Novel Functions of Red Cell Proteins Lu and LW

红细胞蛋白 Lu 和 LW 的新功能

• 批准号: 7729026
• 负责人: JOEL A CHASIS
• 金额: $ 43.05万
• 依托单位: UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-25 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7729026
• 关键词: Abnormal Red Blood Cell; Acute; Address; Adhesions; Adhesives; Affect; Affinity; Amino Acids; Anemia; Antibodies; Area; Autoimmune Diseases; Binding; Binding Sites; Biochemical; Biological Assay; Biology; Blocking Antibodies; Bone Marrow; Cell Adhesion; Cell Adhesion Molecules; Cells; Cytoplasmic Tail; Cytoskeleton; Data; Defect; Disease; ECM receptor; Erythroblasts; Erythrocytes; Erythroid; Erythroid Cells; Erythropoiesis; Erythropoietin; Exhibits; Extracellular Matrix; Fluorescent Probes; Funding; Glycoproteins; Goals; Hemorrhage; Immunofluorescence Microscopy; In Situ; Inherited; Integrins; Island; Knockout Mice; Laminin; Life; Maps; Marrow; Mediating; Mediator of activation protein; Membrane; Molecular; Mus; Mutagenesis; Myelofibrosis; Nature; Output; Pathology; Physiological; Play; Polychromatophilic Erythroblast; Production; Proliferating; Pronormoblasts; Protein Isoforms; Proteins; RNA Splicing; Relative (related person); Research; Reticulocytes; Role; Sickle Cell Anemia; Site; Spectrin; Staging; Stress; Structure; Surface; Techniques; Testing; Thalassemia; Tissues; Vascular Cell Adhesion Molecule-1; Wild Type Mouse; Work; base; bioimaging; biological adaptation to stress; erythroid differentiation; hematopoietic tissue; human disease; in vitro Assay; in vivo; insight; macrophage; migration; mouse model; novel; protein function; public health relevance; reconstitution; research study; response; synthetic peptide

DESCRIPTION (provided by applicant): The long-term objectives of our research are to develop a mechanistic understanding of how cell-cell and cell-extracellular matrix adhesive interactions regulate erythropoiesis. Erythroblasts differentiate in erythroblast islands, composed of erythroblasts surrounding a macrophage. However, little is known regarding the purpose of adhesive attachments in these erythroid niches. The goals of this application are to explore the function of adhesion molecules LW (termed ICAM-4) and Lutheran (Lu) glycoproteins. We have shown that erythroid islands are markedly decreased in ICAM-4 null bone marrow; ICAM-4/1V integrin adhesion mediates erythroblast-macrophage attachment; and normal but not Lu null erythroblasts bind matrix laminin in a differentiation stage-specific manner. We propose to 1) Explore the hypothesis that ICAM-4 is required for amplification of red blood cell production in stress erythropoiesis. After inducing erythropoietic stress, we will examine potential mechanisms for the deficient response in ICAM-4 knockout mice including: decreased proliferative potential; impaired recruitment of early stage erythroblasts to erythroid islands; and decreased island integrity. We will characterize erythroid proliferation and differentiation in the context of bone marrow and splenic islands employing live cell erythroid island assays, as well as colony assays, and erythroid island cultures. 2) Test the hypothesis that ICAM-4 has dual functions in erythroid islands by modulating both erythroblast-macrophage and erythroblast-erythroblast adhesion via interactions with different integrins. Building on preliminary evidence that erythroblasts bind ICAM-4 via 1421, we will identify the ICAM-4 site involved in 1421 binding; determine the effect on wild type and ICAM-4 null erythroid islands of blocking ICAM-4/1421 binding using fluorescent probes and bioimaging; and analyze whether inhibiting ICAM-4/1421 and ICAM-4/1V binding are additive. Mouse models for deficiency of marrow and splenic macrophages will also be employed to study the in vivo importance of erythroblast-macrophage interactions for erythropoiesis. 3) Explore whether Lu-laminin adhesion localizes erythroid islands to Laminin-containing regions in bone marrow and whether it regulates differentiation and/or proliferation. We will map the distribution of 15 laminin in bone marrow sections by immunofluorescence microscopy and determine its physical relationship to islands, comparing wild type and Lu knockout mice; and analyze and contrast proliferation and differentiation in Lu null and wild type mice by characterizing bone marrow islands and performing colony assays and erythroblast cultures in the presence and absence of laminin. The proposed objectives are relevant to obtaining an understanding of how red blood cell production is increased in response to multiple inherited and acquired anemias including sickle cell disease, hereditary membrane disorders, autoimmune red blood cell destruction and blood loss. They are also relevant to diseases associated with bone marrow fibrosis and abnormal red cell production. PUBLIC HEALTH RELEVANCE: These research objectives are relevant to obtaining increased understanding of how red blood cell production is increased in response to multiple inherited and acquired anemias including thalassemia, sickle cell disease, hereditary membrane disorders, autoimmune red blood cell destruction and blood loss. They are also relevant to diseases associated with bone marrow fibrosis and abnormal red blood cell production.

描述（由申请人提供）：我们研究的长期目标是从机制上理解细胞-细胞和细胞-细胞外基质粘附相互作用如何调节红细胞生成。有红细胞在有红细胞岛中分化，该岛由巨噬细胞周围的有红细胞组成。然而，人们对这些红细胞生态位中粘附附着物的目的知之甚少。此应用的目标是探索粘附分子 LW（称为 ICAM-4）和路德 (Lu) 糖蛋白的功能。我们已经表明，ICAM-4 无效骨髓中的红细胞岛明显减少； ICAM-4/1V 整合素粘附介导成红细胞-巨噬细胞附着；正常但非 Lu 无效的成红细胞以分化阶段特异性方式结合基质层粘连蛋白。我们建议 1) 探索 ICAM-4 是应激性红细胞生成过程中红细胞生成放大所必需的假设。诱导红细胞生成应激后，我们将检查 ICAM-4 敲除小鼠反应缺陷的潜在机制，包括： 增殖潜力降低；早期有红细胞向红细胞岛的募集受损；并降低岛屿完整性。我们将采用活细胞红细胞岛测定、集落测定和红细胞岛培养来表征骨髓和脾岛背景下的红细胞增殖和分化。 2) 检验 ICAM-4 在红细胞岛中具有双重功能的假设，通过与不同整合素的相互作用来调节成红细胞-巨噬细胞和成红细胞-成红细胞粘附。基于有红细胞通过 1421 结合 ICAM-4 的初步证据，我们将确定参与 1421 结合的 ICAM-4 位点；使用荧光探针和生物成像确定阻断 ICAM-4/1421 结合对野生型和 ICAM-4 无效红细胞岛的影响；并分析抑制ICAM-4/1421和ICAM-4/1V结合是否具有累加性。骨髓和脾巨噬细胞缺乏的小鼠模型也将用于研究成红细胞-巨噬细胞相互作用对红细胞生成的体内重要性。 3)探讨Lu-层粘连蛋白粘附是否将红系岛定位于骨髓中含有层粘连蛋白的区域，以及它是否调节分化和/或增殖。我们将通过免疫荧光显微镜绘制骨髓切片中15层粘连蛋白的分布，并通过比较野生型和Lu敲除小鼠来确定其与岛的物理关系；通过表征骨髓岛并在存在和不存在层粘连蛋白的情况下进行集落测定和成红细胞培养，分析和对比 Lu 缺失和野生型小鼠的增殖和分化。拟议的目标涉及了解如何增加红细胞生成以应对多种遗传性和获得性贫血，包括镰状细胞病、遗传性膜疾病、自身免疫性红细胞破坏和失血。它们还与骨髓纤维化和红细胞生成异常相关的疾病有关。公共健康相关性：这些研究目标与加深对如何增加红细胞生成以应对多种遗传性和获得性贫血（包括地中海贫血、镰状细胞病、遗传性膜疾病、自身免疫性红细胞破坏和失血）的了解相关。它们还与骨髓纤维化和红细胞生成异常相关的疾病有关。