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Vascular cell adhesion molecule-1 (VCAM-1) is a member of the immunoglobulin superfamily, and as such shares considerable homology with other cell adhesion molecules (CAM's). VCAM-1 was first described on activated endothelium where its interaction with the a4b1 integrin is thought to facilitate the recruitment of lymphocytes during the immune response (Osborn, et al., 1989). The structure of VCAM-1 is composed of an N-terminal extracellular portion, a transmembrane domain, and a short cytoplasmic tail. The extracellular portion of VCAM-1 is made up of seven immunoglobulin domains homologous to the C2 domains of the immunoglobulin heavy chain. In VCAM-1, these domains appear as a tandem repeat with internal homologies between domains 1-3 and 4-6, respectively. The major ligand binding site of VCAM-1 is contained within Domain 1, with minor structural contributions from Domain 2; however, secondary binding to Domain 4 has been reported, further demonstrating the repeated nature of these domains. While direct interactions of specific proteins with the transmembrane or cytoplasmic domains of VCAM-1 have not been reported, ligation of VCAM-1 on the cell surface has been shown to directly affect cellular activities, implying its interaction with some signaling cascade. VCAM-1 shares its greatest homology, functionally as well as structurally, with intercellular cell adhesion molecule-1 (ICAM-1), which like most CAM's contains only five extracellular immunoglobulin domains. Both receptors bind integrins, and neither display homophilic interactions. This contrasts sharply with the other developmentally important CAM's, neural cell adhesion molecule (NCAM) and platelet / epithelial cell adhesion molecule (PECAM), which bind primarily to themselves or to extracellular matrix components (Kiselyov et al., 1997).
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The integrins are a family of heterodimeric receptors that mediate both cell-cell and cell-extracellular matrix interactions. The Very Late Activation, or VLA, group of antigens comprise integrins in which one of several a subunits is coupled to a common b1 (CD29) subunit. The a4b1 (VLA-4; CD49d/CD29) member of this group has known specificity for two ligands: VCAM-1 and fibronectin (FN), a common component of many developmentally important extracellular matrices and basal laminae (Glukhova and Thiery, 1993; reviewed in Hemler, 1990). The a4 integrin subunit can also interact with the b7 subunit; this heterodimer is expressed by lymphocytes, although its ligand, MAdCAM-1, is an addressin expressed only on Peyer's Patch endothelium in the gut (Holzmann and Weissman, 1989). This interaction is responsible for homing of a subpopulation of lymphocytes to the intestinal lymphatic system. Other interactions have been shown between a4b7 and both VCAM-1 and FN, although binding to these ligands is of a much lower affinity than to MAdCAM-1 (Berlin, et al., 1993).
Endothelial cells express VCAM-1 in response to specific cytokines, such as IL-4 and TNFa (Iademarco, et al., 1997), which are produced by lymphocytes at sites of inflammation. The interaction of VCAM-1 with a4b1 at these sites is essential for migration of lymphocytes between endothelial cells of the blood vessel wall during extravasation toward the inflamed tissue, following which expression of a4b1 on the surface of immune cells mediates their interact with FN at sites of injury and inflammation (Wayner, et al., 1989). Binding to VCAM-1 can activate CD4+ T-lymphocytes expressing a4b1 (Dämle and Aruffo, 1991), as can FN (Nojima, et al., 1990), and may be necessary for lymphocyte maturation: VCAM-1 on bone marrow stromal cells and on the dendritic cells at lymph node germinal centers interacts with a4b1 expressed on the surface of maturing hematopoietic cells (Miyake, et al., 1991).
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a4b1 and VCAM-1 are also important in skeletal muscle formation. In early myogenesis, precursor cells express a4b1 as they differentiate into primary myoblasts, and continue to express a4b1 as they fuse to form primary myotubes. Subsequently, secondary myoblasts expressing VCAM-1 use the a4b1-positive primary myotubes as a template for fusion and secondary myotube formation, such that the secondary muscle fibers, which make up the bulk of adult muscle, are aligned along their primary myotube scaffolds (Rosen, et al., 1992). This same interaction is seen during muscle repair, where satellite cells expressing VCAM-1 use the existing muscle fibers to orient the newly forming tubes. In these cases, a4b1 appears to be upregulated on muscle fibers at sites of injury (Jesse et al., 1998).
Also, it is during the early developmental events leading to myogenesis, that interactions between a4b1 and FN appear to play an important role. As the pre-myoblast cell of the myotome begin their migration into the limb, as well as down the body wall, they begin to express a4b1. This expression increases as these cells form the premuscle masses that will give rise to the various muscle groups (Allan Sheppard, unpublished results). Interestingly, there is no FN detectible within these premuscle masses, suggesting a model in which pre-myoblasts use a4b1 to migrate along FN tracts leading to the limb and down the body wall, but then lose their association with FN once they reach their goals. This is supported by work done by Chuanyue Wu comparing Chinese Hamster Ovary (CHO) cells expressing either a4b1 integrin, a5b1 integrin, or neither. In these experiments, CHO cells expressing either integrin were able to migrate over a matrix containing FN: however, only the CHO cells expressing a5b1 integrin were able to assemble a FN matrix (Wu et al., 1995). In fact, further studies have demonstrated that most, if not all, integrins which recognize the RGD site of FN are able to support its assembly (Wu et al., 1996). It has been further suggested that the inability of a4b1 to support FN matrix assembly may be related to its inability to activate the mitogenic signalling pathways through Shc (Wary et al., 1996). Taken together, these findings suggest that the expression of a4b1 on these cells and the exclusion of FN from the premuscle masses are part of the differentiation of these cells into myoblasts.
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The findings of a4b1 integrin expression on developmental tissues - specifically on the developing lymphocytes and skeletal muscle mentioned above, as well as on migrating neural crest cells and their derivatives - suggested that a4b1 may be expressed on other developmental tissues. Furthermore, the importance of its interaction with VCAM-1 during each of these processes, suggested that the interaction between a4b1 and VCAM-1 may be required for some of these other developmental processes. Herein, I describe work done to delineate the expression of a4 integrins and VCAM-1 during mouse embryonic development.
This developmental survey demonstrates the expression of a4 and VCAM-1 on several tissues, including the heart, extraembryonic membranes, and central nervous system (CNS). These findings are especially interesting in light of work from other laboratories examining the phenotypic changes in mice lacking the genes for either a4 (Yang et al., 1995) or VCAM-1 (Gurtner et al., 1995; Kwee et al., 1995). All mouse embryos lacking either a4_or VCAM-1 died in utero from either poor placentation or heart defects. Placental defects involved improper fusion of the chorioallantoic membrane which forms the placental plug (Gurtner et al., 1995; Yang et al., 1995), corresponding to the expression of a4 on the chorion and VCAM-1 on the allantois reported here. One major heart defect was a poorly formed epicardium (Kwee et al., 1995; Yang et al., 1995), corresponding to the expression of a4 on the pre-epicardial cells and VCAM-1 on the myocardium reported here. Of particular interest was the perforate or missing interventricular septum seen in embryos lacking VCAM-1 (Kwee et al., 1995) but not in embryos lacking a4 (Yang et al., 1995), corresponding to the restriction of myocardial VCAM-1 expression to the interventricular septum. Unfortunately, none of these knock-out mice survived long enough to demonstrate any phenotypic changes to the CNS: even so, these studies the importance of a4 and VCAM-1 expression in the tissues reported here.
While other members of our laboratory have characterized the elements controlling expression of VCAM-1 in skeletal muscle (Jesse et al., 1998) and the CNS (Sheppard et al., 1995), I found it more interesting to examine the activities of the VCAM-1 promoter on the developing myocardium, since this was the major source of fatal deformaties in the VCAM-1-deficient mice (Kwee et al., 1995). As reported here, the differential expression of VCAM-1 on the developing myocardium is controled by octamer sites over a kilobase upstream of the VCAM-1 transcription start site, particularly one octamer site at -1180 bp. Furthermore, the control of expression through this octamer site is dependent on binding of either an undefined repressor complex or an activator complex containing the transcription factors Oct-1 and Bob-1.
The work done by our laboratory on the expression of VCAM-1 in the CNS demonstrated the importance of members of the Pit/Oct/unc (POU) family of transcription factors in the activation of VCAM-1 during retinoic acid induced differentiation of P19 teratocarcinoma cells (Sheppard, et al., 1995). POU proteins are functionally analogous to Hox proteins, and have already been implicated in differentiation and pattern formation in several tissues including the heart (Komuro, et al., 1993; Lints, et al., 1993). Oct-1, a member of this family, is found ubiquitously, and has been implicated in B-cell maturation, and neuronal differentiation. Though a weak activator alone, Oct-1 is able to recruit stronger viral activators, including the herpesvirus transcriptional activator VP16, to the promoter. In B-cells, Oct-1 appears to work in conjunction with a similar activator: the B-cell, Oct-1 Binding protein-1 (Bob-1: also called OBF-1 and OCA-B: Babb et al., 1997). Bob-1 has been shown to activate the immunoglobulin heavy chain gene, and, like Oct-1, is thought to be important for B-cell maturation. As yet, Bob-1 has only been describe in B-cells and neural tissues, where its role is less well understood. Experiments creating transgenic mice lacking the Bob-1 gene gave little phenotype outside of reduced lymphatic germinal centers, although the results from these studies are difficult to interpret.
We show here the presence of Oct-1 and Bob-1 in protein complexes bound to the octamer sites, with highest affinity for the octamer site at -1180 bp. Through immunohistological examination of embryonic mouse hearts we demonstrate the colocalization of Bob-1 with VCAM-1 expression. More importantly, we report that overexpression of Bob-1 in murine cardiomyocytic cell lines results in increased VCAM-1 expression, and that this overexpression results in not only derepression of the octamer sites on reporter constructs, but increased activation when these site are present. These data indicate that the expression of VCAM-1 that is essential for formation of the interventricular septum is controled by Bob-1, which had previously not been described outside of lymphocytes and the central nervous system.
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