Extracellular fragment of brain-specific angiogenesis inhibitor 1 suppresses endothelial cell proliferation by blocking αvβ5 integrin
Introduction
Angiogenesis depends on growth factors and their receptors, but is also influenced by receptors for extracellular matrix (ECM) proteins. In general, cell adhesion to the ECM is mediated by integrins, heterodimeric transmembrane proteins composed of over 15α and 8β subunits. Some integrin combinations recognize a single ECM ligand, while others bind several different ECM proteins [1]. During angiogenesis, it is likely that integrins expressed on the surface of activated endothelial cells (ECs) regulate critical adhesive interactions with a variety of ECM proteins. These interactions regulate distinct biological events such as cell migration, proliferation, and differentiation. In addition, angiogenesis in specific tissues, such as in the brain, may depend on specific EC interactions with ECM proteins that vary considerably in their adhesive properties [2], [3], [4].
Two αv integrins, αvβ3 and αvβ5, have been demonstrated to be necessary for cytokine- or tumor-induced angiogenesis [2], [5]. These integrins mediate two distinct pathways of angiogenesis. Particularly, αvβ3 is necessary for the formation, survival, and maturation of newly formed blood vessels [2], [6], [7]. These survival pathways can be blocked either by function-blocking antibodies or by peptide analogues that block the active RGD binding site on integrins αvβ3 and αvβ5, thus inducing apoptosis of the angiogenic ECs [5], [6]. Endothelial apoptosis results in inhibition of new blood vessel formation, inhibition of tumor growth, and tumor regression [8], [9]. Thrombospondins (TSPs) are ECM proteins that inhibit EC proliferation, migration, and angiogenesis. Among the five different thrombospondin genes, only TSP1 and TSP2 contain three thrombospondin type 1 repeats (TSRs), and these two genes inhibit angiogenesis in vitro and in vivo [10], [11], [12], [13].
BAI1 is a novel brain-specific gene that was identified during a screen for genomic DNA fragments containing functional p53 binding sites [14]. Expression of BAI1 is directly induced by wild-type p53 [14]. The predicted BAI1 protein includes a seven-span transmembrane region (STR) flanked by large extracellular and cytoplasmic domains. Two additional human genes homologous to BAI1 have been identified and designated as BAI2 and BAI3. Analysis of the three predicted BAI proteins showed that the TSR and STR regions were well conserved. However, the extracellular and cytoplasmic domains are relatively different among the three proteins [15], [16]. All three BAI genes were specifically expressed in brain, and it is likely that the three are closely related.
The third cytoplasmic loop of an STR-containing G-protein coupled receptor is important to its interaction with G proteins [17]. However, this third loop is very short in BAI1 compared with BAI2, and it appears likely that BAI1 does not perform the function normally mediated by the third cytoplasmic loop. BAI1 possesses two presumably functional elements in its extracellular region, one RGD motif and 5 TSRs [3]. The former is a potential recognition sequence for binding integrins, and the TSRs of BAI1 can inhibit experimental angiogenesis induced by fibroblast growth factor [14], [18]. It is assumed that the functions of the TSR region of BAI1 are similar to those of TSP1 or TSP2. We recently showed that the brain-specific developmental expression pattern of BAI1 and BAI2 was correlated with decreasing neovascularization in the adult brain [16]. BAI1 and BAI2 were involved in the late and early stages, respectively, of neovascularization in the cerebral cortex after ischemia [16]. This finding suggests that BAI1 and BAI2 act as angiostatic factors in ischemia-induced brain angiogenesis, perhaps in concert to angiogenic vascular endothelial growth factor (VEGF). However, BAI1's mechanisms of anti-angiogenic action in the brain are still unknown.
In this study, we investigated BAI1's effects on endothelial cell proliferation to derive an understanding of the mechanisms underlying this protein's anti-angiogenic action. We found that the antiproliferative activity of BAI1 is localized to the TSR region, which binds and functions via αvβ5 integrin. Our results indicate that neurons may inhibit nearby angiogenesis through the paracrine antiproliferative effect of BAI1 on EC.
Section snippets
Cell lines and culture
HUVECs were purchased from Clonetics at passage 3 and used until passage 8. These cells were grown in basic medium (EBM2) containing growth supplements (EGM2MV, Clonetics). SHSY5Y neuroblastoma and U343 glial cells were grown in DMEM and α-MEM, respectively, both supplemented with 10% FBS. Human embryonic kidney 293 (HEK293) cells, which stably expressed human β3 or β5 integrin [19], were kindly provided by Dr. Jeffrey Smith (The Burnham Institute, La Jolla), and were maintained in DMEM
BAI1 inhibited cell proliferation
BAI1 was transiently transfected into SHSY5Y neuroblastoma cells, which express a low level of endogenous BAI1. BAI1 overexpression decreased the number of SHSY5Y to 67% of the control 36 h after gene transfer (Fig. 1A). The vector did not significantly affect the cell number.
BAI1 was also transiently delivered to U343 glial cells and HUVECs, which do not express endogenous BAI1. At 36 h after gene transfer compared to control, BAI1 reduced the survivals of U343 cells by 29% and of HUVECs by
Discussion
In this study, we demonstrated that BAI1 has transferable antiproliferative action. An immunoblotting assay of concentrated conditioned media from BAI1 transfected cells showed fragments of BAI1. Moreover, the addition of anti-BAI1 antibody to the conditioned media neutralized the antiproliferative action of the media. However, there were no changes in other anti-TSR antibody-reactive proteins, such as TSP1, between the vector- and BAI1-transfected conditioned media (data not shown).
Acknowledgements
We thank Jennifer Macke for assistance preparing the text. This work was supported by the Korea Science & Engineering Foundation through the Medical Research Center for Gene Regulation (R13-2002-013-01000-0) at Chonnam National University. H.J. Kee and J.H. Lee were financially supported by a Korea Research Foundation Grant (KRF-99-005-F00014).
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