Gadd153 mRNA Levels Research Articles

Expression of two endoplasmic reticulum stress markers, GRP78 and GADD153, in rat retinal detachment model and its implication

To investigate the expression of endoplasmic reticulum (ER) stress-related genes, glucose-regulated protein 78 (GRP78) and growth arrest DNA damage-inducible gene 153 (GADD153)/CPEBP homologous protein (CHOP), in rat retinal detachment (RD) model. At various time points after RD, the apoptosis of retinal cells was detected by TdT-mediated fluorescein-16-dUTP nick-end labelling (TUNEL) assay; GRP78 and GADD153 mRNA levels were detected by reverse transcription (RT)-PCR; proteins were detected by western blotting analysis; protein distributions in the retinal cells were observed by immunofluorescence using laser-scanning confocal microscope. After RD, the apoptosis was peaked on 2-4 d and then dropped down. The GRP78 mRNA and GADD153 mRNA levels in RD groups on 0.5, 1, 2, and 4 d were all significantly higher than those in the control group (P<0.05). The expression of GRP78 mRNA peaked on 1-2 d after RD. Expression of GRP78 protein was significantly higher than that in the normal control group on 0.5, 1, 2, 4, 8, 16, and 32 d after RD (P<0.05). The expression of GRP78 protein was observed in all the layers of retina in the RD groups, and peaked on 8, 16, and 32 d. The expression of GADD153 protein, mostly in photoreceptor layers, was significantly higher than that in the control group on 0.5, 1, 2, and 4 d after RD (P<0.05). ER stress-related markers, GRP78 and GADD153, are elevated after RD. The elevation of GADD153 is in parallel with the post-RD apoptosis of retinal cells, suggesting that ER stress-mediated death is likely to be activated after RD and involved in post-RD vision loss.

Human respiratory syncytial virus reduces the number of cells in S-phase and increases GADD153 expression in HEp-2 cells

Human respiratory syncytial virus (HRSV) associated with bronchiolitis and asthma is known to replicate actively in ciliated epithelial cells. However, little is known about the influence of HRSV replication on the cell cycle. We found that HRSV infection of HEp-2 cells led to a reduction of the number of cells in S-phase, to an increase in the number of cells in G1-phase, together with an increase of GADD153 mRNA levels and GADD153 protein expression. These results implied that a shorter S-phase supported HRSV replication suggesting possible strategies for interfering with productive HRSV infection.

Genes associated with pro-apoptotic and protective mechanisms are affected differently on exposure of neuronal cell cultures to arsenite. No indication for endoplasmic reticulum stress despite activation of grp78 and gadd153 expression

The effect of arsenite exposure on cell viability, protein synthesis, energy metabolism and the expression of genes coding for cytoplasmic (hsp70) and endoplasmic reticulum (ER; gadd153, grp78, grp94) stress proteins was investigated in primary neuronal cell cultures. Furthermore, signs of ER stress were evaluated by investigating xbp1 mRNA processing. Arsenite levels of 30 and 100 μM induced severe cell injury. Protein synthesis was reduced to below 20% of control in cultures exposed to 30 and 100 μM arsenite for 1 h, and it remained markedly suppressed until 24 h of exposure. Arsenite induced a transient inhibition of energy metabolism after 1 h of exposure, but energy state recovered completely after 3 h. Arsenite exposure affected the expression and translation of genes coding for HSP70 and GRP78, GRP94, GADD153 to different extents. While hsp70 mRNA levels rose drastically, approximally 550-fold after 6 h exposure, HSP70 protein levels did not change over the first 6 h. On the other hand, gadd153 mRNA levels rose only approximately 14-fold after 6 h exposure, while GADD153 protein levels were markedly increased after 3 and 6 h exposure. HSP70 protein levels were markedly increased and GADD153 protein levels decreased to almost control levels in cultures left in arsenite solution for 24 h, i.e. when only a small fraction of cells had escaped arsenite toxicity. Arsenite exposure of neurons thus induced an imbalance between pro-apoptotic and survival-activating pathways. Despite the marked increase in gadd153 mRNA levels, we did not observe signs of xbp1 processing in arsenite exposed cultures, indicating that arsenite did not produce ER stress.

Activation of peroxisome proliferator-activated receptor-γ stimulates the growth arrest and DNA-damage inducible 153 gene in non-small cell lung carcinoma cells

Activation of peroxisome proliferator-activated receptor (PPAR)-gamma by the thiazolidinedione (TZD) class of antidiabetic drugs elicits growth inhibition in a variety of malignant tumors. We clarified the effects of TZDs on growth of human non-small cell lung carcinoma (NSCLC) cells that express endogenous PPAR-gamma. Troglitazone and pioglitazone caused inhibition of cellular growth and induced apoptosis of NSCLC cells in a time- and dose-dependent manner. Subtraction cloning analysis identified that troglitazone stimulated expression of the growth arrest and DNA-damage inducible (GADD)153 gene, and the increased expression of GADD153 mRNA was also confirmed by an array analysis of the 160 apoptosis-related genes. Western blot analysis revealed that troglitazone also increased GADD153 protein levels in a time-dependent manner. Troglitazone did not stimulate GADD153 mRNA levels in undifferentiated 3T3-L1 cells lacking PPAR-gamma expression, whereas its induction was clearly observed in differentiated adipocytes expressing PPAR-gamma. Activity of the GADD153 promoter occurred in a NSCLC cell line in transient transcription assays and was significantly stimulated by troglitazone, although binding of PPAR/retinoid X receptor heterodimer was not detected in the promoter region in gel retardation assays. Inhibition of GADD153 gene expression by an antisense phosphorothionate oligonucleotide attenuated the troglitazone-induced growth inhibition. These findings collectively indicated that activation of PPAR-gamma by TZDs could cause growth inhibition and apoptosis of NSCLC cells and that GADD153 might be a candidate factor implicated in these processes.

Peroxidative stress selectively down-regulates the neuronal stress response activated under conditions of endoplasmic reticulum dysfunction.

Oxidative stress has been implicated in mechanisms leading to neuronal cell injury in various pathological states of the brain. Here, we investigated the effect of peroxide exposure on the expression of genes coding for cytoplasmic and endoplasmic reticulum (ER) stress proteins. Primary neuronal cell cultures were exposed to H(2)O(2) for 6 h and mRNA levels of hsp70, grp78, grp94, gadd153 were evaluated by quantitative PCR. In addition, peroxide-induced changes in protein synthesis and cell viability were investigated. Peroxide treatment of cells triggered an almost 12-fold increase in hsp70 mRNA levels, but a significant decrease in grp78, grp94 and gadd153 mRNA levels. To establish whether peroxide exposure blocks the ER-resident stress response, cells were also exposed to thapsigargin (Tg, a specific inhibitor of ER Ca(2+)-ATPase) which has been shown to elicit the ER stress response. Tg exposure induced 7.2-fold, 3.6-fold and 8.8-fold increase in grp78, grp94 and gadd153 mRNA levels, respectively. However, after peroxide pre-exposure, the Tg-induced effect on grp78, grp94 and gadd153 mRNA levels was completely blocked. The results indicate that oxidative damage causes a selective down-regulation of the neuronal stress response activated under conditions of ER dysfunction. This down-regulation was only observed in cultures exposed to peroxide levels which induced severe suppression of protein synthesis and cell injury, implying a causative link between peroxide-induced down-regulation of ER stress response system and development of neuronal cell injury. These observations could have implications for our understanding of the mechanisms underlying neuronal cell injury in pathological states of the brain associated with oxidative damage, including Alzheimer's disease where the neuronal stress response activated under conditions of ER dysfunction has been shown to be down-regulated. Down-regulation of ER stress response may increase the sensitivity of neurones to an otherwise nonlethal form of stress.

Homocysteine-induced changes in mRNA levels of genes coding for cytoplasmic- and endoplasmic reticulum-resident stress proteins in neuronal cell cultures

Elevated homocysteine levels have been suggested to contribute to various pathological states of the brain. However, the basic mechanisms underlying homocysteine-induced neurotoxicity have not yet been fully elucidated. In the present series of experiments, we investigated the effect of homocysteine on mRNA levels of genes coding for cytoplasmic- or endoplasmic reticulum-resident stress proteins. Primary neuronal cell cultures were exposed to different homocysteine levels for 1–24 h. Cell injury was evaluated using the MTT assay, protein synthesis was studied by measuring the incorporation of l-[4,5- 3H]leucine into proteins, mRNA levels of hsp70, gadd153, grp78, and grp94 were evaluated by quantitative PCR, and changes in protein levels of hsp70, grp78 and grp94 were analyzed by immunoblotting. Exposure of cells to 5 or 10 mM homocysteine for 24 h induced marked cell injury (decrease of viability to 58 or 45% of control respectively). After 6 h treatment, gadd153, grp78 and grp94 mRNA levels increased markedly, but only when cells were exposed to levels of homocysteine high enough to induce cell injury. In addition, hsp70 mRNA levels and protein synthesis were significantly reduced. At earlier (1 or 3 h) or later (12 or 24 h) time intervals, homocysteine exposure induced a marked increase in mRNA levels of all genes studied. GRP78 and GRP94 protein levels were increased in cells exposed to 5 mM homocysteine for 24 h but not in cells exposed to 10 mM homocysteine. HSP70 protein levels, in contrast, were decreased in cells exposed to homocysteine for different periods. The expression of genes coding for ER-resident stress proteins is specifically activated under conditions of ER stress. The close relationship between the extent of cell injury and increase in grp78 mRNA levels suggests that ER dysfunction may contribute to the pathological process. The results imply that the ER is an intracellular target of homocysteine toxicity.

Increase in tumor GADD153 mRNA level following treatment correlates with response to paclitaxel.

We investigated the relationship between the basal and treatment-induced change in the tumor expression of the drug resistance gene MDR1 and the cellular injury response gene GADD153, and clinical response to paclitaxel treatment. MDR1 and GADD153 mRNA levels were measured by reverse transcriptase polymerase chain reaction (RT-PCR) in tumor samples obtained by fine needle aspiration biopsy from 14 patients before and 24 h after paclitaxel infusion. There was no difference between responders and non-responders with respect to either the basal MDR1 mRNA level or the change in MDR1 mRNA level at 24 h after treatment (P = 0.464). Likewise, there was no difference in basal GADD153 mRNA level between responders and non-responders. However, there was a significantly greater increase in GADD153 mRNA at 24 h in responders compared with non-responders (P = 0.005). An increase in GADD153 mRNA level of 1.5-fold or higher predicted response with a sensitivity of 86% and a specificity of 100%. An increase in GADD153 mRNA level reflects chemotherapy-induced damage sufficient to be manifest as a clinically detectable reduction in tumor volume. Measurement of the change in GADD153 mRNA level successfully identified patients destined to respond as early as 24 h post-treatment.

Effect of nitric oxide on endoplasmic reticulum calcium homeostasis, protein synthesis and energy metabolism

It has been suggested that nitric oxide (NO) may contribute to ischemia-induced cell injury. However, the mechanisms underlying NO toxicity have not yet been fully elucidated. In the present study, we investigated the effect of NO on the level of endoplasmic reticulum (ER) calcium stores, on ER Ca2+pump activity, on protein synthesis, on concentrations of high-energy phosphates, and on gadd153 mRNA levels. Primary neuronal cells were exposed to the NO-donor (±)-S-Nitroso-N-acetylpenicillamine (SNAP) for 1 h, 2 h, 6 h or 24 h. The level of ER calcium stores was evaluated by measuring the increase in cytoplasmic calcium activity induced by exposing cells to thapsigargin, an irreversible inhibitor of ER Ca2+-ATPase; the activity of ER Ca2+-ATPase was determined by measuring a phosphorylated intermediate; SNAP-induced changes in gadd153 expression were evaluated by quantitative PCR; SNAP-induced changes in protein synthesis were investigated by measuring the incorporation of L-[4,5-3H]leucine into proteins, and changes in the levels of ATP, ADP, AMP were measured by HPLC. Exposing cells to SNAP for 1 h to 2 h induced a marked depletion of ER calcium stores through an inhibition of ER Ca2+-ATPase (to 58% of control), and a concentration-dependent suppression of protein synthesis which was reversed in the presence of hemoglobin, suggesting NO-related effects. ATP levels and adenylate energy charge were significantly decreased only when cells were exposed to the highest SNAP concentration for 6 h or 24 h, excluding significant effects of NO on the energy state of cells in the acute state, i.e. when ER calcium stores were already completely depleted and protein synthesis severely suppressed. In light of the regulatory role of ER calcium homeostasis in the control of protein synthesis, the results imply that the suppression of protein synthesis resulted from NO-induced inhibition of ER Ca2+-ATPase and depletion of ER calcium stores, and that NO-induced disturbances of energy metabolism are secondary to the effect of NO on ER calcium homeostasis. It is, therefore, concluded that ER calcium stores are a primary target of NO-toxicity.

Stress- and growth-related gene expression are independent of chemical-induced prostaglandin E(2) synthesis in renal epithelial cells.

Cellular stress can initiate prostaglandin (PG) biosynthesis which, through changes in gene expression, can modulate cellular functions, including cell growth. PGA(2), a metabolite of PGE(2), induces the expression of stress response genes, including gadd153 and hsp70, in HeLa cells and human diploid fibroblasts. PGs, gadd153, and hsp70 expression are also influenced by the cellular redox status. Polyphenolic glutathione conjugates retain the ability to redox cycle, with the concomitant generation of reactive oxygen species. One such conjugate, 2,3,5-tris(glutathion-S-yl)hydroquinone (TGHQ), is a potent nephrotoxic and nephrocarcinogenic metabolite of the nephrocarcinogen, hydroquinone. We therefore investigated the effects of TGHQ on PGE(2) synthesis and gene expression in a renal proximal tubular epithelial cell line (LLC-PK(1)). TGHQ (200 microM, 2 h) increases PGE(2) synthesis (2-3-fold) in LLC-PK(1) cells with only minor (5%) reductions in cell viability. This response is toxicant-specific, since another proximal tubular toxicant, S-(1, 2-dichlorovinyl)-L-cysteine (DCVC), stimulates PGE(2) synthesis only after massive (68%) reductions in cell viability. Consistent with the ability of TGHQ to generate an oxidative stress, both deferoxamine mesylate and catalase protect LLC-PK(1) cells from TGHQ-mediated cytotoxicity. Only catalase, however, completely blocks TGHQ-mediated PGE(2) synthesis, implying a major role for hydrogen peroxide in this response. TGHQ induces the early (60 min) expression of gadd153 and hsp70. However, while inhibition of cyclooxygenase with aspirin prevents TGHQ-induced PGE(2) synthesis, it does not affect TGHQ-mediated induction of gadd153 or hsp70 expression. In contrast, a stable PGE(2) analogue, 11-deoxy-16, 16-dimethyl-PGE(2) (DDM-PGE(2)), which protects LLC-PK(1) cells against TGHQ-mediated cytotoxicity, modestly elevates the levels of gadd153 and hsp70 expression. In addition, catalase and, to a lesser extent, deferoxamine mesylate block TGHQ-induced gene expression. Therefore, although TGHQ-induced generation of reactive oxygen species is required for PGE(2) synthesis and stress gene expression, acute TGHQ-mediated increases in gadd153 and hsp70 mRNA levels are independent of PGE(2) synthesis.

Complexes containing activating transcription factor (ATF)/cAMP-responsive-element-binding protein (CREB) interact with the CCAAT/enhancer-binding protein (C/EBP)–ATF composite site to regulate Gadd153 expression during the stress response

Gadd153, also known as chop, encodes a member of the CCAAT/enhancer-binding protein (C/EBP) transcription factor family and is transcriptionally activated by cellular stress signals. We recently demonstrated that arsenite treatment of rat pheochromocytoma PC12 cells results in the biphasic induction of Gadd153 mRNA expression, controlled in part through binding of C/EBPbeta and two uncharacterized protein complexes to the C/EBP-ATF (activating transcription factor) composite site in the Gadd153 promoter. In this report, we identified components of these additional complexes as two ATF/CREB (cAMP-responsive-element-binding protein) transcription factors having differential binding activities dependent upon the time of arsenite exposure. During arsenite treatment of PC12 cells, we observed enhanced binding of ATF4 to the C/EBP-ATF site at 2 h as Gadd153 mRNA levels increased, and enhanced binding of ATF3 complexes at 6 h as Gadd153 expression declined. We further demonstrated that ATF4 activates, while ATF3 represses, Gadd153 promoter activity through the C/EBP-ATF site. ATF3 also repressed ATF4-mediated transactivation and arsenite-induced activation of the Gadd153 promoter. Our results suggest that numerous members of the ATF/CREB family are involved in the cellular stress response, and that regulation of stress-induced biphasic Gadd153 expression in PC12 cells involves the ordered, sequential binding of multiple transcription factor complexes to the C/EBP-ATF composite site.

Activation of gadd153 expression through transient cerebral ischemia: evidence that ischemia causes endoplasmic reticulum dysfunction

The expression of the gene encoding the C/EBP-homologous protein (CHOP), which is also known as growth arrest and DNA-damage-inducible gene 153 (gadd153), has been shown to be specifically activated under conditions that disturb the functioning of the endoplasmic reticulum (ER). To investigate a possible role of ER dysfunction in the pathological process of ischemic cell damage, we studied ischemia-induced changes in gadd153 expression using quantitative PCR. Transient cerebral ischemia was produced in rats by four-vessel occlusion. In the hippocampus, ischemia induced a pronounced increase in gadd153 mRNA levels, peaking at 8 h of recovery (6.4-fold increase, p<0.01), whereas changes in the cortex were less marked (non-significant increase). To elucidate the possible mechanism underlying this activation process, gadd153 mRNA levels were also evaluated in primary neuronal cell cultures under two different conditions, both leading to a depletion of ER calcium pools in the presence or absence of an increase in cytoplasmic calcium activity. The first procedure, exposure to thapsigargin, an irreversible inhibitor of ER Ca 2+-ATPase, caused a marked increase in gadd153 mRNA levels both in cortical and hippocampal neurons, peaking at 12–18 h after treatment. The second procedure, immersion of cells in calcium free medium supplemented with EGTA, caused only a transient increase in gadd153 mRNA levels, peaking at 6 h of recovery, indicating that a depletion of ER calcium stores in the absence of an increase in cytoplasmic calcium activity is sufficient to activate neuronal gadd153 expression. The results imply that transient cerebral ischemia disturbs the functioning of the ER and that these pathological changes are more pronounced in the hippocampus compared to the cortex.

Induction of the growth arrest and DNA damage-inducible gene GADD153 by cisplatin in vitro and in vivo

The inability to assess the extent of tumour damage immediately following treatment is a major clinical obstacle to improving the management of cancer patients. Normally, the effectiveness of chemotherapy or radiation therapy cannot be determined for at least several weeks after treatment. We studied the increase in mRNA of the growth arrest and DNA damage-inducible gene GADD153 in human 2008 ovarian carcinoma cells in vitro and in vivo to determine whether treatment-induced increases in the level of GADD153 mRNA could be used as a marker of the extent of tumour damage. GADD153 mRNA was increased in a transient, dose-dependent manner by cisplatin (DDP) when the tumour cells were grown both in vitro and as tumour xenografts in nude mice. The magnitude of induction of GADD153 mRNA did not vary significantly between different 2008 xenografts treated with equal doses of DDP, and GADD153 mRNA induction correlated with the degree of in vitro cytotoxicity for two different schedules of drug exposure. DDP increased GADD153 mRNA levels in melanoma and head and neck xenograft models as well. We conclude that the increase in GADD153 mRNA can be used to detect tumour injury at time points as short as 24 h after administration of DDP.

Regulation of the C/EBP-related gene gadd153 by glucose deprivation.

gadd153 encodes a CCAAT/enhancer-binding protein (C/EBP)-related protein that lacks a functional DNA-binding domain. Since the gadd153 protein is capable of heterodimerizing with other C/EBPs, gadd153 may function as a negative regulator of these transcription factors. Here we examined the role of glucose in regulating gadd153 expression. We found that glucose deprivation markedly induces gadd153 mRNA levels in both HeLa and 3T3-L1 cells and that addition of D-(+)-glucose resulted in a rapid decrease of gadd153 mRNA. Similar induction and reversal of gadd153 expression were observed at the protein level. Because C/EBP alpha appears to play an important role in regulating genes involved in adipogenesis and energy metabolism, we examined gadd153 expression during the differentiation of 3T3-L1 preadipocytes and as a function of glucose utilization in differentiated adipocytes. Using a standard differentiation protocol that consisted of hormonal stimulation for 2 days followed by medium changes every 2 days thereafter, we observed that both C/EBP alpha and gadd153 mRNAs were elevated. However, C/EBP alpha induction occurred on day 3, while gadd153 expression was not seen until day 4, when the cells were fully differentiated. Frequent addition of fresh medium to the cells during the differentiation process, as well as supplementation of medium with glucose, reduced gadd153 expression without preventing C/EBP alpha expression or interfering with cellular differentiation. Thus, gadd153 expression is not essential for the process of adipocyte differentiation but is significantly influenced by the availability of glucose to the cell.

Regulation of the C/EBP-Related Gene gadd153 by Glucose Deprivation

gadd153 encodes a CCAAT/enhancer-binding protein (C/EBP)-related protein that lacks a functional DNA-binding domain. Since the gadd153 protein is capable of heterodimerizing with other C/EBPs, gadd153 may function as a negative regulator of these transcription factors. Here we examined the role of glucose in regulating gadd153 expression. We found that glucose deprivation markedly induces gadd153 mRNA levels in both HeLa and 3T3-L1 cells and that addition of D-(+)-glucose resulted in a rapid decrease of gadd153 mRNA. Similar induction and reversal of gadd153 expression were observed at the protein level. Because C/EBP alpha appears to play an important role in regulating genes involved in adipogenesis and energy metabolism, we examined gadd153 expression during the differentiation of 3T3-L1 preadipocytes and as a function of glucose utilization in differentiated adipocytes. Using a standard differentiation protocol that consisted of hormonal stimulation for 2 days followed by medium changes every 2 days thereafter, we observed that both C/EBP alpha and gadd153 mRNAs were elevated. However, C/EBP alpha induction occurred on day 3, while gadd153 expression was not seen until day 4, when the cells were fully differentiated. Frequent addition of fresh medium to the cells during the differentiation process, as well as supplementation of medium with glucose, reduced gadd153 expression without preventing C/EBP alpha expression or interfering with cellular differentiation. Thus, gadd153 expression is not essential for the process of adipocyte differentiation but is significantly influenced by the availability of glucose to the cell.

Cell growth inhibition by prostaglandin A 2 results in elevated expression of gadd153 mRNA

Treatment of Hela cells with prostaglandin A 2 (PGA 2) resulted in a marked inhibition of cell proliferation which was associated with a significant induction of gadd153 mRNA, a member of a novel class of genes associated with growth arrest and DNA damage. Induction of gadd153 mRNA was specific to prostaglandins capable of arresting cell growth and was dose-dependent with the maximum effect seen at 36 μ M PGA 2. Induction was rapid, occurring within 2–4 h and reaching a maximum by 8 h. These effects were reversible as removal of PGA 2 resulted in a rapid decline in gadd153 mRNA levels coincident with resumption of cell growth. PGA 2 induction of gadd153 mRNA was completely prevented by the presence of actinomycin D at a concentration sufficient to block transcription and was partially inhibited (50%) by the protein synthesis inhibitor cycloheximide. The presence of the protein kinase inhibitor 2-aminopurine decreased the PGA 2 induction of gadd153 mRNA by greater than 90%, suggesting that cellular kinases play a role in the induction of gadd153 by PGA 2. Thus PGA 2-mediated growth arrest provides a useful model to further define the role of gadd153 in the negative control of cell growth.