Up-frameshift 1 (UPF1), as the most critical factor in nonsense-mediated messenger RNA (mRNA) decay (NMD), regulates tumor-associated molecular pathways in many cancers. However, the role of UPF1 in lung adenocarcinoma (LUAD) amino acid metabolism remains largely unknown. In this study, we found that UPF1 was significantly correlated with a portion of amino acid metabolic pathways in LUAD by integrating bioinformatics and metabolomics. We further confirmed that UPF1 knockdown inhibited activating transcription factor 4 (ATF4) and Ser51 phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), the core proteins in amino acid metabolism reprogramming. In addition, UPF1 promotes cell proliferation by increasing the amino-acid levels of LUAD cells, which depends on the function of ATF4. Clinically, UPF1 mRNA expression is abnormal in LUAD tissues, and higher expression of UPF1 and ATF4 was significantly correlated with poor overall survival (OS) in LUAD patients. Our findings reveal that UPF1 is a potential regulator of tumor-associated amino acid metabolism and may be a therapeutic target for LUAD.
Activating Transcription Factor 4/genetics* ; Adenocarcinoma of Lung ; Amino Acids ; Cell Proliferation ; Eukaryotic Initiation Factor-2 ; Humans ; Lung Neoplasms ; RNA Helicases/metabolism* ; RNA, Messenger/metabolism* ; Trans-Activators/metabolism*

Cyclooxygenase-2 (COX-2) is an enzyme induced by various proinflammatory and mitogenic stimuli. Celecoxib is a selective inhibitor of COX-2 that have been shown to affect cell growth and apoptosis. Lung cancer cells expressing COX-2 is able to be a target of celecoxib, this study focuses on investigating that celecoxib induces apoptosis via endoplasmic reticulum (ER) stress on lung cancer cells. We investigated whether celecoxib induced apoptosis on non-small cell lung cancer cell line, A549 and H460. The 50 µM of celecoxib increased apoptotic cells and 100 µM of celecoxib significantly induced apoptosis. To check involvement of caspase cascade, pretreatment of z-VAD-fmk blocked celecoxib-induced apoptosis. However, caspase-3, -8, and -9 were not activated, but cleavage of non-classical caspase-4 was detected using western blot. As checking ER stress associated molecules, celecoxib did not increase expressions of growth arrest and DNA damage inducible protein 34, activating transcription factor 4, and spliced X-box binding protiens-1, but increase of both glucose-regulated protein 78 (GRP78) and C/EBP homologous transcription factor were detected. Salubrinal, inhibitor of eIF2 and siRNA for IRE1 did not alter celecoxib-induced apoptosis. Instead, celecoxib-induced apoptosis might be deeply associated with ER stress depending on GRP78 because siRNA for GRP78 enhanced apoptosis. Taken together, celecoxib triggered ER stress on lung cancer cells and celecoxib-induced apoptosis might be involved in both non-classical caspase-4 and GRP78.
Activating Transcription Factor 4 ; Apoptosis ; Blotting, Western ; Carcinoma, Non-Small-Cell Lung* ; Caspase 3 ; Celecoxib* ; Cell Death* ; Cell Line ; Cyclooxygenase 2 ; DNA Damage ; Endoplasmic Reticulum Stress* ; Endoplasmic Reticulum* ; Eukaryotic Initiation Factor-2 ; Lung Neoplasms ; RNA, Small Interfering ; Transcription Factors

The present study was performed to evaluate the relationship between the neurotoxicity of acrylamide and the differential gene expression pattern in mice. Both locomotor test and rota-rod test showed that the group treated with higher than 30 mg/kg/day of acrylamide caused impaired motor activity in mice. Based on cDNA microarray analysis of mouse brain, myelin basic protein gene, kinesin family member 5B gene, and fibroblast growth factor (FGF) 1 and its receptor genes were down-regulated by acrylamide. The genes are known to be essential for neurofilament synthesis, axonal transport, and neuro-protection, respectively. Interestingly, both FGF 1 and its receptor genes were down-regulated. Genes involved in nucleic acid binding such as AU RNA binding protein/enoyl-coA hydratase, translation initiation factor (TIF) 2 alpha kinase 4, activating transcription factor 2, and U2AF 1 related sequence 1 genes were down-regulated. More interesting finding was that genes of both catalytic and regulatory subunit of protein phosphatases which are important for signal transduction pathways were down-regulated. Here, we propose that acrylamide induces neurotoxicity by regulation of genes associated with neurofilament synthesis, axonal transport, neuro-protection, and signal transduction pathways.
Acrylamide ; Activating Transcription Factor 2 ; Animals ; Axonal Transport ; Brain ; Fibroblast Growth Factors ; Gene Expression ; Humans ; Kinesin ; Mice ; Motor Activity ; Myelin Basic Protein ; Oligonucleotide Array Sequence Analysis ; Peptide Initiation Factors ; Phosphoprotein Phosphatases ; Phosphotransferases ; RNA ; Signal Transduction

PURPOSE: To investigate the anti-apoptotic mechanism of leptin in non-small cell lung cancer. MATERIALS AND METHODS: The influences of leptin on apoptosis were investigated, analyzing the mechanism that triggers growth of A549 cells. The effects of leptin on cell proliferation were examined by XTT analysis. Leptin, C/EBP homologous protein (CHOP), phosphorylated-PKR-like ER kinase (p-Perk), inositol requiring proteins-1, spliced X-box transcription factor-1 (XBP1), cleaved activating transcription factor-6 (ATF6), eukaryotic translation initiation factor-2alpha, caspase-12 and CHOP protein were detected in four groups by western blot, and endoplasmic reticulum (ER) stress related mRNA were detected by reverse transcription PCR. RESULTS: The expression of leptin in A549 and leptin transfected cells inhibited cisplatin activated ER stress-associated mRNA transcription and protein activation. Two ER stress unfolded protein response pathways, PERK and ATF6, were involved, and XBP1 and tumor necrosis factor receptor-associated factor 2 (TRAF2) were increased significantly when treated with cisplatin in A549-siRNA against leptin cells. Furthermore, CHOP expression was inhibited upon leptin expression in A549, LPT-PeP and LPT-EX cells. CONCLUSION: Leptin serves as an important factor that promotes the growth of A549 cells through blocking ER stress-mediated pathways. This blocking is triggered by p-Perk and ATF6 via inhibition of CHOP expression.
Activating Transcription Factor 6/genetics/*metabolism ; Apoptosis/*drug effects/genetics ; Blotting, Western ; Cell Line, Tumor ; Cell Proliferation/*drug effects ; Humans ; Leptin/*pharmacology ; Reverse Transcriptase Polymerase Chain Reaction ; eIF-2 Kinase/*metabolism

PURPOSE: Lithium-pilocarpine induced status epilepticus (LPSE) causes selective and age-dependent neuronal death, although the mechanism of maturation-related injury has not yet been clarified. The activating transcription factor-2 (ATF-2) protein is essential for the normal development of mammalian brain and is activated by c-Jun N-terminal kinase (JNK). It induces the expression of the c-jun gene and modulates the function of the c-Jun protein, a mediator of neuronal death and survival. Therefore, we investigated the expression of c-Jun and ATF-2 protein in the immature and adult rat hippocampus to understand their roles in LPSE-induced neuronal death. MATERIALS AND METHODS: Lithium chloride was administrated to P10 and adult rats followed by pilocarpine. Neuronal injury was assessed by silver and cresyl violet staining, performed 72 hours after status epilepticus. For evaluation of the expression of ATF-2 and c-Jun by immunohistochemical method and Western blot, animals were sacrificed at 0, 4, 24, and 72 hours after the initiation of seizure. RESULTS: Neuronal injury and expression of c-Jun were maturation-dependently increased by LPSE, whereas ATF-2 immunoreactivity decreased in the mature brain. Since both c-Jun and ATF-2 are activated by JNK, and targets and competitors in the same signal transduction cascade, we could speculate that ATF-2 may compete with c-Jun for JNK phosphorylation. CONCLUSION: The results suggested a neuroprotective role of ATF-2 in this maturation-related evolution of neuronal cell death from status epilepticus.
Activating Transcription Factor 2/*metabolism ; Animals ; Antimanic Agents/pharmacology ; Blotting, Western ; Hippocampus/drug effects/*metabolism ; Immunohistochemistry ; Lithium/pharmacology ; Miotics/pharmacology ; Pilocarpine/pharmacology ; Proto-Oncogene Proteins c-jun/*metabolism ; Rats ; Status Epilepticus/*chemically induced

OBJECTIVE: Autophagy is a highly conserved intracellular degradation pathway. Many picornaviruses induce autophagy to benefit viral replication, but an understanding of how autophagy occurs remains incomplete. In this study, we explored whether coxsackievirus B3 (CVB3) infection induced autophagy through endoplasmic reticulum (ER) stress. METHODS: In CVB3-infected HeLa cells, the specific molecules of ER stress and autophagy were detected using Western blotting, reverse transcription polymerase chain reaction (RT-PCR), and confocal microscopy. Then PKR-like ER protein kinase (PERK) inhibitor, inositol-requiring protein-1 (IRE1) inhibitor, or activating transcription factor-6 (ATF6) inhibitor worked on CVB3-infected cells, their effect on autophagy was assessed by Western blotting for detecting microtubule-associated protein light chain 3 (LC3). RESULTS: CVB3 infection induced ER stress, and ER stress sensors PERK/eIF2α, IRE1/XBP1, and ATF6 were activated. CVB3 infection increased the accumulation of green fluorescent protein (GFP)-LC3 punctuation and induced the conversion from LC3-I to phosphatidylethanolamine-conjugated LC3-1 (LC3-II). CVB3 infection still decreased the expression of mammalian target of rapamycin (mTOR) and p-mTOR. Inhibition of PERK, IRE1, or ATF6 significantly decreased the ratio of LC3-II to LC3-I in CVB3-infected HeLa cells. CONCLUSION CVB3 infection induced autophagy through ER stress in HeLa cells, and PERK, IRE1, and ATF6a pathways participated in the regulation of autophagy. Our data suggested that ER stress may inhibit mTOR signaling pathway to induce autophagy during CVB3 infection.
Activating Transcription Factor 6 ; metabolism ; Autophagy ; Coxsackievirus Infections ; metabolism ; Endoplasmic Reticulum Stress ; Endoribonucleases ; metabolism ; Enterovirus B, Human ; HeLa Cells ; Humans ; Protein-Serine-Threonine Kinases ; metabolism ; Signal Transduction ; eIF-2 Kinase ; metabolism

Expression of protein kinase C-delta (PKC delta) is up-regulated by apoptosis-inducing stimuli. However, very little is known about the signaling pathways that control PKC delta gene transcription. In the present study, we demonstrate that JNK stimulates PKC delta gene expression via c-Jun and ATF2 in response to the anticancer agent doxorubicin (DXR) in mouse lymphocytic leukemia L1210 cells. Luciferase reporter assays showed that DXR-induced activation of the PKC delta promoter was enhanced by ectopic expression of JNK1, c-Jun, or ATF2, whereas it was strongly reduced by expression of dominant negative JNK1 or by treatment with the JNK inhibitor SP600125. Furthermore, point mutations in the core sequence of the c-Jun/ATF2 binding site suppressed DXR-induced activation of the PKC delta promoter. Our results suggest an additional role for a JNK signaling cascade in DXR-induced PKC delta gene expression.
Activating Transcription Factor 2/*physiology ; Animals ; Anthracenes/pharmacology ; Antibiotics, Antineoplastic/*pharmacology ; Apoptosis ; Cell Line, Tumor ; Doxorubicin/*pharmacology ; Mice ; Mitogen-Activated Protein Kinase 8/*physiology ; Mutation ; Promoter Regions, Genetic ; Protein Kinase C-delta/genetics/*metabolism ; Proto-Oncogene Proteins c-jun/antagonists & inhibitors/*physiology ; Signal Transduction/physiology ; Transcription, Genetic

BACKGROUND: Selenoprotein P (SEPP1) and fetuin-A, both circulating liver-derived glycoproteins, are novel biomarkers for insulin resistance and nonalcoholic fatty liver disease. However, the effect of exendin-4 (Ex-4), a glucagon-like peptide-1 receptor agonist, on the expression of hepatokines, SEPP1, and fetuin-A, is unknown. METHODS: The human hepatoma cell line HepG2 was treated with palmitic acid (PA; 0.4 mM) and tunicamycin (tuni; 2ug/ml) with or without exendin-4 (100 nM) for 24 hours. The change in expression of PA-induced SEPP1, fetuin-A, and endoplasmic reticulum (ER) stress markers by exendin-4 treatment were evaluated using quantitative real-time reverse transcription polymerase chain reaction and Western blotting. Transfection of cells with AMP-activated protein kinase (AMPK) small interfering RNA (siRNA) was performed to establish the effect of exendin-4-mediated AMPK in the regulation of SEPP1 and fetuin-A expression. RESULTS: Exendin-4 reduced the expression of SEPP1, fetuin-A, and ER stress markers including PKR-like ER kinase, inositol-requiring kinase 1alpha, activating transcription factor 6, and C/EBP homologous protein in HepG2 cells. Exendin-4 also reduced the expression of SEPP1 and fetuin-A in cells treated with tunicamycin, an ER stress inducer. In cells treated with the AMPK activator 5-aminoidazole-4-carboxamide ribonucleotide (AICAR), the expression of hepatic SEPP1 and fetuin-A were negatively related by AMPK, which is the target of exendin-4. In addition, exendin-4 treatment did not decrease SEPP1 and fetuin-A expression in cells transfected with AMPK siRNA. CONCLUSION: These data suggest that exendin-4 can attenuate the expression of hepatic SEPP1 and fetuin-A via improvement of PA-induced ER stress by AMPK.
Activating Transcription Factor 6 ; alpha-2-HS-Glycoprotein* ; AMP-Activated Protein Kinases* ; Blotting, Western ; Carcinoma, Hepatocellular ; Cell Line ; Endoplasmic Reticulum ; Endoplasmic Reticulum Stress* ; Fatty Liver ; Glucagon-Like Peptide 1 ; Glycoproteins ; Hep G2 Cells ; Humans ; Insulin Resistance ; Palmitic Acid ; Phosphotransferases ; Polymerase Chain Reaction ; Reverse Transcription ; RNA, Small Interfering ; Selenoprotein P ; Transfection ; Tunicamycin ; Biomarkers ; Glucagon-Like Peptide-1 Receptor

OBJECTIVETo study the effect of Jianpi Jiedu Recipe (JJR) on the expression of cyclooxygenase (COX-2) in Helicobacter pylori (Hp) infected gastric cancer cell line MKN 45, and its regulatory mechanism of p38MAPK signal transduction.

METHODSThe expressions of COX-2 mRNA and protein in human gastric cancer cell line MKN 45 infected by Hp type strain NCTC 11637 and the regulatory effect of JJR containing serum were detected using Real-time fluorescent quantitative polymerase chain reaction (RFQ-PCR) and Western blot. The effects of Hp on COX-2 mRNA and protein expressions in human gastric cancer cell line MKN 45 were observed using blocking p38MAPK signal transduction pathway by p38MAPK specific inhibitor SB203580. The effects of JJR on Hp-infection activated p38MAPK signal transduction pathway and its downstream activating transcription factor 2 (ATF-2) were observed.

RESULTSCOX-2 mRNA and protein expressions were obviously higher after human gastric cancer cell line MKN 45 were infected by Hp (P<0.01). After blocking p38MAPK signal transduction pathway, COX-2 mRNA and protein expressions in Hp-induced MKN 45 cell line were obviously down-regulated (P<0.01). JJR containing serum down-regulated Hp-induced COX-2 mRNA and protein expressions in MKN 45 cell line in a dose dependent manner. Besides, it could inhibit the activation of Hp-induced p38MAPK signal pathway. It also showed obvious inhibition on the activity of its downstream transcription factor ATF-2.

CONCLUSIONSHp infection induced COX-2 expressions of gastric cancer cells via p38MAPK signal transduction pathway. JJR inhibited Hp-induced the expression of COX-2 through regulating p38MAPK/ATF-2 signal transduction pathway, which may be one of its mechanisms in prevention and treatment of Hp-induced gastric cancer.

Activating Transcription Factor 2 ; metabolism ; Animals ; Cell Line, Tumor ; Cyclooxygenase 2 ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Gene Expression Regulation, Neoplastic ; Helicobacter pylori ; drug effects ; Humans ; Imidazoles ; pharmacology ; MAP Kinase Signaling System ; drug effects ; Pyridines ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Serum ; Signal Transduction ; drug effects ; Stomach Neoplasms ; metabolism ; microbiology ; p38 Mitogen-Activated Protein Kinases ; antagonists & inhibitors

Formation of the periodontium begins following onset of tooth-root formation in a coordinated manner after birth. Dental follicle progenitor cells are thought to form the cementum, alveolar bone and Sharpey's fibers of the periodontal ligament (PDL). However, little is known about the regulatory morphogens that control differentiation and function of these progenitor cells, as well as the progenitor cells involved in crown and root formation. We investigated the role of bone morphogenetic protein-2 (Bmp2) in these processes by the conditional removal of the Bmp2 gene using the Sp7-Cre-EGFP mouse model. Sp7-Cre-EGFP first becomes active at E18 in the first molar, with robust Cre activity at postnatal day 0 (P0), followed by Cre activity in the second molar, which occurs after P0. There is robust Cre activity in the periodontium and third molars by 2 weeks of age. When the Bmp2 gene is removed from Sp7(+) (Osterix(+)) cells, major defects are noted in root, cellular cementum and periodontium formation. First, there are major cell autonomous defects in root-odontoblast terminal differentiation. Second, there are major alterations in formation of the PDLs and cellular cementum, correlated with decreased nuclear factor IC (Nfic), periostin and α-SMA(+) cells. Third, there is a failure to produce vascular endothelial growth factor A (VEGF-A) in the periodontium and the pulp leading to decreased formation of the microvascular and associated candidate stem cells in the Bmp2-cKO(Sp7-Cre-EGFP). Fourth, ameloblast function and enamel formation are indirectly altered in the Bmp2-cKO(Sp7-Cre-EGFP). These data demonstrate that the Bmp2 gene has complex roles in postnatal tooth development and periodontium formation.
Actins ; analysis ; Activating Transcription Factor 2 ; genetics ; Age Factors ; Ameloblasts ; pathology ; Amelogenesis ; genetics ; Animals ; Bone Morphogenetic Protein 2 ; genetics ; Cell Adhesion Molecules ; analysis ; Cell Differentiation ; genetics ; Cementogenesis ; genetics ; Dental Cementum ; pathology ; Dental Pulp ; blood supply ; Fluorescent Dyes ; Green Fluorescent Proteins ; Male ; Mice ; Mice, Knockout ; Microvessels ; pathology ; Molar ; growth & development ; Molar, Third ; growth & development ; NFI Transcription Factors ; analysis ; Odontoblasts ; pathology ; Odontogenesis ; genetics ; Periodontal Ligament ; growth & development ; Sp7 Transcription Factor ; Stem Cells ; physiology ; Tooth Root ; growth & development ; Transcription Factors ; genetics ; Vascular Endothelial Growth Factor A ; analysis ; Zinc Fingers ; genetics