The p53 tumor suppressor protein does not regulate expression of its own inhibitor, MDM2, except under conditions of stress - PubMed (original) (raw)

The p53 tumor suppressor protein does not regulate expression of its own inhibitor, MDM2, except under conditions of stress

S M Mendrysa et al. Mol Cell Biol. 2000 Mar.

Abstract

MDM2 is an important regulator of the p53 tumor suppressor protein. MDM2 inhibits p53 by binding to it, physically blocking its ability to transactivate gene expression, and stimulating its degradation. In cultured cells, mdm2 expression can be regulated by p53. Hence, mdm2 and p53 can interact to form an autoregulatory loop in which p53 activates expression of its own inhibitor. The p53/MDM2 autoregulatory loop has been elucidated within cultured cells; however, regulation of mdm2 expression by p53 has not been demonstrated within intact tissues. Here, we examine the role of p53 in regulating mdm2 expression in vivo in order to test the hypothesis that the p53/MDM2 autoregulatory loop is the mechanism by which low levels of p53 are maintained. We demonstrate that basal expression of mdm2 in murine tissues is p53 independent, even in tissues that express functional p53. Transcription of mdm2 is induced in a p53-dependent manner following gamma irradiation, indicating that p53 regulates mdm2 expression in vivo following a stimulus. The requirement for a stimulus to activate p53-dependent regulation of mdm2 expression in vivo appeared to differ from the situation in early-passage mouse embryo fibroblasts, where mdm2 expression is enhanced by the presence of p53. Analysis of mdm2 expression in intact and dispersed embryos revealed that establishment of mouse embryo fibroblasts in culture induces p53-dependent mdm2 expression, suggesting that an unknown stimulus activates p53 function in cultured cells. Together, these results indicate that p53 does not regulate expression of its own inhibitor, except in response to stimuli.

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Figures

FIG. 1

RNAs from both the P1 and P2 promoters of mdm2 are expressed in murine tissues. (A) Schematic of exons 1 to 3 of the mdm2 gene and of the S1 nuclease protection assay used to distinguish mdm2 transcripts from the P1 and P2 promoters. Indicated are the predicted sizes of hybridization products following digestion with S1 nuclease. (B) The amounts of mdm2 RNA from the P1 and P2 promoters were compared in spleen (lanes 1 to 3), thymus (lanes 4 to 6), kidney (lanes 7 to 9), heart (lanes 10 to 12), brain (lanes 13 to 15), and liver (lanes 16 to 18). The indicated tissues were isolated from three age-matched wild-type FVB/N animals. Twenty-five micrograms of total RNA from each tissue was analyzed for mdm2 RNA from the P1 and P2 promoters by S1 nuclease digestion following hybridization to a radiolabeled, denatured DNA probe complementary to mdm2 exons 1 to 3. The band that appears sporadically in some samples is an artifact from the probe.

FIG. 2

Expression from the mdm2 P2 promoter is independent of p53. Three adult FVB/N mice were sacrificed that were either wild type (WT) (lanes 1 to 3) or null for p53 (lanes 4 to 6). Twenty-five micrograms of total RNA from the indicated tissues was analyzed for mdm2 P1 and P2 transcripts by S1 nuclease protection assay. (A) Spleen. (B) Kidney. (C) Brain. (D) Thymus. (E) Heart. (F) Liver.

FIG. 3

Induction of the mdm2 P2 transcript following treatment with ionizing radiation (IR). Three adult FVB/N mice were sacrificed that had been either untreated (lanes 1 to 3) or treated 4 h previously with 5 Gy of ionizing radiation (lanes 4 to 6). Twenty-five micrograms of total RNA from the indicated tissues was analyzed for mdm2 P1 and P2 transcripts by S1 nuclease protection assay. (A) Spleen. (B) Kidney. (C) Brain. (D) Thymus. (E) Heart. (F) Liver.

FIG. 4

Requirement for p53 for the induction of mdm2 following treatment with (ionizing radiation IR). Adult FVB/N mice that were either wild type (lanes 3 to 5) or null for p53 (lanes 6 to 8) were sacrificed 4 h following 5 Gy of whole-body gamma irradiation. Twenty-five micrograms of total RNA from the indicated tissues was analyzed for RNAs from the mdm2 P1 and P2 promoters by S1 nuclease protection. For comparison, mdm2 RNA in wild-type (lane 1) and _p53_-null (lane 2) tissues prior to gamma irradiation was also analyzed by S1 nuclease protection. In the gel represented here, multiple start sites at the P2 promoter are distinguishable. (A) Spleen. (B) Kidney. (C) Brain. (D) Thymus. (E) Heart. (F) Liver.

FIG. 5

(A) S1 nuclease protection analysis of mdm2 RNA in MEFs. A comparison of the amounts of mdm2 RNA from the P1 and P2 promoters present in MEFs that were wild type (WT) for p53 (lanes 3 and 5) or null for p53 due to either deletion of p53 exons 2 to 6 (lane 4) or deletion of part of exon 5 (lane 6) is shown. MEFs were all early passage (lanes 3 and 6, passage 2; lanes 4 and 5, passage 1). Ten micrograms of total RNA was protected from S1 nuclease digestion. The negative control (−) was a reaction in which RNA was omitted from the hybridization reaction. As a positive control (+), probe was allowed to hybridize to RNA synthesized in vitro from plasmids designed to reflect transcriptional initiation at the mdm2 P1 or P2 promoter. (B) Levels of mdm2 RNA during establishment of MEFs. Seven wild-type embryos (A to G) were explanted at day 14. Tissue and/or cells were snap frozen in liquid nitrogen at various stages during the establishment of MEFs as follows: embryos A and B, immediately following explantation: C and D, following trypsinization for 15 min at 37°C; E and F, incubated at 37°C overnight following trypsinization; G, treated as embryos E and F, split into two populations (p and p′), and passaged every 3 to 4 days. Twenty-five micrograms of total RNA was analyzed for mdm2 RNAs from the P1 and P2 promoters by S1 nuclease protection.

FIG. 6

Model illustrating the regulation of p53 function and mdm2 expression under unstressed and stressed conditions. Under unstressed conditions, expression of mdm2 is regulated by an unknown factor, Y. The function of p53 is regulated by MDM2 or by unknown factor Z. Under stressed conditions, p53 activates mdm2 expression and MDM2 inhibits p53.

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The p53 tumor suppressor protein does not regulate expression of its own inhibitor, MDM2, except under conditions of stress - PubMed (original) (raw)