MAP KINASES IN OLIGODENDROCYTE CELL SIGNALING

描述（申请人提供）：本项目旨在研究丝裂原活化蛋白激酶（MAPK）家族的两个相关成员的作用，即，细胞外信号调节激酶（ERK）和p38 MAPK在少突胶质细胞（CNS中髓鞘形成和脱髓鞘的关键细胞）的发育和分化中的作用。这些研究对于理解脱髓鞘疾病如多发性硬化症的神经发育、髓鞘形成和髓鞘再生的分子机制具有重要意义。由于少突胶质细胞的发育受到环境信号的严格控制，因此本研究的长期目标是确定调节其分化和髓鞘特异性基因表达的信号转导机制。响应于生长/分化刺激的大鼠脑少突胶质细胞祖细胞的原代培养物将用作实验模型以实现以下特定目标：* ERK和p38 MAPK的特异性抑制剂将用于研究两种激酶在促有丝分裂诱导和谱系进展中的作用（即，阶段特异性抗原的表达）以及关键细胞周期调节因子水平的相互调节，即，细胞周期蛋白D1（细胞周期蛋白依赖性蛋白激酶，cdk的激活剂）和p27（cdk抑制剂）。* p38 MAPK亚型和ERK在少突胶质细胞分化中的作用将使用分子方法进一步表征，即，用p38 MAPK同种型的显性负性和/或组成型活性形式转染（即，α和β）和ERK（即，ERK 1和ERK 2）以及它们的上游激酶，然后分析分化标志物的表达。假定的细胞质和核靶点（即，CREB、ATF2、C/EBP和MEF2C）。* p38 MAPK介导的少突胶质细胞特异性基因的转录激活（即，髓鞘碱性蛋白、原脂蛋白）将使用与激酶的组成型活性形式共转染的启动子-报道基因构建体进行检测。通过缺失和凝胶位移分析确定推定的MAPK响应元件和相关结合活性

DESCRIPTION (provided by applicant): This project is designed to investigate the roles of two related members of the mitogen-activated protein kinase (MAPK) family, i.e., extracellular signal-regulated kinase (ERK) and p38 MAPK in the development and differentiation of oligodendrocytes, the key cells of myelination and demyelination in the CNS. The studies have implications for an understanding of the molecular mechanisms of neural development, myelination and remyelination in demyelinating diseases such as multiple sclerosis. Since the development of oligodendrocytes is stringently controlled by environmental signals, the long-range objective of this research will define signal transduction mechanisms regulating their differentiation and the expression of myelin-specific genes. Primary cultures of rat brain oligodendrocyte progenitors responding to growth/differentiation stimuli will serve as the experimental model to accomplish the following specific aims: * Specific inhibitors of ERK and p38 MAPK will be used to investigate the roles of the two kinases in mitogenic induction and lineage progression (i.e., expression of stage-specific antigens) of oligodendrocyte progenitors as well as in the reciprocal regulation of the levels of key cell cycle regulators, i.e., cyclin D1 (an activator of cyclin-dependent protein kinase, cdk) and p27 (a cdk inhibitor). * The roles of p38 MAPK isoforms and ERKs in oligodendrocyte differentiation will be further characterized using a molecular approach, i.e., transfection with dominant negative and/or constitutively active forms of p38 MAPK isoforms (i.e., alpha and beta) and ERKs (i.e., ERKI and ERK2) as well as their upstream kinases followed by an analysis of the expression of differentiation markers. Putative cytoplastic and nuclear targets (i.e., CREB, ATF2, C/EBP and MEF2C) of p38 MAPK pathway that may signal oligodendrocyte differentiation will be characterized. * p38 MAPK-mediated transcriptional activation of oligodendrocyte-specific genes (i.e., myelin basic protein, protoelipid protein) will be examined using promoter-reporter constructs co-transfected with constitutively active forms of the kinases. Putative MAPK-responsive elements and relevant binding activities will be determined by deletion and gel shift analyses