Abstract 5561: Id1 Regulates Angiogenesis By Interactions With Notch And PKA Signaling
Id1, a helix-loop-helix transcriptional factor, has emerged as a potent contributor of angiogenesis. We have previously reported that Id1 confers in vivo angiogenic property on human vascular endothelial cells (ECs) and that protein kinase A (PKA) regulates Crm1/exportin-dependent nucleo-cytoplasmic localization of Id1 possibly via the phosphorylation of Ser-5 during in vitro angiogenic process on Matrigel. However, the downstream targets of Id1 and its interaction with other signaling pathways involved in angiogenesis are grossly unclear. We here report that a crosstalk among Id1, Notch and PKA might control angiogenesis. In immunocytochemical analysis, we observed that ECs with an Id1-positive nucleus exhibited periodically-patterned distribution in sprouting capillary-like structures from murine aortic explant in type-1 collagen gel under VEGF stimulation. Because this spatial expression pattern was reminiscent of Notch activation during somitogenesis, we then examined expression of Hey1 and Hey2 (Gridlock), direct downstream targets of Notch. Interestingly, we observed that ECs expressing Hey2 but not Hey1 were dotted in the sprouting capillary-like structures, and they appeared to be distributed reciprocally against ECs expressing Id1. These spatial expression patterns lead us to speculate that Id1 and Notch signaling may constitute a regulatory circuit via negative feedback. This possibility was tested by RNAi-induced gene knockdown in cultured human vascular ECs. Expectedly, siRNA-induced Id1-knockdown strikingly enhanced Notch-dependent upregulation of Hey2. Id1 also upregulated the expression of other Notch-downstream genes in a Hey2-dependent manner. Furthermore, PKA stimulation by dbcAMP completely suppressed the Notch-dependent upregulation of Hey2 in both Id1-dependent and -independent manner. These results suggest a crosstalk between the Notch/Hey2 pathway and Id1, the activity of which is modulated by PKA partly through its nucleocytoplasmic shuttling. We propose that the periodic Id1/Notch signaling circuit may be involved in the control of angiogenesis leading to proper branching morphology.