Roles Of SHP-1during The Mouse Early Embryo And The Regular Mechanisms

Organisms can response to the environment change in the body and outside stimuli viathe regulation of protein conformation changes, the protein phosphorylation, as one of themost common and important post-translational modification of proteins is a process ofdynamic balance determined by protein kinases and protein phosphatases. It is not only acritical link which takes part in the expressions and regulations of both prokaryotes andeukaryotes, but also implicated in the regulation of growth, development, differentiation,cytoskeleton, apoptosis, neural activity, muscle contraction, metabolism and tumorgenesisand other process of life activity. SHP-1is a protein tyrosine phosphatase that is implicated inthe regulation of growth, differentiation, survival, apoptosis and proliferation and otherprocess of cell life activities, but its function of embryo development is unclear. Signaltransducer and activator of transcription3(STAT3) participate in the regulation of growth,differentiation, apoptosis and activated by extracellular factor and growth factor and otherpolypeptide ligand, has important role on development, skin renewal, immune response andother physiological process. Nanog, as a critical factor which can maintain the self-renewaland differentiation of ES cells, it also can make the terminally differentiated cells activated toproliferate by promote them enter into new cell cycle events. Therefore, we selected andinvestigated the expression of SHP-1gene and Nanog gene in early embryonic and furtherresearched the function of SHP-1and Nanog in early embryonic development in the mouseand the Mechanisms in F9cell line.1. The expression and functions of SHP-1gene and Nanog gene in the mousepreimplantation embryos. The results of RT-PCR and immunohistochemistry show that theSHP-1is detected from the stages of zygote to8-cell embryo and vanishes in morula andblastocysts, the Nanog was first expressed in the morulae where SHP-1expression wasdown-regulated and its expression in the blastocyst was confined to the inner cell mass butabsent from the trophectoderm；the data suggested that knockdown Nanog in8-cell, theembryos were mainly arrested at the8-cell stage;8-cell stage embryos electroporated with pCMV-MYC-SHP-1-V1/V2appeared to be arrested, whereas embryos in the control groupreached the blastocyst stage. Embryos over-expressing SHP-1were mainly arrested at the8-cell stage and the mRNA of Nanog was also reduced. These results suggested anantagonistic relationship between SHP-1and Nanog during early embryonic developmentand SHP-1is involved in regulation of early embryonic development.2. SHP-1participated in regulation of Nanog expression in F9cell. Usingoverexpression and knockdown strategies, DLR assays were conducted to detect Nanogpromoter activity as previously described, Nanog promoter activity in F9cells was reduced toabout40%when SHP-1was overexpressed. Conversely, Nanog promoter activity wasup-regulated around1.55–1.62-fold when SHP-1was knocked down; The RT-PCR andwestern blotting results show the same change.3. It has been proved that STAT3can regulate the expression of Nanog by interactionwith the promoter of Nanog.Here we found, in F9cells, in the NP-WT group, Nanogpromoter activity was up-regulated1.75-fold when STAT3was overexpressed, in theNP-MUT group, overexpression of STAT3had no effect on Nanog promoter activity;conversely, if endogenous STAT3was degraded by si-STAT3, Nanog promoter activity was35%compared with the control, in the NP-MUT group, knockdown of STAT3had no effecton Nanog promoter activity, western blotting and RT-PCR analysis shown the same change.4. SHP-1can regulates the activation of STAT3in F9cell. The data suggested thatphosphorylated STAT3was significantly down-regulated when SHP-1was overexpressed.Conversely, phosphorylated STAT3was up-regulated when SHP-1was inhibited usingsiRNA.5. SHP-1regulates Nanog expression via STAT3signal pathway. The results has showedthat Nanog activity in F9cells was reduced when SHP-1was overexpressed and Nanogactivity was up-regulated when SHP-1was knocked down; Further more; site mutation ofSTAT3was performed to confirm that SHP-1was responsible for rapid STAT3dephosphorylation and a decrease of Nanog expression in F9cells. With the overexpressed ofSTAT3（Y750F）, on one side, block SHP-1may up-regulated Nanog expression, on the otherside, overexpressed SHP-1can reduced the expression of Nanog, meanwhile, with theoverexpression of STAT3（Y750D）and STAT3, Nanog expression was up-regulated regardlessof the inhibition or overexpression of SHP-1. These findings suggest that SHP-1mayfunction as a key regulator for Nanog.