Mdm2 is a target and mediator of IRP2 in cell growth control

Abstract

Iron is an essential element to all living organisms and plays a vital role in many cellular processes, such as DNA synthesis and energy production. The Mdm2 oncogene is an E3 ligase and known to promote tumor growth. However, the role of Mdm2 in iron homeostasis is not certain. Here, we showed that Mdm2 expression was increased by iron depletion but decreased by iron repletion. We also showed that Iron Regulatory Protein 2 (IRP2) mediated iron-regulated Mdm2 expression. Specifically, Mdm2 expression was increased by ectopic IRP2 but decreased by knockdown or knockout of IRP2 in human cancer cells as well as in mouse embryonic fibroblasts. In addition, we showed that IRP2-regulated Mdm2 expression was independent of tumor suppressor p53. Mechanistically, we found that IRP2 stabilized Mdm2 transcript via binding to an iron response element (IRE) in the 3'UTR of Mdm2 mRNA. Finally, we showed that Mdm2 is required for IRP2-mediated cell proliferation and Mdm2 expression is highly associated with IRP2 in both the normal and cancerous liver tissues. Together, we uncover a novel regulation of Mdm2 by IRP2 via mRNA stability and that the IRP2-Mdm2 axis may play a critical role in cell growth.

Keywords: IRP2; Iron homeostasis; Iron-response element (IRE); Mdm2; mRNA stability.

Figure 1

Mdm2 expression is regulated by iron. (A) HEPG2 cells were mock-treated or treated with various amounts of DFO or FAC, followed by western blot analysis to examine the levels of IRP2, Mdm2 and actin proteins. (B-C) MCF7 (B) and HCT116 (C) cells were mock-treated or treated with DFO (0-300 μM, 24h) for 24 hours and the levels of IRP2, TFR, Mdm2, p53, and actin proteins were examined by western blot analysis. (D-E) HCT116-p53-KO (D) and Mia-PaCa2 (E) cells were mock-treated or treated with DFO (0-300 μM, 24h) for 24 hours and the levels of IRP2, TFR, Mdm2, and actin proteins were examined by western blot analysis.

Figure 2

IRP2, but not IRP1, is required for iron-mediated Mdm2 expression. (A-B) H1299 cells were uninduced or induced to express HA-tagged IRP1 (A) or IRP2 (B) for 24 hours, the levels of IRP1 (A), IRP2 (B), Mdm2, and actin proteins were examined by western blot analysis. (C) The level of IRP1, Mdm2, and GAPDH transcripts was measured in Mia-PaCa2 cells transfected with a scrambled or IRP1 siRNA for 3 days. (D) HCT116 and MCF7 cells were transfected with a scrambled or IRP2 siRNA for 3 days, and the levels of IRP2, Mdm2 and vinculin were examined by western blot analysis. (E-F) Isogenic control and IRP2-KO p53^−/−HCT116 (D) and Mia-PaCa2 (E) cells were used to examine the levels of IRP2, Mdm2, vinculin (D) and actin (E) proteins by western blot analysis.

Figure 3

Loss of IRP2 leads to reduced Mdm2 expression in primary mouse embryonic fibroblasts independent of p53. (A) The levels of Mdm2, p53 and actin proteins were examined in wild-type MEFs treated with or without DFO or DFP. (B) Wild-type and IRP2^−/− MEFs were mock-treated or treated with doxorubicin for 18 hours and the levels of IRP2, Mdm2, and vinculin proteins were determined by western blot analysis. (C) The levels of IRP2, Mdm2, and vinculin proteins were examined by western blot analysis in p53^+/− and p53^+/−;IRP2^−/− MEFs. (D) The levels of IRP2, Mdm2, and actin proteins were examined by western blot analysis in p53^−/− and p53^−/−;IRP2^−/− MEFs.

Figure 4

The level of Mdm2 transcripts is reduced by loss of IRP2. (A-B) H1299 cells were uninduced or induced to express HA-tagged IRP2 for 24 hours and the levels of Mdm2, TFR, and actin transcripts were examined by semi-quantitate (A) or quantitative (B) RT-PCR analysis. * indicates p < 0.05 by paired Student’s t test. (C-D) The levels of Mdm2 and GAPDH transcripts were examined in isogenic control and IRP2-KO p53^−/−HCT116 cells by semi-quantitate (C) or quantitative (D) RT-PCR analysis. * indicates p < 0.05 by paired Student’s t test. (E-G) The levels of Mdm2 and GAPDH transcripts were examined in isogenic control and IRP2-KO Mia-PaCa2 cells treated with or without DFO or DFP by semi-quantitate (E) or quantitative (F-G) RT-PCR analysis. * indicates p < 0.05 by paired Student’s t test. (H-J) The levels of IRP2, Mdm2 and GAPDH transcripts were examined in wild-type and IRP2^−/− MEFs (H), p53^+/− and p53^+/−;IRP2^−/− (I), and p53^−/− and p53^−/−;IRP2^−/− MEFs (J). (K) Quantitative RT-PCR analysis was performed with the samples in H-J. * indicates p < 0.05 by paired Student’s t test.

Figure 5

IRP2 is required for Mdm2 mRNA stability via binding to the IRE in its 3’UTR. (A) The half-life of Mdm2 transcripts was measured in isogenic control and IRP2-KO p53^−/−HCT116 cells treated with DRB for various times. The level of MDM2 transcript was normalized to that of GAPDH control and the relative half-life of MDM2 was calculated. (B) IRP2 associates with MDM2 transcript in vivo. H1299 cells were uninduced or induced to express HA-tagged IRP2, followed by immunoprecipitation with anti-HA or an isotype control IgG. RT-PCR analysis was performed to measure the level of TFR, MDM2 and GAPDH transcripts in the control and IRP2-RNA immunocomplexes. (C) Schematic representation of the Mdm2 transcript, the locations of IREs as well as the reporters containing various lengths of Mdm2 3’UTRs. (D-H) p53^−/− HCT116 cells were transiently transfected with various amounts of IRP2 expression vector and a fixed amount of EGFP expression vector alone (D) or a EGFP vector fused with Mdm2-3’UTR-A (E), Mdm2-3’UTR-B (F), Mdm2-3’UTR-C (G), or Mdm2-3’UTR-D (H). Twenty-four hours posttransfection, the levels of IRP2 (D-H), EGFP (D-H), vinculin (D), and actin (E-H) proteins were examined by western blot analysis. (I-L) The experiments were performed as in (D-H) except that an EGFP vector fused with Mdm2-3’UTR-A1 (I), Mdm2-3’UTR-A2 (J), Mdm2-3’UTR-A3 (K), or Mdm2-3’UTR-A2-M (L) were used.

Figure 6

The IRP2-Mdm2 axis plays a role in cell proliferation. (A) Isogenic control and IRP2-KO p53^−/− HCT116 cells were transiently transfected with a control vector or a vector expressing IRP2 or Mdm2 for 24 hours, followed by colony formation with or without treatment of DFO (100 μM, 24 hours). (B) Quantification of colony formation assays in (A). * indicates p < 0.05 and ** indicates p< 0.01 by paired Student’s t test. (C) Mdm2 expression is associated with IRP2 expression in normal liver tissues (left panel) and liver hepatocellular carcinomas (right panel). The analysis was performed using the GEPIA2 database (http://gepia2.cancer-pku.cn/#correlation). Statistical analysis suggests a strong correlation between IRP2 and Mdm2 expression in normal liver tissues (Pearson’s r=0.67) and cancerous liver tissues (Pearson’s r=0.4). (D) A model proposed to elucidate the interplay among IRP2, Mdm2, and p53.