Rheumatoid arthritis (RA) is a chronic inflammatory disease associated with synovial lining hypertrophy, and infiltration of the sublining region by macrophages, lymphocytes and plasma cells. Activated macrophages in the synovial lining and sublining region produce a variety of cytokines, which are mediators of inflammation, angiogenesis, chemotaxis, and cell activation. Of these cytokines, TNFalpha plays a central role in the pathogenesis of RA. Our recent studies, employing TNFalpha promoter-reporter constructs transiently transfected into myelomonocytic cells and Jurkat T cells, indicated that the bZip family transcription factor C/EBP beta (CCAAT/enhancer binding protein beta, also called NF-IL6) was important in the cell type-specific regulation of TNFalpha. The ability of endogenous C/EBP beta to activate TNFalpha promoter-reporter constructs was inhibited by cotransfection of a dominant negative (DN) version of C/EBP beta, representing the DNA binding and the leucine zipper domains, with the transactivation domain deleted. The ability of C/EBP beta regulate the endogenous TNFalpha gene has yet to be documented. Therefore, this grant proposes to examine the following specific aims: 1) Define the relationship between the nuclear localization of C/EBP alpha and the expression of the endogenous and ectopically expressed TNF alpha genes. 2) Determine if the ectopic expression of C/EBP beta is necessary for expression of the endogenous TNF alpha gene under conditions which do not result in the production of endogenous C/EBP beta. 3) Determine if the DN form of C/EBP beta is capable of suppression of the expression of the endogenous TNF alpha gene in cells which synthesize C/EBP beta, such as PMA stimulated myelomonocytic cells, LPS stimulated monocytes, and synovial fluid macrophages from patients with RA. 4) In Mice: (A) Elucidate the relationship of the expression of C/EBP beta and TNF alpha within the synovial tissue during the development of collagen induced arthritis in mice; and (B) examine the expression of the DN form of C/EBP beta in the synovial lining of normal mice following intraarticular injection of the appropriate replication defective adenovirus vectors. Knowledge of the mechanisms regulating TNF alpha gene expression will enhance our ability to modulate this very important cytokine. The cell type-specific regulation of the TNF alpha gene may provide tremendous therapeutic benefit in a wide variety of diseases. Our approach is unique in three aspects. First, it takes advantage of cell type-specific regulation of TNF alpha by C/EBP beta, which we have recently identified. Second, it will employ the use of a DN C/EBP beta in an attempt to regulate the endogenous TNF alpha gene. Finally, as will be discussed in the body of the grant, studies will employ replication defective adenovirus vectors, which will not only be useful in examining the regulation of the endogenous TNFalpha gene, but may also be readily adaptable for somatic gene therapy in experimental animals.
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