RESUMO
Epigenetic mechanisms such as DNA methylation and histone modification are important in stem cell differentiation. Methylation is principally associated with transcriptional repression, and histone acetylation is correlated with an active chromatin state. We determined the effects of these epigenetic mechanisms on adipocyte differentiation in mesenchymal stem cells (MSCs) derived from bone marrow (BM-MSCs) and adipose tissue (ADSCs) using the chromatin-modifying agents trichostatin A (TSA), a histone deacetylase inhibitor, and 5-aza-2′-deoxycytidine (5azadC), a demethylating agent. Subconfluent MSC cultures were treated with 5, 50, or 500 nM TSA or with 1, 10, or 100 µM 5azadC for 2 days before the initiation of adipogenesis. The differentiation was quantified and expression of the adipocyte genes PPARG and FABP4 and of the anti-adipocyte gene GATA2 was evaluated. TSA decreased adipogenesis, except in BM-MSCs treated with 5 nM TSA. Only treatment with 500 nM TSA decreased cell proliferation. 5azadC treatment decreased proliferation and adipocyte differentiation in all conditions evaluated, resulting in the downregulation of PPARG and FABP4 and the upregulation of GATA2. The response to treatment was stronger in ADSCs than in BM-MSCs, suggesting that epigenetic memories may differ between cells of different origins. As epigenetic signatures affect differentiation, it should be possible to direct the use of MSCs in cell therapies to improve process efficiency by considering the various sources available.
Assuntos
Adulto , Humanos , Pessoa de Meia-Idade , Adipócitos/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Desoxicitidina/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Adipócitos/citologia , Western Blotting , Células Cultivadas , Proliferação de Células/efeitos dos fármacos , Metilação de DNA , Epigenômica , Imunofluorescência , Reação em Cadeia da Polimerase/métodos , Regulação para CimaRESUMO
The process of Trypanosoma cruzi metacyclogenesis involves the transformation of noninfective epimastigotes into metacyclic trypomastigotes, which are the pathogenic form. The analysis of stage-specific genes during T. cruzi metacyclogenesis may provide insight into the mechanisms involved in the regulation of gene expression in trypanosomatids. It may also improve the understanding of the mechanisms responsible for the pathology of Chagas disease, and could lead to the identification of new targets for chemotherapy of this disease. We have demonstrated that during metacyclogenesis the expression of several genes is controlled at the translational level by an alternative regulatory mechanism. This mechanism may involve the mobilization of mRNA to the translation machinery. We have been using self-made T. cruzi microarrays to investigate the role of polysomal mobilization in modulating gene expression during metacyclogenesis