New vessel formation is critical for solid tumor growth and it is primarily stimulated by the most potent angiogenic factor vascular endothelial growth factor (VEGF or VEGF-A165). VEGF promotes endothelial cell proliferation by initiating signaling cascades to increase gene transcription. Recent works showed that VEGF potently and rapidly induces expression of orphan nuclear receptor Nurr1 in endothelial cells. However, the signaling pathway for VEGF-induced Nurr1 expression and its role in VEGF-induced endothelial cell proliferation and angiogenic response have not been examined. In our study, we first show that VEGF significantly induces expression of Nurr1 mRNA, protein and its promoter activity in cultured endothelial cells. Furthermore, the promoter analysis shows that deletion of the putative cAMP-responsive element binding protein (CREB) site in the proximal region of the promoter markedly reduces VEGF-induced promoter activity whereas deletion of the upstream NF-κB site has moderate effect. Transfection of a dominant negative CREB mutant (K-CREB) or mutation of this putative CREB site in the Nurr1 promoter attenuates VEGF-induced Nurr1 expression. VEGF also stimulates the binding of nuclear CREB protein to its site in the Nurr1 promoter in vitro and in vivo. Moreover, using pharmacological inhibitors and molecular approaches, we show that VEGF-induced CREB activation is largely mediated by protein kinase C-dependent protein kinase D activation. Finally, our data indicate that knockdown of endogenous Nurr1 expression attenuates VEGF-induced endothelial cell proliferation, migration and in vivo matrigel angiogenesis, suggesting its potential importance in mediating VEGF-induced tumor angiogenesis.
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