PLATELET REGULATION OF MONOKINE SYNTHESIS

描述：该项目的长期目标是了解如何 血小板调节单核细胞的功能。这一问题尤其突出 重要的是因为血小板与单核细胞在多种情况下相互作用 动脉粥样硬化性疾病申请人最近证明 被激活的血小板，但不是未被刺激的血小板，会立即- 单核细胞早期(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) 单核细胞暴露于活化的血小板后合成和分泌。 其他IE基因，包括肿瘤坏死因子-2(TNF-2)，则不是 已生成。初步结果表明，P-选择素和RANTES (被激活的正常T细胞表达的调节推定为分泌)， IL-8和MCP-1都需要两个血小板衍生分子 单核细胞通过P-选择素和RANTES合成。信令传入 单核细胞通过P-选择素和RANTES将被比作信号 由强效单核细胞激动剂内毒素诱导。在第一个具体目标中， 申请人将进一步描述P-选择素和RANTES如何 调节单核细胞合成。这些研究最初将被定义 在简化的系统中，不同形式的纯化P-选择素和 RANTES被呈递给单核细胞；细胞内的mRNA被保留下来 白介素8、单核细胞趋化蛋白1和肿瘤坏死因子2的蛋白和分泌蛋白 量过了。一个更复杂的涉及血小板-单核细胞的系统 相互作用以确定是否需要P-选择素和RANTES 将检查单核细胞的生产情况。具体目标3将决定 如果P-选择素和RANTES调节单核细胞中的核因子-kappaB活性。核因子- KappaB是一种转录因子，是产生最大IL-8所必需的， MCP-1和肿瘤坏死因子-2的合成，以及由 申请人指出，核因子-kappaB及其抑制因子IkappaB-2， 被P-选择素和RANTES激活。第三个具体目标是 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激酶在翻译上调节许多 蛋白质和其他证据也表明，p70S6激酶可以 调节核因子-kappaB家族成员。初步结果显示，p70 S6 P-2暴露后单核细胞中的激酶活性升高 选择素和RANTES或激活的血小板。此外，对p70的抑制 雷帕霉素激活S6蛋白抑制单核细胞分泌MCP-1 暴露在激活的血小板中。在最终的具体目标中，相互关联 确定P-选择素和RANTES是否定位于破裂的研究 颈动脉斑块将被协调。总而言之，这些结果 将开始定义活化的血小板，通过P-选择素和 RANTES，诱导单核细胞合成。

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.