Influence of metabolic stress on translation of p53 isoforms

ABSTRACT

p53 and its isoforms are integral in modulating transcriptional gene expression programs and maintaining cellular homeostasis. We recently reported that glucose deprivation/caloric restriction induced translational control of p53 mRNA by scaffold/matrix attachment region binding-protein 1 (SMAR1), adding a cytoplasmic role of SMAR1 to its traditional nuclear role as a transcription factor.

Abbreviations

AKR1a4

aldo-keto reductase protein 1a4

HIF1α

hypoxia inducible factor 1 α

IRES

internal ribosome entry site

ITAF

IRES trans-acting factor

MDM2

murine double minute 2 protein

PTB

polypyrimidine-tract binding protein 1

SMAR1

scaffold/matrix attachment region binding-protein 1

TIGAR

TP53 inducible glycolysis and apoptosis regulator

VEGF

vascular endothelial growth factor

Nutrient limitation or starvation induces cellular stress. An adaptive response entails a global shutoff of protein synthesis at both cap-dependent initiation and elongation steps of translation to preserve cellular energy.^1,2 Nevertheless, cap-independent translation supports selective translation under nutrient-deprived conditions.¹

We have recently demonstrated that glucose deprivation of cultured mammalian cells induces p53 internal ribosome entry site (IRES) activities.³ Translational control of p53 mRNA, which has a dual IRES structure, ensures levels of full-length p53 is predominantly regulated by cap-dependent translation initiation while IRES functions more as a back-up in stress conditions, when cap-dependent translation decreases.⁴ Δ40p53 translation, however, is solely IRES dependent. In our recent study, activities of both IRES modules of p53 mRNA were induced in reporter assays upon glucose starvation. There was a striking abrogation of induced p53 and Δ40p53 isoforms upon glucose deprivation in the backdrop of a greater turnover of p53 isoforms. Earlier studies suggest that ER stress induces Δ40p53 translation.⁵ Depleting glucose results in increased eIF2α phosphorylation, initiating ER stress and possibly explaining the upstream mechanism of our recent observation. A ternary complex of p53–murine double minute 2 protein (MDM2)– scaffold/matrix attachment region binding-protein 1 (SMAR1) was recently reported in cells during a post-stress recovery period.⁶ Apart from ubiquitinylating p53 protein, MDM2 binds to the coding region of p53 IRES RNA and promotes translation of full-length p53 and the Δ40p53 isoform.⁷ Moreover, p53 itself has demonstrated RNA-binding properties.⁸ Now, we have shown that p53 IRES is associated with SMAR1, and that the mouse SMAR1 ortholog can directly interact with p53 IRESs. We found that SMAR1 is directly linked to translation of both p53 and Δ40p53 in normal as well as glucose-deprived conditions. Using Δ40p53-mediated transactivation as a model, we showed that SMAR1 is important for IRES-dependent Δ40p53 induction, as well as for the consequent elevation of 14-3-3σ mRNA, an inhibitor of G2/M progression. Therefore, activation of p53 IRES by SMAR1 is likely to contribute more to Δ40p53 function than to that of full-length p53, as translational induction of the shorter isoform can occur through IRES alone. Induction of mRNA levels of 3 p53 transcription targets, p21, MDM2, and TIGAR, was suppressed in SMAR1 knockdown cells. Also, SMAR1 itself was overexpressed upon glucose deprivation in cells possessing endogenous p53 and Δ40p53 but not in null cells, probably suggestive of the need for a functional p53 pathway in this mechanism.

Nuclear-cytoplasmic relocalization of IRES-transacting factors (ITAFs) is essential for cellular IRES function. We found that glucose deprivation induced cellular redistribution of SMAR1 in cultured cells, with higher cytoplasmic SMAR1 levels post-starvation. Polypyrimidine-tract binding protein (PTB) is a critical ITAF for p53 IRESs and had been demonstrated to undergo similar relocalization under genotoxic stress. However, under glucose deprivation there was no such redistribution of PTB, highlighting the importance of SMAR1 for p53 IRESs. SMAR1 exhibited a greater interaction with p53 IRES in glucose-starved cells, as seen in ex vivo studies. VEGF and HIF1α IRES activities were refractory to SMAR1 knockdown, suggesting p53 mRNA-specific SMAR1-mediated translational control.

Preliminarily investigations showed a correlation of Δ40p53 and S-phase duration in glucose deprivation that is contingent on SMAR1 levels. In vivo experiments in mice demonstrated that there was a marked increase in Smar1 levels in liver and thymus after 24 hours of caloric restriction (40% decrease in blood glucose levels) that corresponds to an increase in the levels of p53 and p44 (mouse Δ40p53) protein. In the liver from Smar1 transgenic mice (with a high steady state level of Smar1), the increase in Smar1 was more evident within 12 hours of caloric restriction (20% decrease in blood glucose levels) with a corresponding increase in p44 at this time point, suggesting that glucose sensing might depend on Smar1 levels with at least one of the outputs being a change in the ratio of p53 isoforms. Rescue experiments, in which glucose was replenished in the medium in vitro or mice were fed ad libitum after caloric restriction, restored the levels of SMAR1/Smar1 and p53 isoforms, as well as p53 and Δ40p53/p44 targets, to unstarved states, suggesting a reversible, nutrient-responsive cellular switch.

Cytoplasmic SMAR1 was reported to regulate free radical stress through modulation of the enzyme AKR1a4, a monomeric NADPH-dependent oxidoreductase that is involved in glucose metabolism, ascorbic acid metabolism, and cytochrome P450 activation.⁹ Our study opens up another layer of cytoplasmic SMAR1 function as a mediator of translation. An open question is whether the p53–MDM2–SMAR1 ternary complex regulates the translation of p53 mRNA. Also, apparent changes in cell-cycle phase durations upon glucose deprivation, coupled with the reversibility of the SMAR1-p53/Δ40p53 switch upon stress removal, indicate that altering p53 isoform ratios is a survival-directed adaptation response to nutrient deprivation. Another attractive question is directed to the metabolic consequences of such alterations under continued nutrient deprivation, such as in tumors: does differential translational induction and the altered ratio of p53 isoforms feed into anaplerosis in tumor cells, fueling survival?

One of the hallmarks of tumor cells is a drastic change in the metabolic rate, in which p53 plays an important role. p53 normally drives the regulation of glycolysis, mitochondrial oxidative phosphorylation, and lipid metabolism. Both loss-of-heterozygosity and specific mutations in the hotspots within p53 can globally change cancer cell metabolism. Thus, control of p53 and its isoforms by regulators such as SMAR1 has direct implications for tumor therapy. Interestingly, SMAR1 has been shown to be downregulated in many cancers.¹⁰ Thus, the use of small compounds that can induce or stabilize SMAR1 in cancer cells might have therapeutic implications. It is suggested that the primary cause of cancer is replacement of normal utilization of oxygen within healthy cells via breakdown of glucose. Cells generate most of their energy through conversion of pyruvate and oxygen in the mitochondria; however, in many cancers mitochondrial functions are switched off and cancer cells survive through the use of alternative pathways. Since SMAR1 is a novel tumor suppressor and its expression is turned on upon glucose starvation, it is important to understand the role of SMAR1 in regulating metabolic pathway genes, especially those that are dependent on p53 and its isoforms. It would also be important to correlate causes of SMAR1 downregulation in cancers and its downstream consequences on SMAR-p53 mediated gene regulation.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.