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. 2004 Jan 8;23(1):212–220. doi: 10.1038/sj.emboj.7600045

Figure 4.

Figure 4

FOXO3a mediates Akt-induced growth arrest via the ROS/p53/p21-dependent mechanisms. (A) Whole-cell lysates (30 μg) of pLNCX (Mock)- or AktCA-infected endothelial cells were examined for expression of phospho-FOXO3a (pFOXO3a, Thr32), total FOXO3a (FOXO3a), MnSOD, catalase and tubulin (loading control) by Western blotting. (B) Human endothelial cells infected with pLNCX (Mock) or AktCA were loaded with DCF for 30 min and analyzed by FACS. Representative results from two independent experiments are shown. (C) The luciferase reporter gene plasmid PG13-Luc was transfected into endothelial cells infected with pLNCX (Mock) or AktCA and cultured in the absence or presence of NAC (0.5 mM). At 24 h after transfection, the luciferase activity was measured. The activity in mock-infected cells is set at 100%. *P<0.01 versus Mock, #P<0.001 versus AktCA+NAC, ANOVA, n=4. (D) Human endothelial cells were infected with pBabe (empty vector) or pBabe mutant FOXO3a (FOXO). Infected cell populations were then transduced with pLNCX (Mock) or AktCA and seeded at a density of 3 × 105 cells per 100 mm plate on day 0. Cell number was then counted at indicated time points. *P<0.05 versus Mock, ANOVA, n=3. (E) Morphology of Akt-infected cell populations prepared in (D). (F) Whole-cell lysates (30 μg) prepared in (D) were examined for the expression of p21 and actin (loading control) by Western blotting. Constitutive activation of Akt inhibits the transcriptional activity of FOXO3a and thereby downregulates MnSOD, leading to an increase of ROS that promotes senescence-like growth arrest via the p53/p21-dependent pathway. (G) Proposed signaling pathway of Akt-induced senescence in human endothelial cells compared with that in C. elegans. Akt inactivates FOXO3a and thereby downregulates its target antioxidant gene MnSOD, leading to an increase of ROS. ROS induces p53 activity, resulting in upregulation of p21 expression, which promotes cellular senescence in human endothelial cells. In C. elegans, the PI3K/Akt pathway also negatively regulates longevity by inactivating DAF-16 activity. This regulatory pathway partly involves the decreased expression of anti-stress genes including SOD.