Levels Of Activating Transcription Factor Research Articles

ATF4 inhibits tumor development and mediates p-GCN2/ASNS upregulation in colon cancer

Colon cancer (CC) is a highly malignant tumor with a high incidence and poor prognosis. This study aimed to explore the function and molecular mechanisms of activating transcription factor 4 (ATF4) in CC. The expression levels of ATF4, GCN2, and ASNS in CC tissues were measured using immunohistochemistry (IHC) and reverse transcription quantitative PCR (RT-qPCR). Cell counting kit-8 (CCK-8), clone formation, transwell, and flow cytometry assays were conducted to assess cell viability, clonogenicity, migration, invasion, cell cycle, and apoptosis, respectively, in the ATF4 knockdown and overexpression SW480 cell lines. The effect of ATF4 on the expression of GCN2 and ASNS was detected using RT-qPCR, Chip-qPCR, and western blotting. ATF4, GCN2, and ASNS were expressed at low levels in CC tissues, and all had a significant negative correlation with tumor diameter. ATF4 knockdown promoted cell proliferation, invasion, and S-phase cell cycle and inhibited apoptosis in SW480 cells. In contrast, ATF4 overexpression had the opposite effect. Furthermore, ATF4 overexpression enhanced ATF4 binding to the ASNS promoter region. ATF4 knockdown significantly inhibited the expression of p-GCN2 and ASNS, whereas ATF4 overexpression significantly upregulated their expression. ATF4 inhibited CC cell viability, clone formation ability, migration, and invasion and promoted apoptosis, possibly by regulating the expression of p-GCN2 and ASNS. Our study provides a novel potential therapeutic target for the treatment of CC.

The E3 ubiquitin-protein ligase synoviolin (Syvn1/Hrd1) promotes adaptive decreases in cardiac myocyte protein synthesis via eIF2α/ATF4 pathway activation

As the heart undergoes pathologic hypertrophy in response to many disease conditions, an imbalance between protein synthesis and degradation in cardiac myocytes, due to the increases in protein synthesis relative to protein degradation, happens, which challenges protein folding and endoplasmic reticulum (ER) proteostasis. This leads to ER stress and the activation of the ER stress response. Synoviolin (Syvn1), also known as HMG-CoA reductase degradation protein 1 (Hrd1), is an ER-localized protein that is upregulated in cardiac myocytes during ER stress. We previously found that Syvn1 mediates the ubiquitylation and consequent degradation of toxic misfolded proteins and preserved cardiac function in a model of pressure overload-induced cardiac pathology. The objective of this study was to examine whether Syvn1 is involved in regulating protein synthesis, and if so, what its function is during hypertrophic growth of cardiac myocytes. Given that cardiac hypertrophy is caused by increases in protein synthesis, we hypothesized that Syvn1 provides an adaptive response in part through regulation of protein synthesis and consequent decrease in cardiac hypertrophic response. We examined the effects of Syvn1 on ventricular myocyte growth in vitro and found that overexpression of Syvn1 decreased cardiac myocyte surface area as well as protein synthesis when treated with phenylephrine (PE), a treatment which normally leads to increased protein synthesis and increased cardiac myocyte size, mimicking pathological cardiac hypertrophy. We next focused on identifying the mechanism of Syvn1-mediated growth inhibition and found that increases in Syvn1 lead to activation of the PERK (protein kinase RNA (PKR)-like ER kinase) branch of the ER stress response, which leads to increased phosphorylation of the α-subunit of translation initiation factor 2 (eIF2α) at Ser51, thereby inhibiting global translational initiation. PERK activation also results in selective translation of a subset of mRNAs, including activating transcription factor 4 (ATF4), which activates the transcription of a wide range of genes involved in adaptation to stress conditions. We found increased ATF4 levels and increased ATF4 target gene expression with Syvn1 overexpression during PE treatment, compared to control PE treatment. Inhibition of the PERK pathway blocked Syvn1-mediated decrease in cardiac myocyte size and protein synthesis as well as PERK activation and ATF4 target gene induction. Additionally, we found that an adeno-associated virus encoding Syvn1, administered in a therapeutically-relevant manner after the onset of pathological cardiac hypertrophy induced by transverse-aortic constriction in mice, blunted pathological remodeling and preserved cardiac function. We posit that the Syvn1/eIF2α/ATF4 pathway constitutes a newly-discovered mechanism for balancing the proteome in cardiac myocytes. Here we found that Syvn1 upregulation blunts maladaptive increase in cardiac myocyte size during pathologic hypertrophy. In conclusion, these findings suggest that Syvn1 is a critical regulator of cardiac myocyte protein synthesis and subsequent growth and that increases in Syvn1 lead to the activation of the PERK branch of the ER stress response, which results in inhibition of global protein synthesis, and activation of ATF4. This work was supported by the National Institutes of Health (R01HL157027) and the University of Arizona Health Sciences Career Development Award. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

O-GlcNAc regulates the mitochondrial integrated stress response by regulating ATF4.

Accumulation of mitochondrial dysfunctional is a hallmark of age-related neurodegeneration including Alzheimer's disease (AD). Impairment of mitochondrial quality control mechanisms leading to the accumulation of damaged mitochondria and increasing neuronal stress. Therefore, investigating the basic mechanisms of how mitochondrial homeostasis is regulated is essential. Herein, we investigate the role of O-GlcNAcylation, a single sugar post-translational modification, in controlling mitochondrial stress-induced transcription factor Activating Transcription Factor 4 (ATF4). Mitochondrial dysfunction triggers the integrated stress response (ISRmt), in which the phosphorylation of eukaryotic translation initiation factor 2α results in the translation of ATF4. We used patient-derived induced pluripotent stem cells, a transgenic mouse model of AD, SH-SY5Y neuroblastoma and HeLa cell-lines to examine the effect of sustained O-GlcNAcase inhibition by Thiamet-G (TMG) on ISRmt using biochemical analyses. We show that TMG elevates ATF4 protein levels upon mitochondrial stress in SH-SY5Y neuroblastoma and HeLa cell-lines. An indirect downstream target of ATF4 mitochondrial chaperone glucose-regulated protein 75 (GRP75) is significantly elevated. Interestingly, knock-down of O-GlcNAc transferase (OGT), the enzyme that adds O-GlcNAc, in SH-SY5Y increases ATF4 protein and mRNA expression. Additionally, ATF4 target gene Activating Transcription Factor 5 (ATF5) is significantly elevated at both the protein and mRNA level. Brains isolated from TMG treated mice show elevated levels of ATF4 and GRP75. Importantly, ATF4 occupancy increases at the ATF5 promoter site in brains isolated from TMG treated mice suggesting that O-GlcNAc is regulating ATF4 targeted gene expression. Interestingly, ATF4 and GRP75 are not induced in TMG treated familial Alzheimer's Disease mice model. The same results are seen in a human in vitro model of AD. Together, these results indicate that in healthy conditions, O-GlcNAc regulates the ISRmt through regulating ATF4, while manipulating O-GlcNAc in AD has no effect on ISRmt.

Sivelestat sodium alleviated lipopolysaccharide-induced acute lung injury by improving endoplasmic reticulum stress

Acute lung injury (ALI) is a common inflammatory respiratory disorder characterized by a high incidence and mortality rate. This study aimed to investigate the potential therapeutic effects of the neutrophil elastase inhibitor Sivelestat sodium (SIV) in improving endoplasmic reticulum stress (ERS) while treating lipopolysaccharide (LPS)-induced ALI. An ALI model was established using LPS induction. The effects of SIV on ALI were observed both in vivo and in vitro, along with its impact on ERS. Lung tissue damage was assessed using Hematoxylin-eosin (H&E) staining. Lung edema was measured by the lung wet/dry weight ratio. The expression levels of protein kinase R-like ER kinase (PERK), Phospho-protein kinase R-like ER kinase (p-PERK), activating transcription factor 4 (ATF4), eukaryotic translation initiation factor 2α (EIF2a), phosphorylated α subunit of eukaryotic initiation factor 2α (P-EIF2a), and C/EBP homologous protein (CHOP) were analyzed by Western blotting in vivo and in vitro. The levels of tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) in Lung tissue samples supernatants were measured by ELISA. Oxidative stress markers were measured by ELISA. Apoptosis was measured using the TUNEL assay. Apoptosis-associated proteins B-cell lymphoma-2 (Bcl-2)、Bcl2-associated × (Bax)、caspase-3 were evaluated through Western blotting in vivo and in vitro. The expression levels of ERS-related proteins, including p-PERK, ATF4, P-EIF2a, and CHOP, were significantly increased in the LPS-induced ALI model. However, SIV markedly reduced the expression levels of these proteins, suppressing the LPS-induced ERS response. Further investigations revealed that SIV exerted a protective effect on ALI by alleviating lung tissue damage and apoptosis, improving lung function, and reducing inflammation and oxidative stress levels. However, when SIV was co-administered with Tunicamycin (TUN), TUN blocked the beneficial effects of SIV on ERS and reversed the protective effects of SIV on ALI. In conclusion, SIV alleviated lung tissue damage and apoptosis, improving lung function, and reducing inflammation and oxidative stress in LPS-induced ALI by improving ERS.

Endoplasmic Reticulum Stress in the Pathogenesis of Hyperglycemia Induced by Thiamine-Responsive Megaloblastic Anemia.

Thiamine responsive megaloblastic anemia (TRMA) is a genetic disease caused by SLC19A2 gene mutation. This study aimed to preliminarily explore the relationship between endoplasmic reticulum stress (ERS)-PERK signaling pathway and the pathogenesis of hyperglycemia induced by TRMA. Islet β (INS.1 and β-TC-6) and HEK293T cell line models with stable overexpression of SLC19A2 and SLC19A2 (c.1409insT) were established. The cells were divided into empty virus group (control), wild-type group (overexpressed SLC19A2), and mutation group (overexpressed SLC19A2 (c.1409insT)). Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and western blotting were used to detect the expression levels of ERS-PERK signaling pathway-related proteins, including glucose-regulated protein 78 (GRP78), protein kinase R-like ER kinase (PERK), and eukaryotic initiation factor 2 (eIF2α), activating transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP) in islet β cells. Protein localization was assessed by immunofluorescence staining. Compared with the control group, the mRNA expression levels of SLC19A2 in wild-type and mutant islet β cells (INS.1 and β-TC-6) and HEK293T cells were significantly upregulated (all p < 0.05). Compared with the control group and the wild-type group, the mRNA expression levels of GRP78, PERK, eIF2α, ATF4, and CHOP were increased (all p < 0.05) in the mutant islet β cells; the protein expression levels of PERK, GRP78, and eIF2α were elevated (all p < 0.05). In addition, the results of immunofluorescence staining showed that SLC19A2 (c.1409insT) mutation changed the localization of the proteins in the cells. Thus, they were not located on the cell surface, but in the cytoplasm and nuclei, and protein aggregation occurred in the cytoplasm. 1. Islet β and HEK293Tcell lines, stably overexpressing SLC19A2 and SLC19A2 (c.1409insT) mutations, were successfully constructed. 2. SLC19A2 (c.1409insT) mutation could raise the expression levels of ERS-PERK signaling pathway-related proteins (GRP78, PERK, eIF2α, ATF4, and CHOP), and activate apoptosis pathway. 3. SLC19A2 (c.1409insT) mutation could change the localization of proteins and produce protein aggregation in cells. It could lead to protein misfolding and ERS, which would participate in the pathological mechanism of hyperglycemia induced by TRMA.

ATF4-dependent fructolysis fuels growth of glioblastoma multiforme

Excessive consumption of fructose in the Western diet contributes to cancer development. However, it is still unclear how cancer cells coordinate glucose and fructose metabolism during tumor malignant progression. We demonstrate here that glioblastoma multiforme (GBM) cells switch their energy supply from glycolysis to fructolysis in response to glucose deprivation. Mechanistically, glucose deprivation induces expression of two essential fructolytic proteins GLUT5 and ALDOB through selectively activating translation of activating transcription factor 4 (ATF4). Functionally, genetic or pharmacological disruption of ATF4-dependent fructolysis significantly inhibits growth and colony formation of GBM cells in vitro and GBM growth in vivo. In addition, ATF4, GLUT5, and ALDOB levels positively correlate with each other in GBM specimens and are poor prognostic indicators in GBM patients. This work highlights ATF4-dependent fructolysis as a metabolic feature and a potential therapeutic target for GBM.

Activating transcription factor 4 aggravates angiotensin II-induced cell dysfunction in human vascular aortic smooth muscle cells via transcriptionally activating fibroblast growth factor 21.

Abdominal aortic aneurysm (AAA) is a life-threatening disorder worldwide. Fibroblast growth factor 21 (FGF21) was shown to display a high level in the plasma of patients with AAA; however, its detailed functions underlying AAA pathogenesis are unclear. An in vitro AAA model was established in human aortic vascular smooth muscle cells (HASMCs) by angiotensin II (Ang-II) stimulation. Cell counting kit-8, wound healing, and Transwell assays were utilized for measuring cell proliferation and migration. RT-qPCR was used for detecting mRNA expression of FGF21 and activating transcription factor 4 (ATF4). Western blotting was utilized for assessing protein levels of FGF21, ATF4, and markers for the contractile phenotype of HASMCs. ChIP and luciferase reporter assays were implemented for identifying the binding relation between AFT4 and FGF21 promoters. FGF21 and ATF4 were both upregulated in Ang-II-treated HASMCs. Knocking down FGF21 attenuated Ang-II-induced proliferation, migration, and phenotype switch of HASMCs. ATF4 activated FGF21 transcription by binding to its promoter. FGF21 overexpression reversed AFT4 silencing-mediated inhibition of cell proliferation, migration, and phenotype switch. ATF4 transcriptionally upregulates FGF21 to promote the proliferation, migration, and phenotype switch of Ang-II-treated HASMCs.

Abstract 3178: A stromal integrated stress response activates perivascular cancer-associated fibroblasts to drive angiogenesis and tumor progression

Abstract Despite recent advances in prevention and treatment, including immune checkpoint inhibitors, malignant melanoma remains a particularly aggressive and deadly malignancy, which is partly attributed to its highly heterogeneous TME. However, malignant cells exhibit altered signaling pathways, which enables them to adapt to both cell-intrinsic and extrinsic stressors within TME. The activating transcription factor 4 (ATF4) is a master transcriptional effector of the Integrated Stress Response (ISR), a homeostatic mechanism coupling cell growth and survival to bioenergetic demands. We and others have established a critical tumor cell-intrinsic role of ATF4 which culminates in the promotion of primary tumor growth and in the establishment of micro- and macro-metastases in xenograft, allograft and transgenic models. However, the potential roles of the ISR and particularly of ATF4-mediated responses in host-dependent, tumor-related processes, have not been yet extensively investigated. Using novel conditional knockout ATF4 mouse models, we show that global loss of host ATF4 results in deficient tumor vascularization and a pronounced tumor growth delay in syngeneic melanoma and pancreatic tumor models. Immunofluorescence analysis revealed a severely impaired angiogenic phenotype in tumors grown in ATF4 KO mice which was accompanied by deficiencies in markers of CAF activation. Single-cell transcriptomic analysis of B16F10 melanoma tumors further localized this defect to a distinct CAF population, previously identified as vascular CAFs (vCAFs), and revealed a significant reduction in the expression of extracellular matrix components, primarily type I collagen, in tumors grown in ATF4 KO mice. Intriguingly, we identified a multifaceted impairment of the collagen biosynthetic pathway with the ATF4 to directly regulate the expression of the Col1a1 gene as well as the intracellular levels of glycine and proline, the major amino acids comprising collagen fibers. Moreover, we showed that the ATF4-deficient vCAFs secrete significantly lower levels of angiogenic factors (i.e., VEGF, SDF-1 etc.) in the perivascular area leading to an abnormal angiogenesis and significant attenuation of tumor growth. Specific deletion of ATF4 in the fibroblast compartment (Col1a1 promoter) produced a similar tumor growth delay as in the global ATF4 KO mice, and notably, co-injection of fibroblasts from ATF4-proficient mice led to significant recovery of tumor growth rates in ATF4-deficient mice. Finally, analysis of human melanoma and pancreatic tumor samples revealed a strong correlation between ATF4 and collagen levels and between an ISR gene signature and expression of collagen and CAF activation genes. Our findings uncover a novel role of stromal ATF4 in shaping CAF functionality, a key driver of disease progression, metastasis, and therapy resistance. Citation Format: Ioannis I. Verginadis, Harris Avgousti, Kyle Kim, Giorgos Skoufos, Frank Chinga, Nektaria Maria Leli, Ilias V. Karagounis, Brett I. Bell, Anastasia Velalopoulou, Victoria S. Wu, Yang Li, Jiangbin Ye, David A. Scott, Andrei L. Osterman, Arjun Sengupta, Aalim Weljie, Artemis G. Hatzigeorgiou, Sandra Ryeom, Alan J. Diehl, Serge Y. Fuchs, Ellen Puré, Constantinos Koumenis. A stromal integrated stress response activates perivascular cancer-associated fibroblasts to drive angiogenesis and tumor progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3178.

PRMT5 regulates ATF4 transcript splicing and oxidative stress response

Protein methyltransferase 5 (PRMT5) symmetrically dimethylates arginine residues leading to regulation of transcription and splicing programs. Although PRMT5 has emerged as an attractive oncology target, the molecular determinants of PRMT5 dependency in cancer remain incompletely understood. Our transcriptomic analysis identified PRMT5 regulation of the activating transcription factor 4 (ATF4) pathway in acute myelogenous leukemia (AML). PRMT5 inhibition resulted in the expression of unstable, intron-retaining ATF4 mRNA that is detained in the nucleus. Concurrently, the decrease in the spliced cytoplasmic transcript of ATF4 led to lower levels of ATF4 protein and downregulation of ATF4 target genes. Upon loss of functional PRMT5, cells with low ATF4 displayed increased oxidative stress, growth arrest, and cellular senescence. Interestingly, leukemia cells with EVI1 oncogene overexpression demonstrated dependence on PRMT5 function. EVI1 and ATF4 regulated gene signatures were inversely correlated. We show that EVI1-high AML cells have reduced ATF4 levels, elevated baseline reactive oxygen species and increased sensitivity to PRMT5 inhibition. Thus, EVI1-high cells demonstrate dependence on PRMT5 function and regulation of oxidative stress response. Overall, our findings identify the PRMT5-ATF4 axis to be safeguarding the cellular redox balance that is especially important in high oxidative stress states, such as those that occur with EVI1 overexpression.

Mechanism of Endoplasmic Reticulum Stress Pathway in the Osteogenic Phenotypic Transformation of Aortic Valve Interstitial Cells

Background and PurposeCalcific Aortic Valve Disease (CAVD) is a crucial component of degenerative valvular disease in old age and with the increasing prevalence of the aging population. we hope that by modeling valvular osteogenesis and intervening with endoplasmic reticulum stress inhibitor TUDCA to observe the effect of endoplasmic reticulum stress on valve osteogenesisMethodsIn this study, rabbit heart valvular interstitial cells (VICs) were isolated and cultured. They treated with ox-LDL (Oxidized Low Density Lipoprotein) stimulation to establish a model of valvular osteogenic transformation. BMP2 (Bone Morphogenetic Protein 2), PERK (Protein kinase R-like endoplasmic reticulum kinase), CHOP (CCAAT/enhancer-binding protein homologous protein) and transcriptional regulatory factor ATF4 (Activating Transcription Factor 4 )were recorded after intervention with ER stress inhibitor TUDCA. The effects of er stress on valvular osteogenic transformation were analyzed.ResultAfter stimulation of VICs with ox-LDL, the expression levels of BMP2, PERK, CHOP, and ATF4 increased. However, TUDCA treatment can alleviate the increased expression levels of BMP2, PERK ATF4, and CHOP under ox-LDL stimulation to a certain extent.ConclusionThe endoplasmic reticulum stress signaling pathway is involved in ox-LDL-induced calcification of rabbit valve interstitial cells. Inhibition of endoplasmic reticulum stress using TUDCA can improve the progression of rabbit aortic valve calcification.

Folate ameliorates homocysteine-induced osteoblast dysfunction by reducing endoplasmic reticulum stress-activated PERK/ATF-4/CHOP pathway in MC3T3-E1 cells.

Homocysteine (Hcy) is considered a newly identified risk factor for osteoporosis. Nevertheless, the underlying mechanism of folate (FA), a key factor in the metabolism of Hcy, in protection against osteoblast dysfunction remains unclear. The purpose of this study was to investigate the mechanism by which FA attenuates Hcy-induced osteoblast damage. The Hcy-induced MC3T3-E1 cells were treated with different concentrations of FA. Cell morphology, cell density, cell proliferation ability, alkaline phosphatase (ALP) activity and mineralization capacity were observed and determined; the gene expression of B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (BAX) and ERS-associated factors, including glucose-regulated protein 78 (GRP-78), activating transcription factor 4 (ATF-4) and growth arrest and DNA damage inducible gene 153 (CHOP/GADD153), were assessed by RT-PCR; and protein levels of GRP-78 and ATF-4 were analyzed by western blotting. Hcy suppressed the proliferation, differentiation and mineralization ability of MC3T3-E1 cells in a concentration-dependent manner and activated the ERS signaling pathway. After intervention with different concentrations of FA, the cell viability and density, ALP activity, number of mineralized nodules, calcium content and Bcl-2 gene expression were all significantly increased, whereas the gene expression of GRP-78, CHOP/GADD153, ATF-4 and Bax was markedly downregulated, and protein levels of GRP-78 and ATF-4 were also markedly decreased. The adverse effects of Hcy on osteoblast differentiation are dose dependent. FA not only protects against osteoblasts apoptosis but also has a direct osteogenic effect on Hcy-induced osteoblasts, which could be partially mediated by inhibition of the PERK-activated ERS pathway.

Artificial neuronal network analysis in investigating the relationship between oxidative stress and endoplasmic reticulum stress to address blocked vessels in cardiovascular disease.

Cardiovascular disease is the leading cause of death in the world and is associated with significant morbidity. Atherosclerosis is the main cause of cardiovascular disease (CVD), including myocardial infarction (MI), heart failure, and stroke. The mechanism of atherosclerosis has not been well investigated in different aspects, such as the relationship between oxidative stress and endothelial function. This project aims to investigate whether an oxidative enzyme vascular peroxidase 1 (VPO1) and activating transcription factor 4 (ATF4) can be used as biomarkers in highlighting the pathogenesis of the disease and in evaluating the prognosis of the relationship with endoplasmic reticulum and oxidative stress. This paper used artificial neural network analysis to predict cardiovascular disease risk based on new generation biochemical markers that combine vascular inflammation, oxidative and endoplasmic reticulum stress. For this purpose, 80 patients were evaluated according to the coronary angiography results. hs-CRP, lipid parameters and demographic characteristics, VPO1, ATF4 and Glutathione peroxidase 1(GPx1) levels were measured. We found an increase in VPO1 and hs-CRP levels in single-vessel disease as compared to controls. On the contrary, ATF4 and GPx1 levels were decreased in the same group, which was not significant. Our results showed a significant positive correlation between ATF4 and lipid parameters. A statistically significant positive correlation was also observed for VPO1 and ATF4 (r=0.367, P<0.05), and a negative correlation was found for ATF4 and GPx1 (r=-0.467, P<0.01). A significant negative relationship was noted for GPx1 and hs-CRP in two/three-vessel disease (r=-0.366, P<0.05). Artificial neural network analysis stated that body mass index (BMI) and smoking history information give us an important clue as compared to age, gender and alcohol consumption parameters when predicting the number of blocked vessels. VPO1 and ATF4 might be potential biomarkers associated with coronary artery disease, especially in the follow-up and monitoring of treatment protocols, in addition to traditional risk factors.

Protective effect of ethyl acetate extract from Bidens bipinnata on hepatocyte damage induced by endoplasmic reticulum stress

To explore the protective effect and mechanism of ethyl acetate extract from Bidens bipinnata on hepatocyte damage induced by endoplasmic reticulum stress. Tunicamycin was used to establish the damage model in L02 cells. Methyl thiazolyl tetrazolium(MTT) colorimetric assay was used to investigate the survival rate of ethyl acetate extract from B. bipinnata in L02 cells injury induced by endoplasmic reticulum stress; the protein expressions of endoplasmic reticulum stress-related molecule glucose regulated protein 78(GRP78), PKR-like ER kinase(PERK), eukaryotic initiation factor-2(eIF2α), activating transcription factor 4(ATF4), C/EBP homologous protein(CHOP), B-cell CLL/lymphoma 2(Bcl-2), Bal-2 associated X apoptosis regulator(Bax) were examined by Wes-tern blot. The expressions of the above proteins were also detected after endoplasmic reticulum stress inhibitor(4-phenyl butyric acid) and CHOP shRNA-mediated knockdowns were added. The expressions of GRP78, PERK, CHOP in L02 cells were observed by immunofluorescence method. The results showed that ethyl acetate extract from B. bipinnata could significantly increase the survival rate of L02 cell injury caused by endoplasmic reticulum stress in a dose and time-dependent manner(P&lt;0.05 or P&lt;0.01). The expression levels of GRP78, PERK, eIF2α, ATF4, CHOP and Bax in the drug treatment groups were significantly down-regulated(P&lt;0.05 or P&lt;0.01), while Bcl-2 was significantly up-regulated(P&lt;0.01). After endoplasmic reticulum stress inhibitor and CHOP shRNA-mediated knockdowns were added, the expression levels of GRP78, PERK, eIF2α, ATF4, CHOP, Bax in the drug treatment groups were significantly down-regulated(P&lt;0.01), whereas Bcl-2 was significantly up-regulated(P&lt;0.01). Immunofluorescence results showed that the expressions of GRP78, PERK, CHOP were consistent with the Western blot method. In conclusion, ethyl acetate extract from B. bipinnata has a significant protective effect on the damage of L02 cells caused by endoplasmic reticulum stress. The mechanism may be related to the inhibition of endoplasmic reticulum stress and the down-regulation of apoptosis in cells through the PERK/eIF2α/ATF4/CHOP signaling pathway.

Identification of amino acid response element of SLC38A9 as an ATF4-binding site in porcine skeletal muscle cells

Amino acids can affect protein synthesis by activating mammalian target of rapamycin complex 1 (mTORC1) signaling pathway. Amino acid transporters SLC38A9 on the lysosomal membrane not only transport amino acids, but also can sense amino acids and activate mTORC1 signaling pathway. Activating transcription factor 4 (ATF4) can promote the expression of amino acid transporters by binding with amino acid response element (AARE). In this study, two AAREs were found in the SLC38A9 promoter region of pig, and both of them bound to ATF4. The AARE in the first intron was located in the core promoter region of SLC38A9. ATF4 regulated mRNA expression level of SLC38A9 in porcine skeletal muscle cells. In the absence of amino acids, the expression of ATF4 decreased and the expression of SLC38A9 increased. After leucine addition, the expression levels of ATF4 and SLC38A9 increased. It suggested that in the absence of amino acids, the expression of SLC38A9 was increased via binding of ATF4 to AARE binding factors in SLC38A9 promoter fragment; after the addition of leucine, ATF4 was activated, resulting in the increase of SLC38A9 expression.

Puerarin Inhibits the PERK-eIF2[Formula: see text]-ATF4-CHOP Pathway through Inactivating JAK2/STAT3 Signal in Pancreatic beta-Cells.

Type 1 diabetes (T1D) is an autoimmune and inflammatory disease with excessive loss of pancreatic islet [Formula: see text]-cells. Accumulating evidence indicated that endoplasmic reticulum (ER) stress played a critical role in [Formula: see text]-cells loss, leading to T1D. Therefore, promoting the survival of pancreatic [Formula: see text]cells would be beneficial for patients with T1D. Puerarin is a natural isoflavone that has been demonstrated to be able to decrease blood glucose in patients with T1D. However, it remains unknown whether puerarin improves ER stress to prevent [Formula: see text]-cells from apoptosis. Here, we sought to investigate the role of puerarin in ER stress-associated apoptosis and explore its underlying mechanism in the mouse insulinoma cell line (MIN6). Flow cytometry and cell counting kit-8 (CCK8) experiments showed that puerarin caused a significant increase in the viability of MIN6 cells injured by H2O2. Furthermore, the protein kinase R-like ER kinase (PERK) signal pathway, a critical branch of ER stress response, was found to be involved in this process. Puerarin inhibited the phosphorylation of PERK, subsequently suppressed the phosphorylation of eukaryotic initiation factor 2[Formula: see text] (eIF2[Formula: see text], then decreased the activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP) expression, ultimately attenuating ER stress to prevent MIN6 cells from apoptosis. In addition, puerarin inhibited the activation of Janus kinase 2 (JAK2)/signal transducer and activators of transcription 3 (STAT3), which suppressed the PERK signal cascade with decreased ATF4 and CHOP levels. Taken together, our results firstly demonstrated that puerarin could prevent MIN6 cells from apoptosis at least in part by inhibiting the PERK-eIF2[Formula: see text]-ATF4-CHOP axis under ER stress conditions, which might be mediated by inactivation of the JAK2/STAT3 signal pathway. Therefore, investigating the mechanism underlying the effects of puerarin might highlight the potential roles of puerarin developing into an antidiabetic drug.

Histone deacetylase inhibitor valproic acid attenuates high glucose‑induced endoplasmic reticulum stress and apoptosis in NRK‑52E cells.

Previous studies have demonstrated that valproic acid (VPA), a histone deacetylase inhibitor, alleviates diabetic nephropathy (DN). However, the biological mechanisms underlying this protective effect remains unclear. This study aimed to investigate the effects of histone deacetylase inhibitor VPA on hyperglycemic induction of NRK‑52E cell ERS and apoptosis. Endoplasmic reticulum stress (ERS)‑related apoptosis is involved in DN, and improving ERS may delay the symptoms of DN. Histone deacetylase regulates gene transcription or expression of ERS‑related proteins. The present study established an ERS model by treating the rat renal tubular epithelial cells NRK‑52E with high glucose(HG) and investigated the effects of VPA on the apoptosis of the NRK‑52E cells. HG stimulation significantly increased the protein levels of the ERS‑related proteins including glucose regulated protein78 (GRP78), activating transcription factor 4 (ATF4), C/EBP homologous protein (CHOP), caspase‑12 and phosphorylated (p)‑JNK. VPA treatment further upregulated GRP78 expression and attenuated the levels of ATF4, CHOP, caspase‑12 and p‑JNK. Notably, HG markedly promoted apoptosis of NRK‑52E cells by regulating the protein levels of Bax, cleaved caspase‑3 and Bcl‑2, which was attenuated by simultaneous VPA treatment. Mechanistically, VPA increased the total acetylation levels of histoneH4 in NRK‑52E cells and increased the histoneH4 acetylation of the GRP78 promoter region. In conclusion, VPA attenuated HG‑induced ERS and apoptosis in NRK‑52E cells, which may be due to the regulation of acetylation levels of ERS‑related proteins. In addition, the present study suggested that HDACIs are promising drugs for treating patients with DN.

KLF6 Induces Apoptosis in Human Lens Epithelial Cells Through the ATF4-ATF3-CHOP Axis.

BackgroundMany studies have confirmed that high myopia is related to the high prevalence of cataracts, which results from apoptosis of lens epithelial cells (LECs) due to endoplasmic reticulum stress. Krüppel-like factor 6 (KLF6) is a tumor suppressor that is involved in the regulation of cell proliferation and apoptosis.PurposeIn this study, our purpose was to find the relationship between KLF6-induced apoptosis in LECs and ATF4 (activating transcription factor 4)-ATF3 (activating transcription factor 3)-CHOP (C/EBP homologous protein) signaling pathway.MethodsKLF6, ATF4, ATF3, and CHOP were ectopically expressed using cDNAs subcloned into the pCDNA3.1+ vector. ATF4, ATF3, and CHOP knockdown were performed by small interfering RNA (siRNA). Expression of relative gene was tested using QT-PCR and western-blot. Then, accompanied by UVB stimulation, cell viability was measured by CCK-8 assay; The cell damage was examined by live & dead staining; The apoptotic markers Bax and Bcl-2 were detected by immunoblotting; Quantitative apoptotic levels were measured with the Apoptosis Detection Kit; The expression level of reactive oxygen-free radical (ROS) was analyzed by DCFH-DA` probe.ResultsEctopically expressed ATF4, ATF3, and CHOP-induced apoptosis in cells, whereas ATF4, ATF3, and CHOP knockdown by small interfering RNA (siRNA) blocked KLF6-induced apoptosis. In addition, we determined that ATF4 regulates ATF3 and CHOP expression and that ATF3 silencing reduces CHOP upregulation without changing ATF4 levels; however, ATF4 and ATF3 expression was unaffected by blockade of CHOP, suggesting that KLF6 triggers endoplasmic reticulum stress in LECs by mediating the ATF4-ATF3/CHOP axis. Besides, KLF6 overexpression significantly induced LEC apoptosis under UV radiation, as demonstrated by the elevated Bax/Bcl-2 ratio.ConclusionThe ATF4-ATF3-CHOP pathway plays an important role in KLF6-induced apoptosis in HLECs. Our results increase our understanding of the mechanisms that regulate LEC apoptosis and contribute to the development of a new preventative strategy for cataract.

Erythropoietin attenuates vascular calcification by inhibiting endoplasmic reticulum stress in rats with chronic kidney disease

Previous studies suggested that endoplasmic reticulum (ER) stress induced-apoptosis promoted vascular calcification (VC). Interestingly, erythropoietin (EPO), an endogenous glycoprotein, exerts multiple tissue protective effects by inhibiting ER stress and apoptosis. We investigated the role and potential mechanism of EPO on VC in chronic kidney disease (CKD) rats and cultured vascular smooth muscle cells (VSMCs). The calcification model was established by subtotal nephrectomy in vivo or phosphate overload in vitro. The protein level of EPO receptor (EPOR) was increased in the calcified aortas of CKD rats. EPO prevented the reduction of VSMC phenotypic markers, and reversed the increased calcium content and calcium salt deposition in the aortas of CKD rats and cultured calcified VSMCs. The protein levels of activating transcription factor 4 (ATF4) and glucose-regulated protein 94 (GRP94) were upregulated in aortas and VSMCs under calcifying conditions, indicating ER stress activation. EPO treatment of CKD rats or calcified VSMCs downregulated the protein levels of ATF4 and GRP94. Furthermore, ER stress-mediated apoptosis, determined by the protein levels of CCAAT⁄enhancer-binding protein-homologous protein and cleaved caspase 12, was increased in tunicamycin or calcification media-treated VSMCs, but the increased effect was reversed in EPO-treated groups. The increased apoptotic cells in calcified VSMCs, as indicated by Hoechst staining and flow cytometry, were downregulated by the co-administration of EPO or 4-phenyl butyric acid. In conclusion, EPO might attenuate VC by inhibiting ER stress mediated apoptosis through EPOR signaling.

Activating transcription factor 4 is required for high glucose inhibits proliferation and differentiation of MC3T3-E1 cells

Activating transcription factor 4 (ATF4) promotes bone formation in human bone marrow mesenchymal stem cells. However, the underlying mechanisms of ATF4 in high glucose-induced injury of osteoblast still remain unclear. Small interfering RNA and plasmid targeting ATF4 were used to transfect MC3T3-E1 cells to knock down and overexpress ATF4 using Lipofectamin 3000. Cell viability, alkaline phosphatase (ALP) activity and levels were determined by MTT, ALP kit assay, quantitative real-time (qRT)-PCR and Western blot. Osteocalcin (OCN) expression was determined by ELISA, PCR and Western blot. The mRNA and protein levels of ATF4, glucose regulated protein 78 kDa (GRP78) and C/EBP homologous protein (CHOP) were detected by PCR and Western blot. In the current study, viabilities of MC3T3-E1 cells were inhibited by high glucose. Meanwhile, the mRNA and protein levels of ATF4 were effectively up-regulated in high glucose-incubated MC3T3-E1 cells. By conducting functional experiments, silencing ATF4 induced by small interfering RNA partially reversed the inhibitory effects of high glucose on viabilities of MC3T3-E1 cells. We also found that the expressions of ER stress-related proteins (ATF4, GRP78 and CHOP) were higher in high glucose-treated MC3T3-E1 cells but were inhibited by siATF4. However, overexpression of AFT4 had opposite results, and high glucose attenuated the protein levels of osteogenic marker genes ALP and OCN, which were further inhibited by ATF4 knockout gene. Thus, ATF4 was a necessary gene for high glucose to inhibit the proliferation and differentiation of MC3T3-E1 cells.

The PERK Pathway Plays a Neuroprotective Role During the Early Phase of Secondary Brain Injury Induced by Experimental Intracerebral Hemorrhage.

The protein kinase RNA-like endoplasmic reticulum kinase (PERK) pathway, which is a branch of the unfolded protein response, participates in a range of pathophysiological processes of neurological diseases. However, few studies have investigated the role of the PERK in intracerebral hemorrhage (ICH). The present study evaluated the role of the PERK pathway during the early phase of ICH-induced secondary brain injury (SBI) and its potential mechanisms. An autologous whole blood ICH model was established in rats, and cultured primary cortical neurons were treated with oxyhemoglobin to mimic ICH in vitro. We found that levels of phosphorylated alpha subunit of eukaryotic translation initiation factor 2 (p-eIF2α) and activating transcription factor 4 (ATF4) increased significantly and peaked at 12h during the early phase of the ICH. To further elucidate the role of the PERK pathway, we assessed the effects of the PERK inhibitor, GSK2606414, and the eIF2α dephosphorylation antagonist, salubrinal, at 12h after ICH both in vivo and in vitro. Inhibition of PERK with GSK2606414 suppressed the protein levels of p-eIF2α and ATF4, resulting in increase of transcriptional activator CCAAT/enhancer-binding protein homologous protein (CHOP) and caspase-12, which promoted apoptosis and reduced neuronal survival. Treatment with salubrinal yielded opposite results, which suggested that activation of the PERK pathway could promote neuronal survival and reduce apoptosis. In conclusion, the present study has demonstrated the neuroprotective effects of the PERK pathway during the early phase of ICH-induced SBI. These findings highlight the potential value of PERK pathway as a therapeutic target for ICH.