The effects of DNA damage on NO production have not been completely elucidated. Using ultraviolet (UV) irradiation as a DNA-damaging agent, we studied its effect on NO production in bovine aortic endothelial cells (BAEC). UV irradiation acutely increased NO production, the phosphorylation of endothelial NO synthase (eNOS) at serine 1179, and eNOS activity. No alterations in eNOS expression nor phosphorylation at eNOS Thr 497 or eNOS Ser 116 were found. SB218078, a checkpoint kinase 1 (Chk1) inhibitor, inhibited UV-irradiation-stimulated eNOS-Ser 1179 phosphorylation and NO production. Similarly, ectopic expression of small interference RNA for Chk1 or a dominant-negative Chk1 repressed the UV-irradiation stimulatory effect, whereas wild-type Chk1 increased basal eNOS-Ser 1179 phosphorylation. Purified Chk1 directly phosphorylated eNOS Ser 1179 in vitro. Confocal microscopy and coimmunoprecipitation studies revealed a colocalization of eNOS and Chk1. In basal BAEC, heat shock protein 90 (Hsp90) predominantly interacted with Chk1. This interaction, which decreased significantly in response to UV irradiation, was accompanied by increased interaction of Hsp90 with eNOS. The Hsp90 inhibitor geldanamycin attenuated UV-irradiation-stimulated eNOS-Ser 1179 phosphorylation by dissociating Hsp90 from eNOS. UV irradiation and geldanamycin did not alter the interaction between eNOS and Chk1. Overall, this is the first study demonstrating that Chk1 directly phosphorylates eNOS Ser 1179 in response to UV irradiation, which is dependent on Hsp90 interaction.
|Number of pages||10|
|Journal||Free Radical Biology and Medicine|
|State||Published - 15 Dec 2011|
Bibliographical noteFunding Information:
This work was supported by the Korea Research Council for Fundamental Science and Technology through the Basic Research Project managed by the Korea Research Institute of Standards and Science and RP-Grant 2009 from Ewha Womans University.
- Checkpoint kinase
- Endothelial nitric oxide synthase
- Free radicals
- Heat shock protein
- UV irradiation