Activating Transcription Factor 4 Is Translationally Regulated by Hypoxic Stress

Abstract

Hypoxic stress results in a rapid and sustained inhibition of protein synthesis that is at least partially mediated by eukaryotic initiation factor 2␣ (eIF2␣) phosphorylation by the endoplasmic reticulum (ER) kinase PERK. Here we show through microarray analysis of polysome-bound RNA in aerobic and hypoxic HeLa cells that a subset of transcripts are preferentially translated during hypoxia, including activating transcription factor 4 (ATF4), an important mediator of the unfolded protein response. Changes in mRNA translation during the unfolded protein response are mediated by PERK phosphorylation of the translation initiation factor eIF2␣ at Ser-51. Similarly, PERK is activated and is responsible for translational regulation under hypoxic conditions, while inducing the translation of ATF4. The overexpression of a C-terminal fragment of GADD34 that constitutively dephosphorylates eIF2␣ was able to attenuate the phosphorylation of eIF2␣ and severely inhibit the induction of ATF4 in response to hypoxic stress. These studies demonstrate the essential role of ATF4 in the response to hypoxic stress, define the pathway for its induction, and reveal that GADD34, a target of ATF4 activation, negatively regulates the eIF2␣-mediated inhibition of translation. Taken with the concomitant induction of additional ER-resident proteins identified by our microarray analysis, this study suggests an important integrated response between ER signaling and the cellular adaptation to hypoxic stress.Mammalian cells have the ability to alter their gene expression in order to adapt to a variety of environmental stresses, including nutrient depletion, oxidative stress, UV irradiation, reductive stress, exposure to toxins, and hypoxia, although the exact molecular events controlling the stress response have not been fully elucidated.Hypoxic stress plays a pivotal role in normal human development and physiology, including embryogenesis and wound repair, and has been well studied for its importance in the pathogenesis of several human diseases, namely, heart disease, stroke, diabetes, and cancer (for a detailed review, see reference 52). Hypoxia results when oxygen availability does not meet the demand of the surrounding tissue, resulting in decreased oxygen tension. In cancer this is initiated by the rapid proliferation of tumor cells, which gives rise to abnormal and chaotic vasculature, leading to the development of occlusions, blind ends, and vesicular shunts (4). The presence of hypoxic cells in solid tumors is well documented, and both clinical and experimental evidence suggests that the hypoxic microenvironment of a tumor helps to produce a more aggressive phenotype (62) by functioning as a selective pressure for the clonal expansion of apoptotically insensitive cells (16). The presence of hypoxic regions in solid tumors also correlates with a poor prognosis and has been shown to limit the efficacy of standard anticancer treatments, such as radiotherapy and chemotherapy (5). A key element to cellular survival and adaptation during hypo...

Clock and ATF4 transcription system regulates drug resistance in human cancer cell lines

“…Both expression and function of ATF4 have been reported to be regulated by the post-transcriptional pathways (Blais et al, 2004). Phosphorylation of the a subunit of translation initiation factor (eIF2a) promotes translation of ATF4 and ATF4 phosphorylated by RSK2 increases transactivation ability (Yang et al, 2004).…”

Loss of c/EBP-β activity promotes the adaptive to apoptotic switch in hypoxic cortical neurons

“…While salubrinal lowered caspase-3 activation to control levels, it was less effective in blocking nuclear pyknosis, Reports describing caspase-3-independent induction of EndoG activity by BNIP3 offer one potential explanation for this observation and reinforce the notion that combination therapies for stroke will likely be required (Zhang et al, 2007). It is possible that other non-ER kinases like PKR regulate eIF2α phosphorylation and neuron survival after hypoxic stress (Blais et al, 2004). Likewise, the physiologic stressors that activate PERK also activate the AMPK/TSC2/mTOR pathway (Liu et al, 2006).…”

“…What was most intriguing was the set of interactions uncovered among the bZIP transcription factors ATF4, CHOP-10, c-Jun and c/EBP-β. And while c-Jun, ATF4 and CHOP have been linked with cellular responses to hypoxia, c/EBP-β's role in hypoxic signaling has not been established (Blais et al, 2004;Ron and Habener, 1992). To investigate this further, we analyzed c-Jun and c/EBP-β expression by western blotting and found that hypoxia stimulated the transient induction of Jun and its downstream transcriptional target c/EBP-β before any appreciable induction of ATF4 was seen (Lin et al, 2002).…”

Induction of activating transcription factor 3 by anoxia is independent of p53 and the hypoxic HIF signalling pathway

“…Figure 3a shows that the steady-state level of ATF3 mRNA increases under anoxia after 2 h and persists at 20 h. The increase in ATF3 mRNA level (Figure 3a) precedes the increase in ATF3 protein level (Figure 2a), suggesting that increased ATF3 protein accumulation in anoxia could be due to a later posttranscriptional event. We do not rule out other non-HIF-1 pathways of increased protein translation that could also contribute to ATF3 accumulation under anoxia (Blais et al, 2004). Figure 3b shows that the increase in ATF3 mRNA level under anoxia is independent of HIF-1a.…”

“…Anoxia has been shown to regulate gene expression in an HIF-1-independent manner (Ameri et al, 2004;Blais et al, 2004). To investigate the extent and mechanisms of HIF-independent gene regulation under low oxygen tensions, we considered activating transcription factor 3 (ATF3), a member of the ATF/cAMP-responsive element binding protein family of transcription factors, as a potential master regulator to alter the cellular transcriptional programs under hypoxia and anoxia for the following reasons.…”