Is p53 the Long-Sought Molecular Trigger for Cyclophilin D–Regulated Mitochondrial Permeability Transition Pore Formation and Necrosis?
p53 Opens the Mitochondrial Permeability
Transition Pore to Trigger Necrosis
Vaseva et al
An article recently published in Cell concluded that p53 is necessary and sufficient to induce mitochondrial permeability transition pore (MPTP)–dependent necrosis through inducible p53 translocation to the matrix with cyclophilin D (CypD) binding. The results and implications are very provocative. The physiological significance of the proposed paradigm, however, is uncertain because calcium itself, which is a fundamental regulator of MPTP, is independent of p53, as shown by the authors. In addition, purified mitochondria from any unstimulated cell type or tissue, which presumably lacks p53 given the inducible mechanism proposed, have a fully functional MPTP to all the classic modes of stimulation as analyzed in vitro.
Myocardial infarction produces an area of myocyte loss that is a mixture of apoptotic and necrotic cell death. Recent data from multiple laboratories have shown that necrosis can be a programmed event and that specific molecular inhibitors of this process can be potently cardioprotective. During ischemic injury, both reactive oxygen species (ROS) and calcium levels increase within cells, triggering a type of programmed necrosis through MPTP opening that is CypD-dependent.1–5 Indeed, treatment with cyclosporine A, a CypD inhibitor, as well as genetic deletion of CypD, reduces ischemia/reperfusion injury in both the brain and the heart.3–5 Thus, mitochondria are central regulators of ischemic cell death through a mechanism involving the MPTP, but until recently we have lacked an understanding of how CypD is activated and potentially induces MPTP directly. However, Moll et al6 recently published an interesting article in the journal Cell, which shows that p53 can open the MPTP leading to necrotic cell death (Figure). They propose that upon ROS stimulation, p53 translocates to the matrix of the mitochondria where it binds to CypD and somehow opens the MPTP (Figure). This function of p53 was independent of its transcriptional activity or other functions in the cytosol, because a mitochondrial targeted form of p53 was able to drive MPTP opening by itself and mediate H2O2-dependent cellular necrosis. Surprisingly, this function of p53 seems to be completely independent of the Bcl-2 family members, as Bax and Bak double-knockout (DKO) mouse embryonic fibroblasts (MEFs) were susceptible to a combination of matrix-targeted p53- and H2O2-induced cell death. More surprisingly, calcium-dependent MPTP opening and cellular necrosis occurred independent of p53. Finally, p53+/− mice were protected from ischemia/reperfusion brain injury. This study is provocative because it suggests, for the first time, a molecular mechanism for how CypD senses and induces MPTP formation and subsequent induction of programmed cellular necrosis. It has long been appreciated that oxidative stress, which stabilizes and activates p53, sensitizes the MPTP to open, although calcium is typically also required. Thus, the results of Moll and colleagues suggest a universal integrating point whereby cytoplasmic stress signaling can program mitochondria-triggered cell death, both apoptotic and necrotic, through p53 (p53 was previously reported to induce Bax/Bak activity in programming outer mitochondrial membrane opening leading to apoptosis). Thus, the recent article begs the question, is p53 the long-sought-after trigger mechanism for MPTP formation and programmed necrotic cell death? In short, the authors’ own data show no regulation of calcium-dependent MPTP opening by p53, which is arguably the more important physiological mediator of MPTP opening.
Another issue to consider relates to the relationship of p53 with the proapoptotic Bcl-2 family members Bax and Bak. Moll and colleagues showed that p53 was able to induce mitochondrial swelling and death in Bax/Bak DKO MEFs. The authors suggest that p53 can regulate oxidative stress–induced programmed necrosis through its translocation to the mitochondrial matrix where it interacts with CypD to induce MPTP formation directly, independent of Bax/Bak. Previous work has already demonstrated that p53 can directly bind to multiple Bcl-2 family members and activate mitochondrial outer membrane permeabilization through Bax and Bak.7–9 Also, it is known that the proapoptotic Bcl-2 family members can sensitize MPTP opening.10 Taken together, it is plausible to hypothesize that p53 sensitizes MPTP opening through its ability to manipulate Bcl-2 family members. However, the authors interpret their results to mean that p53-mediated regulation of the MPTP is Bax/Bak-independent. The inconsistency here is the fact that DKO MEFs, which express p53, are known to be highly resistant to H2O2-induced cell death.11 If p53 functioned completely independent of Bax and Bak in this form of cell death, then DKO MEFs should be as susceptible to H2O2 necrosis as wild type MEFs, but this was not the case. Thus, DKO MEFs are protected from an insult that clearly mobilizes p53 and hence should be inducing the proposed mechanism of CypD-dependent necrosis with ROS stimulation, but it clearly does not given past data from multiple laboratories. This inconsistency suggests a problem in the overall model that is proposed whereby p53 directly binds CypD to induce MPTP, devoid of any regulation by Bcl-2 family members.
As alluded to above, Moll and colleagues present data in the supplemental information that calcium-dependent cellular killing and MPTP formation does not involve p53. This simple result already suggests that p53 is not a universal molecular switch in regulating programmed cellular necrosis through the MPTP. What regulates MPTP during calcium-induced mitochondrial swelling when p53 is absent is not known, especially because it remains CypD-dependent and inhibitable with cyclosporine. Such questions are hard to reconcile, with the known regulatory properties already ascribed to the MPTP in which calcium alone can cause swelling and programmed cell death through a CypD-dependent mechanism.
Another related and equally unresolved issue is that purified mitochondria from unstimulated cells or tissue are still fully able to undergo both calcium- and ROS-dependent swelling in vitro (this is well established). However, the authors suggest that purified mitochondria from unstimulated cells completely lack p53. So, if p53 is really serving as a necessary molecular trigger for the MPTP and the classic in vitro assay using purified mitochondria is physiologically meaningful (which has been debated), why does opening occur normally when p53 is apparently absent?
Although the data presented whereby p53 might regulate the MPTP through direct CypD binding are well controlled and convincing, upon further investigation some aspects of the data are less clear. For example, the authors conclude that the p53–CypD complex is only present when ROS are elevated, but the data show a p53–CypD complex (clearly visible) in the untreated fraction as well, which could simply be increased in the treated sample by the same ratio as total p53 accumulation in the cell itself with H2O2 stimulation. If so, it could suggest that the complex may not be inducible in the binary manner that was proposed. It is also perplexing that p53 is equally distributed to the mitochondrial outer membrane, intermembrane space, inner membrane, and matrix. What is p53 doing in the intermembrane space or inner membrane of the mitochondria? Regulation of protein localization in the mitochondria is typically well controlled, making it uncertain as to what machinery allows p53’s equal distribution to all mitochondrial subcompartments. Similarly, how can p53 independently regulate programmed necrosis from the mitochondrial matrix by binding CypD and at the same time regulate apoptosis at both the cell membrane (such as binding and direct activation of caspase 8) and the outer mitochondrial membrane by binding Bcl-2 proteins.12
There are a few other minor issues to consider, such as with the matrix-targeted p53 mutant. The authors show in purified mitochondria that recombinant p53 was sufficient to induce MPTP opening on its own, but adenoviral infection of cells with a matrix-targeted p53 mutant was not capable of driving cell death. Furthermore, p53 translocation to the mitochondria was previously shown to be inhibited by cyclosporine A, so additional experimentation is needed to better address the regulatory relationships between CypD and p53.13 Finally, what is the mechanism whereby p53 presumably activates CypD directly in promoting MPTP? Although there are many unanswered questions, the authors do present a convincing case that p53 has the ability to influence necrosis. The results of Moll and colleagues are important and exciting, but they are only a first step as much additional experimentation is needed to more convincingly solidify how p53 might be regulating programmed necrosis from within the mitochondria.
The opinions expressed in this Commentary are not necessarily those of the editors or of the American Heart Association.
Commentaries serve as a forum in which experts highlight and discuss articles (published here and elsewhere) that the editors of Circulation Research feel are of particular significance to cardiovascular medicine.
Commentaries are edited by Aruni Bhatnagar & Ali J. Marian.
- © 2012 American Heart Association, Inc.
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