The epigenetic modifiers 5-aza-2'-deoxycytidine and trichostatin A influence adipocyte differentiation in human mesenchymal stem cells.

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.

The epigenetic modifiers 5-aza-2'-deoxycytidine and trichostatin A influence adipocyte differentiation in human mesenchymal stem cells

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.

Correlation between cytosolic Ca2+ concentration, protein phosphorylation and platelet secretion.

Addition of the calcium-ionophore ionomycin to acetylsalicylate-treated platelets suspended in a low Ca2+ concentration-containing medium (about 0.1 microM), induced a dose-dependent (range 0.25-3 microM) and transient increase in the cytosolic Ca2+ concentration ([Ca2+]c). Less than 10% of the maximal releasable amount of serotonin was secreted at [Ca2+]c lower than 1 microM, whereas secretion was almost maximal at [Ca2+]c higher than 2 microM. In all cases the secretion stopped after about 1 min even if the [Ca2+]c was kept constant by repeated small additions of CaCl2 (25-40 microM). A rapid phosphorylation of pleckstrin (47 kDa) and myosin light chain (20 kDa) was found in all cases, whereas a weak phosphorylation of a 27 kDa protein occurred at [Ca2+]c lower than 1.5 microM. Addition of 0.2 mM CaCl2 to platelets pretreated for 4 min with 0.5-1 microM ionomycin brought about a serotonin secretion remarkably lower than obtained by the simultaneous addition of CaCl2 and ionophore. Platelets suspended in a low calcium-containing medium and exposed to ionomycin showed a major increase in tyrosine phosphorylation of 60 and 72 kDa proteins and a slight increment in tyrosine phosphorylation of 115 and 130 kDa proteins. Subsequent addition of 0.2 mM CaCl2 induced a widespread phosphotyrosine dephosphorylation, particularly evident in the 60 kDa protein identified as p60c-src kinase. The protein kinase inhibitor genistein caused, together with a marked prevention of the protein tyrosine phosphorylation, a remarkable increase in the ionomycin-elicited secretory activity of platelets All together these results indicate that protein kinase C-dependent pleckstrin phosphorylation is a prerequisite of platelet secretion, but that the latter process is apparently regulated by a network of phosphoproteins, in particular the serine/threonine phosphorylation of 27 and 68 kDa proteins and the tyrosine phosphorylation of the p60c-src were found to be associated with a decrease in the secretory activity.