BMP2 has previously been shown to cause upregulation of Id1 and Id2, and forced expression of either gene can inhibit neurogenesis in telencephalic cultures, suggesting that these two factors play selleck chemical a role in the BMP promoted switch from neurogenesis to astrogliogenesis. In addition, we could demonstrate significant increases in the mRNA and protein levels of Stat3 and also in its phosphorylated, transcriptionally active form. This is of particular relevance for astrogliogenesis as Stat3 has been shown to functionally interact with the BMP2 responsive transcription factor Smad1/5/8 at the p300 transcriptional coactivator and thereby synergistically pro mote astrogliogenesis. How TSA promotes an increase in Stat3 levels is unclear at this point, but we have uncovered evidence that the acetylation of Stat3 is regulated by TSA mediated HDAC inhibition.
The transient activation of Erk2 in response to BMP2 and TSA treatment could play a role in the con trol of the duration of activated Smad1/5/8 signals. Erk2, but also other kinases, including Gsk3 beta, are involved in the control of Smad signals through Smad linker phosphorylation. Phosphorylation of the linker region by Erk2 and Gsk3 beta targets regulatory Smads for ubiquitinylation and proteasomal degradation. The observed activation of Erk2 should lead to a more rapid degradation of activated Smads, which can be fur ther modulated by Gsk3 beta. Thus, induction of Erk2 by phosphorylation would contribute to termination of BMP signals. Analysis of the genes upregulated in response to TSA and BMP2 treatment revealed several genes known to be expressed in neurons.
Most of these genes are not mark ers or regulators of basic neurogenesis, but are rather involved in maturation processes or establishment of the neuronal network, such as neurite outgrowth, axon guidance and synapse maturation and function. The fact that we see an upregulation of these genes can be pos sibly explained by the developmental age of the cultures, which were derived from E15. 5 GE. At this time point neurogenesis has reached its peak, before radial glia cells in GE start to generate astrocytes,neuronal genes in those precursors that have already committed to the neuronal fate or have already been born as neurons. The cultures in our ex periments were treated at 2. 5 DIV, and a small amount of neurogenesis has already occurred at this time point.
In addition, it is known that markers of maturing neurons already begin to be expressed by neuronal pro genitors. TSA and BMP2 treatment results in a drastic downre gulation of genes known to be specific for oligodendro cytes, such as Sox10 and Nkx2 2, a var iety of genes involved in Drug_discovery myelinization Mag, Mal, Mog, Omg, Mbp, Mobp, Gm98, and other genes known to be highly expressed in oligodendrocytes, such as Gpr17, Bcas1, and Enpp6.