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Activation of ATM/Akt/CREB/eNOS Signaling Axis by Aphidicolin Increases NO Production and Vessel Relaxation in Endothelial Cells and Rat Aortas

Biomolecules & Therapeutics 2020년 28권 6호 p.549 ~ 560
박중현, 조두형, Hwang Yun-Jin, 이지영, 이현주, 조인호,
소속 상세정보
박중현 ( Park Jung-Hyun ) - Ewha Womans University School of Medicine Department of Molecular Medicine
조두형 ( Cho Du-Hyong ) - Yeungnam University College of Medicine Department of Pharmacology
 ( Hwang Yun-Jin ) - Yeungnam University College of Medicine Department of Pharmacology
이지영 ( Lee Jee-Young ) - Ewha Womans University School of Medicine Department of Molecular Medicine
이현주 ( Lee Hyeon-Ju ) - Ewha Womans University School of Medicine Department of Molecular Medicine
조인호 ( Jo In-Ho ) - Ewha Womans University School of Medicine Department of Molecular Medicine

Abstract


Although DNA damage responses (DDRs) are reported to be involved in nitric oxide (NO) production in response to genotoxic stresses, the precise mechanism of DDR-mediated NO production has not been fully understood. Using a genotoxic agent aphidicolin, we investigated how DDRs regulate NO production in bovine aortic endothelial cells. Prolonged (over 24 h) treatment with aphidicolin increased NO production and endothelial NO synthase (eNOS) protein expression, which was accompanied by increased eNOS dimer/monomer ratio, tetrahydrobiopterin levels, and eNOS mRNA expression. A promoter assay using 5’-serially deleted eNOS promoters revealed that Tax-responsive element site, located at ?962 to ?873 of the eNOS promoter, was responsible for aphidicolin-stimulated eNOS gene expression. Aphidicolin increased CREB activity and ectopic expression of dominantnegative inhibitor of CREB, A-CREB, repressed the stimulatory effects of aphidicolin on eNOS gene expression and its promoter activity. Co-treatment with LY294002 decreased the aphidicolin-stimulated increase in p-CREB-Ser133 level, eNOS expression, and NO production. Furthermore, ectopic expression of dominant-negative Akt construct attenuated aphidicolin-stimulated NO production. Aphidicolin increased p-ATM-Ser1981 and the knockdown of ATM using siRNA attenuated all stimulatory effects of aphidicolin on p-Akt-Ser473, p-CREB-Ser133, eNOS expression, and NO production. Additionally, these stimulatory effects of aphidicolin were similarly observed in human umbilical vein endothelial cells. Lastly, aphidicolin increased acetylcholine-induced vessel relaxation in rat aortas, which was accompanied by increased p-ATM-Ser1981, p-Akt-Ser473, p-CREB-Ser133, and eNOS expression. In conclusion, our results demonstrate that in response to aphidicolin, activation of ATM/Akt/CREB/eNOS signaling cascade mediates increase of NO production and vessel relaxation in endothelial cells and rat aortas.

키워드

Endothelial nitric oxide synthase; Nitric oxide; Vessel relaxation; Aphidicolin; DNA damage response

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