Alpha4 integrins on leukocytes interact with VCAM-1 on endothelial cells, and this interaction targets leukocytes to sites of inflammation in inflammatory lung diseases. This interaction is particularly important for selective recruitment of eosinophils and lymphocytes that occurs in allergic diseases. During the previously grant period, we have investigated molecular mechanisms that control expression of VCAM-1 on the endotheium and alpha4 integrin on leukocytes. In the course of these studies we found that the alpha4 integrin gene is under negative regulation by a zinc finger/homeodomain protein known as ZEB. Several other important leukocyte genes (e.g., IL-2 and CD4) also have binding sites for ZEB, suggesting a general role for the protein in leukocyte function. Accordingly, in ZEB knockout mice there is severe inhibition of thymocyte differentiation, and in cells that do manage to differentiate, there is an increase in expression of alpha4 and CD4 on peripheral cells (consistent with the removal of a repressor of alpha4 and CD4 genes). Thus, ZEB has a critical role in differentiation and function of T cells, and it regulates expression of important T cell genes such as alpha4 and CD4 in vivo. Understanding the mechanism of action of a protein such as ZEB that is linked to T cell differentiation and function (and probably also to the function of other leukocytes such as eosinophils through regulation of alpha4 integrin) should provide insight into this process. In the new proposal we will undertake a detailed structure/function analysis of ZEB in leukocytes examining repressor activity and proteins that interact with ZEB. Additionally, we propose a series of experiments using transgenic mice to assess the role of ZEB in leukocyte differentiation and function. These studies will not only extend our knowledge of how alpha4 gene expression is regulated, but should provide key information on regulation of other important T cell genes such as IL-2 and CD4 (as well as genes in other leukocyte subsets). It is our aim in the future to apply the basic science information that we gain from the studies on ZEB activity to regulate leukocyte differentiation and function in inflammatory diseases.
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