VCAM1 is a leukocyte adhesion molecule of the immunoglobulin superfamily class, which is expressed on the surface of endothelial cells that have been activated by inflammatory cytokines (TNF-a, IL-1), lipopolysaccharide, double stranded RNA and oxidized lipid species. It specifically binds the integrin VLA4, which is restricted to monocytes, lymphocytes, eosinophils and basophils, but is not expressed on neutrophils. VCAM1 mediates a central role in chronic inflammatory responses and immune cell interactions. Immunohistochemical techniques reveal endothelial VCAM1 expression associated with a variety of inflammatory processes including arthritis, chronic vascular graft rejection, and early atherosclerosis. The induction of VCAM-1 expression occurs at the transcriptional level and is controlled by binding of proteins to a 100 bp region, which includes Rel, IRF and Sp transcription factors. This proposal will show how these transcription factors respond to different signals, assemble on the promoter and interact to control VCAM-1 expression. The hypothesis is that activation, sustained expression and down regulation of VCAM-1 mRNA synthesis is the result of the combinatorial interaction of limited members of transcription factor families. In TNF-a stimulated endothelial cells, these proteins include members of the Rel, Interferon regulatory factor (IRF) and Sp families of transcription factors. This proposal will expand on earlier observations and aims to characterize how the known (and candidate) transcription factors respond to different signals, assemble on the promoter and ultimately interact to control the characteristic pattern of VCAM1 expression. The central hypothesis is that initial activation, sustained expression, and down regulation of VCAM1 mRNA synthesis is the result of combinatorial interactions of limited number of transcription factor families. Additionally, it is hypothesized that the different known inducers of VCAM1 may activate distinct combinations of transcription factors. To test these hypotheses, three specific aims use complementary in vitro and in vivo approaches.
The first aim proposes to directly test transcription factor interactions in vitro using cotransfection assays and DNase1 footprinting to determine which combinations of proteins have positive or negative interactions. The second and third aims will use in vivo methods with cultured endothelial cells. Specifically, the second aim proposes to identify the proteins present in endothelial cells which assemble on the VCAM1 promoter in different functional states and with different inducers.
The third aim plans to utilize in vivo footprinting to explore DNA-protein interactions in living cells. Elucidation of the mechanisms of transcription factor interactions will further our knowledge of how VCAM1 induction is regulated and how this important molecule is controlled by a variety of immuno-inflammatory stimuli.
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