Download Puma joins the battery of BH3-only proteins that promote death and

Am J Physiol Heart Circ Physiol 291: H20 –H22, 2006;
doi:10.1152/ajpheart.00111.2006.
Editorial Focus
Puma joins the battery of BH3-only proteins that promote death and
infarction during myocardial ischemia
Keith A. Webster
Department of Pharmacology and the Vascular Biology Institute, University of Miami School of Medicine, Miami, Florida
Address for reprint requests and other correspondence: K. A. Webster, Dept.
of Pharmacology and Vascular Biology Institute, Rosenstiel Medical Science
Bldg., Rm. 1044C, Univ. of Miami School of Medicine, 1600 NW 10th Ave.,
Miami, FL 33136 (e-mail: [email protected]).
H20
of p53-null mice are similar to those of wild-type mice (16).
Therefore, p53 does not appear to be the principal mediator of
Puma during ischemic or hypoxic stress in the heart. In contrast
to BNIP3 and Noxa, which are induced by hypoxia through
promoter elements that bind the transcription factor hypoxiainducible factor-1␣, Puma has not been reported to possess
such a function and probably does not respond directly to
hypoxia. So how is Puma induced by ischemia? One possibility
is that it is regulated indirectly through components of the ER
stress pathways, which have been described in ischemic heart
and brain (13, 19). ER stress during ischemia is predicted to
occur under conditions of ATP depletion, acidosis, and abnormal ER/sarcoplasmic reticulum Ca2⫹ handling. In isolated
hippocampal neurons, Puma is induced by treatment of cells
with the ER stress mediators tunicamycin, a protein glycosylation inhibitor, and thapsigargin, an ER Ca2⫹-ATPase inhibitor
(13). Transcriptional activation of Puma coincides with induction of the early growth response factor-1 and the ER stressspecific transcription factor C/EBP homologous protein
(CHOP). In forebrain ischemia, Puma activation was reported
to occur late (48 –120 h) after ischemia-reperfusion and coincided with apoptotic death (13). The delayed induction occurred, despite early induction of CHOP, and was attributed to
a requirement for prolonged ER stress. The molecular mediators of Puma induction by such prolonged ER stress have not
been identified. If, as suggested by Toth et al., Puma has a
predominant role in apoptosis and necrosis in the heart, it will
be extremely important to identify the upstream regulation.
Two other factors that have been implicated in the transcriptional activation of Puma are p73, a homolog of p53, and E2F1,
a transcription factor that binds the retinoblastoma protein and
controls cell cycle regulatory genes (5, 9). The relations are
complex: E2F1 and p73 transactivate Puma directly by binding
and activating the promoter. E2F1 also transactivates p73, and
this may amplify the induction of Puma (11) (Fig. 1). Similarly, p73 has multiple and complex roles in apoptosis. In
addition to inducing Puma, p73 also stimulates the transcription of Bax (proapoptotic) and Scotin, a protein required for
initiating the ER stress pathway (3, 15). Brains of E2F-null
mice are resistant to focal ischemia, and at least one study
reported that apoptosis of cardiac myocytes exposed to simulated ischemia also requires active E2F (4, 8).
If E2F and/or p73 contribute to the induction of Puma in the
heart by hypoxia and/or ischemia, these conditions must activate these factor(s). This could occur by changes in the posttranslational regulatory pathways. There is some evidence for
this. Both p53 and p73 are subject to ubiquitination and
proteasomal degradation. Degradation of p53 is determined by
its binding to the murine double minute-2 ubiquitin ligase and
p73 by binding to the Hect ubiquitin ligase Itch (14). Binding
of p73 to Itch promotes ubiquitination and rapid proteasomedependent degradation. This process normally keeps p73 at
very low levels in most cells. Under conditions of stress, such
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(p53-upregulated modulator of apoptosis) is a member of
the Bcl-2 homology 3 (BH3)-only subfamily of Bcl-2-related
proapoptotic proteins that are implicated in numerous apoptotic
pathways responding to developmental signals and cellular
stress. Puma was originally identified as a target for the tumor
suppressor transcription factor p53 and was shown to play the
leading role in p53-mediated apoptosis (for review, see Ref.
20). Puma can bind and sequester all the prosurvival Bcl-2
proteins and initiates the intrinsic death pathway by activating
proapoptotic Bax and Bak (18). Death by Puma typically
involves increased permeability of the outer mitochondrial
membrane, leakage of cytochrome c and apoptosis-inducing
factor-1, and activation of caspases. Puma is activated at the
transcriptional level by p53 and is induced by all stimuli that
activate p53, including DNA damage and oxidative stress (19).
In addition, Puma mediates multiple p53-independent death
pathways initiated by diverse cytotoxic stimuli, including cytokine deprivation and exposure to glucocorticoids, the kinase
inhibitor staurosporine, phorbol esters, and conditions that
induce endoplasmic reticulum (ER) stress and the unfolded
protein response (2, 19). As an essential target for p53, Puma
is credited with the tumor suppressor function of p53 that
protects organs against cancer by eliminating cells that have
suffered DNA damage or proliferate in an uncontrolled manner
(20). As such, Puma has become an important target for
anticancer drugs. Recently, Puma was implicated in the apoptotic death of neurons resulting from ischemia-reperfusion and
copper and arsenite toxicity and as a component in the progression of Parkinson’s disease (13). In this issue of American
Journal of Physiology–Heart and Circulatory Physiology,
Toth et al. (15a) extended Puma’s activity repertoire even
further by making the important discovery that hearts of Pumanull mice are significantly resistant to ischemia-reperfusion
injury. Infarct sizes in the Puma-null hearts were reduced by
50% compared with wild-type or heterozygous mice. Apoptotic indexes were dramatically reduced, and postischemic
recovery was significantly improved. Toth et al. further report
that Puma levels were induced in cardiac myocytes exposed to
hypoxia under glucose starvation, and Puma promoted necrosis
as well as apoptosis if energy was depleted. This study aligns
Puma alongside other BH3-only proteins, including BNIP3,
Bid, Bad, and, possibly, Noxa, which are also implicated in the
death pathways that are activated by ischemia.
Whereas Puma appears to be essential for p53-mediated
apoptosis, p53 is not essential for Puma activation. Indeed,
Toth et al. (15a) observed that Puma-null mice were resistant
to ischemia-reperfusion, but p53-null mice were not. This latter
result confirms previous reports that ischemia-related infarcts
PUMA
Editorial Focus
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as DNA damage or oxidative stress, Itch is downregulated, and
p73 protein levels rise. Because this p73 regulatory pathway
was discovered very recently, it is not yet known whether Itch
or p73 levels change during ischemia in the brain or heart. If
Itch levels do not change, another pathway that could mediate
an increase of p73 and Puma in the ischemic heart is reduced
activity of the proteasome. Proteasomal activity in the heart is
significantly inhibited by ischemia-reperfusion (12).
Toth et al. (15a) report that infarction due to ischemiareperfusion decreased by 50% in mouse hearts with targeted
disruption of Puma. The reduced infarcts presumably involved
protection from Puma-mediated apoptosis and necrosis. These
observations prompt additional questions, in particular, the
contributions of other BH3-only proteins to infarction and the
extent to which each pathway is responsive to cardioprotective
interventions such as ischemic preconditioning and kinase
(PKC and JNK) inhibitors. Other BH3-only proteins that have
been causally linked to ischemic injury include Bid, Bad, and
BNIP3. Additionally, pathways that activate Puma, including
ER stress, EF2, and p73, also activate Noxa (15). If it is
assumed that the Noxa gene is expressed in the heart, it should
be induced in parallel with Puma. Noxa is also directly induced
by hypoxia through hypoxia-inducible factor-1␣ and may be
activated before Puma during ischemia (7). Bid is cleaved and
activated during ischemia-reperfusion, and Chen et al. (1)
report that blocking activation of the caspase-activated form of
AJP-Heart Circ Physiol • VOL
GRANTS
This work was supported by National Heart, Lung, and Blood Institute
Grants HL-44578 and HL-69812.
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Fig. 1. Ischemia-reperfusion mediates hypoxic and oxidative stress in cardiac
myocytes. Multiple pathways linked to hypoxia and reoxygenation may induce
p53-upregulated modulator of apoptosis (Puma). In one pathway, Puma is
induced by endoplasmic reticulum (ER) stress caused by hypoxia and glucose
depletion, reoxygenation, and abnormal Ca2⫹ flux. Transcription factor C/EBP
homologous protein (CHOP) is a major transcription factor activated by ER
stress, but it is not known whether CHOP is directly involved in Puma
induction. Oxidative stress may also inactivate Itch and/or the proteasome,
both of which may promote p73 accumulation. Puma is directly activated by
E2F and p73, and E2F also induces p73, creating a positive-feedback loop.
Scotin is a factor required for activation of the ER stress pathway and is
induced by p73, creating another positive-feedback loop. Once induced, Puma
translocates to mitochondria, where it joins forces with other Bcl-2 homology
3 (BH3)-only proteins to bind and neutralize prosurvival Bcl-2 proteins and
activate proapoptotic Bax and Bak. PKC-␦ also translocates during ischemiareperfusion and activates Bad. BNIP3 may function in combination with or
independently of the other BH3-only proteins. ROS, reactive oxygen species;
AIF-1, apoptosis-inducing factor-1.
Bid (tBid) with calpain inhibitors reduced infarct size by 50%
in rabbits. Similarly, Bad is dephosphorylated during reperfusion and translocates to the mitochondria to activate apoptosis.
Inhibition of Bad by blocking PKC-␦ was reported to reduce
infarction by ⬎80% (6, 10). BNIP3 is induced by hypoxia in
cardiac myocytes and by ischemia in the heart (17). BNIP3
also translocates to mitochondria, where it activates an atypical
programmed death pathway. The contribution of BNIP3 to
reoxygenation damage as determined by small interfering RNA
knockdown might also account for up to 50% of cardiac
myocyte death (unpublished observations). Puma, Bid, Bad,
and Noxa pathways converge on the intrinsic mitochondrial
pathway through direct or indirect interactions with Bax and/or
Bak (18). Puma and Bid bind and neutralize all Bcl-2 prosurvival proteins, whereas Bad and Noxa recognize only a subset
and, as a consequence, are weaker killers; therefore, complementary family members may be required for efficient killing.
To account for the contributions of multiple BH3-only proteins
to death and infarction, it is probable that they act in concert in
wild-type hearts, neutralizing prosurvival Bcl-2 proteins and
maximizing the activation of Bax and Bak. BNIP3 appears to
be capable of activating caspase-dependent and -independent
death pathways, but both involve mitochondrial signaling and
outer membrane puncture, which may complement the activities of the other BH3-only proteins. Further work is required to
determine the precise signaling pathways that mediate Puma
induction during ischemia-reperfusion, to define the interrelations between the different BH3-only proteins, and to identify
the optimal interventions for protecting the heart against these
combined death pathways.
Editorial Focus
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