Regulation of MDMX expression by mitogenic signaling

Abstract

MDMX is an important regulator of p53 transcriptional activity and stress response. MDMX overexpression and gene amplification are implicated in p53 inactivation and tumor development. Unlike MDM2, MDMX is not inducible by p53, and little is known about its regulation at the transcriptional level. We found that MDMX levels in tumor cell lines closely correlate with promoter activity and mRNA level. Activated K-Ras and insulin-like growth factor 1 induce MDMX expression at the transcriptional level through mechanisms that involve the mitogen-activated protein kinase and c-Ets-1 transcription factors. Pharmacological inhibition of MEK results in down-regulation of MDMX in tumor cell lines. MDMX overexpression was detected in approximately 50% of human colon tumors and showed strong correlation with increased extracellular signal-regulated kinase phosphorylation. Therefore, MDMX expression is regulated by mitogenic signaling pathways. This mechanism may protect normal proliferating cells from p53 but also hamper p53 response during tumor development.

FIG. 1.

MDMX overexpression occurs at the transcriptional level. (A) Total RNAs and proteins from tumor cell lines were analyzed by quantitative PCR and Western blotting. The MDMX mRNA level was normalized to 18S rRNA (n = 3). (B) JEG-3, MCF-7, and HCT116 cells were treated with cycloheximide (CHX; 50 μg/ml) for 0, 2, 5, or 8 h and analyzed by Western blotting. (C) JEG-3, MCF-7, and HCT116 cells were treated with MG132 (25 μM) for 4 h and analyzed by Western blotting.

FIG. 2.

MDMX promoter analysis. (A) Cell lines were transfected with the 1.1-kb MDMX promoter-luciferase construct and CMV-LacZ. The luciferase/LacZ activity ratio is shown (n = 3). (B) MCF-7 (high endogenous MDMX expression) and H1299 (low endogenous MDMX expression) cells were transfected with MDMX promoter deletion constructs and normalized by CMV-LacZ expression (n = 3).

FIG. 3.

MDMX promoter mutation analysis. (A) Sequence of the human and mouse MDMX basal promoter (bp −120 to 0) with the positions of putative transcription factor binding sites underlined and mutated nucleotides in bold. (B to D) The full-length MDMX promoter and deletion mutations, as well as single, double, and quadruple point mutations in the full-length MDMX luciferase reporter construct, were tested for activity in H1299 (B), JEG-3 (C), and MCF-7 (D) cells (n = 3).

FIG. 4.

MDMX basal promoter activity requires c-Ets-1 and Elk-1. (A) H1299 cells were transfected with the full-length MDMX promoter and 50 ng of c-Ets-1 plasmid to induce MDMX promoter activity. MDMX promoter mutants were also transfected with 50 ng c-Ets-1 to determine the response of each binding site mutant to c-Ets-1 expression. (B) Synthetic RNA interference oligonucleotides targeted to Elk-1, c-Ets-1, and MDMX mRNA were transfected into U2OS cells using Oligofectamine reagent. After 48 h, cells were treated with actinomycin D for 20 h and analyzed for the expression level of indicated markers by Western blotting.

FIG. 5.

MAPK activity induces MDMX expression. (A) 35.8 (p53-null) and DKO (p53/ARF-double-null) murine embryonic fibroblasts stably infected with pBabe-HA-K-Ras (12V) virus were analyzed by Western blotting for indicated markers. (B) Total RNAs from 35.8 and DKO cells expressing activated K-Ras were analyzed for MDMX mRNA level by quantitative PCR (n = 6). (C) H1299 cells were transiently transfected with HA-K-Ras, HA-MEK1, B-RafV600E, or c-Ets-1. Endogenous expression of MDMX, phospho-ERK, ERK1/2, and actin was analyzed by Western blotting. (D) H1299 cells were transfected with full-length or EE1/EE2 mutant MDMX reporter constructs and expression vectors for HA-K-Ras, HA-MEK1, B-RafV600E, or c-Ets-1. The luciferase reporter activity for each of the transfection conditions is shown (n = 3).

FIG. 6.

Oncogenic K-Ras induces MDMX expression in an ERK-dependent manner. (A) 35.8 cells stably transfected with HA-K-Ras were treated with U0126 for 8 h and analyzed for expression of MDMX and phospho-ERK. (B) MCF-7 cells were treated with 37.5 μM PD98059 at indicated time points followed by Western blot analysis. (C) A panel of cell lines expressing different endogenous levels of MDMX were treated with 30 μM U0126 for 18 h and compared for expression of the indicated proteins and MDMX and MDM2 mRNA. (D) Cells were transfected with MDMX siRNA for 72 h and analyzed for expression of indicated markers by Western blotting.

FIG. 7.

c-Ets-1 and Elk-1 bind to the MDMX promoter in an ERK-dependent manner. (A) JEG-3 (high MDMX expression), U2OS, and H1299 (low MDMX expression) cells were analyzed by ChIP to detect binding of endogenous c-Ets-1 and Elk-1 to the basal MDMX promoter. YY1 was used as a negative control. PCR of a promoter element 3 kb upstream of the basal MDMX promoter was performed as a specificity control. (B) MCF-7 cells treated with 30 μM of MAPK (U0126) or p38 stress kinase inhibitor (SB203580) were compared to H1299 cells by ChIP analysis for c-Ets-1 and Elk-1 binding to the MDMX promoter.

FIG. 8.

IGF-1 induces ERK-dependent MCF-7 expression (A) MCF-7 cells were starved in Dulbecco modified Eagle medium with 0% serum for 24 h. IGF-1 (10 to 100 ng/ml) was added, and cells were analyzed 8 h later by Western blotting. (B) MCF-7 cells were transfected with MDMX promoter constructs for 24 h, serum starved for 24 h, and treated with 100 ng/ml IGF-1 for 8 h. Promoter activity was compared to that for MCF-7 cells in 10% serum (n = 3). (C) MCF-7 cells were serum starved for 24 h and treated with IGF-1 and inhibitors of PI3K (30 μM LY294002), MAPK (37.5 μM PD98059), or p38 kinase (30 μM SB203580) for 8 h. Cell lysate was analyzed by Western blotting. (D) Total RNAs from serum-starved MCF-7 cells treated with IGF-1, 30 μM LY294002, 37.5 μM PD98059, and 30 μM SB203580 were analyzed for MDMX mRNA levels by quantitative PCR (n = 3).

FIG. 9.

MDMX expression increases with tumor stage. (A) Representative MDMX immunohistochemical staining of normal colon mucosa and stage I to III tumors from a colon cancer tissue microarray (brown). An increased staining intensity as a function of tumor stage was observed. (B) Each tumor in the array was manually scored according to MDMX staining intensity from 1 to 3 and displayed according to the stage of colon cancer progression. The correlation between intensity of MDMX staining and the stage of colon cancer was calculated using Spearman's correlation analysis (n = 117; r² = 0.36; P < 0.0001). MDMX staining intensity in each stage was compared to that for normal colon mucosa, and the P value is indicated.

FIG. 10.

MDMX expression correlates with phospho-ERK level in colon cancer. (A) Representative pictures of colon tumors stained for MDMX (left) or phospho-ERK (right). Each pair of pictures is from consecutive sections of the same tumor at the same position. (B) Intensity of MDMX staining in the phospho-ERK-positive and -negative colon carcinomas. The correlation between intensity of MDMX staining and phospho-ERK was calculated using Spearman's correlation analysis (n = 117; r² = 0.24; P = 0.008). (C) A comparison of MDMX and phospho-AKT staining intensities in the colon microarray. There is no significant correlation between intensity of MDMX staining and phospho-AKT as calculated using Spearman's correlation analysis (n = 73; r² = 0.14; P = 0.22).