• BMB Reports
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• v.44 no.3
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• pp.199-204
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• 2011

Ephrin signaling is involved in various morphogenetic events, such as axon guidance, hindbrain segmentation, and angiogenesis. We conducted a yeast two-hybrid screen using the intracellular domain (ICD) of EphrinB1 to gain biochemical insight into the function of the EphrinB1 ICD. We identified the transcriptional co-repressor xTLE1/Groucho as an EphrinB1 interacting protein. Whole-mount in situ hybridization of Xenopus embryos confirmed the co-localization of EphrinB1 and a Xenopus counterpart to TLE1, xTLE4, during various stages of development. The EphrinB1/xTLE4 interaction was confirmed by co-immunoprecipitation experiments. Further characterization of the interaction revealed that the carboxy-terminal PDZ binding motif of EphrinB1 and the SP domain of xTLE4 are required for binding. Additionally, phosphorylation of EphrinB1 by a constitutively activated fibroblast growth factor receptor resulted in loss of the interaction, suggesting that the interaction is modulated by tyrosine phosphorylation of the EphrinB1 ICD.

• Molecules and Cells
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• v.40 no.6
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• pp.426-433
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• 2017

Ephrin-A5 has been implicated in the regulation of brain morphogenesis and axon pathfinding. In this study, we used bacterial homologous recombination to express a LacZ reporter in various ephrin-A5 BAC clones to identify elements that regulate ephrin-A5 gene expression during mesencephalon development. We found that there is mesencephalon-specific enhancer activity localized to a specific +25.0 kb to +30.5 kb genomic region in the first intron of ephrin-A5. Further comparative genomic analysis indicated that two evolutionary conserved regions, ECR1 and ECR2, were present within this 5.5 kb region. Deletion of ECR1 from the enhancer resulted in disrupted mesencephalon-specific enhancer activity in transgenic embryos. We also found a consensus binding site for basic helix-loop-helix (bHLH) transcription factors (TFs) in a highly conserved region at the 3'-end of ECR1. We further demonstrated that specific deletion of the bHLH TF binding site abrogated the mesencephalon-specific enhancer activity in transgenic embryos. Finally, both electrophoretic mobility shift assay and luciferase-based transactivation assay revealed that the transcription factor Ascl1 bound the bHLH consensus binding site in the mesencephalon-specific ephrin-A5 enhancer in vitro. Together, these results suggest that the bHLH TF binding site in ECR1 is involved in the positive regulation of ephrin-A5 gene expression during the development of the mesencephalon.

• Journal of Life Science
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• v.14 no.5
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• pp.874-881
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• 2004

Angiogenesis has been implicated in progression of inflammation, arthritis, psoriasis, atherosclerosis as well as tumor growth and metastasis. Intensive studies have been carried out to develop a strategy for cancer treatment by blocking angiogenesis. During angiogenesis, endothelial proliferation and migration essentially occurs upon activation. In this study, we compared the expression profiles of human umbilical endothelial cells activated by incubating in vitro in the rich medium containing several growth factors, and non-activated ones. cDNA targets derived from total RNAs of HUVEC activated for 13 h in M199 medium containing endothelial cell growth supplement, 20% fetal bovine serum, and heparin, after reaching 70～80% confluency, or non-activated, were hybridized onto oligonucleotide microarrays containing 1,8864 genetic elements. Unsupervised hierarchical clustering analysis resulted in two subgroups on dendrogram exhibiting activated and non-activated HUVECs. We then extracted 122 outlier genes which were shown to be up-regulated or under-expressed by at least 2-folds in activated HUVECs. Among these, 32 annotated genes were up-regulated and 38 were down-regulated in activated HUVECs. Interestingly, genes involved in cell proliferation, motility, and inflammation/ immune response were up-regulated in activated HUVEC, whereas genes for cell adhesion or vessel morphogenesis/function were down-regulated. Unexpectedly, the expression of genes well-characterized as angiogenesis markers was not changed except Eph-B4, which was down-regulated about 4 folds. 52 unknown genes were also up- or down-regulated. Therefore, these results could provide an opportunity to targeting new vascular molecules for the development of anti-angiogenic molecules.