| Budget Amount *help |
¥3,910,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥510,000)
Fiscal Year 2007: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
Fiscal Year 2006: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Research Abstract |
Helix-loop-Helix factors (HLH) Ids lack a basic DNA binding domain and primarily acts as endogenous inhibitors of basic HLH transcriptional factors. In the family, Id1 and Id3 have been implicated in embryonic and postnatal angiogenesis. We have also reported that Id1-overexpression enhanced in vitro and in vivo angiogenesis. However, the molecular mechanisms underlying the involvement of Id in angiogenesis are grossly unclear. To address this issue, we have investigated the behavior and molecular interactions of Id1 in cultured vascular endothelial cells (ECs). We first found that Id1 was localized in the nucleus of HUVECs cultured on uncoated plates, whereas translocated to the cytoplasm on Matrigel along with capillary-like morphogenesis. Treatment with the Crm1/exportin inhibitor leptomycin B and mutagenesis analysis using GFP revealed the Crm1/exportin-dependent nuclear export of Id1. This nuclear export of Id1 was inhibited by protein kinase A (PKA) activator dbcAMP, but was prom
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oted by PKA inhibitor H-89. Mutagenesis analysis of Id1 showed that the phosphorylation of Ser-5 might mediate the effect of PKA. Corresponding to the dynamic behavior of Id1, cells with an Id1-positive nucleus exhibited periodically-patterned distribution in sprouting capillary-like structures from murine aortic explant in type I collagen. Because this spatial pattern was reminiscent of Notch activation in developing vessels, we then examined whether Id1 might interact with Notch signaling. Interestingly, siRNA-induced Id1-knockdown strikingly enhanced Notch-dependent upregulation of Hey2 (Gridlock), a basic HLH factor known as a downstream target of Notch, in cultured HUVECs. Furthermore, Id1 also enhanced the upregulation of other target genes of Notch in a Hey2-dependent manner. These results suggest a crosstalk between Notch/Hey and Id1, the activity of which may be regulated by PKA at the level of subcellular localization. Considering the possible requirement of Hey2 (Gridlock) for arterial EC differentiation, this crosstalk may be involved in the regulation of not only angiogenic process but also arterio-venous characterization in the Ecs. Less
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