Pososome Regulated Monocyte/Macrophage Tissue Infiltration

摘要单核细胞/巨噬细胞（MDs）是先天免疫的重要组成部分，但在某些病理条件下，如慢性炎症性疾病或癌症，它们也会对宿主造成不利影响。有研究表明，足质体是一种富含f -肌动蛋白的腹侧粘连和突出结构，被包括MDs在内的一系列白细胞利用，不仅介导基质降解，而且在MDs趋化性和组织募集中发挥作用。Wiskott-Aldrich综合征蛋白（WASP）是一种仅在造血细胞中表达的肌动蛋白核子Arp2/3复合物的激活剂，是足小体形成所必需的，对适当的MD趋化性和基质降解至关重要。我们最近也发现了WASP和普遍表达的家族成员N-WASP的非冗余作用。这一建议将确定WASP和N-WASP在趋化性、跨内皮迁移（或渗出）和侵袭中的确切作用。我们之前的工作已经证明，WASP的酪氨酸磷酸化是足小体周转和趋化的关键因素，初步数据表明，Src家族酪氨酸激酶Hck介导WASP磷酸化，这是MD跨内皮浸润所必需的。在Specific Aim 1中，我们将确定只与磷酸化WASP结合的蛋白Intersectin 2L在介导WASP功能中的作用。交叉蛋白2L是Cdc42的鸟嘌呤核苷酸交换因子。我们假设WASP在趋化过程中通过募集Intersectin 2L来促进Cdc42活性的维持。包括siRNA在内的各种技术，监测Cdc42在体外和体内的活性，以及创建可光激活形式的Intersectin 2将被用来验证这一假设。我们将在特异性目标2中探讨足质体在趋化、侵袭和迁移中的作用。我们将使用各种新颖的成像方法来确定足质体对趋化性的具体要求，包括使用生物传感器来分析局部活性，利用足质体相关肌动蛋白的光转化来监测趋化过程中肌动蛋白丰富的区室的动力学和贡献，以及通过发色团辅助激光灭活光消融足质体中的WASP或其他成分。除了WASP，我们已经证明了N-WASP在podosome介导的基质降解中也是必需的，初步数据表明在跨内皮迁移过程中需要WASP磷酸化，这可能通过影响内皮上下表面的podosome动力学来实现。我们将利用活细胞成像技术监测足小体的定位和WASP的活性。我们将使用MMP生物传感器和抑制MMPs的药理学和遗传学方法来检测足质体在渗出和侵袭过程中的蛋白水解功能。最后，在Aim 3中，我们将确定Hck、WASP和N-WASP在体内MD募集中的作用。在Rag2-/-小鼠的原位肿瘤中，通过野生型、Hck-或WASP缺陷小鼠中标记GFP的内源性巨噬细胞，或通过尾静脉注射标记有下调或表达WASP、N-WASP或Intersectin2突变的GFP的巨噬细胞，来监测MD脱落。此外，我们将使用一种新的活体成像技术来获得活小鼠乳腺肿瘤中单核细胞迁移的高分辨率图像。表征介导MD趋化性和外渗的分子事件将有助于更好地理解这些细胞向肿瘤的募集。此外，本工作的结果可能适用于MDs似乎起致病作用的慢性炎症性疾病，这表明抑制WASP可能在治疗几种不同的人类疾病中具有治疗益处。

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.