MKP-1 IN Regulation of Inflammatory Cytokine Production

描述(由申请人提供)：这项建议的长期目标是了解巨噬细胞中促炎症细胞因子生物合成终止的机制。促炎症细胞因子，尤其是肿瘤坏死因子-α和白介素1，在许多人类疾病的发病机制中起着重要作用，包括类风湿性关节炎、克罗恩病和感染性休克。巨噬细胞中肿瘤坏死因子-α和白介素1的生物合成受复杂的信号转导途径调节，涉及MAP激酶和核因子-kappaB。我们实验室对RAW264.7细胞的初步研究提供了强有力的证据，支持这一假设，即MKP-1在p38和JNK MAP激酶的反馈控制中发挥关键作用，并负责终止内毒素刺激的巨噬细胞产生促炎细胞因子。此外，糖皮质激素对MKP-1有很强的诱导作用。本方案的第一个具体目的是验证MKP-1在抑制LPS刺激后腹膜和肺泡巨噬细胞中由LPS诱导的前炎性细胞因子的生物合成中起关键作用的假说。第二个具体目的是验证MKP-1在抑制巨噬细胞对革兰氏阳性细菌的反应中也起到关键的负调节作用的假设。第三个特异性目的是验证MKP-1-/-小鼠的原代巨噬细胞对内毒素刺激的反应与从MKP-L+/+小鼠分离的巨噬细胞的反应不同的假设。来自MKP-1基因敲除小鼠的冷冻胚胎来自百时美施贵宝药物研究所，通过材料转移协议获得。杰克逊实验室已经完成了将这些胚胎再生为老鼠的工作。我们将分离MKP-L+/+和MKP-1-/-小鼠的腹膜巨噬细胞，并利用这些巨噬细胞来确定MKP-1在内毒素反应中的作用，即MAPK失活和控制炎症细胞因子的产生。我们还将研究MKP-1基因的缺失是否会削弱糖皮质激素对内毒素诱导的肿瘤坏死因子-α产生的抑制作用。这些研究旨在回答有关在细菌感染期间终止巨噬细胞产生促炎细胞因子的调控机制的关键问题，并揭示开发新的抗炎/抗风湿药物的新靶点。

DESCRIPTION (provided by applicant): The long-term objective of this proposal is to understand the mechanisms responsible for the termination of proinflammatory cytokine biosynthesis in macrophages. Proinflammatory cytokines, TNF-alpha and IL-1 in particular, play an important role in the pathogenesis of a variety of human diseases, including rheumatoid arthritis, Crohn's disease, and septic shock. Biosyntheses of both TNF-a and IL-1 in LPS-stimulated macrophages are regulated by complex signal transduction pathways involving MAP kinases and NF-kappaB. Preliminary studies in our laboratory with RAW264.7 cells provide strong evidence to support the hypothesis that MKP-1 plays a critical role in the feedback control of p38 and JNK MAP kinases and is responsible for the termination of pro-inflammatory cytokine production in LPS-stimulated macrophages. Moreover, MKP-1 is potently induced by glucocorticoids. The First Specific Aim of the present proposal is to test the hypothesis that MKP-1 plays critical roles in restraining the LPS-induced biosynthesis of proinflarnmatory cytokines in both peritoneal and alveolar macrophages after LPS stimulation. The Second Specific Aim is to test the hypothesis that MKP-1 also acts as a critical negative regulator in the restraint of macrophage responses to Gram-positive bacteria. The Third Specific Aim is to test the hypothesis that the responses to LPS stimulation of primary peritoneal macrophages from Mkp-1-/- mice differ from the responses of macrophages isolated from Mkp-l+/+ mice. The cryopreserved embryos derived from the Mkp-1 knockout mice are obtained from Bristol-Myers Squibb Pharmaceutical Research Institute, through a materials transfer agreement. Regeneration of these embryos into mice has been accomplished in The Jackson Laboratory. We will isolate peritoneal macrophages from the Mkp-l+/+ and Mkp-1-/- mice and use these macrophages to determine the role of MKP- 1 in the responses to LPS, with respect to MAP kinase inactivation and control of inflammatory cytokine production. We will also examine whether the lack of Mkp-1 gene will compromise the suppressant effects of glucocorticoids on TNF-alpha production induced by LPS. These studies are designed to answer pivotal questions regarding the regulatory mechanisms responsible for terminating proinflammatory cytokine production in macrophages during bacterial infections, and to reveal novel targets for developing new antiinflammatory/ anti-rheumatic drugs.