Regulation of cell volume is of great importance because persistent swelling or shrinkage leads to cell death. Tissues experience hypertonicity in both physiological (kidney medullar cells) and pathological states (hypernatremia). Hypertonicity induces an adaptive gene expression program that leads to cell volume recovery or apoptosis under persistent stress. We show that the commitment to apoptosis is controlled by phosphorylation of the translation initiation factor eIF2␣, the master regulator of the stress response. Studies with cultured mouse fibroblasts and cortical neurons show that mutants deficient in eIF2␣ phosphorylation are protected from hypertonicity-induced apoptosis. A novel link is revealed between eIF2␣ phosphorylation and the subcellular distribution of the RNA-binding protein heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1). Stress-induced phosphorylation of eIF2␣ promotes apoptosis by inducing the cytoplasmic accumulation of hnRNP A1, which attenuates internal ribosome entry site-mediated translation of anti-apoptotic mRNAs, including Bcl-xL that was studied here. Hypertonic stress induced the eIF2␣ phosphorylation-independent formation of cytoplasmic stress granules (SGs, structures that harbor translationally arrested mRNAs) and the eIF2␣ phosphorylation-dependent accumulation of hnRNP A1 in SGs. The importance of hnRNP A1 was demonstrated by induction of apoptosis in eIF2␣ phosphorylation-deficient cells that express exogenous cytoplasmic hnRNP A1. We propose that eIF2␣ phosphorylation during hypertonic stress promotes apoptosis by sequestration of specific mRNAs in SGs in a process mediated by the cytoplasmic accumulation of hnRNP A1.

A poptosis, or programmed cell death, is important for the normal development of organisms and maintaining tissue homeostasis. Misregulation of apoptosis may lead to neurodegenerative disorders when apoptosis is increased and tumor formation when apoptosis is inhibited; thus, the pathway is tightly controlled, with both pro-and antiapoptotic factors playing a role. Of particular interest are members of the inhibitor of apoptosis (IAP) and Bcl-2 families of proteins that intercept virtually all apoptotic signals in the cell.X chromosome-linked inhibitor of apoptosis (XIAP) is the most potent member of the IAP family; it directly interacts with and inhibits caspases 3, 7, and 9 and is therefore a key regulator of apoptosis (20). In contrast, Bcl-x L controls apoptosis by maintaining mitochondrial membrane homeostasis (14). Interestingly, both the XIAP and Bcl-x L mRNAs contain an internal ribosome entry site (IRES) that allows them to be translated during cellular stress by a cap-independent mechanism when cap-dependent translation is inhibited, which is necessary for their protective roles in the cell (4,11,16,24,49). Under normal growth conditions, translation of cellular mRNAs occurs through a cap-dependent mechanism that requires interaction of specific initiation factors (such as eukaryotic initiation factor 4E [eIF4E]) with the 5= cap of the mRNA, followed by recruitment of ribosomal subunits, recognition of the AUG start codon, and commencement of polypeptide chain elongation (reviewed in reference 26). However, certain cellular stresses such as nutrient deprivation, hypoxia, or low-dose irradiation cause attenuation of cap-dependent translation, and yet, under these conditions, a sizeable proportion of cellular mRNAs, perhaps as much as 10%, have been shown to be translated by a cap-independent mechanism, such as through an IRES (23,28,39). IRES elements are located within the 5= untranslated region (UTR) of some cellular mRNAs and are believed to recruit the ribosome directly, thereby bypassing the requirement for the mRNA 5= cap and eIF4E. Moreover, while IRES-dependent translation requires some canonical translation initiation factors, most (if not all) cellular IRES elements require the activity of auxiliary RNA binding proteins that function as IRES trans-acting factors (ITAFs) (reviewed in references 28 and 32). The mechanism by which ITAFs function is poorly understood, and since different IRESs require different sets of ITAFs, elucidating the identity of all known ITAFs has proven challenging. Furthermore, it has been shown that ITAFs can function as either positive or negative regulators of IRES-mediated translation. For example, heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) was shown to act as a repressor of XIAP IRES-mediated translation (33), whereas La autoantigen (19), hnRNPC1 and hnRNPC2 (18), and HuR (11) have all been shown to have a stimulating effect on XIAP IRES-mediated translation.We have previously demonstrated that treatment of small-cell lung cancer cells with fibroblast gr...

X-chromosome linked inhibitor of apoptosis, XIAP, is cellular caspase inhibitor and a key regulator of apoptosis. We and others have previously shown that XIAP expression is regulated primarily at the level of protein synthesis; the 5′ untranslated region (UTR) of XIAP mRNA contains an Internal Ribosome Entry Site (IRES) that supports cap-independent expression of XIAP protein during conditions of pathophysiological stress, such as serum deprivation or gamma irradiation. Here, we show that XIAP is encoded by two distinct mRNAs that differ in their 5′ UTRs. We further show that the dominant, shorter, 5′ UTR promotes a basal level of XIAP expression under normal growth conditions. In contrast, the less abundant longer 5′ UTR contains an IRES and supports cap-independent translation during stress. Our data suggest that the combination of alternate regulatory regions and distinct translational initiation modes is critical in maintaining XIAP levels in response to cellular stress and may represent a general mechanism of cellular adaptation.