Akt negatively regulates the in vitro lifespan of human endothelial cells via a p53/p21-dependent pathway - PubMed (original) (raw)

Akt negatively regulates the in vitro lifespan of human endothelial cells via a p53/p21-dependent pathway

Hideyuki Miyauchi et al. EMBO J. 2004.

Abstract

The signaling pathway of insulin/insulin-like growth factor-1/phosphatidylinositol-3 kinase/Akt is known to regulate longevity as well as resistance to oxidative stress in the nematode Caenorhabditis elegans. This regulatory process involves the activity of DAF-16, a forkhead transcription factor. Although reduction-of-function mutations in components of this pathway have been shown to extend the lifespan in organisms ranging from yeast to mice, activation of Akt has been reported to promote proliferation and survival of mammalian cells. Here we show that Akt activity increases along with cellular senescence and that inhibition of Akt extends the lifespan of primary cultured human endothelial cells. Constitutive activation of Akt promotes senescence-like arrest of cell growth via a p53/p21-dependent pathway, and inhibition of forkhead transcription factor FOXO3a by Akt is essential for this growth arrest to occur. FOXO3a influences p53 activity by regulating the level of reactive oxygen species. These findings reveal a novel role of Akt in regulating the cellular lifespan and suggest that the mechanism of longevity is conserved in primary cultured human cells and that Akt-induced senescence may be involved in vascular pathophysiology.

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Figures

Figure 1

Akt negatively regulates the lifespan of primary cultured human endothelial cells. (A) Whole-cell lysates (30 μg) of young (passage 4) or senescent (passages 14–15) human endothelial cells were analyzed for the expression of phospho-Akt (pAkt, Ser473) and actin (loading control) by Western blotting. (B) Human endothelial cells were infected with pLNCX (Mock), AktCA or AktDN. After purification, infected cell populations were passaged until they underwent senescence, and the number of cumulative population doublings was determined. Similar results were obtained from three independent experiments. To validate the transduction of AktCA and AktDN, whole-cell lysates (30 μg) of each infected population were examined for the expression of exogenous myc-tagged Akt (c-Myc) and total Akt (Akt). (C) Human endothelial cells infected with pLNCX (Mock), AktCA or AktDN were purified with G418 for 7 days and seeded at a density of 3 × 105 cells per 100 mm plate on day 0. Cell number per 100 mm plate was then counted at indicated time points. *P<0.001 versus Mock, ANOVA, _n_=4. (D) Cell morphology (upper panel) and senescence-associated β-galactosidase staining (lower panel) in endothelial cells infected with pLNCX (Mock), AktCA or AktDN. (E) Independent clones were isolated from pLNCX (Mock)- or AktCA-infected endothelial cells. At 30 days after isolation, the cell number of each clone was counted. Whole-cell lysates (∼10 μg) of isolated clones were also prepared and analyzed for the expression of phospho-Akt by Western blotting (left panel, mock-infected clone for lane 1 and AktCA-infected clones for lanes 2–7). The cell number of each clone was as follows: 16.6 × 105 for lane 1; 6–10 × 105 for lanes 2–4; 0.1–2 × 105 for lanes 5–7. As the availability of samples was limited in the case of most AktCA-infected clones, the lysates used were less than 10 μg (lanes 5–7). Therefore, the levels of phospho-Akt were standardized on the basis of actin expression, and the relative level of phospho-Akt and the cell number of each clone were plotted in the graph (right panel, _r_=0.92, P<0.01). The corrected value of phospho-Akt in mock-infected clones (lane 1) is set at 1.

Figure 2

Upregulation of p21 is essential for Akt-induced growth arrest. (A) Whole-cell lysates (30 μg) of pLNCX (Mock)- or AktCA-infected endothelial cells on day 0 were examined for the expression of phospho-Akt (pAkt), cell cycle regulatory proteins and tubulin (loading control) by Western blotting. (B) MEF derived from wild-type (p21+/+) or _p21_-deficient mice (p21−/−) were infected with pLNCX (Mock) or AktCA, purified with G418 for 7 days and seeded at a density of 1 × 105 cells per 100 mm plate on day 0. Cell number per 100 mm plate was then counted at indicated time points. *P<0.001 versus Mock, ANOVA, _n_=4. (C) Human endothelial cells infected with pLNCX (Mock) or AktCA were treated with cycloheximide (10 μg/ml) for the indicated time interval. Whole-cell lysates (30 μg) were then prepared at each time point and assayed for the expression of p21 and actin (loading control) by Western blotting. The graph indicates the results of densitometric analysis for the levels of p21 protein relative to actin expression. The value at time 0 is set at 100%. (D) Total RNA (30 μg) was extracted from human endothelial cells infected with pLNCX (Mock) or AktCA and analyzed for p21 mRNA levels by Northern blotting (upper panel). Ribosomal RNA was used as an internal control (lower panel). (E) The luciferase reporter gene plasmid controlled by the promoter of the human p21 gene was transfected into endothelial cells infected with pLNCX (Mock) or AktCA 24 h before the luciferase activity was measured. The activity in mock-infected cells is set at 100%. *P<0.05 versus Mock, paired _t_-test, _n_=4.

Figure 3

Critical role of p53 transcriptional activity in Akt-induced growth arrest. (A) The luciferase reporter gene plasmid pPG13-Luc containing the p53-binding sequence or pMG15-Luc containing the mutated p53-binding sequence was transfected into endothelial cells infected with pLNCX (Mock) or AktCA 24 h before the luciferase activity was measured. The activity of PG13-Luc in mock-infected cells is set at 100%. *P<0.005 versus Mock, ANOVA, _n_=4. (B) Human endothelial cells were infected with pBabe (empty vector) or pBabe E6 and purified with puromycin. Infected cells were then transduced with pLNCX or AktCA as described in Figure 1C and seeded at a density of 2 × 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_=4. (C) Morphology of cell populations prepared in (B). (D) Whole-cell lysates (30 μg) were extracted from cells prepared in (B) and examined for the expression of p53, p21 and actin (loading control).

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.

Figure 5

Pathophysiological role of Akt-induced endothelial cell senescence. (A) Immunohistochemistry for phospho-Akt (brown) in the coronary arteries (CA) and the internal mammary arteries (IMA) from the same patients. Scale bar: 10 μm. (B) Tube formation assay. Human endothelial cells infected with pLNCX (Mock) or AktCA were seeded onto Matrigel. After 48 h, the total tube length was estimated by an angiogenesis image analyzer (Kurabo, Osaka, Japan). The graph shows relative tube length in Mock- and AktCA-infected cells. The length in Mock-infected cells is set at 100%. *P<0.005 versus Mock, unpaired _t_-test, _n_=4. (C) Whole-cell lysates (30 μg) of pLNCX (Mock)- or AktCA-infected endothelial cells were examined for the expression of ICAM-1 and tubulin (loading control) by Western blotting.

Figure 6

Insulin promotes endothelial cell senescence. (A) Whole-cell lysates (30 μg) of human endothelial cells treated with insulin (1000 μU/ml) for 30 min were analyzed for the levels of phosphorylated FOXO3a and actin (loading control) by Western blotting. (B) The luciferase reporter gene plasmid PG13-Luc was transfected into endothelial cells in the presence of insulin at the indicated dose. At 24 h after transfection, the luciferase activity was measured. The activity in controls is set at 100%. *P<0.05, **P<0.0001 versus control, #P<0.01, ##P<0.001 versus insulin 30 μU/ml, _n_=4. (C) Human endothelial cells were cultured in the presence of insulin at the indicated dose and passaged. The number of cumulative population doublings was determined (_n_=3).

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Akt negatively regulates the in vitro lifespan of human endothelial cells via a p53/p21-dependent pathway - PubMed (original) (raw)