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，尤其是tnf - α和IL-1，在多种人类疾病的发病机制中发挥重要作用，包括类风湿关节炎、克罗恩病和感染性休克。在lps刺激的巨噬细胞中，TNF-a和IL-1的生物合成均受MAP激酶和nf - κ b等复杂信号转导通路的调控。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MKP-1在lps刺激的巨噬细胞中对p38和JNK MAP激酶的反馈控制中起关键作用，并负责终止促炎细胞因子的产生。此外，MKP-1可被糖皮质激素有效诱导。本研究的第一个具体目的是验证MKP-1在LPS刺激后腹膜和肺泡巨噬细胞抑制LPS诱导的促炎细胞因子的生物合成中起关键作用的假设。第二个特定目的是验证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负调节作用的假设。第三个特定目的是验证Mkp-1-/-小鼠原代腹腔巨噬细胞对LPS刺激的反应与Mkp-1 +/+小鼠分离的巨噬细胞的反应不同的假设。通过材料转让协议，从Mkp-1基因敲除小鼠中获得的冷冻胚胎来自百时美施贵宝制药研究所。杰克逊实验室已经完成了将这些胚胎再生成老鼠的工作。我们将从MKP- 1 +/+和MKP- 1-/-小鼠中分离腹腔巨噬细胞，并利用这些巨噬细胞来确定MKP- 1在LPS反应中关于MAP激酶失活和炎症细胞因子产生控制的作用。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。这些研究旨在回答在细菌感染期间巨噬细胞终止促炎细胞因子产生的调节机制的关键问题，并揭示开发新的抗炎/抗风湿病药物的新靶点。

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.