REGULATION OF CD154 EXPRESSION

描述（由申请人提供）：活化T细胞表达的CD 154（CD 40配体）在免疫应答中起着重要作用。因此，CD 154表达已被认为是治疗风湿性疾病的主要靶点，并通过在风湿性关节炎和系统性红斑狼疮动物模型中证实的CD 154阻断的功效得到验证。此外，CD 154基因表达在系统性红斑狼疮患者中失调。CD 154（CD 40配体）表达相对于细胞因子表现出独特的调节模式。这些研究直接涉及CD 154 mRNA降解。我们已经确定，在CD 154 mRNA的3 'UTR中的富含多嘧啶的区域是必要的，并且足以减少嵌合报告基因在细胞系和正常人CD 4 + T淋巴细胞中的表达，从而将该区域鉴定为顺式作用元件。此外，我们已经确定了两种蛋白质结合到这个多聚嘧啶丰富的区域作为不同的剪接异构体（PTB，PTB-T）的PTB基因。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衰变中的功能。我们称之为PTB-T的新型较小剪接异构体的过表达在正常人CD 4 + T淋巴细胞和细胞系中以CD 154 3 'UWR特异性方式持续抑制报告基因表达。 这些数据表明PTB-T与CD 154 3 'UTR中的多聚嘧啶富集区相互作用以介导mRNA降解的假设。我们建议直接检验这一假设，并确定PTB-T在调节CD 154 mRNA稳定性中的生物学作用。随后，我们将确定PTB-T介导这些效应的途径。通过这种方式，我们将在功能上描述体内调节CD 154 mRNA周转的分子机制，并确定其对CD 154基因调节的独特模式以及免疫应答的贡献。这些研究将产生重要的见解的方法，将选择性地调节CD 154表达的发展。

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.