The Role Of SVCTS In Pluripotency Maintenance Of F9 Cells By VC-JAK-STAT Signaling Pathway

Vitamin C is a kind of natural micromolecular compound.For human and many other species,Vc is an essential nutrient.It has been reported that Vc could promote generation of pluripotent stem cells both from mouse and human sources,which has been proved to be independent of its innate antioxidant capacity.Above all,perhaps VC has additional functions,not just eliminating free radicals.Embryonic Stem Cell(ESCs)are derived from inner cell mass of blastocyst.ESCs are a cluster of pluripotent stem cells,which can further differenciate into three main germ layers including endoderm,mesoderm and ectoderm.F9 cells belong to mouse embryonal carcinoma cells which possess similar pluripotent state with J1 ESCs,thus will usually be used as model cells to investigate ESCs.Nanog is one of protein factorshighly expressing in ESCs.This key factor is also involved in pluripotency maintenance of ESCs.Previously,our research group has found that Vc could enhance Nanog expression through activation of JAK2/STAT2 signalling pathway,leading to pluripotency maintenance of mouse embryonic stem cells.On the basis of preliminary work,this research explored the molecular mechanisms by which Vc activates JAK/STAT signaling pathway.Since JAK2 kinase belongs to membrane peripheralprotein,we presume that Vc activates JAK2/STAT2 signaling pathway followed by thansportation through cell membrane.There have not been any reports about Vc cell surface receptors yet,nevertheless,a group of Vc transpotors,sodium-dependent Vitamin C Transpoters(SVCTs),has aroused our concern.It has been reported that Vitamin A cell surface transportor,STRA6,is a bifunctional protein,simultaneously involving in Vitamin A transportation and signal transduction.Similarly,Vc cell surface transportor SVCT probably also mediates VC signaling pathway.In order to verify the essential role of SVCTs in Vc-induced JAK2-STAT2-Nanog pathway,this study was initiated via methods including Co-Immunoprecipitation(Co-ip),Immunofluorescence(IF)and Laser Confocal,to explore interaction and distribution between SVCTs and core components of JAK/STAT signaling pathway.On the other hand,this research also refered to the influence of abnormal expression of SVCTs on the phosphorylation level of JAK/STAT pathway components and expression level of Nanog.In addition to the expression level,whether the SVCT channel is opening or closing may also be an essential factor in the indexes mentioned above.Here,several methods have been adopted like Quantitative Real-time PCR(qRT-PCR)and Phosphorylated Western blotting.This study preliminary confirmed that SVCTs interacted with JAKs,which simulated the interaction relationship between cytokine receptors and JAK kinase,mediating Vc signaling transduction from SVCTs to JAK/STAT.Moreover,Vc transportation was suggested to induce the phosphorylation of JAKs.Meanwhile,expression change of SVCTs influenced phosphorylation of JAK2 and STAT2,hence altering gene expression level of Nanog.All above,SVCTs seem to be a new type of bifunctional proteins like STRA6 whose capacities combine molecule transpotation and signaling transduction.In mouse F9 embryonal carcinoma cells,SVCTs could be a potential regulation target,elucidating the molecular mechanisms of Vc in stem cell regulation network from the angle of receptor/signaling pathway.Furthermore,presense of SVCTs consolidated the theory basis of Vc functions in stem cell pluripotency maintenance.