Aiken J, Roudier E, Ciccone J, Drouin G, Stromberg A, Vojnovic J, Olfert IM, Haas T, Gustafsson T, Grenier G, Birot O. Phosphorylation of murine double minute-2 on Ser166 is downstream of VEGF-A in exercised skeletal muscle and regulates primary endothelial cell migration and FoxO gene expression. FASEB J. 2016 Mar;30(3):1120-34. doi: 10.1096/fj.15-276964. Epub 2015 Nov 17.
In skeletal muscle tissue, the capillary network matches oxygen and nutrient delivery to the metabolic needs of active muscle cells. Angiogenesis, the growth of new capillaries, therefore plays a key role in muscle adaptation to exercise training. One bout of intense exercise is a powerful stimulus for the release of various pro-angiogenic factors, in particular the Vascular Endothelial Growth Factor-A (VEGF). We have recently demonstrated that Murine Double Minute-2 (Mdm2) was indispensable for exercise-induced VEGF production and angiogenesis in rodent skeletal muscle (Roudier et al. 2012). The current publication suggests that the "classical" representation of Mdm2 as an upstream regulator of VEGF-A expression might in fact be much more complex. We show that in response to a single bout of exercise, both VEGF-A and Mdm2 that is phosphorylated on its serine 166 (p-Ser166-Mdm2) are strongly induced in both rodent and human skeletal muscle, and that this response was impaired in VEGF-A myofibre-specific knockout mice. This suggested that VEGF-A may be an essential regulator of Mdm2 phosphorylation in vivo in response to a single bout of exercise. In order to explore this relationship further, using both primary human and rodent endothelial cells, we demonstrate that recombinant VEGF-A protein stimulates p-Ser166-Mdm2. This recombinant VEGF-A stimulation significantly elevated endothelial cell migration, however, this effect was entirely abrogated by the Mdm2 antagonist Nutlin-3a, suggesting that VEGF-driven endothelial cell migration is dependent on Mdm2 activity. To further study the functional impact of p-Ser166-Mdm2 in endothelial cells, primary human endothelial cells were infected with lentiviruses carrying plasmids encoding for either wild-type Mdm2 (WT-Mdm2) or a mimetic of Mdm2 phosphorylation on serine 166 (S166D-Mdm2). Overexpression of S166D-Mdm2 protein greatly enhances endothelial cell migration, indicating that Mdm2 activation (i.e. phosphorylation) promotes the pro-angiogenic activity of endothelial cells. Mdm2 can interact with the transcription factors HIF-1α, p53 and FoxO1 that regulate both pro- and anti-angiogenic molecules. Using co-immunoprecipitation, we show increased Mdm2 protein binding to FoxO1, but not HIF-1α or p53, in the S166D-Mdm2 compared to WT-Mdm2 overexpressing cells. This corresponded to a reduction in the mRNA expression of hallmark anti-angiogenic FoxO1 transcriptional targets (p27, sprouty-2, TSP-1) and an increased VEGF-A to TSP-1 mRNA ratio, thus promoting a pro-angiogenic environment. These findings clearly establish a novel pro-angiogenic role of p-Ser166-Mdm2 in human skeletal muscle and endothelial cells.