Characterization of the Phosphoinositide 3-Kinase-Dependent Transcriptome in Murine Embryonic Stem Cells: Identification of Novel Regulators of Pluripotency

Abstract

Phosphoinositide 3-kinase (PI3K)-dependent signaling has been implicated in the regulation of embryonic stem (ES) cell fate. To gain further insight into the mechanisms regulated by PI3Ks in murine ES cells, we have performed expression profiling using Affymetrix GeneChips to characterize the transcriptional changes that arise as a result of inhibition of PI3K-dependent signaling. Using filtering of greater than 1.5-fold change in expression and an analysis of variance significance level of p < .05, we have defined a dataset comprising 646 probe sets that detect changes in transcript expression (469 down and 177 up) on inhibition of PI3Ks. Changes in expression of selected genes have been validated by quantitative reverse transcription polymerase chain reaction. Gene ontology analyses reveal significant over-representation of transcriptional regulators within our dataset. In addition, several known regulators of ES cell pluripotency, for example, Nanog, Esrrb, Tbx3, and Tcl-1, are among the downregulated genes. To evaluate the functional involvement of selected genes in regulation of ES cell self-renewal, we have used short interfering RNA-mediated knockdown. These studies identify genes not previously associated with control of ES cell fate that are involved in regulating ES cell pluripotency, including the protein tyrosine phosphatase Shp-1 and the Zscan4 family of zinc finger proteins. Further gain-of-function analyses demonstrate the importance of Zscan4c in regulation of ES cell pluripotency. STEM CELLS 2009;27:764-775 Disclosure of potential conflicts of interest is found at the end of this article.

Akt suppresses DLK for maintaining self-renewal of mouse embryonic stem cells

“…20 Inhibition of PI3K/Akt signaling with LY-294002 reduces mouse ES proliferation, pluripotency, and Nanog expression. 18,19,21,22 Those effects are similar to the effects of overexpressing DLK in mouse ES cells ( Fig. 3C and D).…”

“…[18][19][20][21][22] Many proteins and signaling pathways involved in proliferation or pluripotency are modulated by PI3K/Akt including Gsk3b/Myc, FoxO, mTOR and Tbx3/Nanog pathways. 8,21,22,41-43, In this study, for the first time, DLK was demonstrated to be an Akt substrate.…”

“…[7][8][9][10] Expression level of Nanog is regulated by PI3K/Akt signaling, 8,18,22 and DLK (Figs. 1, 3 and 6).…”

“…16,17 In mouse ES cells, PI3K/Akt signaling is essential for self-renewal and Nanog expression. 8,[18][19][20][21][22] Inhibition of Akt activity with LY-294002 reduces cellular growth, pluripotency and expression amounts of Nanog protein. 18,19,21,22 On the other hand, the increase of Akt activity by overexpression of ERas or knockout of PTEN promotes cell survival and tumorigenic properties of mouse ES cells.…”

“…8,[18][19][20][21][22] Inhibition of Akt activity with LY-294002 reduces cellular growth, pluripotency and expression amounts of Nanog protein. 18,19,21,22 On the other hand, the increase of Akt activity by overexpression of ERas or knockout of PTEN promotes cell survival and tumorigenic properties of mouse ES cells. 23,24 Even in the absence of LIF, Akt overexpression maintains the undifferentiated status of ES cells and Nanog expression levels.…”