The role of catechol-O-methyltransferase (COMT) and the effects of COMT inhibition in brain and in cardiovascular and renal pathophysiology

Catechol-O-methyltransferase (COMT) metabolizes catecholamines such as dopamine (DA), noradrenaline (NA) and adrenaline, which are vital neurotransmitters and hormones that play important roles in the regulation of physiological processes. COMT enzyme has a functional Val158Met polymorphism in humans, which affects the subjects COMT activity. Increasing evidence suggests that this functional polymorphism may play a role in the etiology of various diseases from schizophrenia to cancers. The aim of this project was to provide novel biochemical information on the physiological and especially pathophysiological roles of COMT enzyme as well as the effects of COMT inhibition in the brain and in the cardiovascular and renal system. To assess the roles of COMT and COMT inhibition in pathophysiology, we used four different study designs. The possible beneficial effects of COMT inhibition were studied in double-transgenic rats (dTGRs) harbouring human angiotensinogen and renin genes. Due to angiotensin II (Ang II) overexpression, these animals exhibit severe hypetension, cardiovascular and renal end-organ damage and mortality of approximately 25-40% at the age of 7-weeks. The dTGRs and their Sprague-Dawley controls tissue samples were assessed with light microscopy, immunohistochemistry, reverse transcriptase-polymerase chain reaction (RT-PCR) and high-pressure liquid chromatography (HPLC) to evaluate the tissue damages and the possible protective effects pharmacological intervention with COMT inhibitors. In a second study, the consequence of genetic and pharmacological COMT blockade in blood pressure regulation during normal and high-sodium was elucidated using COMT-deficient mice. The blood pressure and the heart rate were measured using direct radiotelemetric blood pressure surveillance. In a third study, the effects of acute and subchronic COMT inhibition during combined levodopa (L-DOPA) + dopa decarboxylase inhibitor treatment in homocysteine formation was evaluated. Finally, we assessed the COMT enzyme expression, activity and cellular localization in the CNS during inflammation-induced neurodegeneration using Western blotting, HPLC and various enzymatic assays. The effects of pharmacological COMT inhibition on neurodegeneration were also studied. The COMT inhibitor entacapone protected against the Ang II-induced perivascular inflammation, renal damage and cardiovascular mortality in dTGRs. COMT inhibitors reduced the albuminuria by 85% and prevented the cardiovascular mortality completely. Entacapone treatment was shown to ameliorate oxidative stress and inflammation. Furthermore, we established that the genetic and pharmacological COMT enzyme blockade protects against the blood pressure-elevating effects of high sodium intake in mice. These effects were mediated via enhanced renal dopaminergic tone and suggest an important role of COMT enzyme, especially in salt-sensitive hypertension. Entacapone also ameliorated the L-DOPA-induced hyperhomocysteinemia in rats. This is important, since decreased homocysteine levels may decrease the risk of cardiovascular diseases in Parkinson´s disease (PD) patients using L-DOPA. The Lipopolysaccharide (LPS)-induced inflammation and subsequent delayed dopaminergic neurodegeneration were accompanied by up-regulation of COMT expression and activity in microglial cells as well as in perivascular cells. Interestingly, similar perivascular up-regulation of COMT expression in inflamed renal tissue was previously noted in dTGRs. These results suggest that inflammation reactions may up-regulate COMT expression. Furthermore, this increased glial and perivascular COMT activity in the central nervous system (CNS) may decrease the bioavailability of L-DOPA and be related to the motor fluctuation noted during L-DOPA therapy in PD patients.

Transcriptional regulation by the orphan nuclear receptor ERRgamma

The nuclear receptor (NR) superfamily is comprised of receptors for small lipopfilic ligands such as steroid hormones, thyroid hormone, retinoids, and vitamin D. NRs are ligand-inducible transcription factors capable of both activating and repressing their target gene expression. They control a wide range of biological functions connected to growth, development, and homeostasis. In addition to the ligand-regulated receptors, the family includes a large group of receptors whose physiological ligands are unknown. These receptors are referred to as orphan NRs. Estrogen-related receptor gamma (ERRgamma) belongs to the ERR subfamily of orphan NRs together with the related ERRalpha and ERRbeta. ERRs share amino acid sequence homology with the classical estrogen receptors (ERs) but they are unable to bind natural estrogenic ligands. ERRgamma is expressed in several embryonic and adult tissues but its biological role is still largely unknown. ERRgamma activates reporter gene expression in transfected cells independently of added hormones implying that ERRgamma harbors constitutive activity. However, the intrinsic activity of ERRgamma can be inhibited by synthetic compounds such as the selective estrogen receptor modulator 4-hydroxytamoxifen (4-OHT). Ligands of NRs can act as agonists that activate transcription, as antagonists that prevent activation of transcription, or as inverse agonists that antagonize the constitutive transcriptional activity of receptor. Most of the synthetic ERRgamma ligands act as inverse agonists but recently, a synthetic ERRgamma agonist GSK4716 was identified. This demonstrates that it is possible to design and identify agonists for ERRgamma. Prior to this thesis work, the structural and functional characteristics of ERRgamma were largely unknown. The aim of this study was to define the functional requirements for ERRgamma-mediated transcriptional regulation and to examine the cross-talk between ERRgamma and other NRs. Due to the fact that natural physiological ligands of ERRgamma are unknown, another aim of this study was to seek new natural compounds that may affect transcriptional activity of ERRgamma. Plant-derived phytoestrogens have previously been shown to act as ligands for ERs and ERRalpha, and therefore the effects of these compounds were also studied on ERRgamma-mediated transcriptional regulation. This work demonstrated that ERRgamma-mediated transcriptional regulation was dependent on DNA-binding, dimerization and activation function-2. Heterodimerization with ERRalpha inhibited the transcriptional activity of ERRgamma. In addition to 4-OHT, another anti-estrogen, 4-hydroxytoremifene (4-OHtor), was identified as an inverse agonist of ERRgamma. Interestingly, ERRgamma activated transcription in the presence of 4-OHT and 4-OHtor on activator protein-1 binding sites. ERRgamma was found to interact with another orphan NR Nurr1 by repressing the ability of Nurr1 to activate transcription of the osteopontin gene. Transcriptional activity of ERRgamma was shown to be stimulated by the phytoestrogen equol. Structural model analysis and mutational experiments indicated that equol was able to bind to the ligand binding domain of ERRgamma. The growth inhibitory effect of ERRgamma on prostate cancer cells was found to be enhanced by equol. In summary, this study demonstrates that despite the absence of an endogenous physiological ligand, the activity of ERRgamma can be modulated in other ways such as dimerization with related receptors or by cross-talk with other transcription factors as well as by binding some synthetic or natural compounds.

Molecular Mechanisms of Androgen Receptor Interactions

The androgen receptor (AR) mediates the effects of the male sex-steroid hormones (androgens), testosterone and 5?-dihydrotestosterone. Androgens are critical in the development and maintenance of male sexual characteristics. AR is a member of the steroid receptor ligand-inducible transcription factor family. The steroid receptor family is a subgroup of the nuclear receptor superfamily that also includes receptors for the active forms of vitamin A, vitamin D3, and thyroid hormones. Like all nuclear receptors, AR has a conserved modular structure consisting of a non-conserved amino-terminal domain (NTD), containing the intrinsic activation function 1, a highly conserved DNA-binding domain, and a conserved ligand-binding domain (LBD) that harbors the activation function 2. Each of these domains plays an important role in receptor function and signaling, either via intra- and inter-receptor interactions, interactions with specific DNA sequences, termed hormone response elements, or via functional interactions with domain-specific proteins, termed coregulators (coactivators and corepressors). Upon binding androgens, AR acquires a new conformational state, translocates to the nucleus, binds to androgen response elements, homodimerizes and recruits sequence-specific coregulatory factors and the basal transcription machinery. This set of events is required to activate gene transcription (expression). Gene transcription is a strictly modulated process that governs cell growth, cell homeostasis, cell function and cell death. Disruptions of AR transcriptional activity caused by receptor mutations and/or altered coregulator interactions are linked to a wide spectrum of androgen insensitivity syndromes, and to the pathogenesis of prostate cancer (CaP). The treatment of CaP usually involves androgen depletion therapy (ADT). ADT achieves significant clinical responses during the early stages of the disease. However, under the selective pressure of androgen withdrawal, androgen-dependent CaP can progress to an androgen-independent CaP. Androgen-independent CaP is invariably a more aggressive and untreatable form of the disease. Advancing our understanding of the molecular mechanisms behind the switch in androgen-dependency would improve our success of treating CaP and other AR related illnesses. This study evaluates how clinically identified AR mutations affect the receptor s transcriptional activity. We reveal that a potential molecular abnormality in androgen insensitivity syndrome and CaP patients is caused by disruptions of the important intra-receptor NTD/LBD interaction. We demonstrate that the same AR LBD mutations can also disrupt the recruitment of the p160 coactivator protein GRIP1. Our investigations reveal that 30% of patients with advanced, untreated local CaP have somatic mutations that may lead to increases in AR activity. We report that somatic mutations that activate AR may lead to early relapse in ADT. Our results demonstrate that the types of ADT a CaP patient receives may cause a clustering of mutations to a particular region of the receptor. Furthermore, the mutations that arise before and during ADT do not always result in a receptor that is more active, indicating that coregulator interactions play a pivotal role in the progression of androgen-independent CaP. To improve CaP therapy, it is necessary to identify critical coregulators of AR. We screened a HeLa cell cDNA library and identified small carboxyl-terminal domain phosphatase 2 (SCP2). SCP2 is a protein phosphatase that directly interacts with the AR NTD and represses AR activity. We demonstrated that reducing the endogenous cellular levels of SCP2 causes more AR to load on to the prostate specific antigen (PSA) gene promoter and enhancer regions. Additionally, under the same conditions, more RNA polymerase II was recruited to the PSA promoter region and overall there was an increase in androgen-dependent transcription of the PSA gene, revealing that SCP2 could play a role in the pathogenesis of CaP.

Genetic Basis of Pituitary Adenoma Predisposition

Much of the global cancer research is focused on the most prevalent tumors; yet, less common tumor types warrant investigation, since A rare disorder is not necessarily an unimportant one . The present work discusses a rare tumor type, the benign adenomas of the pituitary gland, and presents the advances which, during the course of this thesis work, contributed to the elucidation of a fraction of their genetic background. Pituitary adenomas are benign neoplasms of the anterior pituitary lobe, accounting for approximately 15% of all intracranial tumors. Pituitary adenoma cells hypersecrete the hormones normally produced by the anterior pituitary tissue, such as growth hormone (GH) and prolactin (PRL). Despite their non-metastasizing nature, these adenomas can cause significant morbidity and have to be adequately treated; otherwise, they can compromise the patient s quality of life, due to conditions provoked by hormonal hypersecretion, such as acromegaly in the case of GH-secreting adenomas, or due to compressive effects to surrounding tissues. The vast majority of pituitary adenomas arise sporadically, whereas a small subset occur as component of familial endocrine-related tumor syndromes, such as Multiple Endocrine Neoplasia type 1 (MEN1) and Carney complex (CNC). MEN1 is caused by germline mutations in the MEN1 tumor suppressor gene (11q13), whereas the majority of CNC cases carry germline mutations in the PRKAR1A gene (17q24). Pituitary adenomas are also encountered in familial settings outside the context of MEN1 and CNC, but unlike in the latter syndromes, their genetic background until recently remained elusive. Evidence in previous literature supported the notion that a tumor suppressor gene on 11q13, residing very close to but still distinct from MEN1, causes genetic susceptibility to pituitary tumors. The aim of the study was to identify the genetic cause of a low penetrance form of Pituitary Adenoma Predisposition (PAP) in families from Northern Finland. The present work describes the methodological approach that led to the identification of aryl hydrocarbon receptor interacting protein (AIP) as the gene causing PAP. Combining chip-based technologies (SNP and gene expression arrays) with traditional gene mapping methods and genealogy data, we showed that germline AIP mutations cause PAP in familial and sporadic settings. PAP patients were diagnosed with mostly adenomas of the GH/PRL-secreting cell lineage. In Finland, two AIP mutations accounted for 16% of all patients diagnosed with GH-secreting adenomas, and for 40% of patients being younger than 35 years of age at diagnosis. AIP is suggested to act as a tumor suppressor gene, a notion supported by the nature of the identified mutations (most are truncating) and the biallelic inactivation of AIP in the tumors studied. AIP has been best characterized as a cytoplasmic interaction partner of aryl hydrocarbon receptor (AHR), also known as dioxin receptor, but it has other partners as well. The mechanisms that underlie AIP-mediated pituitary tumorigenesis are to date largely unknown and warrant further investigation. Because AIP was identified in the genetically homogeneous Finnish population, it was relevant to examine its contribution to PAP in other, more heterogeneous, populations. Analysis of pituitary adenoma patient series of various ethnic origins and differing clinical settings revealed germline AIP mutations in all cohorts studied, albeit with low frequencies (range 0.8-7.4%). Overall, PAP patients were typically diagnosed at a young age (range 8-41 years), mainly with GH-secreting adenomas, without strong family history of endocrine disease. Because many PAP patients did not display family history of pituitary adenomas, detection of the condition appeared challenging. AIP immunohistochemistry was tested as a molecular pre-screening tool on mutation-positive versus mutation-negative tumors, and proved to be a potentially useful predictor of PAP. Mutation screening of a large cohort of colorectal, breast, and prostate tumors did not reveal somatic AIP mutations. These tumors, apart from being the most prevalent among men and women worldwide, have been associated with acromegaly, particularly colorectal neoplasia. In this material, AIP did not appear to contribute to the pathogenesis of these common tumor types and other genes seem likely to play a role in such tumorigenesis. Finally, the contribution of AIP in pediatric onset pituitary adenomas was examined in a unique population-based cohort of sporadic pituitary adenoma patients from Italy. Germline AIP mutations may account for a subset of pediatric onset GH-secreting adenomas (in this study one of seven GH-secreting adenoma cases or 14.3%), and appear to be enriched among young (≤25 years old) patients. In summary, this work reveals a novel tumor susceptibility gene, namely AIP, which causes genetic predisposition to pituitary adenomas, in particular GH-secreting adenomas. Moreover, it provides molecular tools for identification of individuals predisposed for PAP. Further elaborate studies addressing the functional role of AIP in normal and tumor cells will hopefully expand our knowledge on endocrine neoplasia and reveal novel cellular mechanisms of pituitary tumorigenesis, including potential drug targets.

The role of AIP and CDKN1B/p27Kip1 in endocrine neoplasia

Identification of genes predisposing to tumor syndromes has raised general awareness of tumorigenesis. Genetic testing of tumor susceptibility genes aids the recognition of individuals at increased risk of tumors. Identification of novel predisposing genes enables further studies concerning the classification of potential associated tumors and the definition of target patient group. Pituitary adenomas are common, benign neoplasms accounting for approximately 15% of all intracranial tumors. Accurate incidence estimation is challenging since a great portion of these adenomas are small and asymptomatic. Clinically relevant adenomas, that cause symptoms due to the expansion of the cell mass or the over-secretion of normally produced hormones, occur in approximately one of 1 000 individuals. Although the majority of pituitary adenomas are sporadic, a minority occur as components of familial syndromes, such as Multiple Endocrine Neoplasia type 1 (MEN1) and Carney complex (CNC). MEN1 syndrome is caused by germ-line mutations in the MEN1 gene, whereas most of the CNC patients carry the mutated protein kinase A (PKA) regulatory subunit-1-α (PRKAR1A) gene. Recently, other conditions predisposing to endocrine tumors have been identified: Pituitary Adenoma Predisposition (PAP) and MEN type 4 (MEN4). PAP was originally identified in a genetically homogeneous Finnish population. In a population based cohort from Northern Finland, aryl hydrocarbon receptor-interacting protein (AIP) gene mutations were found in 16% of all patients diagnosed with growth hormone (GH) producing pituitary adenoma, and in 40% of the subset of patients who were diagnosed under the age of 35 years. Since AIP mutations were originally described in a defined, homogeneous population from Northern Finland, it was relevant to study whether mutations also occur in more heterogeneous populations. In patient cohorts with different ethnic origins and variable clinical phenotypes, germ-line AIP mutations were detectable at low frequencies (range 0.8-7.4%). AIP mutation-positive patients were often diagnosed with a GH-producing adenoma at a young age, and usually had no family history of endocrine tumors. The low frequency of AIP mutations in randomly selected patients, and the lack of any family history of pituitary adenomas create a challenge for the identification of PAP patients. Our preliminary study suggests that AIP immunohistochemistry may serve as a pre-screening tool to distinguish between the AIP mutation-negative and the mutation-positive tumors. Tumors of various endocrine glands are components of MEN1 and CNC syndromes. Somatic MEN1 and PRKAR1A mutations in sporadic pituitary adenomas are rare, but occur in some of the other tumors related to these syndromes. The role of AIP mutations in endocrine neoplasia was studied and our results indicated that somatic AIP mutations are rare or non-existent in sporadic tumors of endocrine glands (0 of 111). Furthermore, germ-line AIP mutations in prolactin producing adenomas (2 of 9) confirmed the role of this pituitary tumor type in the PAP phenotype. Thyroid disorders are common in the general population, and the majority of them are sporadic. Interestingly, it has been suggested that thyroid disorders might be more common in PAP families. For this reason we studied germ-line AIP mutations in 93 index cases from familial non-medullary thyroid cancer (NMTC) families. The underlying gene or genes for familial NMTC have not been identified yet. None of the patients had any potentially pathogenic AIP mutation. This suggests that AIP is unlikely to play a role in familial NMTCs. A novel multiple endocrine syndrome was originally described in rats with phenotypic features of human MEN type 1 and 2. Germ-line mutations of cyclin-dependent kinase inhibitor 1B (CDKN1B also known as p27Kip1) gene were reported later in these rats and a germ-line mutation was also identified in one human family with MEN1-like phenotype (later named MEN4). To confirm the importance of this gene’s mutations in humans, we performed a mutation screening in MEN-like patients and in patients with pituitary adenoma. Our results indicate that CDKN1B/p27Kip1 mutations appear in a small portion of MEN1-like patients (one of 36), and that such mutations are rare or non-existent in both familial (0 of 19) and sporadic pituitary adenoma patients (0 of 50). In conclusion, this work strengthens the tumor susceptibility role of AIP and CDKN1B/p27Kip1 in endocrine neoplasia. Clarifying the PAP phenotype facilitates the identification of potential AIP mutation carriers. Genetic counseling can be offered to the relatives and follow-up of the mutation carriers can be organized, hence an earlier diagnosis is feasible.

Defining Adequate Vitamin D Intake : Cross-sectional and Intervention Studies

D-vitamiini ylläpitää normaalia luun kasvua ja uudistumista koko elämän ajan. Suomessa, kuten monissa muissakin länsimaissa, väestön D-vitamiinitilanne on riittämätön – talvisin osalla jopa puutteellinen. Tässä väitöskirjassa on tutkittu, lisääkö D-vitamiini luumassan kertymistä kasvuiässä, ja ylläpitäkö D-vitamiini luuston tasapainoista aineenvaihduntaa aikuisiällä. Nämä vaikutukset saattavat ehkäisi osteoporoosin kehittymistä eri ikäkausina. Väitöskirjatyössä tutkittiin erisuuruisten D-vitamiinilisäysten vaikutuksia kolmessa eri ikäryhmässä, jotka olivat 11-12 -vuotiaat tytöt (N=228), 21-49 -vuotiaat miehet (N=54) ja 65-85 -vuotiaat naiset (N=52). Tutkittavat satunnaistettiin ryhmiin, jotka nauttivat joko lumevalmistetta tai 5-20 µg D3-vitamiinia vitamiinilisänä. Tutkimukset olivat kaksoissokkoutettuja. Tutkimuksen aikana tutkittavilta otettiin paastoveri- ja virtsanäytteitä. Lisäksi he täyttivät tutkimuslomakkeen taustatietojen kartoittamiseksi sekä frekvenssikyselylomakkeen kalsiumin ja D-vitamiinin saannin selvittämiseksi. Tyttöjen luunmineraalitiheys (BMD) mitattiin DXA–laitteella ja miesten volumetrinen luuntiheys pQCT-menetelmällä. Näytteistä määritettiin mm. seerumin 25-hydroksi-D-vitamiinin (=S-25-OHD), lisäkilpirauhashormonin (=S-PTH) ja luun aineenvaihduntaa kuvaavien merkkiaineiden pitoisuuksia. Murrosikäisten tyttöjen poikkileikkaustutkimuksessa S-25-OHD- ja luun muodostusmerkkiaineen pitoisuudet vaihtelivat kuukausien välillä; suurimmat pitoisuudet mitattiin syyskuussa ja pienimmät maaliskuussa, mikä kuvastaa vuodenaikaisvaihtelua. Vastaava vaihtelu havaittiin lannerangan ja reisiluun BMD:ssä. D-vitamiinilisäyksellä oli myönteinen vaikutus tyttöjen luumassan lisääntymiseen. Suurin D-vitamiinilisä (10 µg/vrk) lisäsi luumassaa 17.2% enemmän reisiluussa ja 12.5% enemmän lannerangassa verrattuna lumevalmistetta nauttivien tyttöjen vastaaviin tuloksiin, mutta tulos riippui hoitomyöntyvyydestä. D-vitamiinin vaikutus luustoon välittyi vähentyneen luun hajotuksen kautta. Tutkimustuloksiin perustuen riittävä D-vitamiinin saanti murrosikäisille tytöille on 15 µg/vrk. D-vitamiinilisän vaikutus 65-85 -vuotiaiden naisten S-25-OHD-pitoisuuteen vakioitui kuudessa viikossa annoksen ollessa 5-20 µg/vrk. Näillä D-vitamiiniannoksilla ei saavutettu tavoiteltavaa S-25-OHD-pitoisuutta, joka on 80 nmol/l. Arvioimme, että 60 nmol/l -pitoisuuden, jota esiintyy kesäisin tämän ikäryhmän suomalaisilla, tämän ikäryhmän naiset saavuttaisivat 24 µg:n päivittäisellä D-vitamiinin saannilla. Terveillä miehillä havaittiin vuodenaikaisvaihtelu S-25-OHD- ja S-PTH-pitoisuudessa sekä luun hajotusta kuvaavassa merkkiainepitoisuudessa. Toisaalta vaihtelua ei havaittu radiuksen volumetrisessä luuntiheydessä eikä luun muodostusmerkkiaineen pitoisuudessa. Vuodenaikaisvaihtelu estettiin 17 µg:n päivittäisellä D-vitamiinin saannilla, mutta tämän ei havaittu vaikuttavan radiuksen luuntiheyteen kuusi kuukautta kestävän tutkimuksen aikana. Yhteenvetona todetaan, että D-vitamiinin saanti on edelleenkin riittämätöntä tutkimusten kohderyhmillä. Tämä näkyy S-25-OHD- ja PTH-pitoisuuden sekä luunaineenvaihduntaa kuvaavien merkkiaineiden vuodenaikaisvaihteluna, mikä on haitallista luuston hyvinvoinnille. D-vitamiinin saantia tulisi lisätä, jotta vähintäänkin riittävä D-vitamiinitilanne (S-25-OHD>50 nmol/l) tai mahdollisesti jopa tavoiteltava D-vitaminitilanne (S-25-OHD≥80 nmol/l) saavutettaisiin. Jotta D-vitamiinin saannin lisääminen olisi kaikissa ikäryhmissä mahdollista, on suunniteltava nykyistä enemmän D-vitamiinilla täydennettyjä elintarvikkeita.

Molecular Modelling of Estrogen-Producing Enzymes CYP450 Aromatase and 17beta-Hydroxysteroid Dehydrogenase Type 1

Breast cancer is the most common cancer in women in the western countries. Approximately two-thirds of breast cancer tumours are hormone dependent, requiring estrogens to grow. Estrogens are formed in the human body via a multistep route starting from cholesterol. The final steps in the biosynthesis include the CYP450 aromatase enzyme, converting the male hormones androgens (preferred substrate androstenedione ASD) into estrogens(estrone E1), and the 17beta-HSD1 enzyme, converting the biologically less active E1 into the active hormone 17beta-hydroxyestradiol E2. E2 is bound to the nuclear estrogen receptors causing a cascade of biochemical reactions leading to cell proliferation in normal tissue, and to tumour growth in cancer tissue. Aromatase and 17beta-HSD1 are expressed in or near the breast tumour, locally providing the tissue with estrogens. One approach in treating hormone dependent breast tumours is to block the local estrogen production by inhibiting these two enzymes. Aromatase inhibitors are already on the market in treating breast cancer, despite the lack of an experimentally solved structure. The structure of 17beta-HSD1, on the other hand, has been solved, but no commercial drugs have emerged from the drug discovery projects reported in the literature. Computer-assisted molecular modelling is an invaluable tool in modern drug design projects. Modelling techniques can be used to generate a model of the target protein and to design novel inhibitors for them even if the target protein structure is unknown. Molecular modelling has applications in predicting the activities of theoretical inhibitors and in finding possible active inhibitors from a compound database. Inhibitor binding at atomic level can also be studied with molecular modelling. To clarify the interactions between the aromatase enzyme and its substrate and inhibitors, we generated a homology model based on a mammalian CYP450 enzyme, rabbit progesterone 21-hydroxylase CYP2C5. The model was carefully validated using molecular dynamics simulations (MDS) with and without the natural substrate ASD. Binding orientation of the inhibitors was based on the hypothesis that the inhibitors coordinate to the heme iron, and were studied using MDS. The inhibitors were dietary phytoestrogens, which have been shown to reduce the risk for breast cancer. To further validate the model, the interactions of a commercial breast cancer drug were studied with MDS and ligand–protein docking. In the case of 17beta-HSD1, a 3D QSAR model was generated on the basis of MDS of an enzyme complex with active inhibitor and ligand–protein docking, employing a compound library synthesised in our laboratory. Furthermore, four pharmacophore hypotheses with and without a bound substrate or an inhibitor were developed and used in screening a commercial database of drug-like compounds. The homology model of aromatase showed stable behaviour in MDS and was capable of explaining most of the results from mutagenesis studies. We were able to identify the active site residues contributing to the inhibitor binding, and explain differences in coordination geometry corresponding to the inhibitory activity. Interactions between the inhibitors and aromatase were in agreement with the mutagenesis studies reported for aromatase. Simulations of 17beta-HSD1 with inhibitors revealed an inhibitor binding mode with hydrogen bond interactions previously not reported, and a hydrophobic pocket capable of accommodating a bulky side chain. Pharmacophore hypothesis generation, followed by virtual screening, was able to identify several compounds that can be used in lead compound generation. The visualisation of the interaction fields from the QSAR model and the pharmacophores provided us with novel ideas for inhibitor development in our drug discovery project.

Stress and developmental responses in Arabidopsis thaliana: regulation through the transcription factor-interacting protein RCD1

Plants constantly face adverse environmental conditions, such as drought or extreme temperatures that threaten their survival. They demonstrate astonishing metabolic flexibility in overcoming these challenges and one of the key responses to stresses is changes in gene expression leading to alterations in cellular functions. This is brought about by an intricate network of transcription factors and associated regulatory proteins. Protein-protein interactions and post-translational modifications are important steps in this control system along with carefully regulated degradation of signaling proteins. This work concentrates on the RADICAL-INDUCED CELL DEATH1 (RCD1) protein which is an important regulator of abiotic stress-related and developmental responses in Arabidopsis thaliana. Plants lacking this protein function display pleiotropic phenotypes including sensitivity to apoplastic reactive oxygen species (ROS) and salt, ultraviolet B (UV-B) and paraquat tolerance, early flowering and senescence. Additionally, the mutant plants overproduce nitric oxide, have alterations in their responses to several plant hormones and perturbations in gene expression profiles. The RCD1 gene is transcriptionally unresponsive to environmental signals and the regulation of the protein function is likely to happen post-translationally. RCD1 belongs to a small protein family and, together with its closest homolog SRO1, contains three distinguishable domains: In the N-terminus, there is a WWE domain followed by a poly(ADP-ribose) polymerase-like domain which, despite sequence conservation, does not seem to be functional. The C-terminus of RCD1 contains a novel domain called RST. It is present in RCD1-like proteins throughout the plant kingdom and is able to mediate physical interactions with multiple transcription factors. In conclusion, RCD1 is a key point of signal integration that links ROS-mediated cues to transcriptional regulation by yet unidentified means, which are likely to include post-translational mechanisms. The identification of RCD1-interacting transcription factors, most of whose functions are still unknown, opens new avenues for studies on plant stress as well as developmental responses.

Regulation of Osteoblast Differentiation and Gene Expression by Phosphodiesterases and Bioactive Peptides

Bone is a mineralized tissue that enables multiple mechanical and metabolic functions to be carried out in the skeleton. Bone contains distinct cell types: osteoblasts (bone-forming cells), osteocytes (mature osteoblast that embedded in mineralized bone matrix) and the osteoclasts (bone-resorbing cells). Remodelling of bone begins early in foetal life, and once the skeleton is fully formed in young adults, almost all of the metabolic activity is in this form. Bone is constantly destroyed or resorbed by osteoclasts and then replaced by osteoblasts. Many bone diseases, i.e. osteoporosis, also known as bone loss, typically reflect an imbalance in skeletal turnover. The cyclic adenosine monophosphate (cAMP) and the cyclic guanosine monophosphate (cGMP) are second messengers involved in a variety of cellular responses to such extracellular agents as hormones and neurotransmitters. In the hormonal regulation of bone metabolism, i.e. via parathyroid hormone (PTH), parathyroid hormone-related peptide (PTHrp) and prostaglandin E2 signal via cAMP. cAMP and cGMP are formed by adenylate and guanylate cyclases and are degraded by phosphodiesterases (PDEs). PDEs determine the amplitudes of cyclic nucleotide-mediated hormonal responses and modulate the duration of the signal. The activities of the PDEs are regulated by multiple inputs from other signalling systems and are crucial points of cross-talk between the pathways. Food-derived bioactive peptides are reported to express a variety of functions in vivo. The angiotensin-converting enzymes (ACEs) are involved in the regulation of the specific maturation or degradation of a number of mammalian bioactive peptides. The bioactive peptides offer also a nutriceutical and a nutrigenomic aspect to bone cell biology. The aim of this study was to investigate the influence of PDEs and bioactive peptides on the activation and the differentiation of human osteoblast cells. The profile of PDEs in human osteoblast-like cells and the effect of glucocorticoids on the function of cAMP PDEs, were investigated at the mRNA and enzyme levels. The effects of PDEs on bone formation and osteoblast gene expression were determined with chemical inhibitors and siRNAs (short interfering RNAs). The influence of bioactive peptides on osteoblast gene expression and proliferation was studied at the mRNA and cellular levels. This work provides information on how PDEs are involved in the function and the differentiation of osteoblasts. The findings illustrate that gene-specific silencing with an RNA interference (RNAi) method is useful in inhibiting, the gene expression of specific PDEs and further, PDE7 inhibition upregulates several osteogenic genes and increases bALP activity and mineralization in human mesenchymal stem cells-derived osteoblasts. PDEs appear to be involved in a mechanism by which glucocorticoids affect cAMP signaling. This may provide a potential route in the formation of glucocorticoid-induced bone loss, involving the down-regulation of cAMP-PDE. PDEs may play an important role in the regulation of osteoblastic differentiation. Isoleucine-proline-proline (IPP), a bioactive peptide, possesses the potential to increase osteoblast proliferation, differentiation and signalling.

Genotype-Phenotype Relationships in Long QT Syndrome: Role of Mental Stress, Adrenergic Activity and a Common KCNH2 Polymorphism

Long QT syndrome is a congenital or acquired arrhythmic disorder which manifests as a prolonged QT-interval on the electrocardiogram and as a tendency to develop ventricular arrhythmias which can lead to sudden death. Arrhythmias often occur during intense exercise and/or emotional stress. The two most common subtypes of LQTS are LQT1, caused by mutations in the KCNQ1 gene and LQT2, caused by mutations in the KCNH2 gene. LQT1 and LQT2 patients exhibit arrhythmias in different types of situations: in LQT1 the trigger is usually vigorous exercise whereas in LQT2 arrhythmia results from the patient being startled from rest. It is not clear why trigger factors and clinical outcome differ from each other in the different LQTS subtypes. It is possible that stress hormones such as catecholamines may show different effects depending on the exact nature of the genetic defect, or sensitivity to catecholamines varies from subject to subject. Furthermore, it is possible that subtle genetic variants of putative modifier genes, including those coding for ion channels and hormone receptors, play a role as determinants of individual sensitivity to life-threatening arrhythmias. The present study was designed to identify some of these risk modifiers. It was found that LQT1 and LQT2 patients show an abnormal QT-adaptation to both mental and physical stress. Furthermore, as studied with epinephrine infusion experiments while the heart was paced and action potentials were measured from the right ventricular septum, LQT1 patients showed repolarization abnormalities which were related to their propensity to develop arrhythmia during intense, prolonged sympathetic tone, such as exercise. In LQT2 patients, this repolarization abnormality was noted already at rest corresponding to their arrhythmic episodes as a result of intense, sudden surges in adrenergic tone, such as fright or rage. A common KCNH2 polymorphism was found to affect KCNH2 channel function as demonstrated by in vitro experiments utilizing mammalian cells transfected with the KCNH2 potassium channel as well as QT-dynamics in vivo. Finally, the present study identified a common β-1-adrenergic receptor genotype that is related a shorter QT-interval in LQT1 patients. Also, it was discovered that compound homozygosity for two common β-adrenergic polymorphisms was related to the occurrence of symptoms in the LQT1 type of long QT syndrome. The studies demonstrate important genotype-phenotype differences between different LQTS subtypes and suggest that common modifier gene polymorphisms may affect cardiac repolarization in LQTS. It will be important in the future to prospectively study whether variant gene polymorphisms will assist in clinical risk profiling of LQTS patients.

Genistein and 17β-estradiol fatty acid esters in blood and adipose tissue and the structure-related antioxidant activity of estrogens on lipoproteins

Soy-derived phytoestrogen genistein and 17β-estradiol (E2), the principal endogenous estrogen in women, are also potent antioxidants protecting LDL and HDL lipoproteins against oxidation. This protection is enhanced by esterification with fatty acids, resulting in lipophilic molecules that accumulate in lipoproteins or fatty tissues. The aims were to investigate, whether genistein becomes esterified with fatty acids in human plasma accumulating in lipoproteins, and to develop a method for their quantitation; to study the antioxidant activity of different natural and synthetic estrogens in LDL and HDL; and to determine the E2 esters in visceral and subcutaneous fat in late pregnancy and in pre- and postmenopause. Human plasma was incubated with [3H]genistein and its esters were analyzed from lipoprotein fractions. Time-resolved fluoroimmunoassay (TR-FIA) was used to quantitate genistein esters in monkey plasma after subcutaneous and oral administration. The E2 esters in women s serum and adipose tissue were also quantitated using TR-FIA. The antioxidant activity of estrogen derivatives (n=43) on LDL and HDL was assessed by monitoring the copper induced formation of conjugated dienes. Human plasma was shown to produce lipoprotein-bound genistein fatty acid esters, providing a possible explanation for the previously reported increased oxidation resistance of LDL particles during intake of soybean phytoestrogens. Genistein esters were introduced into blood by subcutaneous administration. The antioxidant effect of estrogens on lipoproteins is highly structure-dependent. LDL and HDL were protected against oxidation by many unesterified, yet lipophilic derivatives. The strongest antioxidants had an unsubstituted A-ring phenolic hydroxyl group with one or two adjacent methoxy groups. E2 ester levels were high during late pregnancy. The median concentration of E2 esters in pregnancy serum was 0.42 nmol/l (n=13) and in pre- (n=8) and postmenopause (n=6) 0.07 and 0.06 nmol/l, respectively. In pregnancy visceral fat the concentration of E2 esters was 4.24 nmol/l and in pre- and postmenopause 0.82 and 0.74 nmol/l. The results from subcutaneous fat were similar. In serum and fat during pregnancy, E2 esters constituted about 0.5 and 10% of the free E2. In non-pregnant women most of the E2 in fat was esterified (the ester/free ratio 150 - 490%). In postmenopause, E2 levels in fat highly exceeded those in serum, the majority being esterified. The pathways for fatty acid esterification of steroid hormones are found in organisms ranging from invertebrates to vertebrates. The evolutionary preservation and relative abundance of E2 esters, especially in fat tissue, suggest a biological function, most likely in providing a readily available source of E2. The body s own estrogen reservoir could be used as a source of E2 by pharmacologically regulating the E2 esterification or hydrolysis.

Complex trait of acute pancreatitis : Studies of candidate genes and adipokines

Acute pancreatitis (AP) is a common disease. Mild disease resolves spontaneously in a few days. Severe forms of the disease can lead to local complications, necrosis, and abscesses in and around the pancreas. Systemic inflammation in severe AP is associated with distant organ failures. The aim of this study is to identify genetically determined prognostic factors involved in the clinical features of AP. The study employs a candidate-gene approach, and the genes are involved in trysinogen activation in the initiation phase of the disease, as well as in the systemic inflammation as the disease proceeds. The last study examines adipokines, fat-derived hormones characterized with the capacity to modify inflammation. SPINK 1 is a gene coding trypsin activation inhibitor. Mutations N34S and P55N were determined by minisequencing methods in 371 AP patients and in 459 controls. The mutation N34S was more common in AP patients (7.8%) than in controls (2.6%). This suggests that SPINK 1 gene mutation N34S is a risk factor for AP. In the fourth study, in 12 matched pairs of patients with severe and mild AP, levels of adipokines, adiponectin, and leptin were evaluated. Plasma adipokine levels did not differ between patients with mild and severe AP. The results suggest that in AP, adipokine plasma levels are not factors predisposing to organ failures. This study identified the SPINK 1 mutation N34S to be a risk factor for AP in the general population. As AP is a multifactorial disease, and extensive genetic heterogeneity is likely, further identification of genetic factors in the disease requires larger future studies with more advanced genetic study models. Further identification of the patient characteristics associated with organ failures offers another direction of the study to achieve more detailed understanding of the severe form of AP.

The function of NR3B and NR4A orphan nuclear receptors in osteoblasts

Nuclear receptors (NRs) comprise a large family of proteins that mediate the effects of small lipophilic molecules such as steroid hormones. In addition, there are a group of NRs which lack identified natural ligands and are referred as orphan NRs. In this thesis, the function of two such orphan NR families, the NR3B (ERRα, ERRβ and ERRγ) and the NR4A family (NGFI-B, Nurr1 and Nor1), was studied. NR3B and NR4A receptors regulate many biological processes such as energy metabolism and carcinogenesis. In addition, NR3B and NR4A receptors are expressed in bone. Therefore, the signaling and function of NR3B and NR4A orphan nuclear receptors was studied specifically in osteoblasts. NR4A receptors were found to be regulated by NR3B receptors and the Wnt/β-catenin signaling pathway as ERRα, ERRγ and β-catenin repressed the transcriptional activity of NR4A receptors in U2-OS cells. NGFI-B was found to repress the transcriptional activity of ERRγ in HeLa cells. The phytoestrogen equol was identified as a new agonist for ERRγ and ERRβ in PC-3, U2-OS, and SaOS-2 cells. Equol increased the transcriptional activity of ERRγ by increasing ERRγ co-activator binding and by inducing a conformational change in the ligand binding pocket of ERRγ. The growth inhibitory effect of equol on PC-3 prostate cancer cells was decreased by blocking ERRγ expression by siRNA. Therefore, ERRγ could mediate some of the beneficial health effects of equol. The Wnt/β-catenin signaling pathway is important for the differentiation and function of osteoblasts. NR3B and NR4A receptors were found to repress the transcriptional activity mediated by β-catenin in U2-OS cells. The mesenchymal stem cells (MSCs) isolated from ERRα knockout (KO) mice showed diminished proliferation and osteoblastic differentiation compared to the wild-type cells. The overexpression of ERRα in osteoblastic MC3T3-E1 cell line increased their mineralization. Bone sialoprotein (BSP) was shown to be a direct target gene for ERRα and ERRγ as the BSP promoter was activated by ERRα or ERRγ and PGC-1α in HeLa cells. The adipogenic differentiation of ERRα KO MSCs was also decreased and they expressed less adipogenic marker genes. In conclusion, the studies described in this thesis demonstrated that the transcriptional activity of NR3B and NR4A receptors can be regulated by other orphan NRs and signaling pathways in osteoblasts. NR3B receptors can also be regulated by ligands and a new agonist, equol, was identified for ERRβ and ERRγ. New roles for NR3B and NR4A were also identified as they were shown to converge with the Wnt signaling pathway in osteoblasts, ERRγ was shown to mediate the growth inhibitory effect of equol in prostate cancer cells, and ERRα was shown to regulate positively MSC proliferation, osteoblastic differentiation and adipogenesis.