilization was achieved in the liver only with a higher dosage of 60 mg/kg (Figure 1A in I). Stabilization of HIF-2α in the kidney and liver required a dosage of 100 mg/kg (Figure 1A in I). Increases in s-EPO level were seen already with the 20 mg/kg dosage and it increased in a FG-4497 dosedependent manner (Figure 1B and 1C in I). The dosage of 100 mg/kg increased the s-EPO level 40 fold when compared to the vehicle-receiving controls (Figure 1B and 1C in I). In all the following experiments of the study an orally administered dosage of 100 mg/kg was used.

57

5.1.2 Increased HIF-1α and HIF-2α stabilization in the kidneys of P4htm-/- relative to wild-type mice leads to increased EPO mRNA levels after FG-4497 treatment The main results of chapters 5.1.2-5.1.5 are summarized in Table 5 and described below.

58

5 59 9

-/-

No difference





*Dosage on Mondays, Tuesdays and Fridays, ** Only statistically significant differences are given.

Reticulocyte

No difference



Hematocrit

No difference

No difference

No difference

2.5 x





No difference No difference

Hemoglobin

liver**

Hepcidin mRNA

Epo mRNA liver**

2.5 x



difference

No

difference

No

5x

difference

No

No difference

3.5 x

No difference



No difference



No difference

5x

Hif-p4h-3-/-

 No difference No difference

No difference

No difference No difference



No difference

4x

No difference

4.5 x



Epo mRNA kidney**

Hif-2α liver



2.5 x

P4h-tm



difference

No

Hif-p4h-3

5 weeks* -/-

Hif-2α kidney

No difference

Hif-p4h-3

-/-

4 weeks*



4x

Hif-p4h-2

-/-

No difference

2.5 x

P4h-tm

gt/gt

3 weeks*



5x

Hif-p4h-3

-/-



12 x

Hif-p4h-2

-/-

-/-

and Hif-p4h-3 mice relative to wild type.

Hif-1α liver

3x

P4h-tm

gt/gt

6 h after single dose -/-

gt/gt

Hif-1α kidney

Serum Epo

Parameter

Table 5. FG-4497 (100 mg/kg) induced effects on P4h-tm , Hif-p4h-2

P4h-tm-/-, Hif-p4h-2gt/gt, Hif-p4h-3-/- and wild-type mice were given orally either the FG-4497 inhibitor or vehicle 3 times per week for 3-5 weeks and the samples were collected 6 h after the final dose. HIF-1α and HIF-2α stabilization was measured by Western blot analysis of kidney and liver lysates. Transcription of the Epo gene in kidney and liver was measured by quantitative PCR (qPCR). Vehicle treatment did not cause any changes in the measured parameters (Figure 4 in I). The inhibitor stabilized HIF-1α and HIF-2α in all animals, but differences were seen between the genotypes when compared to their wild-type controls. P4h-tm-/- mice had more HIF-1α and HIF-1α stabilization than the wild-type mice at the 5-week point in the kidney, but not in the liver (Figure 4A in I). This led to a statistically higher Epo mRNA level (2-fold) in the kidneys of the P4h-tm-/- mice (Figure 4A in I). A similar trend in the Epo mRNA level between the FG-4497 treated P4h-tm-/- and control mice was already seen at the 3-week time point, but the difference was not statistically significant (Figure 4A in I). In the liver there was no difference in the Epo mRNA level between the P4h-tm-/- and control mice after FG-4497 treatment at either of the time points (Figure 4A in I). Hif-p4h-2gt/gt mice have varying amounts of wild-type Hif-p4h-2 mRNA in different tissues. In the kidneys of the Hif-p4h-2gt/gt mice the expression level of wild-type Hif-p4h-2 mRNA is 35% and in the liver 85% of that in the wild-type mice. Hif-p4h-2gt/gt mice have (as expected) increased HIF-1α stabilization in the kidneys relative to the wild-type mice as seen in the Western blot analysis of the vehicle treated Hif-p4h-2gt/gt and wild-type mice (Figure 4B in I). Stabilization of HIF-1α and HIF-2α by FG-4497 was increased in the kidneys of the Hif-p4h-2gt/gt mice more than in the wild-type mice at the 3-week time point (Figure 4B in I). Similar but smaller differences between the genotypes were seen in the liver (Figure 4B in I). This led to a 4.5-fold increase in Epo mRNA expression level in the kidney, but not in the liver, of Hif-p4h-2gt/gt mice when compared to the wild type (Figure 4B in I). FG-4497 treated Hif-p4h-3-/- mice had no difference in the kidney HIF-α levels when compared to the wild-type mice at the 5-week time point, but differences were seen in the liver between the genotypes (Figure 4C in I). Hifp4h-3-/- livers had increased stabilization of HIF-1α and HIF-2α relative to the wild type (Figure 4C in I). This resulted also in an increase in the Epo mRNA level in the Hif-p4h-3-/- liver (Figure 4C in I). Hif-p4h-3-/- mice had a ~2.5-fold increase in the liver Epo mRNA level relative to the wild type already at the 360

week time point and the difference increased to a 4-5 fold increase through the 4week and 5-week time points (Figure 4C in I).

5.1.3 Increased serum EPO level in the P4h-tm-/- relative to wild-type mice after FG-4497 treatment The s-EPO amount was measured by a Quantikine Mouse Epo Immunoassay kit (R&D Systems). The effect of FG-4497 on the s-EPO level was measured both after a single dosage and after a longer period (3-5 weeks) of repeated dosages. The terminal blood samples were taken 6 h after the last FG-4497 administration. The vehicle treatment did not have any effect on the s-EPO values in any time point analyzed and the vehicle treated P4h-tm-/-, Hif-p4h-2gt/gt, Hif-p4h-3-/- mice did not have changes in their s-EPO levels when compared to the wild type (Figure 2 and 3 in I). A single dosage of FG-4497 (100 mg/kg) caused a steep increase in the sEPO values of the wild-type mice and all three gene-modified mouse lines (Figure 2 in I). In wild-type mice FG-4497 treatment increased the s-EPO value by about 40-fold when compared to the vehicle group (Figure 2 in I). A significant further increase in the s-EPO level was seen in all the FG-4497 treated genetically modified mouse lines, when compared to their FG-4497 treated wildtype controls (Figure 2 in I). The increase in FG-4497 treated P4h-tm-/- mice was 3-fold, in Hif-p4h-2gt/gt mice 12-fold and in Hif-p4h-3-/- mice 5-fold when compared to the FG-4497 wild-type mice (Figure 2 in I). After repeated dosages of 100 mg/kg of FG-4497 for 3-5 weeks the increase in the s-EPO values was also higher in the gene-modified mice than in the wild type (Figure 3 in I). The increase was ~2.5-fold in the P4h-tm-/- mice relative to wild type at the 5-week time point, 4-fold in the Hif-p4h-2gt/gt mice at the 3-week time point and 5-fold in the Hif-p4h-3-/- mice at the 5-week time point (Figure 3 in I). 5.1.4 No difference in blood hemoglobin and hematocrit values between P4h-tm-/- and wild-type mice after FG-4497 treatment Blood counts were measured from the terminal blood samples with Cell-Dyn Sapphire (Abbott). Vehicle treatment had no effect on the blood parameters and no differences were observed between the vehicle treated gene-modified and 61

wild-type mice (Figure 5 in I). FG-4497 administration increased the hemoglobin (Hb) and hematocrit (Hct) values in all animals (Figure 5 in I). No differences were seen in the Hb and Hct values of P4h-tm-/- mice when compared to wild type at any time point measured, even though the reticulocyte count was significantly increased in the P4h-tm-/- mice relative to wild type at the 3-week time point (Figure 5A in I). Hif-p4h-2gt/gt mice had significantly higher Hb and Hct values at the 3-week time point after FG-4497 treatment when compared to the wild type (Figure 5B in I). In Hif-p4h-3-/- mice a higher reticulocyte count was seen at the 4week time point and higher Hct values at the 5-week time point after FG-4497 treatment relative to the wild type (Figure 5C in I). 5.1.5 Hepcidin mRNA levels are decreased more in P4h-tm-/- than wild-type mice after FG-4497 treatment Hepcidin mRNA levels were measured by qPCR from the liver lysates after repeated FG-4497 administrations for 3-5 weeks. Vehicle treatment did not affect Hepcidin mRNA levels and no differences were seen between the mouse genotypes after vehicle treatments (Figure 6 in I). FG-4497 treatment decreased Hepcidin mRNA levels in all genotypes (Figure 6 in I). A 60%-65% decrease was seen in the wild-type mice after the FG-4497 treatment (Figure 6 in I). A significantly larger decrease was seen in the P4h-tm-/- mice at the 5-week time point, a further ~60% decrease when compared to the wild-type FG-4497 treated mice (Figure 6A in I). The Hepcidin mRNA level was also decreased more significantly upon FG-4497 treatment in the Hif-p4h-2gt/gt mice relative to wildtype and a further decrease was observed in the Hif-p4h-3-/- mice, ~70% and 40%, respectively (Figure 6B and 6C in I). 5.1.6 Hif-p4h-2gt/gt/P4h-tm-/- double gene-modified mice have higher hemoglobin and hematocrit values P4h-tm-/- and Hif-p4h-2gt/gt mice were crossed to achieve a double gene-modified mouse line. Only female mice were used in the above analyses and we obtained 6 female Hif-p4h-2gt/gt/P4h-tm-/- mice from the crossings of Hif-p4h-2+/gt/P4h-tm+/or Hif-p4h-2+/gt/P4h-tm-/- mice with Hif-p4h-2+/gt/P4h-tm+/- or Hif-p4h-2+/gt/P4htm-/- mice. However, the number of wild-type females obtained was too low and therefore Hif-p4h-2+/+/P4h-tm+/-, Hif-p4h-2+/gt/P4h-tm+/+ and Hif-p4h-2+/gt/P4htm+/- were also used as controls in addition to the wild types. As we have shown 62

above that homozygous vehicle treated P4h-tm-/- and Hif-p4h-2gt/gt had no changes in hemoglobin and hematocrit values relative to wild type, these wereadditional controls that could be used. In total we had 21 controls. The Hif-p4h-2gt/gt/P4h-tm-/- mice had a small, but significant increase in the Hb and Hct values when compared to controls (Figure 7A and 7Bin I). The Hifp4h-2gt/gt/P4h-tm-/- mice had Hb values ranging between 149-180 g/L with a mean of 159.0 g/L (Figure 7A in I). The controls had Hb values ranging between 131151 g/L with a mean of 142.9 g/L (Figure 7A in I). We noticed a trend of increased Hb values (mean 148.0 g/L, range 136-161 g/L) also in the Hif-p4h2+/gt/P4h-tm-/- mice that are heterozygous for the Hif-p4h-2 hypomorph allele, but the change was not statistically significant (Figure 7A in I). Similar differences were seen in the Hct values (Figure 7B in I). Surprisingly, we saw a significant decrease in the s-EPO values in the Hifp4h-2gt/gt/P4h-tm-/- mice (Figure 7C in I). The lowest values were measured from the mice with the highest Hb values. When Epo mRNA values were measured in the kidney no changes were seen (Figure 7D in I). This was probably because the increases in Hb and Hct were small despite being significant, and the increase in Epo mRNA would probably be so small that it was missed within the variation of the individual values. Epo mRNA expression levels from the liver were low and some even under the detection threshold, but there was no difference between the Hif-p4h-2gt/gt/P4h-tm-/- and wild-type mice. Other tissues have been reported to produce EPO, such as the brain and bone (Rankin et al. 2012, Weidemann et al. 2009). We measured the Epo mRNA levels from the bone marrow and brain, but did not see any differences between the Hif-p4h-2gt/gt/P4h-tm-/- mice and controls. 5.2

Role of LOX in skeletal muscle development (II)

5.2.1 Reduced myofiber content and disorganized connective tissue in Lox-/- limbs Lox-/- pups (P0), that were born alive, were harvested and the limb skeletal muscles were subjected to whole-mount immunostaining of myosin heavy chain (MHC), which binds to myofibers. The analysis revealed abnormally patterned, shorter and smaller muscles particularly in the fore limb zeugopod area (Figure 1A, 1B, 1C and 1D in II). Electron microscopy (EM) of the muscles from the embryos of embryonic day 18.5 (E18.5) revealed, however, that sarcomeric 63

structures were grossly normal in the Lox-/- muscles (Figure S2 in II). To rule out a possible role of abnormalities in the bones and cartilage in the muscle phenotype alcian blue staining for cartilage and alizarin red staining for bone were performed. The results showed no obvious changes between the bones of Lox-/- and wild-type fore limbs and thus the phenotype of the skeletal muscles is not likely the result of skeletal patterning defects. To characterize the muscle phenotype further, muscle sections from E18.5 fore limbs were stained with periodic acid-Schiff and hematoxylin-eosin (H&E). Morphometric analyses revealed a significant decrease in the myofiber number in the Lox-/- muscles when compared to wild-type (Figure 1E and 1F in II). No changes were seen in the fiber diameter or shape (Figure 1E, 1F and S1 in II). The myofiber mass reduction was also confirmed by Western blot analysis using an anti-myosin antibody (Figure 1G in II). To analyze the amount of muscle connective tissue (MCT), Western blot analysis was performed to study the amount of Tcf-4 positive fibroblasts and type I collagen, which is the main component of the ECM of MCT. The results revealed a ~33% increase in type I collagen and a ~53% increase in fibroblasts in the Lox-/- muscle lysates when compared to wild type (Figure 2A in II). When the distribution and appearance of fibrillar collagen were analyzed by the second harmonic generation (SHG) technique, disorganized fibrillar collagen was discovered in the Lox-/- muscles (Figure 2B and 2C in II). Analyses with immunoelectron microscopy (IEM) by using a type I collagen antibody supported the results from the SHG technique (Figure S3 in II). Digital images were captured from the EM and widths of the fibrillar collagens were measured, which revealed a significant decrease in the mean width of the fibers in the Lox-/muscles when compared to wild type (Figure 2D, 2E and 2F in II). Immunohistochemical staining of the MHC in the zeugopod area revealed missing collagen type I lattices sheathing the fibers in the Lox-/- muscles (Figure 2G and 2H in II). Type I collagen was also found inside the Lox-/- muscle fibers, which indicate their contribution to the ECM secretion (Figure 2G and 2H in II). No changes were seen in the localization of the basement membrane type IV collagen type (Figure S4 in II).

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5.2.2 LOX is expressed in myofibers during embryonic myogenesis To determine the Lox expression pattern during muscle development, RNA in situ hybridization was performed. The analysis revealed that Lox expression coincides spatially and temporally with muscle development (Figure S5 in II). To discover which cells produce LOX, wild-type limbs from different embryonic days were dissected and stained with MHC (myofiber), Tcf4 (fibroblasts) or Pax7 (myoblast progenitors) antibodies together with a LOX antibody raised against a synthetic LOX peptide from the active site of the enzyme (EDTSCDYGYHRRFA; GenScript). The results revealed that at E13.5 Lox is expressed widely in the muscles including the myofibers expressing MHC (Figure 3A in II). At E15.5 Lox expression is restricted to MHC expressing cells, and surprisingly Lox was not strongly expressed in the Tcf4 positive fibroblasts in MCT or Pax7 positive myoblast progenitor cells (Figure 3B, 3C and 3D in II). The results were supported by Western blot analysis, where lysates from primary cultured MCT fibroblasts and the murine myoblast cell line C2C12 showed more Lox protein in the myoblast cells than in fibroblasts (Figure 3E in II). These results indicated that Lox is mainly expressed in the differentiated myofibers during muscle development. Myogenesis occurs in four subsequent phases: the first is embryonic myogenesis from E10.5 to E12.5-E13.5, followed by fetal myogenesis from E14.5 to birth (P0). To determine whether Lox affects embryonic myogenesis, E13.5 embryos were dissected and Lox expression in the limb was studied by whole-mount RNA in situ hybridization with myogenin and MyoD markers (Figure 4A and 4B in II). No differences were seen between the Lox-/- and wildtype mice at the end of embryonic myogenesis (Figure 4A and 4B in II). Wholemount immuno-staining of the Lox-/- and wild-type limbs at E14.5 to E16.5 with the My32 antibody against fast MHC was performed and the results revealed that the muscle phenotype is observed at E16.5 at the earliest, which is during the fetal myogenesis (Figure 4C, 4C, 4D and 4F in II). Immunohistochemical staining with a laminin antibody, marking the basal lamina, demonstrated a loss of the organization in the myofibers and a reduced amount of myofibers (Figure 4G and 4H in II).

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5.2.3 Increased TGF-β signaling in Lox-/- mice Previous reports have indicated, that decreased myofibers and increased MCT, which are seen in the Lox-/- mice, are associated with increased TGF-β signalling (Burks & Cohn 2011, Cusella-De Angelis et al. 1994, Heino & Massague 1990, Liu et al. 2004a). Interaction between LOX and TGF-β have also been reported to decrease TGF-β activity (Atsawasuwan et al. 2008). To test whether TGF- β is the mediator of the phenotype seen in the Lox-/- mice, mouse embryonic fibroblasts (MEFs) were isolated from E14.5 Lox-/- and wild-type embryos. MEFs were subjected to different concentrations of recombinant TGF-β (r TGF-β) and the effect of this on the downstream signaling molecules p-Smad2, Smad2, p-Smad3 and Smad3 was studied with Western blot analysis (Figure 5A in II). The amount of activated p-Smad2 was increased in Lox-/- MEFs relative to wild-type MEFs indicating increased TGF-β signaling (Figure 5A in II). p-Smad3 was detected in both the Lox-/- and wild-type cells, but there was no difference in the amount between the genotypes (data not shown). TGF-β signaling activity was also measured by using a reporter gene assay including a plasmid containing Smad2/3 binding sites upstream of a luciferase reporter (Cignal; Qiagen). The results showed significantly increased TGF-β signaling in the Lox-/- MEFs even without the addition of rTGF-β (Figure 5B in II). The same experiment was done with the myogenic C2C12 cell line, from which the endogenous Lox mRNA was targeted by short hairpin RNA viral particles (MISSION shRNA; Sigma Aldrich). The administration of rTGF-β resulted in a 2.5-fold higher activation of TGF-β signaling in mock treated cells, whereas the difference was 8.3-fold in the C2C12shLox cells (Figure 5C in II). To determine whether TGF-β activation was also increased in the Lox-/- skeletal muscles in vivo the expression of genes that are upregulated during increased TGF-β signaling were measured by qPCR. Total mRNAs were isolated from the fore limbs of E18.5 Lox-/- and wild-type mice. The results showed significantly increased mRNA levels of Pai-1, Ctgf and ColI, while the mRNA levels of α-Sma and ColIII were not upregulated (Figure 5D in II). Taken together, these results demonstrate that LOX regulates TGF-β activity and in the Lox-/- muscle the TGF-β signaling is increased. TGF-β signaling has been shown to increase the transcription of Lox and the Loxl genes in different studies (Gacheru et al. 1997, Sethi et al. 2011, Shanley et al. 1997, Xie et al. 2012a). To determine whether this Lox gene regulation by TGF-β signaling occurs during embryonic development, the affect of rTGF-β1 and rTGF-β2 on the developing chick limb was studied. Beads soaked with either 66

rTGF-β1, rTGF-β2 or PBS, were implanted to the chick limb bud and after two days Lox expression was determined by in situ hybridization. The results showed increased Lox expression in the limbs with rTGF-β beads when compared to the PBS treated beads (Figure S7 in II). This indicated that Lox regulation by TGF-β signaling occurs also during development. The mRNA levels of the Loxl genes were also measured from the muscle tissue of Lox-/- embryos by qPCR. Loxl1 and Loxl3 were upregulated by 15% and 25%, respectively, but this obviously was not enough to reverse the effect of Lox deficiency (Figure 5E in II). 5.2.4 Inhibition of TGF-β signaling rescues the Lox-/- muscle phenotype Our results indicate that LOX has a role as a repressor of TGF-β signaling during skeletal muscle development. To establish whether this is the case in vivo, 25 ng of a TGF-β receptor I kinase inhibitor (Calbiochem; 616451) was injected into pregnant Lox+/- mice at E12.5 and E13.5 to block the TGF-β signaling. This resulted in hemorrhages and embryonic lethality and not many embryos survived until birth. Surprisingly, many of the surviving embryos where either Lox-/- or Lox+/- and hardly any wild-types survived. For this reason, the embryos were harvested at E16.5, the earliest point at which the muscle phenotype was seen. Western blot results from the dissected fore limb lysates revealed that the inhibition reversed the amount of Tcf4 positive fibroblasts back to normal (Figure 6A in II). Whole-mount immunostaining with the My32 antibody specific for the fast MHC showed that the inhibition of the TGF-β signaling rescued to a large extent the abnormal Lox-/- skeletal muscle phenotype and showed only minor defects (Figure 6B, 6C and 6D in II).

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6

Discussion

6.1

P4H-TM contributes to regulation of erythropoiesis

Severe anemia can be caused by chronic kidney disease (CKD) or cancer, EPO deficiency being the primary cause. Before recombinant EPO became available for medical purposes, CKD was treated with regular blood transfusions. Recombinant EPO therapy has thus changed the lives of the patients suffering from anemia. Safety concerns have, however, hindered the use of recombinant EPO recently. High dosing has been associated with cardiovascular complications and in cancer patients with tumor growth and mortality. Some CKD patients are also resistant to EPO therapy resulting from decreased iron availability. (Jelkmann 2013, Muchnik & Kaplan 2011, Tanaka & Nangaku 2012) HIF-P4Hs are the main regulators of the HIF pathway and they are seen as potent therapeutic targets in treating disorders such as anemia, ischemia, inflammation and arterial disease (Baker et al. 2013b, Bernhardt et al. 2006, Bosch-Marce et al. 2007, Muchnik & Kaplan 2011, Myllyharju 2008, Myllyharju 2009, Ogle et al. 2012, Rabinowitz 2013, Robinson et al. 2008, Soni 2014). Some HIF-P4H inhibitors targeted to treat anemia are already in clinical trials to establish their safety and effectiveness in treating anemia. For example, treatment of CKD patients with the HIF-P4H antagonists FG-2261 and FG-4592, stabilizes HIF and induces Epo mRNA levels in the liver achieving low s-EPO levels that still are sufficient to activate erythropoiesis (Muchnik & Kaplan 2011). Induction of erythropoiesis via induction of the liver EPO production would be beneficial considering the fact that high EPO levels in the serum with the recombinant EPO treatments are associated with adverse outcomes. HIF also directly regulates factors involved in iron metabolism deficiency (Lee et al. 1997, Mukhopadhyay et al. 2000, Rolfs et al. 1997). All the HIF-P4H isoenzymes work as regulators of HIF, but they do have their distinct roles also. In addition to HIF they may have additional isoenzyme-specific targets (Koditz et al. 2007, Luo et al. 2011, Xie et al. 2009, Zhang et al. 2009). It is thus important to define the individual roles of the HIF-P4H isoenzymes to avoid undesired effects if their inhibition is used as a therapeutic strategy for anemia. The role of P4H-TM in HIF regulation in vivo has not been established before. It has been suggested to have possible targets other than HIF due to its ER localization (Koivunen et al. 2007). The results obtained from a zebrafish model 69

deficient in P4H-TM suggest it to have structural effects (Hyvärinen et al. 2010b, Koivunen et al. 2007). Results obtained in the current work clearly demonstrate the in vivo role of P4H-TM in the regulation of erythropoiesis. Administration of the inhibitor FG4497 to P4h-tm-/- mice resulted in a stronger erythropoietic response when compared to wild-type mice. HIF-1α and HIF-2α protein levels were increased in the P4h-tm-/- kidneys to a higher extent than in the wild-type kidneys upon FG4497 treatment, whereas no differences between the P4h-tm-/- and wild-type mice were seen in the liver. This led to higher upregulation of Epo mRNA in the P4htm-/- kidney relative to wild type. Thedecrease in Hercidin expression after FG4497 treatment was 60% greater in the P4h-tm-/- liver than in the wild-type. P4htm-/- mice had significantly higher s-EPO values (2.5-fold) than the wild-type mice after 5 weeks of dosing and already at the 3-week time point P4h-tm-/- mice tended to have higher s-EPO values. However, despite the higher s-EPO, no difference was seen in the Hb and Hct values between the FG-4497 treated P4htm-/- and wild-type mice even though the reticulocyte count was higher at the 5week time point (Fig. 9). This can result from the high baseline increase in the sEPO values set by the FG-4497 administration. The increase in s-EPO in the wild-type mice at the 5-week point was already 100-fold and the additional 2.5fold increase in the P4h-tm-/- mice might be too small to further elevate Hb and Hct values. Recent reports have shown, that downregulation of Hepcidin is not only HIF-dependent, but also requires an increase in Epo mRNA transcription and a signal from activated erythropoiesis from the bone marrow (Haase 2010, Mastrogiannaki et al. 2012). From this perspective, the decreased Hepcidin mRNA levels after FG-4497 treatment in the liver of P4h-tm-/- mice indicates that they have increased erythropoiesis, even though an increase in the Hb and Hct values was not seen in comparison to the wild-type mice. It cannot be excluded, however, that if the experiment had continued for a longer time, there could have been changes also in the Hb and Hct values. As expected, FG-4497 treatment increased the stabilization of HIF-1α and HIF-2α in the kidneys of the Hif-p4h-2gt/gt mice much more robustly than in the wild-type mice; a smaller difference was seen in the liver. Therefore, a significant increase in the Epo mRNA upregulation in the Hif-p4h-2gt/gt mice relative to wild type was only seen in the kidney. This led to a 5-fold higher increase in the serum EPO values of the the Hif-p4h-2gt/gt mice relative to wild type. These data agree with earlier data, where inactivation of Hif-p4h-2 from the kidney led to increased Epo production and erythrocytosis, whereas deletion from the liver had no effect 70

on erythropoiesis (Minamishima & Kaelin 2010, Takeda et al. 2008). FG-4497 treatment also resulted in differences in the erythropoietic response between the Hif-p4h-3-/- and wild-type mice. More extensive HIF-1α and HIF-2α stabilization was seen in the livers of Hif-p4h-3-/- mice, leading to higher liver Epo mRNA levels and serum EPO values than in the wild-type mice. No differences in HIF stabilization and Epo mRNA levels were seen between the Hif-p4h-3-/- and wildtype kidneys. These data agree with earlier reports that have shown that double deficient Hif-p4h-1-/-/Hif-p4h-3-/- mice have HIF-2α stabilized in the liver leading to increased Epo mRNA levels, whereas no changes were seen in the kidneys (Takeda et al. 2008). The role of P4H-TM in erythropoiesis was confirmed by crossing P4h-tm-/mice with Hif-p4h-2gt/gt mice to produce a double gene-modified mouse line Hifp4h-2gt/gt/P4h-tm-/-. The previous results from our study showed that at baseline Hif-p4h-2gt/gt mice do not have any signs of increased erythropoiesis when compared to wild-type mice. HIF-1α is slightly stabilized in the Hif-p4h-2gt/gt kidneys, but this does not result in Epo mRNA upregulation. Blood analyses revealed small but significant increases in the Hb and Hct values in the Hif-p4h2gt/gt/P4h-tm-/- mice. Surprisingly, the s-EPO values were downregulated in these mice (Fig. 9). A similar situation has been observed before with the Hif-p4h-1-//Hif-p4h-3-/- mice (Takeda et al. 2008). These double deficient mice had increased Hct values, but the s-EPO levels were significantly lower. It was hypothesized that the high amount of erythroid progenitor cells could bind the EPO from the serum via their EPO receptors (Takeda et al. 2008). However, our results do not exclude the possibility that the erythroid lineage of the Hif-p4h-2gt/gt/P4h-tm-/mice could be directly affected. As seen with human HIF-P4H-2 mutations leading to erythrocytosis, some of the patients nonetheless have normal levels of s-Epo (Percy et al. 2006, Percy et al. 2007). The results obtained from the current work together with data from other laboratories (Minamishima et al. 2008, Minamishima & Kaelin 2010, Takeda et al. 2008) support the role of HIF-P4H-2 as the main regulator of erythropoiesis. Our data suggest that simultaneous P4H-TM inhibition leads to a stronger renal erythropoiesis response and thus an inhibitor efficiently targeting these two enzymes could have a better outcome in treatment strategies where induction of the renal EPO pathway is required. Simultaneous activation of the hepatic EPO pathway would require an inhibitor that in addition targets the HIF-P4Hs 1 and 3.

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Fig. 9. P4H-TM cotributes to the regulation of erythropoiesis. Administration of FG4497 increased HIF-1α and HIF-2α stabilization in the kidney leading to increased -/-

serum EPO amounts in P4h-tm mice, when compared to control mice. This resulted in increased erythropoiesis and a decrease in Hepcidin levels in the liver. Crossing of -/-

P4h-tm with Hif-p4h-2

gt/gt

mice resulted in a double gene-modified mouse line, which

had increased hemoglobin and hematocrit values without any FG-4497 administration.

6.2

LOX participates in skeletal muscle development via TGF-β signaling

Developing tissue requires a balance between its constituting members and also the crosstalk between the surroundings. Skeletal muscle is closely in touch with bone tendons and ligaments and consists of muscle fibers, satellite cells, fibroblasts and muscle connective tissue (MCT). MCT is rich especially in proteoglycans and collagen fibers. Generally intrinsic transcription factors are considered to regulate myogenesis, but it has been revealed to be also essentially regulated by extrinsic factors via the MCT. MCT and the limb mesenchyme have important roles in the prepatterning, development and differentiation of the 72

muscle (Hasson 2011, Kardon et al. 2003, Mathew et al. 2011). The signaling between muscle and MCT is not thoroughly known. MCT expresses Tcf4, which is a transcription factor acting downstream of the Wnt signaling pathway and has been shown to regulate muscle patterning. However, it is clear that Tcf4 does not cover all the muscle regulation that originates from the MTC (Kardon et al. 2003). TGF-β signaling has been also shown to participate in myogenesis. It inhibits myoblast differentiation during fetal myogenesis and affects the fibertype formation (McLennan 1993, Murphy & Kardon 2011). Results obtained from the current work have revealed an important role for the interplay between LOX and TGF-β signaling in skeletal muscle development. In mouse embryonic skeletal muscle Lox is mainly produced by the differentiated MHC positive myofibers and only minimally by the TCF4 positive fibroblasts or PAX7 positive myoblast progenitor cells. Lox-/- mice had shorter, smaller and abnormally patterned muscles and the myofiber number was reduced. The amount of MCT components, the TCF-4 positive fibroblasts and type I collagen, was increased in the Lox-/- mice suggesting an increase in the MCT. Previously similar situations have been associated with increased TGF-β signalling (Allen & Boxhorn 1987, Burks & Cohn 2011, Heino & Massague 1990). Muscle defects occurred at E16.5 in the Lox-/- embryos, i.e. during fetal myogenesis (E14.5-P0). This also suggested a role for TGF-β in the developing muscle in Lox-/- embryos since TGF-β has been shown to be expressed from E15 onwards during embryogenesis and is able to inhibit fetal myoblast differentiation, but has no effect during embryonic myogenesis (E10.5-E12.5) (Cusella-De Angelis et al. 1994, McLennan 1993). Increased activation of TGF-β was found also from MEFs that were isolated from the Lox-/- embryos as well as in a myoblast cell line, C2C12, in which Lox was silenced by using shRNA. Increased activation of the TGF-β signaling pathway with recombinant TGF-β was seen in the Lox shRNA cells when compared to wild-type cells. pSmad2 levels were also higher in the Lox-/- MEFs treated with recombinant TGF-β than in wild-type MEFs. Altogether, these findings suggest increased TGF-β signaling in the Lox-/- embryos. Previously LOX has been shown to associate with TGF-β and thus reduce its activity (Atsawasuwan et al. 2008). This interaction requires LOX activity, since the effect on the target genes of TGF-β can be reversed by using the LOX inhibitor BAPN (Atsawasuwan et al. 2008). Whether this is how LOX affects TGF-β signaling in skeletal muscle development remains to be solved (Figure 10). A lack of LOX leads to deficient crosslinking of elastin and collagens in the 73

ECM, which leads to e.g. weak arterial walls, aortic aneurysms, diaphragmatic hernias and pulmonary defects (Mäki et al. 2002, 2005). Impaired crosslinking of ECM leads to increased susceptibility to proteolysis (Hornstra et al. 2003, Mäki et al. 2005). TGF-β is secreted into the ECM as an inactive latent complex. It is stored in the ECM as a latent form bound to the latent TGF-β binding protein (Rifkin 2005). Many loss-of-function mutations in ECM proteins lead to elevated rather than decreased TGF-β signaling (Horiguchi et al. 2012, Neptune et al. 2003). It cannot be excluded that TGF-β can be released more efficiently from the weaker ECM of the Lox-/- mice thus increasing the TGF-β signaling (Fig. 5). TGF-β has also been shown to upregulate the Lox and Loxl genes (Gacheru et al. 1997, Sethi et al. 2011, Shanley et al. 1997, Xie et al. 2012a). This was proved here to also occur during development of the chick limb. In the Lox-/- muscle tissue Loxl1 and Loxl3 were upregulated, but were not able to reverse the Lox-/phenotype. It remains to be established whether the LOXL enzymes can also directly affect the TGF-β activity. Inhibition of TGF-β in various pathological muscle conditions has proven to be beneficial, an example being the DMD related mouse model, mdx (Gosselin et al. 2004, Taniguti et al. 2011). In a similar manner, TGF-β inhibition by a TGF-β receptor I kinase inhibitor rescued the defective muscle phenotype in Lox-/- mice, and normalized the amounts of MCT and muscle fibers. According to our results inhibition of the TGF-β signaling was lethal to many of the wild-type embryos demonstrating the importance of TGF-β during development and normal physiology. Results obtained from the current work show a clear effect of LOX deficiency on mouse skeletal muscle development. The effect is mediated via TGF-β signaling, which has a physiological function in muscle development, but has been also associated with many muscular diseases, such as DMD. In DMD the mutation of the dystrophin gene leads to impaired sarcolemma, which will eventually physically break the membrane. This will lead to release of proteases and is followed by repeated cycles of of degeneration and regeneration, until the muscles are replaced with connective tissue, which leads to weakness. DMD patients have shortened life expectancy and, despite the clinical relevance, the mechanism of the disease is not well known. However, TGF-β signaling activity has been shown to play a role. The ECM released TGF-β can be at least partly the cause of the increased activity of the TGF-β pathway (Burks & Cohn 2011, Muntoni et al. 2003). In a recent work, inhibition of LOXL2 actually reduced lung and liver fibrosis (Barry-Hamilton et al. 2010). Understanding the LOX74

TGF-β feedback loop helps to explain muscle diseases and fibrosis and can lead to the development of treatment strategies.

Fig. 10. Suggested interplay between LOX and TGF-β signaling during muscle -/-

development of LOX embryos. Lox deficiency leads to increased TGF-β signaling via two potential routes: a) loss of the direct interaction between LOX and TGF-β and b) possible increased release of TGF-β from the impaired ECM. Together, this leads to activation of TGF-β signaling, upregulation of MCT and decreased myofiber differentiation.

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7

Conclusions and future prospects

P4H-TM has been previously shown to participate in HIF hydroxylation in vitro and in cellulo, but nothing has been known of its role in mammals in vivo. In the first part of this thesis the role of P4H-TM in regulation of HIF and erythropoiesis was studied in vivo. A small molecule HIF-P4H inhibitor, FG-4497, was administered to three different gene-modified mouse lines, P4h-tm-/-, Hif-p4h-2gt/gt and Hif-p4h-3-/-. No baseline differences were seen in erythropoiesis between the wild-type and the genetically modified mouse lines, but after the administration of the FG-4497, all the genetically modified mouse lines showed a stronger erythropoietic response to the inhibitor when compared to the wild-type in a tissue and HIF isoform specific manner. When P4h-tm-/- mice were crossed with Hif-p4h-2gt/gt mice, both lines having no signs of increased erythropoiesis individually, the double deficient Hif-p4h-2gt/gt/ P4h-tm-/- had increased erythropoiesis. This strongly supports the participation of P4H-TM in the regulation of erythropoiesis. As stated above, the responses of the individual gene-modified mouse lines to FG-4497 treatment were not identical. The HIFP4H isoenzymes and P4H-TM are regulated differentially, and have different expression patterns, target preferences and cellular localizations. These are to be taken into consideration when an individual enzyme is planned for use as a therapeutic target. Much is already known of the erythropoietic roles of HIF-P4H2 and HIF-P4H-3 studied in our experiments and the previous data were supported by the results of our experiments. Our study provided novel information on the fourth HIF hydroxylating enzyme, P4H-TM, that can be very valuable when designing new treatments for hypoxic diseases, such as anemia, that are more effective and do not have undesired side effects. Results show for the first time that P4H-TM has a role in mammalian erythropoiesis, particularly in the regulation of renal the Epo pathway. Based on the localization of P4H-TM in the ER lumen, its ability to hydroxylate also some of the other proline residues in HIF-α that are not hydroxylated by HIF-P4Hs and the study done with zebrafish with a P4H-TM deficiency (Hyvärinen et al. 2010b, Koivunen et al. 2007). P4H-TM is thought to have other roles as well. Further studies to identify the possible substrates for P4H-TM are in progress. In the second part of the thesis, the role of LOX in skeletal muscle development was studied. Muscle development is a delicate process where imbalances between comprising factors can cause drastic defects. LOX deficiency has been earlier shown to cause embryonic lethality due to impaired ECM in the 77

cardiovascular system. We show that in muscle development LOX regulates the activity of TGF-β by decreasing it. When developing muscle does not have sufficient LOX, the TGF-β signaling is increased, which leads to reduced muscle fiber number and increased MCT. This manifests as shorter and abnormally shaped muscles. MEFs isolated from the Lox-/- embryos had increased TGF-β signaling and increased p-Smad2 amounts. The increased TGF-β signaling in the Lox-/- muscle may occur via two routes: loss of direct interaction between LOX and TGF-β and increased release of TGF-β from the impaired Lox-/- ECM. It would be interesting to measure the direct amounts of active TGF-β and total TGF-β, which includes the ECM bound inactive latent form, and compare the results between Lox-/- and wild-type mice. Such data could reveal the role of TGFβ released from the impaired ECM. TGF-β signaling inhibits MRFs, such as MyoD and downstream molecules Smad3 was shown to directly interact with MyoD and Smad2 directly with myocyte enhancer factor-2 (MEF2) proteins (Kollias & McDermott 2008). MEF2 plays an important role as coactivator of MRFs. Studying the effects of LOX deficiency on these transcription factors could clarify the route by which TGF-β mediates the inhibition of myofiber differentiation. TGF-β signaling plays a role also in lung development (Shi et al. 2009) and it has been previously shown in our group that Lox-/- embryos manifest a phenotype of immature lung development (Mäki et al. 2005). It would be interesting to determind whether TGF-β signaling has a role in this phenotype. Wnts, including Wnt3, regule limb development (Thorsteinsdottir et al. 2011), and recently was also shown to upregulate LOX in mesenchymal progenitor cells (Khosravi et al. 2014). Whether it has an effect on LOX expression during muscle development remains to be solved. The data obtained from the Lox-/- embryos provides new perspective on muscle development and regeneration, which can help to explain muscle diseases and more importantly the role of muscle connective tissue in the regulation of skeletal muscle development.

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List of original papers This thesis is based on the following original papers, which are referred to in the text by their Roman numerals: I

II

Anu Laitala, Ellinoora Aro, Gail Walkinshaw, Joni M. Mäki, Maarit Rossi, Minna Heikkilä, Eeva-Riitta Savolainen, Michael Arend, Kari I. Kivirikko, Peppi Koivunen*, and Johanna Myllyharju* (2012) Transmembrane prolyl 4-hydroxylase is a fourth prolyl 4-hydroxylase regulating EPO production and erythropoiesis. Blood 120(16):3336-3344. Liora Kutchuk1*, Anu Laitala*, Sharon Soueid-Bomgarten, Pessia Shentzer, AnnHelen Rosendahl, Shelly Eilot, Moran Grossman, Irit Sagi, Raija Sormunen, Johanna Myllyharju, Joni M Mäki and Peleg Hasson (2014) Muscle composition is regulated by a Lysyl oxidase-Transforming growth factor beta feedback loop. Manuscript

Reprinted with permission from the American Society of Hematology (I) Original publications are not included in the electronic version of the dissertation.

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ACTA UNIVERSITATIS OULUENSIS SERIES D MEDICA

1263. Åström, Pirjo (2014) Regulatory mechanisms mediating matrix metalloproteinase8 effects in oral tissue repair and tongue cancer 1264. Haikola, Britta (2014) Oral health among Finns aged 60 years and older : edentulousness, fixed prostheses, dental infections detected from radiographs and their associating factors 1265. Manninen, Anna-Leena (2014) Clinical applications of radiophotoluminescence (RPL) dosimetry in evaluation of patient radiation exposure in radiology : Determination of absorbed and effective dose 1266. Kuusisto, Sanna (2014) Effects of heavy alcohol intake on lipoproteins, adiponectin and cardiovascular risk 1267. Kiviniemi, Marjo (2014) Mortality, disability, psychiatric treatment and medication in first-onset schizophrenia in Finland : the register linkage study 1268. Kallio, Merja (2014) Neurally adjusted ventilatory assist in pediatric intensive care 1269. Väyrynen, Juha (2014) Immune cell infiltration and inflammatory biomarkers in colorectal cancer 1270. Silvola, Anna-Sofia (2014) Effect of treatment of severe malocclusion and related factors on oral health-related quality of life 1271. Mäkelä, Tuomas (2014) Systemic transplantation of bone marrow stromal cells : an experimental animal study of biodistribution and tissue targeting 1272. Junttila, Sanna (2014) Studies of kidney induction in vitro using gene expression profiling and novel tissue manipulation technique 1273. Häkli, Sanna (2014) Childhood hearing impairment in northern Finland: prevalence, aetiology and additional disabilities 1274. Lehto, Liisa (2014) Interactive two-step training and management strategy for improvement of the quality of point-of-care testing by nurses : implementation of the strategy in blood glucose measurement 1275. Harjunen, Vanessa (2014) Skin stem cells and tumor growth : Functions of collagen XVIII in hair follicle cycling and skin cancer, and Bmx tyrosine kinase in tumor angiogenesis 1276. Savela, Salla (2014) Physical activity in midlife and health-related quality of life, frailty, telomere length and mortality in old age

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HYPOXIA-INDUCIBLE FACTOR PROLYL 4-HYDROXYLASES REGULATING ERYTHROPOIESIS, AND HYPOXIA-INDUCIBLE LYSYL OXIDASE REGULATING SKELETAL MUSCLE DEVELOPMENT DURING EMBRYOGENESIS

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