The focus of this proposed study is to understand the in vivo function of the cellular FLICE-like inhibitory protein, cFLIP, in B lymphocytes. This project has evolved from our longstanding interest in the FADD-mediated apoptotic and proliferation signaling mechanism. We and others have previously demonstrated that FADD is a common signaling adaptor shared by several death receptors including Fas, tumor necrosis factor receptor 1 (TNF-R1), and TNF-related apoptosis-inducing ligand receptors (TRAIL-R). The death domain of FADD binds to the death domain of death receptors, and the death effector domain of FADD associates with the death effector domains of Caspase 8. Death receptor-induced apoptosis plays an important role in suppressing autoimmunity and malignancy; however, they are dispensable during embryonic development. Interestingly, a lack of FADD or Caspase 8 results in early embryonic lethality in mice. Using viable FADD-/-->RAG-1-/- chimeras, we have showed that B cell development is completely blocked when FADD is absent in embryonic stem cells. However, we found that B cell development was not obviously affected when FADD is deleted in pre-B cells using CD19Cre, indicating FADD is essential at earlier stages and/or prior to B lineage commitment. Surprisingly, FADD-deficient B cells are not only defective in apoptosis but also impaired in Toll-like receptor (TLR)-induced proliferation. Similar defects have been observed in conditional Caspase 8-deficient mice. These studies have helped reveal a new paradigm of binary signaling in B cells involving FADD and Caspase 8. How FADD and Caspase 8 regulate two distinct signaling, apoptosis and proliferation in B cells is not well understood. We hypothesize that cFLIP, which can bind to FADD, may play a role in the regulation of the dual function of FADD and Caspase 8 in B cells. cFLIP is homologous to Caspase 8 but lack Caspase activity. In preliminary studies, we showed that cFLIP-deficient mice are embryonic lethal, a phenotype similar to that of mice lacking FADD or Caspase 8. In addition, we found that heterozygous cFLIP T cells are hypoproliferative, whereas transgenic cFLIP overexpression leads to T cell hyperproliferation. There are reports indicating that cFLIP inhibits apoptosis in primary B cells. However, in vitro overexpression of cFLIP was also shown to either enhance or inhibit apoptosis. It is not clear whether cFLIP play a role in FADD/Caspase 8-mediated B cell proliferation induced by TLR stimulation. We propose (1) to analyze the in vivo function of cFLIP by using conditional mutant mice lacking cFLIP in B cells, and (2) to dissect the multiple functions of cFLIP in B lymphocyte by reverse genetics. This study will help identify the in vivo signaling pathways regulated by cFLIP, which would be useful for translational research of various diseases including immune disorders and cancer. In this application, we describe a scientific plan to study the cFLIP protein which plays an essential role in development, immune system functions, and cancer. The results will facilitate the development of effective preventive and therapeutic approaches. ? ? ?
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