The clinical efficacy of TNF-specific antagonists in the treatment of rheumatoid arthritis (RA) points to the critical role of TNF in RA pathogenesis. Binding of TNF to one of its receptors, TNF receptor 1 (TNFR1) can activate NF-kB transcription factors leading to gene expression or the caspase pathway leading to apoptosis. Recent studies have shown that NF-kB induces the expression of anti-apoptotic genes, which then antagonize the apoptosis pathway that is concurrently activated. Therefore, an important question is what are the anti-apoptotic genes regulated by NF-kB and how do they inhibit apoptosis. A Jurkat T cell mutant that is deficient in IKK /NEMO, an essential component of the NF-kB signaling pathway, has been isolated and as a result of this deficiency, this mutant exhibits a profound increase in sensitivity to TNF-induced apoptosis. A20, a NP- B-regulated gene, effectively blocks TNF-induced apoptosis in the mutant cell by blocking either RIP, a death domain kinase in the TNF pathway, or a molecule upstream of RIP.
In Specific Aim 1, the precise molecular target of A20 will be defined by epistasis staging studies, combined with the testing of hypothesis that were formulated based on our current understanding of proximal TNFR1 signal transduction.
In Specific Aim 2, the mechanism utilized by A20 will be examined by identifying proteins that interact with A20. The approaches used will be based on the de novo interaction between A20 with its partners, which can be isolated by anti-A20 antibodies. A number of schemes will be used subsequently to identify the associated partners.
In Specific Aim3, a search for genes that are no longer upregulated by TNF in this mutant cell line will be conducted and then functionally tested for their ability to disrupt the apoptosis pathway. The end result of this project will be a greater knowledge of the pathways and downstream genes regulated by TNF, which may suggest additional avenues for pharmacological modulation in RA.
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