Protein Kinase R Mediates the Inflammatory Response Induced by Hyperosmotic Stress.

High extracellular osmolarity results in a switch from an adaptive to an inflammatory gene expression program. We show that hyperosmotic stress activates the protein kinase R (PKR) independently of its RNA-binding domain. In turn, PKR stimulates nuclear accumulation of nuclear factor κB (NF-κB) p65 species phosphorylated at serine-536, which is paralleled by the induction of a subset of inflammatory NF-κB p65-responsive genes, including inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6), and IL-1ß. The PKR-mediated hyperinduction of iNOS decreases cell survival in mouse embryonic fibroblasts via mechanisms involving nitric oxide (NO) synthesis and posttranslational modification of proteins. Moreover, we demonstrate that the PKR inhibitor C16 ameliorates both iNOS amplification and disease-induced phenotypic breakdown of the intestinal epithelial barrier caused by an increase in extracellular osmolarity induced by dextran sodium sulfate (DSS) in vivo Collectively, these findings indicate that PKR activation is an essential part of the molecular switch from adaptation to inflammation in response to hyperosmotic stress.

Quantitative H2S-mediated protein sulfhydration reveals metabolic reprogramming during the integrated stress response.

The sulfhydration of cysteine residues in proteins is an important mechanism involved in diverse biological processes. We have developed a proteomics approach to quantitatively profile the changes of sulfhydrated cysteines in biological systems. Bioinformatics analysis revealed that sulfhydrated cysteines are part of a wide range of biological functions. In pancreatic ß cells exposed to endoplasmic reticulum (ER) stress, elevated H2S promotes the sulfhydration of enzymes in energy metabolism and stimulates glycolytic flux. We propose that transcriptional and translational reprogramming by the integrated stress response (ISR) in pancreatic ß cells is coupled to metabolic alternations triggered by sulfhydration of key enzymes in intermediary metabolism.

Translational control during endoplasmic reticulum stress beyond phosphorylation of the translation initiation factor eIF2α.

The accumulation of unfolded/misfolded proteins in the endoplasmic reticulum (ER) causes stress to which an unfolded protein response is activated to render cell survival or apoptosis (chronic stress). Transcriptional and translational reprogramming is tightly regulated during the unfolded protein response to ensure specific gene expression. The master regulator of this response is the PERK/eIF2α/ATF4 signaling where eIF2α is phosphorylated (eIF2α-P) by the kinase PERK. This signal leads to global translational shutdown, but it also enables translation of the transcription factor ATF4 mRNA. We showed recently that ATF4 induces an anabolic program through the up-regulation of selected amino acid transporters and aminoacyl-tRNA synthetases. Paradoxically, this anabolic program led cells to apoptosis during chronic ER stress in a manner that involved recovery from stress-induced protein synthesis inhibition. By using eIF2α-P-deficient cells as an experimental system, we identified a communicating network of signaling pathways that contribute to the inhibition of protein synthesis during chronic ER stress. This eIF2α-P-independent network includes (i) inhibition of mammalian target of rapamycin kinase protein complex 1 (mTORC1)-targeted protein phosphorylation, (ii) inhibited translation of a selective group of 5'-terminal oligopyrimidine mRNAs (encoding proteins involved in the translation machinery and translationally controlled by mTORC1 signaling), and (iii) inhibited translation of non-5'-terminal oligopyrimidine ribosomal protein mRNAs and ribosomal RNA biogenesis. We propose that the PERK/eIF2α-P/ATF4 signaling acts as a brake in the decline of protein synthesis during chronic ER stress by positively regulating signaling downstream of the mTORC1 activity. These studies advance our knowledge on the complexity of the communicating signaling pathways in controlling protein synthesis rates during chronic stress.

Phosphorylation of eIF2α at serine 51 is an important determinant of cell survival and adaptation to glucose deficiency.

Various forms of stress induce pathways that converge on the phosphorylation of the alpha (α) subunit of eukaryotic translation initiation factor eIF2 at serine 51 (S51), a modification that results in a global inhibition of protein synthesis. In many cases eIF2α phosphorylation is a biological response that facilitates cells to cope with stressful environments. Glucose deficiency, an important form of stress, is associated with an induction of apoptosis. Herein, we demonstrate that eIF2α phosphorylation is a key step in maintaining a balance between the life and death of a glucose-deficient cell. That is, eIF2α phosphorylation acts as a molecular switch that shifts cells from a proapoptotic to a cytoprotective state in response to prolonged glucose deficiency. This adaptation process is associated with the timely expression of proteins and activation of pathways with significant contributions to cell survival and adaptation including the X-linked inhibitor of apoptosis protein (XIAP). We also show that among the eIF2α kinases GCN2 plays a proapoptotic role whereas PERK and PKR play a cytoprotective one in response to glucose deficiency. Our data demonstrate that eIF2α phosphorylation is a significant determinant of survival and adaptation of glucose-deficient cells with possible important implications in biological processes that interfere with glucose metabolism.

A bifunctional intronic element regulates the expression of the arginine/lysine transporter Cat-1 via mechanisms involving the purine-rich element binding protein A (Pur alpha).

Expression of the arginine/lysine transporter Cat-1 is highly induced in proliferating and stressed cells via mechanisms that include transcriptional activation. A bifunctional INE (intronic element) within the first intron of the Cat-1 gene was identified and characterized in this study. The INE had high sequence homology to an amino acid response element and was shown to act as a transcriptional enhancer in unstressed cells by binding the transcription factor, purine-rich element binding protein A (Pur alpha). During endoplasmic reticulum stress, binding of Pur alpha to the INE decreased; the element acted as a positive regulator in early stress by binding of the transcription factor ATF4 and as a negative regulator in prolonged stress by binding the stress-induced C/EBP family member, CHOP. We conclude that transcriptional control of the Cat-1 gene is tightly controlled by multiple cis-DNA elements, contributing to regulation of cationic amino acid transport for cell growth and proliferation. In addition, we propose that genes may use stress-response elements such as the INE to support basal expression in the absence of stress.

The hnRNA-binding proteins hnRNP L and PTB are required for efficient translation of the Cat-1 arginine/lysine transporter mRNA during amino acid starvation.

The response to amino acid starvation involves the global decrease of protein synthesis and an increase in the translation of some mRNAs that contain an internal ribosome entry site (IRES). It was previously shown that translation of the mRNA for the arginine/lysine amino acid transporter Cat-1 increases during amino acid starvation via a mechanism that utilizes an IRES in the 5' untranslated region of the Cat-1 mRNA. It is shown here that polypyrimidine tract binding protein (PTB) and an hnRNA binding protein, heterogeneous nuclear ribonucleoprotein L (hnRNP L), promote the efficient translation of Cat-1 mRNA during amino acid starvation. Association of both proteins with Cat-1 mRNA increased during starvation with kinetics that paralleled that of IRES activation, although the levels and subcellular distribution of the proteins were unchanged. The sequence CUUUCU within the Cat-1 IRES was important for PTB binding and for the induction of translation during amino acid starvation. Binding of hnRNP L to the IRES or the Cat-1 mRNA in vivo was independent of PTB binding but was not sufficient to increase IRES activity or Cat-1 mRNA translation during amino acid starvation. In contrast, binding of PTB to the Cat-1 mRNA in vivo required hnRNP L. A wider role of hnRNP L in mRNA translation was suggested by the decrease of global protein synthesis in cells with reduced hnRNP L levels. It is proposed that PTB and hnRNP L are positive regulators of Cat-1 mRNA translation via the IRES under stress conditions that cause a global decrease of protein synthesis.

A stress-responsive RNA switch regulates VEGFA expression.

Ligand binding to structural elements in the non-coding regions of messenger RNA modulates gene expression. Ligands such as free metabolites or other small molecules directly bind and induce conformational changes in regulatory RNA elements known as riboswitches. Other types of RNA switches are activated by complexed metabolites-for example, RNA-ligated metabolites such as aminoacyl-charged transfer RNA in the T-box system, or protein-bound metabolites in the glucose- or amino-acid-stimulated terminator-anti-terminator systems. All of these switch types are found in bacteria, fungi and plants. Here we report an RNA switch in human vascular endothelial growth factor-A (VEGFA, also known as VEGF) mRNA 3' untranslated region (UTR) that integrates signals from interferon (IFN)-gamma and hypoxia to regulate VEGFA translation in myeloid cells. Analogous to riboswitches, the VEGFA 3' UTR undergoes a binary conformational change in response to environmental signals. However, the VEGFA 3' UTR switch is metabolite-independent, and the conformational change is dictated by mutually exclusive, stimulus-dependent binding of proteins, namely, the IFN-gamma-activated inhibitor of translation complex and heterogeneous nuclear ribonucleoprotein L (HNRNPL, also known as hnRNP L). We speculate that the VEGFA switch represents the founding member of a family of signal-mediated, protein-dependent RNA switches that evolved to regulate gene expression in multicellular animals in which the precise integration of disparate inputs may be more important than the rapidity of response.

Differential control of the CCAAT/enhancer-binding protein beta (C/EBPbeta) products liver-enriched transcriptional activating protein (LAP) and liver-enriched transcriptional inhibitory protein (LIP) and the regulation of gene expression during the response to endoplasmic reticulum stress.

The accumulation of unfolded proteins in the endoplasmic reticulum (ER) triggers a stress response program that protects cells early in the response and can lead to apoptosis during prolonged stress. The basic leucine zipper transcription factor, CCAAT/enhancer-binding protein beta (C/EBPbeta), is one of the genes with increased expression during ER stress. Translation of the C/EBPbeta mRNA from different initiation codons leads to the synthesis of two transcriptional activators (LAP-1 and -2) and a transcriptional repressor (LIP). The LIP/LAP ratio is a critical factor in C/EBPbeta-mediated gene transcription. It is shown here that the LIP/LAP ratio decreased by 5-fold during the early phase of ER stress and increased by 20-fold during the late phase, mostly because of changes in LIP levels. The early decrease in LIP required degradation via the proteasome pathway and phosphorylation of the translation initiation factor, eIF2alpha. The increased LIP levels during the late phase were due to increased synthesis and increased stability of the protein. It is proposed that regulation of synthesis and degradation rates during ER stress controls the LIP/LAP ratio. The importance of C/EBPbeta in the ER-stress response program was demonstrated using C/EBPbeta-deficient mouse embryonic fibroblasts. It is shown that C/EBPbeta attenuates expression of pro-survival ATF4 target genes in late ER stress and enhances expression of cell death-associated genes downstream of CHOP. The inhibitory effect of LIP on ATF4-induced transcription was demonstrated for the cat-1 amino acid transporter gene. We conclude that regulation of LIP/LAP ratios during ER stress is a novel mechanism for modulating the cellular stress response.