Modulation Of STAT3 Phosphorylation By PTPN2 Inhibits Naive Pluripotency Of Embryonic Stem Cells

STAT3 phosphorylation at tyrosine705(STAT3pY705),triggered by the addition of the leukemia inhibitory factor(LIF),enters the nucleus and stimulates the expression of its target genes to maintain self-renewal status of mouse embryonic stem cells(mESCs).Many reports have demonstrated that activated STAT3 can reprogram primed-state mouse epiblast stem cells(EpiSCs)into naive-state mESCs.The activation of STAT3pY705 occurs mainly through Janus kinases,however,it still remains unclear how STAT3pY705 levels are decreased in mESCs.Overexpression of STAT3 can replace the function of LIF to maintain mESC stemness in the undifferentiated state.However,we found that co-expression of STAT3 and PTPN2 can induce differentiation of mouse embryonic stem cells.While PTPN2 was previously reported to interact with STAT3 protein,we hypothesized that the differentiation of mESCs induced by PTPN2 may be associated with STAT3.In order to investigate the interaction between STAT3 and PTPN2,we conducted the following experiments in vitro and in vitro:First,in order to explore the interaction between STAT3 and PTPTN2 proteins,we overexpressed HA-STAT3 and FLAG-PTPN2 in mouse embryonic stem cells.Then Co-Immunoprecipitation(Co-IP)method was carried out,the results showed that STAT3 and PTPN2 proteins do interact at the protein level.We then performed Co-IP experiments using antibodies specific for STAT3 and PTPN2,further confirming that they do interact at the protein level in mESCs.Next,in order to examine whether PTPN2 can impair the ability of the LIF/STAT3 signaling pathway in maintaining mESC self-renewal,we overexpressed PTPN2 in mESCs.As expected,mESCs differentiated.Western blotting detection of stem cell self-renewal marker genes Esrrb,Klf4,Nanog,the results showed that the expression of these three proteins was reduced.Then we used immunofluorescence and real-time quantitative real-time PCR to detect the expression of pluripotency genes and observed the same phenomena,while the expression of differentiation associated genes T and Cdx2 was up-regulated.mESCs self-renewal is mainly through the phosphorylation of STAT3,and tyrosine of 705 plays a very important role.As a dephosphorylated protein,PTPN2 may function via decreasing STAT3pY705.We then performed western blotting and the results showed that PTPN2 truly reduced the phosphorylation level of STAT3pY705.Furthermore,we also found that LIF could enforce the interaction between phosphorylated STAT3 and PTPN2 protein.Meanwhile,LIF can also stimulate PTPN2 to enter the nucleus from the cytoplasm.Taken together,these results collectively imply that PTPN2 can associate with phosphorylated STAT3 and decrease its phosphorylation level to induce mESC differentiation.Next,to investigate whether suppressing PTPN2 expression can recapitulate the effects of LIF on the maintenance of mESC self-renewal,we used RNA interference technology to reduce the expression of PTPN2,and then cultured these cells in the absence of LIF.The results showed that the decreased expression of PTPN2 can delay the differentiation of mESCs.To confirm these results,we further knocked out the PTPN2 gene in mESCs using the CRISPR/CAS9 system.We observed similar results.These experimental data indicate that the presence of PTPN2 inhibits the ability of mESC self-renewal induced by LIF/STAT3 signaling pathway.Overexpression of STAT3 can reprogram EpiSCs back to the mESCs state,so we finally want to examine whether PTPN2 mediate the function of STAT3 in reprogramming EpiSCs toward naive pluripotency.We knocked down the expression of PTPN2 in EpiSCs lines overexpressed STAT3.These cells were cultured in conventional mESC medium supplemented with LIF,CHIR99021 and PD0325901.After 12 days,downregulation of PTPN2 increased the number of AP-positive colonies generated from STAT3-overexpressing EpiSCs.Therefore,PTPN2 negatively modulates the function of STAT3 in reprogramming EpiSCs to enter naive pluripotency.Finally,we did a converse experiment,PTPN2 and STAT3 were overexpressed together in EpiSCs,and then these cells were cultured in conventional mESC medium supplemented with LIF,CHIR99021 and PD0325901.After 12 days,upregulation of PTPN2 decreased the number of AP-positive colonies generated from STAT3-overexpressing EpiSCs.In conclusion,we characterized PTPN2 and STAT3 as protein partners involved in the complex regulation of STAT transcriptional targets.As a transcriptional repressor,PTPN2 mainly inhibits the STAT3 transcriptional network in mESCs.Therefore,suppression of PTPN2 will be a promising target to promote the development of new ESC culture media for culturing and isolate naive pluripotent state stem cells from different species.