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）：炎症对于维持健康和生命是必要的，并且最常响应伤害或感染而发生。涉及炎症反应的白细胞，例如中性粒细胞，对于侵犯微生物以及伤口愈合和修复的防御至关重要。但是，复发或无法解决的炎症会严重损害宿主组织。引起儿童和成人的急性和慢性疾病。尽管它们固有的好处，但中性粒细胞通常与炎症性疾病的病理生理有关。为了引起组织损伤，中性粒细胞必须通过血管壁迁移并进入间质组织。这个过程要求中性粒细胞在其外部环境中协调趋化剂信号，并重组其肌动蛋白细胞骨架。蛋白质，MARCKS和MARCKS样1（MARCKSL1）的肉豆蔻氨酸富含丙氨酸的C-激酶底物（MARCKS）家族是已知的细胞膜和肌动蛋白结合蛋白，该蛋白质激酶C（PKC）磷酸化和钙/钙调蛋白结合。 MARCKS和/或MARCKSL1对于需要进行肌动蛋白细胞骨架的几个事件至关重要LTK成纤维细胞。该提议将调查Marcks和Marcksl1在体外和体内的人类嗜中性粒细胞迁移方面具有重要和非冗余作用的假设。这三个相关和免费的特定目的是针对理解的：（1）中性粒细胞趋化性中的马克克家族蛋白朝向中间体（IL-8，LTB4）和末端阶段（FMLP，C5A）化学剂的要求； （2）Marcks家庭蛋白调节中性粒细胞迁移的机制； （3）马克克斯或马克克尔1对中性粒细胞贩运和体内招募的敲低或抑制的影响。为了完成特定的目标1和2，将使用从健康志愿者中分离出的原代人性中性粒细胞以及临时性白血病细胞系（HL60S）进行实验，可以诱使看起来像原发性中性粒细胞。在这些实验中，将使用：使用：与每种蛋白质N末端的24个氨基酸相同的细胞渗透性肽，将抑制的细胞固定肽，截短的荧光症状的N-末端MARCKS或MARCKSL1转基因蛋白或sirna介导的敲低的转基因表达。将使用三种不同的炎症动物模型来完成特定的目标3：斑马鱼和TNFA诱导的Cremaster损伤和小鼠LPS介导的急性肺损伤。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，以通过中性粒细胞募集和激活的失调引起或加剧许多疾病。 公共卫生相关性（由申请人提供）：这项研究的目的是调查有希望的细胞靶标，以治疗和预防急性和慢性炎症性气道疾病。这些疾病（即哮喘，慢性支气管炎，急性肺损伤，慢性阻塞性肺疾病）是由称为嗜中性粒细胞的白细胞引起或加剧的，这些白细胞从血液中迁移到肺部。通过鉴定中性粒细胞迁移的关键细胞调节剂，可以开发出疗法，从而减少中性粒细胞在气道中的积累，减少甚至阻止这些毁灭性疾病。