Project description:Farnesol (FOH) and other isoprenoid alcohols induce apoptosis in various carcinoma cells and inhibit tumorigenesis in several in vivo models. However, the mechanisms by which these isoprenoids mediate their effects are not yet fully understood. In this study, we show that FOH is effective inducer of apoptosis in several lung carcinoma cells, including H460 cells. This induction is associated with activation of caspase-3, -9, and -12, and cleavage of PARP. To obtain insight into the mechanism involved in FOH-induced apoptosis, we compared the gene expression profiles of FOH-treated and control H460 cells using microarray analysis. This analysis revealed that many of the genes implicated in endoplasmic reticulum (ER) stress, including ATF3, CHOP/GADD153, HERPUD1, BIP (GRP78), XBP1, PDIA4, and TDAG51, were highly up-regulated within 4 hr of FOH treatment suggesting that FOH-induced apoptosis involves an ER-stress response. This was supported by observations showing that treatment with FOH induces phosphorylation of eIF2ï¡. FOH induces activation of several MAPK pathways, including p38, MEK-ERK, and JNK. Inhibition of MEK1/2 by U0126 inhibited the induction of ER stress-response genes. In addition, knockdown of the MEK1/2 and JNK1/2 expression by short interfering RNA (siRNA) effectively inhibited the induction of apoptosis and activation of caspase-3 and cleavage of PARP by FOH. However, only MEK1/2 siRNAs reduced the expression of ER stress-related genes and inhibited phosphorylation of eIF2ï¡. Our results demonstrate that FOH-induced apoptosis is coupled to ER stress and that activation of MEK1/2 is an upstream event in the FOH-induced ER stress signaling cascade. Vehicle vs. 4h FOH Signature Gene lists (Replicates 1 & 2) are linked as supplementary files to the Series record. Experiment Overall Design: H460 cells were treated for 4 hours with 250 micromolar FOH or vehicle (DMSO) in duplicate experiments. Each FOH treated sample was compared to its matched Vehicle and dye-flips were performed, resulting in 4 arrays (2 replicate sets x 2 technical replicates).

Project description:Bronchopulmonary dysplasia (BPD), a chronic lung disease of prematurity, has been linked to endoplasmic reticulum (ER) stress. To investigate a causal role for ER stress in BPD pathogenesis, we generated mice (cGrp78f/f) with lung epithelial cell-specific knockout (KO) of Grp78, a gene encoding the ER chaperone 78-kDa glucose-regulated protein (GRP78), a master regulator of ER homeostasis and the unfolded protein response (UPR). Lung epithelial-specific Grp78 KO disrupted lung morphogenesis, causing developmental arrest, increased alveolar epithelial type II cell apoptosis and decreased surfactant protein and type I cell marker expression in perinatal lungs. cGrp78f/f pups died immediately after birth, likely due to respiratory distress. Importantly, Grp78 KO triggered UPR activation with marked induction of pro-apoptotic transcription factor C/EBP homologous protein (CHOP). Increased expression of genes involved in oxidative stress and cell death and decreased expression of genes encoding antioxidant enzymes suggest a role for oxidative stress in alveolar epithelial cell (AEC) apoptosis. Increased Smad3 phosphorylation and expression of transforming growth factor-Î² (TGF-Î²)/Smad3 targets Cdkn1a (encoding p21) and Gadd45a suggest that interactions among the apoptotic arm of the UPR, oxidative stress and TGF-Î²/Smad signaling pathways contribute to Grp78 KO-induced AEC apoptosis and developmental arrest. Chemical chaperone taursodeoxycholic acid reduced UPR activation and apoptosis in cGrp78f/f lungs cultured ex vivo, confirming a role for ER stress in observed AEC abnormalities. These results demonstrate a key role for GRP78 in AEC survival and gene expression during lung development through modulation of ER stress and suggest the UPR as a potential therapeutic target in BPD. Whole-genome expression profiling was performed using MouseRef-8 v2.0 Expression BeadChips (Illumina) on RNA isolated from lungs of four Grp78f/f and three cGrp78f/f mice at E18.

Project description:Myeloperoxidase (MPO), oxidative stress (OS), and endoplasmic reticulum (ER) stress are all increased in the lungs of neonatal rat pups raised in hyperoxia (HOX, >90% O2), an established model of bronchopulmonary dysplasia (BPD). However, the relationship between OS, MPO, and ER stress has not been examined in HOX rat pups. To determine the relationship between OS, MPO, and ER stress in BPD we treated Sprague-Dawley neonatal rat pups with Tunicamycin (Tun) or with HOX. Tun directly induces ER stress and simplifies neonatal lung alveolarization. Previously, we showed that HOX induces a cycle of destruction that we hypothesize through increased OS, MPO, and ER stress to induce BPD. To inhibit ER stress specifically, we used tauroursodeoxycholic acid (TUDCA), a molecular chaperone. To break the cycle of destruction and reduce OS and MPO we used N-acetyl-lysyltyrosylcysteine-amide (KYC), a systems pharmacology agent. Lung structure was studied morphometrically. ER stress was detected using immunofluorescence (IF), transcriptomic, proteomic, and electron microscopic analyses. Increased ER stress was observed in the lungs of HOX rat pups and also in human BPD lungs by IF. Morphometric and proteomic studies of rat lungs showed that Tun treatment decreased lung complexity and increased ER stress and BPD severity. The fact that TUDCA improved lung complexity in Tun-treated neonatal rat pups, and decreased BPD induced by HOX provides strong support for the idea that ER stress plays a causal role in BPD. Additional support comes from data showing TUDCA decreased lung myeloid cells and MPO levels in the lungs of both Tun- and HOX-treated neonatal rat pups. These data link OS and MPO to ER stress in the mechanisms mediating BPD. KYC's effective inhibition of ER stress in the lungs of Tun-treated rat pups provides additional support for the idea that MPO-induced ER stress plays a direct causal role in BPD. Thus, ER stress appears to expand our proposed cycle of destruction. Our results suggest ER stress evolves from OS and MPO to increase neonatal lung injury and impaired neonatal lung growth and development. The encouraging effect of TUDCA indicates chemical chaperone has the potential in treating BPD. Using multiomic data to investigate the role of endoplasmic reticulum stress in the development of BPD in rat model.

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Mus musculus

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