Detection of mRNA levels for the estrogen alpha, estrogen beta and androgen nuclear receptor genes in archival breast cancer tissue

Previous studies in our laboratory have shown association of nuclear receptor expression and histological breast cancer grade. To further investigate these findings, it was the objective of this study to determine if expression levels of the estrogen alpha, estrogen beta and androgen nuclear receptor genes varied in different breast cancer grades. RNA extracted from paraffin embedded archival breast tumour tissue was converted into cDNA and cDNA underwent PCR to enable quantitation of mRNA expression. Expression data was normalised against the 18S ribosomal gene multiplex and analysed using ANOVA. Analysis indicated a significant alteration of expression for the androgen receptor in different cancer grades (P=0.014), as well as in tissues that no longer possess estrogen receptor alpha proteins (P=0.025). However, expression of estrogen receptors alpha and beta did not vary significantly with cancer grade (P=0.057 and 0.622, respectively). Also, the expression of estrogen receptor alpha or beta did not change, regardless of the presence of estrogen receptor alpha protein in the tissue (P=0.794 and 0.716, respectively). Post-hoc tests indicate that the expression of the androgen receptor is increased in estrogen receptor negative tissue as well as in grade 2 and grade 3 tumours, compared to control tissue. This increased expression in late stage breast tumours may have implications to the treatment of breast tumours, particularly those lacking expression of other nuclear receptor genes.

Detection of mRNA for nuclear receptor co-activators 1 and 3 in archival breast cancer tissue and surrounding stroma : a tissue expression study

Before the age of 75 years, approximately 10% of women will be diagnosed with breast cancer, one of the most common malignancies and a leading cause of death among women. The objective of this study was to determine if expression of the nuclear receptor coactivators 1 and 3 (NCoA1 and NCoA3) varied in breast cancer grades. RNA was extracted from 25 breast tumours and transcribed into cDNA which underwent semi-quantitative polymerase chain reaction, normalised using 18S. Analysis indicated that an expression change for NCoA1 in cancer grades and estrogen receptor alpha negative tissue (P= 0.028 and 0.001 respectively). NCoA1 expression increased in grade 3 and estrogen receptor alpha negative tumours, compared to controls. NCoA3 showed a similar, but not significant, trend in grade and a non-significant decrease in estrogen receptor alpha negative tissues. Expression of NCoA1 in late stage and estrogen receptor alpha negative breast tumours may have implications to breast cancer treatment, particularly in the area of manipulation of hormone signalling systems in advanced tumours.

Expression of glucocorticoid and progesterone nuclear receptor genes in archival breast cancer tissue.

BACKGROUND: Previous studies in our laboratory have shown associations of specific nuclear receptor gene variants with sporadic breast cancer. In order to investigate these findings further, we conducted the present study to determine whether expression levels of the progesterone and glucocorticoid nuclear receptor genes vary in different breast cancer grades. METHODS: RNA was extracted from paraffin-embedded archival breast tumour tissue and converted into cDNA. Sample cDNA underwent PCR using labelled primers to enable quantitation of mRNA expression. Expression data were normalized against the 18S ribosomal gene multiplex and analyzed using analysis of variance. RESULTS: Analysis of variance indicated a variable level of expression of both genes with regard to breast cancer grade (P = 0.00033 for glucocorticoid receptor and P = 0.023 for progesterone receptor). CONCLUSION: Statistical analysis indicated that expression of the progesterone nuclear receptor is elevated in late grade breast cancer tissue.

The orphan nuclear receptor LRH-1 promotes breast cancer motility and invasion

The orphan nuclear receptor liver receptor homologue-1 (LRH-1) has roles in the development, cholesterol and bile acid homeostasis, and steroidogenesis. It also enhances proliferation and cell cycle progression of cancer cells. In breast cancer, LRH-1 expression is associated with invasive breast cancer; positively correlates with ERα status and aromatase activity; and promotes oestrogen-dependent cell proliferation. However, the mechanism of action of LRH-1 in breast cancer epithelial cells is still not clear. By silencing or over-expressing LRH-1 in ER-positive MCF-7 and ER-negative MDA-MB-231 breast cancer cells, we have demonstrated that LRH-1 promotes motility and cell invasiveness. Similar effects were observed in the non-tumourigenic mammary epithelial cell line, MCF-10A. Remodelling of the actin cytoskeleton and E-cadherin cleavage was observed with LRH-1 over-expression, contributing to increased migratory and invasive properties. Additionally, in LRH-1 over-expressing cells, the truncation of the 120 kDa E-cadherin to the inactive 97 kDa form was observed. These post-translational modifications in E-cadherin may be associated with LRH-1-dependent changes to matrix metalloproteinase 9 expression. These findings suggest a new role of LRH-1 in promoting migration and invasion in breast cancer, independent of oestrogen sensitivity. Therefore, LRH-1 may represent a new target for breast cancer therapeutics.

Transcriptional regulation by the orphan nuclear receptor ERRgamma

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

Orphan nuclear receptor subfamily NR4A their interplay with other nuclear receptors and functions in osteoblasts

Nurr1, NGFI-B and Nor1 (NR4A2, NR4A1 and NR4A3, respectively) belong to the NR4A subfamily of nuclear receptors. The NR4A receptors are orphan nuclear receptors which means that activating or repressing ligands for these receptors have not been found. NR4A expression is rapidly induced in response to various stimuli including growth factors and the parathyroid hormone (PTH). The studies concerning the NR4A receptors in the central nervous system have demonstrated that they have a major role in the development and function of the dopaminergic neurons of the midbrain and in regulating hypothalamus-pituitary-adrenal-axis. However, the peripheral functions of the NR4A family are largely unknown. Cultured mouse primary osteoblasts, a preosteoblastic cell line and several osteoblastic cell lines were used to investigate the role of NR4A receptors in osteoblasts. NR4A receptors were shown to directly bind to and activate the promoter of the osteopontin gene (OPN) in osteoblastic cells, thus regulating its expression. OPN is a major bone matrix protein expressed throughout the differentiation of preosteoblastic cells into osteoblasts. The activation of the OPN promoter was shown to be dependent on the activation function-1 located in the N-terminal part of Nurr1 and to occur in both monomeric and RXR heterodimeric forms of NR4A receptors. Furthermore, PTH was shown to upregulate OPN expression through the NR4A family. It was also demonstrated that the fibroblast growth factor-8b (FGF-8b) induces the expression of NR4A receptors in osteoblasts as immediate early genes. This induction involved phosphatidylinositol-3 kinase, protein kinase C, and mitogen activated protein kinase, which are all major pathways of FGF signalling. Nurr1 and NGFI-B were shown to induce the proliferation of preosteoblastic cells and to reduce their apoptosis. FGF-8b was shown to stimulate the proliferation of osteoblastic cells through the NR4A receptors. These results suggest that NR4A receptors have a role both in the differentiation of osteoblasts and in the proliferation and apoptosis of preosteoblast. The NR4A receptors were found to bind to the same response element on OPN as the members of the NR3B family of orphan receptors do. Mutual repression was observed between the NR4A receptors and the NR3B receptors. This repression was shown to be dependent on the DNA-binding domains of both receptor families, but to result neither from the competition of DNA binding nor from the competition for coactivators. As the repression was dependent on the relative expression levels of the NR4As and NR3Bs, it seems likely that the ratio of the receptors mediates their activity on their response elements. Rapid induction of the NR4As in response to various stimuli and differential expression of the NR3Bs can effectively control the gene activation by the NR4A receptors. NR4A receptors can bind DNA as monomers, and Nurr1 and NGFI-B can form permissive heterodimers with the retinoid X receptor (RXR). Permissive heterodimers can be activated with RXR agonists, unlike non-permissive heterodimers, which are formed by RXR and retinoic acid receptor or thyroid hormone receptor (RAR and TR, respectively). Non-permissive heterodimers can only be activated by the agonists of the heterodimerizing partner. The mechanisms behind differential response to RXR agonists have remained unresolved. As there are no activating or repressing ligands for the NR4A receptors, it would be important to find out, how they are regulated. Permissiviness of Nurr1/RXR heterodimers was linked to the N-terminal part of Nurr1 ligand-binding domain. This region has previously been shown to mediate the interaction between NRs and corepressors. Non-permissive RAR and TR, permissive Nurr1 and NGFI-B, and RXR were overexpressed with corepressors silencing mediator for retinoic acid and thyroid hormone receptors (SMRT), and with nuclear receptor corepressor in several cell lines. Nurr1 and NGFI-B were found to be repressed by SMRT. The interaction of RXR heterodimers with corepressors was weak in permissive heterodimers and much stronger in non-permissive heterodimers. Non-permissive heterodimers also released corepressors only in response to the agonist of the heterodimeric partner of RXR. In the permissive Nurr1/RXR heterodimer, however, SMRT was released following the treatment with RXR agonists. Corepressor release in response to ligands was found to differentiate permissive heterodimers from non-permissive ones. Corepressors were thus connected to the regulation of NR4A functions. In summary, the studies presented here linked the NR4A family of orphan nuclear receptors to the regulation of osteoblasts. Nurr1 and NGFI-B were found to control the proliferation and apoptosis of preosteoblasts. The studies also demonstrated that cross-talk with the NR3B receptors controls the activity of these orphan receptors. The results clarified the mechanism of permissiviness of RXR-heterodimers. New information was obtained on the regulation and functions of NR4A receptors, for which the ligands are unknown.

Endocrine disrupting effects of zearalenone, alpha- and beta-zearalenol at the level of nuclear receptor binding and steroidogenesis.

The mycotoxin zearalenone (ZEN) is a secondary metabolite of fungi which is produced by certain species of the genus Fusarium and can occur in cereals and other plant products. Reporter gene assays incorporating natural steroid receptors and the H295R steroidogenesis assay have been implemented to assess the endocrine disrupting activity of ZEN and its metabolites -zearalenol (-ZOL) and -zearalenol (-ZOL). -ZOL exhibited the strongest estrogenic potency (EC50 0.022 ± 0.001 nM), slightly less potent than 17- estradiol (EC50 0.015 ± 0.002 nM). ZEN was ~70 times less potent than -ZOL and twice as potent as -ZOL. Binding of progesterone to the progestagen receptor was shown to be synergistically increased in the presence of ZEN, -ZOL or -ZOL. ZEN, -ZOL or -ZOL increased production of progesterone, estradiol, testosterone and cortisol hormones in the H295R steroidogenesis assay, with peak productions at 10 M. At 100 M, cell viability decreased and levels of hormones were significantly reduced except for progesterone. -ZOL increased estradiol concentrations more than -ZOL or ZEN, with a maximum effect at 10 M, with -ZOL (562 ± 59 pg/ml) > -ZOL (494 ± 60 pg/ml) > ZEN (375 ± 43 pg/ml). The results indicate that ZEN and its metabolites can act as potential endocrine disruptors at the level of nuclear receptor signalling and by altering hormone production.

Endocrine disrupting effects of ochratoxin A at the level of nuclear receptor activation and steroidogenesis

Ochratoxin A (OTA) is a mycotoxin and extrolite of fungi which has been reported in a range of foods. This study uses mammalian reporter gene assays (RGAs) with natural steroid receptors and the H295R steroidogenesis assay to assess the endocrine disrupting activity of OTA.

At the receptor level, OTA (within a concentration range of 0.25–2500 ng/ml) did not induce an agonistic response in an oestrogen, androgen, progestagen or glucocorticoid RGA. An antagonistic effect was observed in all of the RGAs at the highest concentration tested (2500 ng/ml). However, while there was no significant cytotoxic effect observed in the MTT (thiazolyl blue tetrazolium bromide) cell viability assay at this concentration, there was a corresponding change in cell morphology which may be related to the resulting antagonistic effect.

At the hormone production level, H295R cells were used as a steroidogenesis model and exposed to OTA (within a concentration range of 0.1–1000 ng/ml). Treatment of the cells with 1000 ng/ml OTA increased the production of estradiol (117 ± 14 ng/ml) over 3 times that of the solvent control (36 ± 9 pg/ml). Western blotting confirmed an increase in aromatase protein.

Overall the results indicate that OTA does not appear to interact with steroid receptors but has the potential to cause endocrine disruption by interfering with steroidogenesis. This is the first study identifying the effect OTA may have on production of the steroid hormone estradiol.

Dietary Restriction Induced Longevity is Mediated by Nuclear Receptor NHR-62 in Caenorhabditis elegans

Dietary restriction (DR) extends lifespan in a wide variety of species, yet the underlying mechanisms are not well understood. Here we show that the C. elegans HNF4a- related nuclear hormone receptor NHR-62 is required for metabolic and physiologic responses associated with DR-induced longevity. nhr-62 mediates the longevity of eat- 2 mutants, a genetic mimetic of dietary restriction, and blunts the longevity response of DR induced by bacterial food dilution at low nutrient levels. Metabolic changes associated with DR, including decreased Oil Red O staining, increased autophagy, and changes in fatty acid composition are partly reversed by mutation of nhr-62. Expression profiles reveal that several hundred genes induced by DR depend on the activity of NHR-62, including a putative lipase required for the DR response. This study provides critical evidence that nuclear hormone receptors regulate the DR response, suggesting hormonal and metabolic control of life span.

Effects of the mycotoxin patulin at the level of nuclear receptor transcriptional activity and steroidogenesis in vitro

Patulin (PAT) is a mycotoxin produced by various species of fungi, with Penicillium expansum being the most commonly occurring. Apples and apple products are the main sources of PAT contamination. This mycotoxin has been shown to induce toxic effects in animals, a few of which include reproductive toxicity and interference with the endocrine system. Here the endocrine disrupting potential of PAT has been investigated in vitro to identify disruption at the level of oestrogen, androgen, progestagen and glucocorticoid nuclear receptor transcriptional activity, and to assess interferences in estradiol, testosterone and progesterone steroid hormone production. At the receptor level, 0.5-5000ng/ml (0.0032-32μM) PAT did not appear to induce any specific (ant) agonistic responses in reporter gene assays (RGAs); however, nuclear transcriptional activity was affected. A >6 fold increase in the glucocorticoid receptor transcriptional activity was observed following treatment with 5000ng/ml PAT in the presence of cortisol. At the hormone production level, despite cytotoxicity being observed after treatment with 5000ng/ml PAT, estradiol levels had increased >2 fold. At 500ng/ml PAT treatment, an increase in progesterone and a decrease in testosterone production were observed. The findings of this study could be considered in assessing the health risks following exposure to PAT.

Nuclear receptor Rev-erb alpha (Nr1d1) functions in concert with Nr2e3 to regulate transcriptional networks in the retina.

The majority of diseases in the retina are caused by genetic mutations affecting the development and function of photoreceptor cells. The transcriptional networks directing these processes are regulated by genes such as nuclear hormone receptors. The nuclear hormone receptor gene Rev-erb alpha/Nr1d1 has been widely studied for its role in the circadian cycle and cell metabolism, however its role in the retina is unknown. In order to understand the role of Rev-erb alpha/Nr1d1 in the retina, we evaluated the effects of loss of Nr1d1 to the developing retina and its co-regulation with the photoreceptor-specific nuclear receptor gene Nr2e3 in the developing and mature retina. Knock-down of Nr1d1 expression in the developing retina results in pan-retinal spotting and reduced retinal function by electroretinogram. Our studies show that NR1D1 protein is co-expressed with NR2E3 in the outer neuroblastic layer of the developing mouse retina. In the adult retina, NR1D1 is expressed in the ganglion cell layer and is co-expressed with NR2E3 in the outer nuclear layer, within rods and cones. Several genes co-targeted by NR2E3 and NR1D1 were identified that include: Nr2c1, Recoverin, Rgr, Rarres2, Pde8a, and Nupr1. We examined the cyclic expression of Nr1d1 and Nr2e3 over a twenty-four hour period and observed that both nuclear receptors cycle in a similar manner. Taken together, these studies reveal a novel role for Nr1d1, in conjunction with its cofactor Nr2e3, in regulating transcriptional networks critical for photoreceptor development and function.

The role of the nuclear receptor Nr5a2 in ovulation in mice

Le récepteur nucléaire Nr5a2 est exprimé dans l’ovaire, plus spécifiquement dans les cellules de granulosa et lutéales. Une déplétion conditionnelle de Nr5a2 dans les cellules de granulosa au stade de follicule primaire par croisement de souris Nr5a2-flox et Amhr2-Cre (Nr5a2f/fAmhr2Cre/+) génère des problèmes au niveau de l’expansion du cumulus, de l’ovulation et de la lutéinisation. Ainsi, nous estimons que Nr5a2 régule les connexions intercellulaires dans le follicule ovarien via la connexine 43 (Cx43), une protéine de jonction impliquée dans l’expansion du cumulus. Le premier objectif de l’étude était de déterminer si l’absence d’expansion du cumulus chez les souris Amhr2Cre-cKO est liée à l’absence de communication intercellulaire adéquate entre les cellules de granulosa et de cumulus dans les follicules préovulatoires. À cette fin, des ovaires de souris immatures Amhr2Cre-cKO et non transgéniques ont été prélevés (n=3) après un traitement de superstimulation utilisant les gonadotropines eCG suivie de hCG afin d’induire l’ovulation. Nous avons ainsi démontré, par RT-PCR, une sous-expression de Cx43 avant et au moment du stimulus ovulatoire (0 h et 2 h) chez le groupe Amhr2Cre-cKO (P<0.01), ce qui pourrait mener à un problème dans l’acquisition de la compétence développementale de l’oocyte. D’un autre côté, au moment de l’ovulation (12 h), l’ARNm de Cx43 est surexprimé dans le groupe Amhr2Cre-cKO, ce qui pourrait prévenir les cellules du cumulus de se détacher l’une de l’autre. Nous avons ainsi conclu que Cx43 est un gène sous le contrôle de Nr5a2 et qu’une régulation erronée de ce gène est une cause possible du problème d’expansion du cumulus chez les souris Amhr2Cre-cKO. Afin d’examiner le rôle de Nr5a2 dans l’ovulation et la lutéinisation à différents stades de la maturation folliculaire, nous suggérons que Nr5a2 module la séquence temporelle des événements menant à l’ovulation. En croisant des souris Nr5a2-flox et Cyp19-Cre (Nr5a2f/fCyp19Cre/+), l’expression de Nr5a2 a été interrompue dans les cellules de granulosa des follicules antraux et préovulatoires. Aucune portée n’a été obtenue de ces souris (n=4) durant un essai d’accouplement de 6 mois. Chez les souris Cyp19Cre-cKO on remarque la présence de structures s’apparentant à des cellules de type lutéales et les femelles âgées d’un an présentent des kystes folliculaires hémorragiques et une hypertrophie de l’épithélium en surface de l’ovaire. Les deux modèles transgéniques démontrent donc une absence de l’expansion du cumulus et de l’ovulation. En conclusion, Nr5a2 semble réguler différemment la folliculogenèse et l’ovulation dans les cellules de granulosa des follicules primaires et antraux.

The orphan nuclear receptor NR5A2 regulates peri-ovulatory events and their consequent luteinization in mice

Le récepteur nucléaire Nr5a2, également connu sous le nom de liver receptor homolog-1 (Lrh-1), est exprimé au niveau de l’ovaire chez la souris, exclusivement dans les cellules lutéales et de la granulosa. La perturbation de Nr5a2, spécifique aux cellules de la granulosa chez la souris à partir des follicules primaires dans la trajectoire du développement folliculaire a démontré que Nr5a2 est un régulateur clé de l’ovulation et de la fertilité chez la femelle. Notre hypothèse veut que Nr5a2 régule les évènements péri- et post-ovulatoires dans une séquence temporelle lors de la folliculogénèse. Afin d'étudier l’implication de Nr5a2 lors de l’ovulation et de la lutéinisation à différents stades du développement folliculaire, nous avons généré deux modèles de souris knockout spécifiques aux cellules de la granulosa pour Nr5a2: 1) Nr5a2Amhr2-/-, avec une réduction de Nr5a2 à partir des follicules primaires et subséquents; 2) Nr5a2Cyp19-/-, avec une réduction de Nr5a2 débutant au stade antral de développement en progressant. L’absence de Nr5a2 à partir des follicules antraux a résulté en une infertilité chez les femelles Nr5a2Cyp19-/-, de même qu’en des structures non-fonctionnelles similaires aux structures lutéales au niveau des ovaires, en une réduction des niveaux de progestérone synthétisée ainsi qu’en un échec dans le support d’une pseudo-gestation. La synthèse de progestérone a été entravée suite à l’absence de Nr5a2 par l’entremise d’une régulation à la baisse des gènes reliés au transport du cholestérol, Scarb1, StAR et Ldlr, démontré par qPCR. Les complexes cumulus-oocytes des femelles Nr5a2Cyp19-/- immatures super-stimulées ont subi une expansion in vivo, mais l’ovulation a été perturbée, possiblement par une régulation à la baisse du gène du récepteur de la progestérone (Pgr). Un essai d’expansion du cumulus in vitro a démontré une expansion défectueuse du cumulus chez les Nr5a2Amhr2-/-, associée à un dérèglement de la protéine des jonctions communicantes (Gja1; Cx43). Cependant, l’expansion du cumulus chez les Nr5a2Cyp19-/- n’a pas été autant affectée. Des résultats obtenus par qPCR ont démontré une régulation à la baisse dans l’expression des gènes Areg, Ereg, Btc et Tnfaip6 chez les deux modèles de cellules ovariennes knockout à 2h et 4h post hCG. Nous avons observé que 85% des oocytes, chez les deux génotypes mutants, peuvent subir une rupture de la vésicule germinative, confirmant leur capacité de maturation in vivo. La technique d’injection intra-cytoplasmique de spermatozoïdes a prouvé que les oocytes des deux génotypes mutants sont fertilisables et que 70% des embryons résultants ont poursuivi leur développement vers le stade de blastocyste, et ce, indépendamment du génotype. En conclusion, Nr5a2 régule la fertilité chez les femelles tout au long du processus du développement folliculaire. Il a été démontré que Nr5a2 est essentiel à la lutéinisation et que sa perturbation dans les cellules somatiques ovariennes ne compromet pas la capacité des oocytes à être fertilisés. En vue d’ensemble, nous avons fourni une investigation inédite et complète, utilisant de multiples modèles et techniques afin de déterminer les mécanismes par lesquels Nr5a2 régule les importants processus que sont l’expansion du cumulus, l’ovulation ainsi que la formation du corps jaune.

Study of nuclear receptor dynamics by BRET

Les récepteurs nucléaires (RN) sont des facteurs de transcription ligand dépendants qui contrôlent une grande variété de processus biologiques de la physiologie humaine, ce qui a fait d'eux des cibles pharmacologiques privilégiées pour de nombreuses maladies. L'un de ces récepteurs, le récepteur de l’œstrogène alpha (ERα), peut activer la prolifération cellulaire dans certaines sections de l'épithélium mammaire tandis qu’un autre, le récepteur de l'acide rétinoïque alpha (RARα), peut provoquer un arrêt de la croissance et la différenciation cellulaire. La signalisation de ces deux récepteurs peut être altérée dans le cancer du sein, contribuant à la tumorigénèse mammaire. L’activité d’ERα peut être bloquée par les anti-oestrogènes (AE) pour inhiber la prolifération des cellules tumorales mammaires. Par contre, l’activation des voies de RARα avec des rétinoïdes dans un contexte clinique a rencontré peu de succès. Ceci pourrait résulter du manque de spécificité des ligands testés pour RARα et/ou de leur activité seulement dans certains sous-types de tumeurs mammaires. Puisque les récepteurs nucléaires forment des homo- et hétéro-dimères, nous avons cherché à développer de nouveaux essais pharmacologiques pour étudier l'activité de complexes dimériques spécifiques, leur dynamique d’association et la structure quaternaire des récepteurs des œstrogènes. Nous décrivons ici une nouvelle technique FRET, surnommée BRET avec renforcement de fluorescence par transferts combinés (BRETFect), qui permet de détecter la formation de complexes de récepteurs nucléaires ternaires. Le BRETFect peut suivre l'activation des hétérodimères ERα-ERβ et met en évidence un mécanisme allostérique d'activation que chaque récepteur exerce sur son partenaire de dimérisation. L'utilisation de BRETFect en combinaison avec le PCA nous a permis d'observer la formation de multimères d’ERα fonctionnels dans des cellules vivantes pour la première fois. La formation de multimères est favorisée par les AE induisant la dégradation du récepteur des oestrogènes, ce qui pourrait contribuer à leurs propriétés spécifiques. Ces essais de BRET apportent une nette amélioration par rapport aux tests de vecteurs rapporteur luciférase classique, en fournissant des informations spécifiques aux récepteurs en temps réel sans aucune interférence par d'autres processus tels que la transcription et de la traduction. L'utilisation de ces tests nous a permis de caractériser les propriétés de modulation de l’activité des récepteurs nucléaires d’une nouvelle classe de molécules hybrides qui peuvent à la fois lier ERa ou RAR et inhiber les HDACs, conduisant au développement de nouvelles molécules prometteuses bifonctionnelles telles que la molécule hybride RAR-agoniste/HDACi TTNN-HA.