Study of extracellular release of HMGB1 and its mechanism in lipopolysaccharide-induced bladder cancer T24 cells
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Objective To investigate the extracellular release of high mobility group box 1(HMGB1) in bladder cancer T24 cells induced by lipopolysaccharide(LPS) and its possible mechanism.Methods The changes of HMGB1 concentration in the culture medium and HMGB1 mRNA expression were investigated after T24 cells were stimulated with 100 μg/L LPS for different hours.The HMGB1 concentration and the level of HMGB1 mRNA expression were deter mined by ELISA and real-time fluorescent quantitative PCR,respectively.Inhibitory effect of PI3K/AKT pathway in hibitor was observed on HMGB1 release.Results The HMGB1 concentration in the culture medium and the level of HMGB1 mRNA expression significantly increased in a time-dependent manner after T24 cells after induced by LPS for 12 h.LY294002 partly inhibited extracellular release of HMGB1 in LPS-induced T24 cells.Conclusion LPS induces bladder cancer cells to release HMGB1,which may be related to PI3K/AKT signaling pathway.Keywords:
HMGB1
LY294002
High-mobility group
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To investigate the signaling pathways involved in interleukin (IL)-17A -mediated production of interleukin 8 (CXCL8), chemokine (C-C motif) ligand 2 (CCL2), and interleukin 6 (IL-6) by ARPE-19 cells, a spontaneously arisen cell line of retinal pigment epithelium (RPE).Flow cytometry analysis and western blot were used to detect the phosphorylation of extracellular signal-regulated kinases 1/2 (Erk1/2), p38 mitogen activated protein kinase (MAPK) and protein kinase B (PKB; Akt) in ARPE-19 cells stimulated with IL-17A. These cells were further pretreated with a series of kinase inhibitors and followed by incubation with IL-17A. CXCL8, CCL2, and IL-6 in the supernatant were quantified by enzyme-linked immunosorbent assay (ELISA).Coculture of ARPE-19 cells with IL-17A resulted in significant increases in Erk1/2, p38 MAPK, and Akt phosphorylation. Inhibition of p38MAPK, phosphoinositide 3-kinase (PI3K)-Akt and nuclear factor-kappaB (NF-κB), with the inhibitors SB203580, LY294002 and pyrrolydine dithiocarbamate (PDTC) respectively, reduced IL-17 (100 ng/ml) mediated production of CXCL8, CCL2, and IL-6 in a concentration dependent manner. Inhibition of Erk1/2 with PD98059 decreased the expression of the tested three inflammatory mediators when using low doses of IL-17A (0-10 ng/ml) but not at higher concentrations.IL-17A-induced production of inflammatory mediators by ARPE-19 cells involves Erk1/2, p38MAPK, PI3K-Akt and NF-κB pathways.
Interleukin 8
LY294002
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Objective
To evaluate the role of M3 receptor in penehyclidine hydrochloride(PHC)-induced reduction of increased permeability of human pulmonary microvascular endothelial cells(PMVECs)caused by endotoxin and the relationship with mitogen-activated protein kinase(MAPK)signaling pathway.
Methods
Human PMVECs were seeded in 6-well plates(2 ml/hole)or in culture flasks(4 ml/flask)at the density of 1×105 cells/ml and randomly divided into 6 groups(n=5 each): control group(group C), M3 receptor shRNA transfection group(group shRNA), lipopolysaccharide(LPS)group, penehyclidine plus LPS group(group P+ LPS), LPS plus M3 receptor shRNA transfection group(group LPS+ shRNA)and PHC plus LPS plus M3 shRNA transfection group(group P+ LPS+ shRNA). The cells were transfected with shRNA plasmid containing 2.5 nmol/L M3 receptors in shRNA, LPS+ shRNA and P+ LPS+ shRNA groups.LPS at the final concentration of 0.1 μg/ml was added at 24 h of incubation and then cells were incubated for 1 h in LPS and LPS+ shRNA groups.PHC at the final concentration of 2 μg/ml was added at 24 h of incubation, cells were incubated for 1 h, then LPS at the final concentration of 0.1 μg/ml was added, and cells were incubated for another 1 h in P+ LPS and P+ LPS+ shRNA groups.The permeability of PMVECs was measured using Transwell assay.The expression of phosphorylated p38 MAPK(p-p38 MAPK)and phosphorylated extracellular signal-regulated protein kinase 1/2(p-ERK1/2)was detected by Western blot, the expression of heat shock protein 27(HSP27)using immunofluorescent staining, and the expression of M3receptor mRNA by real-time polymerase chain reaction.
Results
Compared with group C, M3 receptor mRNA expression was significantly down-regulated in group shRNA, and the permeability of cells was significantly increased, and the expression of p-p38 MAPK, p-ERK1/2, HSP27 and M3 receptor mRNA was up-regulated in group LPS(P<0.05). The permeability of cells was significantly decreased, and the expression of p-p38 MAPK, p-ERK1/2, HSP27 and M3 receptor mRNA was down-regulated in P+ LPS, LPS+ shRNA and P+ LPS+ shRNA groups as compared with group LPS, and in group P+ LPS+ shRNA as compared with group LPS+ shRNA(P<0.05).
Conclusion
The mechanism by which PHC reduces endotoxin-caused increased permeability of human PMVECs is related to inhibiting activation of MAPK signaling pathway after down-regulating M3 receptor.
Key words:
Receptor, muscarinic M3; Cholinergic agent; Endothelial cells; Blood capillary; Lung; Endoxemia; p38 MAPKs; Extracellular Signal-Regulated MAP Kinases
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The expression of E-selectin induced by tumor necrosis factor (TNF) on the surface of human umbilical vein endothelial cells (HUVEC) was partially inhibited by an increase in the level of adenosine 3',5'-cyclic monophosphate (cAMP), produced by forskolin or cholera toxin combined with the type IV phosphodiesterase inhibitor rolipram and the protein kinase A agonist phosphorothioate analogue of cAMP SpcAMPS. The same agents had no significant effect on the constitutive and TNF-stimulated expression of intercellular adhesion molecule 1 (ICAM-1), whereas the effect on vascular cell adhesion molecule 1 (VCAM-1) expression was variable depending on cell culture conditions. The stimulatory effects of phorbol 12-myristate 13-acetate and bacterial lipopolysaccharide (LPS) on E-selectin expression were also downregulated by the forskolin-rolipram combination and by SpcAMPS. Inhibition of the surface expression of E-selectin was associated with a decrease of the total amount of the protein in the cell lysate and a reduced mRNA level, with no significant effect on mRNA stability. In anesthetized rats, the terbutaline-rolipram combination reduced the rolling of leukocytes induced by LPS in the mesenteric microcirculation. In addition to their partial inhibitory effect on the TNF-induced surface expression of E-selectin on HUVEC, the forskolin-rolipram combination and SpcAMPS strongly inhibited the release of soluble E-selectin from these cells; the release of soluble ICAM-1 and VCAM-1 was unaffected by these agents. Isoproterenol reduced the release of soluble E-selectin, whereas it had no significant effect on the cell surface expression of the protein. This study underscores the potential anti-inflammatory effect of a rise in the endothelial cAMP level.
Rolipram
Phorbol
Cholera toxin
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Wortmannin
Parthenolide
Proinflammatory cytokine
Interleukin 8
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Objective
To investigate the role of β-arrestin-1 in inhibition of endoxin-induced activation of MAPK signaling pathway in pulmonary microvascular endothelial cells (PMVECs) by penehyclidine hydrochloride (PHC).
Methods
Human PMVECs were seeded in 6-well plates (2 ml/well) or in culture flasks (4 ml/flask) at the density of 1×105 cells/ml, and randomly divided into 5 groups (n=20 each) using a random number table: empty plasmid transfection group (group C), lipopolysaccharide (LPS) + empty plasmid transfection group (LPS group), PHC+ LPS+ empty plasmid transfection group (P+ LPS group), LPS+ β-arrestin-1 short hairpin RNA (shRNA) transfection group (LPS+ shRNA group), and PHC + LPS+ β-arrestin-1 shRNA transfection group (P+ LPS+ shRNA group). After the cells were transfected with empty plasmid 1.5 μg or with plasmid containing 15 nmol/L β-arrestin-1 shRNA, the cells were incubated for 24 h. At 24 h of incubation, LPS with the final concentration of 0.1 μg/ml was added, and the cells were then incubated for 1 h in LPS and LPS+ shRNA groups.In P+ LPS and P+ LPS+ shRNA groups, PHC with the final concentration of 2 μg/ml was added, and the cells were incubated for 1 h, and then LPS with the final concentration of 0.1 μg/ml was added, and the cells were incubated for 1 h. The expression of filamentous actin (F-actin) was detected by flow cytometry.The expression of myosin light chain kinase (MLCK) and vascular endothelial-cadherin (VE-cadherin) was detected by immunofluorescence.The expression of phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) and phosphorylated c-Jun N-terminal kinase (p-JNK) was determined by Western blot.The expression of β-arrestin-1 mRNA was determined by real-time polymerase chain reaction.
Results
Compared with group C, the expression of F-actin, VE-cadherin and β-arrestin-1 mRNA was significantly down-regulated, and the expression of MLCK, p-ERK1/2 and p-JNK was up-regulated in group LPS, and the expression of p-ERK1/2 and p-JNK was significantly up-regulated (P 0.05). Compared with group LPS, the expression of F-actin, VE-cadherin and β-arrestin-1 mRNA was significantly up-regulated, and the expression of MLCK, p-ERK1/2 and p-JNK was down-regulated in group P+ LPS, and the expression of F-actin, VE-cadherin and β-arrestin-1 mRNA was significantly down-regulated, and the expression of MLCK and p-JNK was up-regulated in group LPS+ shRNA (P<0.05). Compared with group P+ LPS, the expression of F-actin, VE-cadherin and β-arrestin-1 mRNA was significantly down-regulated, and the expression of MLCK, p-ERK1/2 and p-JNK was up-regulated in group P+ LPS+ shRNA (P<0.05).
Conclusion
The mechanism by which PHC inhibits endoxin-induced activation of MAPK signaling pathway in PMVECs is partially related to up-regulation of β-arrestin-1 expression.
Key words:
Arrestins; Cholinergic antagonists; Endotoxins; Lung; Capillaries; Endothelial cells; Extracellular signal-regulated MAP kinases; JNK mitogen-activated protein kinases
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To explore the molecular mechanism of inducible nitric oxide synthase (iNOS) gene expression induced by lipopolysaccharide (LPS) in monocytes or macrophages.Luciferase report gene vector pGL2iNOSLuc driven by murine iNOS promoter region was constructed using gene recombination technique. RAW264.7 cells were cultured and transfected with luciferase report gene vector pGL2iNOSLuc. Twenty four hours later, PKC inhibitor H-7, calcium channel blocker verapamil, was added into the culture. LPS or PKC activator PMA was added 2 hours later to stimulate the cells. The activity of beta-galactosidase and luciferase was examined and the relative luciferase activity, representing the iNOS transactivity, was calculated. Modified Griess method was used to measure the concentration of NO(2)(-)/NO(3)(-) in the culture so as to evaluate the effect of PKC inhibitor on NO production induced by LPS. RT-PCR was used to study the effect of LPS on iNOS gene expression in RAW264.7 cells.Thirty minutes after stimulation of RAW264.7 cells by LPS, the activity of PKC in cytomembrane increased and the activity of PKC in cytoplasm decreased significantly in comparison with the baseline levels (P < 0.01). The NO concentration was 30.1 micro mol/L +/- 5.4 micro mol/L 12 hours after the stimulation of LPS on RAW264.7 cells, and was much more higher 24 and 48 hours after, all significantly higher than before (all P < 0.01). The NO concentration in the supernatant with H-7 at any time point was significantly lower than those in corresponding samples without H-7 (all P < 0.01). After stimulation of LPS and PMA, the relative luciferase activity was 25.3 +/- 4.6, significantly higher than the baseline level (P < 0.01). H-7 and verapamil significantly inhibited the LPS-induced iNOS transactivity (both P < 0.01). iNOS mRNA expression was increased greatly, and H-7 significantly inhibited this expression 12, 24, and 48 hours after LPS stimulation.In RAW264.7 cells, LPS stimulation increases intracellular calcium and activates PKC, thus inducing iNOS gene expression that contributes to the production of NO, which is an important signaling mechanism of NO production in septic shock.
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Objective: To investigate the effect of PKGⅡ (cGMP-dependent protein kinase Ⅱ) on cell proliferation-related MAPK (mitogen-activated protein kinase)/ERK (extracellular signal-regulated kinase) downstream targets RSK1 (ribosomal S6 kinase 1) and proto-oncogenes c-Jun and c-Fos in gastric cancer SGC-7901 cells. Methods: Gastric cancer SGC-7901 cells were infected with Ad-LacZ and Ad-PKG Ⅱ adenovirus, and then the PKGⅡ was overexpressed in SGC-7901 cells. These cells were treated with PKGⅡ specific activator — 8-pCPT-cGMP [8-(p-chlorophenylthio)-cyclic GMP], and then they were stimulated with EGF (epidermal growth factor). The proliferation of SGC-7901 cells was examined by MTT method, and the mRNAs expression levels of c-Jun and c-Fos and the protein expression levels of p-RSK1, c-Jun and c-Fos were examined by RT-PCR and Western blotting, respectively. The nuclear accumulation of p-RSK1 was observed under an immunofluorescence microscope. Results: EGF-induced increase of cell proliferation and the elevation of expressions of c-Jun and c-Fos mRNAs and proteins as well as p-RSK1 (Ser380-) protein in SGC-7901 cells which were infected with PKGⅡ were significantly inhibited after treatment with 8-pCPT-cGMP. The phosphorylation of RSK1 (Ser380) was decreased, and the accumulation of p-RSK1 (Ser380) in nuclei of SGC-7901 cells was increased after stimulation with EGF, while it was decreased after treatment with 8-pCPT-cGMP. Conclusion: Activated PKGⅡ can inhibits the proliferation of gastric cancer cells, phosphorylation of RSK1 (Ser380), nuclear accumulation of p-RSK1(Ser380) and the expressions of c-Jun and c-Fos genes which were induced by EGF. DOI:10.3781/j.issn.1000-7431.2012.11.002
Ribosomal s6 kinase
cGMP-dependent protein kinase
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Objective To study the extracellular release of high mobility group box 1(HMGB1) in hepatocytes induced by lipopolysaccharide and its intracellular signaling pathway.Methods The changes of HMGB1 mRNA expression and HMGB1 concentration in the culture medium were investigated after BRL-3A hepatocytes were induced with 100 μg/L lipopolysaccharide for 6-24 hours.The effects of MAPKs pathway inhibitors(SB202190 and U0126) with various concentrations were observed on HMGB1 mRNA expression and extracellular release in lipopolysaccharide-induced BRL-3A hepatocytes.HMGB1 concentration was determined by enzyme-linked immunosorbent assay(ELISA).Results The level of HMGB1 mRNA expression and HMGB1 concentration in the culture medium significantly increased after BRL-3A hepatocytes were induced by lipopolysaccharide for 6 hours(P0.01).The HMGB1 concentration continued to increase as the induction time lengthened.20 μmol/L SB202190 and 20 μmol/L U0126 partly inhibited extracellular release of HMGB1 in lipopolysaccharide-induced BRL-3A hepatocytes.But no effects were observed on HMGB1 mRNA expression for these inhibitors.Conclusion Lipopolysaccharide induces hepatocytes to release HMGB1,which mechanism is involved with intracellular MAPKs signaling pathway.
HMGB1
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Previous studies have shown that exogenously generated nitric oxide (NO) inhibits smooth muscle cell proliferation. In the present study, we stimulated rabbit vascular smooth muscle cells (RVSMC) with E. coli lipopolysaccharide (LPS), a known inducer of NO synthase transcription, and established a connection between endogenous NO, phosphorylation/dephosphorylation-mediated signaling pathways, and DNA synthesis. Non-confluent RVSMC were cultured with 0, 5, 10, or 100 ng/ml of the endotoxin. NO release was increased by 86.6% (maximum effect) in low-density cell cultures stimulated with 10 ng/ml LPS as compared to non-stimulated controls. Conversely, LPS (5 to 100 ng/ml) did not lead to enhanced NO production in multilayered (high density) RVSMC. DNA synthesis measured by thymidine incorporation showed that LPS was mitogenic only to non-confluent RVSMC; furthermore, the effect was prevented statistically by aminoguanidine (AG), a potent inhibitor of the inducible NO synthase, and oxyhemoglobin, an NO scavenger. Finally, there was a cell density-dependent LPS effect on protein tyrosine phosphatase (PTP) and ERK1/ERK2 mitogen-activated protein (MAP) kinase activities. Short-term transient stimulation of ERK1/ERK2 MAP kinases was maximal at 12 min in non-confluent RVSMC and was prevented by preincubation with AG, whereas PTP activities were inhibited in these cells after 24-h LPS stimulation. Conversely, no significant LPS-mediated changes in kinase or phosphatase activities were observed in high-density cells. LPS-induced NO generation by RVSMC may switch on a cell density-dependent proliferative signaling cascade, which involves the participation of PTP and the ERK1/ERK2 MAP kinases.
Dephosphorylation
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Objective To investigate the effect of knockdown of high mobility group protein B1 (HMGB1) on the proliferation of rat mesangial cells (GMCs) cultured in high glucose (HG) and its mechanism. Methods Rat GMCs was cultured and divided into normal group, high glucose treatment group, negative control small interfering RNA combined with high glucose treatment group (siRNA-NC-HG group) and HMGB1 small interference RNA combined with high glucose treatment group (siRNA-HMGB1-HG group). GMCs in the normal group were cultured in normal DMEM medium. GMCs in the HG treatment group were cultured with HG-DMEM medium. The GMCs in the siRNA-HMGB1-HG group, after transfected with siRNA-HMGB1 sequence for 6 hours, were cultured with high glucose medium for 24 hours. GMCs in the siRNA-NC-HG group, after transfected with siRNA-NC sequence for 6 hours, were cultured in HG medium for 24 hours. HMGB1 mRNA expression levels of GMCs were detected by real-time quantitative PCR. MTT assay was used to detect the proliferation of GMCs. Flow cytometry was performed to assess the apoptosis of GMCs. Western blot analysis was used to detect the protein levels of HMGB1, NF-κBp65 and nuclear factor kappa B inhibitor alpha (IκBα). ELISA was used to detect the levels of interleukin-1β (IL-1β), IL-6 and tumor necrosis factor α (TNF-α) in the cell supernatants. Results Compared with the siRNA-NC-HG group or HG treatment group, HMGB1 mRNA level decreased in GMCs in the siRNA-HMGB1-HG group, and after 24-, 48-, 72- and 96-hour treatment, the proliferation activity and apoptosis rate of GMCs decreased. After knock-down of HMGB1 level of GMCs, the level of NF-κBp65 protein decreased, the level of IκBα protein increased, and the levels of IL-1β, IL-6 and TNF-α in the supernatant decreased. Conclusion Knockdown of HMGB1 inhibits proliferation and promotes apoptosis of GMCs induced by HG, which may be related to the inhibition of NF-κB/IκB-α pathway.
HMGB1
High-mobility group
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