Pososome Regulated Monocyte/Macrophage Tissue Infiltration

脂质体调节单核细胞/巨噬细胞组织浸润

• 批准号: 8656354
• 负责人: Dianne Cox
• 金额: $ 27.03万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-02-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8656354
• 关键词: Actinin; Actins; Adhesions; Affect; Basement membrane; Binding; Biochemical; Biological Assay; Biosensor; Cells; Chemotaxis; Chronic; Complex; Data; Disadvantaged; Disease; Down-Regulation; Endothelium; Event; Extravasation; F-Actin; Family member; Feedback; Genetic; Guanine Nucleotide Exchange Factors; Hematopoietic; Image; Imaging Techniques; In Vitro; Infiltration; Inflammatory; Injection of therapeutic agent; Kinetics; Label; Lasers; Lead; Leukocytes; Life; Maintenance; Malignant Neoplasms; Mammary Neoplasms; Mammary gland; Mediating; Metalloproteases; Methods; Molecular; Molecular Conformation; Monitor; Mus; Mutation; Natural Immunity; Pathway interactions; Phosphorylation; Physiological; Play; Process; Proline-Rich Domain; Protein Inhibition; Proteins; Recruitment Activity; Regulation; Resolution; Role; Signal Transduction; Signal Transduction Pathway; Small Interfering RNA; Structure; Surface; Tail; Techniques; Testing; Therapeutic; Tissues; Tyrosine Phosphorylation; Veins; Wiskott-Aldrich Syndrome; Work; cellular imaging; chromophore; human disease; in vivo; intersectin 1; intravital imaging; macrophage; migration; monocyte; novel; photoactivation; protein function; src-Family Kinases; tumor; tumor progression

DESCRIPTION (provided by applicant): Podosome regulated monocyte/macrophage tissue infiltration ABSTRACT While monocyte/macrophages (MDs) are essential components of innate immunity, they can also pose disadvantages to the host in certain pathological conditions such as chronic inflammatory diseases or cancer. It has been proposed that podosomes, F-actin rich ventral adhesion and protrusive structures, are employed by a range of leukocytes including MDs to not only mediate matrix degradation but to also play a role in MD chemotaxis and tissue recruitment. Wiskott-Aldrich syndrome protein (WASP), an activator of the actin nucleator Arp2/3 complex expressed exclusively in hematopoietic cells, is required for podosome formation and is critical for proper MD chemotaxis and matrix degradation. We have also recently identified non- redundant roles for WASP and the ubiquitously expressed family member N-WASP. This proposal will determine the precise role of WASP and N-WASP in chemotaxis, transendothelial migration (or diapedesis) and invasion. Previous work by us has demonstrated that tyrosine phosphorylation of WASP is a key factor in podosome turnover and chemotaxis and preliminary data suggest that the Src family tyrosine kinase Hck mediates WASP phosphorylation, which is required for MD diapedesis across an endothelium. In Specific Aim 1 we will determine the role of Intersectin 2L, a protein that only binds to phosphorylated WASP, in mediating WASP function. Intersectin 2L is a guanine nucleotide exchange factor for Cdc42. We hypothesize that WASP promotes the maintenance of Cdc42 activity by recruitment of Intersectin 2L during chemotaxis. A variety of techniques including siRNA, monitoring Cdc42 activity both in vitro and in vivo, and the creation of a photo- activatable form of Intersectin 2 will be utilized to test this hypothesis. The role of podosomes in chemotaxis, invasion and diapedesis will be explored in Specific Aim 2. We will determine the specific requirement of podosomes for chemotaxis using a variety of novel imaging approaches including the use of biosensors to analyze localized activity, photoconversion of podosome-associated actin to monitor kinetics and contributions of actin-rich compartments during chemotaxis, and photoablation of WASP or other components in podosomes by chromophore-assisted laser inactivation. In addition to WASP, we have demonstrated that N-WASP is also required for podosome-mediated matrix degradation and preliminary data suggest a requirement for WASP phosphorylation during transendothelial migration, potentially by affecting podosome dynamics at both the upper and lower surface of the endothelium. We will monitor the localization of podosomes and WASP activity during diapedesis using live cell imaging. The proteolytic function of podosomes during diapedesis and invasion will be assayed using an MMP biosensor and both pharmacological and genetic methods of inhibiting MMPs. Finally in Aim 3 we will determine the role of Hck, WASP and N-WASP in MD recruitment in vivo. MD diapedesis will be monitored using orthotopic tumors in Rag2-/- mice either with endogenous macrophages labeled with GFP in wild-type, Hck- or WASP-deficient mice or by tail vein injection of GFP labeled macrophages with down-regulation or expressing mutations in WASP, N-WASP or Intersectin2. In addition, we will use a new intravital imaging technique to obtain high resolution images of monocytes migrating into breast tumors in live mice. Characterizing the molecular events mediating MD chemotaxis and extravasation will lead to a better understanding of the recruitment of these cells to tumors. In addition, the results of this work may be applicable to chronic inflammatory diseases where MDs appear to play pathogenic roles, suggesting that WASP inhibition may have therapeutic benefits in the treatment of several different human diseases.

描述（由申请人提供）：足体调节的单核细胞/巨噬细胞组织浸润摘要，而单核细胞/巨噬细胞（MDS）是先天免疫的重要组成部分，它们还可以在某些病理状况（例如慢性炎症性疾病或癌症）的某些病理状况下对宿主构成缺点。已经提出，Podosomes，F-肌动蛋白富腹粘附和凸起结构都由包括MD在内的一系列白细胞使用，不仅介导了基质降解，还可以在MD化疗和组织募集中发挥作用。 Wiskott-Aldrich综合征蛋白（WASP）是肌动蛋白成核ARP2/3复合物的活化剂，仅在造血细胞中表达，对足体形成是必需的，对于适当的MD趋化性和基质降解至关重要。我们最近还确定了黄蜂的非冗余角色和无处不在的家庭成员N-WASP。该建议将确定黄蜂和N-WASP在趋化性，跨内皮迁移（或二尿症）和侵袭中的精确作用。我们以前的工作表明，黄蜂的酪氨酸磷酸化是足病变的关键因素，趋化性和初步数据表明，SRC家族酪氨酸激酶HCK介导WASP磷酸化，这是MD二尿所需要的。在特定的目标1中，我们将确定相交2L的作用，该蛋白仅与磷酸化的黄蜂结合，在介导的黄蜂功能中。 Intersectin 2L是Cdc42的鸟嘌呤核苷酸交换因子。我们假设黄蜂通过在趋化性过程中募集索塞蛋白2L促进CDC42活性的维持。各种技术，包括体外和体内监测Cdc42活性，以及​​创建可激活形式的相交蛋白2的形式，以检验该假设。 The role of podosomes in chemotaxis, invasion and diapedesis will be explored in Specific Aim 2. We will determine the specific requirement of podosomes for chemotaxis using a variety of novel imaging approaches including the use of biosensors to analyze localized activity, photoconversion of podosome-associated actin to monitor kinetics and contributions of actin-rich compartments during chemotaxis, and photoablation of WASP or发色团辅助激光失活中的其他成分。除WASP外，我们还证明了足​​体介导的基质降解还需要N-WASP，并且初步数据表明，在跨内皮迁移过程中需要WASP磷酸化，这可能是通过影响内皮内皮层上层和下表面的足体动力学。我们将使用活细胞成像监测二尿症期间的足体和黄蜂活性的定位。二尿和侵袭过程中足体的蛋白水解功能将使用MMP生物传感器以及抑制MMP的药理学和遗传方法测定。最后，在AIM 3中，我们将确定HCK，WASP和N-WASP在体内MD招募中的作用。 MD二尿症将使用rag2 - / - 小鼠中的原位肿瘤进行监测，野生型巨噬细胞在野生型，HCK-或WASP缺乏小鼠中标记为GFP，或者通过尾静脉注射GFP在wASP，n-n-n-n-n-weScect，n-Interscect sectect，n sectement或Interscect的GFP标记的GFP巨噬细胞注入巨噬细胞。此外，我们将使用一种新的浸润成像技术来获取活小鼠中迁移到乳腺肿瘤中的单核细胞的高分辨率图像。表征介导MD趋化性和渗出的分子事件将使人们更好地了解这些细胞对肿瘤的募集。此外，这项工作的结果可能适用于MD似乎起着致病作用的慢性炎症性疾病，这表明黄蜂抑制作用可能在治疗几种不同的人类疾病方面具有治疗益处。