PERP expression stabilizes active p53 via modulation of p53-MDM2 interaction in uveal melanoma cells

The activation and regulation of target genes by the tumour-suppressor p53 dictates the fate of a cell, with cell cycle arrest or apoptosis being two distinct outcomes. PERP (p53 apoptosis

effector related to PMP-22), a p53 transcriptional target, is induced specifically during apoptosis but not cell cycle arrest. Downregulation of PERP is associated with the aggressive,

monosomy 3-type of uveal melanoma (UM), the most common primary intraocular tumour in adults, and increased PERP expression has a pro-apoptotic effect in UM cells. Here, we identify a novel

effect of PERP expression, as elevated PERP protein positively influences active levels of its own transcriptional regulator, p53. Using fluorescent fusion proteins of PERP, p53 and MDM2, we

demonstrate in single living UM cells that PERP expression significantly enhances p53 activity and its nuclear localization, increases p53-dependent transcription (including that of MDM2)

while allowing oscillatory nucleo-cytoplasmic shuttling of p53/MDM2 complexes. Phosphorylation of p53 serine residues that interfere with the interaction between p53 and its negative

regulator MDM2 and enhance pro-apoptotic gene transcription also occurs subsequent to PERP expression. These results implicate a role for PERP in amplifying functional p53 levels that

promote p53-dependent apoptosis, and reveal a potential target for exploitation in enhancing p53 activity.

The regulation of p53 and of its impressive array of interacting and target genes holds a central place among the molecular mechanism(s) influencing the choice between cell cycle arrest and

apoptosis.1 The ability to engage in apoptosis is critical to the tumour-suppressor role of p53 that is strongly supported by the presence of p53 mutations in over half of human cancers2, 3

and the compromised p53 activity by other mechanisms in the majority of other cancers.3 The latter scenario points towards the deregulation of downstream p53 targets as a mechanism employed

by tumour cells to evade apoptosis. PERP (p53 apoptosis effector related to PMP-22) was identified as a p53 transcriptional target that is distinctively induced during apoptosis, and not

cell cycle arrest,4 and consequently emerged as a prime candidate effector in the p53-dependent apoptotic pathway. Subsequent studies confirmed the pro-apoptotic role for PERP in a variety

of cell types and tissues,5, 6, 7, 8 with reported engagement of caspase-dependent pathways.5, 8 However, the precise function of PERP – a tetraspan protein primarily localized at the plasma

membrane – in eliciting an apoptotic response remains unknown.

Cellular p53 levels are principally determined by the rate of degradation – mostly but not entirely linked to the E3 ligase activity of MDM2 – rather than synthesis.3, 9 Increased p53

stability in response to stress is achieved primarily through decreased MDM2 protein levels and reduced p53-MDM2 interaction, while the ensuing elevated levels of transcriptionally active

p53 result in increased MDM2 transcription.10, 11 This p53-MDM2 regulatory feedback loop is essential in maintaining tight regulation of p53 levels both in unstressed and stressed cells,

with additional control through posttranslational modifications, most notably phosphorylation of key p53 serine (Ser) residues,12 some of which influence the p53-MDM2 interaction13 and the

nature of p53-target genes transcribed.14 (Co)localization and nuclear-cytoplasmic shuttling of p53 and MDM2 also contribute to regulation of p53 activity.15, 16

We have shown that PERP is an important molecular determinant of apoptosis in primary uveal melanoma (UM) tumours that is significantly downregulated in the aggressive monosomy-3 type,

compared with less aggressive disomy-3 type of UM.5, 17 Downregulation of PERP (THW18) was also reported in tumours of the ovary, uterus and breast, and in cutaneous melanoma, pancreas and

mammary carcinoma cell lines, compared with the respective normal tissues and non-metastasizing cell lines.18

The aim of this study was to investigate the effect of increased PERP expression on its own upstream transcriptional regulator, p53. For this purpose, we used the UM cell line MEL20219 that

provides a biological background of low endogenous PERP protein with intact cell death machinery downstream of PERP.5 Our results show that PERP expression causes nuclear localization of p53

and increases the level of transcriptionally active p53 protein, which also presents posttranslational modifications known to influence the p53–MDM2 interaction and to enhance the

pro-apoptotic gene transcription. Together, these results propose a novel role for PERP in enhancing functional p53 levels and reveal a potential new target for exploitation in the

development of new therapeutic agents aimed at increasing the endogenous p53 protein pool in neoplastic cells.

To investigate the effect of increased levels of PERP protein on its transcriptional regulator p53, lysates of MEL202 cells expressing green fluorescent protein (GFP)–PERP were analyzed by

western blotting alongside lysates from control non-transfected (NT) and GFP-only-transfected cells. Significantly increased levels of p53 protein were observed in MEL202 cells expressing

GFP–PERP compared with control cells, in which p53 was barely detectable (Figure 1a). Furthermore, the increased p53 protein levels in response to PERP expression were accompanied by an

increase in the p53-negative regulator MDM2. As PERP is a transcriptional target of p53, the increase in p53 protein in response to PERP expression suggested a role for PERP in the positive

feedback regulation of p53. To determine if the observed increase in p53 and MDM2 proteins was transcription-driven, p53 and MDM2 transcriptional levels were determined by real-time

quantitative PCR (Q-PCR) in control and GFP–PERP-expressing cells. There was no significant increase in p53 mRNA in cells expressing PERP compared with GFP-only-expressing cells (T-test,

P≥0.19). However, MDM2 mRNA was significantly higher in cells expressing PERP at the three time points analyzed (T-test, P≤0.004; Figure 1b). Taken together, the results suggested that the

increased p53 protein levels detected in response to PERP expression may be a consequence of increased p53 protein stability rather than transcription, and that the ensuing p53 pool is

likely to be transcriptionally active resulting in increased MDM2 at both the transcriptional and protein level.

PERP expression augments p53 and MDM2 protein level in MEL202 cells. (a) p53 and MDM2 proteins are increased in cells expressing GFP–PERP. MEL202 cells were transfected with GFP–PERP and

lysates prepared at 24-, 48- and 72-h PT and analyzed by western blotting alongside NT and GFP-only-transfected cells that served as controls. A431 cell lysate served as a positive control

for the respective antibodies. PERP, p53 and MDM2 proteins were detected with appropriate antibodies and their relative levels were quantified by densitometry and normalized to GAPDH. (b)

Elevated PERP expression leads to increased MDM2 transcription. RNA was extracted from MEL202 cells transfected with GFP-only or GFP–PERP expression plasmids at the indicated times PT. The

levels of PERP, p53 and MDM2 mRNA were determined by Q-PCR and normalized to the respective endogenous level of GAPDH. Normalized mRNA levels are expressed relative to the respective levels

in GFP-only-transfected cells that were given an arbitrary value of 1. The mean of three independent experiments along with S.D. is presented. (*T-test, P ≤0.004 compared with

GFP-only-transfected cells). (c) Expression of exogenous PERP increases endogenous PERP protein. Representative western blots showing endogenous PERP protein level in control cells and cells

expressing GFP–PERP in MEL202 and U2OS cell lines; increased levels of endogenous PERP are detectable at 72-h PT in MEL202 cells and 48–72 h in U2OS cells. *a nonspecific protein that is

consistently detected with the anti-PERP antibody

There was also evidence that endogenous PERP protein (21 kDa) was slightly elevated in MEL202 cells at 72 h following transfection with GFP–PERP (Figure 1c). Owing to the low basal level of

endogenous PERP in MEL202 cells and similar intensities of nonspecific proteins, the level of endogenous PERP in response to GFP–PERP expression was further investigated in the wild-type

p53-expressing cell line U2OS. An increase in endogenous PERP was apparent in U2OS cells at 48 and 72 h following transfection with GFP–PERP (Figure 1c). It was conceivable that the observed

increase in endogenous PERP was due to the GFP–PERP-induced increased activity of its transcriptional regulator, p53. Consequently, it was important to characterize the functional status of

the raised p53 protein following GFP–PERP expression.

The effect of increased PERP expression described above was found in transiently transfected cell populations where, on average, the transfection efficiency was 13%. To refine our findings,

we assessed the effect of PERP expression on the subcellular localization and expression of the p53-transcriptional target MDM2 in individual cells by co-transfecting GFP–PERP with an

expression plasmid in which MDM2 was fused to yellow fluorescent protein (YFP) under the control of the human MDM2 native promoter that was previously shown to mirror the kinetic behaviour

of endogenous MDM2.20 The first striking observation was the almost exclusive nuclear localization of MDM2–YFP when co-expressed with GFP–PERP (in 96.8% of co-transfected cells; T-test,

P=0.02 and 0.04 versus MDM2–YFP or MDM2–YFP and GFP-only; Figures 2a and b). In contrast, MDM2–YFP expression alone or in combination with GFP-only expression showed an additional diffuse

cytoplasmic localization of MDM2 in many cells (28 and 31%, respectively; Figures 2a and b). Control cells transfected with YFP-only presented a diffuse YFP expression throughout the

cytoplasm and nucleus, which was maintained following co-expression of GFP–PERP (98% cells; Figures 2a and b).

PERP expression influences the nuclear translocation and the p53-driven expression of MDM2. (a) PERP expression leads to predominantly nuclear localization of MDM2. MEL202 cells transfected

with YFP-only, YFP-only and GFP–PERP, MDM2–YFP, MDM2–YFP and GFP-only, or MDM2–YFP and GFP–PERP were monitored by confocal fluorescence microscopy and the analysis of the intracellular

distribution of proteins of interest was undertaken at 20-h PT. The number of cells exhibiting predominantly nuclear MDM2 localization (N>C) or more even distribution in the nucleus and

cytoplasm (N≤C) were counted. Results are presented as the mean percentage of transfected cells from three independent transfections in each scenario with S.D., with 200 cells counted for

each transfection. MDM2 was predominantly nuclear in a significantly higher proportion of cells when co-expressed with GFP–PERP (*T-test, P=0.02 and P=0.04 versus MDM2–YFP or MDM2–YFP and

GFP-only-transfected cells, respectively). (b) Differential MDM2 subcellular distribution in the presence and absence of GFP-PERP. A predominantly nuclear MDM2–YFP localization (yellow) is

evident in cells co-expressing GFP-PERP, in contrast to the diffuse cytoplasmic/nuclear localization of MDM2–YFP in the absence of GFP–PERP and of YFP alone in control cells. Green

fluorescence shows the characteristic distribution of GFP or GFP–PERP proteins. The phase image of cells lacking GFP fluorescence is shown. Scale bar=50 μm. (c) Single cell analysis verified

that PERP expression leads to increased MDM2 expression. MEL202 cells were transfected and monitored as described in (a). Images were taken from three independent transfection experiments

and YFP fluorescence was measured in cells in arbitrary units (AU). Mean YFP fluorescence is indicated (♦) with S.D. MDM2–YFP fluorescence was significantly higher in cells co-expressing

GFP–PERP (T-test, P