In this study, we have revealed the crucial function of PDK1 in endothelial cells and cardiovascular development through activation of Akt and Snail. Loss of PDK1 in endothelial cells caused impaired vascular integrity, remodeling, and development and embryonic lethality at around E11.5. In the heart, endothelial deletion of PDK1 affected endocardiac development, including EMT in the AVC and valve formation. As a result of poor development of the endothelium, the structure of the trabeculae and myocardium was disrupted. Thus, our study has demonstrated that PDK1 plays an indispensable role in the development of the cardiovascular system.
VEGF is essential for endothelial proliferation, survival, and migration (1
). After binding to its receptor of VEGFR, VEGF activates two essential intracellular signaling pathways of MAPK and PI3K-PDK1-Akt. Similarly, both angiopoietin 1 (Ang1) and its receptor, Tie2, are required for vascular remodeling and vessel integrity. Recently, it was reported that Akt is the major downstream effector of Ang1-Tie2 signaling (18
). Therefore, the developmental and functional defects in the absence of PDK1
in endothelial cells could be a consequence resulting from the disruption of VEGF and Ang1 signaling. This is consistent with a previous report using PDK1
KO ES cells to study cell migration (34
). It was shown that PDK1 plays an essential role in regulating endothelial cell migration in response to VEGF-A stimulation (34
Recently, it was shown that PI3K
KO mice displayed defects in angiogenesis and vascular integrity (19
). In these mice, the activation of Akt was impaired, which is the possible cause for vascular abnormalities (19
). Previously, we studied the Akt1
double knockout (DKO) mice (41
). These mice were embryonically lethal at around E12 and displayed developmental defects in multiple organs and tissues, including the cardiovascular system (43
). Endothelium-specific PDK1
deletion in mice also caused embryonic mortality at around E11.5 and cardiovascular defects. These similarities between the endothelial PDK1
deletion and Akt1
DKO mice suggest that, first, Akt proteins are the major downstream players of PI3K-PDK1 signaling among the AGC kinases in the endothelium and, second, the phenotype in Akt 1
DKO mice could be attributed to disrupted endothelial development and function. Data presented here support this idea because delivery of active Akt to the AVC explant of PDK1
-deficient mice rescued EMT defects, and enhancement of Akt activation by PTEN
deletion resulted in normal AVC development and survival of PDK1
-deficient mice at approximately E12. In the future, it will be of great interest to study whether overexpression of Akt could rescue the phenotype of endothelial PDK1
deletion mice. However, this might be a big challenge as transgenic mice with Akt overexpression in endothelial cells were embryonically lethal and showed excessive aberrant vasculature (33
). On the other hand, this suggests that balanced and refined regulation of PDK1-Akt signaling is crucial for cardiovascular development, as either a higher intensity (in the case of Akt transgenic mice) or less activity (in the case of PDK1
KO mice) of Akt signaling gives rise to developmental abnormalities.
In this study, we have demonstrated that deletion of PDK1
in endothelial cells had nearly no impact on cell proliferation. Instead, we found that PDK1 played a role in promoting cell survival, as loss of PDK1 caused endothelial apoptosis. One of the important functions of Akt is antiapoptosis and prosurvival (12
). Therefore, the increased endothelial cell death might be due to inactivation of Akt.
Our study was consistent with a previous report showing that Akt activation promoted Snail expression (20
). We found that Snail was ubiquitously expressed in the AVC of the control but was absent from a large amount of cells in the PDK1
deletion AVC. In addition, its cellular localization was changed from the nucleus in the control to the cytosol in PDK1
deletion cells. As Snail is a transcription factor and a master gene for EMT that activates mesenchymal gene expression while suppressing the transcription of adhesive molecules of epithelial cells, the changes of Snail expression patterns may reflect the deficit of EMT in the PDK1
deletion AVC. GSK3β was reported to phosphorylate Snail, resulting in Snail translocation to the cytosol and degradation (45
). Because GSK3β has been the best-characterized substrate of the Akt kinase and the phosphorylation of GSK3β by Akt suppresses GSK3β activity, the distribution and cellular localization of Snail in PDK1
deletion cells could be due to loss of activity of PDK1-Akt signaling. Our results have demonstrated that Snail S6A (constitutively active) rescues EMT defects in PDK1
deficiency and confirmed this hypothesis.
Another intriguing finding of this study is that the loss of PTEN can rescue the phenotype of PDK1 deficiency. Mechanistically, PTEN loss in the absence of PDK1 may compensate Akt activity through enhancement of Akt S473 phosphorylation. We found a dramatic increase of Akt S473 phosphorylation in cardiomyocyte-specific PTEN/PDK1 double deletion mice (data not shown). Hence, our study suggests that Akt S473 phosphorylation might be PI3K dependent.
The data presented here could be promising for therapeutic applications. In the future, it will be helpful to modulate Akt signaling to improve cardiovascular complications in human patients.