Activating Transcription Factor 4 Activation Research Articles

Ku Regulates Signaling to DNA Damage Response Pathways through the Ku70 von Willebrand A Domain

The Ku heterodimer (Ku70/Ku80) is a main component of the nonhomologous end-joining (NHEJ) pathway that repairs DNA double-strand breaks (DSBs). Ku binds the broken DNA end and recruits other proteins to facilitate the processing and ligation of the broken end. While Ku interacts with many proteins involved in DNA damage/repair-related functions, few interactions have been mapped to the N-terminal von Willebrand A (vWA) domain, a predicted protein interaction domain. The mutagenesis of Ku70 vWA domain S155/D156 unexpectedly increased cell survival following ionizing radiation (IR) treatment. DNA repair appeared unaffected, but defects in the activation of apoptosis and alterations in the DNA damage signaling response were identified. In particular, Ku70 S155A/D156A affected the IR-induced transcriptional response of several activating transcription factor 2 (ATF2)-regulated genes involved in apoptosis regulation. ATF2 phosphorylation and recruitment to DNA damage-induced foci was increased in Ku70-deficient cells, suggesting that Ku represses ATF2 activation. Ku70 S155A/D156A substitutions further enhanced this repression. S155A substitution alone was sufficient to confer enhanced survival, whereas alteration to a phosphomimetic residue (S155D) reversed this effect, suggesting that S155 is a phosphorylation site. Thus, these findings infer that Ku links signals from the DNA repair machinery to DNA damage signaling regulators that control apoptotic pathways.

Unfolded Protein Response Signaling and MAP Kinase Pathways Underlie Pathogenesis of Arsenic-Induced Cutaneous Inflammation

Arsenic exposure through drinking water is a major global public health problem and is associated with an enhanced risk of various cancers including skin cancer. In human skin, arsenic induces precancerous melanosis and keratosis, which may progress to basal cell and squamous cell carcinoma. However, the mechanism by which these pathophysiologic alterations occur remains elusive. In this study, we showed that subchronic arsenic exposure to SKH-1 mice induced unfolded protein response (UPR) signaling regulated by proteins, inositol-requiring enzyme-1 (IRE1), PKR-like endoplasmic reticulum kinase (PERK) and activating transcription factor 6 (ATF6). Arsenic activated all three UPR regulatory proteins in the skin. Arsenic induced IRE1 phosphorylation which resulted in augmented splicing of X-box binding protein 1 (XBP-1) leading to its migration to the nucleus, and also enhanced transcriptional activation of downstream target proteins. Hyperphosphorylation of PERK which induces eukaryotic translation initial factor 2α (eIF2α) in a phosphorylation-dependent manner enhanced translation of ATF4, in addition to augmenting proteolytic activation of ATF6 in arsenic-treated skin. A similar increase in the expression of CHOP was observed. Enhanced XBP-1s, ATF4, and ATF6 regulated downstream chaperones GRP94 and GRP78. In addition, arsenic induced inflammation-related p38/MAPKAPK-2 MAPK signaling and alterations in Th-1/Th-2/Th-17 cytokines/chemokines and their receptors. Antioxidant N-acetyl cysteine blocked arsenic-induced reactive oxygen species, with a concomitant attenuation of UPR and mitogen-activated protein kinase (MAPK) signaling and proinflammatory cytokine/chemokine signatures. Our results identify novel pathways involved in the pathogenesis of arsenic-mediated cutaneous inflammation which may also be related to enhanced cancer risk in arsenic exposed cohorts.

Abstract 9139: Mitochondrial Aldehyde Dehydrogenase 2*2 Allele Carriers Possess Better Cardioprotection Results After Cardioplegic Arrest During Open-Heart Surgery

Introduction: Previous rodent studies indicated aldehyde dehydrogenase-2 (ALDH2) activation conferred cardioprotection. About 40% East Asians carry an ALDH2*2 allele to result in a reduced ALDH2 activity. The influence of ALDH2*2 on human cardioprotection after cardioplegia has not been studied, and we hypothesized that ALDH2*2 allele could have an effect on it. Methods: A prospective cohort of East Asians with a diagnosis of Tetralogy of Fallot (n=80) undergoing elective surgical repair were genotyped. All subjects followed the same operation and ICU protocol. Myocardium samples were dissected after cardioplegic arrest and detailed clinical information were collected. Myocardium ALDH2 activity, 4-hydroxy-2-nonenal (4-HNE) adducts and glutathione (GSH) levels, and expressions of ALDH2 and activating transcription factor-4 (ATF4), a key transcriptional regulator for GSH formation, were determined by spectrophotography, ELISA, and western blotting respectively. All the experimental and clinical results were compared between ALDH2*2 carriers and non-carriers. Results: ALDH2*2 allele presented in 38.75% of the patients. Myocardium ALDH2 enzyme activity difference among ALDH2*2 carriers and non-carriers disappeared during cardioplegia. ALDH2*2 carriers expressed less ALDH2 and more ATF4, and they got lower 4-HNE adducts levels and higher GSH levels. A lower postoperative inotropic score and troponin I level, and shorter length of hospital stay were observed in ALDH2*2 carriers (Table). Conclusions: In order to antagonize oxidative stress, ALDH2*2 carriers developed a compensatory larger myocardium GSH pool through ATF4 activation. When ALDH2 activity was impaired during cardioplegia, this larger GSH pool leaded to unexpectedly better cardioprotection results on ALDH2*2 carriers. ALDH2 genotype might be served as a genetic predictor for cardioprotection, and new cardioprotective strategies for ALDH2*2 non- carriers should be developed.

Carbon monoxide, a reaction product of heme oxygenase-1, suppresses the expression of C-reactive protein by endoplasmic reticulum stress through modulation of the unfolded protein response

The expression of C-reactive protein (CRP) rises rapidly in response to inflammation. The endoplasmic reticulum (ER) stress has been reported to cause CRP expression. Carbon monoxide (CO), a reaction product of heme oxygenase, exerts anti-inflammatory effects. In this study, we aimed to examine the role of CO in modulating ER stress-induced CRP expression. In HepG2 cells, ER stress triggered by tunicamycin, thapsigargin and homocysteine markedly induced CRP expression and the activation of protein kinase R-like endoplasmic reticulum kinase (PERK), inositol-requiring transmembrane kinase/endonuclease 1α (IRE1α), activating transcription factor 6 (ATF6), and hepatocyte-specific cyclic AMP response element binding protein H (CREBH). A CO-releasing molecule (CORM) inhibited ER stress-induced CRP expression. While CORM attenuated ER stress-induced activation of IRE1α, ATF6 and CREBH, it augmented PERK activation, which was associated with its inhibition of CRP expression. CORM also inhibited CRP expression in response to the pro-inflammatory cytokine IL-6 that was found to induce ER stress response in HepG2 cells. Moreover, in mice treated with the ER stress inducer tunicamycin, CORM administration reduced serum levels of CRP and the expression of CRP mRNA in the liver. Collectively, our findings suggest that CO may attenuate ER stress-induced CRP expression through modulation of the unfolded protein response.

Selective activation of the transcription factor ATF6 mediates endoplasmic reticulum proliferation triggered by a membrane protein

It is well known that the endoplasmic reticulum (ER) is capable of expanding its surface area in response both to cargo load and to increased expression of resident membrane proteins. Although the response to increased cargo load, known as the unfolded protein response (UPR), is well characterized, the mechanism of the response to membrane protein load has been unclear. As a model system to investigate this phenomenon, we have used a HeLa-TetOff cell line inducibly expressing a tail-anchored construct consisting of an N-terminal cytosolic GFP moiety anchored to the ER membrane by the tail of cytochrome b5 [GFP-b(5)tail]. After removal of doxycycline, GFP-b(5)tail is expressed at moderate levels (1-2% of total ER protein) that, nevertheless, induce ER proliferation, as assessed both by EM and by a three- to fourfold increase in phosphatidylcholine synthesis. We investigated possible participation of each of the three arms of the UPR and found that only the activating transcription factor 6 (ATF6) arm was selectively activated after induction of GFP-b(5)tail expression; peak ATF6α activation preceded the increase in phosphatidylcholine synthesis. Surprisingly, up-regulation of known ATF6 target genes was not observed under these conditions. Silencing of ATF6α abolished the ER proliferation response, whereas knockdown of Ire1 was without effect. Because GFP-b(5)tail lacks a luminal domain, the response we observe is unlikely to originate from the ER lumen. Instead, we propose that a sensing mechanism operates within the lipid bilayer to trigger the selective activation of ATF6.

Abstract LB-429: Arsenic induces unfolded protein response signaling, inflammation and tumorigenesis in murine skin

Abstract Arsenic (As) exposure through drinking water is a major public health problem. 100 million humans globally are exposed to As contaminated water. As exposure is associated with an enhanced risk for the development of various cancers including skin cancers. In human skin, As induces precancerous lesions, melenosis and keratosis some of which progress to basal cell (BCC) and squamous cell carcinoma. However, the mechanism by which these pathophysiological alterations occur remains elusive. In this study, we show that chronic As (sodium arsenite) exposure to SKH-1 mice leads to induction of unfolded protein response (UPR) signaling. UPR signaling is controlled by multiple pathways regulated by the proteins, inositol-requiring enzyme-1 (IRE-1), PKR-like endoplasmic reticulum kinase (PERK) and activating transcription factor 6 (ATF6). As activated all three UPR regulatory proteins in the skin. It induced phosphorylation of cutaneous IRE-1, resulting in an augmented splicing of X-box binding protein-1 (XBP-1), which then migrated to the nucleus and activated transcription of its target proteins. Similarly, it induced hyperphosphorylation of PERK which is required to induce eukaryotic translation initial factor 2 α (eIF2α) in a phosphorylation-dependent manner. Hyperphosphorylated eIF2α enhanced translation of ATF4. In addition, As augmented proteolytic activation of ATF6. XBP1, ATF4 and ATF6 regulate overlapping downstream target proteins including GRP94 and GRP78 chaperones. As also mediated the production of Th-1/Th-2 types of inflammatory cytokines, chemokines and receptors in skin. Consistent with these results, As treatment was associated with the enhanced expression of inflammation-associated epidermal MAP kinase signaling. Enhanced levels of p-ERK, p-p38 and p-MAPKAPK2 were observed in As-treated groups as compared to age-matched controls. In addition, we observed a disruption in iron metabolism characterized by the enhancement in hepcidin and ferroportin. This also leads to enhanced production of reactive oxygen species in the skin. Chronic As exposure to Ptch+/−/SKH-1 mice enhanced the growth of BCCs by enhancing sonic hedgehog signaling. An enhanced number of both visible and microscopic BCCs was noted in these animals. We also observed that p53 status determines the tumor-growth-related effects of As in these mice. The tumor growth enhancing effects of As were not observed in Ptch+/−/p53+/−/SKH-1. These animals showed that diminution in p53 gene dose level inhibited tumorigenesis. Our data suggest that As-mediated enhancement in UPR signaling pathways in murine skin underlie the pathogenesis of inflammation and tumor development. However, these effects are dependent on p53 mutational status. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-429. doi:10.1158/1538-7445.AM2011-LB-429

Tumor hypoxia and genetic alterations in sporadic cancers

The cancer genome contains many gene alterations. How cancer cells acquire these alterations is a matter for discussion. One hypothesis is that cancer cells obtain mutations in genome stability genes at an early stage of tumor development, which results in genetic instability and generates a gene pool that enhances cellular proliferation and survival. Another hypothesis puts its emphasis on the natural selection of gene mutations for fitness. Recent data for systematic cancer genome sequencing shows that mutations in stability genes are rare in human sporadic cancers. Instead, many “passenger” mutations that do not drive the carcinogenesis process have been found in the cancer genome. Both the hypotheses mentioned above fall short in explaining recent data. Recently, many studies demonstrate the role of the tumor microenvironment, especially hypoxia and reoxygenation, in genetic instability. In this review, literature will be presented which supports a third hypothesis, i.e. that hypoxia/re-oxygenation induces genetic instability.

Does IGF-1 administration after a mild traumatic brain injury in mice activate the adaptive arm of ER stress?

Mild traumatic brain injury (mTBI) induces along with cognitive impairments, both cell death and survival pathways. Previously, IGF-1 (Insulin like growth factor 1) administration prevented TBI-induced damage. This study was aimed at testing the effect of mTBI on ER (endoplasmic reticulum) stress activation and looking for a possible interaction between IGF-1 and ER stress pathways. Mice were subjected to a weight drop closed head injury. Western blot analysis revealed that mTBI induced activation of ATF6 (activating transcription factor 6), but not of CHOP or grp78. IGF-1 administration following mTBI did not change ATF6 or grp78 levels, but significantly elevated CHOP. These results suggest that IGF-1 may exert its neuroprotection via PERK/CHOP, the adaptive arm of the unfolded protein response.

Role of the Host Cell's Unfolded Protein Response in Arenavirus Infection

Arenaviruses are enveloped RNA viruses with a nonlytic life cycle that cause acute and persistent infections. Here, we investigated the role of the host cell's unfolded protein response (UPR) in infection of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV). In mammalian cells, the endoplasmic reticulum (ER) chaperone protein GRP78/BiP functions as the principal sensor for the induction of the UPR and interacts with three mediators: kinase/endonuclease inositol-requiring protein 1 (IRE1), PKR-like ER kinase (PERK), and activating transcription factor 6 (ATF6). Acute infection with LCMV resulted in a selective induction of the ATF6-regulated branch of the UPR, whereas pathways controlled by PERK and IRE1 were neither activated nor blocked. Expression of individual LCMV proteins revealed that the viral glycoprotein precursor (GPC), but not that of other viral proteins, was responsible for the induction of ATF6. Rapid downregulation of the viral GPC during transition from acute to persistent LCMV infection restored basal levels of UPR signaling. To address a possible role of ATF6 signaling in LCMV infection, we used cells deficient in site 2 protease (S2P), a metalloprotease required for the activation of ATF6. Cells deficient in S2P showed significantly lower levels of production of infectious virus during acute but not persistent infection, indicating a requirement for ATF6-mediated signaling for optimal virus multiplication. In summary, acute LCMV infection seems to selectively induce the ATF6-regulated branch of the UPR that is likely beneficial for virus replication and cell viability, but it avoids induction of PERK and IRE1, whose activation may be detrimental for virus and the host cell.

Evaluation of the endoplasmic reticulum-stress response in eIF2B-mutated lymphocytes and lymphoblasts from CACH/VWM patients

BackgroundEukaryotic translation initiation factor 2B (eIF2B), a guanine nucleotide exchange factor (GEF) and a key regulator of translation initiation under normal and stress conditions, causes an autosomal recessive leukodystrophy of a wide clinical spectrum. EBV-immortalised lymphocytes (EIL) from eIF2B-mutated patients exhibit a decrease in eIF2B GEF activity. eIF2B-mutated primary fibroblasts have a hyper-induction of activating transcription factor 4 (ATF4) which is involved in the protective unfolded protein response (UPR), also known as the ER-stress response. We tested the hypothesis that EIL from eIF2B-mutated patients also exhibit a heightened ER-stress response.MethodsWe used thapsigargin as an ER-stress agent and looked at polysomal profiles, rate of protein synthesis, translational activation of ATF4, and transcriptional induction of stress-specific mRNAs (ATF4, CHOP, ASNS, GRP78) in normal and eIF2B-mutated EIL. We also compared the level of stress-specific mRNAs between EIL and primary lymphocytes (PL).ResultsDespite the low eIF2B GEF activity in the 12 eIF2B-mutated EIL cell lines tested (range 40-70% of normal), these cell lines did not differ from normal EIL in their ATF4-mediated ER-stress response. The absence of hyper-induction of ATF4-mediated ER-stress response in eIF2B-mutated EIL in contrast to primary fibroblasts is not related to their transformation by EBV. Indeed, PL exhibited a higher induction of the stress-specific mRNAs in comparison to EIL, but no hyper-induction of the UPR was noticed in the eIF2B-mutated cell lines in comparison to controls.ConclusionsTaken together with work of others, our results demonstrate the absence of a major difference in ER-stress response between controls and eIF2B-mutated cells. Therefore, components of the ER-stress response cannot be used as discriminantory markers in eIF2B-related disorders.

Regulation of Mouse Brain-Selective Sulfotransferase Sult4a1 by cAMP Response Element-Binding Protein and Activating Transcription Factor-2

Sulfotransferase 4A1 (SULT4A1) is a novel cytosolic sulfotransferase that is primarily expressed in the brain. To date, no significant enzyme activity or biological function for the protein has been identified, although it is highly conserved between species. Mutations in the SULT4A1 gene have been linked to schizophrenia susceptibility, and recently, its stability was shown to be regulated by Pin1, a peptidyl-prolyl cis-trans isomerase implicated in several neurodegenerative diseases. In this study, we investigated the transcriptional regulation of mouse Sult4a1. Using a series of promoter deletion constructs, we identified three cAMP-responsive elements (CREs) that were required for maximal promoter activity. The CREs are located within 100 base pairs of the major transcription start site and are also present in the same region of the human SULT4A1 promoter. Electrophoretic mobility shift assays (EMSAs) identified two specific complexes that formed on each of the CREs. One complex contained cAMP response element-binding protein (CREB), and the other contained activating transcription factor-2 (ATF-2) and c-Jun. Overexpression of CREB or ATF-2 increased not only reporter promoter activity but also endogenous Sult4a1 mRNA levels in Neuro2a cells. Moreover, [d-Ala(2),N-MePhe(4),Gly-ol(5)]enkephalin (DAMGO) treatment increased both reporter promoter activity and Sult4a1 levels in mu-opioid receptor expressing Neuro2a/mu-opioid receptor cells, and EMSAs showed this to be due to increased binding of CREB and ATF-2 to the Sult4a1 promoter. We also show that DAMGO treatment increases Sult4a1 mRNA and protein levels in primary mouse neurons. These results suggest that Sult4a1 is a target gene for the mu-opioid receptor signaling pathway and other pathways involving activation of CREB and ATF-2.

Activation transcription factor-3 activation and the development of spinal cord degeneration in a rat model of amyotrophic lateral sclerosis

It has been reported that an early activation of glial fibrillary acid protein (GFAP) in astroglial cells occurs simultaneously in peripheral nerves and spinal cord from the G93A SOD1 mouse model of amyotrophic lateral sclerosis (ALS), an invariably fatal neurodegenerative disorder. In ALS, the contribute to the pathological process of different cell types varies according to the disease stage, with a florid immune response in spinal cord at end stage disease. In this study, we have mapped in different anatomical sites the process of disease-induced functional perturbation from a pre-symptomatic stage using a marker of cellular distress expressed in neurons and glial cells, the activating transcription factor 3 (ATF-3), and applied large-scale gene expression analysis to define the pattern or transcriptional changes occurring in spinal cord from the G93A SOD1 rat model of ALS in parallel with ATF-3 neuronal activation. From the disease onset onward, transgenic lumbar spinal cord displayed ATF-3 transcriptional regulation and motor cells immunostaining in association with the over-expression of genes promoting cell growth, the functional integrity of cell organelles and involved in the modulation of immune responses. While spinal cord from the pre-symptomatic rat showed no detectable ATF-3 transcriptional regulation, ATF-3 activation was appreciated in large size neurofilament-rich, small size non-peptidergic and parvalbumin-positive neurons within the dorsal root ganglia (DRG), and in ventral roots Schwann cells alongside macrophages infiltration. This pattern of peripheral ATF-3 activation remained detectable throughout the disease process. In the G93A SOD1 rat model of ALS, signs of roots and nerves subtle distress preceded overt clinical-pathological changes, involving both glial cells and neurons that function as receptors of peripheral sensory stimuli from the muscle. In addition, factors previously described to be linked to ATF-3 activation under various experimental conditions of stress, become switched on in spinal cord from the end-stage transgenic rat model of ALS.

EIF2α and caspase-12 activation are involved in oxygen–glucose–serum deprivation/restoration-induced apoptosis of spinal cord astrocytes

Astrocytes play an important role in protecting neurons during ischemia and reperfusion in the central nervous system. Although many studies have shown that oxygen–glucose deprivation (OGD) can induce astrocyte apoptosis, the role of PERK/eIF2α/ATF4 integrated stress response (ISR) in astrocyte apoptosis mediated by oxygen–glucose–serum deprivation (OGSD)/restoration remains uncertain. Astrocytes were subjected to a combination of oxygen, glucose, and serum deprivation for 8 h followed by restoration. Hoechst 33342 staining was performed to quantify apoptotic astrocytes and cell viability was assessed with Cell Counting Kit-8 (CCK8). Immunocytochemical analysis and Western blotting for some related molecules, including pancreatic ER stress kinase (PERK), p-PERK, eukaryotic initiation factor 2 alpha (eIF2α), p-eIF2α, activating transcription factor 4 (ATF4), caspase-12, were examined. Caspase activation and apoptosis were detected in neonatal rat astrocytes from spinal cord subjected to OGSD/restoration. We also observed an increase in cytoplasmic staining of p-eIF2α in astrocytes (8 h OGSD/15 min restoration) compared with that of non-treated cells. In addition, we found the sequential activation of PERK, eIF2α, and ATF4 during OGSD/restoration by Western blotting. These results indicate that both the PERK/eIF2α/ATF4 ISR and activation of caspase-12 may be involved in apoptosis of spinal cord astrocytes induced by OGSD/restoration.

ATF6 is important under both pathological and physiological states in the heart

Accumulation of unfolded proteins in the endoplasmic reticulum (ER) evokes the ER stress response, including activating transcription factor 6 (ATF6), a key transcriptional activator to maintain cellular homeostasis. The ER stress has recently been reported to cause various diseases, but the role of ATF6 in the heart remains unknown. We clarified the role of ATF6 in the heart. The ATF6 activity was increased in the murine heart after myocardial infarction (MI). Treatment of mice with 4-(2-aminoethyl) benzenesulfonyl fluoride, an inhibitor of ATF6, further reduced cardiac function and increased the mortality rate at 14days after MI. Pharmacological inhibition of ATF6 induced dilatation of left ventricle and depression of cardiac function even in sham-operated murine hearts. The transgenic mice that expressed dominant negative mutant of ATF6 showed larger left ventricular dimension and reduced fractional shortening compared with wild-type littermates, resulting in death of heart failure at ∼8weeks of age. In contrast, cardiac function after MI was better in transgenic mice that expressed constitutively active mutant of ATF6, compared with wild-type littermates. These results suggest that activation of the ER stress response factor ATF6 plays a critical role in not only protecting hearts under the pathological state but also maintaining cardiac function under the physiological state.

Nuclear protein 1 induced by ATF4 in response to various stressors acts as a positive regulator on the transcriptional activation of ATF4

Nuclear protein 1 (NUPR1) was originally identified as p8, a member of the family of HMG-I/Y transcription factors induced in response to various cellular stressors. However, the signaling pathway underlying NUPR1 induction by cellular stresses remains to be established. In this study, we found that the expression of NUPR1 by various stresses induced by activating transcription factor 4 (ATF4). Loss of ATF4 using siRNA significantly diminished NUPR1 expression. Overexpression of ATF4 caused NUPR1 levels to rise. NUPR1 expression was associated with enhanced transcriptional activation of genes of ATF4 downstream, suggesting that the protein promoted the transcription of stress-regulated genes via positive feedback on the ATF4 pathway.

House Dust Mite Allergen Der f 2-induced Phospholipase D1 Activation Is Critical for the Production of Interleukin-13 through Activating Transcription Factor-2 Activation in Human Bronchial Epithelial Cells

The purpose of this study was to identify the role of phospholipase D1 (PLD1) in Der f 2-induced interleukin (IL)-13 production. The major house dust mite allergen, Der f 2, increased PLD activity in human bronchial epithelial cells (BEAS-2B), and dominant negative PLD1 or PLD1 siRNA decreased Der f 2-induced IL-13 expression and production. Treatment of Der f 2 activated the phospholipase Cgamma (PLCgamma)/protein kinase Calpha (PKCalpha)/p38 MAPK pathway. Der f 2-induced PLD activation was attenuated by PLCgamma inhibitors (U73122 and PAO), PKCalpha inhibitors (RO320432 and GO6976), and p38 MAPK inhibitors (SB203580 and SB202190). These results indicate that PLCgamma, PKCalpha, and p38 MAPK act as upstream activators of PLD in Der f 2-treated BEAS-2B cells. Furthermore, expression and production of IL-13 increased by Der f 2 were also blocked by inhibition of PLCgamma, PKCalpha, or p38 MAPK, indicating that IL-13 expression and production are related to a PLCgamma/PKCalpha/p38 MAPK pathway. We found that activating transcription factor-2 (ATF-2) was activated by Der f 2 in BEAS-2B cells and activation of ATF-2 was controlled by PLD1. When ATF-2 activity was blocked with ATF-2 siRNA, Der f 2-induced IL-13 expression and production were decreased. Thus, ATF-2 might be one of the transcriptional factors for the expression of IL-13 in Der f 2-treated BEAS-2B cells. Taken together, PLD1 acts as an important regulator in Der f 2-induced expression and production of IL-13 through activation of ATF-2 in BEAS-2B cells.

APS decreased high glucose induced overexpression of PTP1B through inhibiting the activation of ATF6

Protein tyrosine phosphatase 1B(PTP1B) which negatively regulated the insulin signaling was considered as a potential target to prevent and treat type 2 diabetes(T2DM). Endoplasmic reticulum(ER) stress was a key link between obesity, insulin resistance, and T2DM. Interestingly, PTP1B located on the cytosolic surface of ER and played an essential role in potentiating ER stress, we hypothesized that PTP1B may be a chaperon of ER stress. Activating transcription factor 6(ATF6) promoted the synthesis of chaperones when ER stress happened. We have found that astragalus polysaccharide(APS) could increase insulin sensitivity probably through decreasing the expression of PTP1B. In this study, we intended to investigate whether ATF6 was involved in this effect. Firstly, we used MTT assay to analyze the cytotoxicity of different concentration of APS and found that APS in a wide range had little cytotoxicity, approximately from 0‐1.6mg/ml; secondly, we used 25mmol/l D‐glucose to treat on cells for 24 hours, followed by APS with different concentrations mentioned above. Results showed that APS at 0.1‐0.8mg/ml decreased the expression of p50‐ATF6, APS at 0.2‐0.8mg/ml were more potential than 0.1mg/ml; while APS at 0.2‐0.8mg/ml reduced the expression of PTP1B and APS at 0.4mg/ml were more effective than 0.2 and 0.8mg/ml. So APS at 0.2‐0.8mg/ml may be the effective concentration range that can not only decrease the expression of PTP1B but also reduce glucose induced ER stress. This positive correlation provided significant information that APS decreased the expression of PTP1B was mediated at least partly through inhibiting the activation of ATF6.

Astragalus polysaccharides decreased the expression of PTP1B through relieving ER stress induced activation of ATF6 in a rat model of type 2 diabetes

Protein tyrosine phosphatase 1B (PTP1B) was considered as a potential therapeutic target of type 2 diabetes (T2DM) because of its negative regulation of insulin signaling. It located on the cytosolic surface of endoplasmic reticulum (ER) and played an essential role in the ER stress signaling. Activating transcription factor 6 (ATF6) was an ER stress regulated transmembrane transcription factor that activated the transcription of ER molecular chaperones. We hypothesized that the expression of PTP1B may be regulated by ATF6 when ER stress happened. Our previous studies showed that Astragalus polysaccharide (APS) increased the insulin sensitivity through decreasing the overexpression of PTP1B in T2DM animal models. In this study, we intended to investigate the possible mechanisms involved in this effect. A rat model of T2DM was established using high fat diet associated with intraperitoneal injection of 25mg/kg streptozocin; 25mmol/l d-glucose and 5×10−7mol/l insulin were used as in vitro investigations to mimic T2DM-like environment. 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) and pCI-Flag-ATF6(N)(2-366) plasmid were treated separately on human hepatocyte line HL-7702 to observe the effect of ATF6 on the expression of PTP1B. The results suggested that APS not only restored the glucose homeostasis but also reduced the ER stress in this rat model of T2DM; ATF6 was involved in mediating the expression of PTP1B when ER stress happened; APS decreased the expression of PTP1B at least partly through inhibiting the activation of ATF6.

Activating transcription factor 4 and CCAAT/enhancer-binding protein-β negatively regulate the mammalian target of rapamycin via Redd1 expression in response to oxidative and endoplasmic reticulum stress

Regulation of mRNA translation in mammalian cells involves the coordinated control of mammalian target of rapamycin (mTOR) signaling. At present, limited information is available on the potential relevance of mTOR regulation, although translation inhibition during oxidative and endoplasmic reticulum (ER) stress is clearly important. In this study, we show that activating transcription factor 4 (ATF4) and CCAAT/enhancer-binding protein-β (C/EBP-β) negatively regulate mTOR via Redd1 expression in response to oxidative and ER stress. Oxidative and ER stress conditions induce rapid and significant activation of ATF4 downstream of eIF2α phosphorylation, which is responsible for Redd1 expression. In our experiment, overexpression of ATF4 was associated with reduced mTOR activity via Redd1 expression, whereas suppression of ATF4 levels with small interfering RNA led to the recovery of decreased mTOR activity mediated by downregulation of Redd1 during oxidative and ER stress. We additionally identified Redd1 as a downstream effector of C/EBP-β stimulated by ATF4 activated under the stress conditions examined. RNA interference studies provided further evidence of the requirement of C/EBP-β for Redd1 expression. We conclude that the Redd1 gene is transactivated by the ATF4 and C/EBP family of transcription factors, leading to mTOR inhibition in response to oxidative and ER stress.

Down-regulation of Inhibition of Differentiation-1 via Activation of Activating Transcription Factor 3 and Smad Regulates REIC/Dickkopf-3–Induced Apoptosis

REIC/Dickkopf-3 (Dkk-3), a tumor suppressor gene, has been investigated in gene therapy studies. Our previous study suggested that REIC/Dkk-3-induced apoptosis mainly resulted from phosphorylation of c-Jun-NH(2) kinase (JNK) in prostate cancer cells. However, the precise mechanisms, especially the molecular mechanisms regulating JNK phosphorylation, remain unclear. In this study, we investigated the mechanisms participating in JNK phosphorylation in the context of a refractory cancer disease, malignant mesothelioma (MM). Adenovirus-mediated overexpression of REIC/Dkk-3 induced apoptosis mainly through JNK activation in immortalized MM cells (211H cells). Interestingly, transcriptional down-regulation of inhibition of differentiation-1 (Id-1) was detected in REIC/Dkk-3-overexpressed 211H cells. Moreover, restoration of Id-1 expression antagonized REIC/Dkk-3-induced JNK phosphorylation and apoptosis. Mutagenesis experiments with the 2.1-kb human Id-1 promoter revealed that activating transcription factor 3 (ATF3) and Smad interaction, with their respective binding motifs, was essential for REIC/Dkk-3-mediated suppression of Id-1 promoter activity. ATF3 activation was probably induced by endoplasmic reticulum stress. Finally, we showed strong antitumor effects from REIC/Dkk-3 gene transfer into the pleural cavity in an orthotopic MM mouse model. Relative to control tumor tissue, REIC/Dkk-3-treated tumor tissue showed down-regulated expression of Id-1 mRNA, enhanced expression of phosphorylated JNK, and an increased number of apoptotic cells. In summary, we first showed that both ATF3 and Smad were crucially and synergistically involved in down-regulation of Id-1, which regulated JNK phosphorylation in REIC/Dkk-3-induced apoptosis. Thus, gene therapy with REIC/Dkk-3 may be a promising therapeutic tool for MM.