Cellular adaptation to hypoxic stress is a multifaceted process that involves both a shift of cellular metabolism toward anaerobic glycolysis and an inhibition of energy-consuming processes like cell proliferation and macromolecular synthesis (30
). At the multicellular or organ level, additional mechanisms like neoangiogenesis and increased synthesis of erythropoietin are also employed in an attempt to increase the oxygen supply to oxygen-starved tissue (4
). Abnormalities in oxygen homeostasis are associated with pathological disease states, including brain and cardiac muscle ischemia-reperfusion injury and tumor hypoxia (2
). Understanding the molecular events in these types of stress responses is crucial for identifying strategies for therapeutic intervention. In the case of tumor hypoxia, elucidation of adaptive mechanisms of tumor cells to hypoxia-anoxia may provide a mechanism for selective targeting of tumor cells (20
In this report, we demonstrate that translation initiation factor eIF2α plays a critical role in the downregulation of protein synthesis in response to hypoxic stress in normal and transformed cells. The increase in eIF2α phosphorylation is both time and oxygen dependent and shows a strong correlation with decreased rates of protein synthesis. Reoxygenation of hypoxic cells has been previously shown to result in recovery of protein synthesis to levels similar to that found in normoxic cells, and we find that eIF2α becomes dephosphorylated within 30 to 60 min of reoxygenation. This rapid and reversible modification of eIF2α could allow the cell to adapt to the fluctuating O2
concentrations that are known to occur in tumors. While the model of static, chronic hypoxia appears to apply to certain cases of solid tumors and to spheroids, recent evidence suggests that a more dynamic and fluctuating hypoxia model may apply to most solid tumors (35
). This form of transient or intermittent hypoxia has been attributed to the irregular vasoconstriction and vasodilation of newly formed blood vessels, as well as to the leakiness of the vasculature in tumors (5
). In this situation, a rapid on-off mechanism to regulate energy consumption, like eIF2α phosphorylation, would be more advantageous than regulation of gene transcription.
The fast kinetics of phosphorylation-dephosphorylation of eIF2α and inhibition and recovery of protein synthesis are similar to the regulation of other cellular processes during hypoxia-reoxygenation. In addition to the rapid inhibition of firing of replicon initiation in Ehrlich ascites and HeLa cells, a recent report describes the remarkable property of zebra fish embryos to enter a state of suspended animation under anaerobic conditions (44
). The embryos are able to survive hypoxic treatments of up to 24 h by suspending processes like cell cycle progression and cardiac function. These processes resumed and the embryos appeared to function normally when they were returned to normoxic conditions. Therefore, exposure of a cell to a hypoxic environment initiates a fast-acting, reversible program of adaptation that involves downregulation of protein synthesis, DNA replication, and cell cycle arrest. The decrease in protein synthesis rates has been postulated to contribute to energy conservation under a reduced energy supply because of decreased oxidative phosphorylation. This strategy appears to be also employed in other cases of cellular adaptation to stress. For example, Frerichs et al. demonstrated that during mammalian hibernation, protein synthesis in the brain is suppressed to 0.04% of the level in active animals (19
). This remarkable repression is not only compatible with normal brain function and with the absence of any evidence of measurable cell death but is also completely reversible at the end of hibernation. More importantly, phosphorylation of eIF2α was shown to correlate with this marked depression in protein synthesis. Thus, by reducing protein synthesis and energy consumption to a minimum, the cells in the hibernating brain are able to survive under conditions of limited energy supply.
Despite the rapid and relatively extended phosphorylation of eIF2α by hypoxic stress, this phosphorylation is not sustained indefinitely. Longer anoxia incubation times result in decreased eIF2α phosphorylation levels. Sustained eIF2α phosphorylation (and subsequently general translational inhibition) may be incompatible with cell viability. Therefore, it appears that under hypoxic stress, eIF2α phosphorylation levels may be tightly regulated to ensure cellular survival in a low-oxygen environment. This reduction in eIF2α phosphorylation under prolonged hypoxia may be achieved via activation of a negative feedback pathway, and such a pathway may also be involved in the rapid dephosphorylation of eIF2α upon reoxygenation. Recently, it has been reported that the product of the GADD34 gene participates in such a negative feedback loop to dephosphorylate eIF2α following stress (42
). Although there is no evidence that hypoxic stress upregulates GADD34, tumor hypoxia has been shown to induce two other GADD family members, GADD45 and GADD153 (49
). It would be interesting to examine whether hypoxia or hypoxia-reoxygenation induces GADD34 and whether overexpression of this protein can antagonize hypoxia-induced phosphorylation of eIF2α.
The reduction in eIF2α phosphorylation after sustained periods of hypoxia did not correlate with an increase in overall levels of protein translation at those time points. Similarly, protein synthesis remains inhibited following prolonged hypoxia in cells that express the dominant-negative S51A eIF2α protein and in cells that express dominant-negative PERKΔC. This occurs despite no observable increase in the phosphorylation of eIF2α in both of these mutant cell lines. What mechanisms could be responsible for this phenomenon? First, reduced rates of mRNA synthesis have been observed under prolonged hypoxia and may, in part, explain the reduced rates of protein synthesis. Alternatively, other translational control mechanisms may be used during hypoxia, such as those that regulate the assembly mRNA cap-binding complex eIF4F. Indeed, under conditions of prolonged hypoxia, we have observed changes in the regulation of several factors that result in reduced translation initiation, including eIF4E, eIF4G, and the eIF4E binding proteins (B. G. Wouters et al., unpublished observations). The transition from regulation of translation from eIF2α to eIF4F during prolonged hypoxia may facilitate the translation of specific mRNAs that are critical for the phenotypic changes that occur during hypoxia.
The switch of cellular metabolism from mostly aerobic to mostly anaerobic is regulated by several gene products under the control of HIF-1α (58
). Cells with homozygous deletions of this gene are unable to adapt their metabolism to anaerobic glycolysis and are less able to adapt to the new hypoxic environment. Under these conditions, the cell can deplete glucose faster and run out of energy (56
). Recently, eIF2α phosphorylation was shown to be critical for glucose homeostasis. Mice homozygous for a mutant, nonphosphorylatable allele of eIF2α or homozygous for deletion of the PERK gene displayed a deficiency in pancreatic β cells and in the unfolded-protein response (24
). Therefore, a reasonable hypothesis is that HIF-1α might regulate eIF2α phosphorylation directly or indirectly under hypoxic stress via glucose deprivation. However, our data demonstrate that this is not the case. eIF2α was found to be phosphorylated in response to hypoxia in both HIF-1α+/+
MEFs, with similar kinetics. This result indicates that the signal for eIF2α phosphorylation under hypoxia is independent of HIF-1α accumulation and subsequent downstream events and further suggests that hypoxia-induced eIF2α phosphorylation does not involve changes in glucose homeostasis (Fig. ). Rather, coupled with the data showing activation of PERK, the results described above are compatible with an ER-generated signal for activation of eIF2α phosphorylation by hypoxia.
FIG. 9. Model for the regulation of protein synthesis by hypoxia. Hypoxia activates PERK either directly or through the induction of misfolded proteins in the ER. PERK then phosphorylates eIF2α, which leads to translational inhibition and increased cell (more ...)
Although the mechanism of PERK activation by hypoxia remains unknown, a possible mechanism is activation of the unfolded-protein response (Fig. ). Hypoxic stress is known to activate a number of ER-resident proteins, in addition to PERK, like the chaperone protein BiP (GRP78) (37
), the 150-kDa ORP protein (43
), and the stress response protein CHOPP (GADD153) (49
). Hypoxic stress induces BiP accumulation in a number of tumor cells, and this activation was shown to be critical for cell survival, since expression of an antisense BiP construct enhanced the cytotoxic effect of hypoxia on these tumor cells (38
). The increase in BiP levels is postulated to confer a cytoprotective effect on stressed cells via its chaperonin role and inhibition of protein misfolding in the ER. More recently, BiP was shown to bind to PERK and another ER-resident kinase, IRE1, in unstressed cells and to negatively regulate their function, presumably by inhibiting their oligomerization (26
). Upon ER stress, BiP dissociates from PERK and IRE1, relieving its negative regulation, which leads to their activation. BiP levels increase under hypoxic stress and in response to other ER stressors, but this accumulated BiP may not be able to interact with PERK because of competitive binding to misfolded proteins, leading to PERK activation.
Consistent with the protective role of BiP in hypoxic cells is our finding of a protective role for PERK under conditions of prolonged hypoxic stress. The differential survival levels between PERK+/+
cells under extreme hypoxia (~3-fold) are not as dramatic as those observed under conditions of pharmacologically induced acute ER stress (10- to 100-fold), but it is unlikely that such extreme conditions of ER stress occur physiologically in organisms (25
). The difference in clonogenic survival observed under hypoxia in vitro may also extend to in vivo tumor models and may have important ramifications for tumor growth in vivo. In this respect, it will be important to investigate the role of PERK in tumor growth in vivo with xenograft tumor models and correlate any differences in growth rates with hypoxic areas of the tumor. If eIF2α phosphorylation and PERK activity are also found to play critical roles in tumor growth rates, these findings may lead to the design of therapeutic modalities that inhibit this adaptive response and thereby target hypoxic cells within a tumor.