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.

Function and regulation of Cdk7

Understanding the process of cell division is crucial for modern cancer medicine due to the central role of uncontrolled cell division in this disease. Cancer involves unrestrained proliferation as a result of cells loosing normal control and being driven through the cell cycle, where they normally would be non-dividing or quiescent. Progression through the cell cycle is thought to be dependent on the sequential activation of cyclin-dependent kinases (Cdks). The full activation of Cdks requires the phosphorylation of a conserved residue (threonine-160 on human Cdk2) on the T-loop of the kinase domain. In metazoan species, a trimeric complex consisting of Cdk7, cyclin H and Mat1 has been suggested to be the T-loop kinase of several Cdks. In addition, Cdk7 have also been implicated in the regulation of transcription. Cdk7, cyclin H, and Mat1 can be found as subunits of general transcription factor TFIIH. Cdk7, in this context, phosphorylates the Carboxy-terminal domain (CTD) of the large subunit of RNA polymerase II (RNA pol II), specifically on serine-5 residues of the CTD repeat. The regulation of Cdk7 in these and other functions is not well known and the unambiguous characterization of the in vivo role of Cdk7 in both T-loop activation and CTD serine-5 phosphorylation has proved challenging. In this study, the fission yeast Cdk7-cyclin H homologous complex, Mcs6-Mcs2, is identified as the in vivo T-loop kinase of Cdk1(Cdc2). It also identifies multiple levels of regulation of Mcs6 kinase activity, i.e. association with Pmh1, a novel fission yeast protein that is the apparent homolog of metazoan Mat1, and T-loop phosphorylation of Mcs6, mediated by Csk1, a monomeric T-loop kinase with similarity to Cak1 of budding yeast. In addition, Skp1, a component of the SCF (Skp1-Cullin-F box protein) ubiquitin ligase is identified by its interactions with Mcs2 and Pmh1. The Skp1 association with Mcs2 and Pmh1 is however SCF independent and does not involve proteolytic degradation but may reflect a novel mechanism to modulate the activity or complex assembly of Mcs6. In addition to Cdk7, also Cdk8 has been shown to have CTD serine-5 kinase activity in vitro. Cdk8 is not essential in yeast but has been shown to function as a transcriptional regulator. The function of Cdk8 is unknown in flies and mammals. This prompted the investigation of murine Cdk8 and its potential role as a redundant CTD serine-5 kinase. We find that Cdk8 is required for development prior to implantation, at a time that is co-incident with a burst of Cdk8 expression during normal development. The results does not support a role of Cdk8 as a serine-5 CTD kinase in vivo but rather shows an unexpected requirement for Cdk8, early in mammalian development. The results presented in this thesis extends our current knowledge of the regulation of the cell cycle by characterizing the function of two distinct cell cycle regulating T-loop kinases, including the unambiguous identification of Mcs6, the fission yeast Cdk7 homolog, as the T-loop kinase of Cdk1. The results also indicate that the function of Mcs6 is conserved from fission yeast to human Cdk7 and suggests novel mechanisms by which the distinct functions of Cdk7 and Mcs6 could be regulated. These findings are important for our understanding of how progression of the cell cycle and proper transcription is controlled, during normal development and tissue homeostasis but also under condition where cells have escaped these control mechanisms e.g. cancer.

Early growth and adult health: focus on blood pressure, glucose tolerance status and body composition

Theory of developmental origins of adult health and disease proposes that experiences during critical periods of early development may have consequences on health throughout a lifespan. Thesis studies aimed to characterize associations between early growth and some components of the metabolic syndrome cluster. Participants belong to two epidemiological cohorts with data on birth measurements and, for the younger cohort, on serial recordings of weight and height during childhood. They were born as singletons between 1924-33 and 1934-44 in the Helsinki University Central Hospital, and 500 and 2003 of them, respectively, attended clinical studies at the age of 65-75 and 56-70 years, respectively. In the 65-75 year old men and women, the well-known inverse relationship between birth weight and systolic blood pressure (SBP) was confined to people who had established hypertension. Among them a 1-kg increase in birth weight was associated with a 6.4-mmHg (95% CI: 1.0 to 11.9) decrease in SBP. This relationship was further confined to people with the prevailing Pro12Pro polymorphism of the peroxisome proliferator-activated receptor-γ2 (PPARγ2) gene. People with low birth weight were more likely to receive angiotensin-converting enzyme inhibitors/angiotensin-receptor blockers (ACEI/ARB, p=0.03), and, again, this relationship was confined to the carriers of the Pro12Pro (p=0.01 for interaction). These results suggest that the inverse association between birth weight and systolic BP becomes focused in hypertensive people because pathological features of BP regulation, associated with slow fetal growth, become self-perpetuating in adult life. Insulin resistance of the Pro12Pro carriers with low birth weight may interact with the renin-angiotensin system leading to raised BP levels. Habitual physical activity protected men and women who were small at birth, and thus at increased risk for the development of type 2 diabetes, against glucose intolerance more strongly. Among subjects with birth weight ≤3000 g, the odds ratio (OR) for glucose intolerance was 5.2 (95% CI: 2.1 to 13) in those who exercised less than 3 times per week compared to regular exercisers; in those who scored their exercise light compared with moderate exercisers (defined as comparable to brisk walking) the OR was 3.5 (1.5 to 8.2). In the 56-70 year old men a 1 kg increase in birth weight corresponded to a 4.1 kg (95% CI: 3.1 to 5.1) and in women to a 2.9 kg (2.1 to 3.6) increase in adult lean mass. Rapid gain in body mass index (BMI), i.e. crossing from an original BMI percentile to a higher one, before the age of 2 years increased adult lean mass index (LMI, lean mass/height squared) without excess fat accumulation whereas rapid gain in BMI during later childhood, despite the concurrent rise in LMI, resulted in a relatively higher increase in adult body fat mass. These findings illustrate how genes, the environment and their interactions, early growth patterns, and adult lifestyle modify adult health risks which originate from early life.

The Functions and Regulation of Ornithine Decarboxylase

Ornithine decarboxylase (ODC) regulates the synthesis of polyamines which are involved in many cellular functions e.g. proliferation and differentiation. Due to its critical role, ODC is a tightly regulated enzyme by antizymes and antizyme inhibitors. If the regulation fails, the activity of ODC increases and may lead to malignant transformation of a cell. Increased ODC activity is found in many common cancers, including colon, prostate, and breast cancer. In a transformed cell, dynamics of the actin cytoskeleton is disturbed. A small G-protein, RhoA regulates organization of the cytoskeleton, and its overactivity increases malignant potential of the cell. The present results indicate that covalent attachment of polyamines by transglutaminase is a physiological means of regulating the activity of RhoA. The translocation of RhoA to the plasma membrane, where it exerts its activity is dependent on the presence of catalytically active ODC. As the overactivity of ODC and RhoA are implicated in cell transformation, the results provide a mechanistic explanation of the interrelationship between the polyamine metabolism and the reorganization of the actin cytoskeleton occurring in cancer cells. ODC and polyamines have also an important role in the function of central nervous system. They participate in the regulation of brain morphogenesis in embryos. In adult nervous tissue, polyamines regulate K+ and glutamate channels. K+ inward rectifying channels control membrane potentials and NMDA-type glutamate receptors (NMDAR) regulate synaptic plasticity. High ODC activity and polyamine levels are considered important in the development of ischemic brain damage and they are implicated in the pathogenesis of Alzheimer s disease (AD). A homolog of ODC was cloned from a human brain cDNA library, and several alternatively spliced variants were detected in human brain and testis. The novel protein was nevertheless devoid of ODC catalytic activity. It was subsequently found to be a novel inductor of ODC activity and polyamine synthesis, called antizyme inhibitor 2 (AZIN2). The accumulation of AZIN2 in vesicle-like formations along the axons and beneath the plasma membrane of neurons as well as in steroid hormone producing Leydig cells and luteal cells of the gonads implies that AZIN2 plays a role in secretion and vesicle trafficking. An accumulation of AZIN2 was detected also in specimens of AD brains. This increased expression of AZIN2 was specific for AD and was not found in brains with other neurodegenerative diseases including CADASIL or dementia with Lewy bodies.

The Neurofibromatosis 2 tumor suppressor merlin in cytoskeleton organization and cell cycle regulation

Neurofibromatosis 2 (NF2) is a dominantly inherited disorder, which predisposes to multiple tumours of the nervous system, typically schwannomas and meningiomas. Biallelic inactivation of the NF2 gene occurs both in sporadic and NF2-related schwannomas and in most meningiomas. The NF2 gene product merlin (or schwannomin) is structurally related to the ERM proteins, ezrin, radixin and moesin, which act as molecular linkers between the actin cytoskeleton and the plasma membrane. Merlin is a tumor suppressor that participates in cell cycle regulation. Merlin s phosphorylation status appears to be associated with its tumour suppressor activity, i.e. non-phosphorylated merlin functions as a tumour suppressor, whereas protein phosphorylation results in loss of functional activity. This thesis study was initiated to investigate merlin s role as a tumor suppressor and growth inhibitor. These studies show, that like many other tumor suppressors, also merlin is targeted to the nucleus at some stages of the cell cycle. Merlin s nuclear localization is regulated by cell cycle phase, contact inhibition and adhesion. In addition, a potential nuclear binding partner for merlin was identified, Human Enhancer of Invasion 10 (HEI10), a cyclin B interacting protein. Many tumor suppressors interact with microtubules and this thesis work shows that also merlin colocalizes with microtubules in mitotic structures. Merlin binds microtubules directly, and increases their polymerization in vitro and in vivo. In addition, primary mouse Schwann cells lacking merlin displays disturbed microtubule cytoskeleton. Fourth part of this thesis work began from the notion that PKA phosphorylates an unidentified site from the merlin N-terminus. Our studies show that serine 10 is a target for PKA and modulation of this residue regulates cytoskeletal organization, lamellipodia formation and cell migration. In summary, this thesis work shows that merlin s role is much more versatile than previously thought. It has a yet unidentified role in the nucleus and it participates in the regulation of both microtubules and the actin cytoskeleton. These studies have led to a better understanding of this enigmatic tumor suppressor, which eventually will aid in the design of specific drugs for the NF2 disease.