Figure 6. Modulation of TFAP2C Translation.

(A-D) TFAP2C translation is controlled by PPP function. Phosphorylation of threonine 37/46 on 4E-BP1 (p-4E-BP1), a target of mTOR is expressed at high levels in the 8-cell embryo (A). This expression is lost in −G (B) or when PPP is inhibited (C). Quantitation in (D) also shows that blocking HBP does not affect p-4E-BP1.

(E, F) Western blot analysis of members of mTOR signaling pathway and its activation. (E) Total mTOR, TSC2, phosphorylation level of serine 473 on AKT1/2 (p-AKT) and serine 127 on YAP1 (p-YAP1) all remain unchanged in −G embryos, but phosphorylated mTOR targets 4E-BP1 and RPS6 (p-RPS6) levels are significantly reduced. (F) p-RPS6 is eliminated when S1PR₂, Rac1 or mTOR is inhibited.

(G-K) TFAP2C expression requires mTOR and S1P signaling. The expression of TFAP2C (G) is eliminated in the presence of mTOR inhibitors (H, I) and S1PR₂ inhibitor JTE (J). (K) Quantitation of TFAP2C expression in embryos in which the mTOR and S1P pathways are inhibited.

(L) Quantitation shows that the morula to blastocyst transition is acutely sensitive to mTOR inhibitors, inhibitors of the S1P pathway and its downstream component Rac1, but is not affected by PI3K inhibitors.

(M-T) Role of glucose and S1P signaling in controlling Rac1 expression. In control, Rac1-GTP staining is observed only on the apical surface of the polar cells (M). Inhibition of Rac1 (N), S1P receptor (P), and S1P biosynthesis (Q) cause a significant reduction in Rac1-GTP staining. In contrast, inhibition of mTOR (O), lack of glucose during culture (R), or inhibition of specific arms of glucose metabolism (PPP and HBP) (S-T) has no effect on Rac1-GTP levels or localization.