Identification and cloning of an 85-kDa protein homologous to RING3 that is upregulated in proliferating endothelial cells.
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/VEGF165. In this study, a new mAb, LY9, which is highly specific to VPF/VEGF165-activated HDMECs, was isolated. Stimulation of HDMECs by VPF/VEGF165 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/VEGF165. 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 lambdagt11 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/VEGF165 and bFGF that allows endothelial cells to enter the proliferative phase of the angiogenic process.
BelAiba
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INSERM U331, Faculty of Médecine René Laënnec, Lyon, France.
Baril
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Chebloune
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Tabone
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Boukerche
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D000328:Adult; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001483:Base Sequence; D015153:Blotting, Western; D002478:Cells, Cultured; D002868:Chromosomal Proteins, Non-Histone; D003001:Cloning, Molecular; D016228:Endothelial Growth Factors / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000378:metabolism*; D005260:Female; D006801:Humans; D007114:Immunization; D008222:Lymphokines / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000302:isolation & purification*; D014157:Transcription Factors; D015854:Up-Regulation; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D000092468:Bromodomain Containing Proteins
D000911:Antibodies, Monoclonal; D002868:Chromosomal Proteins, Non-Histone; D016228:Endothelial Growth Factors; D008222:Lymphokines; D017346:Protein Serine-Threonine Kinases; D014157:Transcription Factors; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; C098415:BRD2 protein, human; D000092468:Bromodomain Containing Proteins; C490844:Brd2 protein, mouse; C467484:VEGFA protein, human