REGULATION OF CD154 EXPRESSION

描述(由申请人提供)：CD154(CD40配体)由激活的T细胞表达，在免疫反应中发挥核心作用。因此，CD154的表达被认为是风湿性疾病治疗的主要靶点，并在类风湿性关节炎和系统性红斑狼疮的动物模型中被证实了CD154的有效性。此外，系统性红斑狼疮患者CD154基因表达异常。CD154(CD40配体)的表达表现出与细胞因子相关的独特的调节模式。这些研究直接与CD154的mRNA降解有关。我们已经证实，CD154基因3‘非编码区中的多嘧啶富集区对于减少嵌合报告基因在细胞系和正常人T淋巴细胞中的表达是必要且充分的，从而确定该区域是顺式作用元件。此外，我们还鉴定了两种与这个多嘧啶富集区结合的蛋白质，它们是PTB基因的不同剪接异构体(PTB，PTB-T)。这一发现表明，这些蛋白质是反式作用因子，决定了该区域在mRNA衰退中的功能。我们称为PTB-T的新型较小剪接异构体的过表达以CD154 3‘UWR特异的方式在正常人类CD4+T淋巴细胞和细胞系中持续抑制报告基因的表达。 这些数据表明，PTB-T与CD154 3‘非编码区中的多嘧啶富集区相互作用，介导了mRNA的降解。我们建议直接检验这一假说，并确定PTB-T在CD154 mRNA稳定性调节中的生物学作用。随后，我们将确定PTB-T介导这些效应的途径(S)。通过这种方式，我们将从功能上描述在体内调节CD154mRNA周转的分子机制(S)，并确定它们在CD154基因调控的独特模式以及免疫应答中的作用。这些研究将产生对开发选择性调节CD154表达的方法非常重要的见解。

DESCRIPTION (provided by applicant): CD154 (CD40 ligand) expression by activated T cells plays a central role in the immune response. As such, CD154 expression has been implicated as a major target in the treatment of rheumatic diseases and validated by the demonstrated efficacy of CD154 blockade in animal models of Rheumatoid Arthritis and Systemic Lupus Erythematosus. Furthermore, CD154 gene expression is dysregulated in patients with Systemic Lupus Erythematosus. CD 154 (CD40 ligand) expression exhibits a unique pattern of regulation relative to cytokines. These studies have directly implicated CD154 mRNA degradation. We have established that a polypyrimidine-rich region in the 3'UTR of the CD154 mRNA is necessary and sufficient to reduce chimeric reporter gene expression in both cell lines and normal human CD4+ T lymphocytes, thus identifying this region as a cis-acting element. Furthermore, we have identified two proteins that bind to this polypyrimidine-rich region as distinct splice isoforms (PTB, PTB-T) of the PTB gene. This finding suggest that these proteins are trans-acting factors that determine the function of this region in mRNA decay. Overexpression of the novel smaller splice isoform we call PTB-T, consistently inhibits reporter gene expression in a CD154 3'UWR specific manner in normal human CD4+ T lymphocytes and cell lines. These data suggest the hypothesis that PTB-T interacts with the polypyrimidine-rich region in the CD154 3'UTR to mediate mRNA degradation. We propose to directly test this hypothesis as well as determine the biologic role of PTB-T in the modulation of CD154 mRNA stability. Subsequently, we will identify the pathway(s) by which PTB-T mediates these effects. In this manner, we will functionally delineate the molecular mechanism(s) which regulate CD154 mRNA turnover in vivo and determine their contribution to the unique pattern of CD154 gene regulation as well as the immune response. These studies will generate insights important for the development of approaches that will selectively modulate CD154 expression.