Vitamin C Maintain The Nanog Expression Through JAK/sTAT Signaling Pathway In Mouse F9Cells

Vitamin C also known as ascorbic acid and was involved in many important metabolicand physiological reactions in the body. It has been reported that Vitamin C could enhancesthe generation of mouse and human induced pluripotent stem cells, and sufficient evidencesshowed that Vitamin C improve the efficiency of reprogramming has nothing to do with itsantioxidant ability, suggesting that Vitamin C may effect the expression of some crucial genesthat related to reprogramming.To explain this phenomenon at molecular level, firstly we proved that Vitamin C helps tomaintain the undifferentiated state of embryonic stem cells by immunofluorescence staining.Subsequently, we cloned the2.5kb promoter of Nanog gene and found that Vitamin Ctreatment can significantly enhance the activity of Nanog promoter by Dual LuciferaseReporter assay. Next, quantitative PCR and Western blot experiment supported theconclusion that Vitamin C enhances the expression of Nanog on the level of mRNAtranscription and protein translation.The next in-depth study we investigated the mechanism of how did Vitamin C regulateNanog expression. Through signaling pathway profiling system we realized that Vitamin Cmay work via the JAK/STAT pathway, then the JAK/STAT pathway inhibitor was used toconfirm the thought, we found that once the JAK/STAT pathway was inhibited, the effect ofVitamin C on the regulation of Nanog was completely disappeared.According to the reported conserved binding sequence of STATs family, assisted withbioinformatics software prediction, we found a potential STAT binding site at the+272bp-+280bp point of Nanog proximal promoter. The site was confirmed to be the Vitamin Csignaling response site by using the binding site mutation and pathway inhibition methods. Then phospho-western blot analysis proved that STAT2was activated by Vitamin C treatment.Finally, the Electrophoretic Mobility Shift Assay(EMSA) is a direct proof for that STAT2could bind to the Vitamin C signaling response site specifically.In summary, this study put forwad the view of Vitamin C could regulate the expressionof Nanog for the first time, and discovered a brand new transcription factor–that is STAT2of Nanog. We also described the mechanism of Vitamin C work on Nanog, established acomplete Vitamin C signaling pathway, which is “Vitamin C stimulates in vitro—Signalpasses through JAK/STAT—STAT2bingding to the Nanog proximal promoter—Theexpession changes of Nanog”. All these results provide theoretical basises for the applicationof Vitamin C in the embryonic stem cells and induced embryonic stem cells fields, make animportant complement for the Nanog regulation networks, as well as put new insight to theresearch of pluripotency regulation of embryonic stem cells.