Dissertations / Theses on the topic 'Glucocorticoids'

Glucocorticoids - cortisol and corticosterone - are steroid hormones synthesised in the adrenal gland that are important mediators of the stress response. Glucocorticoids are also vital in development to aid in organ maturation. Endogenous glucocorticoid levels rapidly rise before birth in all mammals to promote fetal organ maturation. Because preterm birth occurs before this natural rise in glucocorticoid levels, pregnant women at risk of preterm delivery are administered synthetic glucocorticoids to mature the fetal lung and aid neonatal survival. Mice that globally lack the glucocorticoid receptor (GR) die at birth, attributed to lung immaturity. Effects on tissues other than the lung remain less well characterised. Previous work has shown endogenous glucocorticoid action is also essential to mature the mouse fetal heart. Mice globally lacking GR have small, functionally and structurally immature hearts. Mice with tissue-specific deletion of GR in cardiomyocytes and vascular smooth muscle cells (SMGRKO mice; generated using Sm22α-Cre) also have an increased risk of death around the time of birth, suggesting that glucocorticoid maturation of the cardiovascular system is important for neonatal survival. GR expression within the fetal mouse heart initiates at E10.5 but GR in the myocardium is not activated and localised to the nucleus until E15.5. This suggests that mice can respond to glucocorticoid from E10.5. Here, it was hypothesised that antenatal glucocorticoid exposure, prior to the increase in endogenous glucocorticoid levels, would advance fetal heart maturation and this will depend on cardiovascular GR. To investigate the effects of antenatal glucocorticoid treatment on fetal heart maturation in mid-gestation and identify effects mediated by GR, mice with a conditional deletion of GR in cardiomyocytes and vascular smooth muscle cells were studied (SMGRKO mice). Pregnant mice received dexamethasone (dex) in the drinking water from E12.5-E15.5. Levels of Fkbp5 mRNA (a marker of glucocorticoid action) were unchanged between control and SMGRKO mice at E15.5 or following dex treatment. This suggested a lack of response to dex treatment. However, liquid chromatography mass spectrometry measurement confirmed the presence of dex and its active metabolite 6- hydroxydexamethasone (6OHDex) in livers of E15.5 fetuses from dex treated dams (fetal: Dex 0.46 ± 0.1 ng/g, 6OHDex 13.6 ± 0.35 ng/g; dam: Dex 7.96 ± 3.65 ng/g, 6OHDex 4.75 ± 1.2 ng/g). Livers of fetuses exposed to dex had lower levels of the naturally occurring active glucocorticoid, corticosterone, compared to vehicle treated fetuses. This suggests HPA axis suppression in dex exposed fetuses. Maternal liver showed no significant difference in corticosterone levels between dex and vehicle treated mice, suggesting that whilst dex suppressed the HPA axis in fetuses, it did not in the dams. To determine any persistent effects of early antenatal dex treatment on fetal heart, a later time point in gestation, E17.5, was also assessed. At E17.5, 2-days following cessation of dex treatment, dex and its metabolites were undetectable in the fetal and maternal liver. However, corticosterone levels remained reduced in fetal liver at E17.5 in dex exposed animals (vehicle treated: 4.31 ± 0.47 ng/g, Dex treated: 1.72 ± 0.42 ng/g, p < 0.01), whilst levels in the dam liver did not differ from vehicle treated controls. This suggests prolonged HPA axis suppression following dex treatment, which reduced the natural late-gestation rise in glucocorticoids required for fetal organ maturation. To determine whether early antenatal dex treatment could advance fetal heart function, Doppler imaging with a Vevo 770 high frequency ultrasound imager was used. Isovolumetric contraction time, isovolumetric relaxation time and ejection time of the left ventricle were unaltered by dex treatment. However, at E15.5 the mitral deceleration index (MDI), a measure of diastolic function that takes into account loading conditions, was 1.5 fold lower in vehicle treated SMGRKO mice than control (Cre-) littermates (p < 0.05). This reduction in SMGRKO mice suggests glucocorticoids are required within the fetal cardiomyocytes and/or vascular smooth muscle cells to mature the diastolic function of the fetal heart. Dex exposure had no effect on MDI in SMGRKO fetuses, but reduced the MDI by 1.5 fold in control mice to similar levels as in SMGRKO mice (p < 0.05). RNA analysis revealed a trend (p=0.09) for reduced levels of Nr3c1 mRNA (encoding GR) in hearts of E15.5 control (Cre-) fetuses following dex treatment. Although this requires confirmation at the level of GR protein, this finding together with the lack of induction of the GR target, Fkbp5, suggests dex may cause glucocorticoid resistance through down-regulation of GR. At E17.5, 2-days following cessation of dex there were no changes in systolic parameters and the reduction in MDI found at E15.5, following dex, had normalised. Litter size was reduced (close to a 50% reduction) at E17.5 in dex treated mice. This was similar between SMGRKO and control fetuses. The cause of death was not established, but potentially could be due to the reduction in the natural rise in glucocorticoids at E17.5, previously shown to be important for fetal heart maturation. It is therefore possible that mice with more immature hearts may die before reaching E17.5. RNA analysis was undertaken to determine any mechanistic alterations following dex treatment, which could support fetal heart functional alterations found at E15.5. In contrast to expectation, dex also decreased expression of mRNA encoding the calcium handling proteins SERCA2a, NCX1, and CaV1.2 in E15.5 fetal mouse hearts in both control and SMGRKO mice (p < 0.05), compared with the respective vehicle treated mice. These proteins had previously shown to be induced by glucocorticoid action in cardiomyocytes. However, the similar down-regulation in both genotypes indicates this effect is not dependent on GR in cardiomyocytes. Lowered SERCA2a activity following dex treatment could contribute to the changes in MDI observed in control mice. Similarly, Scnn1a and Kcnj12 mRNA levels, previously found to be induced by glucocorticoids in cardiomyocytes, were down-regulated in the E15.5 fetal heart in vivo following dex. Collectively, these data are consistent with glucocorticoid resistance or down-regulation of glucocorticoid action in E15.5 fetal hearts following dex administration. Mutations in KCNJ12 are associated with long QT syndrome, which is characterised by a delayed repolarisation of the heart following each contraction. An altered relaxation of the fetal heart found in control mice following dex could therefore be due to a prolongation of the cardiac action potential, particularly with a delayed repolarisation, because of lower Kcnj12 expression. At E17.5, there were no significant differences in expression of calcium handling genes or ion channel mRNAs between genotypes or following earlier dex exposure. Thus, effects of dex on mRNA expression level may not persist, which could account for the lack of functional changes observed 2-days following cessation of treatment. Because effects seen in vivo with dex treatment were contrary to those predicted, and to further investigate the effect of dex upon calcium content, an in vitro model of primary fetal E15.5 cardiomyocytes was used. Cardiomyocytes were treated with dex for 24 hours and effects on membrane potential voltage changes and calcium transients measured. Following dex, isolated fetal cardiomyocytes showed an elongated repolarisation phase of the action potential (untreated: 120.45 ± 13.81 ms, Dex: 142.34 ± 12.97 ms, p < 0.01), and duration of calcium transients (untreated: 103.31 ± 13.78 ms, Dex: 120.43 ± 23.36 ms, p < 0.05). This assessment of fetal cardiomyocytes was preliminary work to aid in the understanding of mechanisms of fetal heart functional alterations associated with glucocorticoid regulation. The results suggest glucocorticoids may be important in regulating calcium levels. In summary, dex treatment in mice from E12.5-E15.5 did not advance fetal heart maturation. It reduced litter size at E17.5, irrespective of whether GR was expressed in cardiomyocytes or not. The normal late-gestation increase in endogenous glucocorticoid levels in the fetus was reduced by dex, even after treatment finished.
The suppression of corticosterone levels following antenatal dex may reduce maturation of the heart at E15.5 and could be responsible for the reduction in litter size. Downregulation of GR in the fetal heart, may be a mechanism that results in glucocorticoid resistance following antenatal dex treatment, which could explain the lack of beneficial effects of antenatal dex upon fetal heart maturation in these experiments in mice.

In obese versus lean Zucker rats, hepatic 5α-reductase type 1 mRNA expression and protein levels were increased. They also had increased activity of hepatic 5β-reductase activity. By contrast, 3α-hydroxysteroid dehydrogenase mRNA expression was unchanged in obesity. Greater inactivation of B by A-ring reductases in liver may decrease local corticosterone (B) concentrations in these sites, and increase the metabolic clearance rate of glucocorticoids, thus increasing drive to the hypothalamic-pituitary-adrenal axis (HPA). To investigate whether 5α-reduced metabolites of corticosterone are glucocorticoid receptor agonists, competition binding studies were carried out. In displacing tritiated dexamethasone from binding sites in hepatocytes from male lean Zucker rats, B and 5α-tetrahydrocorticosterone (5αTHB) had similar affinities which were greater than 5α-dihydrocorticosterone (5αDHB) and 5β-reduced metabolites. Binding of B and 5αDHB binding was impaired in obesity whereas 5αTHB binding was unaltered suggesting that 5αTHB may modulate GR activation in obesity. Activation of flucocorticoid receptors was assessed following transient transfection into HeLa cells with an MMTV-luciferase reporter. By comparison with corticosterone, 5αTHB was active and additive. 5β-Reduced metabolites did not activate glucocorticoid receptors. In addition, in H4IIe cells which express endogenous glucocorticoid receptors, 5αTHB induced tyrosine aminotransferase mRNA expression albeit to a lesser extent than corticosterone. 5αTHB was also found to possess glucocorticoid activity in vivo as suppression of plasma ACTH was demonstrated in adrenalectomised lean Zucker rats following i.p. administration of B or 5αTHB. We conclude that hepatic A-ring reduction is enhanced in the obese Zucker rat producing increased concentrations of 5αTHB. Transcription of glucocorticoid regulated genes in tissues which express 5α-reductases will thus be influenced by intracellular levels of both corticosterone and its 5α-reduced metabolites. Manipulation of this enzyme may prove to be a useful therapeutic target in obesity.

Atherosclerotic and restenotic lesions develop as a result of an excess inflammatory response to vascular injury. Glucocorticoid hormones have widely-recognised anti-inflammatory and anti-proliferative properties which appear to make them ideal candidates for inhibition of vascular lesion development. Indeed, administration of glucocorticoids to experimental animals does inhibit the growth of vascular lesions in some models. In addition, glucocorticoids are currently being trialled clinically as anti-restenotic agents. However, glucocorticoid excess in patients, either as a result of Cushing’s syndrome or chronic steroid therapy, is associated with enhanced CVD risk. Therefore, the effects of glucocorticoids on vascular lesion development remain imperfectly understood. The overall objective of these studies was to explore the influence of endogenous and exogenous glucocorticoids on vascular lesion development using murine models of atherosclerosis (ApoE-/- mice fed a “western” diet) and neointimal hyperplasia (wireinduced femoral artery injury). The work described in this thesis addresses the hypothesis that glucocorticoids are pro-atherogenic, yet anti-restenotic. Mice were bilaterally adrenalectomised to investigate the role of endogenous glucocorticoids on vascular lesion development. Removal of the adrenal glands had no influence on atherogenesis or neointima development. The influence of exogenous glucocorticoids on lesion development was assessed by orally administering dexamethasone (0.1 or 0.8mg/kg/day). Atherosclerotic lesion burden was augmented by dexamethasone administration. Conversely, fibro-proliferative neointimal proliferation was inhibited by dexamethasone. However, this effect was obscured by thrombotic lesion development. It was proposed that this thrombotic effect is attributable to increased plasminogen activator inhibitor-1 (PAI-1), which was tested using PAI-1 deficient mice. Although PAI-1 was found to mediate the systemic pro-thrombotic effect of dexamethasone, it is not required for the enhanced development of thrombotic lesions at the site of intra-luminal injury. These results suggest that physiological levels of endogenous glucocorticoids play a limited role in vascular lesion development. Conversely, although exogenous glucocorticoids inhibit fibro-proliferative intimal hyperplasia, they appear to have significant detrimental influences on lesion development, augmenting atherosclerosis and inducing thrombotic neointimal lesion formation following vascular injury. Further research is therefore required to improve the cardiovascular outcome of patients requiring glucocorticoid therapy and for the use of glucocorticoids as antirestenotic agents.

It was hypothesised that generation of endogenous glucocorticoids by 11βHSD1 within the vessel wall regulates angiogenesis. In vitro mouse aortic ring cultures established that physiologically-relevant concentrations of glucocorticoids inhibit angiogenesis in a glucocorticoid receptor-dependent manner.  In addition 11βHSD1 was found to modulate glucocorticoid-induced angiostasis, for 11dehydrocorticosterone (a substrate for 11βHSD1) although angiostatic in C57B16 aortae did not inhibit angiogenesis in llβHSD1 deficient animals. In vivo using subcutaneous sponge implants in mice, endogenous glucocorticoids were found to inhibit angiogenesis: sponges in adrenalectomised mice grew more vessels compared to sponges from sham-operated animals. 11βHSD1 regulated the angiostatic effects of glucocorticoids, for cortisone (the human equivalent of 11dehydrocorticosterone), although angiostatic in controls did not inhibit angiogenesis in 11βHSD1 deficient mice. In pathology in cutaneous wounds and infracted myocardium endogenous glucocorticoids were found to inhibit angiogenesis. RU38486, (a glucocorticoids receptor antagonist) in comparison to placebo enhanced angiogenesis in both tissues. In similar studies in C57B16 or llβHSD1 deficient mice, 11βHSD1 was found to tonically repress angiogenesis and impair left ventricular remodelling post infarction. Thus 11βHSD1 deficient mice had increased myocardial revascularisation and preserved left ventricular function. In conclusion, by using in vitro, in vivo, and pathological models, endogenous glucocorticoids were seen to inhibit angiogenesis. In addition, 11βHSD1 regeneration of glucocorticoids tonically repressed angiogenesis and influenced left ventricular remodelling post myocardial infarction. Thus 11βHSD1 appears to be an attractive therapeutic target for the management of tissue revascularisation.

Glucocorticoids are the most effective anti-inflammatory agents currently available, but a variety of adverse effects limit their clinical usefulness. This work explores further two facets of the interaction between glucocorticoids and the skin, with the aim of identifying means of reducing glucocorticoid toxicity. (a) Metabolism of glucocorticoid by skin: Human skin is active in the terminal metabolism of cortisol to cortisone, but the biological implications of this process in skin are uncertain. Because there are technical difficulties in dealing with human skin, an animal model, the nude mouse, has been evaluated for its suitability to the study of the metabolism of corticosterone to 11β-dehydrocorticosterone (the homologous reaction in rodents of cortisol to cortisone conversion in man); a process mediated by 11β-hydroxysteroid dehydrogenase. In this model, skin 11β-hydroxysteroid dehydrogenase had an apparent Km for corticosterone of 37 uM. Skin 11β-hydroxysteroid dehydrogenase was up-regulated, in-vivo, by active glucocorticoids and was NADP dependent. By comparison, kidney 11β-hydroxysteroid dehydrogenase had a higher apparent Km (120 uM) for corticosterone, used NAD and NADP with equal facility and was not regulated in-vivo by glucocorticoids. These data suggest that the skin may possess an isoform distinct from that of the kidney. (b) Skin vasoconstrictor response (blanching) to topical glucocorticoids: Glucocorticoids applied topically to human skin produce vasoconstriction in dermal vessels, the degree of which correlates closely with the potency and clinical efficacy of these compounds. Although previous works had noted heterogeneity in blanching responses to glucocorticoids, this was never systematically studied. In qualitative studies, it was shown that skin blanching was inducible by RU-28362, a specific glucocorticoid receptor (type II) agonist and blocked by RU-38486, a glucocorticoid antagonist.

La cachexie affecte environ la moitié des patients atteints d’un cancer et est caractérisée par une perte progressive de la masse corporelle résultant principalement d’une perte de masse musculaire squelettique. Cette perte de masse musculaire squelettique associée à une perte de force musculaire contribue fortement à réduire la qualité de vie des patients, l’efficacité des traitements et à terme, la survie des patients. Plusieurs facteurs sont connus pour être impliqués dans la régulation de la masse musculaire. Parmi eux, les glucocorticoïdes sont des hormones stéroïdiennes sécrétées sous le contrôle de l’axe hypothalamo-hypophysaire qui sont connues pour induire l’atrophie musculaire mais aussi pour avoir une action systémique via l’activation ou l’expression de gènes dans plusieurs tissus. Nous faisons l’hypothèse que la voie des glucocorticoïdes pourrait être activée pendant la cachexie associée au cancer dans les souris ApcMin/+, un model murin de cancer intestinal. Nous rapportons ici que l’activation du catabolisme musculaire était associée à une reprogrammation complète du métabolisme du foie. En outre, nous montrons une activation de l’axe hypothalamo-hypophysaire associée à une augmentation du niveau en corticostérone (le glucocorticoïde principal chez les rongeurs) dans le sérum, le muscle quadriceps et le foie des souris à un stade avancé de la cachexie associée au cancer. La signature transcriptionnelle dans le muscle quadriceps et le foie des souris à un stade avancé de la cachexie associée au cancer reflète celle observée dans des souris traitées avec de la dexaméthasone, un analogue des glucocorticoïdes. Il est important de souligner que l’inhibition de la cachexie associée au cancer par l’inactivation du gène de la myostatine dans les souris ApcMin/+ a restauré les niveaux en corticostérone et abolit la reprogrammation génique dans le muscle squelettique et le foie. Ensemble, ces données indiquent que les glucocorticoïdes induisent un programme transcriptionnel pour réguler de façon coordonnée la perte de masse musculaire et le remaniement du métabolisme hépatique. L’inhibition de cette réponse par l’invalidation du gène de la myostatine souligne l’existence d’un dialogue moléculaire entre le muscle squelettique et le foie
Cachexia affects about half of cancer patients and is characterized by a progressive body mass loss mainly resulting from skeletal muscle depletion. This loss of skeletal muscle mass together with a decrease in muscle force strongly contribute to reduce cancer patient quality of life, treatment efficiency and ultimately patient survival. Many factors are known to be involved in the regulation of skeletal muscle homeostasis. Among them, glucocorticoids are steroid hormones secreted under the control of the hypothalamic-pituitary axis that have been well described to promote skeletal muscle atrophy but also to exert systemic actions through activation or repression of gene expression in many tissues. We hypothesized that the glucocorticoid pathway could be activated during cancer cachexia in ApcMin/+ mice, a mouse model of intestinal cancer. Here, we reported that activation of skeletal muscle catabolism was associated with a complete reprogramming of liver metabolism. Moreover, we showed an activation of the hypothalamus-pituitary axis that was associated with an increase in the level of corticosterone (the main glucocorticoid in rodent) in serum, quadriceps muscle and liver of advanced cancer cachectic mice. The transcriptional signature in quadriceps muscle and liver of advanced cancer cachectic mice significantly mirrored that observed in mice treated with dexamethasone, an analog glucocorticoid. Importantly, the inhibition of cancer cachexia by myostatin gene invalidation in ApcMin/+ mice restored corticosterone levels and abolished skeletal muscle and liver gene reprogramming. Together, these data indicate that glucocorticoids drive a transcriptional program to coordinately regulate skeletal muscle mass loss and hepatic metabolism rewiring. The inhibition of this response by myostatin gene invalidation highlights the existence of a molecular dialog between skeletal muscle and liver

Le stress chronique apparait comme une cause importante de troubles de la fertilité chez la femelle. L’hypercorticisme (sécrétion excessive de glucocorticoïdes (GC) surrénaliens) induit par le stress agit sur l’axe de reproduction pour perturber le cycle œstrien et l’ovulation. Chez la femelle, l’axe de reproduction comprend 3 grandes structures : l’hypothalamus, l’hypophyse et l’ovaire. Pour bloquer l’activité de cet axe, les GC agissent via un récepteur spécifique (GR) pour induire une signalisation rapide non génomique et/ou une signalisation génomique tardive. Malgré de nombreuses recherches, les connaissances sur le mode d’action des GC sur cet axe restent fragmentaires. Au cours de ce travail de thèse, l’utilisation de différents modèles cellulaires et animaux nous a permis de mettre en évidence des mécanismes d’action des GC originaux sur l’axe hypothalamo-hypophysaire. Au niveau hypothalamique, nous avons décrit un nouveau dialogue entre les œstrogènes et les GC, qui favorise l’expression d’un neuropeptide hypothalamique inhibiteur, la dynorphine A. Ce neuropeptide pourrait ainsi participer au blocage des sécrétions pulsatiles de la GnRH (Gonadotropin-releasing Hormone) hypothalamique et des gonadotropines hypophysaires indispensables à l’ovulation. Au niveau hypophysaire, nous avons mis en évidence une action paradoxale des GC sur les cellules gonadotropes. En absence de GnRH, les GC stimulent une nouvelle voie de signalisation non génomique, initiée à la membrane plasmique par un GR palmitoylé. Cette signalisation rapide implique la calcium/calmoduline kinase II (CaMKII) ainsi qu’une de ses cibles, la synapsine-Ia. Néanmoins, en présence de GnRH, les GC bloquent la signalisation induite par la GnRH, en empêchant l’activation de la CaMKII, pouvant être à l’origine de l’inhibition de la sécrétion de la gonadotropine LH (Luteinizing Hormone).De nouvelles recherches sont à poursuivre pour approfondir nos connaissances sur les différents modes d’action des GC pouvant être spécifiquement mis en jeu au sein de chacune des cellules, et à l’origine des effets variés des GC au sein de l’organisme. Une meilleure compréhension des mécanismes moléculaires responsables de l’altération de la fertilité, en particulier durant un stress chronique, est essentielle pour envisager l’émergence de nouvelles cibles thérapeutiques innovantes
The chronic stress is an important cause of fertility disorders in female. Stress-induced hypercorticism (excessive secretion of glucocorticoids (GC)) acts on the reproductive axis to disrupt the estrous cycle and ovulation. In female, the reproductive axis comprises 3 structures: the hypothalamus, the pituitary and the ovary. To block the activity of this axis, GC act through a specific receptor (GR) to promote rapid non genomic and/or late genomic signaling. Despite extensive researches, knowledge on the mechanism of action of GC on this axis remains elusive. During my PhD, the use of different cellular and animal models allowed to highlight new mechanisms of GC actions on the hypothalamic-pituitary axis. At the hypothalamic level, we described a new genomic cross-talk between estrogen and GC to promote the expression of an inhibitory hypothalamic neuropeptide, dynorphin A. This neuropeptide could then participate in disrupting the pulsatile secretion of GnRH (Gonadotropin-releasing hormone) and pituitary gonadotropins which are essential for ovulation. At the pituitary level, we demonstrated a paradoxical action of GC on gonadotrope cells. In the absence of GnRH, GC stimulate a new non genomic pathway initiated at the plasma membrane through a palmitoylated GR. This rapid signaling involves calcium/calmodulin kinase II (CaMKII) as well as one of its targets, synapsin-Ia. Nevertheless, in the presence of GnRH, GC interfere with GnRH-induced signaling by preventing CaMKII activation, which may be responsible for the inhibition of LH (Luteinizing Hormone) release.Further researches are required to improve our knowledge on cell-specific mechanisms of action of GC that could explain the diversity of their activities. A better understanding of the molecular mechanisms responsible for fertility dysfunction, especially during chronic stress, is essential for the development of new and innovative therapeutic targets

Orientador: Sérgio Luiz Felisbino
Coorientador: Wellerson Rodrigo Scarano
Banca: Raquel Fantin Domeniconi
Banca: Luis Antonio Justulin Júnior
Banca: Renata Carolina Piffer
Banca: Glaura Scatamburio Alves Fernandes
Resumo: Estudos têm sugerido que o excesso de glicocorticoides (GCs) durante períodos críticos do desenvolvimento pode alterar a função reprodutiva. Apesar da função essencial da próstata no sucesso reprodutivo e de sua alta susceptibilidade ao desenvolvimento de lesões com o avançar da idade, o impacto tardio de corticoterapias precoces sobre a homeostase da glândula ainda é desconhecido. No presente estudo, investigamos os efeitos da corticoterapia prenatal (PRE), durante a instalação da puberdade (PU), e sua associação (PRE+PU=REE), sobre a morfofisiologia da próstata senescente. Ratas Wistar prenhes receberam betametasona (0.1mg/kg/dia, i.m.), ou salina, nos dias gestacionais 12, 13, 18 e 19. Os descendentes machos receberam doses de betametasona (7mg/kg/dia, gavage), ou salina, do dia pós-natal 35 ao 50 (PND35-50). Na idade senil (PND300), todos os animais foram eutanasiados, amostras de sangue foram coletadas para dosagens hormonais, e a próstata ventral (VP) foi dissecada e processada para a análise morfológica, bem como para quantificação e localização de proteínas (AR, GR, PAR-4 e PCNA). Reduzidos níveis de testosterona e insulina foram observados no grupo PRE, enquanto apenas a insulina mostrou-se reduzida no grupo PU, e nenhuma redução adicional foi observada em REE. Uma tendência de aumento no índice apoptótico e incidência de ácinos com epitélio metaplásico foi detectada dentre os grupos. A quantificação de proteínas revelou menor expressão de AR no grupo PRE, maior expressão do marcador de proliferação celular (PCNA) no grupo REE, porém, diferença significativa alguma foi observada na expressão do marcador de morte celular por apoptose (PAR-4). A análise da reação imunoistoquímica para o GR indicou uma maior expressão do receptor em células epiteliais dos grupos que receberam betametasona. Com base nos resultados, sugerimos que a corticoterapia com betametasona durante o final da ...
Abstract: Studies have suggested that glucocorticoids (GCs) excess during critical developmental time windows can alter reproductive parameters. Despite of the key function of the prostate in the reproductive success, and its high susceptibility to develop lesions in an age-dependent manner, the impact of early GCs excess on the gland homeostasis is still unknown. In the present study, we have investigated the effects of prenatal (PRE), peripubertal (PU) corticotherapy, and its combination (PRE+PU=REE), on aging prostate's morphophysiology. Pregnant Wistar rats received betamethasone (0.1mg/kg/day, i.m.), or saline, on the gestational days 12, 13, 18 and 19. Male descendents received betamethasone (7mg/kg/day, gavage), or saline, from 35th to 50th postnatal day (PND35-50). Late in life (PND300), all animals were euthanized, blood samples were taken for hormones levels estimation, and the ventral prostate (VP) was excised and processed for morphology evaluation, and for proteins (AR, GR, PAR-4 and PCNA) quantification and localization as well. Lower testosterone and insulin levels were detected in group PRE, while only insulin serum levels was reduced in group PU, and no additional decrease was seen in REE. An increasing trend in the apoptosis index and metaplastic epithelium acini incidence was observed along the treated groups. The protein quantifications showed a decreased AR expression in PRE, higher proliferation marker (PCNA) expression in REE, and no significant difference in the expression of the apoptosis marker PAR-4 was detected among the groups. The immunolocalization of GR indicated a higher receptor expression in epithelial cells of treated groups, when compared to NE. Based on these results, we suggest that the corticotherapy with betamethasone during late pregnancy can program fetal prostate, resulting in altered androgen and glucocorticoids signaling permanently. Peripubertal corticotherapy deflagrated cell ...
Doutor

Glucocorticoids (GC) are powerful metabolic hormones with anti-inflammatory actions. Despite major side effects they remain widely prescribed therapies. GC regulates gene expression through an intracellular receptor (GR), which is a ligand activated transcription factor. Macrophages are innate immune cells and major targets of GC. Traditionally repression of pro-inflammatory genes in the context of an inflammatory stimulus has been considered the primary mode of action of GC in macrophages. The work described in this thesis has demonstrated that GC act primarily as inducers of gene expression in primary macrophages from both mouse and man, but the set of induced genes is very different between the two species. Chromatin immunoprecipitation and sequencing (ChIP-seq) in each species using anti-GR antibodies revealed candidate enhancers in the vicinity of inducible genes that were generally not shared between mouse and man. The differences in binding were correlated with DNA sequence changes at the enhancer sites between the two species, that caused gain or loss of predicted GR receptor-binding motifs. The mechanism of action of GC was investigated by imaging several different target loci using fluorescence in situ hybridisation in macrophage nuclei. Chromatin at specific GC responsive loci was found to decondense within minutes of exposure of macrophages to the ligand. The apparent decondensation was effect was maintained for at least 24 hours and was not prevented by inhibitors of transcription. The general principles of the GC response were shared between species. However the divergence found underlines the caution that must be used when translating specific findings from mouse to man. Additionally, the data support a role for GR driven changes to chromatin structure in gene regulation in macrophages.

2013/2014
The Hippo signalling pathway is tumour suppressor cascade with a central role in the regulation of fundamental cellular biological processes, such as cell proliferation, apoptosis, organ size control and stem cell functions. The Hippo pathway transduces external signals that come to the cell into the nucleus, where it can control the expression of specific target genes, mainly involved in cell proliferation and differentiation. The Hippo pathway is an inhibitory pathway that control by phosphorylation and inhibition Yes-associated protein (YAP) coactivator, one of the two nuclear effectors of this signalling, involved in the regulation of proliferation and organ size. As consequence, deregulation of Hippo tumor suppressor pathway or hyperactivation of its downstream effectors is often associated with formation, development and tumour dissemination. Consistently, YAP is often over-expressed in a broad range of different tumours and it has aberrant activity in breast cancer as well as in several other human carcinomas. Up-regulation of YAP activity increases stem cell self-renewal in normal and cancer stem cells. In this work we describe the identification of a new hormonal-dependent layer for YAP regulation in breast cancer by the glucocorticoids and we analyze the mechanisms through which this regulation occurs. We found that Glucocorticoid Receptor (GR) binds directly the YAP promoter and induces the transcription of YAP mRNA after GC stimulation in cancer cells. Moreover, GC lead to efficient YAP de-phosphorylation and transcriptional activation, in a transcription-independent manner, by inducing actin cytoskeleton reorganization. Importantly, inhibition of the GR by means of RU486 (GR competitive antagonist) strongly blunted the expansion of the cancer stem cell pool in breast cancer cells by blunting the GR/YAP axis.
XXVII Ciclo
1987

11β-Hydroxysteroid dehydrogenase catalyses the conversion of cortisol to inactive cortisone. In kidney, the enzyme confers aldosterone-specificity on mineralocorticoid receptors by preventing their occupancy by cortisol. The expression of 11β-dehydrogenase in many extra-renal sites suggests that it has a wide role in modulating cortisol sensitivity. When renal 11β-dehydrogenase is defective (as occurs in congenital deficiency and after inhibition of the enzyme by liquorice or carbenoxolone) cortisol-dependent mineralocorticoid excess and hypertension ensue. In this thesis I address the hypotheses that: (i) in addition to its role in the kidney, 11β-dehydrogenase modulates the cortisol sensitivity in vascular smooth muscle; (ii) 11β-dehydrogenase is regulated physiologically by the hypothalamic-pituitary-adrenal axis; and (iii) deficiency of 11β-dehydrogenase, associated with increased cortisol sensitivity either in kidney or in blood vessels, contributes to pathophysiology in other forms of hypertension, specifically ectopic ACTH syndrome and essential hypertension. I show that 11β-dehydrogenase immunoreactivity and mRNA are localised to vascular smooth muscle cells in the rat, and bioactivity is greater in resistance vessels than conduit arteries. In support of a diverse role for the enzyme in modulating vascular tone I demonstrate: (i) potentiation of vasoconstrictor sensitivity of cortisol - and thereby to noradrenaline - in the forearm and dermis of men given enzyme inhibitors or congenitally deficient in 11β-dehydrogenase; and (ii) attentuation of noradrenaline reactivity by glucocorticoids in rat aorta which is increased following in vitro carbenoxolone administration. By studying in vitro affinities of rat vascular 11β-dehydrogenase for its cofactors I demonstrate its similarity to the hepatic isoform of the enzyme, and distinguish it from that in kidney. Moreover, vascular but not renal 11β-dehydrogeanse is induced by in vivo administration of glucorticoids. By contrast, ACTH is without effect on 11β-dehydrogenase activity in adrenalectomised rats, but inhibits the peripheral conversion of cortisol to cortisone in man. By selective venous catheterisation studies I confirm that the major site for this conversion is the kidney. Thus ACTH, in addition to stimulating cortisol synthesis, may stimulate the secretion of an 11β-dehydrogenase inhibitor from the adrenal, and thereby increase renal sensitivity to cortisol.

Several domains of cognitive function show lower mean scores and increasing variability with increasing age. Little is known about the biological mechanisms underlying these changes. There is evidence, largely from animal studies, that prolonged exposure to high levels of glucocorticoids is associated with (a) atrophy of brain regions known to be essential for cognitive functioning, such as the hippocampus, and (b) decrements in cognitive function with ageing. There are few human studies examining these links. The studies in this thesis were aimed at testing the hypotheses that elevated levels in Cortisol are associated with relative cognitive impairment, with relative atrophy of the hippocampus and other brain regions, and also with variations in the levels of brain metabolites, and also that cognitive function is associated with brain size and metabolites. Additionally, measures of glucose homeostasis (fasting glucose and glycosylated haemoglobin (HbAlc)) were hypothesised to be negatively correlated with cognitive function. Ill healthy, unmedicated men aged 65-70 were recruited. Subjects had blood taken for 9am, 2.30pm, and post-dexamethasone (0.25mg) Cortisol levels, fasting glucose, and HbAlc, and did a battery of cognitive tests, including tests of 'premorbid' intelligence, fluid intelligence, verbal and visuospatial memory and processing speed. 100 of the subjects underwent two modalities of neuroimaging: (a) structural magnetic resonance imaging, with intracranial area, hippocampus, temporal lobe and frontal lobe volumes measured, and (b) magnetic resonance spectroscopy (MRS), with N-acetylaspartate (NAA), choline (Cho) and creatine (Cr) levels measured. Principal components analyses showed that a single component (designated the 'general cognitive factor') accounted for 51% of the variance in cognitive performance; rotation yielded two correlated components representing fluid intelligence/visuospatial memory tasks, and verbal memory tasks. Intracranial area and several regional brain volumes correlated positively and significantly with 'premorbid' and fluid intelligence and visuospatial memory. Verbal memory and verbal fluency did not correlate significantly with any brain volumes. Structural equation modelling showed that the relationships between cognitive tests and brain volumes could best be summarised by a significant positive relationship between overall brain size and the general cognitive factor (r=0.42, p < 0.05), and not by associations between individual tests and particular brain regions. Both NAA/Cr and Cho/Cr ratios correlated positively with tests of verbal memory and a verbal memory factor (e.g. NAA/Cr and Logical Memory: r=0.24, p < 0.05). Cho/Cr ratios also correlated positively with visuospatial memory (eg. Visual Reproduction: r=0.21, p < 0.05). There were several small but statistically significant correlations in the predicted (negative) direction between brain volumes and Cortisol levels. Left temporal lobe volumes correlated with 9am Cortisol (r=-0.22) and 2.30pm Cortisol (r=-0.26), right temporal lobe volumes correlated with 9am Cortisol (r=-0.21), right hippocampal volumes correlated with 9am Cortisol (r=-0.22) and postdexamethasone Cortisol (r=-0.24). These correlations were significant at p < 0.05, 2- tailed. There were no significant correlations between Cortisol measures and metabolite ratios (from MRS). Correlations between Cortisol measures and cognitive tests were largely in the predicted direction, though few of these correlations reached conventional levels of statistical significance. The general cognitive factor and the fluid/intelligence factor, adjusted for 'premorbid' intelligence, correlated significantly and negatively with 9am Cortisol levels, at r=-0.23 (p=0.028, 2-tailed). HbAlc was significantly negatively correlated with two measures of verbal memory, but not with other cognitive tests (list-learning: r=-0.24, p=0.01; delayed paragraph recall r=-0.31, p=0.018, 2-tailed). These results demonstrate that in healthy, elderly men, overall brain size and metabolite ratios are significantly related to cognitive ability. A possible mechanistic link between these two domains is variations in Cortisol with ageing. The results of the present studies are supportive of the hypothesis that elevated glucocorticoids are associated with ageing-related changes in brain volumes, and, less clearly, cognitive function. Follow-up studies will help determine whether Cortisol levels are predictive of worsening brain atrophy and cognitive decline.

The purpose of these studies was to determine the effects and mechanisms of action of glucocorticoids on eosinophil and neutrophil apoptosis and to examine the effects of these drugs on the phagocytosis of apoptotic granulocytes by macrophages. Dexamethasone exerts diametrically opposed effects on these two cell types; promoting eosinophil apoptosis and inhibiting neutrophil apoptosis. Similarly, elevation of [Ca²⁺]_i also differentially affects the rate of constitutive granulocyte apoptosis. In contrast, elevation of cAMP inhibits the rate of apoptosis in both granulocytes, whereas inhibition of PKC promotes the rate of granulocyte apoptosis. Moreover, inhibition of protein phosphatases inhibits the rate of granulocyte apoptosis at lower concentrations and promotes the rate of granulocyte apoptosis at higher concentrations. Inhibition of the MAP/ERK kinase cascade inhibits the rate of constitutive eosinophil apoptosis, while having no effect upon the rate of constitutive neutrophil apoptosis. However, inhibition of this cascade selectively blocks the neutrophil survival-promoting effects of LPS, but exerts no effect on the glucocorticoid-mediated neutrophil survival-promoting effects of LPS, but exerts no effect on glucocorticoid-mediated inhibition of neutrophil apoptosis. Dexamethasone mediates inhibition of neutrophil apoptosis by a PKA-dependent mechanism, whereas the pro-apoptotic effect of dexamethasone upon eosinophil apoptosis appears to the PKA-independent. Dexamethasone potentiates macrophage recognition and phagocytosis of apoptotic eosinophils and neutrophils. Demonstration of the marked apoptosis-promoting effects of corticosteroids on eosinophil apoptosis together with the augmentation of macrophage clearance of apoptotic eosinophils, may in part explain the known beneficial therapeutic effects of corticosteroids on established 'eosinophilic' inflammatory diseases such as asthma. The opposite effect on neutrophil apoptosis may underlie the lower efficacy of these drugs in 'neutrophilic' inflammatory diseases such as SRDs. Further dissection of the intracellular mechanisms governing the divergent effects of corticosteroids on eosinophil and neutrophil apoptosis may lead to new therapeutic targets with which selective induction of apoptosis could be achieved.

The efficiency of topical drug delivery is notoriously poor, with typical bioavailabilities of only a few percent of the applied dose. A major reason for this disappointing situation is the absence of a quantitative and validated methodology (apart from the vasoconstrictor assay for topical glucocorticoids) with which to quantify the rate and extent of drug delivery to a target into the skin. In an attempt to address this situation, significant efforts are being directed to the dermatopharmacokinetic (DPK) approach using tape-stripping. The main objective of this thesis, therefore, was to compare the in vivo bioavailability profiles of betamethasone 17-valerate (BMV) assessed using the vasoconstrictor assay and the DPK approach. Furthermore, the ability of these two methods to distinguish between different formulations as well as different concentrations was examined. As the DPK approach is currently under critical re-evaluation, different cleaning procedures of the skin before tape-stripping were compared and evaluated. Moreover, the influence of the viscosity of the formulation on the DPK results was also determined. In addition, the effect of the vehicles on skin hydration was studied. Applying different BMV concentrations resulted in a clear concentration dependence of the skin blanching response until saturation of the response occurred. Upon this saturation effect, any changes between different formulations and between concentrations could no longer be observed. Due to the saturable nature of the skin blanching response, the interpretation of the data has to be considered with care and, therefore, it is important to operate in the linear part of the ‘dose-response’ curve, whenever quantitative conclusions about bioavailability are to be drawn. The DPK approach, on the other hand, showed reasonable reproducibility and distinguished clearly between different formulations and different BMV concentrations applied; it appears to offer a reliable metric, therefore, with which to quantify topical bioavailability. The cleaning procedure, as well as the viscosity of the formulation applied, have a significant influence on the apparent extent of drug delivery into the stratum corneum. Excess formulation, especially from semi-solids, may be trapped in the skin ‘furrows’ and requires an efficient skin cleaning procedure to ensure its complete removal. Overall, the DPK technique merits continued evaluation and optimization as a tool for the quantification of topical bioavailability and bioequivalence. These steps are essential for the ultimate objective of validating the results of DPK experiments against credible clinical data.

Il comportamento è concepito come una relazione dipendente stimolo-risposta tra un input sensoriale e una risposta motoria. Nel passaggio da input a output, l’omeostasi interna è continuamente modellata per mantenere un equilibrio ottimale della spesa energetica. Lo scopo ultimo di mantenere l’omeostasi in relazione al mondo circostante viene raggiunto attraverso la produzione di comportamenti adattativi che permettono di incrementare la fitness alla luce della selezione naturale. L’ambiente circostante può essere sia prevedibile sia imprevedibile. La prima condizione ha portato all’evoluzione del ritmo circadiano che promuove la fase di attività durante il momento più favorevole della giornata, mentre la seconda si serve dell’asse dei glucocorticoidi per affrontare le sfide imprevedibili. Quindi, un dialogo tra il sistema circadiano e il sistema dei glucocorticoidi è mantenuto allo scopo di ottenere una regolazione ottimale dell’attività animale. Il mio obiettivo è quello di capire il dialogo tra i due sistemi monitorando il comportamento giornaliero e circadiano, e la sua controparte molecolare, in fase a differenti cicli di luce e cibo. La mia specie modello è lo zebrafish (Danio rerio), in particolare, ho utilizzato un mutante costruito con la tecnica CRISPR/Cas9 che manca della capacità di coordinare la via di trascrizione dei glucocorticoidi a causa della mancata funzionalità del loro recettore cognato tale che l’interazione ligando recettore non è mantenuta. Di conseguenza, i livelli circolanti di glucocorticoidi restano elevati, conferendo al mutante un fenotipo ansioso. Zebrafish gr-/- è stato costruito e gentilmente fornito dal laboratorio della Prof.ssa Luisa Dalla Valle, Università degli studi di Padova. L’analisi sistematica del comportamento in larve e adulti di gr-/- ha mostrato che l’attività locomotoria sincronizzata alla luce mantiene le sue proprietà oscillatorie endogene. Tuttavia, l’attività locomotoria giornaliera insorge con un ritardo di un giorno nei mutanti rispetto ai wild type. Questa insorgenza ritardata è associata a un rallentamento nello sviluppo del tessuto muscolare striato, la normale densità delle fibre muscolari viene ripristinata nei gr-/- al sesto giorno dopo la fertilizzazione. Inoltre, le larve gr-/- hanno mostrato differenze nei livelli di espressione e nelle relative acrofasi di elementi positivi (arntl1a and clock1a) e negativi (per1, per2a and cry1a) dell’orologio molecolare. Al di là degli elementi del cuore dell’orologio circadiano, un’analisi nel fegato di adullti gr-/- rivela un’abolizione dell’espressione di pck2, un gene implicato nella gluconeogenesi. In aggiunta, srebp1 ha un’acrofase anticipata nei mutanti. La sincronizzazione circadiana al cibo fallisce nei gr-/-, sia larve sia adulti producono profili anomali dell’attività locomotoria. L’analisi molecolare non associa la disfunzionalità comportamentale a quella genetica, infatti i geni orologio non mostrano alterate oscillazioni a eccezione di cry1a. Questi dati suggeriscono l’esistenza di un confine sfuocato tra il sistema circadiano e quello dei glucocorticoidi e una complessa organizzazione dei due ha prodotto un alterato output comportamentale negli zebrafish gr-/-. La causa prossima del disallineamento tra lo stimolo alimentare e la locomozione non è stata chiarita sebbene un passo avanti verso una maggiore comprensione del dialogo tra glucocorticoidi e orologio circadiano getta le basi per un’indagine più profonda.
Behavior is conceived as a stimulus-response dependent relationship between a sensory input and a motor output. While moving from an input to an output, internal homeostasis is continuously shaped to maintain an optimal energies expenditure balance. The ultimate purpose of enabling animals to adjust their homeostasis with the surrounding world is by producing adaptive behaviors in order to increase their fitness in light of natural selection. The environment can be either predictable or unpredictable. The former condition led to the evolution of the circadian rhythm to promote an active behavior at the time you mostly benefit from, while the latter take advantage of glucocorticoids axis to face sudden challenges. Thus, a crosstalk between the circadian and the glucocorticoid systems allows a fine tuning of animal’s activity. My goal is to understand the circadian-glucocorticoids dialogue by monitoring the locomotor daily/circadian behavior and its molecular oscillation counterpart under differentially phased light and feeding cycle. My model species is the zebrafish, particularly, I utilized a CRISPR/Cas9 mutant lacking the capability to coordinate glucocorticoids transcription because it lacks functional receptors which permit a correct ligand-receptor interaction. As a result, level of circulating glucocorticoids stays raised conferring an anxiety-related phenotype to the mutant. Zebrafish gr-/- has been built and kindly provided by Dr. Luisa Dalla Valle, University of Padua. Systematic behavioral analysis in gr-/- larvae and adults showed that the light entrainable locomotor activity is synchronized to the zeitgeber and maintain its oscillatory properties in absence of any cue. The onset of daily locomotor activity occurred one day later in mutants with respects to the wild type. This delay is linked to the slower striated muscle development in the gr-/- which recover regular fiber density at 6 days post fertilization. Furthermore, gr-/- larvae showed differences in the expression levels or in the peak phase of positive (arntl1a and clock1a) and negative (per1, per2a and cry1a) elements of the molecular clock. Outside the core clock network, an analysis on gr-/- adult livers reported an abolished daily expression of pck2, a gene involved in gluconeogenesis. In addition, srebp1 expression level has an anticipated acrophase in gr-/-. Feeding entrainment fails to occur in the mutants. Larvae and adults produced abnormal profiles of circadian locomotor activity. Further molecular investigation revealed this behavioral disruption wasn’t associated with a breakdown of molecular rhythms in the core clock genes. Nevertheless, the molecular phenotypes observed during feeding entrainment underlined a cry1a lack of rhythmicity. These data suggest the existence of a blurred boundary between the circadian-glucocorticoids crosstalk. A complex organization of the two produces an altered behavioral output in a food entrained schedule in gr-/- zebrafish. The proximate cause of input and output misalignment underlying food entrained locomotion has not been provided, but a step towards a more exhaustive comprehension about the circadian-glucocorticoids interaction paves the way for an in-depth investigation.

Orientador: Chung Man Chin
Banca: Iracilda Zeppone Carlos
Banca: Gustavo Henrique Goulart Trossini
Resumo: A inflamação é caracterizada como uma resposta do organismo frente à uma agressão, a qual pode resultar em dor, edema e em alguns casos levar à disfunção do órgão ou tecido. Durante o processo inflamatório ocorre a liberação de citocinas, pequenas proteínas transcritas e traduzidas pelo metabolismo intrínseco celular, que realizam uma série de comunicações intra e intercelulares. Uma importante citocina é o fator de necrose tumoral alfa (TNF-α), que atua na manutenção do quadro inflamatório em situações de inflamação crônica, sendo super expressa em diversas doenças inflamatórias. A terapia anti fator de necrose tumoral α (anti-TNF-α) tornou-se uma abordagem interessante no tratamento de doenças inflamatórias crônicas, principalmente daqueles pacientes que não respondem ao tratamento convencional. Entre os fármacos utilizados no tratamento das doenças inflamatórias crônicas, encontram-se os glicocorticoides (GCs), anti-inflamatórios de natureza esteroide. Estes atuam no processo reacional de defesa do organismo minimizando o dano causado pelo agente infeccioso. Porém o uso prolongado de GCs está associado a diversos efeitos adversos, como osteoporose, síndrome metabólica, doenças cardiovasculares, catarata, entre outros, limitando o uso desses fármacos em terapias prolongadas. Dessa forma, nesse trabalho, buscou-se através da estratégia de hibridação molecular, a síntese de novos derivados anti-inflamatórios esteroides, com propriedades de modulação da citocina TNF-α, a fim de se alcançar um sinergismo de ação útil no tratamento de doenças inflamatórias crônicas. Nesse contexto, foram sintetizados e caracterizados seis compostos, série Lapdesf GL-FT, derivados dos glicocorticoides prednisolona (compostos I-III) e budesonida (compostos IV-VI), ligados ao grupamento ftalimida, responsável pela inibição da síntese de TNF-α. Os compostos ...
Abstract: Inflammation is characterized as a organism response against an aggression, which can result in pain, edema and in some cases lead to organ or tissue dysfunction. During the inflammatory process are released of cytokines, small proteins transcribed and translated by intrinsic cellular metabolism, performing a variety of communication intra and intercellular. An important cytokine is tumor necrosis factor alpha (TNF-α), which acts in maintaining the inflammatory in situations of chronic inflammation, being over expressed in many inflammatory diseases. Therapy anti-tumor necrosis factor α (anti-TNF-α) has become an interesting approach in the treatment of chronic inflammatory diseases, especially those patients who do not respond to conventional treatment. Among the drugs used in the treatment of chronic inflammatory diseases are glucocorticoids (GCs), anti-inflammatory steroids. These act in the reactive process of organism defense minimizing the damage caused by the infectious agent. However, prolonged use of GCs is associated with various side effects such as osteoporosis, metabolic syndrome, cardiovascular disease, cataracts, among others, limiting the use of these drugs in prolonged therapies. Thus, in this work, we realized the viability of design and synthesis by molecular hybridization strategy of novel anti-inflammatory steroids with modulating properties of cytokine TNF-α in order to achieve a synergism of action useful in the treatment of diseases chronic inflammatory diseases. In this context, were synthesized and characterized six compounds Lapdesf series GL-FT derived from glucocorticoids prednisolone (compounds I-III) and budesonide (compounds IV-VI), connected to the phthalimide group responsible for inhibition of TNF-α. The obtained compounds were evaluated for anti-inflammatory activity in a model of paw edema and distal ulcerative colitis. Derivatives I and IV showed higher ...
Mestre

Due to health risks associated to the use of glucocorticoids (GC) and their potential ergogenic effects, systemic GCs are prohibited in sports competitions. However, local GC administrations are allowed for therapeutic purposes. The general reporting level used nowadays to discriminate between prohibited and allowed administrations is not adequate for all GCs. To improve the discrimination, the excretion profiles of different GCs (triamcinolone hexacetonide, triamcinolone acetonide, budesonide and betamethasone) were evaluated after prohibited and allowed administrations, and specific discrimination criteria were proposed for each GC. The urinary elimination times of systemic GCs were also evaluated. Moreover, local intra-articular administration of GCs was demonstrated to produce systemic effect. Finally, the impact of GCs on the steroid profile was evaluated. Due to the inhibition of the hypothalamic-pituitary-adrenal axis, the excretion rates of steroid profile metabolites decreased after systemic GC use. Nonetheless, the ratios between metabolites were not affected. Thus, GC administration does not affect the steroid profile.
Degut a que els glucocorticoides (GCs) presenten riscos per a la salut i poden tenir efectes que milloren el rendiment esportiu, el seu ús per vies sistèmiques esta prohibit en competicions esportives. No obstant, l’administració de GCs per vies locals esta permès per raons terapèutiques. Avui en dia s’utilitza un criteri general per discriminar administracions prohibides i permeses, el qual no és adequat per tots els GCs. Per millorar la discriminació, els perfils d’excreció de diferents GCs (triamcinolona hexacetònid, triamcinolona acetònid, budesonida and betametasona) han sigut avaluats després d’administracions prohibides i permeses. Criteris de discriminació específics han sigut proposats per cada compost. Els temps d’eliminació en orina dels GCs han sigut avaluats després d’administracions sistèmiques. A més a més, s’ha demostrat que l’administració local intraarticular de GCs produeix efecte sistèmic. Finalment, s’ha avaluat l’impacte dels GCs en el perfil esteroïdal. Degut a la inhibició de l’eix hipotalàmic-pituïtari-adrenal, l’excreció dels metabòlits del perfil esteroïdal va disminuir després de l’administració sistèmica de GCs. No obstant, les ratios entre els metabòlits no es van veure afectades. Per tant, l’administració de GCs no afecta el perfil esteroïdal.

Platelets are pivotal in regulating haemostasis, but can precipitate atherothrombosis associated to cardiovascular diseases. The synthetic glucocorticoid prednisolone is widely used as an anti-inflammatory and immunosuppressive drug. Previously it was shown that prednisolone inhibited platelet aggregation and adhesion under conditions of flow, although the mechanisms remained unclear. In the present study we examined the mechanisms responsible for the inhibitory effects of prednisolone on thrombin‐mediated platelet activation. Prednisolone caused concentration‐dependent inhibition of thrombin‐induced platelet aggregation. The inhibition of aggregation by prednisolone was rapid suggesting a non‐genomic mode of action of the glucocorticoid on platelets. Prednisolone also targeted the protease‐activated receptors PAR1 and PAR4 that mediate platelet activation following thrombin stimulation. Thrombin triggers two distinct signalling cascades in platelets resulting in the phosphorylation of myosin light chains (MLC). One of these pathways is calcium‐dependent, while the other is RhoA/ROCK‐dependent. In order to understand the molecular mechanism underpinning the inhibitory effects of prednisolone, we examined the RhoA/ROCK pathway. Stimulation of platelets with thrombin led to the RhoA/RhoA kinase (ROCK)‐dependent phosphorylation of MLC. Pre‐treatment of platelets with prednisolone caused a concentration‐dependent inhibition of MLC‐ser¹⁹ phosphorylation. The inhibition was rapid and transient. Consistent with this observation, prednisolone also reduced the inhibitory phosphorylation of the myosin light chain phosphatase (MLCP) at two key residues thr⁶⁹⁶ and thr⁸⁵³. In all cases the effects of prednisolone were inhibited by the glucocorticoid receptor antagonist RU486. Finally, prednisolone also inhibited RhoA activation in platelets following thrombin stimulation. Thus, prednisolone inhibited platelet activation by targeting RhoA/ROCK‐mediated signalling events following thrombin stimulation. Modulation of the RhoA activity represents one of the non‐genomic effects of this synthetic glucocorticoid on platelets and these might have important clinical implications in the treatment of cardiovascular diseases.

Le muscle strié squelettique régénère ad integrum après une lésion aigüe stérile grâce aux cellules satellites qui sont les cellules souches du muscle strié squelettique. L'inflammation, et notamment les macrophages, joue un rôle important durant ce processus. En effet, après une lésion, les monocytes sanguins infiltrent le tissu et deviennent des macrophages avec un phénotype pro-inflammatoire associé à la lésion. Ces macrophages phagocytent les débris cellulaires et promeuvent la prolifération des cellules souches musculaires. Ensuite, les macrophages changent leur phénotype vers un phénotype anti-inflammatoire associé à la restauration du tissu. Ils promeuvent la différenciation, puis la fusion des cellules souches musculaires et la croissance des myofibres. Cette séquence de phénotypes inflammatoires est essentielle pour une régénération musculaire efficace. Le laboratoire a montré que ce changement de phénotype est dépendant d'un senseur énergétique majeur de la cellule qui contrôle le métabolisme cellulaire, l'AMP kinase (AMPK)al. Par ailleurs, les glucocorticoïdes sont utilisés depuis des décennies pour leurs effets anti-inflammatoires sur l'inflammation. Leur action est médiée par le Récepteur aux Glucocorticoïdes qui induit ou réprime l'expression de gènes par interaction directe ou indirecte à l'ADN. Comme l'AMPKal et les glucocorticoïdes induisent des effets anti-inflammatoires similaires sur les macrophages, nous avons posé l'hypothèse que ces 2 voies de signalisation pourraient être interconnectées dans les macrophages afin de permettre leur changement de phénotype et la régénération musculaire. Les données issues d'un modèle in vitro de lésion musculaire utilisant des macrophages dérivés de la moelle osseuse de souris ont montré que : i) les glucocorticoïdes induisaient la phosphorylation de l'AMPKal ; ii) l'AMPKal était requise pour l'acquisition fonctionnelle du statut anti-inflammatoire des macrophages induit par les glucocorticoïdes puisque des macrophages déficients pour l'AMPKal ne modifiaient pas leur phénotype et ne stimulaient pas la myogenèse. Les expériences in vivo utilisant des souris LysMCre/+;AMPKalfl/fl dans lesquelles l'AMPKal est invalidée uniquement dans les cellules myéloïdes ont montré que l'AMPKal dans les macrophages régulait les effets bénéfiques des glucocorticoïdes au cours de la régénération du muscle strié squelettique. En effet, en absence d'AMPKal dans les macrophages, les glucocorticoïdes induisaient un retard de régénération et une modification de la maturation des fibres attestée par une modification de l'expression des isoformes des chaînes lourdes de myosines. En conclusion, ces données montrent que l'AMPKal est requise pour le changement de phénotype des macrophages induit par les glucocorticoïdes et une régénération musculaire efficace
Skeletal muscle regenerates ad integrum after a sterile acute injury thanks to satellite cells (muscle stem cells). Inflammation, and notably macrophages, plays important roles during this process. Just after injury, monocytes infiltrate the tissue from the blood and convert into pro-inflammatory damaged associated macrophages. These macrophages phagocyte muscle debris and promote the proliferation of muscle stem cells. Then, macrophages switch their phenotype toward an anti-inflammatory restorative profile and promote muscle stem differentiation, fusion and myofiber growth. This sequence of macrophage profile is essential for an efficient skeletal muscle regeneration. The lab has shown that this phenotype switch is dependent of AMP kinase (AMPK)a1, a major energetic sensor in the cell controlling cellular metabolism. Besides, glucocorticoids have been used for decades for their anti-inflammatory effects on inflammation. Their actions are mediated by the Glucocorticoid Receptor which induces or represses gene expression by direct or indirect DNA-binding. As AMPKa1 and glucocorticoids induce similar anti-inflammatory effects on macrophages, we hypothesized that these 2 pathways could be interconnected in macrophages to allow the resolution of inflammation and muscle repair. Data from an in vitro model of skeletal muscle injury using bone marrow derived macrophages showed that: i) glucocorticoids induce AMPK phosphorylation; ii) AMPKa1 is required for the functional acquisition of the anti-inflammatory phenotype induced by glucocorticoids. Indeed, AMPKa1-deficient macrophages did not switch their phenotype and did not sustain myogenesis. In vivo experiments using LysMCre/+;AMPKa1fl/fl mice in which AMPKa1 is depleted only in myeloid cells, showed that macrophagic AMPK drove the beneficial effects of glucocorticoids during skeletal muscle regeneration. Inversely, in absence of AMPK in macrophages, glucocorticoids induced a delayed muscle regeneration and a modification in myofiber maturation, assessed by the alteration of myosin heavy chain expression. Altogether, these data show that glucocorticoids need AMPKa1 in macrophages for the resolution of inflammation and an efficient skeletal muscle regeneration

Bone, a specialized connective tissue, consists of cells and mineralized extracellular matrix. The main cell types of bone tissue are: the osteoblasts, the osteocytes and the osteoclasts. Osteoblasts produce extracellular matrix, osteoclasts are responsible of its resorption, hence bone physiology is a delicate balance between synthesis of new bone and resorption of the old one. Osteoporosis is a disease in which catabolic activity of osteoclasts overtakes anabolic activity of osteoblasts leading to increased bone resorption and progressive bone fragility. Primary osteoporosis is a common disease among post-menopausal female population. Pathologies as diabetes mellitus, hyperparathyroidism and long-term treatment with glucocorticoids cause secondary osteoporosis. Glucocorticoid-induced osteoporosis is the most common type of secondary osteoporosis. Glucocorticoid treatment is a well known method to induce osteoporosis in animal models, hence it could be an example of “translational model” in which injured bone could be repopulated by stem cells or progenitors in clinical trials. The aim of this thesis is to investigate whether preosteoblasts could repopulate injured bone in an animal model treated with glucocorticoids. Preosteoblasts have been isolated from newborn calvariae of GFP mice. In these cells, expression of the osteogenic marker Runx2 has been assessed by Real time PCR, while osteogenic potential has been analysed by cytochemistry assays to detect alkaline phosphatase and mineralized bone nodules (Alizarin Red and Von Kossa staining). To realize the in vivo model, C57BL/6 three months aged mice have been divided into three groups [group I (n=4): mice not treated with drug and not infused with cells, group II (n=4): mice treated with drug and not infused with cells, group III (n=4): mice treated with drug and infused with cells]. Drug (methylprednisolone) has been administered for one month with a dose of 75 mg/Kg/week. In mice of group III, 5 x 105 GFP preosteoblasts, previously expanded in vitro, have been infused with injection into the tail vein. Mice have been sacrificed, tibial and femoral bones have been harvested, processed and analysed by istomorphometry and immunoistochemistry. Expression of Runx2, osteonectin (SPARC) and alkaline phosphatase (ALP) in these tissues has been detected with Real time PCR. In vitro preosteoblasts produce alkaline phosphatase during early time in culture with normal medium, while the level decreases in differentiating conditions with medium containing ascorbic acid and β-glycerophosphate. Preosteoblasts maintained in differentiation medium for 30 days are positive to Alizarin and Von Kossa staining, hence they are able to produce mineralized extracellular matrix that is a feature of functional mature osteoblasts. Runx2 expression increases during differentiating conditions; in cells maintained in differentiation medium for 30 days there is an increase of 50% compared to cells maintained in normal medium (p<0.05). In mice of group III an increased level of parameters concerning osteoid has been detected (O.Th, OS/BS, OV/BV) and an increased number of active osteoblasts (during synthetic activity) has been observed compared to group II. Between these two groups, significant variations of bone volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N) and trabecular separation (Tb.Sp) have not been detected. Microarchitecture parameters (Nd.N/TV, Nd/Tm) have not been affected. Similar results have been obtained from inderect microarchitecture parameters as Marrow Star Volume and Fractal Dimension. Real time PCR analysis revealed a reduction in osteogenic gene expression in group II compared to group I (ALP: -50%, p<0.01; Runx2: -56.75%, p<0.01; SPARC: -44.5%, p<0.05). In group III there is a recovery of expression of osteogenic markers (ALP: +40%, p<0.05; Runx2: +66.28%, p<0.001; SPARC: +55%; p<0.01) compared to group II. Immunoistochemistry is under investigation. In our glucocorticoid-induced osteoporosis model we sacrificed mice only one week after infusion of cells, this preparatory investigation shows that our model induces the engraftment of preosteoblasts in injured bone. However, a longer time, at least of 1-2 months, is needed to investigate if preosteoblasts are able not only to graft onto host tissue, but also to proliferate in vivo and to differentiate in full mature and functional osteoblasts.
L’osso è un tessuto connettivo specializzato costituito da cellule e matrice extracellulare mineralizzata. Le cellule principali sono gli osteoblasti, gli osteociti e gli osteoclasti. Gli osteoblasti depongono la matrice ossea, mentre gli osteoclasti sono i responsabili del suo riassorbimento. La fisiologia dell’osso è quindi il risultato di un delicato equilibrio tra deposizione di matrice ossea e suo riassorbimento. Quando l’azione catabolica degli osteoclasti è maggiore rispetto a quella anabolica degli osteoblasti si produce una progressiva fragilità ossea che porta ad un quadro clinico osteoporotico. L’osteoporosi primaria colpisce soprattutto la popolazione femminile dopo la menopausa. Molte patologie come il diabete mellito, l’iperparatiroidismo ed il trattamento a lungo termine con glucocorticoidi causano l’osteoporosi secondaria. L’osteoporosi indotta da glucocorticoidi è la più comune causa di osteoporosi secondaria. Il trattamento con glucocorticoidi è un noto procedimento di induzione dell’osteoporosi in modelli animali e può dunque rappresentare un primo esempio di modello “traslazionale” potenzialmente applicabile in clinica per indurre un ripopolamento dell’osso con cellule staminali mesenchimali o precursori osteogenici. Lo scopo di questo lavoro è stato pertanto valutare nel modello animale se sia possibile ripopolare l’osso danneggiato con preosteoblasti. I crani di topi neonati transgenici (GFP) sono stati prelevati e messi in coltura per ottenere preosteoblasti. Nelle colture in vitro è stata valutata l’espressione del gene Runx2 con la tecnica di Real time PCR, mentre la capacità osteogenica è stata analizzata con colorazioni citochimiche per la fosfatasi alcalina e per la deposizione di matrice ossea mineralizzata (Alizarin Red e Von Kossa). Per la realizzazione del modello in vivo topi C57BL/6 maschi di 3 mesi sono stati divisi in 3 gruppi [gruppo I (n=4): topi non trattati con farmaco e non infusi con cellule; gruppo II (n=4): topi trattati con farmaco non infusi con cellule; gruppo III (n=4): topi trattati con farmaco ed infusi con cellule]. Il farmaco (metilprednisolone) è stato somministrato per un mese alla dose di 75 mg/Kg/settimana. Negli animali appartenenti al gruppo III sono state infuse, attraverso iniezione nella vena della coda, 5 x 105 preosteoblasti GFP precedentemente espansi in vitro. I topi sono stati sacrificati, le tibie ed i femori sono stati prelevati e processati per l’analisi istomorfometrica e della microarchitettura ossea e per l’ immunoistochimica. In questi tessuti, l’espressione genica di Runx2, osteonectina (SPARC) e fosfatasi alcalina (ALP) è stata valutata tramite Real time PCR. In vitro i preosteoblasti producono fosfatasi alcalina durate i primi giorni di coltura in medium non differenziante, mentre il livello decresce in condizioni differenzianti, cioè in medium contenente acido ascorbico e β-glicerofosfato. I preosteoblasti mantenuti in medium di differenziamento per 30 giorni sono positivi alle colorazioni Alizarin Red e Von Kossa, quindi sono in grado di produrre matrice ossea mineralizzata, caratteristica degli osteoblasti funzionali e maturi. L’espressione del gene Runx2 aumenta durante il differenziamento, si ha un aumento del 50% nelle cellule differenziate per 30 giorni rispetto alle cellule non differenziate (p<0.05). L’inoculazione dei preosteoblasti nei topi del gruppo III ha evidenziato un aumento dei parametri statici di neoformazione ossea relativi all’osteoide (O.Th, OS/BS, OV/BV) ed un aumento del numero di osteoblasti attivi, cioè in corso di deposizione di osteoide, rispetto al gruppo II. Tra questi due gruppi non si sono osservate, invece, variazioni significative in termini di volume osseo (BV/TV), spessore trabecolare (Tb.Th) numero delle trabecole (Tb.N) e separazione fra esse (Tb.Sp). Non sono state rilevate, inoltre, differenze dei parametri di microarchitettura (Nd.N/TV, Nd/Tm). Risultati simili sono emersi dalla valutazione dei parametri indiretti di microarchitettura (Marrow Star Volume e Fractal Dimension). L’espressione genica ha dimostrato che nel gruppo II si ha una riduzione dell’espressione dei geni osteogenici rispetto al gruppo I (ALP: -50%, p<0.01; Runx2: -56.75%, p<0.01; SPARC: -44.5%, p<0.05). Nel gruppo III si è avuto un recupero dell’espressione dei geni osteogenici (ALP: +40%, p<0.05; Runx2: +66.28%, p<0.001; SPARC: +55%, p<0.01) rispetto al gruppo II. I campioni di tessuto per l’ immunoistochimica devono essere processati. Nel nostro modello sperimentale di osteoporosi indotta da glucocorticoidi nel topo, abbiamo sacrificato gli animali solo una settimana dopo l’infusione delle cellule; questi dati preliminari dimostrano che il nostro modello induce l’engraftment dei preosteoblasti nell’osso danneggiato. Tuttavia è richiesto un tempo di osservazione più lungo, di almeno 1-2 mesi per valutare se le cellule trapiantate siano in grado, non solo di integrarsi nel tessuto dell’ospite, ma anche di proliferare in vivo e di differenziare in osteoblasti maturi e funzionali.

Les glucocorticoïdes (GCs) ont des effets diabétogènes avérés. Précédemment, notre équipe a également pu montrer que les GCs, en association avec le corégulateur transcriptionnel PGC-1α, sont impliqués dans la programmation fœtale du diabète de type 2 (DT2). Le DT2 est une maladie métabolique, conséquence à la fois de l’insulinorésistance et d’un défaut de sécrétion d’insuline en partie dû à la diminution de la masse des cellules β. Au laboratoire nous nous intéressons donc d’une part aux mécanismes sous-jacents des effets diabétogènes des GCs et d’autre part, aux mécanismes permettant d’améliorer la sécrétion d’insuline en restaurant une masse fonctionnelle de cellules β. Dans la première partie de cette thèse, nous avons montré que PGC-1α, dont l’expression est stimulée par les GCs dans les cellules β, induit un double stress énergétique et oxydatif impliqué dans l’altération de la sécrétion d’insuline. Dans la deuxième partie, nous avons montré grâce à un model murin d’insulinorésistance sévère par surexposition aux GCs, que l’adaptation compensatrice de la masse fonctionnelle des cellules β se fait par un processus de néogenèse, impliquant la réexpression du facteur Ngn3. Ce processus, indépendant de l’effet des GCs sur le pancréas, alimente l’hypothèse d’un facteur circulant libéré par les organes insulinorésistants pour instruire le pancréas endocrine et initier la néogenèse des cellules β. En conclusion, nos travaux associent indirectement les GCs : 1/ à un effet délétère sur la sécrétion et impliquant PGC-1α et 2/ à un effet bénéfique sur la masse β et impliquant Ngn3. Ces deux voies constituent des perspectives thérapeutiques intéressantes du DT2
Glucocorticoids (GCs) are hormones secreted in response to stress and that display diabetogenic effects. Previously, our team was able to demonstrate that GCs, in combination with the transcriptional co-regulator PGC-1α, are involved in fetal programming of type 2 diabetes (T2D). T2D is a metabolic disease characterized by fasting hyperglycemia, consequence of both insulin resistance and an insulin secretory defect, partly due to the decrease of the mass of β cells. In the laboratory we are therefore interested in understanding the mechanisms underlying diabetogenic effects of GCs, and mechanisms that improve insulin secretion and functional β-cell mass. In the first part of this thesis, we have shown that PGC-1α, whose expression is strongly stimulated by GCs in β cells, induces both energy and oxidative stress involved in impaired insulin secretion. In the second part of this thesis, we demonstrated through a murine model of massive GCs overexposure – which induces severe insulin resistance – that the adaptation of the functional β-cell mass in order to counteract insulin resistance occurs through a neogenesis process, involving the re-expression of Ngn3 factor. This process is independent of the effect of GCs on the pancreas. We hypothesize that a circulating factor released by insulin-resistant organs will instruct the endocrine pancreas to initiate β-cells neogenesis. In conclusion, our work indirectly associate GCs: 1/ to a deleterious effect on the secretion involving PGC-1α and 2/ to a beneficial effect on the β-cell mass and involving Ngn3. These two pathways are interesting therapeutic perspectives for curing T2D

Nous avons développé des nanoparticules d’une prodrogue de glucocorticoïde, la dexaméthasone palmitate (DXP) à visée thérapeutique dans le traitement de la polyarthrite rhumatoïde (PR). Cette maladie auto-immune est caractérisée par une inflammation articulaire, une érosion osseuse et cartilagineuse et une dérégulation du système immunitaire. Parmi les traitements indiqués dans la PR, l'utilisation des glucocorticoïdes est limitée par leurs effets secondaires importants induits par leur pharmacocinétique défavorable. Afin de traiter la PR par voie intraveineuse, la formulation de nanoparticules PEGylées semble indispensable afin d’échapper au phénomène d’opsonisation et d’espérer obtenir une accumulation spécifique au niveau des articulations inflammées. Pour cela, nous avons développé des nanoparticules de DXP (DXP-NP) stabilisées par le DSPE-PEG2000.Les caractéristiques physico-chimiques des nanoparticules obtenues ont été évaluées ainsi que leur stabilité au cours du temps. La structure interne des nanoparticules définie comme amorphe ainsi que leur très fort taux de charge prouvent ainsi l’impact du DSPE-PEG2000 dans l’organisation moléculaire des DXP-NP. In vivo, l’étude de la pharmacocinétique et de la biodistribution des DXP-NP suite à leur administration intraveineuse a démontré une circulation prolongée du système. Dans un modèle murin de polyarthrite rhumatoïde, les DXP-NP ont démontré leur accumulation spécifique dans les articulations inflammées en corrélation avec une supériorité thérapeutique significative en comparaison avec la molécule libre hydrosoluble. Des études histologiques ainsi que l’évaluation du traitement sur l’apparition d’effets indésirables complètent l’étude in vivo
We developed nanoparticles of a glucocorticoid prodrug, dexamethasone palmitate (DXP) for the treatment of rheumatoid arthritis (RA). Joint inflammation, bone and cartilage erosion and dysregulation of the immune system characterize this autoimmune disease. Among the treatments indicated in RA, the use of glucocorticoids is hampered by their side effects induced by their unfavorable pharmacokinetics. To treat RA intravenously, the formulation of PEGylated nanoparticles seems essential in order to escape from opsonization and to obtain a specific accumulation in the joints inflamed. Therefore, we developed DXP nanoparticles (DXP-NPs) stabilized by the DSPE-PEG2000.The physicochemical characteristics of the nanoparticles obtained were evaluated as well as their stability over time. The amorphous internal structure of the nanoparticles and their very high drug loading thus prove the impact of DSPE-PEG2000 in the molecular organization of DXP-NPs. In vivo, the study of the pharmacokinetics and biodistribution of DXP-NPs following intravenous administration demonstrated prolonged circulation of the system. In a mouse model of rheumatoid arthritis, DXP-NPs their demonstrated specific accumulation in inflamed joints in correlation with significant therapeutic superiority in comparison with the water-soluble free molecule. Histological studies as well as adverse events evaluation supplemented the in vivo study

Glucocorticoids (GCs) have been widely used as a therapeutic agent for diverse inflammatory ocular diseases. However, a high percentage of patients undergoing this treatment develop high intraocular pressure (IOP), which if left unsupervised may lead to glaucoma. It is believed that the IOP elevation in response to GC treatment has a genetic determinant. In order to test this hypothesis, we analyzed in 52 patients the presence of single nucleotide polymorphisms (SNPs) in the glucocorticoid receptor gene (GR), the principal mediator of GCs uptake by the cells. We studied six GR SNPs previously reported to be associated with sensitivity and resistance to GCs: GluArg22/23GluLys (codon 22-23), Asn363Ser (codon 363), IVS2+646C>G (intron 2/BclI), IVS3-46G>C (intron 3), IVS4-16G>T (intron 4), Asn766Asn (Codon 766). Nevertheless, the results of this preliminary study did not show any specific correlation between SNPs in the GR gene and IOP elevation. Therefore, we proceeded to perform a whole genome SNP screen with the DNA samples of these patients to search for possible target genes responsible for the elevated IOP after GC treatment. As a result, we identified forty-eight SNPs in thirty-three genes that correlate with the high IOP response. The gene showing the strongest association is a poorly known G-protein coupled receptor. In addition, four SNPs hit a single transporter gene. Other candidate genes identified are a translation elongation factor, an F-box protein, an oxysterol binding protein, and a solute carrier family gene. These results support our hypothesis that IOP elevation following GC treatment is a genetically determined response. GCs are a common treatment for innumerable medical conditions; we believe that a genetic association between GC treatment and its physiological response may be important for improving treatment management and drug development for retinal diseases as well as for other medical ailments. However, further studies need to be performed to analyze in depth the association between the candidate genes identified in this study and the steroid response.

[Truncated abstract] Glucocorticoids are critical for the maturation of the fetus late in pregnancy. Indeed, clinical administration of glucocorticoids is used to accelerate fetal lung maturation in mothers at risk of pre-term delivery. Increased glucocorticoid exposure, however, can have detrimental effects on fetal and placental growth and increase the risk of disease in later life. Many studies have focused on the effect of an increase in the transplacental passage of glucocorticoids on both fetal growth and subsequent postnatal development. But there is a growing body of evidence to suggest that the impact of glucocorticoids on fetal growth is mediated, in part, via their direct effects on the placenta . . . Overall, these studies quantify the labyrinth zone-specific increases in placental expression of PPARG and VEGF in association with a marked increase in vascularisation observed near term. Furthermore, this study demonstrates for the first time that these increases in gene expression are prevented by maternal dexamethasone treatment which also inhibits growth of the fetal capillary network. Elevated expression of SFRP4 in the regressing basal zone late in gestation and in both placental zones after dexamethasone-induced placental growth restriction is consistent with a role for SFRP4 in glucocorticoid-mediated inhibition of wnt signalling. Collectively, the data presented in this thesis show that glucocorticoid inhibition of fetal growth is mediated in large part via effects on the placenta, specifically through inhibition of signals that promote proliferation and vascularisation.

In mammals endogenous, self sustained oscillators, known as circadian clocks, have evolved as a result of day night cycles, with a period close to 24 hours, and are involved in many physiological processes; such as sleep wake cycles, metabolic and hormonal activity. The suprachiasmatic nucleus (SCN), is the central oscillator, and is synchronised to the external environment by light, via the eye. It has been demonstrated that peripheral clocks, too, contain the circadian oscillator, with tissues such as the lung, liver, heart and kidney as well as many isolated cell types remaining rhythmic, in culture, for many days. However, these peripheral oscillators require a signal from the central oscillator in order to co-ordinate a synchronised time. Leading candidates in the relay of this information are the circulating glucocorticoid hormones corticosterone (rodents) or cortisol (man), which are known to have potent effects on the peripheral clock, both in-vivo and in-vitro. Further to this, glucocorticoids have been used for many decades to suppress the symptoms of inflammation, a by product of many human diseases.This thesis aims to address the temporal regulation of the peripheral clock by the endogenous glucocorticoid, corticosterone, using a transgenic mouse harbouring a luciferase conjugated clock reporter, and circadian reporter cell lines. It also aims to address the relative contribution of the two closely related nuclear hormone receptors, the glucocorticoid and mineralocorticoid receptors. A further aim of the work with glucocorticoid signalling was to design a flow-though culture system, in order to address the effects of the endogenous pulsatile release of glucocorticoids on the peripheral oscillator. This thesis also aims to characterise the inflammatory response in relation to its circadian characteristics; its relationship with corticosterone and the effect of inflammation on the central clock components. Finally, this thesis aims to investigate a potential input/output of the clock, a member of the family of C/EBP transcription factors, C/EBP alpha, and whether it is under endogenous circadian control and regulated by glucocorticoids.Work in this thesis has shown that glucocorticoids dynamically regulate the peripheral clock at all phases of the circadian cycle and that this regulation occurs mainly through the glucocorticoid receptor; yet the mineralocorticoid receptor does have a function in the immediate response to glucocorticoid administration. Furthermore, as a result of the initial temporal profile after corticosterone addition, on the clock protein PERIOD2, I have shown transient regulation of the clock through Caveolin-1 based signalling. There is also a significant circadian component to the inflammatory response, which appears, at least in part, to be REV-ERB alpha mediated, and the inflammatory response also has profound effects on circadian gene expression in the periphery. A functional flow-through system was designed and a working model produced, albeit with technical difficulties, to address glucocorticoid pulsing and circadian timing but much more work is needed for effects to be fully understood. C/EBP alpha appears not to be under circadian regulation nor under direct glucocorticoid regulation, at least in peripheral models used here.

Since steroid hormones have well characterised programming properties, excessive fetal exposure to glucocorticoids in early in life may produce lifelong effects. In order to examine the role of glucocorticoids in the programming of glucose metabolism, fetal glucocorticoid load was increased by administration of dexamethasone, a poor substrate for 11β-HSD, during the different trimesters of rat pregnancy. Dexamethasone given selectively in the last week of pregnancy reduced birth weight by 10%, and produced hyperglycaemia and hyperinsulinaemia in the adult offspring. In addition, rats exposed to dexamethasone during the last week of intrauterine life had permanent increases in hepatic expression of glucocorticoid receptor (GR) mRNA and phosphoenolpyruvate carboxykinase (PEPCK) mRNA (and activity). In contrast dexamethasone, when given in the first or second week of gestation, had no effect on offspring insulin/glucose responses or hepatic PEPCK and GR expression. Similarly, administration of dexamethasone to pups postnatally did not alter glucose control in the adult animals. These observations suggest that excessive glucocorticoid exposure late in pregnancy predisposes the offspring to glucose intolerance in adulthood. Programmed hepatic PEPCK overexpression, perhaps mediated by increased GR, may promote this process by increasing gluconeogenesis. In order to examine further the molecular basis for hepatic PEPCK overexpression, levels of liver enriched transcription factors known to co-operate with GR in stimulation of PEPCK gene transcription were analysed. Levels of hepatic nuclear factor (HNF)-1, HNF-4, and CCAAT/enhancer binding protein (C/EBP)α (but not C/EBPβ) did alter with prenatal dexamethasone treatment. However, unlike GR mRNA, expression of none of these factors correlated closely with changes in PEPCK level or hyperglycaemia. These studies have confirmed that in utero overexposure to glucocorticoids can programme hyperglycaemia which may be in part due to glucocorticoid mediated increase in hepatic gluconeogenesis, and provide support for the hypothesis that excessive fetal exposure to glucocorticoids underlies the link between low birth weight and disease in adulthood observed in human epidemiological studies.

We investigated the effect of bypassing (using dexamethasone, DEX) or inhibiting (using carbenoxolone, CBX) feto-placental 11b-HSD in rats, on subsequent offspring HPA axis regulation and stress-induced behaviour. Pregnant Wistar rats were injected with CBX, DEX or vehicle daily throughout pregnancy. A separate group received DEX injections in the last week of pregnancy only (DEX3), as this had previously been shown to contain the critical time window for programming of hypertension and hyperglycaemia. All treatments reduced birth weight (CBX 12%, DEX1-3 15%, DEX3 7%). This was reversed by 4 and 6 weeks of life in DEX3 and CBX offspring, respectively, while offspring of dams treated with DEX throughout pregnancy had reduced body weight throughout life (7% at 5 months of age). Prenatal exposure to glucocorticoids resulted in increased basal corticosterone (CORT) levels (CBX 0.70 mg/dl, DEX3 0.76 mg/dl, control 0.40 mg/dl) and a trend (p=0.07) towards increased corticotropin-releasing hormone (CRH) in the hypothalamic paraventricular nucleus (PVN) in adult offspring. In addition, CBX offspring had reduced PVN glucocorticoid receptor (GR) mRNA and a prolonged stress response. Hippocampal GR and mineralocorticoid receptor (MR) mRNA expression was reduced only in DEX3 offspring, which also showed increased CRH mRNA levels in PVN. CBX and DEX3 rats displayed impaired non-spatial learning. In addition, exploratory behaviour in an open field was affected by prenatal glucocorticoid exposure, especially in DEX3 offspring, which showed reduced locomotion and rearing. The behavioural alternations are associated with increased expression of MR, GR and CRH mRNAs in the amygdala, a structure implicated in the expression of fear and anxiety. These data suggest that disturbance of the feto-placental enzymatic barrier to maternal glucocorticoids, or in utero exposure to DEX, reduces birth weight and produces permanent subtle alterations of the HPA axis combined with behavioural inhibition or impaired coping in aversive situations.

Thesis (Ph.D.) - University of Glasgow, 2007.
Ph.D. thesis submitted to the Faculty of Medicine, Dept. of Developmental Medicine, University of Glasgow. Includes bibliographical references. Print version also available.

Antecedentes: La colitis ulcerosa (CU) es una enfermedad inflamatoria intestinal crónica, de etiología desconocida. Los glucocorticoides (GC) constituyen el tratamiento de elección de brotes moderados y graves de CU, pero a pesar de ello hasta un 40% presenta una respuesta inadecuada a los mismos. No se conocen con exactitud los mecanismos asociados a esta mala respuesta, y tampoco somos capaces de predecirla. El uso de moléculas como los microRNAs (miR) en la predicción de respuesta a diferentes tratamientos, es una realidad en otros campos de la medicina, pero su aplicación en la CU es aún limitada. Esta tesis pretende profundizar en el conocimiento de la refractariedad a GC sistémicos a través del estudio del transcriptoma completo (RNAm y miR) de tejido cólico de pacientes con CU. Objetivos: El objetivo principal fue confirmar la existencia de distintos perfiles en el transcriptoma de tejido cólico asociados a una respuesta inadecuada a GC sistémicos en pacientes con brotes moderados y graves de CU. Los objetivos secundarios fueron caracterizar el efecto inmediato de los GC sistémicos sobre el transcriptoma y los mecanismo de acción relacionados con la adecuada e inadecuada respuesta. Material y Métodos: Se recogieron muestras de tejido cólico de pacientes con brotes moderados y graves de CU, antes de iniciar GC sistémicos, y a los 3º días de iniciado los mismos. Los pacientes fueron clasificados en respondedores o Re (actividad leve o inactividad tras 7 días de tratamiento con GC), y no respondedores o noRe (actividad moderada o grave tras 7 días de tratamiento con GC), Además se recogieron muestras de tejido cólico de controles sanos. Las muestras obtenidas fueron estudiadas mediante secuenciación de miR (kit TruSeq Small RNA Sample de Illumina), y microarrays de genoma completo (kit Human HT-12 Expresión v4.0 BeadChip de Illumina), y los resultados de cada grupo se compararon entre si. Posteriormente se utilizaron diferentes herramientas informáticas con la intención de integrar los resultados del estudio experimental y generar un modelo teórico de respuesta al fármaco. Resultados: Se incluyeron 10 controles, y 24 pacientes con brotes moderados o graves de CU tratados con GC. De ellos 13 fueron clasificados como Re, y 11 como noRe a GC. La integridad de las muestras permitió realizar el estudio experimental en 10 pacientes de cada grupo y en 6 controles. Los resultados de la secuenciación mostraron un perfil diferencial de 2 miR antes de iniciar los GC (miR-625-3p; miR-196b-3p) entre el grupo Re y noRe. La comparación realizada entre pacientes Re antes de GC respecto a Re tras 3º días de GC mostró 6 miR diferenciales (miR-183-5p; miR-584-5p; miR-126-5p; miR-625-3p; miR-1271-5p; miR-409-5p), y un RNAm diferencial (DDIT4/REDD1). Y la comparación entre noRe antes de GC y noRe tras 3º días de GC mostró 13 miR diferenciales (miR-200c-3p; miR-18a-5p; miR-192-5p; miR-183-5p; miR-200a-5p; miR-10a-5p; miR-545-5p; miR-132-5p; miR-194-5p; miR-625-3p; miR-1271-5p; miR-409-5p; miR-504-5p). Además el estudio in silico realizado sugiere una posible asociación de la vía mTOR en la respuesta a GC. Conclusiones: En este estudio se demuestra que existe un perfil diferencial en el transcriptoma de tejido cólico de pacientes con brotes moderados o graves de CU Re y noRe a GC sistémicos antes de iniciar el tratamiento. Por otro lado, que dicho transcriptoma se modifica de forma diferente en pacientes Re y noRe en respuesta al tratamiento con GC sistémicos. Podría existir una asociación entre la regulación de la vía mTOR por miR y la respuesta a GC en la CU.
Background: Ulcerative colitis (UC) is a chronic inflammatory bowel disease of unknown etiology. Glucocorticoids (GC) remain the first-line treatment for moderate and severe active disease. However, 40% of patients do not have an appropriate response. We do not know yet the exact underlying mechanism associated with bad response and we are not able to predict it. The utility of molecules like microRNA (miRNA) in the prediction of response to treatments is a reality in other fields of medicine, for example in oncology, but little is known about the utility of these molecules in Inflammatory Bowel Disease. This thesis pretends to delve into refractoriness to GC by studying transcriptome of colonic tissue of patients with ulcerative colitis flares. Objectives: The main objective was to confirm the presence of different colonic transcriptome profiles associated to inadequate response to systemic GC in patients with moderate and severe flares of UC. Secondary objectives were to characterize the immediate effect of systemic GC treatment on the colonic transcriptome, and to determine the mechanism of action related to adequate and inadequate response. Material and Methods: Colic tissue samples of patients with moderate and severe flares of CU were collected before starting GC, and on the 3rd day of treatment. Patients were grouped in responder (less than moderate activity without need for rescue at day 7 of GC) and non-responder (moderate or severe activity at day 7 of GC). In adition, colic tissue samples from healthy controls were collected. Samples were studied by sequencing methods for miR profiles (TruSeq Small RNA Sample kit Illumina), and with microarrays (Human HT-12 kit v4.0 Expression BeadChip Illumina) for hold transcriptome study. Results of each group were compared. Subsequently bioinformatics was used to integrate results and to generate a theoretical model of response to the drug. Results: 10 controls and 24 patients with moderate or severe of UC flares treated with GC were included (13 responders and 11 non-responders to GC). The integrity of samples allowed experimental study in 10 patients of each group and 6 healthy controls. The sequencing results showed a differential miR profile (miR-625-3p, miR-196b-3p) at baseline between responders and non-responders. The comparison between responders before and after treatment show 6 differential miR (miR-183-5p, miR-584-5p, miR-126-5p, miR-625-3p, miR-1271-5p; miR -409-5p), and one differential RNAm (DDIT4 / REDD1). The comparison between non responders before and after GC treatment show 13 differential miR between groups (miR-200c-3p, miR-18a-5p; miR-192-5p, miR-183-5p, miR-200a-5p; miR 10a-5p; miR-545-5p, miR-132-5p, miR-194-5p, miR-625-3p, miR-1271-5p, miR-409-5p, miR-504-5p). In silico study suggests a possible association of the mTOR signaling pathway with the response to glucocorticoids in patients with UC flares and this pathway was used to generate 2 different models of response to GC. Conclusions: This study shows that there is a different profile in the colonic tissue transcriptome of patients with moderate or severe flares of UC responders and non-responders to systemic GC before starting treatment. This colonic transcriptome change with treatment and those changes were different in responders and non-responders. There may be an association between the regulation of the mTOR pathway by miR and the response to GC in UC.

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Introdução: o uso crônico de glicocorticoides é considerado a principal causa de osteoporose secundária e iatrogênica. Existem poucos estudos associando fraturas ao uso de glicocorticoides na faixa etária pediátrica. Eles poderiam ajudar na criação de abordagens preventivas e terapêuticas. Objetivos: avaliar se o uso de glicocorticoides, nos 12 meses precedentes, associou-se à ocorrência de fraturas em crianças e adolescentes; identificar a frequência de asma e outras doenças; comparar o perfil demográfico, o tipo de trauma, o índice de massa corpórea, a prática de exercício físico, a ingesta de leite e o tabagismo passivo domiciliar nos grupos com e sem fratura; verificar a frequência de deficiência de vitamina D. Métodos: no período de abril a outubro de 2015, um estudo tipo caso controle foi conduzido em crianças e adolescentes vitimadas por trauma, com e sem fratura, a partir da análise dos dados coletados. Resultados: foram estudados 104 pacientes, 50 com fratura e 54 com trauma, mas sem fratura. Ao todo, 80,4% eram meninos e 40,4% estavam na faixa etária de 10 a 14 anos. O uso prévio de glicocorticoides ocorreu em 15,4% do total, sem diferença estatisticamente significante entre os dois grupos. Entre 39 pacientes com fratura e que dosaram a vitamina D, 47,2% tinham níveis séricos < 30ng/ml. A prática de exercício físico associou-se a um aumento em 2,2 vezes no risco para fratura. Conclusões: este estudo não mostrou associação entre o uso prévio de glicocorticoides e a ocorrência de fraturas em crianças e adolescentes. A faixa etária de 10 a 14 anos, o trauma grave e o exercício físico associaram-se com um maior risco para fraturas. Cerca de metade de uma amostra dos pacientes com fratura apresentou níveis insuficientes/deficientes de vitamina D, mesmo em região tropical.
Introduction: Osteoporosis is not exclusive to older adults and manifests by fractures. Chronic glucocorticoid use is considered the main cause of secondary and iatrogenic osteoporosis. Few studies have related fractures to the use of glucocorticoids in children and adolescents. Such studies could be useful for the development of preventive and therapeutic strategies. Objectives: To assess whether glucocorticoid use in the past 12 months is associated with the occurrence of fractures in children and adolescents; to identify the frequency of asthma and other diseases; and to compare the demographic profile, type of trauma, body mass index, physical activity, milk intake, and household exposure to cigarette smoke of groups with and without fractures; to verify the frequency of vitamin D insufficiency/deficiency. Methods: A case-control study, conducted from April to October 2015, analyzed the data of trauma children and adolescents with and without fractures. Results: A total of 104 trauma patients were studied, 50 with and 54 without fractures. In all, 80.4% were males, and 40.4% were aged 10 to 14 years. Previous glucocorticoid use occurred in 15.4% of the sample, without significant difference between the groups. Of the 39 fracture patients with measured serum vitamin D levels, 47.2% had levels < 30ng/ml. Physical activity was associated with a 2.2-fold risk of fractures, but without significance in multivariate analysis. Conclusions: This study did not find an association between previous glucocorticoid use and the occurrence of fractures in children and adolescents. In 10- to 14-year-olds, severe trauma and physical activity were associated with higher risk of fractures. About half the fracture sample had insufficient/deficient vitamin D levels, despite residing in a tropical region.

Lors de la liaison d’un glucocorticoïde (GC) naturel ou synthétique (par exemple, la Dexaméthasone) au récepteur des glucocorticoïdes (GR), les GCs régulent l’expression de gènes cibles soit par (i) transactivation par liaison ‘’directe’’ à un élément de liaison à l’ADN de type ‘’(+)GRE’’, (ii) transrépression ‘’directe’’ par liaison à un élément de type ‘’nGRE’’ ou (iii) transrépression ‘’indirecte’’ par interaction physique directe avec des facteurs de transcription pro-inflammatoires tels que AP-1 et NF-κB. Les effets anti-inflammatoires bénéfiques des GCs sont généralement attribués à la transrépression indirecte, alors que nombre de leurs effets secondaires pathologiques indésirables paraissent liés à la transactivation et/ou à la transrépression directe. Notre laboratoire a récemment découvert qu’un composé non-stéroïdien dénommé CpdX ainsi que ses dérivés deutérés, ne présentent ni la fonction de transactivation, ni celle de transrépression directe du GR, tout en stimulant son activité bénéfique de transrépression indirecte. Notre projet a consisté à caractériser un composé non-stéroïdien dit CpdX, ainsi que ses dérivés, quant à leurs activités thérapeutiques et à démontrer qu’elles sont semblables à celles des glucocorticoïdes anti-inflammatoires, couramment utilisés, tout en étant débarrassés de leurs effets pathologiques secondaires, tels que l’ostéoporose, l’atrophie cutanée et le syndrome métabolique. Pour atteindre nos objectifs, nous avons utilisés des modèles de souris présentant soit les affections cutanées (dermatites de contact ou atopique, psoriasis), l'asthme, l’arthrite rhumatismale, la colite ulcérative ou la conjonctivite allergique, associés à des études d’immunologie et de biologie moléculaire et cellulaire. Mon travail de thèse a démontré que CpdX, et certains de ses dérivés deutérés, présentent une activité anti-inflammatoire dans le traitement de ces modèles ‘’souris’’ (Partie I). Nous avons aussi montré que le traitement par CpdX et ses dérivés n’induit pas les effets secondaires pathologiques des glucocorticoïdes (Partie II), ouvrait ainsi la vue à une nouvelle ère dans le traitement à long-terme de maladies inflammatoires, sans provoquer les effets pathologiques indésirables des traitements actuels aux glucocorticoïdes
Upon binding of natural or synthetic glucocorticoids (GCs) (e.g. Dexamethasone) to their glucocorticoid receptor (GR), GCs regulate the expression of target genes either by (i) direct transactivation through direct binding to “(+)GRE” DNA binding sites (DBS), (ii) direct transrepression through binding to “IR nGRE” DBSs or (iii) tethered indirect transrepression mediated through interaction with transactivators, such as NFkB, AP1, or STAT3 bound to their cognate DBSs. The beneficial anti-inflammatory effects of GCs have been generally ascribed to tethered transrepression, whereas many of their long-term undesirable side-effects could be due to transactivation and/or direct transrepression. Our laboratory recently reported that a non-steroidal compound, named CpdX, selectively lacks both direct transactivation and direct transrepression functions, while still exerting an indirect transrepression activity. The goal of our project was to characterize CpdX and some of its derivatives as effective anti-inflammatory drugs similar to glucocorticoids, but lacking their main deleterious side-effects, e.g. osteoporosis, skin atrophy and metabolic disorders. To this end, we have used experimental mouse model for skin disorders (atopic dermatitis, contact dermatitis, and psoriasis), asthma, rheumatoid arthritis, ulcerative colitis and allergic conjunctivitis, combined with immunology, molecular and cellular biology. My thesis studies have demonstrated that in mouse models, CpdX and its derivatives exhibit anti-inflammatory activities, which are similar to those of glucocorticoids (Part I). Importantly, we further show that CpdX and its derivatives do not exhibit the long-term debilitating side-effects of glucocorticoids (Part II). Thereby paving the way to a new era in the long-term therapy of major inflammatory diseases

Thesis (PhD)--Stellenbosch University, 2011.
See also the post-print version of the article that was published from the PhD - http://hdl.handle.net/10019.1/19557
ENGLISH ABSTRACT: Glucocorticoid receptor (GR) levels, which modulate the response to glucocorticoids (GCs), vary between tissues and individuals and are altered by physiological and pharmacological effectors. In this study we set out to investigate the effects and implications of differences in GR concentration. Firstly, we established conditions that resulted in three statistically different GR populations in transiently transfected COS-1 cells. We demonstrated, using whole cell saturation ligand binding experiments, that high levels of wild type GR, but not of dimerization deficient GR, exhibited positive cooperative ligand binding with a concomitant increased ligand binding affinity. Furthermore, we established, through co-immunoprecipitation and fluorescent resonance energy transfer, that ligand independent dimerization correlates with positive cooperative ligand binding. This is the first time that positive cooperative ligand binding and increased ligand binding affinity have been explicitly correlated and linked to increased ligand independent dimerization of the GR. The downstream consequences of variation in GR concentration and dimerization included modulation of GR import and export rates, as investigated through live cell as well as immunofluorescent analysis. Furthermore, the nuclear distribution of GR was also influenced by GR dimerization. The major function of the GR is as a transcription factor, which mediates the response to GCs via activation or repression of genes. We have revealed direct influences of GR concentration and dimerization in a number of promoter reporter assays as well as in the transactivation of an endogenous gene. Specifically, cooperative ligand binding was found to be responsible for the GR level dependent potency shift in transrepression of an NF B containing promoter reporter construct via dexamethasone and the shift in the bio-character of Compound A, a dissociative GR agonist. Transactivation potency of dexamethasone as well as the partial agonist bio-character of medroxyprogesterone and mifepristone via a multiple GRE containing promoter reporter construct were influenced directly by cooperative ligand binding. Dimerization of the GR was shown to be crucial for ligand dependent transactivation of a single GRE containing promoter reporter construct, while ligand independent transactivation of both single and multiple GRE containing constructs was significantly increased due to an increase in GR concentration. The endogenous GC responsive glucocorticoid induced leucine zipper (GILZ) gene demonstrated significant ligand independent transactivation at GR levels, which displayed ligand independent dimerization. An increase in GR concentration resulted in an increase in efficacy through all promoter reporter constructs as well as the endogenous GILZ gene. Positive cooperative binding and the concomitant increase in ligand binding affinity to the GR at high levels may be a crucial factor in determining both the efficacy and potency of the GC response. Considering the significant differences in GR concentrations expressed by different tissues and by individuals within the same tissue, our findings may explain the interindividual as well as tissue specific responses to GC treatment and suggest an important mechanism of action through which the GR is primed to responsed to subsaturating GC concentrations and displays a significant level of ligand independent activity.
AFRIKAANSE OPSOMMING: Glukokortikoïed reseptor (GR) vlakke, wat die gedrag van glukokortikoïede (GCs) moduleer, wissel tussen weefsels en onder individue en word verander deur fisiologiese en farmakologiese effektore. In hierdie studie ondersoek ons die gevolge en implikasies van verskille in GR konsentrasie. Eerstens het ons die kondisies vasgestel wat benodig word om drie statisties verskillende GR populasies te vestig in kortstondige getransfekteerde COS-1 selle. Ons het getoon, met behulp van die heel sel versadigings ligand bindings eksperimente, dat hoë vlakke van wilde-tipe GR, maar nie van dimeriserings gebrekkige GR, positiewe koöperatiewe ligand binding, met 'n gepaardgaande toename in ligand bindings affiniteit, toon. Verder het ons bevestig, deur ko-immunopresipitasie en fluoressente resonansie energie-oordrag, dat ligand onafhanklike dimerisering korreleer met positiewe koöperatiewe ligand binding. Dit is die eerste keer dat positiewe koöperatiewe ligand binding en verhoogde ligand bindings affiniteit uitdruklik gekorreleer en gekoppel word aan verhoogde ligand onafhanklike dimerisering van die GR. Die daarop nagevolge van variasie in GR konsentrasie en dimerisering sluit in modulasie van die invoer en uitvoer tempo van die GR, soos ondersoek deur lewendige sel sowel as immunofluorescente analise. Verder is die verspreiding van die GR in die kern ook beïnvloed deur GR dimerisering. Die belangrikste funksie van die GR is as 'n transkripsie faktor, wat die respons van GCS bemiddel via aktivering of onderdrukking van gene. Ons het die direkte invloed van GR konsentrasie en dimerisering in 'n aantal promotor verslaggewer essais sowel as in die transaktivering van endogene gene onthul. Spesifiek, is gevind dat koöperatiewe ligand binding verantwoordelik is vir die GR vlak afhanklike verskuiwing in transrepressie potensie van 'n NF B bevattende promotor verslaggewer konstruk via deksametasoon en die verskuiwing van die biokarakter van verbinding A,' dissosiatiewe GR agonis. Transaktiverings potensie van deksametasoon, asook die gedeeltelike agonis bio-karakter van medroksie-progesteroon en mifepristoon, via 'n veelvoudige GRE bevattende promotor verslaggewer konstruk is direk beïnvloed deur koöperatiewe ligand binding. Dimerisering van die GR is getoon om deurslaggewend vir ligand afhanklike transaktivering van 'n enkele GRE bevattende promotor verslaggewer konstruk te wees, terwyl ligand onafhanklike transaktivering van beide enkel-en veelvoudige GRE bevattende konstrukte aansienlik toegeneem het as gevolg van toename in GR konsentrasie. Die endogene GC responsiewe glukokortikoïed geïnduseerde leusien rits (GILZ) gene het beduidende ligand onafhanklike transaktivering gedemonstreer op GR vlakke wat ligand onafhanklike dimerisering toon. 'n toename in GR konsentrasie het gelei tot toename in die effektiwiteit van al die promotor verslaggewer konstrukte, sowel as die endogene GILZ gene. Positiewe koöperatiewe ligand binding en die gepaardgaande toename in ligand bindings affiniteit van die GR by hoë vlakke kan 'n belangrike faktor wees in die bepaling van sowel die effektiwiteit as die potensie van die GC respons. As die aansienlike verskille in GR konsentrasies van verskillende weefsels en tussen verskillende individue in dieselfde weefsel in ag geneem word, kan ons bevindings die inter-individuele sowel as weefsel spesifieke response op GC behandeling verduidelik en stel dit 'n belangrike meganisme van aksie voor waardeur die GR voorberei word om op sub-versadigings konsentrasies van GC te reageer deur 'n beduidende vlak van ligand onafhanklike aktiwiteit te toon.

The fetus is highly responsive to the level of glucocorticoids in the gestational environment. Perturbing glucocorticoids during fetal development could yield long-term consequences. Extending prior research about effects of prenatally exposed synthetic glucocorticoids (sGC) on brain structural development during childhood, we investigated functional brain correlates of cognitive conflict monitoring in term-born adolescents, who were prenatally exposed to sGC. Relative to the comparison group, behavioral response consistency (indexed by lower reaction time variability) and a brain correlate of conflict monitoring (the N2 event-related potential) were reduced in the sGC exposed group. Relatedly, source localization analyses showed that activations in the fronto-parietal network, most notably in the cingulate cortex and precuneus, were also attenuated in these adolescents. These regions are known to subserve conflict detection and response inhibition as well as top-down regulation of stress responses. Moreover, source activation in the anterior cingulate cortex correlated negatively with reaction time variability, whereas activation in the precuneus correlated positively with salivary cortisol reactivity to social stress in the sGC exposed group. Taken together, findings of this study indicate that prenatal exposure to sGC yields lasting impacts on the development of fronto-parietal brain functions during adolescence, affecting multiple facets of adaptive cognitive and behavioral control.

Investigations were undertaken to examine the cyclic AMP signal transduction pathway responsible for delay of neutrophil apoptosis. Despite increasing protein kinase A (PKA) activity, this kinase is unlikely to mediate the effects of cyclic AMP in apoptosis since blockade of PKA activation did not influence the survival effects of cyclic AMP. Furthermore cyclic AMP mediated delay of neutrophil apoptosis is independent of PI-3 kinase and MAP kinase activation. Our results suggest cyclic AMP delays neutrophil apoptosis via a novel, reversible and transcriptionally independent mechanism. We show that proteasome activity in the neutrophil is vitally involved in this process and suggest that a balance of pro-apoptotic and anti-apoptotic proteins play a key role in the powerful ability of cyclic AMP to delay neutrophil death. Additional studies were aimed to elucidating the underlying mechanisms of glucocorticoid regulation of granulocyte apoptosis. Glucocorticoids were found to exert diametrically opposed effects on eosinophils and neutrophils, causing induction of apoptosis in eosinophils while delaying neutrophil cell death. Granulocytes were found to express the glucocorticoid receptor (GR) however the nature of isoforms expressed remains undefined. Glucocorticoid regulation of apoptosis in both cell types was found to require hsp90 activity. The use of pharmacological inhibitors suggested that histone deacetylation was not involved in either glucocorticoid inhibition of neutrophil apoptosis or glucocorticoid induction of eosinophil cell death but may be implicated in regulation of constitutive granulocyte apoptosis. In summary, there is good evidence implicating glucocorticoids and cyclic AMP in the regulation of granulocyte apoptosis. A greater understanding of the signalling mechanisms by which these mediators regulate granulocyte death could potentially lead to the development of novel strategies to therapeutically induce apoptosis for the resolution of inflammation.