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Del1 is a novel extracellular matrix protein encoding three Notch-like epidermal growth factor repeats, an RGD motif, and two discoidin domains. Del1 is expressed in an endothelial cell-restricted pattern during early development. In studies reported here, recombinant baculovirus Del1 protein was shown to promote ␣v␤3-dependent endothelial cell attachment and migration. Attachment of endothelial cells to Del1 was associated with clustering of ␣v␤3, the formation of focal complexes, and recruitment of talin and vinculin into these complexes. These events were shown to be associated with phosphorylation of proteins in the focal complexes, including the time-dependent phosphorylation of p125 FAK , MAPK, and Shc. When recombinant Del1 was evaluated in an in ovo chick chorioallantoic membrane assay, it was found to have potent angiogenic activity. This angiogenic activity was inhibited by a monoclonal antibody directed against ␣v␤3, and an RAD mutant Del1 protein was inactive. Thus Del1 provides a unique autocrine angiogenic pathway for the embryonic endothelium, and this function is mediated in part by productive ligation of integrin ␣v␤3.The study of vascular development is important because of the fundamental embryological principles that underlie this complex and essential process, and the potential for therapeutic manipulation of vascular formation in human disease processes. The assembly of endothelial cells into vascular structures requires specific signals from luminal surface receptors that are activated by soluble ligands, and signals from the extracellular matrix mediated through receptors on the abluminal surface of the endothelial cell (1, 2).Virtually all molecules of the matrix are able to communicate to cells by binding to integrins (3, 4). The integrin receptors are noncovalently associated heterodimeric glycoproteins that reside in the cell membrane where their cytoplasmic domains connect to elements of the cytoskeleton. These molecules support a variety of functions, including cell attachment and migration, and activation of cytoplasmic signaling molecules (4, 5). A number of different integrin receptor complexes have been identified on the surface of endothelial cells, and linked to specific functions (2). Data from recent experiments indicate that interaction of the ␣v␤3 receptor with its ligands is critical for tumor and cytokine-induced angiogenesis, through a cell survival function mediated by this integrin (6). Also, ␣v␤3-mediated signaling through the Ras-mitogen activated protein kinase (MAPK) 1 pathway, specifically through sustained activation of MAPK, appears to be critical for the angiogenic program (7).To search for new molecular pathways of vascular development, we have recently characterized a locus identified by an enhancer trap event in a line of transgenic mice (8). The gene located in this locus, Del1, was shown to be expressed in an endothelial-restricted pattern in early embryonic development. The proteins encoded in the locus were characterized by genomic and cDNA cloning. The...

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The extracellular matrix (ECM) acts as a critical factor during morphogenesis. Because the organization of the ECM directly influences the structure of tissues and organs, a determination of the way that ECM organization is regulated should help to clarify morphogenesis. We have analyzed the assembly of Del1, an ECM protein produced by endothelial cells in embryos, in the ECM. Del1 consists of three epidermal growth factor repeats (E1-E3) at its N-terminus and two discoidin domains (C1, C2) at its C-terminus. Experiments with various deletion mutants of Del1 have revealed that fragments containing the C-terminus of C1, which has a lectin-like structure, direct deposition in the ECM. The efficiency of deposition varies according to the presence of other domains in Del1. A fragment containing E3 and C1 has the strongest deposition activity, whereas fragments containing C2, which is highly homologous to C1, have low deposition activity. Digestion of ECM with hyaluronidase from bovine testis releases Del1 from the ECM, suggesting that glycosaminoglycans are involved in the deposition of Del1. In vivo gene transfer experiments have shown that fusion with the deposition domain of Del1 dramatically alters the distribution of exogenous proteins in mice. Thus, the extent of Del1 deposition may modify the organization of the ECM.

The purpose of this study was to clarify the relationship among X-box-binding protein 1 unspliced, spliced (XBP1u, s), Forkhead box O1 (FoxO1) and autophagy in the auditory cells under endoplasmic reticulum (ER) stress. In addition, the relationship between ER stress that causes unfolded protein response (UPR) and autophagy was also investigated. The present study reported ER stress induction by tunicamycin treatment that resulted in IRE1α-mediated XBP1 mRNA splicing and autophagy. XBP1 mRNA splicing and FoxO1 were found to be involved in ER stress-induced autophagy. This inference was based on the observation that the expression of LC3-II was suppressed by knockdown of IRE1α, XBP1 or FoxO1. In addition, XBP1u was found to interact with XBP1s in auditory cells under ER stress, functioning as a negative feedback regulator that was based on two important findings. Firstly, there was a significant inverse correlation between XBP1u and XBP1s expressions, and secondly, the expression of XBP1 protein showed different dynamics compared to the XBP1 mRNA level. Furthermore, our results regarding the relationship between XBP1 and FoxO1 by small interfering RNA (siRNA) paradoxically showed negative regulation of FoxO1 expression by XBP1. Our findings revealed that the XBP1-FoxO1 interaction regulated the ER stress-induced autophagy in auditory cells.