Growth Arrest And DNA Damage-inducible Protein 34 Research Articles

X-irradiation induces ER stress, apoptosis, and senescence in pulmonary artery endothelial cells

Purpose: The use of clinical radiation for cancer treatment is limited by damage to underlying normal tissue including to the vascular endothelium. We investigated the mechanisms of X-ray-induced cell damage to endothelial cells.Methods: We evaluated necrosis, apoptosis, cellular senescence, and the contribution of endoplasmic reticulum (ER) stress in pulmonary artery endothelial cells (PAEC) irradiated with X-rays (2–50 Gray [Gy]).Results: Clonogenic assays showed that 10 Gy induced ∼99.9% loss of cell viability. No necrosis was detected using lactate dehydrogenase assays, but a low population underwent extrinsic and intrinsic apoptosis, as indicated by the activation of caspases 3, 8, and 9 as well as by neutral comet assay. A majority of PAEC underwent accelerated senescence, as indicated by morphological changes, increased 21 kD cyclin-dependent kinase inhibitor (p21/waf1), decreased sirtuin 1 (SIRT1), and elevated senescence-associated β-galactosidase (SA-β-gal). ER stress was detected by assays for glucose-regulated protein 78 (GRP78), CCAAT/enhancer-binding protein homologous protein (CHOP), and growth arrest and DNA damage-inducible protein 34 (GADD34) mRNA, and transient phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α). The ER stress inhibitor salubrinal blocked ∼50% of apoptosis with no effect on senescence.Conclusions: X-rays primarily induced cellular senescence with limited levels of apoptosis in endothelial cells. ER stress contributed to apoptosis but not to senescence.

Liver-specific deletion of protein tyrosine phosphatase (PTP) 1B improves obesity- and pharmacologically induced endoplasmic reticulum stress

Obesity is associated with induction of the ER (endoplasmic reticulum)-stress response signalling and insulin resistance. PTP1B (protein tyrosine phosphatase 1B) is a major regulator of adiposity and insulin sensitivity. The aim of the present study was to investigate the role of L-PTP1B (liver-specific PTP1B) in chronically HFD (high-fat diet) and pharmacologically induced (tunicamycin and thapsigargin) ER-stress response signalling in vitro and in vivo. We assessed the effects of ER-stress response induction on hepatic PTP1B expression, and consequences of hepatic-PTP1B deficiency, in cells and mouse liver, on components of ER-stress response signalling. We found that PTP1B protein and mRNA expression levels were up-regulated in response to acute and/or chronic ER stress, in vitro and in vivo. Silencing PTP1B in hepatic cell lines or mouse liver (L-PTP1B(-/-)) protected against induction of pharmacologically induced and/or obesity-induced ER stress. The HFD-induced increase in CHOP (CCAAT/enhancer-binding protein homologous protein) and BIP (binding immunoglobulin protein) mRNA levels were partially inhibited, whereas ATF4 (activated transcription factor 4), GADD34 (growth-arrest and DNA-damage-inducible protein 34), GRP94 (glucose-regulated protein 94), ERDJ4 (ER-localized DnaJ homologue) mRNAs and ATF6 protein cleavage were completely suppressed in L-PTP1B(-/-) mice relative to control littermates. L-PTP1B(-/-) mice also had increased nuclear translocation of spliced XBP-1 (X box-binding protein-1) via increased p85α binding. We demonstrate that the ER-stress response and L-PTP1B expression are interlinked in obesity- and pharmacologically induced ER stress and this may be one of the mechanisms behind improved insulin sensitivity and lower lipid accumulation in L-PTP1B(-/-) mice.

Level of Activation of the Unfolded Protein Response Correlates With Paneth Cell Apoptosis in Human Small Intestine Exposed to Ischemia/Reperfusion

In the intestine, Paneth cells participate in the innate immune response. Their highly secretory function makes them susceptible to environmental conditions that cause endoplasmic reticulum (ER) stress. We investigated whether intestinal ischemia/reperfusion (I/R) induces ER stress, thereby activating the unfolded protein response (UPR), and whether excessive UPR activation affects Paneth cells. In addition, we investigated the consequences of Paneth cell compromise during physical barrier damage. Jejunal I/R was studied using a human experimental model (n = 30 patients). Activation of the UPR was assessed using immunofluorescence for binding protein and quantitative polymerase chain reaction analyses for C/EBP homologous protein (CHOP), growth arrest and DNA-damage inducible protein 34 (GADD34), and X-box binding protein 1 (XBP1) splicing. Paneth cell apoptosis was assessed by double staining for lysozyme and M30. Male Sprague-Dawley rats underwent either intestinal I/R to investigate UPR activation and Paneth cell apoptosis, or hemorrhagic shock with or without intraperitoneal administration of dithizone, to study consequences of Paneth cell compromise during physical intestinal damage. In these animals, bacterial translocation and circulating tumor necrosis factor-α and interleukin-6 levels were assessed. In jejunum samples from humans and rats, I/R activated the UPR and resulted in Paneth cell apoptosis. Apoptotic Paneth cells showed signs of ER stress, and Paneth cell apoptosis correlated with the extent of ER stress. Apoptotic Paneth cells were shed into the crypt lumen, significantly lowering their numbers. In rats, Paneth cell compromise increased bacterial translocation and inflammation during physical intestinal damage. ER stress-induced Paneth cell apoptosis contributes to intestinal I/R-induced bacterial translocation and systemic inflammation.

Melanoma Differentiation Associated Gene-7/Interleukin-24 Potently Induces Apoptosis in Human Myeloid Leukemia Cells through a Process Regulated by Endoplasmic Reticulum Stress

Melanoma differentiation associated gene-7 (mda-7)/interleukin-24 (IL-24), a member of the IL-10 cytokine gene family, preferentially induces cell death in neoplastic epithelial cells types while sparing their normal counterparts. The effects of mda-7/IL-24 in acute myeloid leukemia (AML) cells have not been extensively characterized. Treatment with recombinant GST-MDA-7/IL-24 potently induced apoptosis in diverse myeloid leukemia cell types including U937, HL60, MV4-11, EOL-1, and MLL/ENL cells. MDA-7/IL-24 also markedly induced apoptosis in and suppressed the colony-forming capacity of primary AML blasts but exerted minimal toxicity toward normal CD34(+) hematopoietic progenitor cells. MDA-7/IL-24 lethality was associated with pronounced endoplasmic reticulum (ER) stress induction in leukemia cell lines and primary AML blasts, manifested by the accumulation of growth arrest and DNA damage-inducible protein 34 (GADD34), 78-kDa glucose-regulated protein (GRP78)/BiP, inositol-requiring enzyme 1α (IRE1α), and eukaryotic initiation factor 2α phosphorylation. It is noteworthy that short hairpin RNA (shRNA) knockdown of IRE1α, GADD34, or GRP78/BiP significantly enhanced MDA-7/IL-24-mediated apoptosis, indicating a protective role for these molecules against MDA-7/IL-24 lethality. MDA-7/IL-24 also down-regulated the antiapoptotic protein Mcl-1 and sharply increased expression of the proapoptotic proteins Bim and Noxa. Ectopic Mcl-1 expression or shRNA knockdown of Bim or Noxa significantly attenuated MDA-7/IL-24-mediated leukemia cell death. Finally, knockdown of Bax or Bak significantly reduced MDA-7/IL-24 lethality. Together, these findings indicate that MDA-7/IL-24 potently induces apoptosis in human myeloid leukemia cells through a process regulated by ER stress induction, Mcl-1 down-regulation, and Bim and Noxa up-regulation. They also suggest that MDA-7/IL-24 warrants further investigation in myeloid leukemia.

Abstract B3: Potential new mechanism whereby IGFBP-5 contributes to cancer progression: Binding of GADD34 and enhancement of eIF2α dephosphorylation

Abstract Insulin-like Growth Factor Binding Protein (IGFBP-5) has a well-defined role in normal mammary gland physiology and has recently been shown to play an important role in the biology of breast cancer; however the IGF-independent mechanisms of action remain obscure. Over-expression of IGFBP-5, but not the addition of the exogenous protein, has been shown to inhibit the growth of breast cancer cells in vitro. This suggests novel intracellular mechanisms where IGFBP-5 interacts with unidentified binding partners and thereby modulates the proliferation and survival of breast cancer cells. To address this issue, we performed a yeast two-hybrid screen utilizing a normal human mammary gland cDNA library and full-length IGFBP-5 as the bait. The pre-eminent binding partner identified was Growth Arrest and DNA Damage-inducible protein 34 (GADD34), which assembles an eukaryotic initiation factor 2 alpha subunit (eIF2α) dephosphorylation complex at the endoplasmic reticulum (ER). We have confirmed that IGFBP-5 and GADD34 interact through coimmunoprecipitation experiments using ectopically expressed IGFBP-5 and GADD34 in mammalian cells. We have also shown that IGFBP-3, which belongs to the same protein family as IGFBP-5, interacts with GADD34 through coimmunoprecipitation studies. Furthermore, IGFBP-5 or IGFBP-3 expression was found to dramatically increase GADD34 protein levels as demonstrated through immunoblot analysis. However, IGFBP-2, another protein related to IGFBP-5, did not interact with GADD34 or increase its protein expression. Because GADD34-mediated eIF2α dephosphorylation is required to promote resumption of protein translation after periods of ER stress, we next examined eIF2α phosphorylation levels in the presence and absence of IGFBPs. Expression of IGFBP-5 or IGFBP-3 decreased the levels of phosphorylated eIF2α, suggesting that the interactions diminish the magnitude and/or duration of ER stress-induced inhibition of translation. Our results demonstrate not only a physical interaction between IGFBP-5 or IGFBP-3 and GADD34, but also show that these IGFBPs appear to increase the stability and activity of GADD34. This discovery suggests a novel role for IGFBP-5 and IGFBP-3 in the regulation of a protective mechanism induced by ER stress. Since ER stress is one of the hallmarks of solid tumor progression, by modifying the tumor response to hostile microenvironments, it is likely that these IGFBPs can have significant impact on cancer growth, survival, and apoptosis. Citation Information: Cancer Res 2009;69(23 Suppl):B3.

A decrease in cellular energy status stimulates PERK-dependent eIF2α phosphorylation and regulates protein synthesis in pancreatic β-cells

In the present study, we demonstrate that, in pancreatic beta-cells, eIF2alpha (eukaryotic initiation factor 2alpha) phosphorylation in response to a decrease in glucose concentration is primarily mediated by the activation of PERK [PKR (protein kinase RNA activated)-like endoplasmic reticulum kinase]. We provide evidence that this increase in PERK activity is evoked by a decrease in the energy status of the cell via a potentially novel mechanism that is independent of IRE1 (inositol requiring enzyme 1) activation and the accumulation of unfolded nascent proteins within the endoplasmic reticulum. The inhibition of eIF2alpha phosphorylation in glucose-deprived cells by the overexpression of dominant-negative PERK or an N-terminal truncation mutant of GADD34 (growth-arrest and DNA-damage-inducible protein 34) leads to a 53% increase in the rate of total protein synthesis. Polysome analysis revealed that this coincides with an increase in the amplitude but not the number of ribosomes per mRNA, indicating that eIF2alpha dephosphorylation mobilizes hitherto untranslated mRNAs on to polysomes. In summary, we show that PERK is activated at low glucose concentrations in response to a decrease in energy status and that this plays an important role in glucose-regulated protein synthesis in pancreatic beta-cells.