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Lysophosphatidylcholine up-regulates human endothelial nitric oxide synthase gene transactivity by c-Jun N-terminal kinase signalling pathway.

Xing F, Liu J, Mo Y, Liu Z, Qin Q, Wang J, Fan Z, Long Y, Liu N, Zhao K, Jiang Y - J. Cell. Mol. Med. (2008)

Bottom Line: The activation of JNK signalling pathway by overexpression of JNK or its upstream kinase active mutant up-regulated the transactivity of eNOS significantly, but the activation of p38 signalling pathway down-regulated it largely.Additionally using decoy oligonucleotides proved that SP1 was necessary for maintaining the basal or stimulated transactivity, whereas AP1 contributed mainly to the increase of the stimulated transactivity.These findings indicate that the up-regulation of the eNOS gene transactivity by LPC involves the enhancement of SP1 transcription factor by the activation of JNK and ERK1/2 signalling pathways and AP1 transcription factor by the activation of JNK signalling pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Jinan University Guangzhou, China. tfyxing@jnu.edu.cn <tfyxing@jnu.edu.cn>

ABSTRACT
Human endothelial nitric oxide synthase (eNOS) plays a pivotal role in maintaining blood pressure homeostasis and vascular integrity. It has recently been reported that mitogen-activated protein kinases (MAPKs) are intimately implicated in expression of eNOS. However detailed mechanism mediated by them remains to be clarified. In this study, eNOS gene transactivity in human umbilical vein endothelial cells was up-regulated by stimulation of lysophosphatidylcholine (LPC). The stimulation of LPC highly activated both extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK), with differences in the dynamic processes of activation between them. Unexpectedly, p38 MAPK could not be activated by the stimulation of LPC. The activation of JNK signalling pathway by overexpression of JNK or its upstream kinase active mutant up-regulated the transactivity of eNOS significantly, but the activation of p38 signalling pathway down-regulated it largely. The inhibition of either ERK1/2 or JNK signalling pathway by kinase-selective inhibitors could markedly block the induction of the transactivity by LPC. It was observed by electrophoretic mobility shift assay that LPC stimulated both SP1 and AP1 DNA binding activity to go up. Additionally using decoy oligonucleotides proved that SP1 was necessary for maintaining the basal or stimulated transactivity, whereas AP1 contributed mainly to the increase of the stimulated transactivity. These findings indicate that the up-regulation of the eNOS gene transactivity by LPC involves the enhancement of SP1 transcription factor by the activation of JNK and ERK1/2 signalling pathways and AP1 transcription factor by the activation of JNK signalling pathway.

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Effects of the selective inhibitors of MAPKs on human eNOS transcriptional activity induced by LPC. (A) After transfected with pDseNOSRed, HUVEC-12 cells were treated without or with PD98059 (50 μMol/l) or SB203580 (15 μMol/l) or curcumin (30 μMol/l), respectively, for 1.5 hrs before treatment of LPC. (B) After transfected with pDseNOSRed plus pcDNA3 or JNK1 expression vector, the cells were treated without or with curcumin (30 μMol/l) at the same time. 45 hrs after the transfection, the cells were stimulated without or with LPC (40 μMol/l). The treatment of each group is shown as indicated in the figure. The red fluorescence-emitting cells were subjected to dynamic observation under an inverted fluorescence microscope. The fluorescence intensity of RFP expressed by the cells that represents the eNOS transactivity was quantitatively analysed using the fluorescence analysis software, Image-Pro Plus, 96 hrs after transfection. The result represents means ± S.D. of 3 independent experiments. **P< 0.01, compared with pDseNOSRed group; #P< 0.05, ##P< 0.01, compared with pDseNOSRed plus LPC group (A) or compared with pDseNOSRed plus pcDNA3 and LPC group (B); AAP< 0.01, compared with pDseNOSRed plus JNK1 expression vector and LPC group.
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fig04: Effects of the selective inhibitors of MAPKs on human eNOS transcriptional activity induced by LPC. (A) After transfected with pDseNOSRed, HUVEC-12 cells were treated without or with PD98059 (50 μMol/l) or SB203580 (15 μMol/l) or curcumin (30 μMol/l), respectively, for 1.5 hrs before treatment of LPC. (B) After transfected with pDseNOSRed plus pcDNA3 or JNK1 expression vector, the cells were treated without or with curcumin (30 μMol/l) at the same time. 45 hrs after the transfection, the cells were stimulated without or with LPC (40 μMol/l). The treatment of each group is shown as indicated in the figure. The red fluorescence-emitting cells were subjected to dynamic observation under an inverted fluorescence microscope. The fluorescence intensity of RFP expressed by the cells that represents the eNOS transactivity was quantitatively analysed using the fluorescence analysis software, Image-Pro Plus, 96 hrs after transfection. The result represents means ± S.D. of 3 independent experiments. **P< 0.01, compared with pDseNOSRed group; #P< 0.05, ##P< 0.01, compared with pDseNOSRed plus LPC group (A) or compared with pDseNOSRed plus pcDNA3 and LPC group (B); AAP< 0.01, compared with pDseNOSRed plus JNK1 expression vector and LPC group.

Mentions: Specific inhibitors of MAPKs are powerful tools to explore the function of MAPK pathways. To confirm the results above, MEK1-selective inhibitor PD98059, JNK-selective inhibitor curcumin and p38-specific inhibitor SB203580 were used to determine effect of MAPK inhibition on the eNOS gene expression induced by LPC. The results showed that the human eNOS promoter activity induced by LPC was dramatically blocked by both PD98059 and curcumin. However, SB203580 lacked the ability to repress the induction of the promoter transactivity by treatment with LPC. These results showed that ERK1/2 and JNK signal pathways might be involved in the activation of the promoter transactivity induced by LPC (Fig. 4A).


Lysophosphatidylcholine up-regulates human endothelial nitric oxide synthase gene transactivity by c-Jun N-terminal kinase signalling pathway.

Xing F, Liu J, Mo Y, Liu Z, Qin Q, Wang J, Fan Z, Long Y, Liu N, Zhao K, Jiang Y - J. Cell. Mol. Med. (2008)

Effects of the selective inhibitors of MAPKs on human eNOS transcriptional activity induced by LPC. (A) After transfected with pDseNOSRed, HUVEC-12 cells were treated without or with PD98059 (50 μMol/l) or SB203580 (15 μMol/l) or curcumin (30 μMol/l), respectively, for 1.5 hrs before treatment of LPC. (B) After transfected with pDseNOSRed plus pcDNA3 or JNK1 expression vector, the cells were treated without or with curcumin (30 μMol/l) at the same time. 45 hrs after the transfection, the cells were stimulated without or with LPC (40 μMol/l). The treatment of each group is shown as indicated in the figure. The red fluorescence-emitting cells were subjected to dynamic observation under an inverted fluorescence microscope. The fluorescence intensity of RFP expressed by the cells that represents the eNOS transactivity was quantitatively analysed using the fluorescence analysis software, Image-Pro Plus, 96 hrs after transfection. The result represents means ± S.D. of 3 independent experiments. **P< 0.01, compared with pDseNOSRed group; #P< 0.05, ##P< 0.01, compared with pDseNOSRed plus LPC group (A) or compared with pDseNOSRed plus pcDNA3 and LPC group (B); AAP< 0.01, compared with pDseNOSRed plus JNK1 expression vector and LPC group.
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fig04: Effects of the selective inhibitors of MAPKs on human eNOS transcriptional activity induced by LPC. (A) After transfected with pDseNOSRed, HUVEC-12 cells were treated without or with PD98059 (50 μMol/l) or SB203580 (15 μMol/l) or curcumin (30 μMol/l), respectively, for 1.5 hrs before treatment of LPC. (B) After transfected with pDseNOSRed plus pcDNA3 or JNK1 expression vector, the cells were treated without or with curcumin (30 μMol/l) at the same time. 45 hrs after the transfection, the cells were stimulated without or with LPC (40 μMol/l). The treatment of each group is shown as indicated in the figure. The red fluorescence-emitting cells were subjected to dynamic observation under an inverted fluorescence microscope. The fluorescence intensity of RFP expressed by the cells that represents the eNOS transactivity was quantitatively analysed using the fluorescence analysis software, Image-Pro Plus, 96 hrs after transfection. The result represents means ± S.D. of 3 independent experiments. **P< 0.01, compared with pDseNOSRed group; #P< 0.05, ##P< 0.01, compared with pDseNOSRed plus LPC group (A) or compared with pDseNOSRed plus pcDNA3 and LPC group (B); AAP< 0.01, compared with pDseNOSRed plus JNK1 expression vector and LPC group.
Mentions: Specific inhibitors of MAPKs are powerful tools to explore the function of MAPK pathways. To confirm the results above, MEK1-selective inhibitor PD98059, JNK-selective inhibitor curcumin and p38-specific inhibitor SB203580 were used to determine effect of MAPK inhibition on the eNOS gene expression induced by LPC. The results showed that the human eNOS promoter activity induced by LPC was dramatically blocked by both PD98059 and curcumin. However, SB203580 lacked the ability to repress the induction of the promoter transactivity by treatment with LPC. These results showed that ERK1/2 and JNK signal pathways might be involved in the activation of the promoter transactivity induced by LPC (Fig. 4A).

Bottom Line: The activation of JNK signalling pathway by overexpression of JNK or its upstream kinase active mutant up-regulated the transactivity of eNOS significantly, but the activation of p38 signalling pathway down-regulated it largely.Additionally using decoy oligonucleotides proved that SP1 was necessary for maintaining the basal or stimulated transactivity, whereas AP1 contributed mainly to the increase of the stimulated transactivity.These findings indicate that the up-regulation of the eNOS gene transactivity by LPC involves the enhancement of SP1 transcription factor by the activation of JNK and ERK1/2 signalling pathways and AP1 transcription factor by the activation of JNK signalling pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Jinan University Guangzhou, China. tfyxing@jnu.edu.cn <tfyxing@jnu.edu.cn>

ABSTRACT
Human endothelial nitric oxide synthase (eNOS) plays a pivotal role in maintaining blood pressure homeostasis and vascular integrity. It has recently been reported that mitogen-activated protein kinases (MAPKs) are intimately implicated in expression of eNOS. However detailed mechanism mediated by them remains to be clarified. In this study, eNOS gene transactivity in human umbilical vein endothelial cells was up-regulated by stimulation of lysophosphatidylcholine (LPC). The stimulation of LPC highly activated both extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK), with differences in the dynamic processes of activation between them. Unexpectedly, p38 MAPK could not be activated by the stimulation of LPC. The activation of JNK signalling pathway by overexpression of JNK or its upstream kinase active mutant up-regulated the transactivity of eNOS significantly, but the activation of p38 signalling pathway down-regulated it largely. The inhibition of either ERK1/2 or JNK signalling pathway by kinase-selective inhibitors could markedly block the induction of the transactivity by LPC. It was observed by electrophoretic mobility shift assay that LPC stimulated both SP1 and AP1 DNA binding activity to go up. Additionally using decoy oligonucleotides proved that SP1 was necessary for maintaining the basal or stimulated transactivity, whereas AP1 contributed mainly to the increase of the stimulated transactivity. These findings indicate that the up-regulation of the eNOS gene transactivity by LPC involves the enhancement of SP1 transcription factor by the activation of JNK and ERK1/2 signalling pathways and AP1 transcription factor by the activation of JNK signalling pathway.

Show MeSH