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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 MAPK signalling pathways on MAPK activities stimulated by LPC. HUVEC-12 cells were pretreated with selective inhibitors including PD98059 (50 μm/l), SB203580 (15 μm/l) and curcumin (30 μm/l) for 1.5 hrs, respectively, followed by stimulation with or without 40 μm/l of LPC (lanes 1–5). The cells were harvested, lysed and cytoplasmic proteins of them were extracted 2 hrs after the treatment of LPC, then Western blot was performed to detect unphosphorylated (upper panel) and phosphorylated (lower panel) levels of ERK1/2 (A, B), JNK1/2 (C, D) and p38 (E, F), respectively. The phosphorylated levels of them were subjected to densitometric analysis. Each bar is means ± S.D. of 3 independent experiments. M, molecular mass markers; Pur, purified ERK1/2 protein that bacterially expressed was phosphorylated or unphosphorylated by MEK as a positive control of ERK1/2; UV, HEK293 cells were treated with or without ultraviolet ray as a positive control of JNK1/2; Ani, C6 glioma cells were treated with or without anisomycin as a positive control of p38; control, HUVEC-12 cells were untreated with LPC as a control. *P< 0.05, **P< 0.01, compared with the control; #P< 0.05, ##P< 0.01, compared with LPC group.
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fig02: Effects of the selective inhibitors of MAPK signalling pathways on MAPK activities stimulated by LPC. HUVEC-12 cells were pretreated with selective inhibitors including PD98059 (50 μm/l), SB203580 (15 μm/l) and curcumin (30 μm/l) for 1.5 hrs, respectively, followed by stimulation with or without 40 μm/l of LPC (lanes 1–5). The cells were harvested, lysed and cytoplasmic proteins of them were extracted 2 hrs after the treatment of LPC, then Western blot was performed to detect unphosphorylated (upper panel) and phosphorylated (lower panel) levels of ERK1/2 (A, B), JNK1/2 (C, D) and p38 (E, F), respectively. The phosphorylated levels of them were subjected to densitometric analysis. Each bar is means ± S.D. of 3 independent experiments. M, molecular mass markers; Pur, purified ERK1/2 protein that bacterially expressed was phosphorylated or unphosphorylated by MEK as a positive control of ERK1/2; UV, HEK293 cells were treated with or without ultraviolet ray as a positive control of JNK1/2; Ani, C6 glioma cells were treated with or without anisomycin as a positive control of p38; control, HUVEC-12 cells were untreated with LPC as a control. *P< 0.05, **P< 0.01, compared with the control; #P< 0.05, ##P< 0.01, compared with LPC group.

Mentions: To further confirm the inducing action of LPC on the phosphorylation of MAPKs above, MEK1-selective inhibitor PD98059, JNK-selective inhibitor curcumin and p38-specific inhibitor SB203580 were administered to HUVEC-12 cells before treatment of LPC, respectively. The results showed that the increase of the phosphorylation level of ERK1/2 by stimulation of LPC might be abolished by PD98059 (Fig. 2A and B), and curcumin might also abrogate the induction of the JNK1 phosphorylation by LPC (Fig. 2C and D). But the phosphorylation level of p38 MAPK was not altered by treatment with LPC no matter in the presence or absence of SB203580 (Fig. 2E and F).


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 MAPK signalling pathways on MAPK activities stimulated by LPC. HUVEC-12 cells were pretreated with selective inhibitors including PD98059 (50 μm/l), SB203580 (15 μm/l) and curcumin (30 μm/l) for 1.5 hrs, respectively, followed by stimulation with or without 40 μm/l of LPC (lanes 1–5). The cells were harvested, lysed and cytoplasmic proteins of them were extracted 2 hrs after the treatment of LPC, then Western blot was performed to detect unphosphorylated (upper panel) and phosphorylated (lower panel) levels of ERK1/2 (A, B), JNK1/2 (C, D) and p38 (E, F), respectively. The phosphorylated levels of them were subjected to densitometric analysis. Each bar is means ± S.D. of 3 independent experiments. M, molecular mass markers; Pur, purified ERK1/2 protein that bacterially expressed was phosphorylated or unphosphorylated by MEK as a positive control of ERK1/2; UV, HEK293 cells were treated with or without ultraviolet ray as a positive control of JNK1/2; Ani, C6 glioma cells were treated with or without anisomycin as a positive control of p38; control, HUVEC-12 cells were untreated with LPC as a control. *P< 0.05, **P< 0.01, compared with the control; #P< 0.05, ##P< 0.01, compared with LPC group.
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Related In: Results  -  Collection

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fig02: Effects of the selective inhibitors of MAPK signalling pathways on MAPK activities stimulated by LPC. HUVEC-12 cells were pretreated with selective inhibitors including PD98059 (50 μm/l), SB203580 (15 μm/l) and curcumin (30 μm/l) for 1.5 hrs, respectively, followed by stimulation with or without 40 μm/l of LPC (lanes 1–5). The cells were harvested, lysed and cytoplasmic proteins of them were extracted 2 hrs after the treatment of LPC, then Western blot was performed to detect unphosphorylated (upper panel) and phosphorylated (lower panel) levels of ERK1/2 (A, B), JNK1/2 (C, D) and p38 (E, F), respectively. The phosphorylated levels of them were subjected to densitometric analysis. Each bar is means ± S.D. of 3 independent experiments. M, molecular mass markers; Pur, purified ERK1/2 protein that bacterially expressed was phosphorylated or unphosphorylated by MEK as a positive control of ERK1/2; UV, HEK293 cells were treated with or without ultraviolet ray as a positive control of JNK1/2; Ani, C6 glioma cells were treated with or without anisomycin as a positive control of p38; control, HUVEC-12 cells were untreated with LPC as a control. *P< 0.05, **P< 0.01, compared with the control; #P< 0.05, ##P< 0.01, compared with LPC group.
Mentions: To further confirm the inducing action of LPC on the phosphorylation of MAPKs above, MEK1-selective inhibitor PD98059, JNK-selective inhibitor curcumin and p38-specific inhibitor SB203580 were administered to HUVEC-12 cells before treatment of LPC, respectively. The results showed that the increase of the phosphorylation level of ERK1/2 by stimulation of LPC might be abolished by PD98059 (Fig. 2A and B), and curcumin might also abrogate the induction of the JNK1 phosphorylation by LPC (Fig. 2C and D). But the phosphorylation level of p38 MAPK was not altered by treatment with LPC no matter in the presence or absence of SB203580 (Fig. 2E and F).

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
Related in: MedlinePlus