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

  • Park, Jung-Hyun (Department of Molecular Medicine, Ewha Womans University College of Medicine) ;
  • Cho, Du-Hyong (Department of Pharmacology, Yeungnam University College of Medicine) ;
  • Hwang, Yun-Jin (Department of Pharmacology, Yeungnam University College of Medicine) ;
  • Lee, Jee Young (Department of Molecular Medicine, Ewha Womans University College of Medicine) ;
  • Lee, Hyeon-Ju (Department of Molecular Medicine, Ewha Womans University College of Medicine) ;
  • Jo, Inho (Department of Molecular Medicine, Ewha Womans University College of Medicine)
  • Received : 2020.01.16
  • Accepted : 2020.04.06
  • Published : 2020.11.01


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 dominant-negative 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.



  1. Bae, S. W., Kim, H. S., Cha, Y. N., Park, Y. S., Jo, S. A. and Jo, I. (2003) Rapid increase in endothelial nitric oxide production by bradykinin is mediated by protein kinase A signaling pathway. Biochem. Biophys. Res. Commun. 306, 981-987.
  2. Brauweiler, A., Garl, P., Franklin, A. A., Giebler, H. A. and Nyborg, J. K. (1995) A molecular mechanism for human T-cell leukemia virus latency and Tax transactivation. J. Biol. Chem. 270, 12814-12822.
  3. Bruckdorfer, R. (2005) The basics about nitric oxide. Mol. Aspects Med. 26, 3-31.
  4. Carrassa, L. and Damia, G. (2017) DNA damage response inhibitors: mechanisms and potential applications in cancer therapy. Cancer Treat. Rev. 60, 139-151.
  5. Cho, D. H., Choi, Y. J., Jo, S. A. and Jo, I. (2004) Nitric oxide production and regulation of endothelial nitric-oxide synthase phosphorylation by prolonged treatment with troglitazone: evidence for involvement of peroxisome proliferator-activated receptor (PPAR) gamma-dependent and PPARgamma-independent signaling pathways. J. Biol. Chem. 279, 2499-2506.
  6. Cho, D. H., Park, J. H., Lee, E. J., Won, K. J., Lee, S. H., Kim, Y. H., Hwang, S., Kwon, K. J., Shin, C. Y., Song, K. H., Jo, I. and Han, S. H. (2014) Valproic acid increases NO production via the SH-PTP1-CDK5-eNOS-Ser(116) signaling cascade in endothelial cells and mice. Free Radic. Biol. Med. 76, 96-106.
  7. Ciccia, A. and Elledge, S. J. (2010) The DNA damage response: making it safe to play with knives. Mol. Cell 40, 179-204.
  8. Du, K. and Montminy, M. (1998) CREB is a regulatory target for the protein kinase Akt/PKB. J. Biol. Chem. 273, 32377-32379.
  9. Fleming, I. (2010) Molecular mechanisms underlying the activation of eNOS. Pflugers Arch. 459, 793-806.
  10. Forstermann, U., Boissel, J. P. and Kleinert, H. (1998) Expressional control of the 'constitutive' isoforms of nitric oxide synthase (NOS I and NOS III). FASEB J. 12, 773-790.
  11. Forstermann, U. and Sessa, W. C. (2012) Nitric oxide synthases: regulation and function. Eur. Heart J. 33, 829-837, 837a-837d.
  12. Forstermann, U., Xia, N. and Li, H. (2017) Roles of vascular oxidative stress and nitric oxide in the pathogenesis of atherosclerosis. Circ. Res. 120, 713-735.
  13. Gonzalez, G. A. and Montminy, M. R. (1989) Cyclic AMP stimulates somatostatin gene transcription by phosphorylation of CREB at serine 133. Cell 59, 675-680.
  14. Heiss, E. H. and Dirsch, V. M. (2014) Regulation of eNOS enzyme activity by posttranslational modification. Curr. Pharm. Des. 20, 3503-3513.
  15. Hwang, S., Lee, D. H., Lee, I. K., Park, Y. M. and Jo, I. (2014) Farinfrared radiation inhibits proliferation, migration, and angiogenesis of human umbilical vein endothelial cells by suppressing secretory clusterin levels. Cancer Lett. 346, 74-83.
  16. Inoue, N., Venema, R. C., Sayegh, H. S., Ohara, Y., Murphy, T. J. and Harrison, D. G. (1995) Molecular regulation of the bovine endothelial cell nitric oxide synthase by transforming growth factor-beta 1. Arterioscler. Thromb. Vasc. Biol. 15, 1255-1261.
  17. Kim, H. P., Lee, J. Y., Jeong, J. K., Bae, S. W., Lee, H. K. and Jo, I. (1999) Nongenomic stimulation of nitric oxide release by estrogen is mediated by estrogen receptor alpha localized in caveolae. Biochem. Biophys. Res. Commun. 263, 257-262.
  18. Kleinert, H., Wallerath, T., Euchenhofer, C., Ihrig-Biedert, I., Li, H. and Forstermann, U. (1998) Estrogens increase transcription of the human endothelial NO synthase gene: analysis of the transcription factors involved. Hypertension 31, 582-588.
  19. Kou, R., Greif, D. and Michel, T. (2002) Dephosphorylation of endothelial nitric-oxide synthase by vascular endothelial growth factor. Implications for the vascular responses to cyclosporin A. J. Biol. Chem. 277, 29669-29673.
  20. Mazouzi, A., Stukalov, A., Muller, A. C., Chen, D., Wiedner, M., Prochazkova, J., Chiang, S. C., Schuster, M., Breitwieser, F. P., Pichlmair, A., El-Khamisy, S. F., Bock, C., Kralovics, R., Colinge, J., Bennett, K. L. and Loizou, J. I. (2016) A comprehensive analysis of the dynamic response to aphidicolin-mediated replication stress uncovers targets for ATM and ATMIN. Cell Rep. 15, 893-908.
  21. Min, J., Jin, Y. M., Moon, J. S., Sung, M. S., Jo, S. A. and Jo, I. (2006) Hypoxia-induced endothelial NO synthase gene transcriptional activation is mediated through the tax-responsive element in endothelial cells. Hypertension 47, 1189-1196.
  22. Nagane, M., Kuppusamy, M. L., An, J., Mast, J. M., Gogna, R., Yasui, H., Yamamori, T., Inanami, O. and Kuppusamy, P. (2018) Ataxiatelangiectasia mutated (ATM) kinase regulates eNOS expression and modulates radiosensitivity in endothelial cells exposed to ionizing radiation. Radiat. Res. 189, 519-528.
  23. Nguyen, G. H., Dexheimer, T. S., Rosenthal, A. S., Chu, W. K., Singh, D. K., Mosedale, G., Bachrati, C. Z., Schultz, L., Sakurai, M., Savitsky, P., Abu, M., McHugh, P. J., Bohr, V. A., Harris, C. C., Jadhav, A., Gileadi, O., Maloney, D. J., Simeonov, A. and Hickson, I. D. (2013) A small molecule inhibitor of the BLM helicase modulates chromosome stability in human cells. Chem. Biol. 20, 55-62.
  24. Park, J. H., Kim, W. S., Kim, J. Y., Park, M. H., Nam, J. H., Yun, C. W., Kwon, Y. G. and Jo, I. (2011) Chk1 and Hsp90 cooperatively regulate phosphorylation of endothelial nitric oxide synthase at serine 1179. Free Radic. Biol. Med. 51, 2217-2226.
  25. Poehlmann, A. and Roessner, A. (2010) Importance of DNA damage checkpoints in the pathogenesis of human cancers. Pathol. Res. Pract. 206, 591-601.
  26. Searles, C. D. (2006) Transcriptional and posttranscriptional regulation of endothelial nitric oxide synthase expression. Am. J. Physiol. Cell Physiol. 291, C803-C816.
  27. Seo, J., Cho, D. H., Lee, H. J., Sung, M. S., Lee, J. Y., Won, K. J., Park, J. H. and Jo, I. (2016) Citron Rho-interacting kinase mediates arsenite-induced decrease in endothelial nitric oxide synthase activity by increasing phosphorylation at threonine 497: mechanism underlying arsenite-induced vascular dysfunction. Free Radic. Biol. Med. 90, 133-144.
  28. Seo, J., Lee, J. Y., Sung, M. S., Byun, C. J., Cho, D. H., Lee, H. J., Park, J. H., Cho, H. S., Cho, S. J. and Jo, I. (2014) Arsenite acutely decreases nitric oxide production via the ROS-protein phosphatase 1-endothelial nitric oxide synthase-Thr(497) signaling cascade. Biomol. Ther. (Seoul) 22, 510-518.
  29. Sessa, C., Zucchetti, M., Davoli, E., Califano, R., Cavalli, F., Frustaci, S., Gumbrell, L., Sulkes, A., Winograd, B. and D'Incalci, M. (1991) Phase I and clinical pharmacological evaluation of aphidicolin glycinate. J. Natl. Cancer Inst. 83, 1160-1164.
  30. Sessa, W. C., Pritchard, K., Seyedi, N., Wang, J. and Hintze, T. H. (1994) Chronic exercise in dogs increases coronary vascular nitric oxide production and endothelial cell nitric oxide synthase gene expression. Circ. Res. 74, 349-353.
  31. Shaywitz, A. J. and Greenberg, M. E. (1999) CREB: a stimulus-induced transcription factor activated by a diverse array of extracellular signals. Annu. Rev. Biochem. 68, 821-861.
  32. Shen, W., Zhang, X., Zhao, G., Wolin, M. S., Sessa, W. and Hintze, T. H. (1995) Nitric oxide production and NO synthase gene expression contribute to vascular regulation during exercise. Med. Sci. Sports Exerc. 27, 1125-1134.
  33. Sun, D., Huang, A., Koller, A. and Kaley, G. (2002) Decreased arteriolar sensitivity to shear stress in adult rats is reversed by chronic exercise activity. Microcirculation 9, 91-97.
  34. Tanaka, T., Huang, X., Halicka, H. D., Zhao, H., Traganos, F., Albino, A. P., Dai, W. and Darzynkiewicz, Z. (2007) Cytometry of ATM activation and histone H2AX phosphorylation to estimate extent of DNA damage induced by exogenous agents. Cytometry A 71, 648-661.
  35. Vesela, E., Chroma, K., Turi, Z. and Mistrik, M. (2017) Common chemical inductors of replication stress: focus on cell-based studies. Biomolecules 7, E19.
  36. Viniegra, J. G., Martinez, N., Modirassari, P., Hernandez Losa, J., Parada Cobo, C., Sanchez-Arevalo Lobo, V. J., Aceves Luquero, C. I., Alvarez-Vallina, L., Ramon y Cajal, S., Rojas, J. M. and Sanchez-Prieto, R. (2005) Full activation of PKB/Akt in response to insulin or ionizing radiation is mediated through ATM. J. Biol. Chem. 280, 4029-4036.
  37. Wright, G. E., Hubscher, U., Khan, N. N., Focher, F. and Verri, A. (1994) Inhibitor analysis of calf thymus DNA polymerases alpha, delta and epsilon. FEBS Lett. 341, 128-130.
  38. Yang, Y. M., Huang, A., Kaley, G. and Sun, D. (2009) eNOS uncoupling and endothelial dysfunction in aged vessels. Am. J. Physiol. Heart Circ. Physiol. 297, H1829-H1836.
  39. Yin, M. J. and Gaynor, R. B. (1996) Complex formation between CREB and Tax enhances the binding affinity of CREB for the human T-cell leukemia virus type 1 21-base-pair repeats. Mol. Cell. Biol. 16, 3156-3168.
  40. Zhang, Y., Lee, T. S., Kolb, E. M., Sun, K., Lu, X., Sladek, F. M., Kassab, G. S., Garland, T., Jr. and Shyy, J. Y. (2006) AMP-activated protein kinase is involved in endothelial NO synthase activation in response to shear stress. Arterioscler. Thromb. Vasc. Biol. 26, 1281-1287.

Cited by

  1. Rational Application of β-Hydroxybutyrate Attenuates Ischemic Stroke by Suppressing Oxidative Stress and Mitochondrial-Dependent Apoptosis via Activation of the Erk/CREB/eNOS Pathway vol.12, pp.7, 2021,