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Tumor necrosis factor-α enhances hyperbaric oxygen-induced visfatin expression via JNK pathway in human coronary arterial endothelial cells.

Wang BW, Lin CM, Wu GJ, Shyu KG - J. Biomed. Sci. (2011)

Bottom Line: Visfatin, a adipocytokine with insulin-mimetic effect, plays a role in endothelial angiogenesis.However, the molecular mechanism of beneficial effects of HBO is poorly understood.HBO significantly increased secretion of TNF-α from cultured human CAECs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Cardiology, Shin Kong Wu Ho-Su Memorial Hospital, and School of Medicine, Fu-Jen Catholic University, New Taipei City, Taipei, Taiwan.

ABSTRACT

Background: Visfatin, a adipocytokine with insulin-mimetic effect, plays a role in endothelial angiogenesis. Hyperbaric oxygen (HBO) has been used in medical practice. However, the molecular mechanism of beneficial effects of HBO is poorly understood. We sought to investigate the cellular and molecular mechanisms of regulation of visfatin by HBO in human coronary arterial endothelial cells (CAECs).

Methods: Human CAECs were exposed to 2.5 atmosphere absolute (ATA) of oxygen in a hyperbaric chamber. Western blot, real-time polymerase chain reaction, and promoter activity assay were performed. In vitro glucose uptake and tube formation was detected.

Results: Visfatin protein (2.55-fold) and mRNA (2.53-fold) expression were significantly increased after exposure to 2.5 ATA HBO for 4 to 6 h. Addition of SP600125 and JNK siRNA 30 min before HBO inhibited the induction of visfatin protein. HBO also significantly increased DNA-protein binding activity of AP-1 and visfatin promoter activity. Addition of SP600125 and TNF-α monoclonal antibody 30 min before HBO abolished the DNA-protein binding activity and visfatin promoter activity induced by HBO. HBO significantly increased secretion of TNF-α from cultured human CAECs. Exogenous addition of TNF-α significantly increased visfatin protein expression while TNF-α antibody and TNF-α receptor antibody blocked the induction of visfatin protein expression induced by HBO. HBO increased glucose uptake in human CAECs as HBO and visfatin siRNA and TNF-α antibody attenuated the glucose uptake induced by HBO. HBO significantly increased the tube formation of human CAECs while visfatin siRNA, TNF-α antibody inhibited the tube formation induced by HBO.

Conclusions: HBO activates visfatin expression in cultured human CAECs. HBO-induced visfatin is mediated by TNF-α and at least in part through JNK pathway.

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HBO increases AP-1-binding activity and visfatin promoter activity. A, Representative EMSA showing protein binding to the AP-1 oligonucleotide in nuclear extracts of human CAECs after HBO treatment in the presence or absence of inhibitors and TNF-α antibody. Similar results were found in another two independent experiments. Cold oligo means unlabeled AP-1 oligonucleotides. B, Constructs of visfatin promoter gene. Positive +1 demonstrates the initiation site for the visfatin transcription. Mutant visfatin promoter indicates mutation of AP-1 binding sites in the visfatin promoter as indicated. C, Quantitative analysis of visfatin promoter activity. The luciferase activity in cell lysates was measured and was normalized with renilla activity (n = 4 per group). *P < 0.001 vs. control.
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Figure 6: HBO increases AP-1-binding activity and visfatin promoter activity. A, Representative EMSA showing protein binding to the AP-1 oligonucleotide in nuclear extracts of human CAECs after HBO treatment in the presence or absence of inhibitors and TNF-α antibody. Similar results were found in another two independent experiments. Cold oligo means unlabeled AP-1 oligonucleotides. B, Constructs of visfatin promoter gene. Positive +1 demonstrates the initiation site for the visfatin transcription. Mutant visfatin promoter indicates mutation of AP-1 binding sites in the visfatin promoter as indicated. C, Quantitative analysis of visfatin promoter activity. The luciferase activity in cell lysates was measured and was normalized with renilla activity (n = 4 per group). *P < 0.001 vs. control.

Mentions: Treatment of HBO for 2 h to 6 h significantly increased the DNA-protein binding activity of AP-1 (Figure 6A). An excess of unlabeled AP-1 oligonucleotide competed with the probe for binding AP-1 protein, whereas an oligonucleotide containing a 2-bp substitution in the AP-1 binding site did not compete for binding. Addition of SP600125 and TNF-α monoclonal antibody 30 min before HBO stimulation abolished the DNA-protein binding activity induced by HBO. Exogenous addition of TNF-α significantly increased the DNA-protein binding activity. To study whether the visfatin expression induced by HBO is regulated at the transcriptional level, we cloned the promoter region of human visfatin (-889~+16), and constructed a luciferase reporter plasmid (pGL3-Luc). The visfatin promoter construct contains AP-1, HIF-1α, and Stat-4 binding sites. As shown in Figure 6B and 6C, transient transfection experiment in human CAECs using this reporter gene revealed that HBO for 6 h significantly induced visfatin promoter activation. This result indicates that visfatin expression is induced at transcriptional level by HBO. When the AP-1 binding sites were mutated, the increased promoter activity induced by HBO was abolished. Moreover, addition of SP600125 and TNF-α antibody caused an inhibition of transcription. The increased promoter activity induced by exogenous addition of TNF-α was similar to that induced by HBO at 2.5 ATA. These results suggested that AP-1 binding site in the visfatin promoter is essential for the transcriptional regulation by HBO and that HBO regulates visfatin promoter via TNF-α and JNK pathways.


Tumor necrosis factor-α enhances hyperbaric oxygen-induced visfatin expression via JNK pathway in human coronary arterial endothelial cells.

Wang BW, Lin CM, Wu GJ, Shyu KG - J. Biomed. Sci. (2011)

HBO increases AP-1-binding activity and visfatin promoter activity. A, Representative EMSA showing protein binding to the AP-1 oligonucleotide in nuclear extracts of human CAECs after HBO treatment in the presence or absence of inhibitors and TNF-α antibody. Similar results were found in another two independent experiments. Cold oligo means unlabeled AP-1 oligonucleotides. B, Constructs of visfatin promoter gene. Positive +1 demonstrates the initiation site for the visfatin transcription. Mutant visfatin promoter indicates mutation of AP-1 binding sites in the visfatin promoter as indicated. C, Quantitative analysis of visfatin promoter activity. The luciferase activity in cell lysates was measured and was normalized with renilla activity (n = 4 per group). *P < 0.001 vs. control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3113732&req=5

Figure 6: HBO increases AP-1-binding activity and visfatin promoter activity. A, Representative EMSA showing protein binding to the AP-1 oligonucleotide in nuclear extracts of human CAECs after HBO treatment in the presence or absence of inhibitors and TNF-α antibody. Similar results were found in another two independent experiments. Cold oligo means unlabeled AP-1 oligonucleotides. B, Constructs of visfatin promoter gene. Positive +1 demonstrates the initiation site for the visfatin transcription. Mutant visfatin promoter indicates mutation of AP-1 binding sites in the visfatin promoter as indicated. C, Quantitative analysis of visfatin promoter activity. The luciferase activity in cell lysates was measured and was normalized with renilla activity (n = 4 per group). *P < 0.001 vs. control.
Mentions: Treatment of HBO for 2 h to 6 h significantly increased the DNA-protein binding activity of AP-1 (Figure 6A). An excess of unlabeled AP-1 oligonucleotide competed with the probe for binding AP-1 protein, whereas an oligonucleotide containing a 2-bp substitution in the AP-1 binding site did not compete for binding. Addition of SP600125 and TNF-α monoclonal antibody 30 min before HBO stimulation abolished the DNA-protein binding activity induced by HBO. Exogenous addition of TNF-α significantly increased the DNA-protein binding activity. To study whether the visfatin expression induced by HBO is regulated at the transcriptional level, we cloned the promoter region of human visfatin (-889~+16), and constructed a luciferase reporter plasmid (pGL3-Luc). The visfatin promoter construct contains AP-1, HIF-1α, and Stat-4 binding sites. As shown in Figure 6B and 6C, transient transfection experiment in human CAECs using this reporter gene revealed that HBO for 6 h significantly induced visfatin promoter activation. This result indicates that visfatin expression is induced at transcriptional level by HBO. When the AP-1 binding sites were mutated, the increased promoter activity induced by HBO was abolished. Moreover, addition of SP600125 and TNF-α antibody caused an inhibition of transcription. The increased promoter activity induced by exogenous addition of TNF-α was similar to that induced by HBO at 2.5 ATA. These results suggested that AP-1 binding site in the visfatin promoter is essential for the transcriptional regulation by HBO and that HBO regulates visfatin promoter via TNF-α and JNK pathways.

Bottom Line: Visfatin, a adipocytokine with insulin-mimetic effect, plays a role in endothelial angiogenesis.However, the molecular mechanism of beneficial effects of HBO is poorly understood.HBO significantly increased secretion of TNF-α from cultured human CAECs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Cardiology, Shin Kong Wu Ho-Su Memorial Hospital, and School of Medicine, Fu-Jen Catholic University, New Taipei City, Taipei, Taiwan.

ABSTRACT

Background: Visfatin, a adipocytokine with insulin-mimetic effect, plays a role in endothelial angiogenesis. Hyperbaric oxygen (HBO) has been used in medical practice. However, the molecular mechanism of beneficial effects of HBO is poorly understood. We sought to investigate the cellular and molecular mechanisms of regulation of visfatin by HBO in human coronary arterial endothelial cells (CAECs).

Methods: Human CAECs were exposed to 2.5 atmosphere absolute (ATA) of oxygen in a hyperbaric chamber. Western blot, real-time polymerase chain reaction, and promoter activity assay were performed. In vitro glucose uptake and tube formation was detected.

Results: Visfatin protein (2.55-fold) and mRNA (2.53-fold) expression were significantly increased after exposure to 2.5 ATA HBO for 4 to 6 h. Addition of SP600125 and JNK siRNA 30 min before HBO inhibited the induction of visfatin protein. HBO also significantly increased DNA-protein binding activity of AP-1 and visfatin promoter activity. Addition of SP600125 and TNF-α monoclonal antibody 30 min before HBO abolished the DNA-protein binding activity and visfatin promoter activity induced by HBO. HBO significantly increased secretion of TNF-α from cultured human CAECs. Exogenous addition of TNF-α significantly increased visfatin protein expression while TNF-α antibody and TNF-α receptor antibody blocked the induction of visfatin protein expression induced by HBO. HBO increased glucose uptake in human CAECs as HBO and visfatin siRNA and TNF-α antibody attenuated the glucose uptake induced by HBO. HBO significantly increased the tube formation of human CAECs while visfatin siRNA, TNF-α antibody inhibited the tube formation induced by HBO.

Conclusions: HBO activates visfatin expression in cultured human CAECs. HBO-induced visfatin is mediated by TNF-α and at least in part through JNK pathway.

Show MeSH
Related in: MedlinePlus