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的生物合成受到复杂的信号转导通路的调节，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假设，即MKP-1在抑制另外两种具有腹膜和肺泡巨噬细胞特性的巨噬细胞系中抑制内毒素诱导的促炎细胞因子的生物合成方面起关键作用。这一建议的第二个特定目的是验证这一假说，即内毒素通过ERK途径调节的组蛋白H3磷酸化的染色质重塑过程来刺激MKP-1的转录。我们将使用RAW264.7巨噬细胞模型来确定组蛋白H3磷酸化/乙酰化在脂多糖刺激的MKP-1转录诱导中的作用。第三个具体目的是测试假设，即MKP-1在抑制巨噬细胞对革兰氏阳性细菌的反应中也起着关键的负面调节作用。第四个特定目的是验证MKP-1-/-小鼠的原代腹膜巨噬细胞对内毒素刺激的反应与从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，MKP-1基因敲除小鼠可从百时美施贵宝药物研究所获得。我们将分离MKP-1+/+和MKP-1-/-小鼠的腹膜巨噬细胞，并使用这些巨噬细胞来确定MKP-1在内毒素反应中的作用，即MAP-1在MAP激酶失活和控制炎症细胞因子产生方面的作用。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-alpha 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 proinflammatory cytokines in two additional macrophage cell lines with characteristics of peritoneal and alveolar macrophages. The Second Specific Aim of this proposal is to test the hypothesis that LPS stimulates Mkp-1 transcription through a chromatin remodeling process involving histone H3 phosphoacetylation regulated by the ERK pathway. We will determine the role of histone H3 phosphorylation/acetylation in the transcriptional induction of Mkp-1 stimulated by LPS, using the RAW264.7 macrophage model. The Third 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 Fourth 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-1+/+ mice. The Mkp-1 knockout mice are available from Bristol-Myers Squibb Pharmaceutical Research Institute, through a materials transfer agreement. We will isolate peritoneal macrophages from the Mkp-1+/+ 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 of terminating cytokine production in macrophages during bacterial infections, and to reveal novel targets for developing new anti-inflammatory/anti-rheumatic drugs.