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Objective:To explore whether the lipopolysaccharide (LPS)-induced modification of O-linked N-acetylglucosamine (O-GlcNAc) is involved in the inflammatory signaling pathway of endothelial cells.Methods:Human umbilical vein endothelial cells (HUVEC) were cultured in vitro, and cells in logarithmic growth phase were used for experiments. Cells were divided into blank control group, LPS group (2 000 mg/L LPS), O-GlcNAc transferase (OGT) overexpression (OGT-OE)+LPS group (plasmid transfection OGT+2 000 mg/L LPS), protein kinase C (PKC) inhibitor+LPS group (10 μmol/L Go 6983+2 000 mg/L LPS), RhoA inhibitor+LPS group (40 μmol/L Rhoin hydrochloride+2 000 mg/L LPS), phosphatidylinositol-3-kinase (PI3K) inhibitor+LPS group (1 μmol/L SL-2052+2 000 mg/L LPS), serine/threonine kinase (Akt) inhibitor+LPS group (10 μmol/L PP2+2 000 mg/L LPS) and small interfering RNA (siRNA) treated Akt (si-AKT)+LPS group (si-Akt+2 000 mg/L LPS). After 24 hours of LPS treatment, real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was used to detect the transcription levels of inflammatory cytokines [interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1)]. The protein expression or phosphorylation of OGT, O-GlcNAc, Akt, extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (p38MAPK), nuclear factor-κB p65 (NF-κB p65), and signal transducer and activator of transcription 3 (STAT3) were determined by Western blotting. Results:Compared with the blank control group, the expression of OGT and the modification of O-GlcNAc in the LPS group were decreased, while the expressions of phosphorylated ERK, p38MAPK, and STAT3 were increased, and the transcript levels of inflammatory cytokines were also significantly increased [IL-6 mRNA (2 -ΔΔCt): 4.71±0.60 vs. 1.03±0.29, TNF-α mRNA (2 -ΔΔCt): 1.89±0.11 vs. 1.04±0.35, ICAM-1 mRNA (2 -ΔΔCt): 2.06±0.18 vs. 1.02±0.21, VCAM-1 mRNA (2 -ΔΔCt): 2.94±0.57 vs. 1.01±0.17, all P < 0.05], indicating that LPS could decrease O-GlcNAc modification, activate inflammatory signaling pathways and increase inflammatory cytokines expression. Compared with the LPS group, the expressions of phosphorylated ERK, p38MAPK, NF-κB p65, and STAT3 in the endothelial cells of the OGT-OE+LPS group were decreased, and the expression of inflammatory factors were significantly decreased [IL-6 mRNA (2 -ΔΔCt): 0.12±0.01 vs. 0.90±0.17, TNF-α mRNA (2 -ΔΔCt): 0.31±0.01 vs. 0.91±0.14, ICAM-1 mRNA (2 -ΔΔCt): 0.64±0.02 vs. 1.13±0.16, VCAM-1 mRNA (2 -ΔΔCt): 0.11±0.01 vs. 0.93±0.11, all P < 0.05], indicating that the increase of OGT level could inhibit the partial activation of the endothelial inflammatory signal pathway under the LPS stimulation. Compared with the blank control group, the phosphorylation level of Akt in the LPS group was increased. Compared with the LPS group, both OGT expression and O-GlcNAc modification were down-regulated after pretreatment of PKC inhibitor, RhoA inhibitor, PI3K inhibitor, or Akt inhibitor. Compared with the LPS group, the transcript levels of IL-6, TNF-α and ICAM-1 in the PP2+LPS group were significantly decreased [IL-6 mRNA (2 -ΔΔCt): 1.46±0.16 vs. 3.55±0.87, TNF-α mRNA (2 -ΔΔCt): 0.98±0.14 vs. 1.76±0.10, ICAM-1 mRNA (2 -ΔΔCt): 1.39±0.24 vs. 2.04±0.13, all P < 0.05], but there was no significant change in VCAM-1. Compared with the LPS group, the expression of OGT and O-GlcNAc modification in the si-Akt+LPS group were decreased, while the transcript levels of inflammatory cytokines were also significantly decreased [IL-6 mRNA (2 -ΔΔCt): 0.75±0.03 vs. 0.99±0.09, TNF-α mRNA (2 -ΔΔCt): 0.69±0.01 vs. 1.10±0.08, ICAM-1 mRNA (2 -ΔΔCt): 0.76±0.01 vs. 0.99±0.02, VCAM-1 mRNA (2 -ΔΔCt): 0.93±0.08 vs. 1.20±0.21, all P < 0.05], indicating that Akt participated in the action process of LPS on OGT and affected the inflammatory factor expression. Conclusions:The decreased level of O-GlcNAc modification in endothelial cells stimulated with LPS promotes partial activation of inflammatory signaling pathways, mainly involving ERK, p38MAPK, and STAT3, and affects the expression of inflammatory factors. AKT may be involved in the effect of LPS on the inhibition of O-GlcNAc modification.
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O-linked-N-acetylglucosamine (O-GlcNAc) modification is a unique post-translational modification that plays a regulatory role in many cellular processes, such as transcription, intracellular signaling, endocytosis, and protein stability. Epidermal growth factor (EGF) domain-specific O-GlcNAc transferase (EOGT) is an endoplasmic reticulum (ER) resident protein which can glycosylate the residues of Ser or Thr of secreted or membrane (transmembrane) glycoproteins containing EGF domain. Notch signaling pathway is involved in cell-to-cell communication which regulates cell biological processes through interactions between adjacent cells. To date, EOGT-mediated O-GlcNAc modification has been found to be involved in many human diseases, and shown significant relation with Notch signaling pathway. However, the specific molecular mechanisms have not been fully elucidated. In this review, we briefly introduce recent studies regarding to the roles of EOGT-mediated O-GlcNAc modification and its correlation with Notch signaling pathway in human diseases.
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Caveolin-1 (Cav-1), a major structural protein of caveolae, is implicated in the vesicular uptake processes of transcytosis and cell signaling. However, its role in modulating protein glycosylation and tumor metastasis remains to be further elucidated. In the present study, it was shown that Cav-1 promotes the expression of O-GlcNAcylation and O-GlcNAc transferase (OGT), and triggers the invasion and metastasis of hepatocellular carcinoma (HCC) cells. The results of RT-qPCR, Western blot and dual lucif-erase reporter assay showed that Cav-1 negatively regulated the expression of transcription factor RUNX2 in HCC. Subsequently, this results in attenuate RUNX2-induced transcription of miR24. miR24 suppresses mouse HCC cells invasion and metastasis via directly targeting Ogt mRNA 3′UTR. This research provides evidence of Cav-1-mediated OGT expression and O-GlcNAc (O-linked N-acetylglucosamine) elevation. These data give insight into a novel mechanism of HCC occurrence and development.
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Objective:To investigate the effect of resveratrol (Res) on the fat synthesis in the liver cancer HepG2cells, and to elucidate its possible mechanism.Methods:The HepG2cells were cultured in vitro and divided into Res group (treated with 40μmol·L-1 DMSO-diluted Res for 24h) and control group (treated with the same concentration of DMSO for 24h) .The cell supernatant was collected, and the levels of triglyceride (TG) and total cholesterol (TC) in the cells in various groups were measured by ELISA.The mRNA and protein expression levels of lipase synthase acetyl-CoA carboxylase (ACC1) , fatty acid synthetase (FASN) and stearoyl-CoA desaturase (SCD1) in the cells in various groups were detected by qRT-PCR and Western blotting method.The levels of O-linked N-acetylglucosamine (O-GlcNAc) glycosylation in the cells in various groups were detected by Western blotting method.Results:Compared with control group, the levels of TG and TC in the cells in Res group were decreased, but the difference was not statistically significant (t1=1.886, P＞0.05;t2=2.457, P＞0.05) .Compared with control group, the levels of expressions of ACC1, FASN and SCD1mRNA and proteins in the cells in Res group were significantly decreased (P＜0.05or P＜0.01) ;the O-GlcNAc glycosylation level in the cells in Res group was significantly decreased (t=2.87, P＜0.05) .Conclusion:Res has the effect of inhibiting the fat synthesis in the liver cancer HepG2 cells.Its mechanism may be related to the reduction of cellular O-GlcNAc glycosylation level and the reduction of the expression of FASN.
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Objective: To investigate the effect of resveratrol (Res) on the fat synthesis in the liver cancer HepG cells, and to elucidate its possible mechanism. Methods: The HepG cells were cultured in vitro and divided into Res group (treated with 40 umol • L-1 DMSO- diluted Res for 24 h) and control group (treated with the same concentration of DMSO for 24 h). The cell supernatant was collected, and the levels of triglyceride (TG) and total cholesterol (TO in the cells in various groups were measured by ELISA. The mRNA and protein expression levels of lipase synthase acetyl-CoA carboxylase (ACCl), fatty acid synthetase (FASN) and stearoyl-CoA desaturase (SCD1) in the cells in various groups were detected by qRT-PCR and Western blotting method. The levels of O-linked N-acetylglucosamine (O-GIcNAc) glycosylation in the cells in various groups were detected by Western blotting method. Results: Compared with control group, the levels of TG and TC in the cells in Res group were decreased, but the difference was not statistically significant (t1=1.886, P>0.05; t2=2.457,P>0.05). Compared with control group, the levels of expressions of ACCl, FASN and SCD1 mRNA and proteins in the cells in Res group were significantly decreased ( P<0.05 or P<0. 01); the O-GlcNAc glycosylation level in the cells in Res group was significantly decreased (t=2. 87, P<0.05). Conclusion: Res lias the effect of inhibiting the fat synthesis in the liver cancer HepG cells. Its mechanism may be related to the reduction of cellular O-GlcNAc glycosylation level and the reduction of the expression of FASN.
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BACKGROUND AND OBJECTIVES: The benefit of high glucose-insulin-potassium (GIK) solution in clinical applications is controversial. We established a neonatal rat ventricular myocyte (NRVM) in vitro coverslip ischemia/reperfusion (I/R) model and investigated the effects of GIK solution on suppressing reactive oxygen species (ROS) and upregulating O-GlcNacylation, which protects cells from ischemic injury. MATERIALS AND METHODS: NRVMs were isolated from postnatal day 3-4 Sprague-Dawley rat pups and grown in Dulbecco's modified Eagle's medium containing high glucose (4.5 g/L), fetal bovine serum, and penicillin/streptomycin. The effects of the GIK solution on ROS production, apoptosis, and expression of O-GlcNAc and O-GlcNAc transferase (OGT) were investigated in the coverslip I/R model. RESULTS: Covering the 24-well culture plates for 3 hr with 12 mm diameter coverslips resulted in the appropriate ischemic shock. Glucose and insulin synergistically reduced ROS production, protected NRVM dose-dependently from apoptosis, and altered O-GlcNAc and OGT expression. CONCLUSION: The high GIK solution protected NRVM from I/R injury in vitro by reducing ROS and altering O-GlcNacylation.
Subject(s)
Animals , Rats , Apoptosis , Glucose , Insulin , Muscle Cells , Myocytes, Cardiac , Rats, Sprague-Dawley , Reactive Oxygen Species , Shock , TransferasesABSTRACT
O-linked beta-N-acetylglucosamine modification is an important post-translational modification, emerging as a novel regulatory mechanism in various cellular events. Recently, several studies have shown that O-GlcNAcylation plays an essential role in human breast, lung, and colon cancers. With regard to skin cancers, the role of O-GlcNAcylation has yet to be elucidated. To investigate whether O-GlcNAcylation is linked to human skin tumor development, immunohistochemical analysis was performed to investigate the presence of O-GlcNAcylation in various skin tumors. We evaluated the levels of O-GlcNAcylation, O-GlcNAc transferase, and O-GlcNAcase in 29 benign tumors, 12 premalignant tumors, and 26 malignant tumors in skin. Compared to the benign tumors, premalignant and malignant tumors had increased patterns of O-GlcNAcylation. In addition, the O-GlcNAc transferase and O-GlcNAcase levels were higher in premalignant and malignant tumors than in benign tumors. Interestingly, O-GlcNAcase levels were significantly increased in premalignant tumors compared to benign and malignant tumors. These results suggest that O-GlcNAcylation of proteins may play an important role in the development of human skin tumors.
Subject(s)
Humans , Breast , Colonic Neoplasms , Immunohistochemistry , Lung , Protein Processing, Post-Translational , Skin Neoplasms , Skin , TransferasesABSTRACT
Objective To evaluate the role of O-GlcNAc protein modification in attenuation of brain damage by glutamine in septic rats.Methods Sixty male SD rata weighing 180-240 g were randomly divided into 4 groups:sham operation group(group S,n =12),sepsis group(group CLP,n =16),glutamine group(group G,n =16),an inhibitor of O-linked-N-acetyl glucosamine transferase Alloxan + glutamine group(group G + A,n =16).Rats were submitted to sepsis by cecal ligation and perforation(CLP).Glutamine(Gln)0.75 g/kg was injected iv after CLP in group G.Gln 0.75 g/kg was injected iv and Alloxan 90 mg/kg was injected ip after CLP in group G + A.Equal volume of normal saline was given in group S and group CLP.A1 24 h afler CLP,the neural reflex score was evaluated,then rat was sacrificed.The brain was removed for measurement of brain water content,observation of histopathology and determination of O-GlcNAc-modified protein expression.Results Compared with group S,neural reflex score and brain water content were significantly increased in groups CLP,G and G + A(P ＜ 0.05).Compared with group CLP,neural reflex score and brain water content were significantly decreased in groups G and G + A(P ＜ 0.05),and the expression of O-GlcNAc-modified protein was upregulated in group G(P ＜ 0.05),Compared with groups G,neural reflex score and brain water content were significantly increased,and the expression of O-GlcNAc-modified protein downregulated in group G + A(P ＜ 0.05).There was no significant difference in O-GlcNAc-modified protein expression among groups S,CLP and G + A.Conclusion Glutamine attenuates brain damage through O-GlcNAc protein modification in septic rats.
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It has been known that O-linked beta-N-acetylglucosamine (O-GlcNAc) modification of proteins plays an important role in transcription, translation, nuclear transport and signal transduction. The increased flux of glucose through the hexosamine biosynthetic pathway (HBP) and increased O-GlcNAc modification of protein have been suggested as one of the causes in the development of insulin resistance. However, it is not clear at the molecular level, how O-GlcNAc protein modification results in substantial impairment of insulin signaling. To clarify the association of O-GlcNAc protein modification and insulin resistance in rat primary adipocytes, we treated the adipocytes with O-(2-acetamido-2deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc), a potent inhibitor of O-GlcNAcase that catalyzes removal of O-GlcNAc from proteins. Prolonged treatment of PUGNAc (100 micrometer for 12 h) increased O-GlcNAc modification on proteins in adipocytes. PUGNAc also drastically decreased insulin-stimulated 2-deoxyglucose (2DG) uptake and GLUT4 translocation in adipocytes, indicating that PUGNAc developed impaired glucose utilization and insulin resistance in adipocytes. Interestingly, the O-GlcNAc modification of IRS-1 and Akt2 was increased by PUGNAc, accompanied by a partial reduction of insulin-stimulated phosphorylations of IRS-1 and Akt2. The PUGNAc treatment has no effect on the expression level of GLUT4, whereas O-GlcNAc modification of GLUT4 was increased. These results suggest that the increase of O-GlcNAc modification on insulin signal pathway intermediates, such as IRS-1 and Akt2, reduces the insulin-stimulated phosphorylation of IRS-1 and Akt2, subsequently leading to insulin resistance in rat primary adipocytes.