Activation of PLCγ1 by receptor tyrosine kinases (RTKs) leads to hydrolysis of phosphoinositides to generate the second messengers inositol 1,4,5-trisphosphate (IP
3) and diacylglycerol (DAG). IP
3 increases intracellular Ca
2+ levels and DAG activates PKCs and other C1 domain-containing proteins.
16 . To test whether activation of PKCs contributes to the tubulogenesis of endothelial cells, we analyzed the ability of TPA (12-
O-tetradecanoylphorbol 13-acetate), an analog of diacylglycerol, to stimulate tube formation of PAE cells. The results showed that treatment of PAE cells with different concentrations of TPA stimulates tube formation of PAE cells, suggesting that activation of PKCs by PLCγ1 affects its ability to mediate tube formation of endothelial cells (
Fig. 2B). To further support the hypothesis that activation of PKCs is important in tube formation by endothelial cells, we attempted to inhibit their activation by a pharmacologic approach with GFX and GÖ6976, potent inhibitors of PKCs. It is known that GFX and GÖ6976 inhibit various isozymes of PKC, including classic PKC (α, βI, βII, and γ), novel PKC (δ, ε, η, and θ), and PKD (protein kinase D). As shown (
Figs. 2C,
2D), both GFX and GÖ6976 inhibited VEGFR-2-mediated tubulogenesis of endothelial cells in a dose-dependent manner. Finally, we decided to inhibit activation of PKCs by a myristoylated PKC peptide inhibitor, which specifically inhibits calcium- and phospholipid-dependent PKCs. This inhibitor is based on the pseudosubstrate region of PKC-α and -β.
17 Treatment of cells with the PKC peptide inhibitor almost completely inhibited the sprouting and tube formation of the PAE cells (
Fig. 2E), further supporting the idea that the PLCγ1/PKC pathway plays a critical role in mediating the angiogenic signaling of VEGF.