The integrin alpha4beta1 (also known as VLA-4) and its counter receptor vascular cell adhesion molecule-1 (VCAM-1) mediate well described cell- cell interactions that are critical for immune function. However, we have found recently that these receptors are expressed in a developmental- specific pattern on other tissues. In skeletal muscle their interaction appears to be important for myogenesis. We propose a series of studies to further our understanding of the role of alpha4beta1 and VCAM-1 in muscle differentiation including: in situ hybridization to confirm which cell types in muscle express these genes, and the effect of antibodies and antisense RNAs that block the activity and expression of these receptors on the fusion of myoblasts into mouse muscle in vivo. The pattern of alpha4beta1 and VCAM-1 expression will also be examined in other developing tissues in the mouse. These results will guide subsequent studies. It is our intention to examine the role of alpha4beta1 and VCAM-1 in tissues here they are expressed using model culture systems as we have done with skeletal muscle. One tissue that is already of interest is bone, where alpha4beta1 and VCAM-1 are expressed in a developmental-specific pattern and their interaction appears important for differentiation. We propose to examine the role of these receptors in differentiation of mouse bone marrow macrophages into osteoclasts-this process requires stromal cells which express VCAM-1, and alpha4beta1 expression is activated on the macrophages in response to macrophage-colony stimulating factor which triggers the differentiation process. Finally, we propose a detailed analysis of mechanisms that control alpha4 and VCAM-1 expression. To this end, we have cloned the promoter regions of the genes that encode these proteins. Transfection studies indicate that a unique position-specific enhancer causes contrasting patterns of VCAM-1 expression in endothelial and muscle cells. The enhancer is inactive in endotheliaI cells. In these cells octamers normally restrict VCAM- 1 expression, but in response to inflammatory cytokines, expression is activated through two tissue- specific NFkappaB sites. In muscle cells the enhancer is active and it overrides the other promoter elements causing constitutive expression of VCAM-1. We propose a detailed characterization of this enhancer and its nuclear binding protein. Expression of alpha4 is controlled at least in part by a tissue-specific silencer located between position -600 and -800 of the alpha4 gene. Experiments to characterize this element are proposed. Additionally, we show that alpha4 expression is activated during muscle differentiation and we present evidence that the alpha4 promoter may contain the same position-specific enhancer as the VCAM-1 gene. However, the alpha4 promoter contains an additional element(s) between position -2 and -10 kb that restricts alpha4 expression in myoblasts. This element(s) is clearly upstream of the silencer described above. We propose to localize and characterize this element. Together, these studies should provide insight into the roles of alpha4beta1 and VCAM-1 during development and the mechanisms controlling their expression.
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