Egr-1 Levels Research Articles

METTL7B contributes to the malignant progression of glioblastoma by inhibiting EGR1 expression

Glioblastoma (GBM), a predominant central nervous system (CNS) malignancy, is correlated with high mortality and severe morbidity. Mammalian methyltransferase-like 7B (METTL7B) as a methyltransferase has been identified to participate in cancer progression. However, its function in GBM is elusive. Accordingly, we aimed to explore the effect of METTL7B on GBM. The expression of METTL7B and EGR2 in GBM patients and GBM cells were detected by qPCR, western blots and immunohistochemical staining. Cell viability was assessed by CCK-8 assays. Cell proliferation was determined by EdU, colony formation, and tumor sphere formation assays. METTL7B shRNA was injected into the Balb/c nude mice. The size and weight of isolated tumor was measured. And the expression levels of Ki67, METTL7B and EGR1 were examined by immunohistochemical staining. METTL7B was significantly elevated, while EGR1 was downregulated in clinical GBM tissues. METTL7B upregulation was associated with the low overall survival of GBM patients. Moreover, METTL7B depletion remarkably attenuated GBM cell proliferation. Mechanistically, METTL7B overexpression inhibited EGR1 expression in GBM cells. EGR1 knockdown rescued the inhibitory effect of METTL7B depletion on GBM cell proliferation. Meanwhile, METTL7B depletion arrested more GBM cells at the G0/G1, but fewer cells at the S phase, which EGR1 knockdown reversed these effects. Furthermore, tumorigenicity analysis revealed that METTL7B promotes tumor growth of GBM cells in vivo. METTL7B contributes to the malignant progression of GBM by inhibiting EGR1 expression. METTL7B and EGR1 may be utilized as the treatment targets for GBM therapy.

Egr1 confers protection against acetaminophen‑induced hepatotoxicity via transcriptional upregulating of Acaa2.

Background: Acetaminophen (APAP)-induced liver injury (AILI) is a common cause of drug-induced liver injury (DILI). The mechanism underlying protection in AILI or DILI remains to be elucidated, and the role of early growth response 1 (Egr1) in AILI and potential mechanisms remain to be known.Methods: The role of Egr1 was studied both in vivo and in vitro. Liver-specific Egr1-knockout (Egr1LKO) mice and those overexpressing Egr1 via tail vein injection of Egr1-expressing adenovirus (Ad-Egr1) were utilized with AILI. Chromatin immunoprecipitation-sequencing, RNA-sequencing, seahorse XF analysis, and targeted fatty acid analysis were performed. EGR1 levels were also studied in liver tissues and serum samples from AILI/DILI patients.Results: In this study, we have demonstrated that Egr1 was upregulated in AILI models in vivo and in vitro. liver-specific Egr1 knockout aggravated AILI; however, Ad-Egr1 treatment ameliorated this. Mechanistically, Egr1 deficiency inhibited, whereas overexpression promoted, mitochondrial respiratory function and fatty acid β-oxidation (FAO) activity in AILI. Egr1 transcriptionally upregulated FAO-related genes in hepatocytes. Notably, the knockdown of acetyl-coenzyme A acyltransferase 2 (Acaa2), a key gene involved in FAO, diminished this protective effect of Egr1. Clinically, EGR1 was markedly increased in liver tissues from AILI patients. Interestingly, EGR1 levels of liver tissues and serum samples were also obviously higher in idiosyncratic DILI patients.Conclusions: Egr1 confers adaptive protection in AILI, mediated via the transcriptional upregulation of Acaa2, which improves mitochondrial FAO, and might be a potential biomarker and novel therapeutic target for AILI.

Roles of c-Fos, EGR-1, PKA, and PKC in cognitive dysfunction in rats after propofol anesthesia

This study aimed to investigate possible changes in the spatial memory of rats and the expression or activity of EGR-1, c-Fos, PKA, and PKC after propofol anesthesia. Thirty-six Sprague-Dawley rats aged 20 months and 36 Sprague-Dawley rats aged three months were each randomly divided into three groups: the control group, the Morris Water Maze (MWM) group, and the propofol group. In the propofol groups of both young and aged rats, the rats were anesthetized by propofol for two or four hours and then performed the MWM test two days or two weeks after anesthesia to assess cognitive function. EGR-1, c-Fos, PKA, and PKC expressions in the rat hippocampus were determined via immunohistochemistry. For the older rats, the escape latency in the P4h/2d group was significantly prolonged (P < 0.05), and the learning curve was right-shifted in the P4h/2w group (P < 0.05). The expression levels of EGR-1, c-Fos, PKA, and PKC in the MWM groups were significantly higher than those in the control groups (P < 0.05). In the P4h/2d group of aged rats, the expression levels of both PKA and PKC were decreased compared with those of the MWM groups. The decreased expression of both protein kinases may be responsible for the observed impairment after propofol anesthesia.

Analysis of hub genes and molecular mechanisms in non-alcoholic steatohepatitis based on the gene expression omnibus database

Objective: To explore the hub genes and mechanisms in the pathological process of non-alcoholic steatohepatitis (NASH) by bioinformatics methods. Methods: Microarray datasets GSE89632 were downloaded from the Gene Expression Omnibus (GEO) database, which including 20 simple non-alcoholic fatty liver disease patients, 19 NASH patients and 24 healthy control individuals. The differentially expressed genes (DEGs) in patients with simple non-alcoholic fatty liver disease and NASH were compared with healthy control individuals respectively, and the intersection of the two groups of DEGs was taken. GO functional annotation and KEGG pathway enrichment analysis of DEGs were performed with DAVID 6.8 and KOBAS 3.0 separately. Protein-protein interaction network (PPI) was constructed by STRING database, then the mRNA hub genes were selected by Cytoscape software. The Attie Lab Diabetes database was used to verify the relative expression of hub genes mRNA in the liver of 4 groups of C57BL/6 mice (4-week-old normal group, 4-week-old obese group, 10-week-old normal group and 10-week-old obese group, 5 mice in each group). Spearman's correlation analysis was performed to analyze the correlation between hub gens and prognostic clinical parameters. Results: From the GSE89632 dataset, 365 common DEGs (115 up-regulated genes and 250 down-regulated genes) were identified in patients with simple non-alcoholic fatty liver disease and NASH patients compared with control individuals. GO analysis showed that DEGs were mainly enriched in biological processes such as inflammatory response and immune response. KEGG pathway analysis showed that up-regulated genes were mainly enriched in cholesterol metabolism, bile secretion and fat digestion and absorption signal pathways. Down-regulated genes were mainly enriched in interleukin-17 signaling pathway, tumor necrosis factor signaling pathway, advanced glycation end products and their receptors of diabetic complications. Seven key hub genes were identified by PPI analysis, which were FOS, EGR1, FOSB, JUNB, FOSL1, MYC and NR4A1.The mRNA relative expression levels of EGR1 and JUNB in the liver of 10-week-old obese mice were lower than those of normal mice (P<0.05).The relative expression levels of NR4A1 in the liver of obese mice at 4-and 10-week-old were lower than those of normal mice at the same age (P values<0.05). Spearman's correlation analysis showed that the expression of EGR1 was negatively correlated with the degree of hepatic steatosis (r=-0.785, P<0.001).The expression levels of FOSB, MYC and NR4A1 were negatively correlated with the level of alanine aminotransferase (r=-0.649, -0.597 and-0.580 respectively, all P values<0.001). Conclusion: EGR1, FOSB, MYC, JUNB and NR4A1 might be the hub genes in the pathological process of NASH and the inflammatory and immune response in hepatocytes, IL-17 signaling pathway and TNF signaling pathway might be the key molecular mechanisms in the occurrence and development of NASH.

EGR1-CCL2 Feedback Loop Maintains Epithelial-Mesenchymal Transition of Cisplatin-Resistant Gastric Cancer Cells and Promotes Tumor Angiogenesis.

The mechanism of cisplatin resistance in gastric cancer (GC) is still elusive; several recent evidences proposed that chemoresistant tumor cells acquired aggressive behaviors. This study was aimed to investigate the mechanism of epithelial-mesenchymal transition (EMT) and angiogenesis in chemoresistant GC. Bioinformatics analysis and function or mechanism experiments including RT-qPCR, immunofluorescence, Western blot, luciferase reporter assay, Chromatin immunoprecipitation, Chicken chorioallantoic membrane assay and animal experiments were applied to evaluate the role of EGR1-CCL2 feedback loop. Compared with the parental cell line SGC7901, cisplatin resistant SGC7901R cells underwent EMT and showed increased angiogenic capabilities. Mechanistically, SGC7901R cells showed increased levels of EGR1, which could transcriptionally activate the angiogenic factor CCL2 and EMT regulator ZEB2. Reciprocally, CCL2 activated the CCR2-ERK-ELK1-EGR1 pathway, thus forming a positive feed-forward loop. Moreover, CCL2 in culture medium of SGC7901R cells promoted angiogenesis of Human Umbilical Vein Endothelial Cells (HUVECs). EGR1 expression was positively correlated with CCL2 and ZEB2 in clinical GC tissues, and the depletion of ERG1 could also decrease microvessel density and ZEB2 expression in metastatic nodules of nude mice. EGR1-CCL2 feedback loop might exert critical roles on EMT and angiogenesis of chemoresistant GC.

MiR-23a-3p regulates the inflammatory response and fibrosis in diabetic kidney disease by targeting early growth response 1

Diabetic kidney disease (DKD) has become the most common cause of chronic kidney disease. Proteinuria is generally considered one of the clinical indicators of renal damage, and it is also closely related to the progression of DKD. Accumulating evidence indicates that proteinuria induces an upregulation of the expression levels of inflammatory cytokines and fibrosis markers in renal tubular epithelial cells, but the mechanism remains unclear. Previously, we showed that early growth response 1 (Egr1) played a key role in renal tubular injury. However, the upstream mechanism of Egr1 in the development of DKD is poorly understood. In this study, we found that albumin stimulation significantly increased the expression levels of Egr1, interleukin 6 (IL-6), tumor necrosis factor-α (TNF-α), and fibronectin (FN) in HK-2 cells but decreased miR-23a-3p levels. We then identified that miR-23a-3p targeted the 3′ untranslated region (UTR) of Egr1 and directly suppressed the expression of Egr1. Moreover, we found that overexpression and inhibition of miR-23a-3p in HK-2 cells attenuated and promoted the expression of IL-6, TNF-α, and FN, respectively. Additionally, Egr1 silencing reversed the inflammation and fibrosis caused by the miR-23a-3p inhibitor. Thus, we conclude that miR-23a-3p attenuates the development of DKD through Egr1, suggesting that targeting miR-23a-3p may be a novel therapeutic approach for DKD.

Gfi1-Mediated Repression of C-Fos, Egr1 and Egr2, and Inhibition of ERK1/2 Signaling Contribute to the Role of Gfi1 in Granulopoiesis

Gfi1 is a transcriptional repressor that plays an important role in hematopoiesis. In the myeloid system, Gfi1 promotes granulopoiesis and antagonizes the alternative monocyte development in part through repressing PU.1 and CSF1 , which promote monopoiesis. However, the molecular mechanism by which Gfi1 favors neutrophil over monocyte development remains incompletely understood. G-CSF is a lineage-specific cytokine that plays a dominant role in granulopoiesis. We recently showed that a G-CSFR mutant in which tyrosine 729 was mutated to phenylalanine (Y729F) promoted monocyte rather than neutrophil development in myeloid precursors, which was associated with prolonged activation of ERK1/2 and augmented activation of downstream targets c-Fos and Egr1. To address whether Gfi1 may promote granulopoiesis independent of its repression of Csf1 , we established murine myeloid 32D and multipotent FDCP-mix cells that constitutively expressed G-CSFR Y729F and inducibly expressed Gfi1 in response to doxycycline (Dox; 32D/Y729F/Gfi1 and FDCP/Y729F/Gfi1). 32D and FDCP-mix cells showed no response to CSF1. In the absence of Dox, 32D/Y729F/Gfi1 and FDCP/Y729F/Gfi1 differentiated into monocytes when cultured in G-CSF. Interestingly, addition of Dox to induce Gfi1 expression rescued neutrophil development in both cell lines. Induction of Gfi1 expression with Dox also markedly attenuated the induction of c-Fos, Egr1 and Egr2 expression in response to G-CSF. In contrast, the mRNA levels of c-Fos, Egr1 and Egr2 were dramatically elevated in lineage marker negative (Lin-) bone marrow (BM) cells from Gfi1-/- mice as compared to BM cells from Gfi1+/+ mice. Chromatin immunoprecipitation (ChIP) and luciferase reporter assays demonstrated that Gfi1 bound to and repressed the activities of c-Fos , Egr1 and Egr2 promoters. c-Fos , Egr1 and Egr2 are immediate early genes that are activated by the ERK1/2 pathway. Notably, G-CSF-stimulated activation of ERK1/2 was stronger in Lin- BM cells from Gfi1-/- mice than in cells from Gfi1+/+ mice. It has been shown that BM cells from Gfi1-/- mice are unable to differentiate into mature neutrophils in vitro , but instead give rise to atypical monocytes. Interestingly, the MEK1/2 inhibitor U0126 or PD0325901 reduced the mRNA levels of c-Fos, Egr1 and Egr2, and partially rescued G-CSF-induced neutrophil development in Gfi1-/- BM cells. Together, our data indicate that Gfi1 inhibits the expression of c-Fos, Egr1 and Egr2 through direct transcriptional repression of their genes and indirect inhibition of ERK1/2 signaling pathway, and that Gfi1-mediated downregulation of c-Fos, Egr1 and Egr2 may contribute to the role of Gfi1 in granulopoiesis. DisclosuresNo relevant conflicts of interest to declare.

Analysis of Long Noncoding RNAs in Aila-Induced Non-Small Cell Lung Cancer Inhibition.

Non-small cell lung cancer (NSCLC) has the highest morbidity and mortality among all carcinomas. However, it is difficult to diagnose in the early stage, and current therapeutic efficacy is not ideal. Although numerous studies have revealed that Ailanthone (Aila), a natural product, can inhibit multiple cancers by reducing cell proliferation and invasion and inducing apoptosis, the mechanism by which Aila represses NSCLC progression in a time-dependent manner remains unclear. In this study, we observed that most long noncoding RNAs (lncRNAs) were either notably up- or downregulated in NSCLC cells after treatment with Aila. Moreover, alterations in lncRNA expression induced by Aila were crucial for the initiation and metastasis of NSCLC. Furthermore, in our research, expression of DUXAP8 was significantly downregulated in NSCLC cells after treatment with Aila and regulated expression levels of EGR1. In conclusion, our findings demonstrate that Aila is a potent natural suppressor of NSCLC by modulating expression of DUXAP8 and EGR1.

Liraglutide Regulates the Kidney and Liver in Diabetic Nephropathy Rats through the miR-34a/SIRT1 Pathway.

Purpose To explore the regulatory effects of liraglutide on the kidney and liver through the miR-34a/SIRT1 pathway with related factors in diabetic nephropathy (DN) rats. Methods DN rats were randomly divided into two groups (n = 10) and were injected with liraglutide or normal saline twice a day. The 24-hour urine microalbumin content and biochemical index levels were measured. qRT-PCR was performed to detect the expression of miR-34a in the kidney and liver tissues. The levels of SIRT1, HIF-1a, Egr-1, and TGF-β1 in kidney and liver tissues were determined using qRT-PCR, western blot, and immunohistochemistry. Electron microscopy and HE staining were used to observe the ultrastructure and pathological changes. Results Liraglutide treatment in DN rats decreased blood glucose, 24-hour urine microalbumin, TC, TG, LDL-C, UA, Cr, UREA, ALT, and AST levels and increased the level of HDL-C (P < 0.05). Compared with the control group, the miR-34a levels were significantly decreased in kidney and liver tissues followed by liraglutide treatment (P < 0.05). The levels of SIRT1 in the liraglutide group are significantly higher than those in the control group with the kidney and liver tissues (P < 0.05). Conversely, the contents of HIF-1a, Egr-1, and TGF-β1 were significantly lower in the liraglutide group than in the control group (P < 0.05). Electron microscopy showed that the kidney of the liraglutide-treated group exhibited minor broadening of the mesangial areas, fewer deposits, and a well-organized foot process. HE staining revealed that the kidney of the liraglutide-treated rats had a more regular morphology of the glomerulus and Bowman sac cavity and lighter tubular edema. Additionally, the liraglutide-treated DN rats had a clear hepatic structure, a lower degree of steatosis, and mild inflammatory cell infiltration. Conclusion Liraglutide, through its effect on the miR-34a/SIRT1 pathway, may have a protective role in the kidney and liver of DN rats.

Intravitreal Interleukin-2 modifies retinal excitatory circuits and retinocollicular innervation

Interleukin-2 is a classical immune cytokine whose neural functions have received little attention. Its levels have been found to be increased in some neuropathologies, such as Alzheimer's disease, multiple sclerosis and uveitis. Mechanistically, it has been demonstrated the role of IL-2 in regulating glutamate and acetylcholine transmission, thus being relevant for CNS physiology. In fact, our previous work showed that an acute intravitreal IL-2 injection during retinotectal development promoted contralateral eye axonal plasticity in the superior colliculus, but the involved mechanisms were not explored. So, our present study aimed to investigate the effect of increased intravitreal IL-2 levels on the retinal glutamatergic and cholinergic signalling required for retinotectal normal development. We showed through HRP neuronal tracing that intravitreal IL-2 also induces ipsilateral eye axonal sprouting. Protein level and/or immunolocalization analysis in the retina confirmed IL-2 pathway activation by increased expression of phospho-STAT-3, coupled to transient (24h) reduced levels of Egr1, PSD-95 and nicotinic acetylcholine receptor β2 subunit, suggesting reduced neural activity and synaptic sites. Also, AChE activity and GluN2B and GluA2 contents were reduced within 96h after IL-2 treatment. Therefore, IL-2-induced retinotectal plasticity might be driven by changes in cholinergic and glutamatergic pathways of the retina.

Curcumin promotes osteogenic differentiation of human periodontal ligament stem cells by inducting EGR1 expression

ObjectiveHuman periodontal ligament stem cells (hPDLSCs) attract attention for the periodontal regeneration therapy. Curcumin may promote osteogenic differentiation of hPDLSCs. This research aims to elucidate whether Curcumin displays promoting osteogenic differentiation and its mechanism. MethodsThe hPDLSCs were isolated from human periodontal ligament by immunomagnetic beads, identified with immumofluorescence. hPDLSCs were treated with 0, 5, 10, 20, 50, 100 μmol/L Curcumin. The early growth response gene 1 (EGR1) siRNA or plasmind were tranfected into the hPDLSCs. The viability, Alkaline Phosphatase (ALP) activity and mineralizaiton level of hPDLSCs were measured with 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay, ALP Assay Kit or Alizarin Red staining. The expression of EGR1, RUNX family transcription factor 2 (Runx2), bone gamma-carboxyglutamate protein (OC), secreted phosphoprotein 1 (OPN) and collagen type I alpha 1 chain (Collagen I), in hPDLSC were determined by Western blotting and quantitative reverse transcription-polymerase chain reaction. ResultsThe isolated hPDLSCs were spindle or irregular, arranged in radial shape and shown positive expression of STRO-1, CD146 and Vimentin. Curcumin 10 μmol/L treatment maximal promoting the cells viability, ALP activities, mineralization, and levels of Runx2, OC, OPN, Collagen I and EGR-1 in hPDLSCs. EGR-1 siRNA transfection inversed Curcumin’s promoting effect on ALP activities, mineralization, and levels of Runx2, OC, OPN, Collagen I and EGR-1 in hPDLSCs. While the EGR-1 plasmid transfection enhanced Curcumin’s promoting effect on these parameters of hPDLSCs. ConclusionCurcumin promotes the osteogenic differentiation of hPDLSCs, which may work through the EGR1. Curcumin may be a promising medicine for periodontitis treatment and periodontal regeneration.

PPARγ Activation-Mediated Egr-1 Inhibition Benefits Against Brain Injury in an Experimental Ischaemic Stroke Model

BackgroundInflammatory response is a critical contributor to cerebral ischaemia injuries and blood-brain barrier (BBB) dysfunction. Early growth response-1 (Egr-1), an oxygen-sensing transcription factor which is rapidly and markedly triggered in ischaemic events, acts as a master switch coordinating the upregulation of multiple target proinflammatory genes. Here, we explored whether peroxisome proliferator-activated receptor-gamma (PPARγ) activation by telmisartan can modulate Egr-1 expression and the subsequent inflammatory responses in a rat model of cerebral ischaemia. MethodsCerebral ischaemia was induced in rats by middle cerebral artery occlusion (MCAO). Brain injury was evaluated by brain water content, infarct volume, and Evans blue dye extravasation. Egr-1 and claudin-5 levels were assessed by western blot and real-time polymerase chain reaction. ResultsMCAO-provoked Egr-1 expression was time dependent, peaking at 24 h and continuing to 72 h. The elevation in Egr-1 was coupled with a reduction in claudin-5. Telmisartan treatment significantly corrected the alterations of Egr-1 and claudin-5, alleviated the neurological deficits, and reduced brain water content, infarct volume, and Evans blue dye extravasation 24 h after MCAO. However, all the benefits of telmisartan were reversed by antagonising PPARγ with GW9662. ConclusionEgr-1, a proinflammatory factor, is positively associated with post-ischaemic inflammation and the associated BBB dysfunction. PPARγ serves as an upstream transcription factor of the Egr-1 cascade. Targeting Egr-1 may emerge as a potential strategy to suppress inflammatory responses following ischaemic stroke.

Role of PAR1-Egr1 in the Initiation of Thoracic Aortic Aneurysm in Fbln4-Deficient Mice.

Remodeling of the extracellular matrix plays a vital role in cardiovascular diseases. Using a mouse model of postnatal ascending aortic aneurysms (termed Fbln4SMKO), we have reported that abnormal mechanosensing led to aneurysm formation in Fbln4SMKO with an upregulation of the mechanosensitive transcription factor, Egr1 (Early growth response 1). However, the role of Egr1 and its upstream regulator(s) in the initiation of aneurysm development and their relationship to an aneurysmal microenvironment are unknown. Approach and Results: To investigate the contribution of Egr1 in the aneurysm development, we deleted Egr1 in Fbln4SMKO mice and generated double knockout mice (DKO, Fbln4SMKO; Egr1-/-). Aneurysms were prevented in DKO mice (42.8%) and Fbln4SMKO; Egr1+/- mice (26%). Ingenuity Pathway Analysis identified PAR1 (protease-activated receptor 1) as a potential Egr1 upstream gene. Protein and transcript levels of PAR1 were highly increased in Fbln4SMKO aortas at postnatal day 1 before aneurysm formed, together with active thrombin and MMP (matrix metalloproteinase)-9, both of which serve as a PAR1 activator. Concordantly, protein levels of PAR1, Egr1, and thrombin were significantly increased in human thoracic aortic aneurysms. In vitro cyclic stretch assays (1.0 Hz, 20% strain, 8 hours) using mouse primary vascular smooth muscle cells induced marked expression of PAR1 and secretion of prothrombin in response to mechanical stretch. Thrombin was sufficient to induce Egr1 expression in a PAR1-dependent manner. We propose that thrombin, MMP-9, and mechanical stimuli in the Fbln4SMKO aorta activate PAR1, leading to the upregulation of Egr1 and initiation of ascending aortic aneurysms.

Ischemia/hypoxia inhibits cardiomyocyte autophagy and promotes apoptosis via the Egr-1/Bim/Beclin-1 pathway.

BackgroundMyocardial injury caused by microvascular obstruction (MVO) is characterized by persistent ischemia/hypoxia (IH) of cardiomyocytes after microembolization. Autophagy and Egr-1 were closely associated with various cardiovascular diseases, including MVO. Bim and Beclin-1 are the important genes for autophagy and apoptosis. We aimed to explore whether the Egr-1/Bim/Beclin-1 pathway is involved in regulating autophagy and apoptosis in IH-exposed cardiomyocytes.MethodsNeonatal rat cardiomyocytes exposed to the IH environment in vitro were transfected with lentivirus expressing Egr-1 or Egr-1 shRNA, or further treated with 3-methyladenine (3-MA). The expressions of autophagy and apoptosis-associated genes were evaluated using RT-qPCR and Western blots assays. Autophagic vacuoles and autophagic flux were detected by transmission electron microscopy (TEM) and confocal microscope, respectively. Cell injury was assessed by lactate dehydrogenase (LDH) leakage, and apoptosis was determined by flow cytometry.ResultsIH exposure elevated Egr-1 and Bim expressions, and decreased Beclin-1 expression in rat cardiomyocytes. Egr-1 overexpression in IH-exposed cardiomyocytes significantly up-regulated the levels of Egr-1 and Bim, and down-regulated the level of Beclin-1. Egr-1 knockdown resulted in down-regulated expressions of Egr-1 and Bim, as well as up-regulated expression of Beclin-1. In addition, Egr-1 knockdown induced autophagy was suppressed by 3-MA treatments. TEM and autophagic flux experiments also confirmed that Egr-1 inhibited autophagy progression in IH-exposed cardiomyocytes. Egr-1 suppression protected cardiomyocytes from IH-induced injury, as evidenced by the positive correlations between Egr-1 expression and LDH leakage or apoptosis index in IH-exposed cardiomyocytes.ConclusionsIH-induced cardiomyocyte autophagy and apoptosis are regulated by the Egr-1/Bim/Beclin-1 pathway, which is a potential target for treating cardiomyocyte injury caused by MVO in the IH environment.

Using CRISPR/Cas9 to Investigate the Role of Egr1 and NAB2 in Neuronal Differentiation of PC12 Cells

PC12 cells are a common model system used to study the molecular events underlying neuronal differentiation due to their ability to differentiate into neuronal cells following treatment with nerve growth factor (NGF). NGF induces neuronal differentiation through activation of the Ras/Raf/MEK/ERK signaling pathway, which in turn activates a transcriptional program leading to up‐regulation of neuronal genes, neurite outgrowth, and establish electrical excitability. Interestingly, treatment with epidermal growth factor (EGF) also activates Ras/Raf/MEK/ERK signaling, but induces proliferation rather than differentiation. The differential effects of NGF versus EGF are due to differences in ERK signal duration; EGF induces more transient ERK signaling that lasts 30–60 min, whereas NGF induces more sustained ERK signaling that lasts 4–6 h. Work in our lab and others indicate that sustained ERK signaling in response to NGF drives differentiation in part through sustained activity of the transcription factor Egr1 and, in turn, sustained expression of Egr1 target genes. Egr1 activity in response to EGF and NGF is regulated at two levels. First, Egr1 levels rapidly rise to peak levels by 1 h after treatment and then decline to baseline levels; similar to ERK signaling, Egr1 levels are sustained longer in response to NGF versus EGF. Second, one Egr1 target gene is its corepressor NAB2, which binds and represses Egr1 transactivation once expressed. This project was designed to further test our hypothesis that sustained Egr1 activity is a major driver of PC12 neuronal differentiation by evaluating whether knockout of NAB2 will lead to sustained Egr1 activity in response to EGF and, in turn, neuronal differentiation. To acquire PC12 cells that lack NAB2 protein, we used CRISPR/Cas9 technology to knockout the NAB2 gene. More specifically, a CRISPR NAB2‐KO plasmid was purchased from Santa Cruz Biotechnology, which encodes a green fluorescent protein (GFP) selectable marker, the Cas9 protein, and the guide RNA (gRNA). The plasmid had three variations for the gRNA which target different locations in the NAB2 gene. One day after transfection, GFP‐positive cells were sorted into 96‐well plates to isolate clones. Following clonal expansion, clones were first screened for NAB2 expression by Western blot, which identified eight putative knockout clones. Genomic DNA was then extracted from the eight clones, the NAB2 gene amplified by PCR, and the PCR project then subjected to Sanger sequencing to identify the INDELs responsible for NAB2 knockout. Ongoing work is examining the effects of NAB2 knockout on Egr1 levels and activity in response to EGF and NGF, as well as neuronal differentiation based on neurite outgrowth with the prediction that NAB2 knockout will result in sustained Egr1 activity and neuronal differentiation in response to both growth factors.Support or Funding InformationBridgewater State University Undergraduate Research Program

Improved FGF21 Sensitivity and Restored FGF21 Signaling Pathway in High-Fat Diet/Streptozotocin-Induced Diabetic Rats After Duodenal-Jejunal Bypass and Sleeve Gastrectomy.

Objective: Bariatric surgery can profoundly improve glucose and lipid metabolism in diabetic rats. Fibroblast growth factor 21 (FGF21) is an important hormone with multiple metabolic beneficial effects. Alteration in serum FGF21 level after bariatric surgery has been reported with conflicting results. Here, we investigated the effect of bariatric surgeries on FGF21 expression and sensitivity.Methods: We performed duodenal-jejunal bypass (DJB), sleeve gastrectomy (SG) and sham surgery in diabetic rats induced by high fat diet and streptozotocin. Metabolic parameters, including body weight, food intake, glucose tolerance, and lipid profiles, were monitored. FGF21 levels in both serum and liver were measured after surgery. FGF21 signaling pathway including FGF receptor 1 (FGFR1), β-klotho (KLB), and phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) was detected in the liver and white adipose tissue (WAT). We also determined FGF21 sensitivity post-operatively by acute recombinant human FGF21 injection. Oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) were conducted immediately after FGF21 injection. Serum triglyceride (TG) and non-esterified fatty acid (NEFA) were measured and the mRNA levels of early growth response 1 (Egr1) and c-Fos in the liver and WAT were detected after FGF21 injection.Results: Improvements in glucose tolerance, insulin sensitivity, and lipid profiles were observed after bariatric surgeries along with ameliorated lipid metabolism in the liver and WAT. Serum and hepatic FGF21 levels decreased in both DJB and SG groups. FGFR1 and phosphorylated ERK1/2 levels increased in both DJB and SG groups 8 weeks after surgery. The expression of KLB was downregulated only in the WAT after DJB and SG. Significant alteration of OGTT and ITT were observed after acute FGF21 administration in DJB and SG groups. Serum TG and NEFA in DJB and SG groups also decreased after FGF21 administration. And increased mRNA levels of Egr1 and c-Fos were detected in the liver and WAT after DJB and SG surgeries.Conclusions: DJB and SG surgeries can downregulate hepatic expression of FGF21, restore FGF21 signaling pathway and improve FGF21 sensitivity in high-fat diet/streptozotocin-induced diabetic rats.

Oct4 upregulates osteopontin via Egr1 and is associated with poor outcome in human lung cancer

BackgroundRoles of cancer stem cells and early growth response gene 1 (Egr1) in carcinogenesis have been extensively studied in lung cancer. However, the role of Egr1 in the metastasis of lung cancer remains undetermined, especially in regard to stem cell-related pathways.MethodsEgr1, osteopontin (OPN) and Oct4 expression in human lung cancer was determined by performing immunohistochemistry. Immunoblotting, ELISA, luciferase reporter assay, chromatin immunoprecipitation assay and RT-PCR were performed to validate the regulation of Oct4-Egr1-OPN axis. Moreover, the effect of Oct4-Egr1-OPN axis on lung cancer progression was evaluated by cell migration assay and mice study.ResultsWe detected Oct4, Egr1, and OPN expression in clinical specimens from 79 lung cancer patients, including 72 adenocarcinomas and 7 squamous cell carcinomas. High expression of Oct4, Egr1, and OPN accounted for 53, 51, and 57% of the patients, respectively. All of the three biomarkers were positively correlated in clinical human lung cancer. Patients with high expression of OPN were significantly associated with shorter disease-free survivals than those with low expression of OPN (p < 0.05). In lung cancer cells, Oct4 transactivated the Egr1 promoter and upregulated Egr1 expression. In a human lung cancer xenograft model, Oct4-overexpressing tumors expressed elevated levels of Egr1. Furthermore, overexpression of Oct4 in lung cancer cells increased the metastatic potential.ConclusionsEgr1 exerts a promoting effect on cancer metastasis in Oct4-overexpressing lung cancer. Thus, therapeutic strategies targeting the Oct4/Egr1/OPN axis may be further explored for the treatment of lung cancer, especially when lung cancer is refractory to conventional treatment due to cancer stem cells.

Simvastatin downregulates adipogenesis in 3T3-L1 preadipocytes and orbital fibroblasts from Graves' ophthalmopathy patients.

BackgroundSmoking is a strong risk factor for the development of Graves’ ophthalmopathy (GO). Immediate early genes (IEGs) are overexpressed in patients with active GO compared to healthy controls. The aim of this study was to study the effects of tobacco smoking and simvastatin on preadipocytes and orbital fibroblasts (OFs) in the adipogenic process.MethodsCigarette smoke extract (CSE) was generated by a validated pump system. Mouse 3T3-L1 preadipocytes or OFs were exposed to 10% CSE with or without simvastatin. Gene expression was studied in preadipocytes and OFs exposed to CSE with or without simvastatin and compared to unexposed cells or cells treated with a differentiation cocktail.ResultsIn 3T3-L1 preadipocytes, Cyr61, Ptgs2, Egr1 and Zfp36 expression levels were two-fold higher in cells exposed to CSE than in unexposed cells. Simvastatin downregulated the expression of these genes (1.6-fold, 5.5-fold, 3.3-fold, 1.4-fold, respectively). CSE alone could not stimulate preadipocytes to differentiate. Scd1, Ppar-γ and adipogenesis were downregulated in simvastatin-treated preadipocytes compared to nontreated preadipocytes 18-, 35- and 1.7-fold, respectively. In OFs, similar effects of CSE were seen on the expression of CYR61 (1.4-fold) and PTGS2 (3-fold). Simvastatin downregulated adipogenesis, PPAR-γ (2-fold) and SCD (27-fold) expression in OFs.ConclusionCSE upregulated early adipogenic genes in both mouse 3T3-L1 preadipocytes and human OFs but did not by itself induce adipogenesis. Simvastatin inhibited the expression of both early and late adipogenic genes and adipogenesis in preadipocytes and human OFs. The effect of simvastatin should be investigated in a clinical trial of patients with GO.

Localization of the ultraviolet-sensor Opn5m and its effect on myopia-related gene expression in the late-embryonic chick eye

Recent studies show that exposure to ultraviolet (UV) light suppresses ocular elongation, which causes myopia development. However, the specific mechanisms of this process have not been elucidated. A UV-sensor, Opsin 5 (Opn5) mRNA was shown to be present in extraretinal tissues. To test the possibility that UV-signals mediated by Opn5 would have a direct effect on the outer connective tissues of the eye, we first examined the expression patterns of a mammalian type Opn5 (Opn5m) in the late-embryonic chicken eye. Quantitative PCR showed Opn5m mRNA expression in the cornea and sclera. The anti-Opn5m antibody stained a small subset of cells in the corneal stroma and fibrous sclera. We next assessed the effect of UV-A (375 nm) irradiation on the chicken fibroblast cell line DF-1 overexpressing chicken Opn5m. UV-A irradiation for 30 min significantly increased the expression of Early growth response 1 (Egr1), known as an immediate early responsive gene, and of Matrix metalloproteinase 2 (Mmp2) in the presence of retinal chromophore 11-cis-retinal. In contrast, expression of Transforming growth factor beta 2 and Tissue inhibitor of metalloproteinase 2 was not significantly altered. These results indicate that UV-A absorption by Opn5m can upregulate the expression levels of Egr1 and Mmp2 in non-neuronal, fibroblasts. Taken together with the presence of Opn5m in the cornea and sclera, it is suggested that UV-A signaling mediated by Opn5 in the extraretinal ocular tissues could influence directly the outer connective tissues of the chicken late-embryonic eye.

Ferulic acid improves self-renewal and differentiation of human tendon-derived stem cells by upregulating early growth response 1 through hypoxia.

We aimed to investigate the potential beneficial effect of ferulic acid (FA) on stemness of human tendon-derived stem cells (hTSCs) in vitro and to elucidate the underlying molecular mechanism. The self-renewal ability of hTSCs was evaluated by colony formation and cell proliferation was determined by CCK-8 kit. Adipogenesis, osteogenesis, and chondrogenesis were determined by Oil Red O, Alizarin Red, and Alcian Blue stainings, respectively. Relative mRNA levels of PPARγ, Col2A1, Acan, Runx2, HIF1α, and EGR1 were measured with real-time PCR. Protein levels of HIF1α and EGR1 were detected by western blot. Direct binding of HIF1α with EGR1 promoter was analyzed by ChIP assay. Hypoxia-induced expression of EGR1 was interrogated by luciferase reporter assay. We demonstrated that FA treatment improved both self-renewal ability and multi-differentiation potential of hTSCs. FA induced hypoxia which in turn upregulated EGR1 expression via direct association with its hypoxia response element consensus sequence. Furthermore, we showed that both HIF1α and EGR1 were required for the enhancing effects of FA on hTSC self-renewal and differentiation. We hereby characterize the beneficial effect of FA on the stemness of hTSCs and highlight the critical role of HIF1α-EGR1 axis in this process.