Dissecting the molecular mechanisms of how proliferation and apoptosis are controlled is the key to understanding tumorigenesis. T cell development involves a fine balance of apoptosis and cell proliferation and is thus an ideal system to probe the molecular mechanisms of these two fundamental processes. Many aspects of T cell development have been studied extensively but several major questions remain to be answered. In particular, pathways underlying negative selection are yet to be elucidated and it is still not clear why CD4+CD8+ thymocytes are exquisitely sensitive to apoptosis. Nur77 orphan steroid receptor, the Bcl-2 pro-apoptotic member Bim, MAP kinases and PTEN tumor suppressor have all been implicated in negative selection but their mechanisms of action and inter-connection remain unclear. The role of MAP kinases in negative selection is also controversial. Studies of Nur77 upstream signaling proteins have shown PTEN-PI3kinase-AKT pathway as a modulator of Nur77 activity and MEK5-ERK5 kinase-cascade as an activator of Nur77 transcription. Nur77 can initiate apoptosis by transcriptional up-regulation of pro-apoptotic molecules FasL, TRAIL and NDG1, a novel molecule. However, a recent report showed that Nur77 could also interact with Bcl-2 in mitochondria of tumor cell lines and convert Bcl-2 into a pro-apoptotic molecule. In this application, we propose several aims designed to probe the Nur77 pathway and to elucidate the mechanism of thymocyte apoptosis.
In aim 1, the role of ERK5 MAP kinase in T cell development will be examined by generating T-cell specific ERK5-/- mice and by expressing dominant active mutants in thymic organ culture. Additional ERK5 target genes will be identified and analyzed.
In aim 2, the differential contribution of transcriptional versus mitochondria pathway in Nur77-mediated apoptosis will be assessed using transgenic and knockout mouse approach. We propose that apoptosis of CD4+CD8+ thymocytes is dependent on activation of the cell cycle machinery. This will be tested in aim 3 by examining transgenic mice over-expressing cell cycle inhibitors and by analyzing PTEN downstream target genes in thymocytes of T cell-specific PTEN-deficient mice. Successful completion of these aims should lead to a significant understanding of how apoptosis proceeds in thymocytes and how tumorigenesis might arise.
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