Inactivation of Foxo3a and Subsequent Downregulation of PGC-1α Mediate Nitric Oxide-Induced Endothelial Cell Migration

Abstract

In damaged or proliferating endothelium, production of nitric oxide (NO) from endothelial nitric oxide synthase (eNOS) is associated with elevated levels of reactive oxygen species (ROS), which are necessary for endothelial migration. We aimed to elucidate the mechanism that mediates NO induction of endothelial migration. NO downregulates expression of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), which positively modulates several genes involved in ROS detoxification. We tested whether NO-induced cell migration requires PGC-1α downregulation and investigated the regulatory pathway involved. PGC-1α negatively regulated NO-dependent endothelial cell migration in vitro, and inactivation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway, which is activated by NO, reduced NO-mediated downregulation of PGC-1α. Expression of constitutively active Foxo3a, a target for Akt-mediated inactivation, reduced NO-dependent PGC-1α downregulation. Foxo3a is also a direct transcriptional regulator of PGC-1α, and we found that a functional FoxO binding site in the PGC-1α promoter is also a NO response element. These results show that NO-mediated downregulation of PGC-1α is necessary for NO-induced endothelial migration and that NO/protein kinase G (PKG)-dependent downregulation of PGC-1α and the ROS detoxification system in endothelial cells are mediated by the PI3K/Akt signaling pathway and subsequent inactivation of the FoxO transcription factor Foxo3a.

This work was supported by the Spanish Ministry of Science and Innovation (grants SAF2006-01619 and SAF2009-07599 to M.M., grant CSD 2007-00020 to M.M. and S.L., and grants SAF2006-02410 and SAF2009-07520 to S.L.), the Spanish Society of Nephrology (grant-in-aid to S.L.), a CNIC-Bancaja predoctoral fellowship to I.V., and an Instituto de Salud Carlos III (ISCIII) contrato de apoyo a la investigación to F.M.-G. The CNIC is supported by the Spanish Ministry of Science and Innovation and the Pro-CNIC Foundation.

Editorial support was provided by S. Bartlett. The FoxO reporter pGL3 construct (3×IRE-luc) was a gift from L. del Peso. The Foxo3a expression vector and adenovirus were gifts from K. Walsh. The PGC-1α^−/− mice were a gift from B. Spiegelman. We thank Elvira Arza and Raquel Nieto for technical support; Enrique Samper, Mariano Redondo, and Alicia Garcia Arroyo for technical advice; and Ramón Bartroons and Eva Cano for careful reading of the manuscript.