Thesis (Ph.D. (Zoology))--North-West University, Potchefstroom Campus, 2005.

Thesis (Ph.D. (Zoology))--North-West University, Potchefstroom Campus, 2005

Hypoxia is a common feature of solid tumors and one of the hallmarks of tumor microenvironment. Tumor hypoxia plays an important role in angiogenesis, malignant progression, metastatic development, chemo-radio resistance and favours immune evasion by the emergence of tumor variants with increased survival and anti-apoptotic potential. There is very little work done on the impact of tumor hypoxia on the regulation of tumor susceptibility to the lysis induced by cytotoxic antitumor response. Therefore, we asked whether hypoxia confers tumor resistance to cytotoxic T lymphocyte (CTL)-mediated killing. We demonstrated that exposure of target cells to hypoxia has an inhibitory effect on the CTL-mediated autologous target cell lysis. Such inhibition was not associated with an alteration of CTL reactivity and tumor target recognition. We also showed that the concomitant hypoxic induction of Signal transducer and activator of transcription 3 (STAT3) phosphorylation on tyrosine 705 residue (pSTAT3) and hypoxia inducible factor 1 alpha (HIF-1α) is functionally linked to the alteration of Non small cell lung carcinoma (NSCLC) target susceptibility to CTL-mediated killing. We also showed that hypoxia-induced resistance of lung tumor to CTL-mediated lysis was associated with autophagy induction in target cells. Inhibition of autophagy resulted in impairment of pSTAT3 (via inhibition Src kinase) and restoration of hypoxic tumor cell susceptibility to CTL-mediated lysis. Moreover, in vivo inhibition of autophagy by hydroxychloroquine (HCQ) in B16F10 tumor bearing mice and mice vaccinated with TRP2 peptide dramatically increased tumor growth inhibition. Collectively, the current study establishes a novel functional link between hypoxia-induced autophagy and the regulation of antigen specific T cell lysis and points to a major role of autophagy in the control of in vivo tumor growth.Finally, as resistance of tumor targets to killer cells is likely to be regulated by multiple factors, we further aimed to identify the microRNA's regulated by hypoxia in NSCLC and melanoma and their putative involvement in the regulation of tumor susceptibility to antigen-specific CTL-mediated killing. MicroRNA-210 (miR-210) was significantly induced in a HIF-1α dependent manner in NSCLC and melanoma cells and miR-210 was expressed in hypoxic zones of human NSCLC tissues. Moreover, we demonstrated that hypoxia-induced miR-210 regulates tumor cell susceptibility to CTL-mediated lysis in part by suppressing PTPN, HOXA1 and TP53I11 expression indicating that miR-210 plays a potential role in the regulation of anti-tumor immune response.

Thesis (Ph.D. (Zoology))--North-West University, Potchefstroom Campus, 2005

Submitted by Ronildo Prado (ronisp@ufscar.br) on 2017-08-22T17:21:37Z No. of bitstreams: 1 TeseRNS.pdf: 3515617 bytes, checksum: 251005e22df508ed87d98457bd4a5d3d (MD5) / Approved for entry into archive by Ronildo Prado (ronisp@ufscar.br) on 2017-08-22T17:21:47Z (GMT) No. of bitstreams: 1 TeseRNS.pdf: 3515617 bytes, checksum: 251005e22df508ed87d98457bd4a5d3d (MD5) / Approved for entry into archive by Ronildo Prado (ronisp@ufscar.br) on 2017-08-22T17:21:54Z (GMT) No. of bitstreams: 1 TeseRNS.pdf: 3515617 bytes, checksum: 251005e22df508ed87d98457bd4a5d3d (MD5) / Made available in DSpace on 2017-08-22T17:22:00Z (GMT). No. of bitstreams: 1 TeseRNS.pdf: 3515617 bytes, checksum: 251005e22df508ed87d98457bd4a5d3d (MD5) Previous issue date: 2016-09-15 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Hyperammonemia impairs skeletal muscle protein synthesis and induces autophagy by upregulating myostatin via nuclear factor-kappaB (NF-kB). Skeletal muscle ammonia metabolism occurs via synthesis of glutamate and glutamine via critical TCA intermediate α-KG, that regulates increase expression of hypoxia inducible factor 1α (HIF-1α). Furthermore, there is a interaction between HIF-1α and NF-kB. Objective: This study evaluated the effects of hyperammonemia in NF-kB and HIF-1α cross-talking. Methods: To examine the effects of ammonium acetate intervention under HIF-1α signaling through NF-kB pathway it has generated a stable knockdown cell line for NFkB and the subunits α and β of the I kappa B kinase (IKK) complex (IKKα and IKKβ) evaluating the HIF-1α and myostatin activities. Results: The protein expression of HIF- 1α was significantly higher in C2C12 murine myotubes under ammonium acetate intervention compared to control. Once the deletion of NF-kB, IKKα and IKKβ occurs, the HIF-1α is not expressed, suggesting a cross-talking between them. The protein expression of myostatin was significantly higher in C2C12 IKKα deletion under ammonium acetate intervention compared to C2C12 random suggesting that myostatin is not IKKα dependent. Conclusion: We conclude that hyperammonemia is a normoxemic activator of HIF-1α through NF-kB pathway that results in sarcopenia via up-regulation of myostatin expression. / Um dos grandes mediadores de sarcopenia em cirrose é a hiperamonemia. Esta anormalidade metabólica é frequente em doenças hepáticas promovendo conversão danificada de amônia em uréia prejudicando a síntese protéica e induzindo autofagia do músculo esquelético pela up-regulação de miostatina via fator nuclear kappa B (NFkB). O metabolismo de amônia no músculo esquelético ocorre via síntese de glutamato e glutamina através do intermediário metabólico α-cetoglutarato do ciclo de Krebs, que regula o Fator Induzido por Hipóxia (HIF-1α). Além disso, existe um interação entre os dois fatores de transcrição HIF-1α and NF-kB. Objetivo: Este estudo avalia os efeitos da hiperamonemia no cross-talking entre NF-kB and HIF-1α. Métodos: Para examinar os efeitos do aumento da concentração de amônia na expressão de HIF-1α através da via NF-kB foi realizado um knockdown estável de NF-kB, e as subunidades IKKα e IKKβ do complexo I kappa B quinase (IKK) em células C2C12 avaliando as atividades de HIF- 1α e miostatina. Resultados: A expressão protéica de HIF-1α foi significativamente alta em células C2C12 sob tratamento com acetato de amônia comparado com controle. Uma vez que ocorre o silenciamento de NF-kB, IKKα and IKKβ em células C2C12, não é observada a expressão protéica de HIF-1α, sugerindo um cross-talking entre eles. A expressão protéica de miostatina foi significativamente alta em células C2C12 silenciadas para IKKα sob tratamento com acetato de amônia comparado com células C2C12 random (controle), sugerindo que miostatina não é dependente da quinase IKKα. Conclusão: Foi concluído que hiperamonemia é um ativador normóxico de HIF-1α através da via de sinalização NF-kB resultando em sarcopenia via up-regulação de miostatina.

A galectina-3 (gal-3) pertence a uma família de proteínas com domínios de ligação a beta-galactosídeos e está relacionada com diversos aspectos tumorais, como proliferação e adesão celular, angiogênese e proteção contra morte celular. Estudos mostram sua relação com o fenômeno da hipóxia, característica de diversos tumores sólidos que apresentam altas taxas de proliferação celular. A adaptação à hipóxia é mediada principalmente pelo Fator Induzido por Hipóxia (HIF-1), a qual atua na indução de diversos genes de sobrevivência em ambientes com baixas concentrações de oxigênio. Além de HIF, outros fatores são importantes nesse processo, como NF-kB, por exemplo, sendo um fator de transcrição responsivo a diversos estresses celulares, entre eles, a hipóxia. Alguns modelos tumorais apresentam-se ideais para o estudo dos efeitos da hipóxia no microambiente tumoral, como os glioblastomas. Estes são tumores do sistema nervoso central com altas taxas de letalidade, são refratários aos principais métodos de tratamento por sua plasticidade, crescimento infiltrativo e heterogeneidade. Histologicamente, estes tumores apresentam atipia nuclear, altas taxas de mitose e áreas de pseudopaliçada. Postula-se que estas áreas sejam compostas por células migrantes de ambientes necróticos, os quais são também hipóxicos devido a sua distância de vasos sanguíneos e é demonstrado que estas células expressam tanto HIF-1alfa quanto gal-3. Ensaios in vitro realizados por nosso grupo demonstraram que a gal-3 é positivamente regulada pela hipóxia em uma linhagem de glioma híbrido, NG97ht, além de demonstrar que esta proteína é um fator chave na proteção destas células contra a morte celular induzida pela privação de oxigênio e nutrientes, mimetizando condições necróticas de pseudopaliçada in vivo, destacando-se as habilidades antiapoptóticas desta proteína. Embora uma de suas possíveis funções tenha sido elucidada, os mecanismos de atuação e de indução da gal-3 ainda são obscuros. Deste modo, este projeto visa explorar os papéis pró-tumorais da gal-3, podendo torná-la um possível alvo em terapias anti-neoplásicas, entendendo melhor seus mecanismos de proteção contra a morte celular e controle de expressão em ambientes hipóxicos, além de estudar suas possíveis funções in vivo no desenvolvimento de tumores, e também estendendo seus estudos para outras linhagens de glioblastoma. Nossos resultados demonstraram que a gal-3 está co-localizada com mitocôndrias nestas linhagens de glioma, podendo sofrer alterações pós-traducionais em hipóxia, como a fosforilação e que houve acúmulo de HIF-1alfa nuclear nestas células em hipóxia. Vimos também que a gal-3 na linhagem NG97ht apresentou-se proveniente de dois alelos diferentes e que fatores intermediários deveriam ser expressos previamente pela célula antes da indução de gal-3 em hipóxia. Também demonstramos que houve dependência de NF-kB na indução transcricional de gal-3 nestas condições. Estes experimentos também demonstraram que a exposição de células à hipóxia e privação de nutrientes é capaz de induzir tanto espécies reativas de oxigênio como o aumento da autofagia nestas células, fatores importantes na indução da morte celular, além de demonstrar que na linhagem NG97ht a indução da morte nestas condições ocorreu por necrose, sem apresentar apoptose celular. Expandimos esta teoria da participação da gal-3 como molécula protetora contra a morte em hipóxia e privação de nutrientes para outra linhagem de glioma humano, a T98G. E finalmente, demonstramos que a diminuição da expressão de gal-3 em células tumorais da linhagem U87MG levou a diminuição das taxas de estabelecimento e crescimento tumoral in vivo / Galectin-3 (gal-3) belongs to a family of proteins with beta-galactoside binding domains and is related to various tumoral aspects, such as cell proliferation and adhesion, angiogenesis and protection against cell death. Studies show its relationship with the hypoxia phenomenon, a characteristic of many solid tumors that have high cell proliferation rates. The adaptation to hypoxia is mainly mediated by Hypoxia Induced Factor (HIF-1), which acts in the induction of several survival genes in environments with low oxygen concentrations. In addition to HIF, other factors are important in this process, such as NF-kB, for example, which is a transcription factor responsive to various cellular stresses, including hypoxia. Some tumor models are ideal for studying the effects of hypoxia in the tumor microenvironment, e.g. glioblastomas. These central nervous system tumors with high mortality rates are refractory to the main treatment methods due to their plasticity, heterogeneity and infiltrative growth. Histologically, these tumors exhibit nuclear atypia, high mitotic rates and pseudopalisading areas. It is postulated that these areas are composed of migrating cells out of necrotic microenvironments, which are also hypoxic due to their distance from the blood vessels and it is shown that these cells express both HIF-1alfa and gal-3. In vitro assays performed by our group demonstrated that gal-3 is positively regulated by hypoxia in a hybrid glioma cell line, NG97ht, and demonstrated that this protein is a key factor in protecting these cells against cell death induced by oxygen and nutrient deprivation conditions mimicking necrotic pseudopalisading areas in vivo, highlighting the pro-survival abilities of this protein. Although one of its possible functions has been elucidated, gal-3 mechanisms of action and induction are still unclear. Thus, this project aims to explore the gal-3 pro-tumoral effects, which may make it a possible target for anti-neoplastic therapies, better understanding the mechanisms of protection against cell death and expression in hypoxic environments, and also study its possible functions in vivo, extending these studies to other glioma cell lines. Our results demonstrated that gal-3 is located within the mitochondria in these glioma cell lines and may undergo posttranslational modifications in hypoxia, such as phosphorylation and that there is accumulation of nuclear HIF-1alfa in these cells under hypoxia. We have also seen that gal-3 in the NG97ht cell line presents two different alleles and that intermediate factors must be expressed previously by the cell before gal-3 induction in hypoxia. We also demonstrated that there is dependence on the NF-kB transcriptional factor for the gal-3 induction under these conditions. These experiments also demonstrated that exposure of cells to hypoxia and nutrient deprivation is capable of inducing reactive oxygen species and increased autophagy in these cells, which are important factors in the induction of cell death. In addition, we demonstrated that the induction of the NG97ht cell death in these conditions is due to necrosis. We expanded this theory of the participation of gal-3 as a protective molecule against cell death in hypoxia and nutrient deprivation to another human glioma cell line, T98G. And finally, we demonstrated that decreased expression of gal-3 in the U87MG glioma cell line leads to lower tumor establishment rates and decreased growth in vivo

Abstract Collagens are subject to extensive post-translational modifications, including the formation of 4-hydroxyproline, 3-hydroxyproline and hydroxylysine. These reactions are catalyzed by collagen prolyl 4-hydroxylases (C-P4Hs), prolyl 3-hydroxylases (P3Hs) and lysine hydroxylases (LHs), which belong to the 2-oxoglutarate-dependent dioxygenase family and require oxygen for their reaction. 4-Hydroxyproline residues have for a long time been known to be required for the stability of the collagen triple helix, but the role of prolyl 3-hydroxylation was revealed only a few years ago when mutations in P3H1 and the consequent loss of a single 3-hydroxyproline in collagen I was shown to cause recessive osteogenesis imperfecta. In this thesis the human P3H isoenzymes were expressed as recombinant enzymes, and analyses of their tissue expression and kinetic properties revealed that P3H2 is located in tissues rich in basement membranes and that it hydroxylates collagen IV, the major basement membrane collagen. The roles of the collagen hydroxylases and collagen IV in basement membrane formation were further studied using Madin-Darby canine kidney (MDCK) epithelial cells as an in vitro model for cell polarization. 4-Hydroxyproline also has a pivotal role in the system of cellular response to reduced oxygen levels (hypoxia). At a normal oxygen concentration, two proline residues in the α subunit of the hypoxia-inducible factor (HIF) are 4-hydroxylated by the HIF-P4Hs, which target HIF-α for proteasomal degradation. In hypoxia, the HIF-P4Hs are inactive, and the α subunit thus escapes degradation, dimerizes with a β subunit and after recruiting transcriptional coactivators induces the transcription of hypoxia-responsive genes in order to adapt the cell to hypoxia. Three human HIF-α subunits have been characterized to date, of which the third is known to be subject to extensive alternative splicing, with one of the splicing variants acting as a negative regulator of the hypoxia responsive system. Four novel splicing variants generated from the human HIF-3α locus are characterized here, and the expression of HIF-3α variants has been shown to be upregulated by hypoxia in a HIF-1 dependent manner. Further studies on the binding partners and transcriptional activity of HIF-3α revealed that this subunit has a more complex role in the adaptation of cells to hypoxia than had been expected. / Tiivistelmä Kollageenit ovat valkuaisaineita, joihin kohdistuu useita synteesin jälkeisiä muokkauksia kuten 4-hydroksiproliinin, 3-hydroksiproliinin ja hydroksilysiinin muodostuminen. Näitä reaktioita katalysoivat kollageeniprolyyli-4-hydroksylaasit (C-P4H:t), prolyyli-3-hydroksylaasit (P3H:t) ja lysyylihydroksylaasit (LH:t), jotka kuuluvat 2-oksoglutaraattidioksygenaasien entsyymiperheeseen ja tarvitsevat happea reaktioonsa. 4-hydroksiproliinitähteiden on kauan tiedetty stabiloivan kollageeninrakenteen, kun taas 3-hydroksiproliinitähteiden merkitys on selvinnyt vasta viime vuosina. Mutaatiot P3H1-isoentsyymiä koodittavassa geenissä ja sen seurauksena yhden ainoan 3-hydroksiproliinitähteen puuttuminen kollageenissa I johtavat vaikeaan luustosairauteen, osteogenesis imperfectaan. Tässä väitöskirjassa ihmisen P3H:t tuotettiin rekombinanttiproteiineina. Tulokset paljastivat, että P3H2 ilmentyy erityisesti kudoksissa, joissa on paljon tyvikalvorakenteita ja että P3H2 hydroksyloi tehokkaasti kollageeni IV:n kaltaisia synteettisiä peptidejä. Lisäksi koiran munuaisten epiteelisoluihin pohjautuvaa in vitro-mallia käytettiin apuna tutkiessamme kollageeneja hydroksyloivien entsyymien ja kollageenin IV roolia tyvikalvon muodostumisessa sekä solujen polarisaatiossa. Kollageenia stabiloivan tehtävänsä lisäksi 4-hydroksiproliinilla on myös merkittävä rooli solujen vasteessa vähähappisille olosuhteille (hypoksia). Normaalissa happiosapaineessa (normoksia), hypoksiaindusoituvan tekijän (HIF) α-alayksikköön muodostuu HIF-P4H entsyymien katalysoimana kaksi 4-hydroksiproliinitähdettä, jotka kohdistavat α-alayksikön proteasomaaliseen hajotukseen. Hypoksiassa HIF-P4H:t eivät kykene toimimaan, jolloin α-alayksikkö säästyy hajotukselta, muodostaa kompleksin β-alayksikön kanssa ja sitoo transkriptiokofaktoreita. HIF-kompleksi kykenee tällöin lisäämään hypoksiassa tarvittavien geenien luentaa. Tänä päivänä tunnetaan kolme HIF α-alayksikköä, joista HIF-3α:sta tiedetään esiintyvän useita erilaisia silmukointimuotoja ja yhden näistä muodoista tiedetään toimivan negatiivisena säätelijänä hypoksiavasteessa. Tässä väitöskirjatyössä on tunnistettu neljä uutta HIF-3α:n silmukointimuotoa ja osoitettu että HIF-3α:n määrä kasvaa hypoksiassa HIF-1:n säätelemänä. Lisäksi sitoutumis- ja transkriptiokokeet paljastivat, että HIF-3α:n rooli hypoksiavasteessa on monimutkaisempi kuin aikaisemmin kuviteltiin.

Abstract Adequate oxygen supply is necessary for aerobic cell survival. Cellular oxygen deprivation, also known as hypoxia, leads to various responses that aim to increase cellular oxygen delivery and reduce oxygen consumption. Oxygen homeostasis is mainly regulated by the hypoxia-inducible factor (HIF), which regulates the expression of over 300 genes in response to hypoxia. The stability of HIF is regulated by the HIF prolyl 4-hydroxylases (HIF-P4Hs), enzymes that catalyze the hydroxylation of proline residues in HIFα subunits and target them towards proteasomal degradation. HIF-P4Hs require oxygen as a cosubstrate for the reaction, allowing for hypoxic HIF stabilization and target gene induction at low oxygen concentrations. In this study we investigated the role of HIF-P4H-2 in the regulation of red blood cell production, erythropoiesis. We showed that Tibetans living at high altitude have genetically adapted to their hypoxic environment via mutations in the gene encoding for HIF-P4H-2. The Tibetan HIF-P4H-2 D4E,C127S variant showed enhanced hydroxylation of HIFα at low oxygen concentrations, resulting in reduced HIFα protein stabilization under hypoxia. In other studies we used a genetically modified HIF-P4H-2 hypomorphic mouse line which expresses a reduced amount of wild-type Hif-p4h-2 mRNA in tissues. We showed that these mice develop mild age-dependent erythrocytosis due to splenic extramedullary erythropoiesis, which is independent of serum erythropoietin concentration. In addition, these mice were protected against inflammation-induced anemia, a condition commonly seen in patients with inflammatory diseases. The HIF-P4H-2 hypomorphic mice also had altered basal metabolism in their skeletal muscles, which, together with an increase in mean capillary area, reduced their infarct size after skeletal muscle ischemia-reperfusion injury. These studies suggest that pharmacological HIF-P4H-2 inhibition may provide a novel treatment for EPO-resistant anemias and peripheral artery disease. / Tiivistelmä Riittävä hapensaanti on välttämätöntä aerobisten solujen selviytymiselle. Solun alentunut hapen määrä, toiselta nimeltään hypoksia, johtaa useisiin vasteisiin joiden tarkoituksena on turvata solun hapensaanti ja vähentää hapenkulutusta. Happitasapainoa säätelee hypoksiassa indusoituva tekijä (HIF), joka aktivoi yli 300 geenin luentaa hypoksisissa oloissa. HIFα:n määrää soluissa säätelevät HIF prolyyli-4-hydroksylaasientsyymit (HIF-P4H:t), jotka hydroksyloivat proliini-aminohappotähteitä HIFα-alayksiköissä ja ohjaavat ne proteasomaaliseen hajotukseen. HIF-P4H:t tarvitsevat reaktiossa happea mahdollistaen HIF:n stabilisaation ja kohdegeenien lisääntyneen luennan matalassa hapen osapaineessa. Tässä tutkimuksessa selvitimme HIF-P4H-2-entsyymin roolia punasolujen muodostuksen eli erytropoieesin säätelyssä. Osoitimme, että korkealla vuoristossa asuvat tiibetiläiset ovat geneettisesti sopeutuneet hypoksiseen elinympäristöönsä johtuen HIF-P4H-2-entsyymiä tuottavan geenin mutaatiosta. Tiibetiläisiltä löytynyt HIF-P4H-2D4E,C127S variantti hydroksyloi tehokkaammin HIFα-alayksiköitä matalassa hapen osapaineessa johtaen vähäisempään HIFα-alayksiköiden stabiloitumiseen hypoksiassa. Muissa tutkimuksissamme käytimme geneettisesti muunneltua HIF-P4H-2-hiirikantaa, joka tuottaa alentunutta määrää villityypin Hif-p4h-2 lähetti-RNA:ta kudoksissaan. Näille hiirille kehittyi ikäriippuvaisesti lievä punasoluylimäärä eli erytrosytoosi johtuen pernan kiihtyneestä punasolutuotannosta riippumatta seerumin erytropoietiinikonsentraatiosta. Lisäksi nämä hiiret olivat suojassa tulehduksen aiheuttamalta anemialta, joka on yleinen ilmiö tulehduksellisista sairauksista kärsivillä potilailla. HIF-P4H-2-muuntogeenisten hiirten lihasten energia-aineenvaihdunta oli muuttunut siten, että se yhdessä suurentuneen keskimääräisen kapillaaripinta-alan kanssa pienensi vaurioituneen kudoksen pinta-alaa alaraajaiskemia-altistuksen jälkeen. Nämä tutkimukset osoittavat, että lääkkeellinen HIF-P4H-2-entsyymin estäminen on mahdollinen uusi hoitomuoto erytropoietiinille resistenteissä anemioissa sekä alaraajojen valtimoahtaumataudissa.

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 oxygen-dependent 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 HIF-P4H 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. / 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.

Abstract Oxygen is essential for aerobic organisms, as shortage of oxygen (hypoxia) can induce cellular dysfunctions and even cell death, leading to tissue damage and decreased viability of the organism. Oxygen homeostasis is regulated delicately by several mechanisms, the major one being the hypoxia-inducible factor (HIF) pathway that is evolutionarily conserved. HIFα subunits are regulated in an oxygen-dependent manner via three HIF prolyl 4-hydroxylases (HIF-P4Hs). In the presence of oxygen HIF-P4Hs modify HIFα, which leads to its degradation, whereas in hypoxia the HIF-P4H enzymes cannot function and HIFα is stabilized. HIF regulates more than 300 genes that enhance oxygen delivery from the lungs to tissues and reduce oxygen consumption in tissues, such as those for erythropoietin and vascular endothelial growth factor. When a tissue suffers from hypoxia caused by a circulatory restriction, the situation is called ischemia. In this study we used a genetically modified HIF-P4H-2 hypomorph mouse line that expresses 8% of the wild-type Hif-p4h-2 mRNA in the heart and 19% in skeletal muscle, and has HIFα stabilization in both tissues. We showed that chronic HIF-P4H-2 deficiency leads to protection against acute ischemic injury both in the heart and in skeletal muscle. The protection was mainly due to enlarged capillaries and better perfusion in both tissues. Hypoxia is known to decrease body weight. The observation of the HIF-P4H-2 deficient mice being leaner than their wild-type littermates led us to study their body constitution, metabolism and adipose tissue in detail. We discovered that chronic HIF-P4H-2 deficiency protects against obesity and several metabolic dysfunctions including diabetes and metabolic syndrome. These beneficial outcomes were mimicked when a pharmacological pan-HIF-P4H inhibitor was administered to wild-type mice. In these studies we showed that pharmacological HIF-P4H-2 inhibition may provide a novel treatment for diseases such as acute myocardial infarction, peripheral artery disease and metabolic disorders. / Tiivistelmä Happi on edellytys aerobisen eliön, kuten ihmisen, elämälle; hapen niukkuus (hypoksia) voi johtaa monenlaisiin solun toimintahäiriöihin, jotka voivat edelleen aiheuttaa solun kuoleman, kyseisen kudoksen vaurion, ja lopulta eliön elinkyvyn heikkenemisen. Happitasapainoa säädellään monilla menetelmillä, joista merkittävin on hypoksiassa indusoituvasta tekijästä (HIF) riippuvainen reitti, joka on evoluutiossa säilynyt. HIFα alayksiköitä säätelee hapesta riippuvaisesti kolme HIF prolyyli 4-hydroksylaasia (HIF-P4Ht). Hapen läsnä ollessa HIF-P4H on aktiivinen ja johtaa HIFα:n hajottamiseen, kun taas hypoksiassa HIF-P4H entsyymit eivät voi toimia ja siten HIFα stabiloituu. HIF säätelee yli 300 geeniä, jotka edistävät hapen kuljetusta ja pääsyä keuhkoista kudoksiin sekä vähentävät hapenkulutusta. Näitä geenejä ovat mm. erytropoietiini sekä vaskulaarinen endoteelikasvutekijä. Kudoksen heikentyneestä verenkierrosta johtuvaa hapenpuutetta kutsutaan iskemiaksi. Tässä tutkimuksessa käytimme geneettisesti muunneltua HIF-P4H-2 hypomorfi-hiirikantaa, joka tuottaa Hif-p4h-2 lähetti-RNA:ta sydämessä vain 8 % ja luurankolihaksessa 19 % villityypin määrästä, ja jolla on HIFα stabiloituneena molemmissa kudoksissa. Osoitimme, että krooninen HIF-P4H-2:n puute suojaa sekä sydäntä että luurankolihasta akuutissa iskemiassa. Vaikutus johtui pääasiassa suuremmista kapillaareista ja paremmasta perfuusiosta molemmissa kudoksissa. Aikaisempien tutkimusten perusteella tiedetään, että hypoksia alentaa painoa. Huomio siitä, että HIF-P4H-2 puutteiset hiiret ovat hoikempia kuin villityypin sisaruksensa, johti meidät tutkimaan hiirten kehon koostumusta, aineenvaihduntaa ja rasvakudosta tarkemmin. Tutkimuksissamme selvisi, että krooninen HIF-P4H-2:n puute suojaa lihavuudelta ja monelta aineenvaihdunnan häiriöltä kuten sokeritaudilta ja metaboliselta oireyhtymältä. Nämä edulliset vaikutukset toistuivat, kun annoimme villityypin hiirille pan-HIF-P4H inhibiittoria. Kaiken kaikkiaan, näissä tutkimuksissa osoitimme, että lääkkeellinen HIF-P4H-2:n estäminen voi tarjota uuden keinon sydäninfarktin, luurankolihasiskemian ja aineenvaihdunnan häiriöiden hoitoon.