Background: Sox10, a member of the Sry-related HMG-Box gene family, is a critical transcription factor for several important cell lineages, most notably the neural crest stem cells and the derivative peripheral glial cells and melanocytes. Thus far, only a handful of direct target genes are known for this transcription factor limiting our understanding of the biological network it governs.
The regulation of megakaryocytic differentiation is poorly understood. Using K562 cells, which can partly recapitulate the process in response to phorbol 12-myristate 13-acetate (PMA), we performed microarray-based gene expression profiling to identify genes that play significant roles in megakaryopoiesis. Here, we describe the function of FosB, an AP-1 transcription factor. FosB is induced in PMA treated K562 cells in a sustained manner and forms an active AP-1 protein-DNA complex. Down-regulation of FosB with specific shRNAs inhibited the induction of CD41, a specific cell surface marker of megakaryocytes. We also show that activation of the PKC-MEK-ERK signaling pathway is required for induction of FosB and CD41. Finally, we cross-examined the microarray data in conjunction with gene function annotation data to identify additional target genes of FosB. We define 3 genes, INHBA, CD9, and ITGA2B as regulatory targets of FosB and show that CD9, in particular, is a direct target of FosB.
In this study we showed that 2-(trimethylammonium)ethyl (R)-3-methoxy-3-oxo-2-stearamidopropyl phosphate [(R)-TEMOSPho], a derivative of an organic chemical identified from a natural product library, promotes highly efficient differentiation of megakaryocytes. Specifically, (R)-TEMOSPho induces cell cycle arrest, cell size increase and polyploidization from K562 and HEL cells, which are used extensively to model megakaryocytic differentiation. In addition, megakaryocyte-specific cell surface markers showed a dramatic increase in expression in response to (R)-TEMOSPho treatment. Importantly, we demonstrated that such megakaryocytic differentiation can also be induced from primary human CD34(+) haematopoietic stem cells. Activation of the PI3K-AKT pathway and, to a lesser extent, the MEK-ERK pathway appears to be required for this process, as blocking with specific inhibitors interferes with the differentiation of K562 cells. A subset of (R)-TEMOSPho-treated K562 cells undergoes spontaneous apoptosis and produces platelets that are apparently functional, as they bind to fibrinogen, express P-selectin and aggregate in response to SFLLRN and AYPGFK, the activating peptides for the PAR1 and PAR4 receptors, respectively. Taken together, these results indicate that (R)-TEMOSPho will be useful for dissecting the molecular mechanisms of megakaryocytic differentiation, and that this class of compounds represents potential therapeutic reagents for thrombocytopenia.
A factor stimulating a mitogenic activity of peritoneal macrophages is purified from bovine udder. It is identified as a triglyceride, 1-palmitoyl-2-linoleoyl-3-acetyl-rac -glycerol (rac -MADG). In this study, its enantiomers, R-(+)-and S-(-)-1-palmitoyl-2-linoleoyl-3-acetylglycerol (R-(+)-MADG, S-(-)-MADG) are synthesized. Among them, R-(+)-MADG enantiomer turns out to increase a mitogenic activity in mouse peritoneal macrophages. Also, (S)-(-)-MADG shows a low mitogenic activity. Treatment of a macrophage with R-(+)-MADG increases reactive oxygen species(ROS). Furthermore, treatment of macrophages with antioxidant, N-acetyl-L-cysteine (NAC), suppresses the R-(+)-MADG-dependent macrophage proliferation. Results show that the generation of ROS induces in R-(+)-MADG-dependent cell signaling. Treatment of a macrophage with R-(+)-MADG increases the activity of protein kinase C (PKC). Treatment of macrophages with calphostin C inhibits R-(+)-MADG-induced macrophage proliferation. Results suggest that R-(+)-MADG enhances the activity of protein kinase C (PKC) and stimulates the macrophage growth. In conclusions, R-(+)-MADG accelerates the production of ROS and increases the activity of PKC to eventually stimulate macrophage cell growth. The existence of rac -MADG in bovine udder and milk provides passive protection for the neonate and immunostimulatory capabilities.
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