RESUMO
Transforming growth factor ß (TGFß) is widely recognised as an important factor that regulates many steps of normal mammary gland (MG) development, including branching morphogenesis, functional differentiation and involution. Tif1γ has previously been reported to temporally and spatially control TGFß signalling during early vertebrate development by exerting negative effects over SMAD4 availability. To evaluate the contribution of Tif1 γ to MG development, we developed a Cre/LoxP system to specifically invalidate the Tif1g gene in mammary epithelial cells in vivo. Tif1g-null mammary gland development appeared to be normal and no defects were observed during the lifespan of virgin mice. However, a lactation defect was observed in mammary glands of Tif1g-null mice. We demonstrate that Tif1 γ is essential for the terminal differentiation of alveolar epithelial cells at the end of pregnancy and to ensure lactation. Tif1 γ appears to play a crucial role in the crosstalk between TGFß and prolactin pathways by negatively regulating both PRL receptor expression and STAT5 phosphorylation, thereby impairing the subsequent transactivation of PRL target genes. Using HC11 cells as a model, we demonstrate that the effects of Tif1g knockdown on lactation depend on both SMAD4 and TGFß. Interestingly, we found that the Tif1γ expression pattern in mammary epithelial cells is almost symmetrically opposite to that described for TGFß. We propose that Tif1γ contributes to the repression of TGFß activity during late pregnancy and prevents lactation by inhibiting SMAD4.
Assuntos
Diferenciação Celular/genética , Células Epiteliais/citologia , Lactação/genética , Glândulas Mamárias Animais/citologia , Proteína Smad4/antagonistas & inibidores , Fatores de Transcrição/fisiologia , Animais , Células Epiteliais/fisiologia , Feminino , Masculino , Glândulas Mamárias Animais/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Gravidez , Transdução de Sinais/genética , Proteína Smad4/fisiologiaRESUMO
TIF1γ, a new regulator of TGFß signaling, inhibits the Smad4-mediated TGFß response by interaction with Smad2/3 or ubiquitylation of Smad4. We have shown that TIF1γ participates in TGFß signaling as a negative regulator of Smad4 during the TGFß-induced epithelial-to-mesenchymal transition (EMT) in mammary epithelial cells, and during terminal differentiation of mammary alveolar epithelial cells and lactation. We demonstrate here that TIF1γ is sumoylated and interacts with Ubc9, the only known SUMO-conjugating enzyme. Four functional sumoylation sites lie within the middle domain of TIF1γ, the Smad interaction domain. We show that a sumoylation-defective TIF1γ mutant significantly reduces TIF1γ inhibition of Smad complexes and that of the Smad-mediated TGFß transcriptional response. Moreover, chromatin immunoprecipitation experiments indicate that TIF1γ sumoylation is required to limit Smad4 binding on the PAI-1 TGFß target gene promoter. Ectopic expression of TIF1γ in mammary epithelial cells inhibits TGFß-induced EMT, an effect relieved by expression of non-sumoylated TIF1γ. Taken together, our results identify a new TGFß regulatory layer, whereby sumoylation strengthens the TIF1γ repressive action on canonical TGFß signaling.
Assuntos
Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Sequência de Aminoácidos , Animais , Diferenciação Celular/fisiologia , Humanos , Dados de Sequência Molecular , Inibidor 1 de Ativador de Plasminogênio/genética , Regiões Promotoras Genéticas , Transdução de Sinais , Proteínas Smad/genética , Proteínas Smad/metabolismo , Sumoilação , TransfecçãoRESUMO
TGF-ß is a potent inducer of epithelial-to-mesenchymal transition (EMT), a process involved in tumour invasion. TIF1γ participates in TGF-ß signalling. To understand the role of TIF1γ in TGF-ß signalling and its requirement for EMT, we analysed the TGF-ß1 response of human mammary epithelial cell lines. A strong EMT increase was observed in TIF1γ-silenced cells after TGF-ß1 treatment, whereas Smad4 inactivation completely blocked this process. Accordingly, the functions of several TIF1γ target genes can be linked to EMT, as shown by microarray analysis. As a negative regulator of Smad4, TIF1γ could be crucial for the regulation of TGF-ß signalling. Furthermore, TIF1γ binds to and represses the plasminogen activator inhibitor 1 promoter, demonstrating a direct role of TIF1γ in TGF-ß-dependent gene expression. This study shows the molecular relationship between TIF1γ and Smad4 in TGF-ß signalling and EMT.
Assuntos
Células Epiteliais/metabolismo , Glândulas Mamárias Humanas/metabolismo , Proteína Smad4/metabolismo , Fatores de Transcrição/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Células Epiteliais/citologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Humanos , Glândulas Mamárias Humanas/citologia , Proteína Smad4/genética , Fatores de Transcrição/genética , Fatores de Transcrição/farmacologia , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genética , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Eukaryotic ribosome biogenesis requires the function of a large number of trans-acting factors which interact transiently with the nascent pre-rRNA and dissociate as the ribosomal subunits proceed to maturation and export to the cytoplasm. Loss-of-function mutations in human trans-acting factors or ribosome components may lead to genetic syndromes. In a previous study, we have shown association between the SBDS (Shwachman-Bodian-Diamond syndrome) and NIP7 proteins and that downregulation of SBDS in HEK293 affects gene expression at the transcriptional and translational levels. In this study, we show that downregulation of NIP7 affects pre-rRNA processing, causing an imbalance of the 40S/60S subunit ratio. We also identified defects at the pre-rRNA processing level with a decrease of the 34S pre-rRNA concentration and an increase of the 26S and 21S pre-rRNA concentrations, indicating that processing at site 2 is particularly slower in NIP7-depleted cells and showing that NIP7 is required for maturation of the 18S rRNA. The NIP7 protein is restricted to the nuclear compartment and co-sediments with complexes with molecular masses in the range of 40S-80S, suggesting an association to nucleolar pre-ribosomal particles. Downregulation of NIP7 affects cell proliferation, consistently with an important role for NIP7 in rRNA biosynthesis in human cells.
Assuntos
Proteínas Nucleares/fisiologia , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Ribossômico/metabolismo , Linhagem Celular , Estruturas do Núcleo Celular/química , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Poli A-U/metabolismo , Poli U/metabolismo , Polirribossomos/química , RNA/química , RNA/metabolismo , Precursores de RNA/química , RNA Ribossômico/químicaRESUMO
The participation of BRCA1 (breast cancer 1) in DNA repair is well established, especially in mammary and ovarian cells. Our purpose was to develop a new in vivo radio-sensitizing therapy for melanoma. We therefore investigated the effect of downregulation of BRCA1 on irradiated melanoma cells using an anti-BRCA1 ribozyme. Our results show that BRCA1 downregulation increased radio-sensitivity of the A375 cell line, suggesting that BRCA1 could act as a caretaker in melanoma; however, as BRCA1 functions are not limited to maintaining genomic integrity but also regulate transcription and the cell cycle, we confirmed that the proliferative rate of BRCA1 downregulated clones did not change. We also demonstrate that: (1) among the major pro-angiogenic genes, FGF-2 was not increased before or after irradiation and vascular endothelial growth factor strongly inhibited after irradiation; (2) expression of two important metalloproteinases, matrix metalloproteinase 2 and 9, involved in melanoma metastasis were decreased before and after irradiation; (3) expression of their major inhibitor, tissue inhibitor of metalloproteinase, was mainly upregulated; and (4) that invasion of BRCA1 downregulated cells was modified. Together these data suggest that BRCA1 downregulation in melanoma cells did not make them more aggressive and could lead to new therapeutic strategies for this tumor, which is so difficult to control once metastasized.
Assuntos
Proteínas de Transporte/genética , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Melanoma/genética , Neovascularização Patológica/genética , Neoplasias Cutâneas/genética , Benzotiazóis , Divisão Celular/fisiologia , Divisão Celular/efeitos da radiação , Linhagem Celular Tumoral/fisiologia , Linhagem Celular Tumoral/efeitos da radiação , Sobrevivência Celular/fisiologia , Sobrevivência Celular/efeitos da radiação , Diaminas , Regulação para Baixo , Fator 2 de Crescimento de Fibroblastos/genética , Corantes Fluorescentes , Humanos , Kisspeptinas , Metaloproteinase 1 da Matriz/genética , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 9 da Matriz/genética , Compostos Orgânicos , Proteínas/genética , Quinolinas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Trombospondina 1/genética , Trombospondinas/genética , Inibidor Tecidual de Metaloproteinase-1/genética , Proteínas Supressoras de Tumor , Ubiquitina-Proteína Ligases , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Azide-tagged Cu(I)-NHC reacts in an 'auto-click' process to furnish complexes functionalized by 1,2,3-triazoles bearing diverse substituents. The resulting Cu(I) complexes are amenable to further transmetallation to Au(I). The whole strategy proceeds with mild conditions and constitutes an efficient entry to functionalised metal-NHCs with biorelevant moieties.
Assuntos
Cobre/química , Ouro/química , Compostos Heterocíclicos/química , Linhagem Celular Tumoral , Química Click , Complexos de Coordenação/síntese química , Complexos de Coordenação/química , Complexos de Coordenação/metabolismo , Cristalografia por Raios X , Humanos , Microscopia Confocal , Conformação Molecular , Triazóis/químicaRESUMO
The facile synthesis of functionalized luminescent nanoparticles from LnL3 lanthanide complexes is described. The luminescence properties of the lanthanide chelates and of the corresponding nanohybrids are reported and compared. For a further application in bioimaging, the cytotoxicity of the nano-objects was investigated.
RESUMO
Epithelial-to-mesenchymal transition (EMT) is a transdifferentiation process that converts epithelial cells into highly motile mesenchymal cells. This physiologic process occurs largely during embryonic development but is aberrantly reactivated in different pathologic situations, including fibrosis and cancer. We conducted a siRNA screening targeted to the human kinome with the aim of discovering new EMT effectors. With this approach, we have identified mTOR complex 1 (mTORC1), a nutrient sensor that controls protein and lipid synthesis, as a key regulator of epithelial integrity. Using a combination of RNAi and pharmacologic approaches, we report here that inhibition of either mTOR or RPTOR triggers EMT in mammary epithelial cells. This EMT was characterized by the induction of the mesenchymal markers such as fibronectin, vimentin, and PAI-1, together with the repression of epithelial markers such as E-cadherin and ZO-3. In addition, mTORC1 blockade enhanced in vivo migratory properties of mammary cells and induced EMT independent of the TGF-ß pathway. Finally, among the transcription factors known to activate EMT, both ZEB1 and ZEB2 were upregulated following mTOR repression. Their increased expression correlated with a marked reduction in miR-200b and miR-200c mRNA levels, two microRNAs known to downregulate ZEB1 and ZEB2 expression. Taken together, our findings unravel a novel function for mTORC1 in maintaining the epithelial phenotype and further indicate that this effect is mediated through the opposite regulation of ZEB1/ZEB2 and miR-200b and miR-200c. Furthermore, these results suggest a plausible etiologic explanation for the progressive pulmonary fibrosis, a frequent adverse condition associated with the therapeutic use of mTOR inhibitors.
Assuntos
Transição Epitelial-Mesenquimal/fisiologia , Complexos Multiproteicos/fisiologia , Serina-Treonina Quinases TOR/fisiologia , Animais , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Células Cultivadas , Embrião de Galinha , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Humanos , Células MCF-7 , Alvo Mecanístico do Complexo 1 de Rapamicina , MicroRNAs/metabolismo , Complexos Multiproteicos/antagonistas & inibidores , Complexos Multiproteicos/genética , Invasividade Neoplásica , Inibidores de Proteínas Quinases/farmacologia , Interferência de RNA , Proteínas Repressoras/metabolismo , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/genética , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/fisiologia , Homeobox 2 de Ligação a E-box com Dedos de Zinco , Homeobox 1 de Ligação a E-box em Dedo de ZincoRESUMO
The Shwachman-Bodian-Diamond syndrome (SDS) is an autosomal disorder with pleiotropic phenotypes including pancreatic, skeletal and bone marrow deficiencies and predisposition to hematological dysfunctions. SDS has been associated to mutations in the SBDS gene, encoding a highly conserved protein that was shown to function in ribosome biogenesis in yeast. In this work, we show that SBDS is found in complexes containing the human Nip7 ortholog. Analysis of pre-rRNA processing in a stable SBDS knock-down HEK293-derivative cell line revealed accumulation of a small RNA which is a further indication of SBDS involvement in rRNA biosynthesis. Global transcription and polysome-bound mRNA profiling revealed that SBDS knock-down affects expression of critical genes involved in brain development and function, bone morphogenesis, blood cell proliferation and differentiation, and cell adhesion. Expression of a group of growth and signal transduction factors and of DNA damage response genes is also affected. In SBDS knock-down cells, 34 mRNAs showed decreased and 55 mRNAs showed increased association to polysomes, among which is a group encoding proteins involved in alternative splicing and RNA modification. These results indicate that SBDS is required for accurate expression of genes important for proper brain, skeletal, and blood cell development.