TARGETING THE MARCKS-FAMILY PROTEINS TO INHIBIT NEUTROPHIL MIGRATION

描述（由申请人提供）：炎症是维持健康和生命所必需的，最常见于损伤或感染。参与炎症反应的白细胞，如中性粒细胞，对宿主防御入侵微生物和伤口愈合和修复都是必不可少的。然而，反复发作或无法消退的炎症会严重损害宿主组织；造成儿童和成人急性和慢性疾病。尽管它们固有的益处，中性粒细胞经常参与炎症性疾病的病理生理。为了引起组织损伤，中性粒细胞必须通过血管壁迁移到间质组织。这一过程需要中性粒细胞通过重组其肌动蛋白细胞骨架来协调外部环境中的趋化信号。MARCKS家族蛋白，MARCKS和MARCKS样1 （MARCKSL1）是已知的细胞膜和肌动蛋白结合蛋白，受蛋白激酶C （PKC）磷酸化和钙/钙调蛋白结合的调节。MARCKS和/或MARCKSL1对肌动蛋白细胞骨架的动态重组至关重要，包括：t细胞微管组织中心的极化，RAW 264.7细胞中b2整合素的扩散和聚集，神经元样细胞系PC12中PIP2细胞膜微域的维持和动态肌动蛋白结构的形成，以及Ltk成纤维细胞的膜褶皱和片层的形成。该提案将研究MARCKS和MARCKSL1在体外和体内调节人类中性粒细胞迁移中具有必要和非冗余作用的假设。三个相关和互补的特定目的是为了理解：(1)中性粒细胞趋化对中间（IL-8, LTB4）和终末期（fMLP, C5a）趋化剂的需求；(2) marks -家族蛋白调控中性粒细胞迁移的机制；(3)敲除或抑制MARCKS或MARCKSL1对体内中性粒细胞运输和募集的影响。为了实现特定的目标1和2，实验将使用从健康志愿者中分离的原代人中性粒细胞以及可诱导其外观和行为类似原代中性粒细胞的早幼粒细胞白血病细胞系（HL60s）进行。在这些实验中，MARCKS或MARCKSL1抑制将通过以下方式实现：与每种蛋白n端24个氨基酸相同的细胞渗透肽，转基因表达荧光标记的n端截断的MARCKS或MARCKSL1转基因蛋白或siRNA介导的敲除。特异性目标3将使用三种不同的炎症动物模型来完成：斑马鱼的尾巴损伤和TNFa诱导的cremaster损伤和lps介导的小鼠急性肺损伤。该项目的成功结果将直接应用于针对中性粒细胞迁移的治疗策略，以治疗和预防由中性粒细胞募集和激活失调引起或加剧的许多疾病。

DESCRIPTION (provided by applicant): Inflammation is necessary to the maintenance of health and life and occurs most commonly in response to injury or infection. White blood cells involved in the inflammatory response, such as neutrophils, are essential both to host defense from invading microorganisms and wound healing and repair. However, inflammation that is recurrent or fails to resolve can severely damage host tissue; causing both acute and chronic diseases in children and adults. Despite their inherent benefit, neutrophils are often involved in the pathophysiology of inflammatory diseases. In order to cause tissue damage, neutrophils must migrate through the blood vessel wall and into the interstitial tissue. This process requires that neutrophils coordinate chemoattractant signals in their external environment with reorganization of their actin cytoskeleton. The Myristolated Alanine-Rich C-Kinase Substrate (MARCKS) family of proteins, MARCKS and MARCKS-like 1 (MARCKSL1), are known cell membrane and actin binding proteins regulated by protein kinase C (PKC) phosphorylation and calcium/calmodulin binding. MARCKS and/or MARCKSL1 are essential to several events requiring dynamic reorganization of the actin cytoskeleton including: polarization of T-cell microtubule-organizing center, b2 integrin diffusion and clustering in RAW 264.7 cells, maintenance of PIP2 cell membrane microdomains and dynamic actin structure formation in the neuron-like cell line PC12 and membrane ruffling and lamellae formation in Ltk fibroblasts. This proposal will investigate the hypothesis that both MARCKS and MARCKSL1 have essential and non-redundant roles in the regulation of human neutrophil migration in vitro and in vivo. The three related and complimentary specific aims are directed at understanding: (1) the requirement for MARCKS-family proteins in neutrophil chemotaxis toward intermediate (IL-8, LTB4) and end-stage (fMLP, C5a) chemoattractants; (2) the mechanism by which MARCKS-family proteins regulate neutrophil migration; and (3) the effect of knock-down or inhibition of MARCKS or MARCKSL1 on neutrophil trafficking and recruitment in vivo. To accomplish specific aims 1 and 2, experiments will be performed using primary human neutrophils isolated from healthy volunteers as well as a promyelocitic leukemia cell line (HL60s) that can be induced to look and behave like primary neutrophils. In these experiments, MARCKS or MARCKSL1 inhibition will be accomplished using: cell permeant peptides identical to the 24 amino acids of the N-terminus of each protein, transgenic expression of a truncated fluorescently-tagged N-terminus MARCKS or MARCKSL1 transgenic protein or siRNA mediated knockdown. Specific Aim 3 will be accomplished using three different animal models of inflammation: tail-wounding in zebrafish and TNFa induced cremaster injury and LPS-mediated acute lung injury in mice. The successful outcome of this project will have direct application to therapeutic strategies targeting neutrophil migration for the treatment and prevention of numerous diseases caused or exacerbated by a dysregulation in neutrophil recruitment and activation. PUBLIC HEALTH RELEVANCE (provided by applicant): The goal of this research is to investigate promising cellular targets for the treatment and prevention of acute and chronic inflammatory airway diseases. These diseases (i.e. asthma, chronic bronchitis, acute lung injury, chronic obstructive pulmonary disease) are caused or exacerbated by white blood cells known as neutrophils, which migrate from the blood stream into the lungs. By identifying key cellular regulators of neutrophil migration, therapeutics can be developed that decrease neutrophil accumulation in the airways, diminishing and even preventing these devastating diseases.