Abstract 3663: Git1 Is A Novel Mediator Of Notch1 Signaling Via Vegf
Notch1 is essential for angiogenisis and embryonic development. Upon ligand binding, Notch1 is proteolytically cleaved by γ-secretase to generate the Notch intracellular domain (N1-ICD). N1-ICD translocates to the nucleus to stimulate target gene expression. G-protein-coupled receptor-kinase-2 interacting protein 1 (GIT1) plays an important role in postnatal lung development by regulating the PLCgamma-ERK1/2 pathway. Recently it was shown that ERK1/2 is a negative regulator of gamma- secretase activity. Therefore, our hypothesis is that GIT1 deficiency should activate Notch1 signaling by inhibiting PLCgamma-ERK1/2 activation. In HUVECs, stimulation by VEGF caused Notch1 cleavage and N1-ICD formation. In ECs treated with GIT1 siRNA cleavage of Notch1 by VEGF was dramatically increased. A similar effect was observed in the presence of U73122 (PLCgamma inhibitor) or PD98059 (MEK1 inhibitor). Furthermore, knockdown of GIT1 resulted in a time dependent increase in Notch1 target genes Hey1 and Delta like-4 (Dll4) expression by VEGF stimulation. The Notch signaling pathway is involved in a feedback loop with VEGF, where Notch lies downstream of VEGF, and activation of Notch signaling can downregulate expression of the VEGF receptor. Therefore we assessed the effect of GIT1 on VEGF receptor expression. VEGFR1 expression was significantly induced by VEGF after knockdown of GIT1, whereas VEGFR3 expression decreased, and VEGFR2 did not change. In vivo, the target gene expression was also examined in lungs of GIT1 WT and KO mice at postnatal day 5. Dll4 and VEGFR1 mRNA expression were significantly increased in lungs of KO mice, while VEGFR3 mRNA expression decreased compared to GIT1 WT mice (P<0.05, n=3). In conclusion, GIT1 regulates Notch1 signaling by a PLCgamma-ERK1/2 pathway that postnatally turns off VEGF signaling by decreasing expression of VEGFR3 and increasing VEGFR1. These data establish GIT1 as a novel mediator of angiogenesis in the lung that modulates Notch and VEGF signaling.