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，即细胞外信号调节激酶(ERK)和p38MAPK在少突胶质细胞发育和分化中的作用，少突胶质细胞是中枢神经系统髓鞘形成和脱髓鞘的关键细胞。这些研究对于理解多发性硬化症等脱髓鞘疾病中神经发育、髓鞘形成和重新髓鞘形成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，本研究的长期目标将确定调节其分化和髓鞘特异性基因表达的信号转导机制。对生长/分化刺激作出反应的大鼠脑少突胶质前体细胞的原代培养将作为实现以下特定目标的实验模型：*ERK和p38MAPK的特定抑制剂将用于研究这两种激酶在少突胶质前体细胞的有丝分裂诱导和谱系发展(即阶段特异性抗原的表达)中的作用，以及在关键细胞周期调节因子，即细胞周期调节蛋白D1(细胞周期蛋白依赖蛋白激酶的激活物，CDK)和p27(CDK抑制物)水平的相互调节中的作用。*p38MAPK异构体和ERKs在少突胶质细胞分化中的作用将进一步用分子方法来表征，即用p38MAPK异构体(即α和β)和ERKs(即Erki和ERK2)及其上游激酶的主要阴性和/或成分活性形式(即α和β)和它们的上游激酶进行转染，然后分析分化标志物的表达。P38MAPK途径可能的胞质和核靶点(即CREB、ATF2、C/EBP和MEF2C)可能是少突胶质细胞分化的信号。*p38 MAPK介导的少突胶质细胞特异性基因(即髓鞘碱性蛋白、原磷脂蛋白)的转录激活将使用与构成活性形式的激酶共转染的启动子-报告结构来检测。假定的MAPK反应元件和相关的结合活性将通过缺失和凝胶位移分析来确定

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