PLATELET REGULATION OF MONOKINE SYNTHESIS

描述：本项目的长期目标是了解如何 血小板调节单核细胞功能。 这个问题尤其 这一点很重要，因为血小板与单核细胞在多种 动脉粥样硬化性疾病申请人最近证明， 激活的血小板，而不是未受刺激的血小板，诱导立即- 单核细胞中的早期（IE）基因表达。 在表达的IE基因中， 白细胞介素-8（IL-8）和单核细胞趋化蛋白-1（MCP-1）， 由暴露于活化血小板的单核细胞合成和分泌。 其他IE基因，包括肿瘤坏死因子-2（TNF-2）， 生成的. 初步结果表明，P-选择素和RANTES （活化后调节正常T细胞表达假定分泌）， IL-8和MCP-1都需要两种血小板衍生分子 通过P-选择素和RANTES在单核细胞中合成。信令 单核细胞通过P-选择素和RANTES将比较信号转导 由LPS诱导，一种有效的单核细胞激动剂。在第一个具体目标中， 申请人将进一步表征P-选择素和RANTES 调节单核因子的合成。这些研究最初将被定义为 在还原系统中，不同形式的纯化P-选择素和 RANTES被呈递给单核细胞;细胞mRNA，细胞保留 蛋白质，以及IL-8、MCP-1和TNF-2的分泌蛋白质将被 测定了 涉及血小板-单核细胞的更复杂的系统 相互作用，以确定是否需要P-选择素和RANTES 将检测单核因子的产生。 第3章将决定 如果P-选择素和RANTES调节单核细胞中NF-κ B活性。 NF- κ B是最大IL-8所需的转录因子， MCP-1和TNF-2的合成，以及 申请人指出，NF-κ B及其抑制因子I κ B-2， 被P-选择素和RANTES激活。 第三个具体目标将 确定P-选择素和RANTES是否调节p70 S6激酶活性， 单核细胞 已知p70 S6激酶对多种细胞因子的表达具有抑制性调节作用， 蛋白质和其他证据也表明，p70 S6激酶可以 调节NF-κ B家族成员。初步结果表明，p70 S6 在暴露于P- 选择素和RANTES或活化血小板。 此外，抑制p70 雷帕霉素引起的S6激酶活性减弱单核细胞分泌MCP-1 暴露在活化的血小板中 在最后的具体目标，相关 研究以确定P-选择素和RANTES是否位于破裂的 协调颈动脉斑块。 这些结果一起 将开始定义如何激活血小板，通过P-选择素和 RANTES，诱导单核因子合成。

DESCRIPTION: The long term goal of this project is to understand how platelets regulate monocyte function. This issue is particularly important since platelets interact with monocytes in a variety of atherosclerotic disorders. The applicant recently demonstrated that activated platelets, but not unstimulated platelets, induce immediate- early (IE) gene expression in monocytes. Among the IE genes expressed, interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) were synthesized and secreted by monocytes exposed to activated platelets. Other IE genes, including tumor necrosis factor-2 (TNF-2), were not generated. Initial results indicate that P-selectin and RANTES (Regulated upon Activation Normal T Cell Expressed presumed Secreted), two platelet-derived molecules, are required for both IL-8 and MCP-1 synthesis in monocytes through P-selectin and RANTES. Signaling in monocytes through P-selectin and RANTES will be compared to signaling induced by LPS, a potent monocyte agonist. In the first Specific Aim, the applicant will further characterize how P-selectin and RANTES regulate monokine synthesis. These studies will initially be defined in a reduced system where different forms of purified P-selectin and RANTES are presented to monocytes; and cellular MRNA, cellular retained protein, and secreted protein for IL-8, MCP-1, and TNF-2 will be measured. A more complex system involving platelet-monocyte interactions to determine if P-selectin and RANTES are required for monokine production will be examined. Specific Aim 3 will determine if P-selectin and RANTES regulate NF-kappaB activity in monocytes. NF- kappaB is a transcription factor that is required for maximal IL-8, MCP-1, and TNF-2 synthesis, and initial results developed by the applicant indicate that NF-kappaB, and its inhibitory factor IkappaB-2, are activated by P-selectin and RANTES. The third Specific Aim will determine if P-selectin and RANTES regulate p70 S6 kinase activity in monocytes. p70 S6 kinase is known to translationally regulate many proteins and other evidence also indicates that p70 S6 kinase can regulate NF-kappaB family members. Initial results suggest that p70 S6 kinase activity is increased in monocytes following exposure to P- selectin and RANTES or activated platelets. Moreover, inhibition of p70 S6 kinase activity by rapamycin attenuates MCP-1 secretion by monocytes exposed to activated platelets. In the final Specific Aim, correlative studies to determine if P-selectin and RANTES are localized in ruptured carotid arterial plaques will be coordinated. Together, these results will begin defining how activated platelets, through P-selectin and RANTES, induce monokine synthesis.