Biomolecules & Therapeutics  https://doi.org/10.4062/biomolther.2020.007
Activation of ATM/Akt/CREB/eNOS Signaling Axis by Aphidicolin Increases NO Production and Vessel Relaxation in Endothelial Cells and Rat Aortas
Jung-Hyun Park1,†, Du-Hyong Cho2,†, Yun-Jin Hwang2, Jee Young Lee1, Hyeon-Ju Lee1 and Inho Jo1,*
1Department of Molecular Medicine, Ewha Womans University College of Medicine, Seoul 07804,
2Department of Pharmacology, Yeungnam University College of Medicine, Daegu 42415, Republic of Korea
*E-mail: inhojo@ewha.ac.kr
Tel: +82-2-6986-6267, Fax: +82-2-6986-7014
The first two authors contributed equally to this work.
Received: January 16, 2020; Revised: March 26, 2020; Accepted: April 6, 2020; Published online: May 12, 2020.
© The Korean Society of Applied Pharmacology. All rights reserved.

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.
Keywords: Endothelial nitric oxide synthase, Nitric oxide, Vessel relaxation, Aphidicolin, DNA damage response


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