Reprogramming somatic cells to induced pluripotent stem (iPS) cells has been accomplished by expressing pluripotency factors and oncogenes1–8, but the low frequency and tendency to induce malignant transformation9 compromise the clinical utility of this powerful approach. We address both issues by investigating the mechanisms limiting reprogramming efficiency in somatic cells. We show that reprogramming factors can activate the p53 pathway. Reducing signaling to p53 by expressing a mutated version of one of its negative regulators, by deleting or silencing p53 or its target gene, p21, or by antagonizing apoptosis enhanced three factor (Oct4/Sox2/Klf4)-mediated reprogramming of mouse fibroblasts. Notably, decreasing p53 protein levels enabled fibroblasts to give rise to iPS cells capable of generating germline transmitting chimeric mice using only Oct4 and Sox2. Furthermore, silencing of p53 significantly increased the reprogramming efficiency of human somatic cells. These results provide insights into reprogramming mechanisms and suggest new routes to more efficient reprogramming while minimizing the use of oncogenes.

The activities of p53 cover diverse aspects of cell biology, including cell cycle control, apoptosis, metabolism, fertility, differentiation and cellular reprogramming. Although loss of p53 function engenders tumor susceptibility, hyperactivation of p53 is lethal. Therefore, p53 activity must be strictly regulated to maintain normal tissue homeostasis. Critical for the control of p53 function are its two main negative regulators: Mdm2 and Mdmx. Recent reports have provided insight into the complex mechanisms that regulate these two proteins and have revealed novel functions for each. Here, we review and evaluate models of Mdm2- and Mdmx-dependent regulation of p53 activity. Both Mdm2 and Mdmx receive input from numerous signaling pathways and interact with many proteins in addition to p53. Therefore, we also consider roles for Mdm2 and Mdmx in additional cancer-related networks, including Notch signaling and the epithelial-to-mesenchymal transition.

P53 regulates numerous downstream targets to induce cell cycle arrest, senescence, apoptosis, and DNA repair in response to diverse stresses. Hdm2 and Hdmx are critical negative regulators of P53 because Hdm2 regulates P53 abundance, and both can antagonize P53 transactivation. Modest changes in Hdm2 or Hdmx abundance affect P53 regulation, yet quantitative information regarding their endogenous intracellular concentrations and subcellular distributions during a stress response are lacking. We analyzed these parameters in normal and cancer cells after DNA damage. Our data show that the nuclear abundance of Hdm2 and Hdmx relative to P53 limits P53 activity in cells growing in culture. Upon DNA damage, P53 nuclear abundance increases, whereas Hdm2 and Hdmx stability decreases, which greatly limits their ability to antagonize P53, regardless of their levels. These data indicate that the damage-activated switch in Hdm2 ubiquitin ligase preference from P53 to itself and Hdmx is central to P53 activation. The mechanisms by which P53 is activated after DNA damage have been widely studied (1). Two models that incorporate key roles for damage-activated kinases have emerged to explain how Hdm2-and Hdmx-mediated inhibition of P53 is overcome. The first proposes that activation of the ATM kinase leads to a kinase cascade resulting in phosphorylation of highly conserved serine and threonine residues in P53 within and flanking the Hdm2/ Hdmx-binding region (6). This induces a conformational change in P53, leading to Hdm2 dissociation, P53 stabilization and accumulation, and binding of P53 transcriptional coactivators (7,8). Histone acetyl transferase binding acetylates P53 C-terminal lysines and chromatin and promotes transactivation (9). The situation is likely more complex, because P53 in which highly conserved C-terminal lysines are replaced by arginines has basal and stress-induced stability and activity comparable with wildtype P53 (10, 11). Additionally, stabilization of P53 in the absence of detectable N-and C-terminal phosphorylation engenders full P53 activation (12). Thus, posttranslational P53 modifications fine-tune P53 transcription responses but do not act as on-off switches.A second model is supported by accumulating evidence that damage-activated kinases also phosphorylate Hdm2 to switch its E3 ligase specificity from P53 to itself and Hdmx (13-17). In part, the switch in substrate specificity is mediated by posttranslational modifications of Hdm2 and Hdmx that promote dissociation of the deubiquitinating enzyme HAUSP from Hdm2 and Hdmx (18). This increases Hdm2 and Hdmx degradation, resulting in P53 stabilization, accumulation, and transcriptional activation. Whether increasing Hdm2 levels is required for efficient Hdmx degradation after DNA damage remains unclear (13, 19). Together these data suggest a model in which destabilization and degradation of Hdm2 and Hdmx are critical for P53 stabilization and activation.Subtle perturbations in Hdm2 and Hdmx stoichiometry profoundly alter P53 activity and tumor sup...

Summary Mdmx is a critical negative regulator of the p53 pathway that is stoichiometrically limiting in some tissues. Post-translational modification and degradation of Mdmx after DNA damage have been proposed to be essential for p53 activation. We tested this model in vivo, where critical stoichiometric relationships are preserved. We generated an Mdmx mutant mouse in which three conserved serines (S341, S367, S402) targeted by DNA damage-activated kinases were replaced by alanines to investigate whether modifications of these residues are important for Mdmx degradation and p53 activation. The mutant mice were remarkably resistant to radiation, and very susceptible to Myc-induced lymphomagenesis. These data demonstrate that Mdmx down-regulation is crucial for effective p53-mediated radiation responses and tumor suppression in vivo. Significance P53 function is sensitive to the levels of its negative regulators, Mdm2 and Mdmx. Cell culture studies have suggested the importance of post-translational modifications in Mdm2 and Mdmx for p53 activation, but this has not been rigorously tested in vivo. This work shows that DNA damage and activated c-Myc both require phosphorylation of Mdmx in residues targeted by the damage kinases ATM and Chk2 for robust p53 activation. Preventing Mdmx post-translational modification stabilizes this negative regulator, which mitigates p53 activation, and presumably enables c-Myc to drive tumor cells with defective genomes into cycle in vivo. The data also stress the relevance of Mdmx as a potential therapeutic target.

h AMP-activated protein kinase (AMPK) has been shown to activate p53 in response to metabolic stress. However, the underlying mechanisms remain unclear. Here we show that metabolic stresses induce AMPK-mediated phosphorylation of human MDMX on Ser342 in vitro and in cells, leading to enhanced association between MDMX and 14-3-3. This markedly inhibits p53 ubiquitylation and significantly stabilizes and activates p53. By striking contrast, no phosphorylation of MDM2 by AMPK was noted. AMPK-mediated MDMX phosphorylation, MDMX-14-3-3 binding, and p53 activation were drastically reduced in mouse embryo fibroblasts harboring endogenous MDMX with S341A (mouse homologue of human serine 342), S367A, and S402A (mouse homologue of human serine 403) mutations. Moreover, deficiency of AMPK prevented MDMX-14-3-3 interaction and p53 activation. The activation of p53 through AMPK-mediated MDMX phosphorylation and inactivation was further confirmed by using cell and animal model systems with two AMPK activators, metformin and salicylate (the active form of aspirin). Together, the results unveil a mechanism by which metabolic stresses activate AMPK, which, in turn, phosphorylates and inactivates MDMX, resulting in p53 stabilization and activation.T he p53 tumor suppressor executes its antitumor functions primarily via its transcriptional activity to induce the expression of protein-encoding genes responsible for p53-dependent apoptosis, cell growth arrest, differentiation, and senescence (1) as well as its ability to induce apoptosis and autophagy by transcriptionindependent mechanisms (2). Since these cellular functions are detrimental to cells, p53 is often tightly monitored by a pair of partner proteins, MDM2 (called HDM2 in humans) and MDMX (also called MDM4), in normally growing cells (3-5). MDM2 and MDMX act as a complex during early embryogenesis (6-10) to ubiquitylate p53 and mediate its proteosomal turnover as well as inactivate its activity in a negative-feedback fashion (10-12), and cooperatively or individually restrain the p53 level to maintain the normal development and function of different tissues (13-16), by binding to p53, inhibiting its transcriptional activity and/or enhancing its ubiquitination. Hence, to activate p53, cells need to trigger different cellular mechanisms or pathways that block the MDM2-MDMX-p53 feedback loop through modifications of one of these proteins in response to a variety of stresses (17, 18). For instance, DNA damage signals can induce p53 by activating the ATM-Chk2 or ATR-Chk1 pathway that leads to phosphorylation of p53, MDMX, and MDM2 (19-21). Of relevance to MDMX, Ser367 phosphorylation by Chk2 or Chk1 triggers interaction between MDMX and 14-3-3, leading to MDMX inactivation and p53 activation (19,21,22). The importance of 14-3-3 binding to Ser367-phosphorylated MDMX for p53 activation by DNA damage was further emphasized in an animal knock-in study in which three serines, including Ser341, Ser367, and Ser402 (23), were mutated into alanines. This mutant MDMX exhibits substantial...