Abstract
Glycogen synthase kinase-3 (Gsk-3) activity is an important regulator of numerous signal transduction pathways. Gsk-3 activity is the sum of two largely redundant proteins, Gsk-3α and Gsk-3β, and in general, Gsk-3 is a negative regulator of cellular signaling. Genetic deletion of both Gsk-3α and Gsk-3β in mouse embryonic stem cells (ESCs) has previously been shown to lead to the constitutive activation of the Wnt/β-catenin signaling pathway. However, in addition to Wnt signaling, all Gsk-3-regulated pathways, such as insulin signaling, are also affected simultaneously in Gsk-3α−/−; Gsk-3β−/−ESCs. In an effort to better understand how specific signaling pathways contribute to the global pattern of gene expression in Gsk-3α−/−; Gsk-3β−/−ESCs, we compared the gene expression profiles in Gsk-3α−/−; Gsk-3β−/− ESCs to mouse ESCs in which either Wnt/β-catenin signaling or phosphatidylinositol 3-kinase (PI3K)-dependent insulin signaling are constitutively active. Our results show that Wnt signaling has a greater effect on up-regulated genes in the Gsk-3α−/−; Gsk-3β−/−ESCs, whereas PI3K-dependent insulin signaling is more responsible for the down-regulation of genes in the same cells. These data show the importance of Gsk-3 activity on gene expression in mouse ESCs, and that these effects are due to the combined effects of multiple signaling pathways.
Highlights
Glycogen synthase kinase-3 (Gsk-3) is an intracellular serine/threonine kinase activity that was originally identified as the rate-limiting step in the glycogen synthesis pathway [1]
We have previously shown that stable expression of a constitutively active form of the p110α catalytic subunit of phosphatidylinositol 3-kinase (PI3K) [8] in WT mouse embryonic stem cells (ESCs) can effectively lead to the phosphorylation of Gsk-3 [9], and expression of p110* has been shown to activate insulin signaling in mouse ESCs [10]
To find that Brachyury and Axin2 were the exception of the 65 Wnt putative target genes; we identified in our microarray data from Gsk-3 DKO ESCs that only seven additional genes (Sp5, Cdx1, Stra6, Lef1, Cyclin D1, Pituitary tumor transforming gene (PTTG), and Fgf18) had their expression increased more than twofold (Table 1)
Summary
Glycogen synthase kinase-3 (Gsk-3) is an intracellular serine/threonine kinase activity that was originally identified as the rate-limiting step in the glycogen synthesis pathway [1]. Several signaling pathways, including protein kinase A (PKA), Hedgehog, transforming growth factor-β (TGF-β), nuclear factor of activated T-cells (NFAT), and phosphatidylinositol 3-kinase (PI3K)-dependent insulin signaling, all affect Gsk-3 activity via phosphorylation of the amino-terminal serines [4,5,6]. Insulin binding to its receptor triggers the activation of PI3K, which phosphorylates and activates Akt, which in turn phosphorylates and inhibits Gsk-3 activity [7]. We have previously shown that stable expression of a constitutively active form of the p110α catalytic subunit of PI3K (termed p110*) [8] in WT mouse ESCs can effectively lead to the phosphorylation of Gsk-3 [9], and expression of p110* has been shown to activate insulin signaling in mouse ESCs [10]
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