SUN Yuan-yuan, LI Wei, YE Shou-dong, LIU Da-hai
Embryonic stem cells (ESCs) are isolated from the inner cell mass of pre-implantation blastocyst, which can proliferate spontaneously and infinitely under appropriate culture conditions in vitro (self-renewal), while maintaining the potential to differentiate into various types of cells derived from the three germ layers, and therefore have important significance not only for tissue repair and regeneration, but also can provide a powerful tool for modeling disease and understanding biological development. Growth arrest and DNA damage inducible protein 45 gamma (Gadd45g) is one of the three members of the GADD45 protein family. It is also known as the cytokine response gene 6 (CR6), which can affect the cell cycle and regulates cell growth negatively. In addition, as a well-known emergency response gene,Gadd45g has been studied in neural precursor cell differentiation and in tumors. There are evidences which have shown that Gadd45g can be methylated in a variety of tumor cells and has become a potential target for clinical therapy of cancers. Because tumor cells and ESCs have similarity in proliferation characteristic, it will be significant to investigate the effects of Gadd45g on mouse embryonic stem cells (mESCs). The recombinant plasmid containing Flag tagged,Gadd45g was constructed and transfected into mESCs to overexpress the target gene. The qRT-PCR and Western blot were then used to determine the expression level of,Gadd45g in transcriptional and translational aspects respectively. Subsequently,the self-renewal status of,Gadd45g -overexpressing mESCs were detected under the serum culture condition, containing leukemia inhibitory factor (LIF), via cell counting, alkaline phosphatase staining, qRT-PCR,and immunofluorescence staining. Among these strategies, the cell counting was used to measure the growth rate of the cells, alkaline phosphatase staining was used to detect the self-renewal degree of the cells, qRT-PCR was used to analyze the expression of self-renewal marker genes and differentiation marker genes which represent different germ layers in the cells and immunofluorescence staining was used to demonstrate the self-renewal marker proteins in the cells. The results shown that compared with control group, overexpression of,Gadd45g can make mESCs growth rate slow down and lead to alkaline phosphatase activity and expression levels of self-renewal marker genes (Oct4, Nanog and Klf2) decline in the cells, meanwhile the expression levels of mesendoderm marker genes, such as,Sox17, Foxa2, T, GSC and Mixl1, were up-regulated obviously, which means these cells tend to exit from self-renewal status and differentiate into mesendodermal stage. As LIF/Stat3 signal pathway is important to mESC maintenance and is able to inhibit mesendoderm formation in mESCs, subsequent experiments were conducted to examine whether,Gadd45g could regulate the activity of LIF/STAT3 signal pathway, and the result shown that overexpression of,Gadd45g could reduce the phosphorylation level of STAT3 in both the serum condition added LIF and the serum-free condition with 2i containing two small molecules:CHIR99021 and PD0325901, which inhibit glycogen synthase kinase 3(GSK3) and mitogen-activated protein kinase (MAPK) kinases(MEK proteins), respectively. This result suggested that Gadd45g may induce mesendodermal formation partially via inhibiting STAT3 activity. The overall results will not only extend the understanding of molecular mechanisms of embryonic stem cell fate determination, but also will be beneficial to ESC basic research and safe application in the future.
