Departments of Pediatrics and Communicable Diseases, Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan 48109-0212
PULMONARY DYSFUNCTION resulting from acute oxygen toxicity is due at least in part to the death of lung epithelial cells. Pulmonary epithelial cells are essential to the maintenance of the alveolar-capillary barrier, and compromise may lead to leakage of fluid and proteins in the airways and alveoli, with consequent clinical deterioration.
In animal models, prolonged exposure to hyperoxia first induces capillary endothelial cell death, followed by that of the alveolar type II cells (6). Recent studies have addressed whether the observed epithelial cell death is due to apoptosis or necrosis. Necrosis is associated with disruption of the cell membrane, with resulting loss of cytoplasm and random nuclear degeneration. In apoptosis, the plasma membrane is intact, and therefore DNA degradation is the result of specific endonucleases. In neonatal and mature mice, both necrosis and apoptosis contribute to cell death during hyperoxia (2, 12).
Epithelial cell necrosis with disruption of the cell membrane may result from the direct oxidation of membrane lipids. Products of lipid oxidation have been assessed in adults with respiratory distress syndrome and premature infants undergoing mechanical ventilation. Exhaled breath condensate isoprostanes, prostanoid compounds primarily formed nonenzymatically via lipid peroxidation, are elevated in patients with acute lung injury or adult respiratory distress syndrome (4). Plasma concentrations of the lipid aldehydes heptanal 2-nonenal and 4-hydroxynonenal were higher in the first day of life in infants who developed bronchopulmonary dysplasia (BPD) than in those that did not (16). High exhaled pentane has also been associated with several adverse outcomes in premature infants, including BPD (15).
Apoptosis can also be triggered by oxidative stress. The signaling pathways and transcription factors responsible for apoptotic cell death in hyperoxic lung injury have been reviewed elsewhere (11, 13) and will not be discussed here. However, it is important to note that, although apoptosis may not necessarily be the predominant form of epithelial cell death in lung-injured patients, strategies aimed at preventing apoptosis may protect against lung injury. Administration of PD-98059, a chemical inhibitor of signaling through the extracellular signal-regulated pathway, and diphenyl iodonium, a reactive oxygen species inhibitor, each attenuates lung epithelial cell death in mice undergoing hyperoxic exposure (27). A constitutively active form of the serine-threonine kinase Akt introduced intratracheally into the lungs of mice by adenovirus gene transfer is protective against hyperoxic pulmonary damage and delays death (10). In a tetracycline-inducible, lung-specific transgenic system, it has been shown in mice that keratinocyte growth factor protects the lung epithelium against hyperoxia (18). On the other hand, mortality and pulmonary epithelial cell apoptosis were increased in Jun amino-terminal kinase-1(-/-) mice compared with wild-type mice after exposure to continuous hyperoxia (14). Most recently, it has been demonstrated that hyperoxic exposure markedly increases mortality and apoptosis of respiratory epithelial cells and macrophages in mice with Legionella pneumonia and that mortality is attenuated in Fas-deficient mice resistant to apoptosis (21). Together, these results strongly suggest that manipulation of the signaling pathways regulating respiratory epithelial cell apoptosis represents a promising strategy for the reduction of lung injury.
Mammalian cells themselves possess an innate strategy for preventing hyperoxia-induced DNA damage and epithelial cell death (whether apoptotic or necrotic): delay or arrest of cells in G1 or G2/M of the cell cycle. This delay facilitates DNA repair and avoids the replication and subsequent propagation of potentially hazardous mutations. As shown by Helt and colleagues in one of the current articles in focus (Ref. 8, see p. L506 in this issue), hyperoxia-induced G1 cell cycle arrest is mediated by the cyclin-dependent kinase (Cdk) inhibitor p21Waf1/Cip1 (also called CDKN1A).
p21Waf1/Cip1 belongs to the Cip/Kip family of Cdk inhibitors (p21Waf1/Cip1, p27Kip1 and p57Kip2), which share sequence homology in their amino-terminal region, which is responsible for binding to cyclin and Cdk targets. The unique carboxy-terminal domain of p21Waf1/Cip1 associates with proliferating nuclear antigen (PCNA), a cofactor required for DNA synthesis and repair. Expression of p21 is induced by mitogenic stimulation and also by the tumor suppressor protein p53.
Helt and colleagues (8) studied the mechanisms underlying hyperoxia-induced cell cycle arrest. First, they found that p53-deficient human lung adenocarcinoma H1299 cells failed to induce p21 or G1 arrest when exposed to 95% oxygen, proving the requirement of p53 for p21 induction and hyperoxia-induced G1 arrest. Instead, p53-deficient cells arrested in S and G2, demonstrating the presence of p53-independent pathways leading to G2/M arrest. Furthermore, stable expression of p53 restored induction of p21 and G1 arrest without affecting mRNA expression of another Cdk inhibitor, p27, indicating the sufficiency of p53 for p21 expression and hyperoxia-induced G1 arrest.
However, the above observations do not prove the sufficiency of p21 for hyperoxia-induced responses, nor do they address the molecular mechanisms underlying p21's effect on cell cycle traversal. To address this, the authors established an elegant set of tetracycline-inducible stable cell lines expressing p21, the p21 amino-terminal Cdk-binding domain, or the p21 carboxy-terminal PCNA binding domain. Expression of each construct restored G1 arrest during hyperoxia, demonstrating that p21 is sufficient to induce G1 arrest and may do so either by inhibition of Cdks or by inhibition of PCNA-facilitated DNA synthesis. Because PCNA also participates in DNA repair, these results raise the possibility that p21 also affects repair of oxidized DNA.
The potential importance of p21 and the delay of cell cycle progression following hyperoxia-induced oxidative DNA damage has been previously demonstrated by O'Reilly and colleagues (17), who showed that mortality and epithelial cell death are significantly increased in p21-deficient mice. These data imply that upregulation of p21 might also improve survival in patients with lung injury.
To put these results in context, I would like to briefly summarize the cell cycle response to hyperoxic exposure: In response to DNA damage due to such factors as ionizing radiation, ultraviolet light, chemotherapeutic drugs, and hyperoxia, the tumor-suppressive transcription factor p53 is post-translationally modified on at least 18 sites (1). Such modifications, which include acetylation, phosphorylation, glycosylation, and sumoylation, regulate the stability, subcellular localization, and transcriptional activation of the protein. p53 levels are usually maintained at a low level by murine double minute-2 (MDM2), a ubiquitin ligase that allows recognition and degradation by the proteosome. Phosphorylation of the p53 MDM2 binding region in response to DNA damage, which is mediated by the kinases checkpoint kinase (Chk) 1, Chk2, ataxia-telangiectasia mutant (ATM), and ATM and Rad3-related (ATR), impairs the interaction with MDM2 and stabilizes the protein (3, 25). These and other upstream activators are also required for nuclear localization and maximal transcriptional activity. Once activated, p53 induces the expression of over 150 genes, most of which are related to cell cycle arrest and apoptosis. p53 can differentially drive cell cycle arrest or apoptosis, depending which target genes it chooses to activate, which in turn is determined by interaction of p53 with specific transcriptional cofactors including p300/CREB binding protein, Zac1, YB-1, AMF1, ASPP, and JMY. Among the cell cycle proteins regulated by p53 are Cdk4-activating kinase (CAK), PCNA, Cdc2, cyclin B1, Gadd45, 14-3-3, Reprimo, topoisomerase II (22, 24), and, as shown by Helt and colleagues (8), p21Waf1/Cip1. It should be noted that these cell cycle intermediates are important not only for G1/S phase traversal but also for regulation of the G2/M transition, and, therefore, p53 may cause G2/M arrest independently of p21 (22).
There are two classes of Cdk inhibitors. The first class, which includes the 12- to 20-kDa INK4a proteins, binds only to Cdks 4 and 6 but not to cyclins and, therefore, is specific only for early G1 phase. The second family is composed of Cip/Kip proteins such as p21 and p27 that inhibit all cyclin-Cdk complexes and is not specific for a particular phase. On the basis largely of in vitro experiments and in vivo overexpression studies, Cdk inhibitors of the Cip/Kip family were initially thought to interfere with the activities of cyclin D-, E-, and A-dependent kinases. It is now thought that Cip/Kip proteins are potent inhibitors of cyclin E- and A-dependent Cdk2, but positive regulators of cyclin D-dependent kinases cdk4 and -6.
To review, G1 of the cell cycle (19), cyclin D1, along with Cdk4, PCNA, and p21Waf1/Cip1, are induced as part of the delayed early response to mitogenic stimulation (26) (Fig. 1). On the other hand, the levels of p27Kip1 in quiescent cells are relatively high. The cyclin D1/Cdk4 dimer titrates p27Kip1 and also enters into complexes with PCNA and p21Waf1/Cip1. At this stage, p21Waf1/Cip1 acts as an assembly factor rather than Cdk inhibitor, promoting binding of cyclin D1 with Cdk4. Once enough cyclin D1 and Cdk4 are synthesized, steric inhibition by p27Kip1 is exceeded, leading to phosphorylation and activation of cdk4 by CAK. CAK is a multiunit enzyme composed of a catalytic subunit (Cdk7) and cyclin H. Activation of Cdk4, in turn, leads to hyperphosphorylation of the p110 retinoblastoma protein (Rb). Rb phosphorylation in mid-G1 of the cell cycle releases the transcription factors E2 promoter binding protein (E2F)1-3, which activate genes required for DNA replication including thymidine kinase, thymidylate synthetase, DNA polymerase-, and cyclins E and A. Expression of cyclin E is periodic, maximal at the G1/S transition, and associated with the formation of cyclin E-Cdk2 complexes. Cyclin E-Cdk2 holoenzyme activation completes the process of S phase entry by phosphorylating Rb on additional sites. Once cells enter S phase, cyclin E is degraded and Cdk2 associates with cyclin A. Active cyclin A-Cdk2 complexes maintain Rb in a hyperphosphorylated form until the cells exit mitosis and Rb is returned to a hypophosphorylated state in the next G1 phase. This is because, as noted above, Cip/Kip proteins are potent inhibitors of cyclin E- and A-dependent Cdk2, but positive regulators of cyclin D-dependent kinases Cdk4 and -6. Titration of p21 and p27 into nascent cyclin D-Cdk complexes progressively relieves cyclin E-Cdk2 from its main inhibitory constraint, thereby inducing Cdk2 activity and cell cycle progression.
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Both p21 and p27 bind to cyclins and Cdks via specific sites in their amino-terminal regions; the Cdk site inserts deep inside the catalytic cleft of the Cdk, blocking ATP loading. Nevertheless, low concentrations of Cip/Kip proteins do not appear to block Cdk4/6, just Cdk2. Indeed, Cip/Kip proteins act as a bridge between the two subunits to enhance the binding of cyclin D1 to Cdk. Cytoplasmic Cip/Kip proteins also promote the nuclear import of D-type complexes that do not possess signal motifs for nuclear localization. Recently, other functions for p21 have been proposed (5). Studies suggest that p21 might function as a transcriptional coactivator. p21 regulates the activity of NF-B, STAT3, Myc, CAAT enhancer binding protein (C/EBP), and E2F, and inhibits the gene expression of cell cycle proteins such as DNA polymerase-
, topoisomerase II, Cdk1, and cyclin B1. Also, in colon carcinoma cells, p21 protects from prostaglandin A2-induced apoptosis, and knockout of p21 promotes apoptosis following DNA damage (7). p21 interacts with procaspase-3 to inhibit caspase-3 activation and to resist Fas-mediated cell death (20). This antiapoptotic function of p21 is consistent with its role in cell cycle delay, which promotes DNA repair.
Finally, as noted above, during G1 of the cell cycle, p21 also enters into complexes with PCNA via a binding domain near the carboxy terminus. What is the function of PCNA, and the interaction between it and p21? PCNA forms a homotrimeric ring that encircles or "clamps" double-stranded DNA and tethers DNA polymerase- to its template, thus dramatically increasing processive DNA replication (9). Thus in the absence of p53, PCNA facilitates DNA synthesis. However, in the presence of p53, as noted by Helt and colleagues (8), high levels of p21 expression (or expression of the p21 PCNA-binding domain) would inhibit DNA synthesis by making PCNA unavailable for DNA polymerase-
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In addition to DNA replication, DNA polymerase- plays a role in several DNA repair pathways including nucleotide excision repair, mismatch repair, and long patch base excision repair. Also, PCNA functions as an accessory protein not only for DNA polymerase-
but also for other repair/replication proteins such as DNA ligase I and flap endonuclease I (9, 23). Therefore, perhaps paradoxically, p21, by competing for PCNA, has the potential to not only inhibit DNA synthesis but DNA repair. Whether this occurs or not in vivo remains unclear. The need for longer periods of uninterrupted interaction between DNA polymerase-
and PCNA during replication compared with repair might make replication more sensitive to p21 than repair. Why would DNA repair be sensitive to p21, which simultaneously functions to promote repair by delaying S phase? Presumably, when a cell is heavily damaged, inhibition of both DNA replication and repair to promote cell death would be preferred. As Helt and colleagues (8) suggest, further study into the physiological role of p21 in DNA repair is needed.
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ACKNOWLEDGMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-54685, HL-63314, and HL-56399 and a grant from the Cystic Fibrosis Foundation.
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FOOTNOTES |
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REFERENCES |
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