Abstract 820: A Novel Regulatory Mechanism of Angiogenesis by Nucleo-Cytoplasmic Shuttling of Id1, a Helix-Loop-Helix Transcriptional Factor
Recently Id1, a helix-loop-helix transcriptional factor, has emerged as a potent contributor of angiogenesis. In this context, we have previously reported that Id1 confers in vivo angiogenic property on human vascular endothelial cells (HUVECs) via angiopoietin-1 upregulation (Circulation 112:2840 –2850, 2005). However, the molecular mechanisms underlying the role of Id1 in angiogenesis remain largely unclear. We here report that nucleo-cytoplsamic shuttling of Id1 may be involved in a process of angiogenesis in vivo and in vitro. In an immunohistochemial analysis using murine embryonic brain, we observed that Id1 was localized both in the nucleus and cytoplasm of endothelial cells (ECs) of developing vessels. We further observed that endogenous Id1 and overexpressed Id1 tagged with GFP was predominantly localized in the cytoplasm of HUVECs during capillary-like morphogenesis in an in vitro angiogenesis assay using Matrigel. Treatment with nuclear export inhibitor Leptomycin B and mutagenesis of the region containing a putative nuclear export sequence revealed the nucleo-cytoplasmic shuttling of Id1 in cultured HUVECs, and further demonstrated that Id1 was exportin-dependently exported into the cytoplasm of HUVECs during capillary-like morphogenesis on Matrigel. In addition, H-89, an inhibitor of protein kinase A (PKA) and MDL-12, 330A, an inhibitor of adenylate cyclase, promoted exportin-dependent nuclear export of Id1 in HUVECs. Finally, mutational analysis revealed that phosphorylation of the N-terminus (Ser-5) by PKA may affect nuclear accumulation of Id1 in HUVECs. Taken together with previous reports showing that PKA activity of ECs is suppressed during vascular morphogenesis, the present findings indicate that Id1 is exportin-dependently exported into the cytoplasm of ECs at least partly via an inhibition of PKA during angiogenesis. We propose that nuclear-cytoplasmic shuttling of Id1 is a novel mechanism regulating angiogenesis and a promising target for vascular regeneration.