OBJECTIVE: To investigate the expression of EGR1 gene and the localization of EGR1 protein in bovine skeletal muscle-derived satellite cells (MDSCs), as well as to investigate the mechanism that EGR1 protein enters the nucleus. METHODS: Bovine MDSCs were cultured in differentiation medium for 1 day, 3 days and 5 days, respectively, and each group was triplicate. The expression of EGR1 gene and the localization of EGR1 protein were studied at different differentiation period in MDSCs by qRT-PC and Western blot. Moreover, the changes on the expression of endogenous EGR1 gene and EGR1 proteins were explored by CRISPRi, site-directed mutagenesis and laser confocal method. RESULTS: The results from the qRT-PCR and Western blot showed that the expressions of EGR1 gene on transcription level and translation level were significantly higher in differentiated cells than those in undifferentiated cells. The highest expression was found on the third day after the differentiation, and then began to decline. Immunofluorescence assays showed that EGR1 proteins were preferentially expressed in differentiated MDSCs, and increased along with the increase of number of myotubes. Confocal observation revealed that some EGR1 proteins were transferred into the nucleus in the differentiation of cells, however, the EGR1 proteins would not be detected in the differentiated MDSCs nuclei if a site directed mutagenesis (serine) on EGR1 protein occurred. CONCLUSION During the differentiation of bovine skeletal muscle satellite cells, the transcriptional level of EGR1 gene is increased, and some EGR1 proteins are transferred into the nucleus. The serine phosphorylation at position 533 of the C terminal of EGR1 protein is necessary for the nucleus transfer.
Animals ; Cattle ; Cell Differentiation ; Cell Nucleus ; Cells, Cultured ; Early Growth Response Protein 1 ; genetics ; metabolism ; Muscle Fibers, Skeletal ; Satellite Cells, Skeletal Muscle ; metabolism

OBJECTIVETo investigate the effects of the DPP4 inhibitor sitagliptin on the expressions of early growth response-1 (Egr-1) and fibronectin in the kidney of ApoE gene knockout mice.

METHODSEight-week-old male ApoE gene knockout mice were randomly divided into sitagliptin + apoE(-/-) group and apoE(-/-) group (n=6), with 6 C57BL mice as the normal control group. After feeding with high-fat diet and drug treatment for 16 weeks, the mice underwent intraperitoneal glucose tolerance test (IPGTT) and were measured for 24-h urinary albumin using ELISA. All the mice were then sacrificed to examine the changes of blood lipid profile and for detection of Egr-1 and fibronectin mRNA and proteins in the renal tissue using real-time PCR and Western blotting.

RESULTSThe mice in both apoE(-/-) group and sitagliptin+apoE(-/-) group all showed prominently increased blood lipids as compared with the control group (P<0.05) without significant differences between the two apoE(-/-) groups. The level of HDL was significantly higher in sitagliptin +apoE(-/-) group than in apoE(-/-) group (P<0.001) and control group (P<0.001). IPGTT showed no significant differences in the levels of blood glucose among the 3 groups. The excretion of urinary albumin was increased in apoE(-/-) group compared with the control group (P<0.01), but was significantly lower in sitagliptin+ apoE(-/-) group than in apoE(-/-) group (P<0.01). Real-time PCR and Western blotting showed significantly decreased mRNA and protein expressions of renal cortical Egr-1 and fibronectin in sitagliptin+apoE(-/-) group compared with apoE(-/-) group.

CONCLUSIONSitagliptin can reduce the renal expression of fibronectin by regulating the expression of Egr-1 to achieve renal protection.

Animals ; Apolipoproteins E ; genetics ; Diet, High-Fat ; Dipeptidyl-Peptidase IV Inhibitors ; pharmacology ; Early Growth Response Protein 1 ; metabolism ; Fibronectins ; metabolism ; Gene Knockout Techniques ; Kidney ; metabolism ; Lipids ; blood ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Real-Time Polymerase Chain Reaction ; Sitagliptin Phosphate ; pharmacology

OBJECTIVETo investigate the mechanism of high transcription of the glial cell-line derived neurotrophic factor (gdnf) gene induced by hyperacetylation of histone H3 lysine 9 (H3K9) at its promoter region II in rat C6 glioma cells.

METHODSThe acetylation level of H3K9 at Egr-1 binding site in gdnf gene promoter region II and the binding capacity of Egr-1 to its binding site in gdnf promoter were examined by ChIP-PCR in C6 astroglioma cells and normal rat astrocytes, and its changes were investigated in C6 astroglioma cells after treatment with histone acetyltransferase inhibitor curcumin or deacetylase inhibitor trichostatin A.

RESULTSCompared normal astrocytes, C6 astroglioma cells showed significantly increased acetylation level of H3K9 at Egr-1 binding site in gdnf gene promoter region II and Egr-1 binding capacity (P<0.01). Curcumin treatment significantly reduced H3K9 acetylation level at Egr-1 binding site and decreased both the binding of Egr-1 to promoter region II and gdnf mRNA levels in C6 astroglioma cells (P<0.05). Conversely, increased H3K9 acetylation at the Egr-1 binding site induced by trichostatin A significantly increased the binding of Egr-1 to promoter region II and gdnf mRNA expression levels (P<0.05).

CONCLUSIONH3K9 hyperacetylation induces increased Egr-1 binding to gdnf gene promoter II, which might be the reason for the high transcription level of gdnf gene in rat C6 glioma cells.

Acetylation ; Animals ; Astrocytes ; metabolism ; Binding Sites ; Cell Line, Tumor ; Early Growth Response Protein 1 ; metabolism ; Glial Cell Line-Derived Neurotrophic Factor ; genetics ; Glioma ; metabolism ; Histones ; chemistry ; Promoter Regions, Genetic ; Protein Processing, Post-Translational ; RNA, Messenger ; Rats ; Transcription, Genetic

OBJECTIVETo explore the combined anti-tumor effect of radiation therapy and gene-targeted suppression of tumor neovasculature in lung adenocarcinoma in vivo, and to explore the feasibility of micro-PET/CT in dynamic evaluation of treatment effectiveness.

METHODSThirty 5-6-week old male BALB/c nude mice were used in this study. The mouse models of xenotransplanted human lung adenocarcinoma were divided into 5 groups at random, six mice in each group: the control group, radiation treatment alone group and three groups of recombinant baculovirus plus radiation treatment (intratumoral injection, tail vein injection, and intramuscular injection). The tumor volume was measured every 2 days. Growth delay time (GD) and growth inhibition rate was calculated. FDG metabolism was evaluated by micro-PET-CT before and after treatment. The expressions of VEGF, CD31 and Ki-67 were detected by immunohistochemistry (IHC).

RESULTSThe tumor growth delay was >12 days, and the tumor inhibition rate was >45% in the recombinant baculovirus combined with radiotherapy groups, significantly higher than that of the radiotherapy alone group (P < 0.05). Immunohistochemical analysis showed that the expressions of VEGF, CD31 and Ki-67 were significantly lower than that in other groups (P < 0.05). The micro-PET-CT assessment showed that the FDG-metabolism in the recombinant baculovirus combined with radiotherapy groups was significantly reduced (P < 0.05), and the SUVmax (FDG metabolism) of transplanted tumors after treatment was also markedly decreased in comparison with that of the control group. The tumor volume after treatment was significantly correlated with SUVmax in the recombinant baculovirus intratumoral injection + radiotherapy group(r = 0.976), recombinant baculovirus intravenous injection + radiotherapy group (r = 0.954), recombinant baculovirus intramuscular injection + radiotherapy group (r = 0.929), and radiotherapy alone group (r = 0.871, P < 0.05).

CONCLUSIONSThe recombinant baculovirus containing Egr1 promoter and K5 gene combined with radiotherapy enhances the suppressing effect on the growth of lung adenocarcinoma in the tumor-bearing nude mice. The inducibility of Egr1 promoter by radiation allows the targeting and controllability of treatment. Micro-PET-CT results have a good correlation with the treatment effectiveness. Therefore, it can be used in real-time evaluation of tumor metabolic function in vivo.

Adenocarcinoma ; metabolism ; pathology ; radiotherapy ; Animals ; Baculoviridae ; genetics ; Cell Line, Tumor ; Combined Modality Therapy ; Early Growth Response Protein 1 ; genetics ; physiology ; Fluorodeoxyglucose F18 ; Genetic Therapy ; Genetic Vectors ; Humans ; Ki-67 Antigen ; metabolism ; Lung Neoplasms ; metabolism ; pathology ; radiotherapy ; Male ; Mice, Inbred BALB C ; Mice, Nude ; Molecular Targeted Therapy ; Neoplasm Transplantation ; Peptide Fragments ; genetics ; physiology ; Plasminogen ; genetics ; physiology ; Platelet Endothelial Cell Adhesion Molecule-1 ; metabolism ; Positron-Emission Tomography ; Promoter Regions, Genetic ; Random Allocation ; Recombinant Proteins ; genetics ; Tomography, X-Ray Computed ; Tumor Burden ; Vascular Endothelial Growth Factor A ; metabolism

Animals ; Cadherins ; metabolism ; Cell Cycle Proteins ; genetics ; metabolism ; Cell Differentiation ; Cell Line, Tumor ; Early Growth Response Protein 1 ; genetics ; metabolism ; Estradiol ; physiology ; Eukaryotic Initiation Factor-3 ; metabolism ; Genes, Tumor Suppressor ; physiology ; HSP90 Heat-Shock Proteins ; metabolism ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; Intracellular Signaling Peptides and Proteins ; genetics ; metabolism ; Iron ; metabolism ; Neoplasms ; metabolism ; pathology

OBJECTIVETo study influence of lanthanum chloride (LaCl(3)) on the expression of immediate early genes (IEGs) including c-jun, early growth response gene 1 (Egr1) and activity-regulated cytoskeletal gene (Arc) in the hippocampus of rats, and discuss the mechanism of LaCl(3) undermining learning and memory capability.

METHODSForty female Wistar adult rats were divided into control group, low LaCl(3)-contaminated group (0.25%), medium LaCl(3)-contaminated group (0.50%), and high LaCl(3)-contaminated group (1.00%) by randomized design. Each group had ten female rats along with five male rats and mated by the ratio of 2:1. The amounts of pups in the above four groups were 80, 83, 78 and 75 separately. The pups in respective group were La-dyed by lactation, and then the pups in LaCl(3)-contaminated groups drank 0.25%, 0.50% and 1.00% LaCl(3) separately for one month. Learning and memory capability of pups were measured in jumping stairs experiment. Hippocampal lanthanum content was determined by inductively coupled plasma mass spectrometry (ICP-MS). Hippocampal c-jun, Egr1 and Arc mRNA expression was detected by RT-PCR, and corresponding protein expression was measured by Western blotting method.

RESULTSIn the jumping stairs experiment, pups in 0.25%, 0.50% and 1.00% LaCl(3)-contaminated groups respectively made (1.75 ± 0.71), (2.38 ± 0.92) and (3.00 ± 0.76) mistakes; significantly higher than control group (1.25 ± 0.46) (q values were 4.386, 6.793, P < 0.05). However, the incubation period of 0.25%, 0.50% and 1.00% LaCl(3)-contaminated groups were (174.13 ± 33.72), (139.25 ± 45.83) and (75.50 ± 18.56) respectively, which were all significantly lower than that of control group (206.75 ± 20.47) (q values were 2.958, 6.121, 11.902, P < 0.05). Hippocampal c-jun mRNA expression were (0.89 ± 0.08), (0.77 ± 0.12), (0.58 ± 0.14) and (0.29 ± 0.10); while the c-jun protein expression were (0.72 ± 0.13), (0.64 ± 0.11), (0.43 ± 0.11) and (0.31 ± 0.14), and the Egr1 mRNA expression were (0.78 ± 0.09), (0.61 ± 0.13), (0.53 ± 0.10) and (0.22 ± 0.08), Egr1 protein expression were (0.65 ± 0.18), (0.40 ± 0.15), (0.32 ± 0.13) and (0.14 ± 0.09) in 0.25%, 0.50% and 1.00% LaCl(3)-contaminated groups; and all of which presented a dose-effect relationship that the correlation coefficients of these parameters with dose were -0.900 (t = 11.309, P = 0.000), -0.969 (t = 7.058, P = 0.000), -0.898 (t = 11.179, P = 0.000) and -0.962 (t = 6.739, P = 0.000).

CONCLUSIONLaCl(3) undermines the learning and memory capability of rats, which is possibly related to lower expression of c-jun and Egr1 gene and protein induced by lanthanum in hippocampus.

Animals ; Early Growth Response Protein 1 ; metabolism ; Female ; Gene Expression ; Genes, Immediate-Early ; drug effects ; genetics ; Hippocampus ; drug effects ; metabolism ; Lanthanum ; pharmacology ; Learning ; drug effects ; Male ; Memory ; drug effects ; Proto-Oncogene Proteins c-jun ; metabolism ; Rats ; Rats, Wistar

Sex-determining region Y box 18 (Sox18/SOX18) gene is an important regulator of vascular development playing a role in endothelial cell specification or differentiation, angiogenesis and atherogenesis. The aim of this study was to perform comprehensive functional characterization of the human SOX18 promoter, including determination of transcription start point (tsp) and identification of control elements involved in the regulation of SOX18 gene expression, with an emphasis on angiogenesis-related transcription factors. Analyses were performed in HeLa cells, representing a tumor cell line, and in EA.hy926 cells used as an endothelial model system. We have determined unique tsp of SOX18 gene, located 172 nucleotides upstream from ATG codon. Further, we have shown that SOX18 promoter region, -726 to -89 bp relative to tsp, contains positive cis-regulatory element(s) that stimulates SOX18 promoter activity, while region -89 to + 166 represents the minimal promoter. Within this region we have recognized the presence of essential element(s), positioned from -89 to +29, which harbors cluster of three putative early growth response 1 (EGR1) binding sites. By in vitro binding assays and functional analyses we have shown that these three putative binding sites are functionally relevant and sufficient for EGR1-induced SOX18 transcription. Mutations of these binding sites significantly impaired activity of the SOX18 promoter, particularly in EA.hy926 cells, indicating the importance of these regulatory elements for SOX18 promoter activity in endothelial setting. By data presented in this study, we have established SOX18 as a novel target gene regulated by EGR1 transcription factor, thus providing the first functional link between two transcription factors previously shown to be involved in the control of angiogenesis.
Early Growth Response Protein 1/genetics/*metabolism ; Electrophoretic Mobility Shift Assay ; Endothelium/*metabolism/pathology ; Gene Expression Regulation ; Hela Cells ; Humans ; Mutagenesis, Site-Directed ; Neovascularization, Physiologic/genetics ; Promoter Regions, Genetic ; Protein Binding/genetics ; SOXF Transcription Factors/genetics/*metabolism ; Transcription Initiation Site ; Transcriptional Activation

OBJECTIVETo explore the therapeutic effect and mechanism of emodin on cholestatic hepatitis.

METHODSRats were divided into 5 groups: 1 group was untreated, the other 4 groups were treated with alpha-naphthylisothiocyanate (ANIT), ANIT and emodin, ANIT and ursodeoxycholic acid, or ANIT and dexamethasone, respectively. At 24 h, 48 h and 72 h after the treatment, NF-kappa B, early growth response factor-1 (Egr-1), cytokine-induced neutrophil chemoattractant 1 (CINC-1), macrophage inflammatory protein 2 (MIP-2), intercellular adhesion molecule 1 (ICAM-1),tumor necrosis factor alpha (TNF alpha) and interleukin-6 (IL-6) were assayed by immunohistochemistry, real-time PCR , western-blot and ELISA. The level of malondialdehyde (MDA), superoxide Dismutase(SOD) and myeloperoxidase (MPO) were assayed by thiobarbituric acid method, xanthine oxidase method and colorimetric method, respectively.

RESULTS(1) Compared to the controls, emodin had a notable effect on total bilirubin (TB), direct bilirubin (DB), alanine aminotransferase (ALT) at all time points (all P less than 0.05). Compared to ursodeoxycholic acid, emodin had a notable effect on TB and DB at 24 h after the treatments, however, after 48 h, emodin had a notable effect only on TB (all P less than 0.05). Compared to Dexamethasone, emodin had a notable effect on TB at 48 h time point, and it had a notable effect on ALT at all time points (all P less than 0.05). (2) The nuclei NF-kappa B p65 staining was significantly increased at 24 h and 48 h after ANIT treatment (all P less than 0.05), and emodin treatment could block the increase (all P less than 0.05). (3) Egr-1 mRNA level was not affected by emodin treatment (P more than 0.05); levels of CINC-1, MIP-2 mRNA and ICAM-1 protein were significantly decreased after emodin treatment (all P less than 0.05). (4) The levels of TNF alpha and IL-6 were decreased after emodin treatment(all P less than 0.05). (5) The levels of MDA at all time points and MPO at 24 h, 48 h time points were notably down-regulated by emodin treatment, while the level of SOD was markedly elevated at all time points after emodin treatment (all P less than 0.05).

CONCLUSIONSEmodin treatment can reduce the levels of TB, DB and ALT in ANIT induced-cholestatic hepatitis. The effect may be due to inhibition of NF-kappa B signal pathway.

1-Naphthylisothiocyanate ; Animals ; Anti-Inflammatory Agents ; pharmacology ; therapeutic use ; Chemical and Drug Induced Liver Injury ; drug therapy ; metabolism ; Cholestasis, Intrahepatic ; chemically induced ; drug therapy ; metabolism ; Early Growth Response Protein 1 ; genetics ; metabolism ; Emodin ; pharmacology ; therapeutic use ; Immunohistochemistry ; Intercellular Adhesion Molecule-1 ; metabolism ; Interleukin-6 ; metabolism ; Liver ; metabolism ; pathology ; Liver Function Tests ; Male ; NF-kappa B ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo investigate the relationship between activities of early growth response gene 1 (EGR-1) of p38 mitogen-activated protein kinase (MAPK) pathway and in the epirubicin resistance of breast carcinoma cells.

METHODSProtein expression of phosphorylated p38MAPK was detected by confocal spectral microscopy. Using specific inhibitor SB203580, the effect of p38MAPK on cell apoptosis was analyzed by FITC-Annexin-V/PI double staining. The concentration of epirubicin was detected by flow cytometry (FCM). The 50% inhibition concentration (IC50) of epirubicin on MCF-7/Adr cells was determined by MTT method. Electrophoretic motility shift assay (EMSA) was performed to examine the affinity of EGR-1. EGR-1 mRNA was assessed by RT-PCR. The expression levels of p-glycoprotein, phosphorylated p53 and p38 were detected by Western blot.

RESULTSAfter treatment with SB203580 (15 micromol/L) 24 h and 48 h, (1) the early and late apoptosis of MCF-7/Adr cells expressing the phosphorylated p38MAPK protein was (25.36 +/- 1.17)% and (38.21 +/- 1.25)%, respectively, P < 0.05. And the tendency was in a time-dependent manner. (2) The average fluorescence intensity of MCF-7/Adr cells expressing the phosphorylated p38MAPK protein was (32.45 +/- 2.36) and (41.66 +/- 3.12), higher than the blank group (14.17 +/- 1.45) and DMSO group (16.28 +/- 0.63), P < 0.01. The epirubicin resistance of MCF-7/Adr cells significantly decreased. (3) SB203580 demonstrated a significantly higher level of EGR-1 activity. The IC50 was (21.53 +/- 2.17) and (8.77 +/- 1.02), lower than the DMSO group (40.74 +/- 2.56). MCF-7/Adr cells treated with SB203580 down-regulated the p38MAPK pathway activity, but up-regulated the EGR-1 mRNA expression. SB203580 significantly increased the cellular phosphorylated p53 protein level, but decreased the p-glycoprotein level in MCF-7/Adr cells.

CONCLUSIONSThere is a close relationship between p38MAPK pathway activity and the epirubicin resistance of breast carcinoma cells. The activation of EGR-1 mediated by p38MAPK pathway plays a critical role in epirubicin resistance.

ATP-Binding Cassette, Sub-Family B, Member 1 ; metabolism ; Apoptosis ; drug effects ; Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Drug Resistance, Neoplasm ; Early Growth Response Protein 1 ; genetics ; metabolism ; Enzyme Inhibitors ; pharmacology ; Epirubicin ; pharmacology ; Female ; Humans ; Imidazoles ; pharmacology ; Phosphorylation ; Pyridines ; pharmacology ; RNA, Messenger ; metabolism ; Signal Transduction ; p38 Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; metabolism

AIMTo investigate the dynamic changes of Egr-1 expression in the lungs of acute pulmonary embolism of rats by infusion of autoblood thrombs.

METHODSThe model of pulmonary embolism by infusion of autoblood thrombs in the pulmonary artery of rats was established and the mean pulmonary arterial pressure was continuously monitored by computer, and the results were evaluated by lung perfusion scan and pathological changes. Expression of Egr-1 proteinum and mRNA were measured by immunohistochemistry and reverse transcription polymerase chain reaction.

RESULTSThe mPAP of rats was increased significantly after infusion of autoblood thrombs at the half hour, and reached high level at the second hour, then remained the high level to four hours compared with group control at the same time point (P < 0.01). ECT image was showed significantly filling defect after infusion of autoblood thrombs at the first hour. The infused thromb was witnessed by hematoxylin and eosin stain. In the tracheal epithelium cells, alveolar epithelium cells and vascular smooth muscle cells of embolism rats, Egr-1 protein expression was increased significantly after embolization at the second hour compared with group control at the same time point (P<0.01), and was decreased slowly at the fourth hour. Egr-1 mRNA expression was showed the similar changes.

CONCLUSIONExpression of Egr-1 was low level in group control, but increased significantly after infusion of autoblood thromb at the second hour in the specificity of cells, suggesting that Egr-1 expression might be an important link of pathological changes in the acute pulmonary embolism.

Animals ; Early Growth Response Protein 1 ; genetics ; metabolism ; Gene Expression ; Lung ; metabolism ; Male ; Pulmonary Embolism ; genetics ; metabolism ; pathology ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley