Abstract
Recent studies have determined that mTOR mediates the activation of the protein kinase Akt in several cell types, but little is known about the association between mTOR and Akt in vascular endothelial cells. Furthermore, the functional significance of mTOR/Akt signaling has not been characterized in the endothelium. In these studies we treated endothelial cells with the mTOR inhibitor rapamycin, and we found that it decreases Akt phosphorylation and activity, as determined by phosphorylation of its substrate glycogen synthase kinase-3. This effect of rapamycin on Akt phosphorylation could not be demonstrated in endothelial cells transfected with a rapamycin-resistant mTOR construct. Also, in the presence of rapamycin, vascular endothelial growth factor, tumor necrosis factor, and insulin failed to phosphorylate Akt, further indicating that mTOR regulates Akt activation in endothelial cells. The activation of Akt is well established to mediate pro-survival signals. In part this is mediated via the phosphorylation and inactivation of the pro-apoptotic Akt substrates Foxo1 and Foxo3a. We find that rapamycin totally blocks vascular endothelial growth factor and Akt-inducible phosophorylation of these transcription factors in endothelial cells. Furthermore, inhibition of Akt activity by rapamycin increased the number of endothelial cells undergoing apoptosis after serum withdrawal as well as after stimulation by vascular endothelial growth factor or tumor necrosis factor. Taken together these observations demonstrate first, that mTOR regulates the phosphorylation and activation of Akt in endothelial cells and, second, that a major effect of mTOR inhibition in endothelial cells is to suppress Akt-inducible pro-survival signals.
Highlights
Upon activation Akt mediates pro-survival and anti-apoptosis in part via the phosphorylation and inhibition of the Bcl-2 homolog BAD, the phosphorylation and inactivation of the FoxO subfamily of forkhead transcription factors, and the transcriptional co-activator Yes-associated protein [11,12,13]
As expected, this brief expoadded to the upper chamber of the Transwell, and after 3 h the sure to rapamycin (20 – 60 min) inhibited mTORC1 and filters were fixed in 2% paraformaldehyde and stained in 0.5% resulted in hypophosphorylation of 4E-BP1 in endothelial cells (EC) (Fig. 1C)
We found that rapamycin potentiated serum withdrawal-mediated apoptosis and attenuated the protective effects of cytokine and growth factor-inducible responses in EC
Summary
Upon activation Akt mediates pro-survival and anti-apoptosis in part via the phosphorylation and inhibition of the Bcl-2 homolog BAD, the phosphorylation and inactivation of the FoxO subfamily of forkhead transcription factors, and the transcriptional co-activator Yes-associated protein [11,12,13]. MTOR Function in Vascular Endothelial Cells ribosome biogenesis, and other growth and proliferation events [22, 23]. Another mTOR complex consisting of mTOR, rictor, Sin, and mLST8 (called mTORC2) has been found to regulate Akt activity and actin polymerization [8, 10, 24, 25]. Depletion of available intracellular mTOR by rapamycin may biologically result in an inhibition of mTORC2 and, inhibition of phosphorylation and activation of Akt. In this study we have evaluated mTOR-Akt interactions and signaling pathways in EC using rapamycin to regulate mTOR activity. We find that the ability of rapamycin to inhibit the migration and proliferation of EC is independent of its effects on mTORC2 activity
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