MKP-1 IN Regulation of Inflammatory Cytokine Production

描述（由申请人提供）：本提案的长期目标是了解巨噬细胞中促炎细胞因子生物合成终止的机制。促炎细胞因子，特别是TNF-α和IL-1，在多种人类疾病的发病机制中起重要作用，包括类风湿性关节炎、克罗恩病和感染性休克。LPS刺激的巨噬细胞中TNF-α和IL-1的生物合成受涉及MAP激酶和NF-κ B的复杂信号转导途径调节。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以下假设：MKP-1在p38和JNK MAP激酶的反馈控制中起关键作用，并负责终止LPS刺激的巨噬细胞中促炎细胞因子的产生。此外，MKP-1可被糖皮质激素有效诱导。本提案的第一个具体目的是检验以下假设：LPS刺激后，MKP-1在抑制腹腔和肺泡巨噬细胞中LPS诱导的促炎细胞因子生物合成中起关键作用。第二个具体目的是检验MKP-1在抑制巨噬细胞对革兰氏阳性菌的反应中也充当关键负调节剂的假设。第三个具体目的是检验来自Mkp-1-/-小鼠的原代腹膜巨噬细胞对LPS刺激的反应不同于从Mkp-1 +/+小鼠分离的巨噬细胞的反应的假设。来自Mkp-1敲除小鼠的冷冻保存胚胎通过材料转移协议从Bristol-Myers Squibb Pharmaceutical Research Institute获得。在杰克逊实验室完成了这些胚胎在小鼠中的再生。我们将从Mkp-1 +/+和Mkp-1-/-小鼠中分离腹腔巨噬细胞，并使用这些巨噬细胞确定MKP- 1在LPS应答中的作用，涉及MAP激酶失活和炎症细胞因子产生的控制。我们还将研究Mkp-1基因的缺失是否会损害糖皮质激素对LPS诱导的TNF-α产生的抑制作用。这些研究旨在回答有关细菌感染期间终止巨噬细胞中促炎细胞因子产生的调控机制的关键问题，并揭示开发新的抗风湿/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.