Abstract 5587: Id1 Regulates Angiogenesis by Determining the Specification and the Timing of Notch 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 nucleo-cytoplasmic localization of Id1 might control angiogenesis. However, the downstream targets of Id1 and its interaction with other signaling pathways involved in angiogenesis are grossly unknown. To unveil the issue, we first examined the expression pattern of Id1 in ECs during angiogenesis in vitro and in vivo. We observed that Id1 expression levels were more prominent in sprouting ECs as compared with sheet-forming ECs at the proximal end of the sprouts in murine aortic ring assay. In the sprouts, furthermore, Id1 exhibited a mosaic expression pattern, mixed with weakly or no Id1-expressing ECs. We also identified similar expression pattern immunohistochemically in developing vessels of murine embryonic brain. Because the mosaic pattern of Id1 expression was reminiscent of Notch activation pattern in developing vessels, we next verified a possible crosstalk between Id1 and Notch signaling by RNAi-induced gene knockdown in cultured human ECs. Expectedly, siRNA-induced Id1-knockdown strikingly and specifically enhanced Notch-dependent upregulation of Hey2 (Gridlock) and to a less extent Hey1 but not Hes1. Id1-knockdown also upregulated the expression of a Notch-downstream effector Ephrin B2 in a Hey2-dependent manner. We finally examined how Id1 exhibited the mosaic expression pattern in ECs of developing vessels. Interestingly, Western blot analysis (hourly sampled for 12 hours) showed that total protein levels of Id1 appeared to oscillate periodically in cultured human ECs under confluent condition with no evident relation to cell cycle. This finding partially explains the mosaic expression pattern of Id1 during angiogenesis. Collectively, these results suggest a novel crosstalk between Id1 and Notch signaling, where Id1 might regulate the specification and the timing of Notch/Hey2 activation. We propose that the periodic expression of Id1 may be involved in the control of angiogenesis leading to proper branching morphology.