C/EBP Homologous Protein Knockdown Research Articles

Endoplasmic Reticulum Stress Signaling Is Involved in Mitomycin C(MMC)-Induced Apoptosis in Human Fibroblasts via PERK Pathway

Endoplasmic reticulum (ER) stress-mediated cell apoptosis has been implicated in various cell types, including fibroblasts. Previous studies have shown that mitomycin C (MMC)-induced apoptosis occurs in fibroblasts, but the effects of MMC on ER stress-mediated apoptosis in fibroblasts have not been examined. Here, MMC-induced apoptosis in human primary fibroblasts was investigated by exposing cells to a single dose of MMC for 5 minutes. Significant inhibition of cell proliferation and increased apoptosis were observed using a cell viability assay, Annexin V/propidium iodide double staining, cell cycle analysis, and TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) staining. Upregulation of proapoptotic factors, including cleaved caspase-3 and poly ADP-ribose polymerase (PARP), was detected by Western blotting. MMC-induced apoptosis was correlated with elevation of 78-kDa glucose-regulated protein (GRP78) and C/EBP homologous protein (CHOP), which are hallmarks of ER stress. Three unfolded protein response (UPR) sensors (inositol-requiring enzyme 1, IRE1; activating transcription factor 6, ATF6; and PKR-like ER kinase, PERK) and their downstream signaling pathways were also activated. Knockdown of CHOP attenuated MMC-induced apoptosis by increasing the ratio of BCL-2/BAX and decreasing BIM expression, suggesting that ER stress is involved in MMC-induced fibroblast apoptosis. Interestingly, knockdown of PERK significantly decreased ER stress-mediated apoptosis by reducing the expression of CHOP, BIM and cleaved caspase-3. Reactive oxygen species (ROS) scavenging also decreased the expression of GRP78, phospho-PERK, CHOP, and BIM. These results demonstrate that MMC-induced apoptosis is triggered by ROS generation and PERK activation.

C/EBP homologous protein drives pro-catabolic responses in chondrocytes

IntroductionExcess C/EBP homologous protein (CHOP) expression is one feature of the unfolded protein response (UPR) to endoplasmic reticulum (ER) stress. Here, we focused on CHOP expression and function in chondrocytes.MethodsWe studied human knee osteoarthritis (OA) cartilage, bovine chondrocytes cultured in alginate and subjected to sub-lethal biomechanical injury, and knee chondrocytes of human autopsy donors. We performed siRNA knockdown and transfection.ResultsUPR activation was increased in human knee OA cartilage in situ, and in biomechanically injured cultured chondrocytes in vitro. In normal human chondrocytes, CHOP “gain of function” sensitized chondrocytes to IL-1β induced nitric oxide (NO) and matrix metalloproteinase (MMP)-3 release without inducing these responses by itself. Excess CHOP expression, by itself, induced superoxide production and apoptosis. Conversely, siRNA knockdown of CHOP and the UPR-specific mediator X-box binding protein (XBP1) inhibited NO release by >80% (P <0.0005) in response to IL-1β, and blunted MMP-3 release, whereas there were only minimal effects of the UPR mediator GRP78 on these responses. The anti-inflammatory metabolic “super-regulator” AMP kinase (AMPK) is known to limit UPR activation in vascular muscle cells. Here, CHOP supported the capacity of IL-1β to suppress AMPK activity in chondrocytes. We also observed that inhibition of AMPK activity promoted an increase in chondrocyte CHOP expression. Conversely, pharmacologic activation of AMPK by 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR) blunted chondrocyte CHOP expression in response to biomechanical injury.ConclusionsBiomechanical injury and IL-1 signaling stimulate UPR activation in chondrocytes. CHOP mediates chondrocyte catabolic and apoptotic responses to IL-1β, and does so partly by inhibiting AMPK activity. Conversely, development of excess CHOP activity is limited by AMPK activity in chondrocytes. Our findings suggest a mechanism for potential chondroprotection by AICAR and other AMPK activators. The work is of translational relevance for OA, since several drugs that activate AMPK are already in the clinic for arthritis (for example, allosteric AMPK activators sodium salicylate and high dose aspirin, and methotrexate, which activates AMPK by generating AICAR).

C/EBP homologous protein contributes to cytokine-induced pro-inflammatory responses and apoptosis in β-cells

Induction of the C/EBP homologous protein (CHOP) is considered a key event for endoplasmic reticulum (ER) stress-mediated apoptosis. Type 1 diabetes (T1D) is characterized by an autoimmune destruction of the pancreatic β-cells. Pro-inflammatory cytokines are early mediators of β-cell death in T1D. Cytokines induce ER stress and CHOP overexpression in β-cells, but the role for CHOP overexpression in cytokine-induced β-cell apoptosis remains controversial. We presently observed that CHOP knockdown (KD) prevents cytokine-mediated degradation of the anti-apoptotic proteins B-cell lymphoma 2 (Bcl-2) and myeloid cell leukemia sequence 1 (Mcl-1), thereby decreasing the cleavage of executioner caspases 9 and 3, and apoptosis. Nuclear factor-κB (NF-κB) is a crucial transcription factor regulating β-cell apoptosis and inflammation. CHOP KD resulted in reduced cytokine-induced NF-κB activity and expression of key NF-κB target genes involved in apoptosis and inflammation, including iNOS, FAS, IRF-7, IL-15, CCL5 and CXCL10. This was due to decreased IκB degradation and p65 translocation to the nucleus. The present data suggest that CHOP has a dual role in promoting β-cell death: (1) CHOP directly contributes to cytokine-induced β-cell apoptosis by promoting cytokine-induced mitochondrial pathways of apoptosis; and (2) by supporting the NF-κB activation and subsequent cytokine/chemokine expression, CHOP may contribute to apoptosis and the chemo attraction of mononuclear cells to the islets during insulitis.

Luteolin induces apoptosis through endoplasmic reticulum stress and mitochondrial dysfunction in Neuro-2a mouse neuroblastoma cells

Luteolin, a dietary flavonoid, induces apoptosis in various types of cancer cells. However, its role in neuroblastoma and the underlying mechanisms remain to be elucidated. In the present study, we investigated the molecular mechanisms of the anti-cancer effect of luteolin in Neuro-2a mouse neuroblastoma cells. Luteolin induced apoptotic cell death and activation of caspase-12, -9, and -3, and knockdown of caspase-12 by siRNA transfection reduced luteolin-induced cell death. Luteolin also induced expression of endoplasmic reticulum (ER) stress-associated proteins, including C/EBP homologous protein (CHOP) and glucose-regulated proteins (GRP) 94 and 78, cleavage of ATF6α, and phosphorylation of eIF2α. CHOP knockdown or ER stress inhibitor, 4-phenylbutyric acid, reduced luteolin-induced cell death. These results suggest involvement of ER stress in luteolin-induced neuroblastoma cell death. We then showed that luteolin induced accumulation of reactive oxygen species and that the anti-oxidant N-acetylcysteine reduced luteolin-induced cell death and expression of CHOP and GRP78. We also demonstrated rapid reduction of mitochondrial membrane potential by luteolin, and N-acetylcysteine, as well as 4-phenylbutyric acid or CHOP siRNA transfection ameliorated luteolin-induced late loss, but not early loss of mitochondrial membrane potential. Finally, we showed that luteolin induced activation of mitogen-activated protein kinases such as JNK, p38, and ERK, and inhibitors of mitogen-activated protein kinases reduced luteolin-induced cell death and CHOP expression, as well as mitochondrial Bax translocation and cytochrome c release. Collectively, our results suggest that luteolin induces apoptosis through ER stress and mitochondrial dysfunction in Neuro-2a mouse neuroblastoma cells.

A Novel Monocarbonyl Analogue of Curcumin, (1E,4E)-1,5-Bis(2,3-dimethoxyphenyl)penta-1,4-dien-3-one, Induced Cancer Cell H460 Apoptosis via Activation of Endoplasmic Reticulum Stress Signaling Pathway

Endoplasmic reticulum (ER) stress-induced cancer cell apoptosis has become a novel signaling target for development of cancer therapeutic drugs. Curcumin exhibits growth-suppressive activity against a variety of cancer cells. We previously synthesized a series of monocarbonyl analogues of curcumin with strong cytotoxicity against tumor cells. In this study, we found that only compound 19 [(1E,4E)-1,5-bis(2,3-dimethoxyphenyl)penta-1,4-dien-3-one] can induce C/EBP-homologous protein (CHOP) expression in human lung cancer H460 cells. Treatment with 19 induced H460 cell apoptosis in a dose-responsive manner, and this effect was associated with corresponding increases in a series of key components in ER stress-mediated apoptosis pathway, followed by caspase cleavage and activation. However, curcumin at the same concentrations does not display such properties. CHOP knockdown by specific siRNA attenuated 19-induced cell apoptosis, further indicating that the apoptotic pathway is ER stress-dependent. In vivo, 19 showed a dramatic 53.5% reduction in H460 xenograft tumor size after 22 days of treatment. Taken together, these mechanistic insights on the novel compound 19, with nontoxicity, may provide us with a novel anticancer candidate.

Proteasome inhibitor-I enhances tunicamycin-induced chemosensitization of prostate cancer cells through regulation of NF-κB and CHOP expression

Although endoplasmic reticulum (ER) stress induction by some anticancer drugs can lead to apoptotic death of cancer cells, combination therapy with other chemicals would be much more efficient. It has been reported that proteasome inhibitors could induce cancer cell death through ER-stress. Our study, however, showed a differential mechanism of proteasome inhibitor-I (Pro-I)-induced cell death. Pro-I significantly enhanced apoptotic death of PC3 prostate cancer cells pretreated with tunicamycin (TM) while other signaling inhibitors against p38, mitogen activated kinase (MEK) and phosphatidyl-inositol 3-kinase (PI3K) did not, as evidenced by cell proliferation and cell cycle analyses. NF-κB inhibition by Pro-I, without direct effect on ER-stress, was found to be responsible for the TM-induced chemosensitization of PC3 cells. Moreover, TM-induced/enhancer-binding protein (C/EBP) homologous protein (CHOP) expression was enhanced by Pro-I without change in GRP78 expression. CHOP knockdown by siRNA also showed a significant decrease in Pro-I chemosensitization. All these data suggest that although TM could induce both NF-κB activation and CHOP expression through ER-stress, both NF-κB inhibition and increased CHOP level by Pro-I are required for enhanced chemosensitization of PC3 prostate cancer cells. Thus, our study might contribute to the identification of anticancer targets against prostate cancer cells.

Neuronal Apoptosis Induced by Endoplasmic Reticulum Stress Is Regulated by ATF4–CHOP-Mediated Induction of the Bcl-2 Homology 3-Only Member PUMA

An increasing body of evidence points to a key role of endoplasmic reticulum (ER) stress in acute and chronic neurodegenerative conditions. Extensive ER stress can trigger neuronal apoptosis, but the signaling pathways that regulate this cell death remain unclear. In the present study, we demonstrate that PUMA, a Bcl-2 homology 3 (BH3)-only member of the Bcl-2 family, is transcriptionally activated in cortical neurons by ER stress and is essential for ER-stress-induced cell death. PUMA is known to be a key transcriptional target of p53, but we have found that ER stress triggers PUMA induction and cell death through a p53-independent mechanism mediated by the ER-stress-inducible transcription factor ATF4 (activating transcription factor 4). Specifically, we demonstrate that ectopic expression of ATF4 sensitizes mouse cortical neurons to ER-stress-induced apoptosis and that ATF4-deficient neurons exhibit markedly reduced levels of PUMA expression and cell death. However, chromatin immunoprecipitation experiments suggest that ATF4 does not directly regulate the PUMA promoter. Rather, we found that ATF4 induces expression of the transcription factor CHOP (C/EBP homologous protein) and that CHOP in turn activates PUMA induction. Specifically, we demonstrate that CHOP binds to the PUMA promoter during ER stress and that CHOP knockdown attenuates PUMA induction and neuronal apoptosis. In summary, we have identified a key signaling pathway in ER-stress-induced neuronal death involving ATF4-CHOP-mediated transactivation of the proapoptotic Bcl-2 family member PUMA. We propose that this pathway may be an important therapeutic target relevant to a number of neurodegenerative conditions.

Endoplasmic reticulum stress-induced transcription factor, CHOP, is crucial for dendritic cell IL-23 expression

The endoplasmic reticulum (ER) stress response detects malfunctions in cellular physiology, and microbial pattern recognition receptors recognize external threats posed by infectious agents. This study has investigated whether proinflammatory cytokine expression by monocyte-derived dendritic cells is affected by the induction of ER stress. Activation of ER stress, in combination with Toll-like receptor (TLR) agonists, markedly enhanced expression of mRNA of the unique p19 subunit of IL-23, and also significantly augmented secretion of IL-23 protein. These effects were not seen for IL-12 secretion. The IL-23 gene was found to be a target of the ER stress-induced transcription factor C/EBP homologous protein (CHOP), which exhibited enhanced binding in the context of both ER stress and TLR stimulation. Knockdown of CHOP in U937 cells significantly reduced the synergistic effects of TLR and ER stress on IL-23p19 expression, but did not affect expression of other LPS-responsive genes. The integration of ER stress signals and the requirement for CHOP in the induction of IL-23 responses was also investigated in a physiological setting: infection of myeloid cells with Chlamydia trachomatis resulted in the expression of CHOP mRNA and induced the binding of CHOP to the IL-23 promoter. Furthermore, knockdown of CHOP significantly reduced the expression of IL-23 in response to this intracellular bacterium. Therefore, the effects of pathogens and other environmental factors on ER stress can profoundly affect the nature of innate and adaptive immune responses.

Endoplasmic reticulum stress mediates γ-tocotrienol-induced apoptosis in mammary tumor cells

gamma-Tocotrienol, a member of the vitamin E family of compounds, induces apoptosis in a variety of cancer cell types. However, previous studies have clearly demonstrated that gamma-tocotrienol-induced apoptosis in neoplastic mouse +SA mammary epithelial cells is not mediated through mitochondrial stress or death receptor apoptotic signaling. Therefore, studies were conducted to determine the role of endoplasmic reticulum (ER) stress in mediating gamma-tocotrienol-induced apoptosis in +SA mammary tumor cells. Treatment with 15-40 microM gamma-tocotrienol induced +SA cell death in a dose-responsive manner, and these effects were associated with a corresponding increase in poly (ADP-ribose) polymerase (PARP)-cleavage and activation of protein kinase-like endoplasmic reticulum kinase/eukaryotic translational initiation factor/activating transcription factor 4 (PERK/eIF2alpha/ATF-4) pathway, a marker of ER stress response. These treatments also caused a large increase in C/EBP homologous protein (CHOP) levels, a key component of ER stress mediated apoptosis that increases expression of tribbles 3 (TRB3). Knockdown of CHOP by specific siRNAs attenuated gamma-tocotrienol-induced PARP-cleavage, CHOP and TRB3 expression. gamma-Tocotrienol treatment also reduced full-length caspase-12 levels, an indication of caspase-12 cleavage and activation. Intracellular levels of 3-hydroxy-3-methylglutaryl-coenzyme A (HMGCoA) reductase, an ER-transmembrane enzyme catalyzing the synthesis of mevalonate, decreased following gamma-tocotrienol treatment, but combined treatment with mevalonate did not reverse gamma-tocotrienol-induced apoptosis, suggesting that a decrease in HMGCoA reductase activity is not required for gamma-tocotrienol induced apoptosis. These results demonstrate that ER stress apoptotic signaling is associated with gamma-tocotrienol-induced apoptosis in +SA mammary tumor cells.

N-acetyl cysteine and penicillamine induce apoptosis via the ER stress response-signaling pathway

N-acetyl cysteine (NAC) and penicillamine (PEN) have been shown to induce apoptosis in multiple types of human cancer cells; however, the molecular mechanism underlying this activity is unclear. This study was designed to identify the genes responsible for apoptosis induction by NAC and PEN. We found that glucose-regulated protein 78 (GRP78) was upregulated in HeLa cells following treatment with NAC or PEN. GRP78 is a central regulator of endoplasmic reticulum (ER) stress and has been used as a marker of ER stress. Additionally, both the activating transcription factor 6 (ATF6) protein and X box-binding protein 1 (XBP1) mRNA were processed, which facilitates the expression of C/EBP homologous protein (CHOP), a key-signaling component of ER stress-induced apoptosis. Furthermore, the PERK-ATF4 pathway, which also induces the expression of CHOP, was activated in NAC-treated cells. The role of the ER stress pathway was further confirmed through the small interfering RNA (siRNA)-mediated knockdown of CHOP, which attenuated NAC and PEN-induced apoptosis. These results demonstrate that NAC- and PEN-induced apoptosis in HeLa cells is mediated by the ER stress pathway.

CHOP Mediates Endoplasmic Reticulum Stress-Induced Apoptosis in Gimap5-Deficient T Cells

Gimap5 (GTPase of the immunity-associated protein 5) has been linked to the regulation of T cell survival, and polymorphisms in the human GIMAP5 gene associate with autoimmune disorders. The BioBreeding diabetes-prone (BBDP) rat has a mutation in the Gimap5 gene that leads to spontaneous apoptosis of peripheral T cells by an unknown mechanism. Because Gimap5 localizes to the endoplasmic reticulum (ER), we hypothesized that absence of functional Gimap5 protein initiates T cell death through disruptions in ER homeostasis. We observed increases in ER stress-associated chaperones in T cells but not thymocytes or B cells from Gimap5−/− BBDP rats. We then discovered that ER stress-induced apoptotic signaling through C/EBP-homologous protein (CHOP) occurs in Gimap5−/− T cells. Knockdown of CHOP by siRNA protected Gimap5−/− T cells from ER stress-induced apoptosis, thereby identifying a role for this cellular pathway in the T cell lymphopenia of the BBDP rat. These findings indicate a direct relationship between Gimap5 and the maintenance of ER homeostasis in the survival of T cells.

Apoptosis induced by t10,c12-conjugated linoleic acid is mediated by an atypical endoplasmic reticulum stress response

Conjugated linoleic acid (CLA) inhibits rat mammary carcinogenesis, in part by inducing apoptosis of preneoplastic and neoplastic mammary epithelial cells. The current study focused on the mechanism by which apoptosis is induced. In TM4t mammary tumor cells, trans-10,cis-12 (t10,c12)-CLA induced proapoptotic C/EBP-homologous protein (CHOP) concurrent with the cleavage of poly(ADP-ribose) polymerase. Knockdown of CHOP attenuated t10,c12-CLA-induced apoptosis. Furthermore, t10,c12-CLA induced the cleavage of endoplasmic reticulum (ER)-resident caspase-12, and a selective inhibitor of caspase-12 significantly alleviated t10,c12-CLA-induced apoptosis. Using electron microscopy, we observed that t10,c12-CLA treatment resulted in marked dilatation of the ER lumen. Together, these data suggest that t10,c12-CLA induces apoptosis through ER stress. To further explore the ER stress pathway, we examined the expression of the following upstream ER stress signature markers in response to CLA treatment: X-box binding protein 1 (XBP1) mRNA (unspliced and spliced), phospho-eukaryotic initiation factor (eIF) 2 alpha, activating transcription factor 4 (ATF4), and BiP proteins. We found that t10,c12-CLA induced the expression and splicing of XBP1 mRNA as well as the phosphorylation of eIF2 alpha. In contrast, ATF4 was induced modestly, but not significantly, and BiP was not altered. In summary, our data demonstrate that apoptosis induced by t10,c12-CLA is mediated, at least in part, through an atypical ER stress response that culminates in the induction of CHOP and the cleavage of caspase-12.