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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 JNK signalling pathway on the DNA binding activities of SP1 and AP1 in HUVEC-12 cells by treatment of LPC. The nuclear extracts from HUVEC-12 cells treated without or with 40 μMol/l of LPC for 2 hrs were used for EMSA to detect effect of a specific inhibitor of JNK on SP1 and AP1 DNA binding activity enhanced by overexpression of JNK1 in the presence or absence of stimulation of LPC. Lane 1, free probe; lane 2, treated without LPC as a control; lanes 3∼4 (A, B), 50- and 100-fold molar excess of cold probe of SP1 was used to determine SP1-specific DNA binding; lane 3 (C, D) 100-fold molar excess of cold probe of AP1 was done; lanes 5 (A, B) and 4 (C, D), transfected alone with a JNK1 expression vector; lanes 6 (A, B) and 5 (C, D), treated with 40 μMol/l of LPC 45 hrs after tansfected with the JNK1 expression vector; lanes 7∼9 (A, B) and 6∼8 (C, D), transfected with the JNK1 expression vector, then pretreated with PD98059 (50 μMol/l), SB203580 (15 μMol/l) and curcumin (30 μMol/l) for 1.5 hrs, respectively, followed by stimulation with LPC (40 μMol/l). The data represent means ± S.D. of 3 independent experiments. *P< 0.05, **P< 0.01, compared with the control; #P< 0.05, compared with the alone transfected JNK1 group; ΔΔP< 0.01, compared with the transfected JNK1 plus LPC group.
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fig07: Effects of JNK signalling pathway on the DNA binding activities of SP1 and AP1 in HUVEC-12 cells by treatment of LPC. The nuclear extracts from HUVEC-12 cells treated without or with 40 μMol/l of LPC for 2 hrs were used for EMSA to detect effect of a specific inhibitor of JNK on SP1 and AP1 DNA binding activity enhanced by overexpression of JNK1 in the presence or absence of stimulation of LPC. Lane 1, free probe; lane 2, treated without LPC as a control; lanes 3∼4 (A, B), 50- and 100-fold molar excess of cold probe of SP1 was used to determine SP1-specific DNA binding; lane 3 (C, D) 100-fold molar excess of cold probe of AP1 was done; lanes 5 (A, B) and 4 (C, D), transfected alone with a JNK1 expression vector; lanes 6 (A, B) and 5 (C, D), treated with 40 μMol/l of LPC 45 hrs after tansfected with the JNK1 expression vector; lanes 7∼9 (A, B) and 6∼8 (C, D), transfected with the JNK1 expression vector, then pretreated with PD98059 (50 μMol/l), SB203580 (15 μMol/l) and curcumin (30 μMol/l) for 1.5 hrs, respectively, followed by stimulation with LPC (40 μMol/l). The data represent means ± S.D. of 3 independent experiments. *P< 0.05, **P< 0.01, compared with the control; #P< 0.05, compared with the alone transfected JNK1 group; ΔΔP< 0.01, compared with the transfected JNK1 plus LPC group.

Mentions: From the results above, it is reasonable to speculate that JNK plays a key role in the regulation of eNOS gene expression in LPC-induced physiological or pathological processes. We would like to check effect of JNK on the SP1 and AP1 DNA binding activities in HUVEC-12 cells. It was found that the overexpression of JNK1 in the cells increased the both SP1 and AP1 DNA binding activities, and also further boosted the LPC-induced SP1 and AP1 DNA binding activities (Fig. 7A and B). However, an experiment with MAPK inhibitors showed the DNA binding activities of SP1 and AP1 were not in parallel, i.e. the LPC-induced SP1 DNA binding activity was sensitive to PD98059 or curcumin, while the AP1 DNA binding activity was only inhibited by curcumin. These results further sup-ported the conclusion that JNK signal pathway was associated with the AP1 DNA binding activity induced by LPC (Fig. 7C and D).


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 JNK signalling pathway on the DNA binding activities of SP1 and AP1 in HUVEC-12 cells by treatment of LPC. The nuclear extracts from HUVEC-12 cells treated without or with 40 μMol/l of LPC for 2 hrs were used for EMSA to detect effect of a specific inhibitor of JNK on SP1 and AP1 DNA binding activity enhanced by overexpression of JNK1 in the presence or absence of stimulation of LPC. Lane 1, free probe; lane 2, treated without LPC as a control; lanes 3∼4 (A, B), 50- and 100-fold molar excess of cold probe of SP1 was used to determine SP1-specific DNA binding; lane 3 (C, D) 100-fold molar excess of cold probe of AP1 was done; lanes 5 (A, B) and 4 (C, D), transfected alone with a JNK1 expression vector; lanes 6 (A, B) and 5 (C, D), treated with 40 μMol/l of LPC 45 hrs after tansfected with the JNK1 expression vector; lanes 7∼9 (A, B) and 6∼8 (C, D), transfected with the JNK1 expression vector, then pretreated with PD98059 (50 μMol/l), SB203580 (15 μMol/l) and curcumin (30 μMol/l) for 1.5 hrs, respectively, followed by stimulation with LPC (40 μMol/l). The data represent means ± S.D. of 3 independent experiments. *P< 0.05, **P< 0.01, compared with the control; #P< 0.05, compared with the alone transfected JNK1 group; ΔΔP< 0.01, compared with the transfected JNK1 plus LPC group.
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Related In: Results  -  Collection

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fig07: Effects of JNK signalling pathway on the DNA binding activities of SP1 and AP1 in HUVEC-12 cells by treatment of LPC. The nuclear extracts from HUVEC-12 cells treated without or with 40 μMol/l of LPC for 2 hrs were used for EMSA to detect effect of a specific inhibitor of JNK on SP1 and AP1 DNA binding activity enhanced by overexpression of JNK1 in the presence or absence of stimulation of LPC. Lane 1, free probe; lane 2, treated without LPC as a control; lanes 3∼4 (A, B), 50- and 100-fold molar excess of cold probe of SP1 was used to determine SP1-specific DNA binding; lane 3 (C, D) 100-fold molar excess of cold probe of AP1 was done; lanes 5 (A, B) and 4 (C, D), transfected alone with a JNK1 expression vector; lanes 6 (A, B) and 5 (C, D), treated with 40 μMol/l of LPC 45 hrs after tansfected with the JNK1 expression vector; lanes 7∼9 (A, B) and 6∼8 (C, D), transfected with the JNK1 expression vector, then pretreated with PD98059 (50 μMol/l), SB203580 (15 μMol/l) and curcumin (30 μMol/l) for 1.5 hrs, respectively, followed by stimulation with LPC (40 μMol/l). The data represent means ± S.D. of 3 independent experiments. *P< 0.05, **P< 0.01, compared with the control; #P< 0.05, compared with the alone transfected JNK1 group; ΔΔP< 0.01, compared with the transfected JNK1 plus LPC group.
Mentions: From the results above, it is reasonable to speculate that JNK plays a key role in the regulation of eNOS gene expression in LPC-induced physiological or pathological processes. We would like to check effect of JNK on the SP1 and AP1 DNA binding activities in HUVEC-12 cells. It was found that the overexpression of JNK1 in the cells increased the both SP1 and AP1 DNA binding activities, and also further boosted the LPC-induced SP1 and AP1 DNA binding activities (Fig. 7A and B). However, an experiment with MAPK inhibitors showed the DNA binding activities of SP1 and AP1 were not in parallel, i.e. the LPC-induced SP1 DNA binding activity was sensitive to PD98059 or curcumin, while the AP1 DNA binding activity was only inhibited by curcumin. These results further sup-ported the conclusion that JNK signal pathway was associated with the AP1 DNA binding activity induced by LPC (Fig. 7C and D).

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