This research programme investigated the expression of prolyl hydroxylase (PHD) proteins in rodent tissues. The importance of PHD enzymes lies in their ability to render oxygen sensitivity to Hypoxia inducible factor (HIF), the principal mediator of intracellular oxygen homeostasis. The first part of this study focused on developing and validating anti-sera capable of detecting PHD proteins in rodent tissues. With these reagents, it was possible to assess the relative expression of each PHD protein in a number of different rat tissues. PHD2 was the most abundant isoform in all tissues studied. In contrast, an abundance of PHD1 was observed only in testis and skeletal muscle. A number of different tissue species of PHD3 were identified and their abundance was found to vary between different tissues. These observations provide further evidence of the principal role of PHD2 in regulating HIF in vivo, but also point towards additional roles for PHD1 and PHD3 in selected tissues. They highlight the potential for there being a complex interplay between different PHD enzymes which could, in the future, prove potential targets for therapeutic manipulation. This study also provides additional insights into the mechanisms underlying the phenotypes observed in PHD deletional mouse models which appear, in many cases, to be directly related to the abundance of a given PHD isoform. The emerging role of PHD3 as a promoter of sympathetic lineage apoptosis prompted further study of PHD3 expression in rat neuronal tissues. An abundance of PHD3 was demonstrated throughout the rat sympathetic nervous system, a finding which appeared at odds with its known role as a promoter of neuronal apoptosis and resulted in a series of collaborative studies which demonstrated a sympatho-adrenal phenotype in wild type compared to PHD3-/- mice. Further collaborative studies utilising wild type mice and those deleted of specific PHD isoforms, were carried out to assess the significance of the abundance of PHD3 and PHD1 noted here in rat hippocampus and testis respectively. While neither study demonstrated statistically significant phenotypes, these observations remain of interest and areas for future research.

Factor inhibiting HIF (FIH) negatively regulates hypoxia inducible factor-1 (HIF-1) transcriptional activity, selectively controlling certain HIF-1 target genes, such as vascular endothelial growth factor (VEGF) and prolyl hydroxylase domain 3 (PHD3), but not others. PHD3 and VEGF are important for placental development and function and are overexpressed in preeclampsia (PE). The purpose of this study was to examine FIH in both normal and pathological human placentae. I hypothesized that FIH regulates VEGF and PHD3 in the placenta and that this rheostat is altered in PE. Results show that FIH suppresses PHD3 and VEGF in JEG-3 cells; this effect was abrogated by FIH gene silencing. Moreover, my data indicate that seven in absentia homologue-1 (Siah-1) targets FIH for degradation in the placenta; this degradation is enhanced in PE and likely contributes to aberrant VEGF and PHD3 expression. Overall, my data suggest an important role for FIH in the pathogenesis of PE.

Factor inhibiting HIF (FIH) negatively regulates hypoxia inducible factor-1 (HIF-1) transcriptional activity, selectively controlling certain HIF-1 target genes, such as vascular endothelial growth factor (VEGF) and prolyl hydroxylase domain 3 (PHD3), but not others. PHD3 and VEGF are important for placental development and function and are overexpressed in preeclampsia (PE). The purpose of this study was to examine FIH in both normal and pathological human placentae. I hypothesized that FIH regulates VEGF and PHD3 in the placenta and that this rheostat is altered in PE. Results show that FIH suppresses PHD3 and VEGF in JEG-3 cells; this effect was abrogated by FIH gene silencing. Moreover, my data indicate that seven in absentia homologue-1 (Siah-1) targets FIH for degradation in the placenta; this degradation is enhanced in PE and likely contributes to aberrant VEGF and PHD3 expression. Overall, my data suggest an important role for FIH in the pathogenesis of PE.

Posttranslational modifications are of great importance in the mediation of transcriptional effects, necessary for signalling in cancer. A characteristic example of such modifications is acetylation of the p53 tumour suppressor, a transcription factor involved in several crucial cellular functions including cell-cycle arrest and apoptosis. p53 is stabilised under hypoxic and DNA damaging-conditions. However, only in the latter scenario is p53 fully capable of inducing the expression of its proapoptotic targets through acetylation. The hypoxia inducible factor 1 (HIF-1) transcription factor is stabilised at low oxygen levels to mediate a cellular adaptive response under these conditions, promoting cell survival. As these two opposing transcription factors share a common transcriptional regulator, p300/CBP, this study focused on deciphering the p300/CBP complex components under differential stress to determine its composition required for cellular responses elicited in response to DNA damage or hypoxia, in an effort to investigate a possible link between differential posttranslational modifications and the resulting cell fate. Hence, the aim of this study was to investigate the roles of p300/CBP components in dictating transcriptional regulation of both HIF-1 and p53 in hypoxic conditions. To carry out this study, the proapoptotic BID gene was the system used, as its promoter contains a p53 response element and a HIF-1 response element (HRE). The p300/CBP associated factors PCAF and Strap were appointed as potent candidates for posttranslational modifications under differential conditions, as they are stress-responsive cofactors. Under DNA damage, PCAF acetylates p53 at K320 and Strap augments p300 binding to p53, both of which amplify the p53 response. Evidence from this study demonstrates that under hypoxia-mimicking conditions PCAF-mediated p53 acetylation at K320 is reduced to a greater extent compared to p300/CBP acetylation at K382. The limited amounts of acetylated p53 at K320 are preferentially recruited to the promoter of the cell cycle arrest p21WAF-1/CIP-1 gene that appears to be unaffected by hypoxia, but fail to be recruited to the BID promoter, rendering p53 incapable of upregulating proapoptotic BID in hypoxic conditions. In addition, under the same conditions, PCAF was found to acetylate, and direct HIF-1 to a particular subset of its targets, leading to alterations in the net physiological effect. Moreover, the intrinsic acetyl transferase activity of PCAF was shown to increase the stability of HIF-1. An additional role was attributed to PCAF in relation to apoptosis, albeit from another angle. BID protein translocation to the cytoplasm in hypoxic conditions was facilitated by ectopically expressed PCAF.Strap was found to be preferentially recruited to the HRE of the BID promoter in hypoxic conditions, and to exert a transrepression effect that appeared to be p53-dependent. Strap also interacted with specific PCAF isoforms depending on the type of cellular stress. Contrary to PCAF, ectopically expressed Strap did not have any effect on BID subcellular distribution. This study has provided additional insight in the mechanisms by which cofactors are involved in cell fate, either by affecting activity and stability of HIF-1 and p53, or having a direct effect on Bcl-2 member subcellular distribution.

Abstract Breast cancer is the most common cancer affecting the female population of the Western world. It is a heterogeneous disease entity that encompasses tumors with remarkably different forms of behaviour, and it is therefore vital to distinguish patients with good and poor prognoses. The classical prognostic and predictive factors for breast cancer serve as tools for clinical oncologists when planning treatment, but the growing awareness of breast cancer biology is bringing about a need for novel prognostic and predictive biomarkers. This thesis examines the prognostic significance of hypoxia response and cell cycle regulators in ductal breast cancer and in triple-negative breast cancer (negative for hormone receptors and human epidermal growth factor receptor 2), concluding that PHD2 and PHD3 are associated with a good prognosis, while the role of PHD1 is controversial, as it is associated with proliferation in ductal breast cancer but with node-negative status in triple-negative breast cancer. In our experiments HIF-1α redeemed its role as a marker of an adverse prognosis, whereas the role of HIF-2α appeared to be the opposite. Our data suggest that PHDs can have other targets than the HIF-αs, and that triple-negative breast tumors express more HIF-1α and less HIF-2α and PHD3 than those with a good prognosis. Furthermore, we identified cyclin D1 as a biomarker with independent prognostic significance in ductal breast cancer, being associated with good prognostic factors and a better outcome, whereas the opposite was seen in triple-negative breast cancer. CDK4 was associated with high proliferation in triple-negative breast cancer. In addition, high levels of p16 correlated with increased survival in breast cancer patients independently of receptor status. / Tiivistelmä Rintasyöpä on naisten yleisin syöpä läntisessä maailmassa. Rintasyöpä on heterogeeninen tautiryhmä, jossa kasvaimet vaihtelevat biologiselta käyttäytymiseltään huomattavasti. Tästä syystä on tärkeää erottaa hyvä- ja huonoennusteiset potilaat. Syöpälääkärit käyttävät klassisia ennustetekijöitä hoitopäätöksiä tehdessään, mutta lisääntynyt tieto rintasyövän biologiasta on saanut aikaan tarpeen löytää uusia ennustetekijöitä. Tässä väitöskirjatyössä tutkimme hypoksiavasteen ja solusyklin säätelijöiden ennusteellisuutta duktaalisessa rintasyövässä sekä kolmoisnegatiivisessa (ei ilmennä hormonireseptoreita eikä epidermaalikasvutekijäreseptoria) rintasyövässä. PHD2 ja PHD3:n vahva ilmentyminen liittyi parempaan ennusteeseen, mutta PHD1:n esiintymisen vaikutus oli ristiriitainen. PHD1:n ilmentyminen liittyi lisääntyneeseen solujakautumiseen duktaalisessa rintasyövässä, mutta kolmoisnegatiivisessa rintasyövässä sen esiintyminen liittyi vähentyneeseen imusolmukemetastasointiin. Tutkimuksessamme HIF-1α osoittautui huonon ennusteen merkiksi. Sitä vastoin HIF-2α:n ilmentymisen vaikutus näytti liittyvän parempaan ennusteeseen. Tuloksemme osoittavat, että PHD-entsyymeillä on mahdollisesti muitakin kohteita kuin HIF-α:t. Osoitimme myös, että HIF-1α:n ilmentyminen on yleisempää ja HIF-2α:n sekä PHD3:n ilmentyminen vähäisempää kolmoisnegatiivisessa kuin duktaalisessa rintasyövässä. Lisäksi totesimme, että sykliini D1 on itsenäinen ennustetekijä liittyen parempaan ennusteeseen duktaalisessa rintasyövässä. Huomioitavaa on kuitenkin, että kolmoisnegatiivisessa rintasyövän alaryhmässä sykliini D1:n esiintyminen oli huonon ennusteen merkki. CDK4 osoittautui voimakkaan proliferaation merkiksi kolmoisnegatiivisessa rintasyövässä. Lisäksi osoitimme, että p16:n ilmentyminen liittyy parempaan ennusteeseen sekä duktaalisessa rintasyövässä että kolmoisnegatiivisessa rintasyövässä.

Adaptation to chronic hypoxia is characterized by a variety of functional changes in order to maintain metabolic and energy homeostasis. It has been known for many years that both humans and animals indigenous or adapted to high-altitude hypoxia are more tolerant to an acute ischemic injury of the heart. HIF1α is found as a primary transcriptional regulator of adaptive response to hypoxia in all tissues, whereas HIF2α is more tissue specific. The activity of HIFα is regualted by prolyl hydoxylases (EGLN). The EGLN1 was shown to be more efficient in HIF1α hydroxylation than in HIF2α. As well as the EGLN3 is more specific for HIF2α . Under hypoxic conditions, HIF activates the transcription of about 70 genes. Hence, HIF1α and HIF2α can play an essential role in pathophysiology of ischemic heart disease. The aim of our study was to determine myocardial expression of HIFs and its regulatory hydroxylases in adult male Wistar rats adapted to continuous normobaric hypoxia (H; FIO2 = 0.1) for 3 weeks. Another two groups of rats were exposed to normobaric hypoxia intermittently for either 8 h/day (INH) or 23 h/day (RH) during the 3-week adaptation period. While H induces protective cardiac phenotype, the later regimen (RH) does not. The protein expression of HIFs, PHDs and FIH were then determined by...

By undertaking a number of different experimental approaches at the genetic, cellular/ molecular and integrative physiology levels, I investigated functional variation in the Hypoxia-Inducible Factor (HIF) transcription pathway in humans. My studies focused on Tibetan natives. Tibetan highlanders are adapted to life in a hypoxic environment and exhibit distinct physiological traits at high altitude. Recent studies identified positive selection at two genetic loci, EPAS1 (HIF2α) and EGLN1 (PHD2), in Tibetan highlanders and demonstrated an association of EGLN1/EPAS1 genotype with haemoglobin concentration. Both are genes of the HIF pathway, which coordinates an organism’s response to hypoxia. Patients living at sea level with genetic diseases of the HIF pathway have characteristic phenotypes at both the integrative physiology and cellular levels. I investigated whether Tibetans living at sea level also possess distinct phenotypic characteristics, and whether these may be related to underlying variation within the HIF pathway. I compared Tibetans living at sea level with Han Chinese, their most closely-related major ethnic group, and found that Tibetans possess a significantly different integrative physiology phenotype. Tibetans had a lower haemoglobin concentration and haematocrit, a higher pulmonary ventilation relative to metabolism, and blunted pulmonary vascular responses to both acute (minutes) and sustained (8 hours) hypoxia. Regarding genotype- phenotype relationships within the Tibetans, I found a significant correlation between both EPAS1 and EGLN1 genotype and the induction of erythropoietin by systemic hypoxia. At an intermediate cellular level, the relative expression and the hypoxic induction of HIF- regulated genes were significantly lower in peripheral blood lymphocytes from Tibetans compared with Han Chinese. I also investigated whether the genetic variation in EPAS1 selected for in Tibetans may be functional at the molecular level by affecting transcription of EPAS1 in cells and whether certain coding variants in <e,>EGLN1 found in Tibetans affect protein (PHD2) activity in cells and in vitro. A small supplementary study was undertaken in patients with idiopathic erythrocytosis, who have elevated or inappropriately normal erythropoietin levels, to investigate if they have genetic alterations in the HIF system.

Abstract Oxygen is vital for human survival. To ensure its sufficient supply, the body has an intricate system, which involves the circulatory, respiratory and neuroendocrine systems. When oxygen is lacking, a state of hypoxia occurs, and adaptive changes in gene expression increase oxygen delivery to promote survival. The key regulator of the transcriptional hypoxia response is hypoxia-inducible factor (HIF) which targets over 1000 genes. The HIF prolyl 4-hydroxylases (HIF-P4Hs) govern the stability of HIF in an oxygen-dependent fashion. In our studies we investigated whether activation of the hypoxia response through inhibition of either of two distinct HIF-P4Hs, HIF-P4H-2 or P4H-TM would reduce atherosclerosis in mice. We found that inhibition of HIF-P4H-2 led to reductions in numbers of atherosclerotic plaques, and levels of serum cholesterol and inflammation in white adipose tissue and aortic plaques. In addition, HIF-P4H-2 deficient mice had elevated levels of modified LDL-targeting, atheroprotective circulating autoantibodies. The P4H-TM knockout mice also had reduced numbers of atherosclerotic plaques and increased levels of atheroprotective autoantibodies in their sera, but in contrast to the HIF-P4H-2 deficient mice, they also showed a reduction in serum triglyceride levels. To determine how hypoxia alters maternal glucose and lipid metabolism in pregnancy, we studied pregnant mice that were predisposed to a hypoxic condition (15% ambient O2). We found that they had enhanced glucose metabolism due to reduced insulin resistance and an increased flux of glucose to maternal tissues. The hypoxic dams also failed to gain weight and store adipose tissue in the anabolic phase to the same extent as normoxic control dams. These results implicate HIF-P4H inhibition as a novel therapeutic mechanism for atherosclerosis, and suggest that the small molecule HIF-P4H inhibitors currently in clinical trials for renal anemia may have further possible therapeutic applications. In addition, greater understanding of the changes in maternal metabolism that underly reduced fetal growth in hypoxic conditions, and the development of targeted interventions may allow the preservation of fetal growth in cases of maternal hypoxia. / Tiivistelmä Happi on ihmiselle elintärkeää. Tämän vuoksi meille on kehittynyt pitkälle jalostunut verenkierto-, hengitys- ja neuroendokriininen järjestelmä sekä sellaisten geenien ilmentymisen muutoksia, jotka joko lisäävät hapen kuljetusta tai auttavat selviytymään hypoksisissa oloissa, jotta taataan riittävä hapen saanti. Hapen puutteessa hypoksiavaste, jonka tärkein säätelijä on hypoksiassa indusoituva transkriptiotekijä (HIF), aktivoituu. HIF:lla on yli 1000 kohdegeeniä joiden kautta sen vaikutukset välittyvät. HIF-prolyyli-4-hydroksylaasit (HIF-P4H:t) säätelevät HIF:n stabiilisuutta hapesta riippuvaisesti. Tutkimuksessamme selvitimme, vähentääkö hypoksiavasteen aktivointi HIF-P4H-2:n tai P4H-TM:n inhibition kautta ateroskleroosia hiirillä. Tuloksena oli, että HIF-P4H-2:n inhibitio vähensi ateroskleroottisia plakkeja, seerumin kolesterolia ja inflammaatiota valkoisessa rasvakudoksessa sekä plakeissa. Lisäksi hiirillä, joilta puuttui HIF-P4H-2, oli lisääntynyt määrä ateroskleroosilta suojaavia muokattua LDL:ää sitovia autovasta-aineita seerumissa. P4H-TM-poistogeenisillä hiirillä todettiin vastaavasti vähemmän ateroskleroottisia plakkeja ja lisääntynyt määrä ateroskleroosilta suojaavia autovasta-aineita seerumissa. Poiketen HIF-P4H-2-puutteisista hiiristä, niillä oli matalammat seerumin triglyseridi-tasot. Tutkimme raskaina olevia hiiriä, jotka altistimme hypoksisille olosuhteille (15% O2), jotta pystyisimme määrittämään, kuinka hypoksia vaikuttaa äidin sokeri- ja rasva-aineenvaihduntaan. Hypoksiassa raskaana olevilla hiirillä todettiin tehostunut sokeriaineenvaihdunta, joka oli seurausta alentuneesta insuliiniresistenssistä sekä lisääntyneestä sokerin sisäänotosta äidin kudoksiin. Hypoksiassa eivät raskaana olevien hiirten paino eivätkä rasvavarastot lisääntyneet samassa suhteessa normoksiassa raskaana olevien hiirten kanssa. Nämä tulokset tarjoavat uusia mahdollisuuksia HIF-P4H-inhibition käyttämiseen terapeuttisena vaihtoehtona ateroskleroosin hoidossa ja ehkäisemisessä. Kliinisissä kokeissa munuaisperäisen anemian hoidossa olevat HIF-P4H-estäjät voisivat näin ollen saada lisää indikaatioita. Lisäksi korkean ilmanalan aiheuttaman pienipainoisuuden takana olevien aineenvaihdunnan muutoksien ymmärtäminen voi mahdollistaa sikiön kasvun turvaamisen spesifein interventioin.

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