Regulation Of SB431542 On Mouse Embryonic Stem Cells Pluripotency

Embryonic stem cells(ES cells) are a type of multipotent cells isolated from the inner cell mass of the blastocyst-stage embryos. Because of the characteristics of that can self-renewal indefinitely and differentiate into almost all types of cells, ES cells become a focus in life science research: ES cells are promising models for embryonic development research, and they can also be used to produce regenerative materials. Today, a tremendous progress has been made in ES cells research. However, the exactly mechanisms of self-renewal and differentiation of ES cells remain unknown, which limit the application of ES cells severely.It has been reported that Tgf-? signaling pathway can accelerate the process of embryonic development. However, there is still a controversy on whether inhibition of Tgf-? signaling pathway would contribute to the pluripotent maintenace of ES cells. 4-(5-Benzol[1,3]dioxol-5-yl-4-pyrldin-2-yl-1H-imidazol-2-yl)-benzamide hydrate(SB431542) is a specific inhibitor of Tgf-?-Smad2/3 signaling pathway. It can specifically inhibit the phosphorylation of Smad2/3 and block the transduction of Tgf-? signaling pathway through occupying the ATP-binding domain of Alk4, Alk5 and Alk7. As previously reported that SB431542 facilitates the pluripotent maintenance of in vitro cultivated ES cells, however, the specific mechanisms of SB431542 are not well documented. The following studies were conducted in J1 mouse ES cells to investigate the functional role of Tgf-? signaling pathway, as well as the specific mechanism of SB431542 in pluripotent maintenace, the detailed contents and results of this research are as follows:1. The results of CCK-8 assay and phosphorylated protein Western blot indicated that SB431542 could inhibit the phosphorylation of Smad2/3, and had no significant influence on the proliferation ability of J1 ES cells. As previously reported, three small moleculars, namely, BIO, CHIR99021 and SC1 are able to promote self-renewal of ES cells, we also found that these small moleculars could enhance the adhesion of cells and promote ES cells growing in a typical colony morphology. The same phenomenon was observed when ES cells were treated with SB431542. Besides, the alkaline phosphatase activity of ES cells was increased, and the genes refered to differentiation marker of ES cells were down-regulated by SB431542 treatment. These results showed that SB431542 contributes ES cells pluripotency maintenance.2. In order to know the specific mechanism of SB431542 on ES cells pluripotency, we examined the global gene expression changes of ES cells that treated with SB431542 by microarray. We found that SB431542 maintains ES cells pluripotency through multiple mechanisms. First of all, a part of self-renewal genes were up-regulated, and most of differential-related genes were down-regulated by SB431542 treatment. Second, GO analysis showed that SB431542 maintains ES cells pluripotency through regulating cell connection and proliferation. Finally, KEGG analysis showed that SB431542 was involved in regulating MAPK, Erb B, VEGF and cell connection signaling pathways.3. SB431542 enhances ES cells pluripotency by activiating Tgf-?-Smad1/5/8 signaling pathway, as well as inhibiting Fgf/Erk and retinoic acid related signaling pathways. Smad7 is a inhibitor of Tgf-?-Smad1/5/8 signaling pathway. When ES cells were under SB431542 treatment, downregulation of Smad7 could be rescued by overexpression of Smad2/3; and upregulation of phosphorylation of Smad1/5/8 and Id gene expression could be suppressed by overexpression of Smad7. These results indicated that SB431542 could activate Tgf-?-Smad1/5/8 signaling pathway indirectly by decreasing the expression of Smad7. Further, when ES cells were induced to differentiate into neural cells by Retinoic acid(RA) treatment, we found that the addition of SB431542 could not only inhibit the process of RA induced differentiation, but also compromise the downregulation of Hoxb caused by RA inducement.4. We also performed deep-sequencing to detect the expression changes of mi RNA in ES cells treated by SB431542. Among the 114 mi RNAs that affected by SB431542 treatment, only 4 of them were upregulated, while the others were downregulated. Except the mi RNAs involved with Tgf-?-Smad2/3 signaling pathway, mi RNAs that downregulated by SB431542 mainly refer to inhibition of pluripotency and promotion of differentiation. All of these results indicated that SB431542 maintain pluripotency by inhibiton of transcription in ES cells.In conclusion, the results suggest that SB431542 is a pluripotent molecular can be used to maintain the undifferentiated state of mouse ES cells. Besides, SB431542 contributes to mouse ES cells pluripotency maintenance mainly by inhibiting ES cells differential processes.