Tesi sul tema "Nr5a1"

Orientador: Marcilda Palandi de Mello
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: O termo Distúrbio da Diferenciação do Sexo (DDS) caracteriza-se pelo desenvolvimento genital ou gonadal incompleto ou desordenado. Os DDS com cariótipo 46,XY são caracterizados por genitália externa ambígua ou feminina, em alguns casos com gônadas disgenéticas, e presença ou ausência de derivados de Müller. Os mais frequentes são a insensibilidade androgênica, deficiência da 5-alfa-redutase tipo 2, disgenesia gonadal e DDS ovário-testicular. Vários são os genes que participam dos processos de determinação e diferenciação do sexo. Alterações no gene NR5A1, que codifica o fator de transcrição SF- 1, é responsável por diferentes fenótipos de DDS. A proteína SF-1 é expressa principalmente em tecidos esteroidogênicos (gônadas, adrenais e placenta), nas células de Sertoli, nas células de Leydig e nos ovários; é o principal regulador do metabolismo do colesterol nas células esteroidogênicas. Além disso, regula a atividade de outros genes, como os CYPs, HSD3B, StAR, SOX9, DAX1, entre outros. Na literatura são descritas alterações no gene NR5A1 associadas à DDS 46,XY, anorquia bilateral, amenorréia primária, falência ovariana precoce, hipospádia, infertilidade masculina, e alguns casos de tumores adrenais e endometrioses. Neste trabalho foi realizada a análise molecular do gene NR5A1 em 86 pacientes com DDS 46,XY, incluindo-se disgenesia gonadal completa (n = 7), disgenesia gonadal parcial (n = 18), DDS 46,XY idiopático (n = 41) e outros (n = 20). Doze alterações foram identificadas neste trabalho, sendo: sete na região codificante (p.Ser32Asn, p.Arg39Cis, p.Lis38*, p.Cis65Tir, p.L80Wfs*8, p.Cis247*, and p.Asp364Trefs*18), uma em sítio de splicing (c.1138+1G>T), duas no exon 1 nãocodificante (c.-133G>A e c.-156_-136ins18pb), três na região 5'UTR (c.-413G>A, c.- 208C>A, e c.-762C>T) e uma na região 3'UTR (c.*1286C>T). As variações aqui descritas, não foram identificadas em controles saudáveis. As análises in silico demonstraram o possível efeito deletério de cada alteração e, suas relações com o fenótipo dos indivíduos. Embora estes resultados demonstrem a importância de cada alteração para o fenótipo, haverá ainda a necessidade de se investigar os efeitos funcionais in vitro. As alterações com potencial deletério foram identificadas em maior frequência nos casos dos distúrbios da diferenciação gonadal (20%) e DDS 46,XY idiopático (22%)
Abstract: The term Disorders of Sex Differentiation (DSD) characterize incomplete or disorganized genital or gonadal development. The DSD with 46, XY karyotype may present either ambiguous or female genitalia and also dysgenetic gonads in some cases, with presence or absence of Müllerian derivatives. The most frequent are androgen insensitivity, 5-alpha-reductase type 2 deficiency, gonadal dysgenesis and ovarian-testicular DSD. There are several genes that participate in both sex determination and differentiation processes. Mutations in NR5A1 gene, which encoding SF-1, a transcription factor, are responsible for different phenotypes of DSD. The protein SF-1, which is expressed mainly in steroidogenic tissues (gonads, adrenal glands and placenta), is also express in Sertoli and Leydig cells, in the ovaries, and is the major regulator of cholesterol metabolism in steroidogenic cells. Moreover, it regulates the activity of other genes, such as CYPs, HSD3B, StAR, SOX9, DAX1, among others. The literature describes the association of changes in NR5A1 gene with 46, XY DSD, bilateral anorchia, primary amenorrhea, premature ovarian failure, hypospadias, male infertility, and some cases of adrenal tumors and endometriosis. The present work involved the molecular analysis of NR5A1 gene in 86 patients with 46, XY DSD including complete gonadal dysgenesis (n = 7), partial gonadal dysgenesis (n = 18), idiopathic 46, XY DSD (n = 41) and others (n = 20). Twelve variations had been identified: seven in the coding region (p.Ser32Asn, p.Arg39Cis, p.Lis38*, p.Cis65Tir, p.L80Wfs*8, p.Cis247*, and p.Asp364Trefs*18), one at a splice site (c.1138+1 G>T), two in the noncoding exon 1 (c.-133G>A and c.-156_-136ins18pb), three in the 5'UTR region (c.- 413G>A, c.-208C>A and c.-762C>T) and one in the 3'UTR (c.*1286C>T). The variations herein described, have not been identified in healthy controls. In silico analysis showed possible deleterious effects for each change and its correlations to individual phenotypes. Although those results demonstrate the importance of each change for the phenotype, there in vitro functional effects must be investigated. The potentially deleterious changes were identified more frequently in cases of disorders of gonadal development (20%) and idiopathic 46, XY DSD (22%)
Mestrado
Genetica Animal e Evolução
Mestra em Genética e Biologia Molecular

Monocytes are haematopoietic stem cell-derived, immune effector cells. In mice, monocytes consist of two principal subsets: the classical Ly6C-expressing subset and the patrolling Ly6Clow subset. The origin and fate of Ly6Clow monocytes remains a topic of debate, as does their interrelationship with Ly6C+ monocytes. The current model of Ly6Clow monocyte development suggests that Ly6C+ monocytes are the obligate, steady state precursors of the Ly6Clow subset, undergoing conversion or maturation within the blood. However, several studies have reported differing genetic dependencies of monocyte subsets that are in conflict with this model of Ly6Clow monocyte development. To re-investigate monocyte precursor-product relationships, this project combined genetic, kinetic and adoptive transfer studies to probe the interrelationships between bone marrow precursors, blood monocytes and tissue macrophages. This study firstly confirms the existence of a third, minor monocyte subset that expresses MHC II and varying levels of Ly6C and is found in the blood, spleen and bone marrow monocyte compartments. Secondly, this study establishes that all three monocyte subsets arise in the bone marrow from a proliferating common pro-monocyte via two genetically distinct lineages. This study demonstrates that Ly6Clow MHC II- monocytes arise from a novel Nr4a1-dependent, Irf8-independent intermediate population in the bone marrow compartment under steady state conditions, whereas. Thirdly, Nr4a1-dependent monocytes have a long half-life and circulate within the systemic and splenic vasculature, contributing minimally to tissue macrophage populations under steady state conditions. The findings in this thesis clarify and extend our understanding of monocyte genetic and functional heterogeneity.

Vitamin A and its derivatives, collectively termed retinoids, are essential for proper growth and development as well as maintenance of homeostasis in the adult. Retinoic acid (RA), the major biologically active vitamin A metabolite, is well characterized for its crucial roles in gene activation during embryogenesis. Our lab had previously performed a microarray analysis to identify genes induced by exogenous RA in the tailbud of early mouse embryos. Three genes were chosen from the microarray results for further investigation; Germ Cell Nuclear Factor (GCNF/NR6A1), Prickle1 (Pk1) and Ror2, the latter of which are known members of the planar cell polarity (PCP) pathway. These genes were further examined for RA regulation by embryo culture and RT-PCR, which strongly supported a direct regulatory mechanism of NR6A1 by RA. Further analysis aiming to identify a functional response element in the promoter of the targets was attempted, including chromatin immunoprecipitation (ChIP), made possible by the generation and characterization of a highly specific antibody against RARγ. This antibody was used in a ChIP promoter walk, which identified regions on target gene promoters that are occupied by RARγ in vivo, and therefore likely harbor RA response elements.

The orphan nuclear receptor NR5A2 is implicated in a multitude of biological processes including cholesterol homeostasis and development. Its role in cholesterol metabolism and cell proliferation is now well established in vitro and in vivo. Both in vitro and gene expression studies have suggested a role for NR5A2 in ovarian function. In this study, we provide in vivo evidence for its involvement in reproductive function by demonstrating that heterozygosity for a null mutation of NR5A2 leads to a reduction in female fertility. Furthermore, we showed that NR5A2+/- females display a severe reduction in ovarian progesterone production and that progesterone supplementation can rescue the NR5A2+/- subfertility phenotype. We also provide evidence that one of the mechanisms by which NR5A2 regulates ovarian progesterone production is through modulating the expression of SCAR, which controls one of the rate-limiting steps of progesterone synthesis.
A targeted disruption of the NR5A2 gene in the mouse leads to early lethality in utero between embryonic days 6.0 and 7.5, showing that NR5A2 plays a crucial role during early embryogenesis. The molecular mechanisms underlying this early lethality, however, are poorly understood. In this study, we used a morphological and marker gene analysis to characterize the NR5A2-/- embryonic phenotype and showed that although initial axis specification occurs in NR5A2-/- embryos, primitive streak and mesoderm fail to form. Using a chimeric approach, we demonstrated a requirement for NR5A2 function in the visceral endoderm (VE), an extra-embryonic tissue, for proper primitive streak morphogenesis and gastrulation. Our results also indicate a reduction in the expression of VE marker genes involved in the nutritive function of this tissue, suggesting that NR5A2 play a dual role in the VE, being implicated in the mediation of both its patterning and nutritive activity.
Taking advantage of the LacZ knock-in approach used to inactivate the NR5A2 gene, we also demonstrated that NR5A2 is expressed during craniofacial and nervous system development, suggesting a novel role for NR5A2 in head formation and neural development.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Nesta tese de mestrado, apresentamos, de acordo com as recomendações da comissão especial do programa de Pós-Graduação em Endocrinologia da Universidade Federal de São Paulo (UNIFESP), um trabalho em forma de manuscrito que será submetido à revista Clinical Endocrinology. Identificamos genes cuja expressão estava diminuída nas bibliotecas de carcinoma folicular de tiróide (FTC): TPO, TFF3, NR4A1 e IYD. O NR4A1 foi selecionado para uma análise mais profunda, por apresentar expressão potencialmente tecido-específica. Passamos então à validação do padrão diferencial de expressão em tecidos neoplásicos e não-neoplásicos, pela técnica de PCR em tempo real, assim como a realização de experimentos em linhagens celulares, com o uso de lítio. Estes são os focos desta tese e estão descritos no manuscrito “Downregulation of NR4A1 in follicular thyroid carcinoma cell line is restored after lithium treatment”, tendo como objetivos: 1) Validar a expressão de NR4A1 nos tecidos benignos e malignos da tiróide. 2) Avaliar a correlação dos genes CCND1 e FOSB com a expressão de NR4A1. 3) Verificar a expressão dos genes NR4A1, CCDN1 e FOSB nas linhagens de carcinoma tiroidiano disponíveis no laboratório, com objetivo de indetificar um modelo semelhante a expressão observada nos tecidos. 4) Verificar se a expressão dos genes NR4A1, FOSB e CCDN1 poderia ser restabelecida quando tratadas com lítio..
Introduction: The identification of follicular thyroid adenoma-associated transcripts will lead to a better understanding of the events involved in pathogenesis and progression of follicular tumors. Using Serial Analysis of Gene Expression, we identified five genes that are absent in a malignant follicular thyroid carcinoma (FTC) library, but expressed in follicular adenoma (FTA) and normal thyroid libraries. Methods: NR4A1, one of the five genes, was validated in a set of 27 normal thyroid tissues, 10 FTAs and 14 FTCs and three thyroid carcinoma cell lines by real time PCR. NR4A1 can be transiently increased by a variety of stimuli, including lithium, which is used as adjuvant therapy of thyroid carcinoma with 131I. We tested if lithium could restore NR4A1 expression. The expression of other genes potentially involved in the same signaling pathway was tested. To this end, lithium was used at different concentration (10mM or 20mM) and time (2h and 24 h) and the level of expression was tested by quantitative PCR. We next tested if Lithium could affect cell growth and apoptosis. Results: We observed that NR4A1 expression was under-expressed in most of the FTCs investigated, compared to expression in normal thyroid tissues and FTAs. We also found a positive correlation between NR4A1 and FOSB gene expression. Lithium induced NR4A1 and FOSB expression, reduced CCDN1 expression, inhibited cell growth and triggered apoptosis in a FTC cell line. Conclusions: NR4A1 is under-expressed in most of FTCs. The loss of expression of both NR4A1 and the Wnt pathway gene FOSB was correlated with malignancy. This is consistent with the hypothesis that its loss of expression is part of the transformation process of FTCs, either as a direct or indirect consequence of Wnt pathway alterations. Lithium restores NR4A1 expression, induces apoptosis and reduces cell growth. These findings may explain a possible molecular mechanism of lithium’s therapeutic action.
FAPESP: 05/60330-8
BV UNIFESP: Teses e dissertações

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
It is well established that locally produced factors exert pivotal roles during dominant follicle selection, oocyte maturation, ovulation and luteolysis. However, the identification of these factors and pathways involved in these processes are not yet established. In the present study, we focused on the in vivo bovine models to study reproductive physiology, which were used to identify receptors and intracellular signaling pathways involved in follicle selection and luteolysis. In the first study, it was reviewed the in vivo models used in our lab, describing and discussing the different bovine models and techniques currently used to study ovarian physiology in this mono-ovulatory specie. In a second study, it was evaluated the expression of estrogen receptors (ESRs) before (day 2 of follicular wave), during (day 3) and after (day 4) follicular deviation in cattle. ESR1 and ESR2 transcripts levels were higher in dominant (F1) than subordinate (F2) follicle after follicular deviation. FSH treatment maintained mRNA levels of both ESR1 and ESR2 in F2 follicles at similar levels observed in F1 follicles. Intrafollicular injection of 100 μM fulvestrant (an antagonist of ESRs) inhibited follicular growth and decreased CYP19A1 mRNA levels. Transcript levels of both ESR1 and ESR2 were not affected by fulvestrant injection. In the third study, our objective was to demonstrate the role of the transcription factor signal transducer and activator of transcription 3 (STAT3) and the nuclear receptor 5A2 (NR5A2) in luteolysis. Luteal and blood samples were collected from separate groups of cows on Day 10 of the estrous cycle 0, 2, 12, 24, and 48 hours after prostaglandin F2 alpha (PGF) treatment. Serum progesterone concentrations decreased (P < 0.05) within 2h and the histological examination of the corpus luteum at 24 and 48h after PGF treatment confirmed functional and morphological luteolysis, respectively. The abundance of STAR mRNA and protein decreased at 12h after PGF treatment. The abundance of NR5A2 mRNA and protein decreased (P < 0.05) at 12 and 24h post-PGF, respectively. Levels of STAT3 mRNA remained constant (P > 0.05) throughout the time-points evaluated. However, the abundance of phosphorylated isoform of STAT3, normalized to total STAT3, increased reaching a peak at 12h and remaining high until 48h after PGF treatment. In conclusion, bovine in vivo models provide a valuable system to study reproductive events under physiological endocrine environment while keeping intact the communication between follicular cells through autocrine and paracrine signaling, without the need to perform ovariectomy or euthanaze the animals. Our results suggest that both ESR1 and ESR2 are regulated during follicular deviation and dominance and in response to FSH treatment in cattle, ESRs are required for normal gene expression and development of the dominant follicle. PGF treatment results in decreased expression of the nuclear receptor NR5A2 and activation of STAT3 by phosphorylation in bovine luteal cells.
É bem estabelecido que fatores produzidos localmente exercem papel essencial durante a seleção do folículo dominante, maturação oocitária, ovulação e luteólise. No entanto, os fatores e vias envolvidas nestes processos não estão totalmente estabelecidos. No presente estudo, enfatizou-se o uso de modelos bovinos in vivo para o estudo da fisiologia reprodutiva, sendo aqui utilizados para identificar receptores e vias de sinalização intracelular envolvidas na seleção do folículo e luteólise. No primeiro estudo, revisaram-se os modelos in vivo utilizados em nosso laboratório, descreveram-se e discutiram-se os diferentes modelos em bovinos e técnicas atualmente utilizadas para estudar fisiologia ovariana nesta espécie monovulatória. Em um segundo estudo, avaliou-se a expressão de receptores de estradiol (ESRS) antes (dia 2 da onda folicular), durante (dia 3) e após (dia 4) a divergência folicular em bovinos. Os níveis dos transcritos ESR1 e ESR2 foram maiores no folículo dominante (F1) que no subordinado (F2) após a divergência folicular. O tratamento com FSH manteve os níveis de RNAm de ambos ESR1 e ESR2 nos folículos F2 em níveis semelhantes aos observados em folículos F1. A injeção intrafolicular de 100 uM de fulvestrant (um antagonista de ESRs) inibiu o crescimento folicular e causou uma diminuição dos níveis de RNAm de CYP19A1. Os níveis de transcritos, tanto para ESR1 e ESR2, não foram afetados pela injeção de fulvestrant. Num terceiro estudo, o nosso objetivo foi demonstrar o papel do Transdutor de sinais e ativador de transcrição 3 (STAT3) e do receptor nuclear 5A2 (NR5A2) na luteólise. Amostras de corpo lúteo (CL) e sangue foram coletadas dos grupos de vacas 0, 2, 12, 24 e 48 horas após o tratamento com prostaglandina F2 alpha (PGF) no dia 10 do ciclo estral. A concentração de progesterona sérica diminuiu (P < 0.05) em 2 horas e o exame histológico do CL às 24h e 48h após o tratamento com PGF confirmou a ocorrência de luteólise funcional e morfológica, respectivamente. A abundância de RNAm e proteína de STAR diminuiu às 12h após o tratamento com PGF. A abundância de RNAm e proteína de NR5A2 diminuiu (P < 0.05) às 12 e 24 horas pós-PGF, respectivamente. Os níveis de RNAm de STAT3 permaneceram constantes (P> 0.05) ao longo do tempo avaliado. No entanto, a abundância da isoforma fosforilada de STAT3, normalizados para STAT3 total, aumentou, atingindo um pico às 12h e permaneceu elevada até 48h após o tratamento com PGF. Em conclusão, os modelos bovinos in vivo fornecem um sistema valioso para estudar os eventos reprodutivos sob ambiente fisiológico, mantendo intacta a comunicação entre as células foliculares através de sinalização autócrina e parácrina, reduzindo a necessidade de realizar ovariectomia ou realizar a eutanásia dos animais. Nossos resultados sugerem que tanto ESR1 como ESR2 são regulados durante a divergência e dominância folicular em bovinos e em resposta ao tratamento com FSH, e ESRs são necessários para a expressão gênica e para o desenvolvimento do folículo dominante. O tratamento com PGF resulta em diminuição da expressão do receptor nuclear NR5A2 e ativação de STAT3 por fosforilação em células luteais bovinas.

La stéroïdogenèse dans les cellules de Leydig est liée au récepteur nucléaire NR4A1. Ce récepteur nucléaire régule la transcription du gène Star qui code pour une protéine essentielle à la stéroïdogenèse. L’expression de Nr4a1 est régulée par trois promoteurs distincts dont l’activité est hormonalement régulée. Deux de ces promoteurs ont été récemment décrits et ont été nommés Nr4a1 IB et Nr4a1 IC (le promoteur original a été renommé Nr4a1 IA). Des analyses qPCR ont montré que ces promoteurs sont actifs dans les cellules de Leydig MA-10 et qu’ils répondent à une stimulation hormonale. Ces promoteurs ont été isolés et fusionnés au gène rapporteur luciférase, et des délétions 5’ progressives ont été produites. Par transfections transitoires dans les cellules de Leydig MA-10 et MLTC-1, deux zones régulatrices importantes ont été localisées pour le promoteur Nr4a1 IB : l’une pour l’activité basale et l’autre, pour l’activation hormonale par l’AMPc et la LH. Bien que la transcription de Nr4a1 soit principalement activée par la voie de l’AMPc, une stimulation par la LH active d’autres voies qui peuvent influencer son profil d’expression. La majorité des études publiées n’ont pas exploré ces voies. À cet égard, la voie PKC est en mesure d’augmenter la quantité d’ARNm et l’activité promotrice de Nr4a1. Par ailleurs, l’inhibition des kinases PKA et PKC suite à une stimulation par la LH montre un profil d’ARNm qui peut être expliqué par l’existence du promoteur Nr4a1 IB récemment identifié dans les cellules de Leydig. Ces données supportent l’existence d’une régulation de l’expression de Nr4a1 impliquant d’autres seconds messagers que l’AMPc et le calcium. Par ailleurs, de nouveaux facteurs de transcriptions ont été testés comme régulateurs des promoteurs alternatifs de Nr4a1. Ces facteurs de transcription son SRF, CLOCK et BMAL1, et c-MAF. Ces informations contribuent à une meilleure compréhension de la régulation de la stéroïdogenèse.

The renin angiotensin system (RAS) is critical for the regulation of blood pressure, electrolyte/fluid, and metabolic homeostasis. Regulation of the RAS is important in the development and treatment of hypertension. As part of the rate-limiting step in a cascade of events ending in the production of angiotensin II, renin is a major regulator of the RAS. Its expression is localized to the juxtaglomerular (JG) cells of the JG apparatus where it is exquisitely located to respond to various physiological cues. Understanding the regulation of renin expression and development of the juxtaglomerular cells is critical. Two regulatory elements, the enhancer and proximal promoter, have been found to be important in controlling cell- and tissue- specific baseline expression of the renin gene. Within the enhancer is a hormone response element (HRE) which confers a high level of activity to the enhancer. Nuclear receptors that bind this element have been found to bind the HRE and regulate renin promoter transcriptional activity. We have previously characterized the role of the orphan nuclear receptor Nr2f6 as a negative regulator of renin expression that mediates its effects through the HRE. However, gel shift assays indicate that there are other transcription factors binding this element. We have identified other orphan nuclear receptors that regulate renin expression. The first, Nr2f2 acts as a negative regulator of renin promoter activity but does not appear to affect baseline expression of the endogenous renin gene. The other, Nr4a1, is a positive regulator of renin expression, but it does not appear to mediate its effects through the HRE. The transcriptional regulation of gene expression is controlled by regulatory elements separated by large distances from promoters. We and others have found that short transgenes of the human renin (hREN) locus are not sufficient to protect them from positional effects that can be exerted upon them by neighboring regulatory elements. We discovered a random truncation in a large genomic construct of the hREN gene that resulted in ubiquitous expression of renin not seen with the intact form. By locating the genomic insertion site of that transgene in the Zbtb20 gene, we found that the hREN promoter had come under control of that gene's regulatory elements. The gene downstream of renin however maintained its tissue-specific expression. We found that CCCTC-binding factor (CTCF) bound to chromatin in and around the renin locus. The presence of CTCF suggests that insulator elements are present in the renin locus, and their loss likely explains the results above. Finally, we assessed the role of microRNAs in the development of renin expressing cells in the mouse kidneys by cell-specific deletion of the processing enzyme Dicer. This resulted in reduction of renin expression and a decrease in the number of renin expressing cells in the kidney. Mice were hypotensive and had several kidney abnormalities including a hypertrophied vasculature and striped fibrosis. These results indicate that Dicer and the miRNAs it processes are critical for the development and maintenance of renin expressing cells that contribute to normal kidney development.

Platelet-derived growth factor (PDGF) is a family of signaling molecules that stimulates cell growth, survival and migration. PDGF is recognized by specific transmembrane proteins, the PDGF receptors, which relay the signals to the cell activating the Mitogen-activated protein (MAP) kinases and other signaling pathways. Aberrant activation of these pathways is frequently detected in cancer. Hence, the study of these processes is essential for identifying potential drug targets or diagnostic markers. In paper I, we identified Receptor Subfamily 4 Group A Member 1 NR4A1 to be regulated by PDGF via MAP kinases, clarifying the role of Extracellular signal–regulated kinases (Erk) 1/2, Erk5 and Nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) in its regulation. NR4A1 was found to be important for the tumorigenic potential, measured as anchorage-independent growth, of glioblastoma cells. Since the cellular responses elicited by PDGF result from the balance between phosphorylation and dephosphorylation events, we investigated the role of the dual specificity phosphatases DUSP4/MKP-2 and DUSP6/MKP-3. In paper II, we describe the crucial role of Erk1/2 and p53 in the expression of DUSP4/MKP2. Moreover, we observed that DUSP4/MKP-2 downregulation decreases Erk5 activation and accelerates PDGFRβ internalization and downregulation resulting in a specific inhibition of Signal transducers and activators of transcription (Stat) 3, Src and protein kinase C (PKC), and partially of p38, Stat1/5 and Phoshoplipase Cγ (PLCγ). In paper III, we report that DUSP6/MKP-3 creates a negative cross-talk between Erk1/2 and Erk5 and an auto-inhibitory feedback loop on the PI3-kinase/Akt pathway. In paper IV, we identify a new regulative mechanism of the PDGF pathway. PDGF induces Erk5 expression and activation that modulates the PDGFRβ activity. After Erk5 downregulation, the receptor undergoes to a faster and stronger activation that results in a faster internalization and degradation. In conclusion, we present a mechanism through which the PDGF/MAP kinases support tumor growth, and elucidate different regulatory pathways involved in PDGF signaling.

Lee, Kai Chuen.
Thesis M.Phil. Chinese University of Hong Kong 2015.
Includes bibliographical references (leaves 71-77).
Abstracts also in Chinese.
Title from PDF title page (viewed on 06, December, 2016).
Lee, Kai Chuen.

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 nuclear receptor Nr5a2 is expressed in the ovary, exclusively in granulosa and luteal cells. Conditional disruption of Nr5a2 in granulosa cells beginning with primary follicles by means of Nr5a2-floxed and Amhr2-Cre mice (Nr5a2f/fAmhr2Cre/+) results in failure in cumulus expansion, ovulation and luteinization. We hypothesize that Nr5a2 regulates intercellular connections in ovarian follicles through connexin 43 (Cx43), a gap-junctional protein related to cumulus expansion. The first objective of this study was to determine whether the lack of cumulus expansion in Amhr2Cre-cKO mice is related to the absence of normal cell-to-cell communication in cumulus/granulosa cells of preovulatory follicles. To address this, immature ovaries of Amhr2Cre-cKO and non-transgenic littermates mice were collected (n=3) after superstimulation to induce follicle development and ovulation. Using RT-PCR, the Cx43 mRNA levels were shown to be downregulated prior to and at the time of the ovulatory stimulus (0 h and 2 h) in the Amhr2Cre-cKO group (P<0.01), which may lead to the failure in the acquisition of oocyte developmental competence. On the other hand, by the time of ovulation (12 h), mRNA levels of Cx43 are upregulated in Amhr2Cre-cKO group, which may prevent the cumulus cells to detach one from another. We conclude that Cx43 is one of the downstream genes under Nr5a2 control and its dysregulation can be one reason for the defect in cumulus expansion in Amhr2Cre-cKO females. To examine the role of Nr5a2 in ovulation and luteinization in different stages of the follicle maturation, we hypothesized that Nr5a2 modulates the events leading to ovulation in a temporal sequence. By crossing Nr5a2-floxed and Cyp19-Cre mice (Nr5a2f/fCyp19Cre/+), Nr5a2 was disrupted in granulosa cells of antral and preovulatory follicles. No litters were born to Cyp19Cre-cKO females (n=4) during a 6 months breeding trial. Cyp19Cre-cKO enabled the development of a luteal-like structure, and 1-year-old females presented hemorrhagic follicular cysts and hypertrophic ovarian surface epithelium. Both knockout models display lack of cumulus expansion and ovulation. We conclude that Nr5a2 differentially regulates folliculogenesis and ovulation in granulosa cells of small and antral follicles.

La période de réceptivité endométriale chez l’humain coïncide avec la différentiation des cellules stromales de l’endomètre en cellules hautement spécifiques, les cellules déciduales, durant le processus dit de décidualisation. Or, on sait qu’une transformation anormale des cellules endométriales peut être à l’origine de pertes récurrentes de grossesses. LRH-1 est un récepteur nucléaire orphelin et un facteur de transcription régulant de nombreux évènements relatif à la reproduction et comme tout récepteur, son activation promouvoit l’activité transcriptionnelle de ses gènes cibles. Nous avons déjà montré que LRH-1 et son activité sont essentiels pour la décidualisation au niveau de l’utérus chez la souris et nous savons qu’il est présent dans l’utérus chez l’humain au moment de la phase de prolifération mais aussi de sécrétion du cycle menstruel, et que son expression augmente dans des conditions de décidualisation in vitro. Notre hypothèse est alors la suivante : LRH-1 est indispensable à la décidualisation du stroma endométrial, agissant par le biais de la régulation transcriptionnelle de gènes requis pour la transformation de cellules stromales en cellules déciduales. Afin d’explorer le mécanisme moléculaire impliqué dans la régulation transcriptionnelle effectuée par l’intermédiaire de ce récepteur, nous avons mis en place un modèle de décidualisation in vitro utilisant une lignée de cellules stromales de l’endomètre, cellules humaines et immortelles (hESC). Notre modèle de surexpression développé en transfectant les dites cellules avec un plasmide exprimant LRH-1, résulte en l’augmentation, d’un facteur 5, de l’abondance du transcriptome de gènes marqueurs de la décidualisation que sont la prolactine (PRL) et l’insulin-like growth factor binding protein-1 (IGFBP-1). En outre, la sous-régulation de ce récepteur par l’intermédiaire de petits ARN interférents (shRNA) abolit la réaction déciduale, d’un point de vue morphologique mais aussi en terme d’expression des deux gènes marqueurs cités ci-dessus. Une analyse par Chromatin ImmunoPrécipitation (ou ChIP) a démontré que LRH-1 se lie à des régions génomiques se trouvant en aval de certains gènes importants pour la décidualisation comme PRL, WNT 4, WNT 5, CDKN1A ou encore IL-24, et dans chacun de ces cas cités, cette capacité de liaison augmente dans le cadre de la décidualisation in vitro. Par ailleurs, des études structurelles ont identifié les phospholipides comme des ligands potentiels pour LRH-1. Nous avons donc choisi d’orienter notre travail de façon à explorer les effets sur les ligands liés à LRH-1 de traitements impliquant des agonistes et antagonistes à notre récepteur nucléaire. Les analyses par q-PCR et Western blot ont montré que la modulation de l’activité de LRH-1 par ses ligands influait aussi sur la réaction déciduale. Enfin, des études récentes de Salker et al (Salker, Teklenburg et al. 2010) ont mis en évidence que les cellules stromales humaines décidualisées sont de véritables biocapteurs de la qualité embryonnaire et qu’elles ont la capacité de migrer en direction de l’embryon. La série d’expériences que nous avons réalisée à l’aide de cellules hESC placées en co-culture avec des embryons de souris confirme que la migration cellulaire est bien dirigée vers les embryons. Cette propriété quant à l’orientation de la migration cellulaire est notoirement diminuée dans le cas où l’expression de LRH-1 est déplétée par shRNA dans les hESC. Nos données prouvent donc que LRH-1 régule non seulement la transcription d’un ensemble de gènes impliqués dans le processus de décidualisation mais agit aussi sur la motilité directionnelle de ces cellules hESC décidualisées in vitro.
The period of endometrial receptivity in humans coincides with the differentiation of endometrial stromal cells into highly specialized decidual cells through a process known as decidualization. This transformation of endometrial cells is abnormal in recurrent pregnancy loss patients. Liver homolog receptor 1 (LHR-1, NR5A2) is an orphan nuclear receptor and a transcription factor that regulates many reproductive events. The activation of this receptor leads to transcriptional activation of its target genes. We have previously shown that it is essential for decidualization in the mouse uterus. LRH-1 is expressed in the human uterus in both proliferative and secretory phases of the menstrual cycle and its expression increases during in vitro decidualization. We hypothesize that LRH-1 is indispensable for proper decidualization of the endometrial stroma, acting through the transcriptional regulation of genes required for transformation of stromal cells into decidual cells. To explore the molecular mechanism of transcriptional regulation mediated by this receptor, we established an in vitro model of decidualization, using an immortal human endometrial stromal cell line (hESC). An overexpression model developed by transfecting the cells with a plasmid constitutively expressing Lrh-1, resulted in 5 fold increases in abundance of transcripts for the decidualization marker genes prolactin (PRL) and insulin-like growth factor binding protein-1 (IGFBP-1). Furthermore, the downregulation of the receptor using short hairpin RNA (shRNA) abrogates the decidual reaction, from both a morphological point of view and in terms of expression of the two marker genes. Chromatin immunoprecipitation (ChIP) analysis showed that LRH-1 binds to genomic regions upstream of genes important for decidualization such as PRL, wingless-type MMTV integration site family, member 4 (WNT4), wingless-type MMTV integration site family, member 5 (WNT5), cyclin-dependent kinase inhibitor 1A (p21, CDKN1A) and interleukin-24(IL-24). For each of these genes, the binding increased during in vitro decidualization. Structural studies have identified phospholipids as potential LRH-1 ligands. We therefore explored the effect of ligand treatment on LRH-1 with an agonist and an inverse agonist for the nuclear receptor. Analysis by quantitative polymerase chain reaction (qPCR) and Western blot demonstrated that the modulation of LRH-1 activity by its ligands also affects the decidual reaction. Recent studies have shown that decidualized human stromal cells are biosensors of embryo quality and that they have the capacity to migrate towards the embryo. Our time-lapse evaluation of hESC cells co-cultured with mouse embryos indicates directed migration of the cells toward the embryo. This effect is markedly diminished when LRH-1 is depleted by shRNA in hESC. Our data provide evidence that LRH-1 regulates not only the transcription of a set of genes involved in decidualization but also the directional motility of these cells in vitro.

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.
The nuclear receptor Nr5a2, also known as liver receptor homolog-1 (Lrh-1), is expressed in the mouse ovary, exclusively in granulosa and luteal cells. Granulosa-specific disruption of Nr5a2 in mice from primary follicles onward in the follicle development trajectory has shown that Nr5a2 is a key regulator of ovulation and female fertility. We hypothesized that Nr5a2 modulates peri- and post-ovulatory events in a temporal sequence during folliculogenesis. To examine the role of Nr5a2 in ovulation and luteinization at different stages of the follicular development, we generated two Nr5a2 granulosa-specific knockout mice models: 1) Nr5a2Amhr2-/-, with Nr5a2 depletion from primary follicles forward; and 2) Nr5a2Cyp19-/-, with Nr5a2 depletion from the antral stage of development forward. The lack of Nr5a2 beginning in antral follicles resulted in infertility in Nr5a2Cyp19-/- females, with ovaries displaying non-functional luteal-like structures, synthesizing reduced progesterone levels and failing in supporting pseudopregnancy. Progesterone synthesis was affected by the lack of Nr5a2 through the downregulation of the cholesterol transport-related genes, Scarb1, StAR and Ldlr, as shown by qPCR. The cumulus-oocyte complexes of superstimulated Nr5a2Cyp19-/- immature females underwent expansion in vivo, but ovulation was disrupted, likely due to the downregulation of the progesterone receptor (Pgr) gene. An in vitro cumulus expansion assay showed defective cumulus expansion in Nr5a2Amhr2-/- associated with a dysregulation in the gap junction alpha-1 (Gja1; Cx43). In vitro cumulus expansion in Nr5a2Cyp19-/- was less affected than in Nr5a2Amhr2-/- cumulus-oocyte complexes. Data from qPCR showed a downregulation in the gene expression of Areg, Ereg, Btc and Tnfaip6 in both knockout ovarian cells at 2 h and 4 h post hCG. We found that 85% of the oocytes in both mutant genotypes can undergo germinal vesicle breakdown, confirming their capability to mature in vivo. Intracytoplasmic sperm injection (ICSI) showed the oocytes in both mutant models to be fertilizable and 70% of the resulting embryos proceeded to a blastocyst stage, independent of the genotype. In conclusion, Nr5a2 regulates female fertility along the entire process of the follicular development. Nr5a2 is shown to be essential for luteinization and its disruption in ovarian somatic cells does not compromise oocyte fertilizability. In overview, we provided a novel and comprehensive investigation, using multiple models and techniques to determine the mechanisms by which Nr5a2 regulates the important processes of cumulus expansion, ovulation and formation of the corpus luteum.

碩士
國立臺灣海洋大學
生物科技研究所
96
Black porgy, Acanthopagrus schlegeli, a marine protandrous hermaphrodite fish, is functionally male for the first and second year of life, but begins to sexually change to female after the third year. The objectives of the study were to investigate the expression of the sex-related genes. The major study was to investigate the interaction of nr5a1a (steroidogenic factor 1, sf-1) and nr0b1 (dosage sensitive sex-reversal, adrenal hypoplasia congenita critical region on the X-chromosome, gene 1, dax-1) in steroidogenic genes during sex development and sex change in black porgy. Quantification of nr5a1a, nr0b1 and steroidogenic genes transcripts were conducted by real-time PCR. nr5a1a transcripts were high in testicular tissue in 1+ to 2+-yr-old black porgy. nr0b1 transcripts were high during spermatogenesis in 1+ to 2-yr-old black porgy but were low in 2+-yr-old black porgy, and Nr0b1 was expressed in spermatogonia. Some of the steroidogenic genes( star, cyp11a1 and cyp11b2) transcripts were low in testicular tissue in 1+ to 2+-yr-old black porgy. nr5a1a and some of steroidogenic genes may be related to testicular tissue development and nr0b1 may be related to spermatogenesis. nr0b1 transcripts were high in ovarian tissue and Nr0b1 was expressed in oogonia. nr5a1a and some of steroidogenic genes transcripts were low in ovarian tissue. nr0b1 may be related to development of inactive ovarian tissue and its relation with nr5a1a and some of steroidogenic genes were negative. cyp19a1a transcripts were high in the ovarian tissue, it may be related with development of ovarian tissue.

碩士
高雄醫學大學
口腔衛生學系碩士班
105
Background: The out of control in cell proliferation was a hall marker of cancer. The activation of Hox transcript antisense intergenic RNA (HOTAIR) and Nuclear Receptor Subfamily 5 Group A Member 2 (NR5A2) genes played important roles of cancer cell proliferation, invasion, progression, and metastasis as well as poor survival in a variety of human cancers. In Taiwan, the incidence rate and mortality rate was still increased in past decade. However, the roles of HOTAIR and NR5A2 A>G polymorphisms in the development of oral cancer and oral potentially malignant disorder (OPMD), and prognosis of oral cancer patients remains unclear. Objective: Our aims were to investigate the association between polymorphisms in HOTAIR rs920778T>C, NR5A2 rs3790844A>G genes in OPMD development, OSCC development, malignant potential from OPMD to oral cancer, survival in oral cancer patients. Material and Methods: Patients with OPMD and OSCC were collected from Kaohsiung Medical University Hospital. Healthy controls were collected from people who were invited from Community Health examinations. After the inform consent was obtained. Potential confounding factors and demographic variables were collected using a standardized structure questionnaire and 8 ml of peripheral blood was collected for genomic DNA extraction. Genetic polymorphisms were determined using real-time PCR TaqMan probe method. SPSS statistical software version 19.0 was used to analyze the association. Result: More than 80% of the study subjects were already recruited in our previous studies. After the IRB was obtained, total of 267 oral cancer patients, 160 OPMD patients and 276 healthy controls were recruited into this study. The frequency distributions of NR5A2 rs3790844 polymorphisms was significant different between oral cancer group and OPMD group (χ2 = 5.37， p = 0.002) but no such association was found for HOTAIR rs920778 polymorphisms. Polymorphisms in HOTAIR genes were not associated with the development of oral cancer but NR5A2 polymorphisms were associated with OPMD turn into oral cancer. The logistic regression analysis only showed that NR5A2 polymorphisms were significantly associated with the OPMD turn into oral cancer (GG+GA vs.AA，odds ratio (OR) = 2.31，95%CI = 1.02-5.24) but no such association was found for HOTAIR rs920778 polymorphisms.. Survival curve analysis showed that HOTAIR rs920778 were not associated with the overall survival of oral cancer patients but NR5A2rs3790844 polymorphisms were (HOTAIR: log rank = 0.084，p = 0.772; NR5A2: log-rank = 3.887，p = 0.049). Disease free survival curve analysis showed that both HOTAIR rs920778 and NR5A2rs3790844 polymorphisms were not associated with the disease free survival of oral cancer patients (HOTAIR: log-rank = 0.309，p = 0.578; NR5A2: log-rank = 0.472，p = 0.492). However, multivariate Cox proportional hazard model analysis showed that HOTAIR rs920778 and NR5A2 rs3790844 polymorphisms were not associated with the low overall survival in oral cancer patients (HOTAIR: adjust hazard ratio (aHR) = 1.20，95%CI = 0.37-3.89；NR5A2:aHR = 2.05，95%CI = 0.98-4.29). According to backward stepwise analysis, HOTAIR rs920778 polymorphisms were not associated with the low overall survival in oral cancer patients but NR5A2 rs3790844 polymorphisms were (HOTAIR: aHR = 1.20，95%CI = 0.37-3.89；NR5A2: aHR = 2.05，95%CI = 0.98-4.29).Both HOTAIR rs920778 and NR5A2rs3790844 polymorphisms were not associated with low disease free survival in oral cancer patients (HOTAIR: aHR = 0.66，95%CI = 0.16-2.73；NR5A2: aHR = 0.78，95%CI = 0.28-2.18). Conclusion: These results suggested that NR5A2 rs3790844 polymorphisms can confer the genetic susceptibility to the high potential of malignant transformation from OPMD to oral cancer, but no such relationship was found for HOTAIR polymorphisms.