TARGETING THE MARCKS-FAMILY PROTEINS TO INHIBIT NEUTROPHIL MIGRATION

靶向马克斯家族蛋白抑制中性粒细胞迁移

• 批准号: 8354247
• 负责人: Mary Katherine Sheats
• 金额: $ 12.69万
• 依托单位: NORTH CAROLINA STATE UNIVERSITY RALEIGH
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8354247
• 关键词: Actin-Binding Protein; Actins; Acute; Acute Disease; Acute Lung Injury; Adhesions; Adult; Adult Respiratory Distress Syndrome; Affect; Alanine; Amino Acids; Animal Model; Asthma; Binding; Binding Proteins; Blood; Blood Vessels; Calcium; Calmodulin; Cell Line; Cell Maintenance; Cell membrane; Cells; Cessation of life; Chemotactic Factors; Chemotaxis; Child; Chronic; Chronic Bronchitis; Chronic Disease; Chronic Obstructive Airway Disease; Cognitive deficits; Complement 5a; Country; Critical Illness; Cytoskeleton; Data; Diagnosis; Diffusion; Disease; Disease Outcome; Disease Progression; Environment; Event; Fibroblasts; Functional disorder; Goals; Health; Hemorrhage; Host Defense; Human; IL8 gene; Immigration; Impaired health; In Vitro; Infection; Inflammation; Inflammation Mediators; Inflammatory; Inflammatory Response; Injury; Integrins; Invaded; Knock-out; Leukocytes; Leukotriene B4; Life; Lung; MARCKS gene; MARCKS-related protein; Maintenance; Mediating; Mediator of activation protein; Membrane; Membrane Microdomains; Microtubule-Organizing Center; Mus; N-terminal; Neurons; Neutrophil Activation; Neutrophil Infiltration; Outcome; Pathogenesis; Patients; Peptides; Persons; Phase; Phosphatidylinositol 4,5-Diphosphate; Phosphorylation; Phosphotransferases; Pneumonia; Population; Prevention; Prevention strategy; Process; Protein Family; Protein Inhibition; Protein Kinase C; Proteins; Recurrence; Regulation; Research; Respiratory Failure; Role; Sepsis; Signal Transduction; Small Interfering RNA; Staging; Stomach; Stream; Structure; Structure of parenchyma of lung; T-Lymphocyte; Tail; Therapeutic; Tissues; Transfusion; Transgenic Organisms; Trauma; United States; Vital capacity; Wound Healing; Zebrafish; airway inflammation; cellular targeting; direct application; health related quality of life; healthy volunteer; in vivo; inhibitor/antagonist; interstitial; knock-down; leukemia; lung injury; microorganism; migration; mortality; mouse model; mutant; neutrophil; prevent; repaired; research study; response to injury; trafficking

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.

描述（由申请人提供）：炎症是维持健康和生命所必需的，最常见于对损伤或感染的反应。参与炎症反应的白色血细胞，如中性粒细胞，对于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，反复发作或未能解决的炎症会严重损害宿主组织;导致儿童和成人的急性和慢性疾病。尽管它们具有固有的益处，但中性粒细胞通常参与炎性疾病的病理生理学。为了引起组织损伤，中性粒细胞必须穿过血管壁并进入间质组织。这一过程需要中性粒细胞在其外部环境中协调化学引诱物信号，并重组其肌动蛋白细胞骨架。富含豆蔻酸的丙氨酸C-激酶底物（MARCKS）蛋白家族MARCKS和MARCKS样1（MARCKSL 1）是已知的细胞膜和肌动蛋白结合蛋白，其由蛋白激酶C（PKC）磷酸化和钙/钙调蛋白结合调节。MARCKS和/或MARCKSL 1对需要动态重组肌动蛋白细胞骨架的几个事件是必不可少的，包括：T细胞微管组织中心的极化，RAW 264.7细胞中的b2整联蛋白扩散和聚集，神经元样细胞系PC 12中PIP 2细胞膜微区的维持和动态肌动蛋白结构的形成，以及Ltk成纤维细胞中的膜皱褶和层形成。本提案将调查的假设，即MARCKS和MARCKSL 1有必要的和非冗余的作用，在体外和体内的人中性粒细胞迁移的调节。这三个相关的和互补的具体目标是为了理解：（1）中性粒细胞趋化性对MARCKS家族蛋白的需求，（IL-8，LTB 4）和终末期（2）MARCKS家族蛋白调节中性粒细胞迁移的机制;和（3）MARCKS或MARCKSL 1的敲低或抑制对体内中性粒细胞运输和募集的影响。为了实现特定目标1和2，将使用从健康志愿者中分离的原代人中性粒细胞以及可被诱导为外观和行为类似于原代中性粒细胞的原代骨髓性白血病细胞系（HL 60）进行实验。在这些实验中，MARCKS或MARCKSL 1抑制将使用与每种蛋白质的N-末端的24个氨基酸相同的细胞渗透肽、截短的荧光标记的N-末端MARCKS或MARCKSL 1转基因蛋白的转基因表达或siRNA介导的敲低来实现。具体目标3将使用三种不同的炎症动物模型来实现：斑马鱼中的尾损伤和小鼠中TNF α诱导的提睾肌损伤和LPS介导的急性肺损伤。该项目的成功结果将直接应用于靶向中性粒细胞迁移的治疗策略，用于治疗和预防由中性粒细胞募集和激活失调引起或加重的多种疾病。 公共卫生相关性（由申请人提供）：本研究的目标是研究用于治疗和预防急性和慢性炎症性气道疾病的有前途的细胞靶点。这些疾病（即哮喘、慢性支气管炎、急性肺损伤、慢性阻塞性肺病）是由称为嗜中性粒细胞的白色血细胞引起或加重的，所述白细胞从血流迁移到肺部。通过鉴定嗜中性粒细胞迁移的关键细胞调节因子，可以开发减少气道中嗜中性粒细胞积聚的治疗方法，从而减少甚至预防这些毁灭性疾病。