Mon projet de thèse s’inscrit dans le cadre des études visant à comprendre comment les cellules embryonnaires intègrent les différents signaux auxquels elles sont exposées pour s’engager dans une voie de différenciation définie. Il est plus particulièrement centré sur le rôle des protéines Smad dans ces processus et peut se diviser en deux axes de recherche. Le premier a trait au rôle de Mad (Smad1) dans les interactions entre signaux Wnt (Wg) et BMP chez la drosophile. Nous avons pu démontrer que la forme Mad non phosphorylée par le récepteur BMP se lie au complexe transcriptionnel ß-catenin/dTCF et est requise pour le signal Wnt canonique. La phosphorylation de Mad par le récepteur BMP dirige Mad vers la voie BMP, créant la possibilité d’une compétition entre ces deux classes de signaux. Le second axe de recherche concerne le facteur de transcription Smad4 qui est requis pour la transduction des signaux TGF-ß et BMP. J’ai pu identifier trois sites potentiels de phosphorylation par la kinase GSK3 dans la séquence primaire de Smad4. En utilisant de nombreuses techniques de biochimie, j’ai pu montrer que Smad4 est phosphorylé par la kinase Erk, puis par GSK-3 en réponse à un signal FGF. Lorsque Smad4 est doublement phosphorylé, il est reconnu par une E3-ligase, beta-TrCP, ce qui entraine sa polyubiquitination et sa dégradation. La voie Wnt étant capable d’inhiber GSK-3, j’ai pu montrer que Smad4 est stabilisé par des signaux Wnt. Ce mécanisme augmente la sensibilité des cellules aux signaux TGF-beta lorsqu’elles reçoivent également un signal Wnt. / During my PhD I studied how cells receive and integrate multiple signals from the extracellular milieu. I focused on Smad proteins and my project can be divided into two parts. My first project was centered on the transcription factor Mad (Smad1) and its requirement for the BMP and Wg pathways. Using a combination of genetic and biochemistry experiments, we showed that Mad is required for Wg signaling both in Tcf reporter gene assays and in vivo in Drosophila. We found that the choice for Mad to transduce Dpp or Wg signals is controlled by C-terminal phosphorylations so that Mad binds to Pangolin and participates in Wg target genes transcription only when not phosphorylated at its C-terminus. This results in a competition between Dpp and Wg controlled by the phosphorylation state of Mad. My second project was focused on the tumor suppressor Smad4. When I first joined the lab, I identified three new potential GSK3 phosphorylation sites in Smad4 primary sequence. I used a home-made phospho-specific antibody to demonstrate that FGF or EGF stimulation trigger Erk-mediated phosphorylation of Smad4 which primes subsequent GSK3 phosphorylations. These phosphorylations regulate a transcription activation domain located in Smad4 linker region and generate a Wnt-regulated phosphodegron recognized by the E3 ligase beta-TrCP. This mechanism provides a means of integrating distinct pathways which would otherwise remain insulated, allowing cells to sense FGF and Wnt inputs and adapt TGF-beta outcome to their context. It provides a molecular explanation of the long-standing mystery of the “competence modifier” effect of Wnt on Nodal signals discovered 20 years ago.

Le développement d’un biomatériau contrôlant précisément le comportement de cellules souches serait un avantage considérable pour des applications de médecine régénératrice et d’ingénierie tissulaire. Actuellement, le vieillissement de la population mondiale est associé à de nombreux problèmes de dégénérescence des tissus, en particulier dans le contexte osseux. Le coût annuel des opérations chirurgicales orthopédiques pratiquées aux États-Unis s’élève à 8.2 milliards de dollars US. Afin de trouver des alternatives aux greffes traditionnelles, des biomatériaux mimant la physiologie osseuse ont été conçus. Une des stratégies consiste à préparer des matériaux biomimétiques en fonctionnalisant, par exemple, la surface des matériaux par des peptides dérivés des protéines d’adhésion de la matrice extracellulaire (ECM) tel que la fibronectine pour favoriser l’ancrage des cellules osseuses. D’autres molécules comme les protéines morphogénétiques osseuses (BMPs) jouent un rôle essentiel dans le processus physiologique de la réparation osseuse. Ces BMPs peuvent être combinées aux biomatériaux afin d’orienter la réponse des cellules osseuses et ainsi améliorer la régénération des tissus. L’utilisation de peptides dérivés des BMPs, 300 fois moins dispendieux, ajoute un avantage considérable dans le développement des matériaux biomimétiques. En outre, des relations croisées entre les récepteurs cellulaires des protéines de l’ECM et ceux des BMPs peuvent influencer la signalisation et la différenciation des cellules osseuses, d’où l’intérêt de fonctionnaliser les biomatériaux non seulement avec des molécules d’adhésion, mais aussi avec des BMPs ou leurs peptides dérivés. Dans ce projet, un biomatériau biomimétique de 3eme génération a été développé afin de permettre l’adhésion et l’orientation des cellules souches vers la lignée ostéoblastique. L’étude a consisté à analyser le potentiel de la co-immobilisation d’un peptide d’adhésion dérivé de la fibronectine (pFibro) et d’un peptide dérivé de la BMP-9 (SpBMP-9) sur un film de polycaprolactone (PCL). L’utilisation d’un peptide négatif du SpBMP-9, le NSpBMP-9, a permis de vérifier les effets synergiques potentiels de cette co-immobilisation. Dans un premier temps, l’attachement et l’organisation du cytosquelette des cellules ont été déterminés par un immunomarquage des protéines du cytosquelette. Puis la cinétique d’activation de la voie de signalisation Smad impliquée dans la différenciation ostéoblastique a été analysée par immunobuvardage de type Western. Afin de vérifier la différenciation des cellules souches vers un phénotype défini, l’expression de gènes codant pour des marqueurs de différenciation ostéogénique (Runx2, Ostérix), chondrogénique (Sox9) et adipogénique (PPARγ) a également été évaluée par des immunobuvardages de type Western. Ce projet de recherche a amélioré les connaissances actuelles sur les interactions entre les biomatériaux co-fonctionnalisés par des peptides d’adhésion et dérivés de la BMP-9 et les cellules osseuses, afin de potentialiser les propriétés ontéoconductive, ostéointégrative et ostéoinductive essentielles pour obtenir un matériau biomimétique efficace.

BACKGROUND: Calcification is a natural process of bone formation or osteogenesis. However, calcium is able to be deposited abnormally in soft tissues such as the aorta, adipose tissue and liver, causing these to harden. Abnormal calcification in arteries is a common factor contributing to high blood pressure and, further, many severe cardiovascular diseases such as atherosclerosis and coronary disease. In liver and adipose tissue, calcification always takes place accompanied by excess extracellular matrix (ECM) accumulation which is called fibrosis, contributing to cirrhosis and metabolic disorders including insulin resistance. In addition, it is documented that severe calcification in adipose tissues is able to cause damage to the micro-vascular system, and calcification in perivascular adipose tissue (PVAT) is a key effector of arterial stiffness. Dystrophic calcification, one of the most common types of abnormal calcification, usually occurs as a reaction to tissue damage such as obesity-induced inflammation. Increasing numbers of studies indicate that abnormal calcification is the result of re-differentiation towards osteogenesis which occurs in the nascent resident cells under the stimulation of multiple factors. The BMP/Smad signaling pathway is commonly known to participate in bone formation and is implicated in mineralization as well as local induction of inflammation. Importantly, BMP/Smad signaling as an inducer of the osteochondrogenic phenotype in vascular calcification is fully appreciated. However, the molecular events of dystrophic calcification triggered by obesity-induced chronic inflammation still remain unclear. Our previous studies have identified that imbalance with increased activity of neutrophil elastase (NE), a Ser protease mainly released by neutrophils during inflammation, and decreased serum levels of the NE inhibitor α1-antitrypsin A1AT, contributes to the development of obesity-related metabolic complications including insulin resistance, fatty liver and chronic inflammation. This study explored the effects of NE on abnormal calcification in soft tissues, which may be mediated by BMP/Smad signaling pathway, and, furthermore, the molecular mechanism by which NE activates the BMP/Smad signaling pathway. METHODS: Wild-type mice were fed with either a high-fat high-fructose diet (HFHFD), a high-fat diet (HFD) alone or a normal chow diet (NCD), and NE-knockdown mice were fed with a HFHFD. Adipose tissue and liver were extracted from all mice. H&E staining and immunofluorescence staining (IF) detected the inflammation condition. Alizarin staining and von kossa staining were used to detect calcium deposits. 3,3′-Diaminobenzidine (DAB) staining was used to examine active phospho-Smad1/5 signaling. Regarding nascent resident cells which have potential ability of osteogenic re-differentiation, 3t3l1 fibroblast and human hepatic stellate cell (hHSC) were cultured in dishes and 6-well plates with coverslips. In our previous research, mouse aortic smooth muscle cells (mASMC) seeded in 6-well plates grew in an osteogenic medium (10mM β-glycerophosphate and 10mM Calcium chloride) in the presence or absence of NE (10nM). Calcium deposits were detected by Alizarin staining. 3t3l1 and hHSC was treated with NE (20nM, 30nM, 40nM), BMP2, TGFβ1 or NE combined with BMP2, TGFβ1 or NE inhibitor GW311616A (Axon). Further, we used specific chemical inhibitors, LDN-193189, BMP-ALK2/3 inhibitor, SB525334, TGFβ-ALK5 inhibitor, and I-191, PAR2 antagonist to investigate the molecular mechanism of NE’s effects on Smad signaling pathways. Cells in dishes were harvested, and the proteins were measured by western blot. Coverslips in 6-well plates were used for immunofluorescence. RESULTS: The most severe calcification was found in the adipose tissue of HFHFD fed wild-type mice and moderate calcification took place in the HFD mouse group while NCD mice rarely had calcium deposits. NE-knockdown significantly prevented calcium deposits in adipose tissue compared with HFHFD wild-type mice. Consistently, we found increased phospho-Smad1/5 (p-Smad1/5) signaling in the adipose tissues of mice on the HFHFD and HFD mice while p-Smad1/5 was prevented in the NE-knockout group. Furthermore, NE enhanced calcium deposits in mASMC cultured in osteogenic medium. NE significantly activated p-Smad1/5 signaling in hHSC in the dose-effect relationship and contributes to an additive effect on p-Smad1/5 in the presence of BMP2 or TGFβ1. Although p-Smad1/5 was only slightly aroused by NE in 3t3l1 fibroblast, NE was able to promote p-Smad1/5 activation tremendously and specifically in the presence of BMP2 or TGFβ1 but not p-Smad2/3 which is the main downstream signaling of TGFβ1. Chemical inhibition of ALK2/3, not ALK5 or PAR2, was able to completely block NE’s effects in hSHC on p-Smad1/5 activation. In addition, the cleavage of osteoblast-cadherin or CDH11 (OB-cadherin) was observed in hHSC, which may indicate a lower beta-catenin abundance in the hHSC cells which were treated with NE. CONCLUSION: NE has the potential to contribute to abnormal calcification in soft tissues including the liver, adipose tissue and aorta via activating canonical ALK2/3-BMP-Smad1/5 signaling pathway in the mesenchymal stem cell/MSC-lineage cells. In addition, NE is likely to break cell-cell adhesion which may contribute to cell proliferation and re-differentiation towards osteogenesis and fibrosis. / 2024-01-28T00:00:00Z

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19396号 / 医博第4047号 / 新制||医||1012(附属図書館) / 32421 / 京都大学大学院医学研究科医学専攻 / (主査)教授 長船 健二, 教授 野田 亮, 教授 萩原 正敏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DGAM

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A identidade na segmentação do corpo de diversos organismos, durante o desenvolvimento, é devida, em grande parte, à ação das proteínas homeóticas. Em especial, dois grupos de proteínas, Trithorax (trxG) e Polycomb (PcG) têm um papel fundamental na manutenção, respectivamente, da ativação e da repressão da transcrição gênica, associando-se à cromatina. A importância das PcG nos estimulou a buscar a caracterização das proteínas humanas ortólogas ao \"Enhancer of Polycomb\" (Epc) de Drosophila, até então não descritas no genoma humano. Para tanto, buscamos: - obter a sequência completa e mapear o cDNA do novo gene humano homólogo ao \"Enhancer of Polycomb\" de Drosophila; - analisar sua expressão em tecidos fetais, adultos e tumorais e fazer estudos buscando sua caracterização funcional. Encontramos, mapeamos e obtivemos a seqüência completa de dois genes humanos, ortólogos de Epc1 (10p11-22) e de Epc2 (2q21-23) de camundongo, publicando estes dados em 2001 (Camargo et al., 2001). Ambos os genes são bastante conservados entre várias espécies, sendo que o cDNA de hEPC2 humano, por exemplo, é 94% idêntico ao Epc2 de camundongo e possui 96% de identidade ao nível de proteína, sugerindo que a função do gene deve ter sido mantida durante a evolução. No entanto, as seqüências protéicas de hEPC1 e hEPC2 humanos possuem apenas 68% de identidade entre si. Portanto, é provável que após a duplicação dos parálogos, estes tenham divergido funcionalmente. A expressão de ambos os genes foi avaliada utilizando \"dot-blots\" contendo 76 mRNAs de amostras de tecidos fetais, adultos e tumorais, mostrando-se fraca e ubíqua. Análises in silico sugeriram a existência de 4 isoformas de splicing para hEPC2, as quais foram validadas por RT-PCR ou \"Northern blots\". Uma das isoformas (de 2.7 Kpb) se mostrou mais abundante em todas as linhagens tumorais estudadas através de análises de \"Northern blot\", principalmente nas linhagens de linfoma de Burkitt\'s Raji e na linhagem de leucemia pró-mielocítica HL-60. Esta isoforma é gerada através de um sítio alternativo de poli-adenilação, que reduz sua porção 3\'UTR, retirando 4 dos 5 \"elementos ricos em adenilatos e uridilatos\" (AREs), envolvidos com a degradação de mRNAs lábeis que codificam proteínas regulatórias. Estes resultados se encontram em um manuscrito recentemente submetido à publicação (anexo à tese). Interação entre hEPC2 e SMADs e sua modulação por TGF-β. Durante a montagem da seqüência completa de hEPC2, verificamos que duas ESTs patenteadas mostravam alta identidade com o gene. Estas seqüências foram descritas como sendo parte de uma nova proteína de interação com as proteínas da família SMAD, envolvidas com transdução de sinais desencadeados por TGF-β. Esta citocina por sua vez, regula a proliferação, diferenciação e morte celular. Partimos para a avaliação da possível interação entre hEPC2 e as SMADs, em colaboração com o grupo do Dr. Aristidis Moustakas, do Ludwig Institute for Cancer Research de Uppsala, Suécia. Os resultados de co-imunoprecipitação sugeriram que as SMADs 2, 3, 4, 7 e 8 interagem com hEPC2, sendo que a interação entre as SMAD2, SMAD3, SMAD4 e hEPC2 nas células tratadas com TGF-β1, mostraram uma redução na co-imunoprecipitação. Este resultado sugere que TGF-β1 modula negativamente a interação entre essas proteínas. Da mesma maneira, foi observada uma redução na interação de hEPC2 com SMAD8 após o tratamento com BMP-7. Esse resultado é ainda mais destacado para as SMADs 2 e 3. Estes dados foram observados para ambas as construções de hEPC2, o que sugere fortemente a veracidade da interação entre estas proteínas. A localização celular de hEPC2, e também sua co-localização com SMAD2 foram investigadas através de imunofluorescência indireta e confirmaram a predição do programa PSORTII, de que hEPC2 se localiza no núcleo. No entanto, não foi possível observar a co-localização entre hEPC2 e SMAD2. É possível que hEPC2 não se ligue diretamente ao DNA, necessitando se associar como parceiro de um fator de transcrição. Esta foi uma das hipóteses para a atuação de hEPC2, como um co-fator que se associe com uma das SMADs e se ligue a um elemento específico de ligação a SMAD (SBE). Para investigar essa hipótese um ensaio de gene repórter foi feito utilizando uma construção de um repórter contendo 12 repetições da seqüência CAGA (seqüência específica de ligação das SMADs 2,3 e 4) fusionado com o gene da luciferase. No entanto, este ensaio não demonstrou que a transcrição de SMAD2 é dependente de hEPC2 e o experimento deverá ser repetido. Para confirmar a interação entre hEPC2 e as SMADs, será feito um experimento de \"pull-down\". Para tal o cDNA de hEPC2 foi clonado no vetor pET-32A de expressão indutível em bactérias. A proteína recombinante já foi produzida, tendo sido induzida e posteriormente purificada em condições desnaturantes. Apesar de dezenas de genes PcG terem sido caracterizados em Drosophila, poucos destes genes foram estudados em mamíferos. Portanto, a descrição do gene hEPC2 e seus transcritos alternativos, contribui para o conhecimento de PcG humanos, indicando a associação de maior expressão de uma de suas isoformas em linhagens celulares tumorais. Em relação à interação de hEPC2 com as SMADs, é interessante observar que nenhuma outra proteína foi descrita por possuir a particularidade de interagir com as SMADs de diferentes categorias. Talvez este seja um dado importante, que indique o papel singular de hEPC2 na sinalização de TGF-β1. / The identity of body segmentation in several organisms during development is, to a large extent, due to the action of the homeotic proteins. In particular, two groups of proteins, the Trithorax (trxG) and Polycomb (PcG), have a major role in maintenance of respectively, transcription activation and repression, when associated to the chromatin. The importance of PcGs has motivated us to pursue the isolation and characterization of two new human proteins that are orthologs of the \"Enhancer of Polycomb\" (Epc) of Drosophila. To achieve this goal we undertook the task of the cloning and mapping of complete cDNA sequence of the novel genes hEPC1 and hEPC2, analyzing its expression in fetal, adult and tumoral tissues and functionally characterizing the hEPC2 protein. In 2001, we published the mapping and cloning of the complete cDNA sequences of both genes, as being orthologs of the mouse Epc1 (10p11-22) and Epc2 (2q21-23), together with the strategy used to obtain the full-length cDNAs (Camargo et al., 2001). Both genes are shown to be highly conserved among several species. Thus, the human hEPC2 cDNA is 94% identical to the mouse Epc2 and displays 96% identity at the protein level, suggesting maintenance of its function during the evolution. However, the protein sequences of the human hEPC1 and hEPC2 display only 68% identity. Therefore, it is likely that they have undergone a functional divergence after their duplication. The expression of both genes was evaluated using \"dot-blots\" containing 76 mRNAs samples from fetal, adult and tumoral tissues and is shown to be weak and ubiquitous. \"In silico\" analysis suggested the existence of 4 hEPC2 splicing isoforms that were validated by RT-PCR and/or Northern-blots. One of the isoforms (of 2.7 Kbp) is shown to be more abundant in all of the tumoral cell lines evaluated using Northern-blot analysis, mainly in the Burkit\'s Raji lymphoma and in the promyelocytic leukemia HL-60. This isoform results from the use of an alternative polyadenylation site that reduces the 3\'UTR, abolishing 4 of 5 \"adenylates and urilates rich elements\" (AREs), involved in the degradation of labile mRNAs that codify to regulatory proteins. These results have been recently submitted to publication (manuscript attached to this thesis). Interaction between the hEPC2/SMADs and its modulation by TGF-β. During the assembly of the hEPC2 full-length cDNA sequence, we found two patented ESTs that tagged a portion of the gene. These sequences were described as partial sequences of a \"new SMAD interacting protein\", involved in signal transduction of TGF-β, a cytokine that regulates cell proliferation, differentiation and death. To evaluate this putative interaction between hEPC2 and the SMADs proteins, we begun a collaboration with the TGF-β signalling group of the Dr. Aristidis Moustakas, from the Uppsala Ludwig Institute for Cancer Research, Sweden. The results of co-imunoprecipitation assays suggested that SMADs 2, 3, 4, 7 e 8 interact with hEPC2. Moreover, the interaction among SMAD2, SMAD3, SMAD4 and hEPC2 in cells treated with TGF-β1 showed decreased co-imunoprecipitation. This result suggests that TGF-β1 negatively modulates the interaction of these proteins. Likewise, we observed a reduction in hEPC2 interaction with SMAD8 upon BMP-7 treatment. This effect was even more dramatic for SMADs 2 and 3. These data were observed for both hEPC2 plasmid constructs, which strongly suggest the veracity of these proteins interaction. The cell localization of the hEPC2 protein, as well as its co-localization with the SMAD2, were investigated through indirect immunofluorescence assay, confirming the predicted localization of hEPC2 in the cell nucleus using the PSORTII program. However, we were not able to confirm the co-localization of hEPC2 and SMAD2. It is possible that hEPC2 does not bind directly to the DNA, requiring an association with a partner such as a transcription factor. This raises the hypothesis of hEPC2 having a role as a co-factor associated to one of the SMADs and binding to a \"SMAD binding element\" (SBE). To investigate this hypothesis, gene reporter assays were undertaken using a reporter construct containing 12 CAGA sequence repetitions (specific binding sequence of the SMADs 2, 3 and 4) fused to the luciferase gene. However, this assay could not demonstrate that the transcription of the SMAD is dependent on hEPC2. This experiment must be repeated. To confirm the interaction of hEPC2 and SMADs, a pull-down assay will be performed. To this end, the coding region of hEPC2 was cloned into the pET-32A bacterial inducible expression vector. The recombinant protein was already produced, having been induced and purified under denaturing conditions. Despite the dozens of PcG genes that were described in Drosophila, only a few of these genes have been characterized in mammals. Therefore, the description of the hEPC2 and its alternative transcripts is a contribution to better knowledge of the human PcGs. Regarding the hEPC2 and SMADs interaction, it\'s it is noteworthy that this is the first protein described to interact with SMADs of distinct categories. This may be an important indication of a unique role for hEPC2 in the TGF-β1 signaling pathway.

No description available.

A identidade na segmentação do corpo de diversos organismos, durante o desenvolvimento, é devida, em grande parte, à ação das proteínas homeóticas. Em especial, dois grupos de proteínas, Trithorax (trxG) e Polycomb (PcG) têm um papel fundamental na manutenção, respectivamente, da ativação e da repressão da transcrição gênica, associando-se à cromatina. A importância das PcG nos estimulou a buscar a caracterização das proteínas humanas ortólogas ao \"Enhancer of Polycomb\" (Epc) de Drosophila, até então não descritas no genoma humano. Para tanto, buscamos: - obter a sequência completa e mapear o cDNA do novo gene humano homólogo ao \"Enhancer of Polycomb\" de Drosophila; - analisar sua expressão em tecidos fetais, adultos e tumorais e fazer estudos buscando sua caracterização funcional. Encontramos, mapeamos e obtivemos a seqüência completa de dois genes humanos, ortólogos de Epc1 (10p11-22) e de Epc2 (2q21-23) de camundongo, publicando estes dados em 2001 (Camargo et al., 2001). Ambos os genes são bastante conservados entre várias espécies, sendo que o cDNA de hEPC2 humano, por exemplo, é 94% idêntico ao Epc2 de camundongo e possui 96% de identidade ao nível de proteína, sugerindo que a função do gene deve ter sido mantida durante a evolução. No entanto, as seqüências protéicas de hEPC1 e hEPC2 humanos possuem apenas 68% de identidade entre si. Portanto, é provável que após a duplicação dos parálogos, estes tenham divergido funcionalmente. A expressão de ambos os genes foi avaliada utilizando \"dot-blots\" contendo 76 mRNAs de amostras de tecidos fetais, adultos e tumorais, mostrando-se fraca e ubíqua. Análises in silico sugeriram a existência de 4 isoformas de splicing para hEPC2, as quais foram validadas por RT-PCR ou \"Northern blots\". Uma das isoformas (de 2.7 Kpb) se mostrou mais abundante em todas as linhagens tumorais estudadas através de análises de \"Northern blot\", principalmente nas linhagens de linfoma de Burkitt\'s Raji e na linhagem de leucemia pró-mielocítica HL-60. Esta isoforma é gerada através de um sítio alternativo de poli-adenilação, que reduz sua porção 3\'UTR, retirando 4 dos 5 \"elementos ricos em adenilatos e uridilatos\" (AREs), envolvidos com a degradação de mRNAs lábeis que codificam proteínas regulatórias. Estes resultados se encontram em um manuscrito recentemente submetido à publicação (anexo à tese). Interação entre hEPC2 e SMADs e sua modulação por TGF-β. Durante a montagem da seqüência completa de hEPC2, verificamos que duas ESTs patenteadas mostravam alta identidade com o gene. Estas seqüências foram descritas como sendo parte de uma nova proteína de interação com as proteínas da família SMAD, envolvidas com transdução de sinais desencadeados por TGF-β. Esta citocina por sua vez, regula a proliferação, diferenciação e morte celular. Partimos para a avaliação da possível interação entre hEPC2 e as SMADs, em colaboração com o grupo do Dr. Aristidis Moustakas, do Ludwig Institute for Cancer Research de Uppsala, Suécia. Os resultados de co-imunoprecipitação sugeriram que as SMADs 2, 3, 4, 7 e 8 interagem com hEPC2, sendo que a interação entre as SMAD2, SMAD3, SMAD4 e hEPC2 nas células tratadas com TGF-β1, mostraram uma redução na co-imunoprecipitação. Este resultado sugere que TGF-β1 modula negativamente a interação entre essas proteínas. Da mesma maneira, foi observada uma redução na interação de hEPC2 com SMAD8 após o tratamento com BMP-7. Esse resultado é ainda mais destacado para as SMADs 2 e 3. Estes dados foram observados para ambas as construções de hEPC2, o que sugere fortemente a veracidade da interação entre estas proteínas. A localização celular de hEPC2, e também sua co-localização com SMAD2 foram investigadas através de imunofluorescência indireta e confirmaram a predição do programa PSORTII, de que hEPC2 se localiza no núcleo. No entanto, não foi possível observar a co-localização entre hEPC2 e SMAD2. É possível que hEPC2 não se ligue diretamente ao DNA, necessitando se associar como parceiro de um fator de transcrição. Esta foi uma das hipóteses para a atuação de hEPC2, como um co-fator que se associe com uma das SMADs e se ligue a um elemento específico de ligação a SMAD (SBE). Para investigar essa hipótese um ensaio de gene repórter foi feito utilizando uma construção de um repórter contendo 12 repetições da seqüência CAGA (seqüência específica de ligação das SMADs 2,3 e 4) fusionado com o gene da luciferase. No entanto, este ensaio não demonstrou que a transcrição de SMAD2 é dependente de hEPC2 e o experimento deverá ser repetido. Para confirmar a interação entre hEPC2 e as SMADs, será feito um experimento de \"pull-down\". Para tal o cDNA de hEPC2 foi clonado no vetor pET-32A de expressão indutível em bactérias. A proteína recombinante já foi produzida, tendo sido induzida e posteriormente purificada em condições desnaturantes. Apesar de dezenas de genes PcG terem sido caracterizados em Drosophila, poucos destes genes foram estudados em mamíferos. Portanto, a descrição do gene hEPC2 e seus transcritos alternativos, contribui para o conhecimento de PcG humanos, indicando a associação de maior expressão de uma de suas isoformas em linhagens celulares tumorais. Em relação à interação de hEPC2 com as SMADs, é interessante observar que nenhuma outra proteína foi descrita por possuir a particularidade de interagir com as SMADs de diferentes categorias. Talvez este seja um dado importante, que indique o papel singular de hEPC2 na sinalização de TGF-β1. / The identity of body segmentation in several organisms during development is, to a large extent, due to the action of the homeotic proteins. In particular, two groups of proteins, the Trithorax (trxG) and Polycomb (PcG), have a major role in maintenance of respectively, transcription activation and repression, when associated to the chromatin. The importance of PcGs has motivated us to pursue the isolation and characterization of two new human proteins that are orthologs of the \"Enhancer of Polycomb\" (Epc) of Drosophila. To achieve this goal we undertook the task of the cloning and mapping of complete cDNA sequence of the novel genes hEPC1 and hEPC2, analyzing its expression in fetal, adult and tumoral tissues and functionally characterizing the hEPC2 protein. In 2001, we published the mapping and cloning of the complete cDNA sequences of both genes, as being orthologs of the mouse Epc1 (10p11-22) and Epc2 (2q21-23), together with the strategy used to obtain the full-length cDNAs (Camargo et al., 2001). Both genes are shown to be highly conserved among several species. Thus, the human hEPC2 cDNA is 94% identical to the mouse Epc2 and displays 96% identity at the protein level, suggesting maintenance of its function during the evolution. However, the protein sequences of the human hEPC1 and hEPC2 display only 68% identity. Therefore, it is likely that they have undergone a functional divergence after their duplication. The expression of both genes was evaluated using \"dot-blots\" containing 76 mRNAs samples from fetal, adult and tumoral tissues and is shown to be weak and ubiquitous. \"In silico\" analysis suggested the existence of 4 hEPC2 splicing isoforms that were validated by RT-PCR and/or Northern-blots. One of the isoforms (of 2.7 Kbp) is shown to be more abundant in all of the tumoral cell lines evaluated using Northern-blot analysis, mainly in the Burkit\'s Raji lymphoma and in the promyelocytic leukemia HL-60. This isoform results from the use of an alternative polyadenylation site that reduces the 3\'UTR, abolishing 4 of 5 \"adenylates and urilates rich elements\" (AREs), involved in the degradation of labile mRNAs that codify to regulatory proteins. These results have been recently submitted to publication (manuscript attached to this thesis). Interaction between the hEPC2/SMADs and its modulation by TGF-β. During the assembly of the hEPC2 full-length cDNA sequence, we found two patented ESTs that tagged a portion of the gene. These sequences were described as partial sequences of a \"new SMAD interacting protein\", involved in signal transduction of TGF-β, a cytokine that regulates cell proliferation, differentiation and death. To evaluate this putative interaction between hEPC2 and the SMADs proteins, we begun a collaboration with the TGF-β signalling group of the Dr. Aristidis Moustakas, from the Uppsala Ludwig Institute for Cancer Research, Sweden. The results of co-imunoprecipitation assays suggested that SMADs 2, 3, 4, 7 e 8 interact with hEPC2. Moreover, the interaction among SMAD2, SMAD3, SMAD4 and hEPC2 in cells treated with TGF-β1 showed decreased co-imunoprecipitation. This result suggests that TGF-β1 negatively modulates the interaction of these proteins. Likewise, we observed a reduction in hEPC2 interaction with SMAD8 upon BMP-7 treatment. This effect was even more dramatic for SMADs 2 and 3. These data were observed for both hEPC2 plasmid constructs, which strongly suggest the veracity of these proteins interaction. The cell localization of the hEPC2 protein, as well as its co-localization with the SMAD2, were investigated through indirect immunofluorescence assay, confirming the predicted localization of hEPC2 in the cell nucleus using the PSORTII program. However, we were not able to confirm the co-localization of hEPC2 and SMAD2. It is possible that hEPC2 does not bind directly to the DNA, requiring an association with a partner such as a transcription factor. This raises the hypothesis of hEPC2 having a role as a co-factor associated to one of the SMADs and binding to a \"SMAD binding element\" (SBE). To investigate this hypothesis, gene reporter assays were undertaken using a reporter construct containing 12 CAGA sequence repetitions (specific binding sequence of the SMADs 2, 3 and 4) fused to the luciferase gene. However, this assay could not demonstrate that the transcription of the SMAD is dependent on hEPC2. This experiment must be repeated. To confirm the interaction of hEPC2 and SMADs, a pull-down assay will be performed. To this end, the coding region of hEPC2 was cloned into the pET-32A bacterial inducible expression vector. The recombinant protein was already produced, having been induced and purified under denaturing conditions. Despite the dozens of PcG genes that were described in Drosophila, only a few of these genes have been characterized in mammals. Therefore, the description of the hEPC2 and its alternative transcripts is a contribution to better knowledge of the human PcGs. Regarding the hEPC2 and SMADs interaction, it\'s it is noteworthy that this is the first protein described to interact with SMADs of distinct categories. This may be an important indication of a unique role for hEPC2 in the TGF-β1 signaling pathway.