15-deoxy-Delta 12,14-prostaglandin J2 induces apoptosis of human hepatic myofibroblasts. A pathway involving oxidative stress independently of peroxisome-proliferator-activated receptors

Hepatic myofibroblasts (hMFs) play a key role in the development of liver fibrosis associated with chronic liver diseases. Apoptosis of these cells is emerging as a key process in the resolution of liver fibrosis. Here, we examined the effects of cyclopentenone prostaglandins on apoptosis of human hMFs. Cyclopentenone prostaglandins of the J series markedly reduced hMF viability, with 15-deoxy-Delta(12,14)-prostaglandin J2 (15-d-PGJ2) being the most potent. This effect was independent of peroxisome-proliferator-activated receptors (PPARs), because PPARgamma and PPARalpha agonists did not affect hMF cell viability, and PPARgamma, the nuclear receptor for 15-d-PGJ2, was not expressed in hMFs. Moreover, 15-d-PGJ2 did not act via a cell surface G protein-coupled receptor, as shown in guanosine-5'-O-(3-thiotriphosphate) binding assays. Cell death resulted from an apoptotic process, because 15-d-PGJ2-treated hMFs exhibited condensed nuclei, fragmented DNA, and elevated caspase-3 activity. Moreover, the caspase inhibitor Z-Val-Ala-Asp(OCH3)-fluoromethyl ketone blocked the cytotoxic effect of 15-d-PGJ2. The apoptotic effects of 15-d-PGJ2 were reproduced by H2O2 and blocked by the antioxidants N-acetylcysteine (NAC), N-(2-mercapto-propionyl)-glycine (NMPG) and pyrrolidine dithiocarbamate (PDTC). Accordingly, 15-d-PGJ2 generated rapid production of reactive oxygen species in hMFs, via a NAC/NMPG/PDTC-sensitive pathway. In conclusion, 15-d-PGJ2 induces apoptosis of human hMFs via a novel mechanism involving oxidative stress and unrelated to activation of its nuclear receptor PPARgamma. These data underline the antifibrogenic potential of 15-d-PGJ2.     

Cyclopentenone prostaglandins induce lymphocyte apoptosis by activating the mitochondrial apoptosis pathway independent of external death receptor signaling

15-Deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) is a naturally occurring cyclopentenone metabolite of PGD(2) that possesses both peroxisome proliferator-activated receptor gamma (PPAR-gamma)-dependent and PPAR-gamma-independent anti-inflammatory properties. Recent studies suggest that cyclopentenone PGs may play a role in the down-regulation of inflammation-induced immune responses. In this study, we report that 15d-PGJ(2) as well as synthetic PPAR-gamma agonists inhibit lymphocyte proliferation. However, only 15d-PGJ(2), but not the specific PPAR-gamma activators, induce lymphocyte apoptosis. We found that blocking of the death receptor pathway in Fas-associated death domain(-/-) or caspase-8(-/-) Jurkat T cells has no effect on apoptosis induction by 15d-PGJ(2). Conversely, overexpression of Bcl-2 or Bcl-x(L) completely inhibits the initiation of apoptosis, indicating that 15d-PGJ(2)-mediated apoptosis involves activation of the mitochondrial pathway. In line with these results, 15d-PGJ(2) induces mitochondria disassemblage as demonstrated by dissipation of mitochondrial transmembrane potential (Deltapsi(m)) and cytochrome c release. Both of these events are partially inhibited by the broad spectrum caspase inhibitor benzyloxycarbonil-Val-Ala-Asp-fluoromethylketone, suggesting that caspase activation may amplify the mitochondrial alterations initiated by 15d-PGJ(2). We also demonstrate that 15d-PGJ(2) potently stimulates reactive oxygen species production in Jurkat T cells, and Deltapsi(m) loss induced by 15d-PGJ(2) is prevented by the reactive oxygen species scavenger N-acetyl-L-cysteine. In conclusion, our data indicate that cyclopentenone PGs like 15d-PGJ(2) may modulate immune responses even independent of PPAR-gamma by activating the mitochondrial apoptosis pathway in lymphocytes in the absence of external death receptor signaling.     

Glutathione S-transferases (GSTs) inhibit transcriptional activation by the peroxisomal proliferator-activated receptor gamma (PPAR gamma) ligand, 15-deoxy-delta 12,14prostaglandin J2 (15-d-PGJ2)

15-Deoxy-Delta(12,14)prostaglandin J(2) (15-d-PGJ(2)), a terminal metabolite of the J-series cyclopentenone prostaglandins, influences a variety of cellular processes including gene expression, differentiation, growth, and apoptosis. As a ligand of peroxisomal proliferator-activated receptor gamma (PPAR gamma), 15-d-PGJ(2) can transactivate PPAR gamma-responsive promoters. Previously, we showed that multidrug resistance proteins MRP1 and MRP3 attenuate cytotoxic and transactivating activities of 15-d-PGJ(2) in MCF7 breast cancer cells. Attenuation was glutathione-dependent and was associated with formation of the glutathione conjugate of 15-d-PGJ(2), 15-d-PGJ(2)-SG, and its active efflux by MRP. Here we have investigated whether the glutathione S-transferases (GST) can influence biological activities of 15-d-PGJ(2). MCF7 cells were stably transduced with human cytosolic GST isozymes M1a, A1, or P1a. These GSTs had no effect on 15-d-PGJ(2) cytotoxicity when expressed either alone or in combination with MRP1. However, expression of any of the three GSTs significantly inhibited 15-d-PGJ(2)-dependent transactivation of a PPAR gamma-responsive reporter gene. The degree of inhibition correlated with the level of GST expressed. Under physiologic conditions, the nonenzymatic rate of 15-d-PGJ(2) conjugation with glutathione was significant. Of the three GST isozymes, only GSTM1a-1a further stimulated the rate of 15-d-PGJ(2)-SG formation. Moreover, GSTM1a-1a rate enhancement was only a transient burst that was complete within 15 s. Hence, catalysis plays little, if any, role in GST inhibition of 15-d-PGJ(2)-dependent transactivation. In contrast, inhibition of transactivation was associated with strong GST/15-d-PGJ(2) interactions. Potent inhibition by 15-d-PGJ(2) and 15-d-PGJ(2)-SG of GST activity was observed with K(i) in the 0.15-2.0 microM range for the three GST isozymes, results suggesting avid associations between GST and 15-d-PGJ(2) or 15-d-PGJ(2)-SG. Electrospray ionization mass spectrometry (ESI/MS) studies revealed no stable adducts of GST and 15-d-PGJ(2) indicating that GST/15-d-PGJ(2) interactions are primarily noncovalent. These results are consistent with a mechanism of GST-mediated inhibition of transactivation in which GST binds 15-d-PGJ(2) and 15-d-PGJ(2)-SG thereby sequestering the ligands in the cytosol away from their nuclear target, PPAR gamma.     

Contribution of cyclopentenone prostaglandins to the resolution of inflammation through the potentiation of apoptosis in activated macrophages

Activation of the macrophage cell line RAW 264.7 with LPS and IFN-gamma induces apoptosis through the synthesis of high concentrations of NO due to the expression of NO synthase-2. In addition to NO, activated macrophages release other molecules involved in the inflammatory response, such as reactive oxygen intermediates and PGs. Treatment of macrophages with cyclopentenone PGs, which are synthesized late in the inflammatory onset, exerted a negative regulation on cell activation by impairing the expression of genes involved in host defense, among them NO synthase-2. However, despite the attenuation of NO synthesis, the percentage of apoptotic cells increased with respect to activated cells in the absence of cyclopentenone PGs. Analysis of the mechanisms by which these PGs enhanced apoptosis suggested a potentiation of superoxide anion synthesis that reacted with NO, leading to the formation of higher concentrations of peroxynitrite, a more reactive and proapoptotic molecule than the precursors. The effect of the cyclopentenone 15-deoxy-Delta(12,14)-PGJ(2) on superoxide synthesis was dependent on p38 mitogen-activated protein kinase activity, but was independent of the interaction with peroxisomal proliferator-activated receptor gamma. The potentiation of apoptosis induced by cyclopentenone PGs involved an increase in the release of cytochrome c from the mitochondria to the cytosol and in the nitration of this protein. These results suggest a role for cyclopentenone PGs in the resolution of inflammation by inducing apoptosis of activated cells.     

15-deoxy-Delta 12,14-PGJ2 induces IL-8 production in human T cells by a mitogen-activated protein kinase pathway.

Mast cells, platelets, and some macrophages are abundant sources of PGD(2) and its active metabolite 15-deoxy-Delta(12,14)-PGJ(2) (15-d-PGJ(2)). The lipid mediator 15-d-PGJ(2) regulates numerous processes, including adipogenesis, apoptosis, and inflammation. The 15-d-PGJ(2) has been shown to both inhibit as well as induce the production of inflammatory mediators such as TNF-alpha, IL-1beta, and cyclooxygenase, mostly occurring via a nuclear receptor called peroxisome proliferator-activated receptor-gamma (PPAR-gamma). Data concerning the effects of 15-d-PGJ(2) on human T cells and immune regulation are sparse. IL-8, a cytokine with both chemotactic and angiogenic effects, is produced by T lymphocytes following activation. Whether 15-d-PGJ(2) can regulate the production of IL-8 in T cells in unknown. Interestingly, 15-d-PGJ(2) treatment of unstimulated T cells induces cell death. In contrast, in activated human T lymphocytes, 15-d-PGJ(2) does not kill them, but induces the synthesis of IL-8. In this study, we report that 15-d-PGJ(2) induced a significant increase in both IL-8 mRNA and protein from activated human T lymphocytes. The induction of IL-8 by 15-d-PGJ(2) did not occur through the nuclear receptor PPAR-gamma, as synthetic PPAR-gamma agonists did not mimic the IL-8-inducing effects of 15-d-PGJ(2). The mechanism of IL-8 induction was through a mitogen-activated protein kinase and NF-kappaB pathway, as inhibitors of both systems abrogated IL-8 protein induction. Therefore, 15-d-PGJ(2) can act as a potent proinflammatory mediator in activated T cells by inducing the production of IL-8. These findings show the complexity with which 15-d-PGJ(2) regulates T cells by possessing both pro- and anti-inflammatory properties depending on the activation state of the cell. The implications of this research also include that caution is warranted in assigning a solely anti-inflammatory role for 15-d-PGJ(2).     

A potential role of 15-deoxy-delta(12,14)-prostaglandin J2 for induction of human articular chondrocyte apoptosis in arthritis

The cyclopentenone prostaglandin (PG) J2 is formed within the cyclopentenone ring of the endogenous prostaglandin PG D2 by a nonenzymatic reaction. The PG J family is involved in mediating various biological effects including the regulation of cell cycle progression and inflammatory responses. Here we demonstrate the potential role of 15-deoxy-Delta(12,14)-prostaglandin J2 (15d-PG J2) in human articular chondrocyte apoptosis. 15d-PG J2 was released by human articular chondrocytes and found in joint synovial fluids taken from osteoarthritis or rheumatoid arthritis patients. Proinflammatory cytokines such as interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) up-regulated chondrocyte release of 15d-PG J2. PG D2 synthase mRNA expression was up-regulated by IL-1beta, TNF-alpha, or nitric oxide. 15d-PG J2 induced apoptosis of chondrocytes from osteoarthritis or rheumatoid arthritis patients as well as control nonarthritic subjects in a time- and dose-dependent manner and in a peroxisome proliferator-activated receptor gamma-dependent manner. Peroxisome proliferator-activated receptor gamma expression was up-regulated by IL-1beta and TNF-alpha. Inhibition of NF-kappaB, and the activation of p38 MAPK were also found to be involved in 15d-PG J2-induced chondrocyte apoptosis. Such signal pathways led to the activation of the downstream pro-apoptotic molecule p53 and caspase cascades. Together, these results suggest that 15d-PGJ2 may play an important role in the pathogenesis of arthritic joint destruction via a regulation of chondrocyte apoptosis.     

Differential effects of serum constituents on apoptosis induced by the cyclopentenone prostaglandin 15-deoxy-delta12,14-prostaglandin J2 in WISH epithelial cells

Cyclopentenone prostaglandins, delta12-PGJ2 and 15d-PGJ2, have potent anti-tumour and anti-inflammatory activities, and have been shown to induce apoptosis in amnion-derived WISH cells. In this study, we have investigated the protective effects of serum and its constituents (growth factors and albumin) on delta12-PGJ2 and 15d-PGJ2-induced apoptosis in WISH cells. Serum (0.5% w/v) was protective against both delta12-PGJ2 and 15d-PGJ2-induced apoptosis. This was not due to the presence of serum-derived growth factors (EGF, IGF-1 and IGF-2), since they had no significant effect on 15d-PGJ2-induced cell death. In contrast, IGF-1 partially inhibited etoposide-induced apoptosis, confirming the presence of a functional IGF-1 receptor signalling system. Albumin was identified as the key survival factor in serum, since albumin and delipidated albumin exhibited the same level of protection from 15d-PGJ2-induced apoptosis as serum itself. The potential for serum albumin to regulate the bioactivity of cyclopentenone PGs may be of considerable importance in pathological conditions where roles for cyclopentenone PGs have been identified.     

Induction of 68,000-dalton heat shock proteins by cyclopentenone prostaglandins. Its association with prostaglandin-induced G1 block in cell cycle progression

Cyclopentenone prostaglandins (PGs) such as PGA2 and delta 12-PGJ2 act specifically on cells in the G1 phase and induce block of cell cycle progression (Ohno, K., Sakai, T., Fukushima, M., Narumiya, S., and Fujiwara, M. (1988) J. Pharmacol. Exp. Ther. 245, 294-298). In this study, we characterized proteins induced by these PGs in HeLa S3 cells of synchronized growth and examined its association with the cell cycle block. HeLa S3 cells transiently expressed two 68-kDa proteins of isoelectric points of 5.5 and 5.6 in the G1 phase of cell cycle. When G1-enriched cells were incubated with either PGA2 or delta 12-PGJ2, synthesis of these proteins was markedly enhanced. Enhancement by delta 12-PGJ2 was persistent and irreversible, whereas that by PGA2 was reversible. delta 12-PGJ2 also enhanced the synthesis of two additional 68-kDa proteins with isoelectric points of 5.8 and 5.9. On two-dimensional gel electrophoresis, these proteins overlapped exactly with the 68-kDa heat shock proteins induced in cells treated at 43 degrees C for 90 min. They were also indistinguishable from the heat shock proteins in limited proteolysis. When delta 12-PGJ2 was incubated with G2/M phase cells, it induced only a small and transient increase in the 68-kDa proteins. These results suggest that cyclopentenone PGs extensively induce 68-kDa heat shock proteins in the G1 phase HeLa S3 cells and this induction is closely associated with the G1 block of cell cycle progression caused by these PGs.     

Sprouty2 and Spred1-2 proteins inhibit the activation of the ERK pathway elicited by cyclopentenone prostanoids

Sprouty and Spred proteins have been widely implicated in the negative regulation of the fibroblast growth factor receptor-extracellular regulated kinase (ERK) pathway. In considering the functional role of these proteins, we explored their effects on ERK activation induced by cyclopentenone prostanoids, which bind to and activate Ras proteins. We therefore found that ectopic overexpression in HeLa cells of human Sprouty2, or human Spred1 or 2, inhibits ERK1/2 and Elk-1 activation triggered by the cyclopentenone prostanoids PGA(1) and 15d-PGJ(2). Furthermore, we found that in HT cells that do not express Sprouty2 due to hypermethylation of its gene-promoter, PGA(1)-provoked ERK activation was more intense and sustained compared to other hematopoietic cell lines with unaltered Sprouty2 expression. Cyclopentenone prostanoids did not induce Sprouty2 tyrosine phosphorylation, in agreement with its incapability to activate tyrosine-kinase receptors. However, Sprouty2 Y55F, which acts as a defective mutant upon tyrosine-kinase receptor stimulation, did not inhibit cyclopentenone prostanoids-elicited ERK pathway activation. In addition, Sprouty2 did not affect the Ras-GTP levels promoted by cyclopentenone prostanoids. These results unveil both common and differential features in the activation of Ras-dependent pathways by cyclopentenone prostanoids and growth factors. Moreover, they provide the first evidence that Sprouty and Spred proteins are negative regulators of the ERK/Elk-1 pathway activation induced not only by growth-factors, but also by reactive lipidic mediators.     

Cyclopentenone prostaglandins as potential inducers of intracellular oxidative stress

In the present study, we find that cyclopentenone prostaglandins (PGs) of the J(2) series, naturally occurring derivatives of PGD(2), are potential inducers of intracellular oxidative stress that mediates cell degeneration. Based on an extensive screening of diverse chemical agents on induction of intracellular production of reactive oxygen species (ROS), we found that the cyclopentenone PGs, such as PGA(2), PGJ(2), Delta(12)-PGJ(2), and 15-deoxy-Delta(12,14)-PGJ(2), showed the most potent pro-oxidant effect on SH-SY5Y human neuroblastoma cells. As the intracellular events that mediate the PG cytotoxicity, we observed (i) the cellular redox alteration represented by depletion of antioxidant defenses, such as glutathione and glutathione peroxidase; (ii) a transient decrease in the mitochondrial membrane potential (Deltapsi); (iii) the production of protein-bound lipid peroxidation products, such as acrolein and 4-hydroxy-2-nonenal; and (iv) the accumulation of ubiquitinated proteins. These events correlated well with the reduction in cell viability. In addition, the thiol compound, N-acetylcysteine, could significantly inhibit the PG-induced ROS production, thereby preventing cytotoxicity, suggesting that the redox alteration is closely related to the pro-oxidant effect of cyclopentenone PGs. More strikingly, the lipid peroxidation end products, acrolein and 4-hydroxy-2-nonenal, detected in the PG-treated cells potently induced the ROS production, which was accompanied by the accumulation of ubiquitinated proteins and cell death, suggesting that the membrane lipid peroxidation products may represent one of the causative factors that potentiate the cytotoxic effect of cyclopentenone PGs by accelerating intracellular oxidative stress. These data suggest that the intracellular oxidative stress, represented by ROS production/lipid peroxidation and redox alteration, may underlie the well documented biological effects, such as antiproliferative and antitumor activities, of cyclopentenone PGs.     

15d-PGJ2 inhibits oxidized LDL-induced macrophage proliferation by inhibition of GM-CSF production via inactivation of NF-kappaB

Macrophage-derived foam cells play an important role in atherosclerotic lesions. Oxidized low-density lipoprotein (Ox-LDL) induces macrophage proliferation via production of GM-CSF in vitro. This study investigated the effects of 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), a natural ligand for peroxisome proliferator-activated receptor gamma, on macrophage proliferation. Mouse peritoneal macrophages and RAW264.7 cells were used for proliferation study and reporter gene assay, respectively. Twenty microgram per milliliter of Ox-LDL induced [3H]thymidine incorporation in mouse peritoneal macrophages, and 15d-PGJ(2) inhibited Ox-LDL-induced [3H]thymidine incorporation in a dose-dependent manner. Ox-LDL increased GM-CSF release and GM-CSF mRNA expression, and activated GM-CSF gene promoter, all of which were prevented by 15d-PGJ(2) or 2-cyclopenten-1-one, a cyclopentenone ring of 15d-PGJ(2). The suppression of GM-CSF promoter activity by 15d-PGJ(2) and 2-cyclopenten-1-one was mediated through reduction of NF-kappaB binding to GM-CSF promoter. These results suggest that 15d-PGJ(2) inhibits Ox-LDL-induced macrophage proliferation through suppression of GM-CSF production via NF-kappaB inactivation.     

Molecular Mechanisms of Exopolysaccharide from Aphanothece halaphytica (EPSAH) Induced Apoptosis in HeLa Cells

The present study aims to investigate the pharmacological effect of the exopolysaccharides from Aphanothece halophytica GR02 (EPSAH) on the HeLa human cervical cancer cell line. HeLa cells were cultured in RPMI-1640-10% FBS medium containing with or without different concentrations of EPSAH. Cell viability was assessed by methylthiazol tetrazolium (MTT) assay. Cell apoptosis was elevated with Wright-Giemsa staining, AO/EB double staining, and DNA fragmentation assay. Apoptosis-associated molecules from cultured HeLa cells were quantified using Western blot analysis. Our results suggest that EPASH induces apoptosis in HeLa cells by targeting a master unfolded protein response (UPR) regulator Grp78. Grp78 further promotes the expression of CHOP and downregulates expression of survivin, which leads to activate mitochondria-mediated downstream molecules and p53-survivin pathway, resulting in caspase-3 activation and causing apoptosis. These findings provide important clues for further evaluating the potential potency of EPSAH for use in cancer therapy.     

PMA induces expression from the herpes simplex virus thymidine kinase promoter via the activation of JNK and ERK in the presence of adenoviral E1A proteins

The herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) promoter contains elements involved in both constitutive and induced expression. We determined that phorbol 12-myristate 13-acetate (PMA) induces the HSV-1 TK promoter in HEK293 cells. However, PMA did not induce expression from the promoter in HeLa cells and did not result in a globally increased gene expression in HEK293 cells. Induction of HSV-1 TK promoter required activation of both of JNK and ERK pathways. However, activation of the two pathways alone was not sufficient for induction of HSV-1 TK promoter. By transiently transfecting into HeLa cells the adenoviral E1A gene, which exists as an integrant in HEK293 genome, we demonstrated that E1A proteins are necessary for induction of HSV-1 TK promoter by PMA. We propose mechanisms by which signaling pathways activated by the tumor-promoter PMA cooperate with the oncogene E1A to stimulate a eukaryotic promoter, namely the HSV-1 TK promoter.     

Cyclopentenone isoprostanes are novel bioactive products of lipid oxidation which enhance neurodegeneration

Oxidative stress and subsequent lipid peroxidation are involved in the pathogenesis of numerous neurodegenerative conditions, including stroke. Cyclopentenone isoprostanes (IsoPs) are novel electrophilic lipid peroxidation products formed under conditions of oxidative stress via the isoprostane pathway. These cyclopentenone IsoPs are isomeric to highly bioactive cyclopentenone prostaglandins, yet it has not been determined if these products are biologically active or are formed in the brain. Here we demonstrate that the major cyclopentenone IsoP isomer 15-A2t-IsoP potently induces apoptosis in neuronal cultures at submicromolar concentrations. We present a model in which 15-A2t-IsoP induced neuronal apoptosis involves initial depletion of glutathione and enhanced production of reactive oxygen species, followed by 12-lipoxygenase activation and phosphorylation of extracellular signal-regulated kinase 1/2 and the redox sensitive adaptor protein p66shc, which results in caspase-3 cleavage. 15-A2t-IsoP application also dramatically potentiates oxidative glutamate toxicity at concentrations as low as 100 nm, demonstrating the functional importance of these molecules in neurodegeneration. Finally, we employ novel mass spectrometric methods to show that cyclopentenone IsoPs are formed abundantly in brain tissue under conditions of oxidative stress. Together these findings suggest that cyclopentenone IsoPs may contribute to neuronal death caused by oxidative insults, and that their activity should perhaps be addressed when designing neuroprotective therapies.     

ERK/ribosomal S6 kinase (RSK) signaling positively regulates death receptor 5 expression through co-activation of CHOP and Elk1

Death receptor 5 (DR5) is a death domain-containing transmembrane receptor that triggers apoptosis upon binding to its ligand or when overexpressed. Its expression is induced by certain small molecule drugs, including celecoxib, through mechanisms that have not been fully elucidated. The current study has revealed a novel ERK/ribosomal S6 kinase (RSK)-dependent mechanism that regulates DR5 expression primarily using celecoxib as a DR5 inducer. Both C/EBP homologous protein (CHOP) and Elk1 are required for celecoxib-induced DR5 expression based on promoter deletion and mutation analysis and siRNA-mediated gene silencing results. Co-expression of both CHOP and Elk1 exhibited enhanced effects on increasing DR5 promoter activity and DR5 expression, indicating that CHOP and Elk1 co-operatively regulate DR5 expression. Because Elk1 is an ERK-regulated protein, we accordingly found that celecoxib increased the levels of phosphorylated ERK1/2, RSK2, and Elk1. Inhibition of either ERK signaling with a MEK inhibitor or ERK1/2 siRNA, or RSK2 signaling with an RSK2 inhibitor or RSK2 siRNA abrogated DR5 up-regulation by celecoxib as well as other agents. Moreover, these inhibitions suppressed celecoxib-induced CHOP up-regulation. Thus, ERK/RSK-dependent, CHOP and Elk1-mediated mechanisms are critical for DR5 induction. Additionally, celecoxib increased CHOP promoter activity in an ATF4-dependent manner, and siRNA-mediated blockade of ATF4 abrogated both CHOP induction and DR5 up-regulation, indicating that ATF4 is involved in celecoxib-induced CHOP and DR5 expression. Collectively, we conclude that small molecules such as celecoxib induce DR5 expression through activating ERK/RSK signaling and subsequent Elk1 activation and ATF4-dependent CHOP induction.