Iron-dependent regulation of MDM2 influences p53 activity and hepatic carcinogenesis

Abstract

Iron overload is a risk factor for hepatocarcinoma, but the pathways involved are poorly characterized. Gene expression analysis in immortalized mouse hepatocytes exposed to iron or the iron chelator deferoxamine revealed that iron downregulated, whereas deferoxamine upregulated, mRNA levels of mouse double minute gene 2 (MDM2), the ubiquitin ligase involved in the degradation of the oncosuppressor p53. Regulation of MDM2 by iron status was observed at protein levels in mouse hepatocytes and rat liver, and was associated with specular changes in p53 expression. Iron dependent regulation of MDM2/p53 was confirmed ex-vivo in human monocytes, by manipulation of iron pool and in a genetic model of iron deficiency, leading to modulation of p53 target genes involved in the antioxidant response and apoptosis. Iron status influenced p53 ubiquitination and degradation rate, and the MDM2 inhibitor nutlin increased p53 levels in iron-depleted cells. Furthermore, nutlin enhanced the antiproliferative activity of deferoxamine in HepG2 hepatoblastoma cells. The MDM2 -309T > G promoter polymorphism, determining increased MDM2 and lower p53 activity, was associated with higher risk of hepatocarcinoma in cirrhotic patients with hemochromatosis, and with HFE mutations in patients with hepatocarcinoma without hemochromatosis, suggesting an interaction between MDM2 and iron in the pathogenesis of hepatocarcinoma. In conclusion, iron status influences p53 activity and antioxidant response by modulating MDM2 expression. MDM2 inhibitors may enhance the antiproliferative activity of iron chelators.

Figure 1

IRP activity as detected by IRP binding assay with relative densitometric quantification (top) and cell viability as detected by Trypan blue exclusion (bottom) according to iron status. AU: arbitrary units. SV40 hepatocytes were treated for 24 hours with 100 μmol/L Dfo (Dfo), 150 μmol/L FAC (FAC) or vehicle (Control). Cell viability was detected by Trypan blue exclusion and represents the mean ± SD of three independent experiments.

Figure 2

Effect of iron status on the MDM2/p53 pathway in SV40 mouse hepatocytes and in rat liver. A: Effect of Dfo and FAC on MDM2 and p53 protein levels in SV40 hepatocytes. Twenty-four hours after plating, cells were treated with 100 μmol/L Dfo (Dfo) or 150 μmol/L FAC (FAC) or vehicle for 24 hours and then lysed. MDM2, p53, and H-ferritin were detected in cell extracts by immunoblot analysis. The blots were reprobed with the antibody against β-actin as a loading control. B: Dose response relationship between iron availability and p53 protein levels in SV40 hepatocytes. Ct: control, untreated hepatocytes. Cells were treated for 24 hours with the indicated concentrations of FAC and Dfo. The upper part of the figure indicates the p53/β-actin ratio as detected by densitometry. AU: arbitrary units. Results are representative of two independent experiments. In the lower part of the figure the corresponding β-actin normalized MDM2 mRNA levels are shown. Results are representative of two independent experiments. *P < 0.05 vs. untreated cells. C: Effect of iron status on p53 protein and mRNA levels and cell viability in differentiating SV40 hepatocytes, at different days from cell plating. AU: arbitrary units. p53 AU indicates the p53/β-actin ratio as detected by densitometry. p53 mRNA levels were normalized for β-actin. *P < 0.05 vs. untreated cells 24 hours after plating (controls). Results are representative of two independent experiments. D: Effect of iron depletion by Dfo on serum iron, hepatic iron concentration, MDM2 and p53 protein levels in rat liver. White bars: control rats (n = 5), black bars: iron depleted rats (n = 4). Serum iron and hepatic iron concentration were measured by atomic absorption spectrometry. Protein expression of p53 and MDM2, was evaluated by densitometry and normalized for β-actin. AU: arbitrary units. *P ≤ 0.05 vs. control rats.

Figure 3

Effect of antioxidants on p53 protein levels in SV40 hepatocytes. After plating, cells were treated for 24 hours with or without NAC and α-Tocopherol, and then 150 μmol/L FAC was added to the culture medium for additional 24 hours. Ct: control, untreated cells, NAC: N-acetyl-cysteine 10 mmol/L, α-Toc: α-Tocopherol 60 μmol/L (+), 120 μmol/L (++). p53 levels AU (arbitrary units): indicates the p53/β-actin ratio as detected by densitometry in two independent experiments. *P < 0.05 vs. untreated cells. **P < 0.05 vs. cells treated with FAC 150 μmol/L alone. The Western blot shown in the lower part of the figure is representative of two independent experiments.

Figure 4

Effect of iron status on the MDM2/p53 pathway in human monocytes. A: Effect of iron status on p53 protein levels. Twenty-four hours after plating, after removal of lymphocytes, cells were treated as indicated for 24 hours. Ct: control, untreated monocytes. H₂O₂ is shown as positive control. β-actin is shown as a loading control. AU (arbitrary units): indicates the p53/β-actin ratio as detected by densitometry. Results are representative of two independent experiments. B: Expression of mRNAs of transferrin receptor-1 (up-regulated by iron depletion), MDM2, p53, and p53 target genes (including Bax, Aldh4a1, Sod2) in monocytes of subjects with C282Y +/+ HH (n = 6, black bars), and subjects with normal iron parameters negative for HFE mutations (n = 6, white bars). *P < 0.05 vs. control subjects. AU: mRNA levels, arbitrary units. C: MDM2 and p53 protein expression, as detected by Western blotting, in subjects with C282Y +/+ HH, and in subjects with normal iron parameters negative for HFE mutations. Three representative subjects are shown for each group. β-actin is shown as a loading control.

Figure 5

Effect of iron status on p53 expression is mediated through MDM2. A: Effect of Nutlin-3 on iron mediated regulation of p53 levels. The effect of manipulation of iron status with FAC and Dfo on p53 is compared in cells exposed or not to Nutlin-3. Human monocytes isolated from a healthy subject with normal iron parameters and negative for HFE mutations were cultured for 24 hours after removal of lymphocytes in the presence or in the absence of 25 μmol/L Nutlin-3, 100 μmol/L Dfo, and 150 μmol/L FAC. β-actin is shown as a loading control. p53 levels normalized for β-actin, as detected by densitometry, are shown in the upper part of the figure. Results are representative of two independent experiments. B: Effect of iron status on p53 polyubiquitination during inhibition of proteasomal activity. Human monocytes isolated from a healthy subject with normal iron parameters and negative for HFE mutations were cultured for 24 hours in the presence or not of 25 μmol/L Nutlin-3, 100 μmol/L Dfo, and 150 μmol/L FAC in the presence of the proteasomal inhibitor MG132. Proteins were immunoprecipitated with p53 antibody and pull-downs blotted with antibodies against ubiquitin (upper panel) and p53 (lower panel). The histogram shows the average levels of ubiquitylated/total p53 as determined by densitometric analysis of two independent experiments. *P < 0.05. C: Iron status affects p53 degradation kinetics. Human monocytes isolated from a healthy subject with normal iron parameters and negative for HFE mutations were cultured for 24 hours after removal of lymphocytes in the presence or not of 120 μmol/L Dfo, and 150 μmol/L FAC for 24 hours and then treated with CHX 40 μg/ml for 1 to 8 hours. Western blot analysis and densitometric analysis were performed for p53 levels, which were normalized for β-actin. Thus, relative expression >1 compared with baseline means that protein degradation was slower, whereas relative expression <1 means that protein degradation was faster than that of β-actin. Results represent means and SDs of two independent experiments. *P < 0.05 compared with controls.

Figure 6

Effect of iron status and of modulation of MDM2/p53 on cell proliferation in HepG2 human hepatoblastoma cells. A: Effect of iron status on H³-Thymidine incorporation in HepG2 cells untreated (Control) and treated for 24 hours with 150 μmol/L FAC (FAC) or 100 μmol/L Dfo (Dfo) in the presence or absence of insulin 0.33 μmol/L. Results are mean ± SD of three independent experiments. cpm: counts per minute. *P < 0.05 vs. untreated cells in the absence of insulin. B: Effect of iron status and of MDM2/p53 modulation on cell number in HepG2 cells untreated or treated for 24 hours with 150 μmol/L FAC (FAC) or 80/120 μmol/L Dfo (+/++ Dfo) in the presence or absence of 25 μmol/L nutlin (Nutlin). The horizontal line indicated the number of cells plated at the beginning of the experiment. *P < 0.05 vs. untreated cells in the absence of nutlin, **P < 0.05 vs. untreated cells in the presence of nutlin. C: Effect of iron status and of MDM2/p53 modulation on p53 protein levels in HepG2 cells untreated or treated for 24 hours with 150 μmol/L FAC (FAC) or 120 μmol/L Dfo (Dfo) in the presence or absence of 25 μmol/L nutlin (Nutlin). AU: protein levels normalized for β-actin, arbitrary units. Results are representative of two independent experiments. *P < 0.05 vs. untreated cells in the absence of nutlin, **P < 0.05 vs. untreated cells in the presence of nutlin.