mTOR-mediated activation of p70 S6K induces differentiation of pluripotent human embryonic stem cells - PubMed (original) (raw)

mTOR-mediated activation of p70 S6K induces differentiation of pluripotent human embryonic stem cells

Charles A Easley 4th et al. Cell Reprogram. 2010 Jun.

Abstract

Deciding to exit pluripotency and undergo differentiation is of singular importance for pluripotent cells, including embryonic stem cells (ESCs). The molecular mechanisms for these decisions to differentiate, as well as reversing those decisions during induced pluripotency (iPS), have focused largely on transcriptomic controls. Here, we explore the role of translational control for the maintenance of pluripotency and the decisions to differentiate. Global protein translation is significantly reduced in hESCs compared to their differentiated progeny. Furthermore, p70 S6K activation is restricted in hESCs compared to differentiated fibroblast-like cells. Disruption of p70 S6K-mediated translation by rapamycin or siRNA knockdown in undifferentiated hESCs does not alter cell viability or expression of the pluripotency markers Oct4 and Nanog. However, expression of constitutively active p70 S6K, but not wild-type p70 S6K, induces differentiation. Additionally, hESCs exhibit high levels of the mTORC1/p70 S6K inhibitory complex TSC1/TSC2 and preferentially express more rapamycin insensitive mTORC2 compared to differentiated cells. siRNA-mediated knockdown of both TSC2 and Rictor elevates p70 S6K activation and induces differentiation of hESCs. These results suggest that hESCs tightly regulate mTORC1/p70 S6K-mediated protein translation to maintain a pluripotent state as well as implicate a novel role for protein synthesis as a driving force behind hESC differentiation.

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Figures

FIG. 1.

FIG. 1.

H7 hESCs exhibit decreased protein translation rates compared to differentiated cells. (A) Phase images of H7 hESCs, teratoma-derived, and differentiated H7 hESCs (H7 Diffs). Scale bar, 100 μm. (B) hESCs exhibit immature Golgi and rough ER compared to differentiated cells. TEM images with arrows indicating Golgi and rough ER in H7 hESCs and H7 Diffs. Scale bar, 500 nm. (C) hESCs exhibit decreased rates of global protein translation compared to differentiated cells. Autoradiograph representative of four separate trials showing reduced protein synthesis in cells grown in the presence of [35S]methionine. Western blot analysis for Actin is shown as a loading control. (D) Graphical analysis of four separate [35S]methionine incorporation assays illustrating reduced rates of global protein synthesis in hESCs compared to differentiated cells (p < 0.01).

FIG. 2.

FIG. 2.

H7 hESCs show reduced levels of p70 S6K signaling compared to their differentiated progeny. (A) Representative Western blots from four separate experiments indicating decreased p70 S6K activation in hESCs compared to differentiated, fibroblast-like cells. (B) Representative images showing reduced S6 phosphorylation in H7 hESCs compared to H7 Diffs. S6 phosphorylation appears to be increased along the outer edges of hESC colonies. Scale bar, 40 μm.

FIG. 3.

FIG. 3.

Suppression of mTORC1-mediated translation does not induce cell death or disrupt pluripotency in H7 hESCs. (A) Rapamycin treatment causes cell death in differentiated, but not undifferentiated cells. Representative images of untreated and treated H7 hESCs and H7 Diffs are shown. Scale bar, 100 μm. (B) Neither treatment with rapamycin nor siRNA against p70 S6K impairs colony formation or affects expression of pluripotency markers in H7 hESCs. Representative images for hESCs treated for 3 days with vehicle control or 20 nM rapamycin are shown. Also shown are representative images from H7 hESCs nucleofected with either control, nonspecific siRNA, or siRNA directed against p70 S6K. Typically, hESC colonies are smaller post nucleofection due to the requirement of single-cell suspensions. Staining for Oct4 and Nanog pluripotency markers are shown. Scale bar, 100 μM. Western blot insert confirms p70 S6K knockdown in hESCs with Actin serving as a loading control.

FIG. 4.

FIG. 4.

Prolonged treatment with rapamycin reduces expression of eIF4B and eIF2Bɛ. (A) Rapamycin treatment slightly reduces global protein translation in H7 hESCs. Representative autoradiograph and Western blots from three separate experiments indicating that rapamycin only slightly decreases protein synthesis compared to global translation inhibitors puromycin and emetine as determined by [35S]methionine incorporation. Arrow indicates an ∼85-kDA band that shows reduced new synthesis in rapamycin-treated samples. Western blots for two proteins that fall within this size range, eIF4B and eIF2Bɛ, are shown. (B) Six-day treatment of 20 nM rapamycin significantly decreases expression of eIF4B and eIF2Bɛ. Representative Western blots from three separate experiments showing eIF4B and eIF2Bɛ expression in response to rapamycin treatment.

FIG. 5.

FIG. 5.

Expression of constitutively active p70 S6K alters cell morphology and causes a loss of Oct4 expression in H7 hESCs. Constitutively active p70 S6K induces differentiation of H7 hESCs. (A) Representative fluorescent imaging of H7 hESC nucleofected with GFP, GFP + p70 S6K WT, or GFP + p70 S6K CON and stained for Oct4. Arrows indicate nucleofected cells. Scale bar, 50 μm. (B) Graphical analysis of the percentage of GFP + cells expressing Oct4. For each condition, approximately 300 cells were analyzed. Statistical significance, p < 0.01.

FIG. 6.

FIG. 6.

Knockdown of Rictor and TSC2 expression elevates p70 S6K activation and alters hESC morphology. Activation of p70 S6K by Rictor and TSC2 siRNA-mediated knockdown leads to changes in cell morphology to a more differentiated phenotype. (A) Representative Western blots from multiple experiments showing expression of proteins associated with mTORC1 (mammalian target of rapamycin complex 1) and mTORC2 signaling in H7 hESCs and H7 Diffs. (B) Western blot of immunoprecipitations of mTOR or TSC1 probed for Raptor, Rictor, or TSC2, respectively, in H7 hESCs and H7 Diffs. (C) Representative phase contrast images of H7 hESCs nucleofected with either control siRNA or Rictor/TSC2 siRNA mixture. Scale bar, 250 μm. (D) Western blot analysis confirming knockdown of TSC2 and Rictor as well as expression of p70 S6K, activated p70 S6K (p-T389), Oct4, and Actin.

FIG. 7.

FIG. 7.

Diagram depicting mTORC signaling in hESCs and differentiated cells. In pluripotent hESCs (left panel), mTOR protein is mostly associated in mTORC2 (mTOR-Rictor), whereas mTORC1 (mTOR-Raptor) activity is suppressed by the heterodimer TSC1/TSC2. In differentiated cells (right panel), mTOR is highly associated with Raptor over Rictor leading to elevated p70 S6K activity. Increased protein translation, in turn, induces differentiation.

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