JAK and TYK2 are dispensable for the maintenance of self renewal but synergestically affect on the differentiation of mouse embryonic stem cells

Abstract

Jak/STAT signaling pathway mediated by leukemia inhibitory factor (LIF) is known as one of the most critical factors to maintain self-renewal of mouse embryonic stem cells (mESCs). Among seven known mammalian STATs, STAT3 is important to maintain the undifferentiated state of mESCs. There are four mammalian Jaks including Jak1, Jak2, Jak3, and Tyk2. mESCs express Jak1, Jak2, and Tyk2. In mESCs, Jak1, Jak2, and Tyk2 are known to be expressed. Jak1 is known as an important molecule to activate STAT3, but the functional roles role of Jak2 and Tyk2 in self-renewal and differentiation of mESCs are is not clear. To gain insight into the functional role of Jak2 in mESCs, we established two Jak2 knockdown cell lines using Jak2 RNAi strategy. To avoid unexpected differentiation due to the down-regulation of Jak2, the estrogen receptor fusion form of STAT3 (STAT3ER) expressing mESC line, which can maintain the undifferentiated state without Jak signaling by treating 4-hydroxytamoxifen, was employed in this study. Western blot analyses revealed that the levels of Jak2 are almost abolished in both lines. Interestingly, in addition to Jak2, Tyk2 was also missing in one line. When these cells were cultured in the different varying concentrations of LIF, they could maintain the undifferentiated state even in a very low concentration of LIF as the parental STAT3ER cells did, indicating that Jak2/Tyk2 are not involved in Jak1/STAT3 mediated LIF dependent self-renewal of mESCs. Embryoid bodies generated from Jak2/Tyk2 knockdown clones underwent faster differentiation in comparison with control. Indeed, RT-PCR analyses revealed that the expression of three germ layer marker genes onsets earlier in Jak2/Tyk2 knockdown clones than in the control clone. In addition, as analyzed by cDNA microarray, many genes specifically expressed in ectodermal and mesodermal lineage cells were differentially expressed in Jak2/Tyk2 knockdown clones. These results suggest that there is an interplay between Jak2 and Tyk2 to evoke unique patterns of gene expression during differentiation in addition to their involvement in the independent signaling pathway in mESCs. To investigate the potential pathways in which Jak2 and Tyk2 might be involved in for regulating differentiation state of mESCs, we treated Jak2/Tyk2 knockdown clones with various inhibitors for different signaling pathways in the presence of LIF. Results demonstrated that the reduced level of Jak2/Tyk2 tends to induce the differentiation of mESCs when the Wnt signaling is blocked. Inhibition of MEK, ERK, or PTK facilitated LIF-dependent self-renewal of all the mESCs clones, indicating that MEK, ERK and PTK are not involved in LIF induced self-renewal regardless of Jak2/Tyk2 expression. Ras/Raf/MEK/ERK pathway. In addition, we identified that PI3K and PKC pathways, and Ser/Thr phosphatase activity are crucial for maintaining self-renewal of mESCs.