Abstract
VEGF is the key modulator of normal vessel generation being also involved in the pathogenesis of angiogenic diseases, such as cancer. Therefore, unraveling its signaling cascades in endothelial cell might provide molecular targets for the development of anti-angiogenic therapies. The present thesis was based on the observation, by cDNA microarrays, that two genes participating in the unfolded protein response (UPR) of ER, HERPUD1 and DNAJB9, were transcriptionally activated by VEGF. The over-expression of the two genes was specific for VEGF not being induced by FGF2, another potent angiogenic factor. Additional screening revealed that VEGF induces not only the HERPUD1 and DNAJB9 genes, but also the majority of the genes regulated by the three UPR branches (IRE1, ATF6 και PERK). The generalized transcriptional activation of UPR genes was not caused by increased load of proteins in the ER lumen due to activation of the secretory pathway by VEGF. This conclusion was based on several exper ...
VEGF is the key modulator of normal vessel generation being also involved in the pathogenesis of angiogenic diseases, such as cancer. Therefore, unraveling its signaling cascades in endothelial cell might provide molecular targets for the development of anti-angiogenic therapies. The present thesis was based on the observation, by cDNA microarrays, that two genes participating in the unfolded protein response (UPR) of ER, HERPUD1 and DNAJB9, were transcriptionally activated by VEGF. The over-expression of the two genes was specific for VEGF not being induced by FGF2, another potent angiogenic factor. Additional screening revealed that VEGF induces not only the HERPUD1 and DNAJB9 genes, but also the majority of the genes regulated by the three UPR branches (IRE1, ATF6 και PERK). The generalized transcriptional activation of UPR genes was not caused by increased load of proteins in the ER lumen due to activation of the secretory pathway by VEGF. This conclusion was based on several experimental data. First, inhibition of protein synthesis by cycloheximide exhibited a minor impact on the transcriptional activation of the HERPUD1 and DNAJB9 genes by VEGF. Secondly, VEGF leads to a rapid (within 15-30 min) but short-term activation of the three branches of UPR (IRE1, ATF6 and PERK), contrary to Tunicamicin (Tm), a N-linked glycosylation inhibitor that leads to ER overload with misfolded proteins, that induces activation of the IRE1, ATF6 and PERK pathways in a delayed and long-term manner. Finally, using a series of specific inhibitors of VEGF pathways and siRNA gene silencing, we have identified that the PLCγ pathway of VEGF was responsible for the activation of the tree UPR branches. The mechanism by which the PLCγ pathway activates IRE1, ATF6 and PERK is still unknown however, our data indicate that is Ca2+-independent. Activation of the three UPR pathways of ER appeared to be critical for the VEGF-induced endothelial cell survival. In particular, silencing of the ATF6 and eIF2a genes reduced the VEGF-induced survival of endothelial cells by 50%. Indeed, activation of the ER-mediated cascades by VEGF was crucial for its anti-apoptotic effect on endothelial cells. VEGF-induced phosphorylation of Akt by the PI3K pathway required concomitantly active PLCγ-ER-ATF6/PERK and PLCγ-IP3-ER-Ca2+-calmodulin-CaMKII cascades. Interestingly, VEGF administration to endothelial cells was associated with undetected levels of the pro-apoptotic protein CHOP despite the transcriptional activation of its gene via the PERK/ATF4 pathway. This is compatible with previous studies reporting that the mRNA and protein of CHOP are unstable being rapidly degraded during mild (adaptive) UPR. Thus, VEGF incorporates the ER-dependent cascades in its signaling machinery to achieve a maximal anti-apoptotic effect on endothelial cells. This study contributes novel knowledge in the signal transduction of VEGF and elucidates unknown aspects of the ER function in cell homeostasis. ER emerges as key component of the signal transducing machinery of VEGF in endothelial cells as the latter activates the three UPR branches of ER through the PLCγ pathway. We named the activation of IRE1, ATF6 and PERK (and their following transcriptional induction) by signaling cascades of extracellular ligands as Extracellular Signaling Response (ESR) of ER to distinguish it from activation due to overloading of ER with unfolded proteins (UPR).
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