Bcl-2 and Bcl-X(L) block thapsigargin-induced nitric oxide generation, c-Jun NH(2)-terminal kinase activity, and apoptosis.

The proteins Bcl-2 and Bcl-X(L) prevent apoptosis, but their mechanism of action is unclear. We examined the role of Bcl-2 and Bcl-X(L) in the regulation of cytosolic Ca(2+), nitric oxide production (NO), c-Jun NH(2)-terminal kinase (JNK) activation, and apoptosis in Jurkat T cells. Thapsigargin (TG), an inhibitor of the endoplasmic reticulum-associated Ca(2+) ATPase, was used to disrupt Ca(2+) homeostasis. TG acutely elevated intracellular free Ca(2+) and mitochondrial Ca(2+) levels and induced NO production and apoptosis in Jurkat cells transfected with vector (JT/Neo). Buffering of this Ca(2+) response with 1, 2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM) or inhibiting NO synthase activity with N(G)-nitro-L-arginine methyl ester hydrochloride (L-NAME) blocked TG-induced NO production and apoptosis in JT/Neo cells. By contrast, while TG produced comparable early changes in the Ca(2+) level (i.e., within 3 h) in Jurkat cells overexpressing Bcl-2 and Bcl-X(L) (JT/Bcl-2 or JT/Bcl-X(L)), NO production, late (36-h) Ca(2+) accumulation, and apoptosis were dramatically reduced compared to those in JT/Neo cells. Exposure of JT/Bcl-2 and JT/Bcl-X(L) cells to the NO donor, S-nitroso-N-acetylpenacillamine (SNAP) resulted in apoptosis comparable to that seen in JT/Neo cells. TG also activated the JNK pathway, which was blocked by L-NAME. Transient expression of a dominant negative mutant SEK1 (Lys-->Arg), an upstream kinase of JNK, prevented both TG-induced JNK activation and apoptosis. A dominant negative c-Jun mutant also reduced TG-induced apoptosis. Overexpression of Bcl-2 or Bcl-X(L) inhibited TG-induced loss in mitochondrial membrane potential, release of cytochrome c, and activation of caspase-3 and JNK. Inhibition of caspase-3 activation blocked TG-induced JNK activation, suggesting that JNK activation occurred downstream of caspase-3. Thus, TG-induced Ca(2+) release leads to NO generation followed by mitochondrial changes including cytochrome c release and caspase-3 activation. Caspase-3 activation leads to activation of the JNK pathway and apoptosis. In summary, Ca(2+)-dependent activation of NO production mediates apoptosis after TG exposure in JT/Neo cells. JT/Bcl-2 and JT/Bcl-X(L) cells are susceptible to NO-mediated apoptosis, but Bcl-2 and Bcl-X(L) protect the cells against TG-induced apoptosis by negatively regulating Ca(2+)-sensitive NO synthase activity or expression.     

Activation of c-Jun N-terminal kinase 1 by UV irradiation is inhibited by wortmannin without affecting c-iun expression

Activation of c-Jun N-terminal kinases (JNKs)/stress-activated protein kinases is an early response of cells upon exposure to DNA-damaging agents. JNK-mediated phosphorylation of c-Jun is currently understood to stimulate the transactivating potency of AP-1 (e.g., c-Jun/c-Fos; c-Jun/ATF-2), thereby increasing the expression of AP-1 target genes. Here we show that stimulation of JNK1 activity is not a general early response of cells exposed to genotoxic agents. Treatment of NIH 3T3 cells with UV light (UV-C) as well as with methyl methanesulfonate (MMS) caused activation of JNK1 and an increase in c-Jun protein and AP-1 binding activity, whereas antineoplastic drugs such as mafosfamide, mitomycin C, N-hydroxyethyl-N-chloroethylnitrosourea, and treosulfan did not elicit this response. The phosphatidylinositol 3-kinase inhibitor wortmannin specifically blocked the UV-stimulated activation of JNK1 but did not affect UV-driven activation of extracellular regulated kinase 2 (ERK2). To investigate the significance of JNK1 for transactivation of c-jun, we analyzed the effect of UV irradiation on c-jun expression under conditions of wortmannin-mediated inhibition of UV-induced stimulation of JNK1. Neither the UV-induced increase in c-jun mRNA, c-Jun protein, and AP-1 binding nor the activation of the collagenase and c-jun promoters was affected by wortmannin. In contrast, the mitogen-activated protein kinase/ERK kinase inhibitor PD98056, which blocked ERK2 but not JNK1 activation by UV irradiation, impaired UV-driven c-Jun protein induction and AP-1 binding. Based on the data, we suggest that JNK1 stimulation is not essential for transactivation of c-jun after UV exposure, whereas activation of ERK2 is required for UV-induced signaling leading to elevated c-jun expression.     

Protection of renal epithelial cells against oxidative injury by endoplasmic reticulum stress preconditioning is mediated by ERK1/2 activation

We investigated the role of the endoplasmic reticulum (ER) stress response in intracellular Ca2+ regulation, MAPK activation, and cytoprotection in LLC-PK1 renal epithelial cells in an attempt to identify the mechanisms of protection afforded by ER stress. Cells preconditioned with trans-4,5-dihydroxy-1,2-dithiane, tunicamycin, thapsigargin, or A23187 expressed ER stress proteins and were resistant to subsequent H2O2-induced cell injury. In addition, ER stress preconditioning prevented the increase in intracellular Ca2+ concentration that normally follows H2O2 exposure. Stable transfection of cells with antisense RNA targeted against GRP78 (pkASgrp78 cells) prevented GRP78 induction, disabled the ER stress response, sensitized cells to H2O2-induced injury, and prevented the development of tolerance to H2O2 that normally occurs with preconditioning. ERK and JNK were transiently (30-60 min) phosphorylated in response to H2O2. ER stress-preconditioned cells had more ERK and less JNK phosphorylation than control cells in response to H2O2 exposure. Preincubation with a specific inhibitor of JNK activation or adenoviral infection with a construct that encodes constitutively active MEK1, the upstream activator of ERKs, also protected cells against H2O2 toxicity. In contrast, the pkASgrp78 cells had less ERK and more JNK phosphorylation upon H2O2 exposure. Expression of constitutively active ERK also conferred protection on native as well as pkAS-grp78 cells. These results indicate that GRP78 plays an important role in the ER stress response and cytoprotection. ER stress preconditioning attenuates H2O2-induced cell injury in LLC-PK1 cells by preventing an increase in intracellular Ca2+ concentration, potentiating ERK activation, and decreasing JNK activation. Thus, the ER stress response modulates the balance between ERK and JNK signaling pathways to prevent cell death after oxidative injury. Furthermore, ERK activation is an important downstream effector mechanism for cellular protection by ER stress.     

Endoplasmic Reticulum Stress Upregulates the Chondroitin Sulfate Level which thus Prevents Neurite Extension in C6 Glioma Cells and Primary Cultured Astrocytes

Chondroitin sulfate (CS), which is known to be a neurite-preventing molecule, is a major component of the extracellular matrix (ECM) in the central nervous system (CNS). The CS expression is upregulated around damaged areas. Endoplasmic reticulum (ER) stress causes neuronal cell death in numerous neurodegenerative diseases. However, the effects of ER stress on glial cells remain to be clarified. The present study examined whether direct ER stress to glial cells can upregulate CS expression in C6 glioma cells and primary cultured mouse astrocytes, and also whether the expression of CS prevents neurite extension. ER stressors tunicamycin (TM) and thapsigargin (TG) significantly increased CS expression in both C6 cells and primary cultured astrocytes, while NO donor sodium nitroprusside (SNP) did not significantly alter the CS expression. The dosage of TM and TG treatment used in this study did not significantly induce cell death but upregulated the ER chaperone molecule Grp78 in C6 glioma cells and primary astrocytes. The ECM of glial cells exposed to ER stress prevented neurite extension in primary cultured mouse cortical neurons, and chondroitinase ABC (ChABC) treatment diminished the inhibitory effect on neurite extension. These findings suggest that direct ER stress to glial cells increases the CS expression, which thus prevents neurite extension.     

Ethanol rapidly causes activation of JNK associated with ER stress under inhibition of ADH

Acute ethanol loading causes oxidative stress to activate cell-death signaling via c-Jun NH2-terminal kinase (JNK) in livers. JNK are stimulated under conditions of endoplasmic reticulum (ER) stress which causes programmed cell death. However, no remarked cell death was observed in acute ethanol intoxication. Akt, one of the cell survival protein kinases, may be activated under ethanol loading. The aim of this study was to estimate activation of JNK and ER stress, role of ethanol metabolism on the activation, and association of JNK with Akt under acute ethanol loading using the perfused rat liver system. Activation of JNK or Akt and association of JNK and Akt with JNK interacting protein 1 were estimated by immunoprecipitation and immunoblotting. Expression of 78 kDa glucose-regulated protein (GRP78) mRNA, a biomarker of ER stress, was detected by quantitative real-time RT-PCR. Activations of JNK and Akt were enhanced by co-treatment with ethanol and a classical inhibitor of alcohol dehydrogenase (ADH). Addition of an antioxidant reduced the activation of JNK. Ethanol loading with ADH inhibition causes down-regulation of GRP78 mRNA levels. Therefore, these findings suggest first revelation that inhibition of ethanol metabolism complicates oxidative and ER stresses produced by ethanol.     

Metformin prevents endoplasmic reticulum stress-induced apoptosis through AMPK-PI3K-c-Jun NH2 pathway

Type 2 diabetes mellitus is thought to be partially associated with endoplasmic reticulum (ER) stress toxicity on pancreatic beta cells and the result of decreased insulin synthesis and secretion. In this study, we showed that a well-known insulin sensitizer, metformin, directly protects against dysfunction and death of ER stress-induced NIT-1 cells (a mouse pancreatic beta cell line) via AMP-activated protein kinase (AMPK) and phosphatidylinositol-3 (PI3) kinase activation. We also showed that exposure of NIT-1 cells to metformin (5mM) increases cellular resistance against ER stress-induced NIT-1 cell dysfunction and death. AMPK and PI3 kinase inhibitors abolished the effect of metformin on cell function and death. Metformin-mediated protective effects on ER stress-induced apoptosis were not a result of an unfolded protein response or the induced inhibitors of apoptotic proteins. In addition, we showed that exposure of ER stressed-induced NIT-1 cells to metformin decreases the phosphorylation of c-Jun NH(2) terminal kinase (JNK). These data suggest that metformin is an important determinant of ER stress-induced apoptosis in NIT-1 cells and may have implications for ER stress-mediated pancreatic beta cell destruction via regulation of the AMPK-PI3 kinase-JNK pathway.     

Calphostin C-induced apoptosis is mediated by a tissue transglutaminase-dependent mechanism involving the DLK/JNK signaling pathway

A role for tissue transglutaminase (TG2) and its substrate dual leucine zipper-bearing kinase (DLK), an upstream component of the c-Jun N-terminal kinase (JNK) signaling pathway, has been previously suggested in the apoptotic response induced by calphostin C. In the current study, we directly tested this hypothesis by examining via pharmacological and RNA-interference approaches whether inhibition of expression or activity of TG2, DLK and JNK in mouse NIH 3T3 fibroblasts and human MDA-MB-231 breast cancer epithelial cells affects calphostin C-induced apoptosis. Our experiments with the selective JNK inhibitor SP600125 reveal that calphostin C is capable of causing JNK activation and JNK-dependent apoptosis in both cell lines. Small interfering RNA-mediated depletion of TG2 alone strongly reduces calphostin C action on JNK activity and apoptosis. Consistent with an active role for DLK in this cascade of event, cells deficient in DLK demonstrate a substantial delay of JNK activation and poly-ADP-ribose polymerase (PARP) cleavage in response to calphostin C, whereas overexpression of a recombinant DLK resistant to silencing, but sensitive to TG2-mediated oligomerization, reverses this effect. Importantly, combined depletion of TG2 and DLK further alters calphostin C effects on JNK activity, Bax translocation, caspase-3 activation, PARP cleavage and cell viability, demonstrating an obligatory role for TG2 and DLK in calphostin C-induced apoptosis.     

Increased expression of interleukin-1β in triglyceride-induced macrophage cell death is mediated by p38 MAP kinase

Triglycerides (TG) are implicated in the development of atherosclerosis through formation of foam cells and induction of macrophage cell death. In this study, we report that addition of exogenous TG induced cell death in phorbol 12-myristate 13-acetate-differentiated THP-1 human macrophages. TG treatment induced a dramatic decrease in interleukin-1β (IL-1β) mRNA expression in a dose- and time-dependent manner. The expression of granulocyte macrophage colony-stimulating factor and platelet endothelial cell adhesion molecule remained unchanged. To identify signaling pathways involved in TG-induced downregulation of IL-1β, we added p38 MAPK, protein kinase C (PKC) or c-Raf1 specific inhibitors. We found that inhibition of p38 MAPK alleviated the TG-induced downregulation of IL-1β, whereas inhibition of PKC and c-Raf1 had no effect. This is the first report showing decreased IL-1β expression during TG-induced cell death in a human macrophage line. Our results suggest that downregulation of IL-1β expression by TG-treated macrophages may play a role during atherogenesis.     

Chronic overexpression of the calcineurin inhibitory gene DSCR1 (Adapt78) is associated with Alzheimer's disease

The DSCR1 (Adapt78) gene was independently discovered as a resident of the "Down syndrome candidate region"and as an "adaptive response"shock or stress gene that is transiently induced during oxidative stress. Recently the DSCR1 (Adapt78) gene product was discovered to be an inhibitor of the serine/threonine phosphatase, calcineurin, and its signaling pathways. We hypothesized that DSCR1 (Adapt78) might also be involved in the development of Alzheimer's disease. To address this question we first studied DSCR1 (Adapt78) in multiple human tissues and found significant expression in brain, spinal cord, kidney, liver, mammary gland, skeletal muscle, and heart. Within the brain DSCR1 (Adapt78) is predominantly expressed in neurons within the cerebral cortex, hippocampus, substantia nigra, thalamus, and medulla oblongata. When we compared DSCR1 (Adapt78) mRNA expression in post-mortem brain samples from Alzheimer's disease patients and individuals who had died with no Alzheimer's diagnosis, we found that DSCR1 (Adapt78) mRNA levels were about twice as high in age-matched Alzheimer's patients as in controls. DSCR1 (Adapt78) mRNA levels were actually three times higher in patients with extensive neurofibrillary tangles (a hallmark of Alzheimer's disease) than in controls. In comparison, post-mortem brain samples from Down syndrome patients (who suffer Alzheimer's symptoms) also exhibited DSCR1 (Adapt78) mRNA levels two to three times higher than controls. Using a cell culture model we discovered that the amyloid beta(1-42) peptide, which is a major component of senile plaques in Alzheimer's, can directly induce increased expression of DSCR1 (Adapt78). Our findings associate DSCR1 (Adapt78) with such major hallmarks of Alzheimer's disease as amyloid protein, senile plaques, and neurofibrillary tangles.     

Coordination of JNK1 and JNK2 is critical for GADD45alpha induction and its mediated cell apoptosis in arsenite responses

Arsenite is a well documented environmental pathogen, whereas it has also been applied as medication to treat various neoplasmas. The pathogenic and therapeutic effects of arsenite are associated with cellular apoptotic responses. However, the molecular mechanisms of arsenite-induced apoptosis are not very well understood. Our previous study has shown that arsenite exposure is able to activate JNKs, which subsequently mediate the apoptotic outcome. The present study further revealed that the coordination of JNK1 and JNK2 was critical for the arsenite-induced expression of GADD45alpha (growth arrest and DNA damage 45alpha), which in turn mediated the cellular apoptosis. The arsenite-induced apoptosis and GADD45alpha expression were significantly impaired in mouse embryonic fibroblasts deficient in either jnk1 (JNK1-/-) or jnk2 (JNK2-/-). Knockdown of GADD45alpha by its specific small interfering RNA also dramatically reduced the apoptotic responses, and overexpression of GADD45alpha in either JNK1-/- or JNK2-/- mouse embryonic fibroblasts partially resensitized the cell death. Furthermore, it was found that the regulation of GADD45alpha by JNK1 and JNK2 was achieved through mediating the activation of c-Jun, since in the JNK1-/- and JNK2-/- cells the c-Jun activation was impaired, and overexpression of the dominant negative mutant of c-Jun (TAM67) in wild type cells could also block GADD45alpha induction as well as cellular apoptosis. Our results demonstrate that the coordination of JNK1 and JNK2 is critical for c-Jun/GADD45alpha-mediated cellular apoptosis induced by arsenite.     

Lycopene Protects against Hypoxia/Reoxygenation Injury by Alleviating ER Stress Induced Apoptosis in Neonatal Mouse Cardiomyocytes

Endoplasmic reticulum (ER) stress induced apoptosis plays a pivotal role in myocardial ischemia/reperfusion (I/R)-injury. Inhibiting ER stress is a major therapeutic target/strategy in treating cardiovascular diseases. Our previous studies revealed that lycopene exhibits great pharmacological potential in protecting against the I/R-injury in vitro and vivo, but whether attenuation of ER stress (and) or ER stress-induced apoptosis contributes to the effects remains unclear. In the present study, using neonatal mouse cardiomyocytes to establish an in vitro model of hypoxia/reoxygenation (H/R) to mimic myocardium I/R in vivo, we aimed to explore the hypothesis that lycopene could alleviate the ER stress and ER stress-induced apoptosis in H/R-injury. We observed that lycopene alleviated the H/R injury as revealed by improving cell viability and reducing apoptosis, suppressed reactive oxygen species (ROS) generation and improved the phosphorylated AMPK expression, attenuated ER stress as evidenced by decreasing the expression of GRP78, ATF6 mRNA, sXbp-1 mRNA, eIF2α mRNA and eIF2α phosphorylation, alleviated ER stress-induced apoptosis as manifested by reducing CHOP/GADD153 expression, the ratio of Bax/Bcl-2, caspase-12 and caspase-3 activity in H/R-treated cardiomyocytes. Thapsigargin (TG) is a potent ER stress inducer and used to elicit ER stress of cardiomyocytes. Our results showed that lycopene was able to prevent TG-induced ER stress as reflected by attenuating the protein expression of GRP78 and CHOP/GADD153 compared to TG group, significantly improve TG-caused a loss of cell viability and decrease apoptosis in TG-treated cardiomyocytes. These results suggest that the protective effects of lycopene on H/R-injury are, at least in part, through alleviating ER stress and ER stress-induced apoptosis in neonatal mouse cardiomyocytes.     

Oxidative and calcium stress regulate DSCR1 (Adapt78/MCIP1) protein

DSCR1 (adapt78) is a stress-inducible gene and cytoprotectant. Its protein product, DSCR1 (Adapt78), also referred to as MCIP1, inhibits intracellular calcineurin, a phosphatase that mediates many cellular responses to calcium. Exposure of human U251 and HeLa cells to hydrogen peroxide led to a rapid hyperphosphorylation of DSCR1 (Adapt78). Inhibitor and agonist studies revealed that a broad range of kinases were not responsible for DSCR1 (Adapt78) hyperphosphorylation, including ERK1/2, although parallel activation of the latter was observed. Phosphorylation of both DSCR1 (Adapt78) and ERK1/2 was attenuated by inhibitors of tyrosine phosphatase, suggesting the common upstream involvement of tyrosine dephosphorylation. The hyperphosphorylation electrophoretic shift in DSCR1 (Adapt78) mobility was also observed with other oxidizing agents (peroxynitrite and menadione) but not nonoxidants. Calcium ionophores strongly induced the levels of both hypo- and hyper-phosphorylated DSCR1 (Adapt78) but did not alter phosphorylation status. Calcium-dependent growth factor- and angiotensin II-stimulation also induced both DSCR1 (Adapt78) species. Phosphorylation of either or both serines in a 13-amino acid peptide made to a calcineurin-interacting conserved region of DSCR1 (Adapt78) attenuated inhibition of calcineurin. These data indicate that DSCR1 (Adapt78) protein is a novel, early stage oxidative stress-activated phosphorylation target and newly identified calcium-inducible protein, and suggest that these response mechanisms may contribute to the known cytoprotective and calcineurin-inhibitory activities of DSCR1 (Adapt78).