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First published online 28 February 2007
doi: 10.1242/dev.02820

Development 134, 1443-1451 (2007)
Published by The Company of Biologists 2007
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Met acts on Mdm2 via mTOR to signal cell survival during development

Anice Moumen1, Salvatore Patané1, Almudena Porras2, Rosanna Dono1 and Flavio Maina1,*

1 Developmental Biology Institute of Marseille-Luminy (IBDML) UMR 6216, CNRS-INSERM-Université de la Méditerrannée, Campus de Luminy-Case 907, 13288 Marseille Cedex 09, France.
2 Dpto. Bioquimica y Biologia Molecular II, Facultad de Farmacia, Universidad Complutense, Ciudad Universitaria, 28040 Madrid, Spain.


Figure 1
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  		Fig. 1. An intact PI3K pathway is required for Hgf-induced Mdm2 translocation. (A) Upon Hgf stimulation, wild-type (+/+) Met induces Mdm2 translocation from the cytoplasm into the nucleus of cultured primary embryonic hepatocytes. By contrast, Met2P, which has intact PI3K activity but reduced Akt signaling (met2P/2P), does not trigger Mdm2 translocation. (B) Quantitative analysis of Mdm2 translocation in wild-type (+/+) and mutant (2P/2P) embryonic hepatocytes. Mdm2 translocation in wild-type hepatocytes is abolished in the presence of the PI3K inhibitor LY 294002 (10 µM). (C) In vivo inactivation of p53 restores hepatocyte survival in met2P/2P mutant embryos. TUNEL staining of liver cryosections from E12.5 wild-type (+/+), met2P/2P treated (met2P/2P+PFT) or not (met2P/2P) with the p53 inhibitor pifithrin-{alpha} and double-homozygous met2P/2P; p53-/- embryos. (D) Quantitative analysis of TUNEL-positive cells in E12.5 liver sections of the indicated genotypes. The proportion of apoptotic cells is represented as fold increase compared with wild-type livers. The numbers of embryos analyzed in these studies are indicated (n). Arrows indicate nuclei. **P<0.001.

 

Figure 2
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  		Fig. 2. Met triggers translation of Mdm2 in cultured embryonic hepatocytes without affecting protein stability. (A) Time-course analysis of Mdm2 protein levels upon Hgf stimulation. Total extracts from wild-type embryonic hepatocytes were blotted and probed with {alpha}-Mdm2 (top) and {alpha}-tubulin (bottom). (B) Levels of newly synthesized Mdm2 in {alpha}-Mdm2 immunoprecipitates (top panel) of 35S-methionine/cysteine labeled embryonic hepatocytes treated or not with Hgf (H). The middle and lower panels show levels of Mdm2 and tubulin, respectively, on total lysates. (C) Rate of decay of Mdm2 protein in embryonic hepatocytes with high Mdm2 levels (Hgf stimulation for 24 hours; top panel) as determined by cycloheximide (CHX) treatment. Hgf treatment (H) does not change Mdm2 protein stability. CHX does not affect the half-life of tubulin. The decrease in Mdm2 protein levels was quantified after normalization with tubulin (bottom panel).

 

Figure 3
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  		Fig. 3. PI3K and its downstream signal Akt are required for Met-triggered Mdm2 translation in embryonic hepatocytes. (A) Hgf-mediated Mdm2 induction and Akt phosphorylation in wild-type hepatocytes is inhibited by LY 294002 in a dose-dependent manner. (B) Western blot analysis showing that Akt siRNA causes a reduction in Mdm2 protein levels in a concentration-dependent manner (100 and 200 pmol). Levels of phospho-S473 Akt and Akt, but not ERKs, were reduced in the presence of Akt siRNA. Control siRNA did not alter protein levels. (C) Hgf-induced Mdm2 translation is prevented by the Akt inhibitor A-443654 in a dose-dependent manner. Inhibition of Akt by A-443654 was confirmed following GSK3{alpha} phosphorylation.

 

Figure 4
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  		Fig. 4. The mTOR pathway is activated upon Hgf stimulation and is required for Met-triggered Mdm2 translation in embryonic hepatocytes. (A) Hgf activates mTOR-p70s6k signaling. p70s6k is phosphorylated on T389 upon Hgf stimulation. Because this phosphorylation is abolished in the presence of LY 294002 (5 µM) or rapamycin (rapa: 1 µM), it must require intact PI3K and mTOR signaling. (B) Hgf stimulation induces phosphorylation of the following mTOR downstream signals: p70s6k on residues T421/S424, S6 ribosomal protein on S235/236 and 4EBP1 on S65. (C) Rapamycin prevents the upregulation of Mdm2 by Hgf in embryonic hepatocyte cultures. (D) Newly synthesized Mdm2 and p53 proteins were analyzed by the immunoprecipitation of 35S-methionine/cysteine-labeled protein extracts from embryonic hepatocytes treated or not with Hgf and/or rapamycin. Rapamycin inhibits the translation of Mdm2, but not of p53. The bottom panels show levels of Mdm2 on total lysates.

 

Figure 5
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  		Fig. 5. Inhibition of Mdm2 and mTOR prevents Met-triggered cell survival in vitro. (A) Death of primary hepatocytes was triggered by actinomycin D (ActD) plus TNF{alpha}. Hgf-mediated cell survival was prevented by blocking Mdm2 function using the Mdm2 inhibitor Nutlin-3 or Mdm2 siRNA. The Hgf survival effects were also impaired by the mTOR inhibitor rapamycin (1 µM). (B) Quantitative analysis of cell death in primary embryonic hepatocyte cultures treated or not with actinomycin D plus TNF{alpha} (AT), Hgf (H), actinomycin D plus TNF{alpha} plus Hgf (ATH), Nutlin3, Mdm2 RNA interference and rapamycin. Data are representative of three independent experiments performed in duplicate. **P<0.001; Student's t-tests. (C) Western blot analysis showing that Mdm2 siRNA causes a reduction in Mdm2 protein levels in a concentration-dependent manner (50, 100 and 170 pmol). A non-silencing siRNA was used as control at concentration of 100 and 170 pmol. Control siRNA did not affect Mdm2 protein levels.

 

Figure 6
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  		Fig. 6. mTOR inhibition causes p53-dependent cell death by affecting Mdm2 protein levels in developing livers. (A) Analysis of Mdm2 (top) and mTOR (bottom) inhibition in E12.5 wild-type (+/+) and p53-/- mutant livers. Panels show TUNEL-positive cells in representative liver sections treated with Nutlin-3 or rapamycin. Cell death for each genotype is shown (right) as a percentage increase in the number of apoptotic cells compared with non-injected mice (*P<0.01; **P<0.001; Student's t-tests). Basal levels of TUNEL labeling were indistinguishable in non-injected wild-type and p53-/- mutant embryos. Analysis on p53 mutants indicated that death of embryonic hepatocytes is also controlled by unknown p53-independent pathways. (B) Western blot analysis of total lysates of E12.5 livers of wild-type embryos treated or not with rapamycin. Rapamycin treatment causes a reduction in the levels of Mdm2 (top), which correlates with an increased phosphorylation of p53 on the S18 residue (pS18-p53; middle), whereas tubulin levels are unchanged. Levels of Mdm2 protein and p53 phosphorylation were quantified (right) after normalization with tubulin. *P<0.01; **P<0.001; Student's t-tests. (C) Immunohistochemical analysis of p53 phosphorylation on the S18 residue of untreated (control), wild-type and p53-/- mice injected with rapamycin. Quantitative analysis (right) is reported as the percentage increase of cells positive for p53 phosphorylation in rapamycin-injected wild-type mice over the number of positive cells in non-injected mice. *P<0.01; Student's t-tests. The numbers of embryos analyzed in these studies are indicated (n).

 

Figure 7
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  		Fig. 7. A novel pathway involving mTOR and Mdm2 signals cell survival. Upon Hgf stimulation, Met triggers the nuclear translocation of Mdm2 by activating the PI3K-Akt pathway. In addition, Met enhances Mdm2 translation by acting on Akt-mTOR via PI3K. The relevance of this mechanism for Met-triggered cell survival has been genetically and pharmacologically demonstrated. Blocking a single step of this pathway alters the cell survival/death balance in embryonic cells.

 

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© The Company of Biologists Ltd 2007