期刊
EUROPEAN JOURNAL OF BIOCHEMISTRY
卷 268, 期 16, 页码 4398-4407出版社
BLACKWELL SCIENCE LTD
DOI: 10.1046/j.1432-1327.2001.02355.x
关键词
antibody screening; endothelial; growth factors; subtractive immunization; lambda gt11 cDNA expression library
A central event in angiogenesis is proliferation of blood vessels, which plays a major role in the progression of a number of inflammatory and neoplastic diseases. It is responsible for the switch of endothelial cells from an antiangiogenic to an angiogenic phenotype. To identify novel activated/proliferating-related proteins in human endothelial cells, a subtractive immunization approach was used to elicit a host antibody response against human dermal microvascular endothelial cells (HDMECs) stimulated with a potent angiogenic cytokine such as VPF/VEGF(165). In this study, a new mAb, LY9, which is highly specific to VPF/VEGF(165)-activated HDMECs, was isolated. Stimulation of HDMECs by VPF/VEGF(165) or basic fibroblast growth factor (bFGF) resulted in a dose-dependent and time-dependent increase in the binding of LY9. On Western-blot analysis, LY9 identified an 85-kDa protein (p85) in the lysates of several endothelial cells derived from microvascular or large vessel sources, the expression of which is dramatically increased by VPF/VEGF(165). The mAb also identified p85 in primary cell cultures of human foreskin keratinocytes but failed to recognize human fibroblasts (MRC5) and a number of different human tumor cell lines, including MG63 osteosarcoma and MCF7 breast carcinoma cells. Immunological screening of a human keratinocyte lambda gt11 cDNA expression library with LY9 identified a partial cDNA clone of 750 bp. DNA sequencing of this clone and predicted amino acids showed more than 93% homology to RING3 kinase, a member of a newly described family of bromodomain-containing proteins that transactivates in the nucleus the promoters of a number of the E2F family of transcription factors. This molecule may represent a new signaling target activated by VPF/VEGF(165) and bFGF that allows endothelial cells to enter the proliferative phase of the angiogenic process.
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