Adaptation to environmental stress is essential for long-term cell survival. Mechanisms that control cellular responses to stress are often exploited by tumor cells, allowing them to survive and thrive in unfavorable environments. As an example, metabolic changes that provide a growth advantage to tumor cells exposed to a hypoxic microenvironment are mediated in large part by the heterodimeric transcription factor HIF-1. The shift in cellular metabolism toward the energy conserving anaerobic glycolysis is facilitated by the HIF-1-regulated glycolytic enzymes (phosphoglycerate kinase 1 and aldolase A) and glucose transporters (GLUT-1 and GLUT-3) (14
). Tolerance to hypoxia is also promoted through changes in protein translation. Translation is the second largest consumer of ATP (22
) in the cell, and its coordinated shutdown may be one strategy to ensure energy conservation in the cell when ATP levels are limiting. It has been well established that hypoxic stress results in a profound inhibition in translation (30
), thereby causing the majority of mRNA transcripts to experience diminished translational efficiencies. Our microarray analysis revealed that 95% of cellular mRNA species that do not change or decrease in abundance during hypoxia experience a decrease in their translational efficiency. PERK has been implicated in the inhibition of translation during hypoxia through the phosphorylation of eIF2α at Ser-51 (28
). However, despite PERK-mediated inhibition, the translation of some genes remains unaffected, whereas others, e.g., HIF-1α and its downstream gene products (31
) are translated more efficiently. Consequently, cells must be capable of maintaining and inducing gene expression in order to mediate cellular adaptation and cell viability during hypoxia.
A collective analysis of data generated by microarray and Q-PCR facilitated the identification of three classes of gene expression that likely contribute to this strategy. The first category includes mRNAs that are transcriptionally induced and are more efficiently translated under hypoxic stress; both BiP and ATF3 are examples of this method of gene regulation (Fig. to F; additional candidate genes are listed in Table ). A second category includes genes that are transcriptionally induced and whose translational efficiencies are either unchanged or diminished in response to hypoxia. CA9 and P4H2, both HIF-1-regulated gene products, demonstrate a decrease in their translational efficiencies during hypoxia (Fig. to F; additional candidate genes are listed in Table ). The third class of gene expression includes genes that are more efficiently translated during hypoxia without an associated transcriptional induction in total mRNA. Genes that correspond to this category include eIF5, txbp151, ATF6, and ATF4 (Fig. to C; additional candidate genes are listed in Table ). Similar analyses have been previously used to identify translationally regulated genes involved in host cell response to poliovirus infection (26
), T-cell activation (38
), VHL expression (14
), and most recently in oncogenic signaling through Ras and Akt (46
). Thus, a combination of changes in steady-state mRNA and translational changes in gene expression can modulate the cellular responses to stress.
The selective inhibition of cap-dependent translation could regulate the translational expression of important hypoxia-responsive genes with IRESs. IRES-mediated initiation was first discovered in picornaviruses such as poliovirus (25
). Many cellular IRES elements—namely, PDGF2 (2
), c-sis (51
), APAF-1 (8
), and XIAP (23
)—function under stress conditions when rates of protein synthesis via cap-dependent translation are reduced. Specifically, VEGF (58
), HIF-1α (31
), BiP (36
), and ODC (45
) have demonstrated functional IRES activity during hypoxic stress. The importance of IRES expression as a key regulator of translation during hypoxia currently remains undetermined. We are undertaking efforts to determine which, if any, of the translationally regulated transcripts reported possess functional IRESs.
ATF4, an important mediator of the UPR, is ubiquitously expressed at low basal levels, although poorly translated in unstressed cells due to the presence of three uORFs in its 5′UTR (18
). This regulatory mechanism resembles that of the yeast protein GCN4. In yeast, global inhibition of translation occurs upon amino acid depletion. Active GCN2 phosphorylates eIF2α and preferentially promotes the synthesis of GCN4, a transcription factor that controls the expression of genes involved in amino acid biosynthesis (21
Translation of ATF4 rapidly follows eIF2α phosphorylation during times of ER stress, arsenite treatment, and amino acid starvation (17
). During hypoxia PERK activation results in the phosphorylation of eIF2α (28
); taken together, this suggests an important UPR-integrated response to hypoxic stress. Microarray analysis of total cellular mRNAs revealed the transcriptional induction of many other ER-resident proteins, including GADD34, GRP58, HSP70, Grp78/BiP, and HSP28 (Table ), as well as CHOP/Gadd153, ORP150, and Gadd45 (C. Koumenis, unpublished data), further supporting the connection between hypoxia and the UPR.
Our studies with PERK−/−
MEFs have shown that this kinase is absolutely required for ATF4 translation (Fig. ). Interestingly, even in the absence of efficient translation, ATF4 mRNA is still recruited, to some extent, into the polysome fraction. Since we and others have shown that the initiation of translation at the authentic start codon of ATF4 is dependent on PERK phosphorylation of eIF2α (18
), our results (Fig. ) suggest that ATF4 mRNA can be recruited to the polysome fraction in the absence of PERK activity through initiation and translation of upstream ORFs.
Although others have suggested that anoxic induction of ATF4 is regulated at the level of protein stability (1
), our experiments with CHX treatment of HeLa cells (Fig. ) suggest a requirement for de novo protein synthesis. Although we cannot exclude that increased protein stability of ATF4 may contribute to its posttranslational regulation, our data demonstrate that ATF4 is regulated, at least in part, by an increase in its translational efficiency during hypoxic stress.
Our microarray analysis revealed an induction of GADD34 gene expression in the total cellular mRNA by 3.4-fold accompanied by a 6.1-fold increase in its translational efficiency. GADD34 directs the catalytic subunit of protein phosphatase 1 to eIF2α (9
), leading to its dephosphorylation and promoting resolution of the translational repression stage of the UPR (41
). ATF4 is known to directly bind and activate an ATF site upstream of the GADD34 promoter (35
); however, the role of GADD34 expression in the attenuation of hypoxia-induced eIF2α phosphorylation has not been addressed. Phosphorylation of eIF2α occurs upon acute exposure to hypoxic stress yet eIF2α phosphorylation appears to subside by 8 h (28
). Our data demonstrate that induction of GADD34 begins after 2 h of hypoxic stress and is strongly induced by 4 h (Fig. ). Furthermore, over expression of a COOH-truncated GADD34, which constitutively dephosphorylates eIF2α, demonstrated a severe inhibition in the induction of ATF4 during hypoxia (Fig. ). This suggests an important role for GADD34 as a negative feedback regulator, mediating eIF2α dephosphorylation and inhibiting ATF4 expression during hypoxic stress (Fig. ). Protein synthesis inhibition and phosphorylation of eIF2α are transient in response to ER stress (44
). Sustained eIF2α phosphorylation is lethal both in cultured cells and in the in vivo neuronal response to ischemic stress (11
). It has been suggested that the GADD34 negative feedback loop is necessary for cellular recovery after ER stress (41
), as seen by a decrease in cell survival in cells expressing a GADD34 mutant lacking the COOH-terminal domain necessary for PP1 activation and eIF2α dephosphorylation. In addition, it has been demonstrated that expression of a nonphosphorylatable eIF2α partially protects cells from apoptosis. Conversely, the expression of an eIF2α phosphormimetic (S51D) increased apoptotic cell death (57
). The observation that the mechanism of translational inhibition during prolonged hypoxic exposure changes from a global inhibition through eIF2α phosphorylation to a selective inhibition of cap-dependent translation by preventing the formation of eIF4F complexes (B. G. Wouters, unpublished data), presents the possibility of a molecular switch responsible for this response. The attenuation of eIF2α phosphorylation by GADD34 during hypoxia may play a role in this molecular switch.
FIG. 8. Model depicting the role of ATF4 during Hypoxic stress. Hypoxia and oxidative stress activate the eIF2α kinase PERK. Phosphorylation of eIF2α results in the global inhibition of protein translation. Some transcripts, such as ATF4, ATF6, (more ...)
Our experiments show that in response to severe hypoxic stress, the result of translational inhibition promotes the selective translation of several hypoxia responsive genes. The increase in the translational efficiency of these genes at a time of global protein synthesis inhibition indicates the importance of rapid and reversible gene expression in response to hypoxic stress. The preferential translation of ATF4 and the analogous translational and transcriptional induction of other ER-resident proteins suggests an integrated stress response involving an ER-generated signal and the cellular adaptation to hypoxia (Fig. ). Furthermore, induction of GADD34 and its role in the dephosphorylation of eIF2α may be an essential intermediate signal for the switch from the inhibition of global translation to a more selective inhibition of cap-dependent translation.
Understanding the molecular events that support the development of tumor cell resistance to hypoxia is imperative for the discovery of effective therapies. The ability of hypoxic tumor cells to simultaneously promote angiogenesis, metastasis, and glycolysis, substantiates the need of cellular adaptation for continued cell viability. Whether the ATF4 signaling pathway is a key element that promotes cell survival during hypoxia still needs to be determined. Interestingly, the overexpression of a novel hypoxia regulated gene, SKIP3
, has been implicating in destabilization of ATF4 protein in multiple primary human tumors (3
). The occurrence of deregulated ATF4 expression in primary cancers therefore implicates ATF4 signaling in tumor progression.