The F-BAR protein CIP4 in WAS-dependent thrombocytopenia

描述（由申请人提供）：血小板通过其数量、激活以及与炎症和血管系统的相互作用，在心血管、中风和出血性疾病中发挥核心作用。Wiskott - aldrich综合征（WAS）是一种以血小板减少、免疫缺陷、湿疹以及淋巴瘤和自身免疫风险增加为特征的X连锁疾病。受影响的基因编码细胞骨架蛋白WASP。通过RhoGTPase Cdc42， WASp发生构象变化，导致肌动蛋白聚合。WASp如何导致血小板减少症（WAS最常见的表现）仍不清楚。Cdc42、Src激酶、膜磷脂和SH3结构域控制WASP的功能。本实验室以Src激酶Lyn为诱饵，在酵母双杂交筛选中发现了一个新的WASP调控因子CIP4 （Cdc42相互作用蛋白4）。CIP4是F-BAR蛋白家族的一员，它重塑质膜并促进肌动蛋白聚合。为了确定CIP4的功能，我们创建了一个CIP4为空的鼠标。我们发现CIP4-/-小鼠表现出血小板减少，与WASP-/-小鼠相似。WAS患者血小板减少的机制尚不清楚。一些研究证实了自身免疫的基础；另一些则是血小板生成缺陷。由于我们的小鼠没有表现出自身免疫性疾病的迹象，我们支持CIP4-WASP形成促进血小板产生的信号通路的假设（“缺陷血小板形成假说”），CIP4或WASP的缺乏会扰乱巨核细胞中的肌动蛋白聚合，从而导致缺陷血小板形成。另一种“免疫破坏假说”认为，CIP4-WASP通路的缺陷促进了自身免疫和免疫介导的血小板破坏。我们提出通过以下两个具体目标来解决这些假设：1)通过培养巨核细胞前体和野生型、CIP4-/-、WASP-/-和CIP4-/- -WASP-/-小鼠的巨核细胞，并分析它们的前血小板形成情况，来验证血小板缺陷假说。我们将利用这些独特的小鼠品系分析血小板前形成、巨核细胞中CIP4与微管的相互作用以及血小板的超微结构特征；2)通过标记血小板并测量其在野生型、CIP4-/-、WASP-/-和CIP4-/- -WASP-/-小鼠中的清除率以及测量其T、B和NK细胞功能来验证免疫破坏假说。我们将使用这些独特的小鼠品系来测量血小板存活率和与免疫功能改变的相关性。

DESCRIPTION (provided by applicant): Platelets play a central role in cardiovascular, stroke, and bleeding disease through their quantity, activation, and interaction with the inflammatory and vascular systems. The Wiskott -Aldrich syndrome (WAS) is an X- linked disorder characterized by thrombocytopenia, immunodeficiency, eczema and an increased risk of lymphoma and autoimmunity. The affected gene encodes the cytoskeletal protein, WASP. Through the RhoGTPase, Cdc42, WASp undergoes conformational change that leads to actin polymerization. How WASp results in thrombocytopenia, the most common manifestation of WAS, remains unknown. Cdc42, Src kinases, membrane phospholipids, and SH3 domains control WASP function. Our lab has discovered a new regulator of WASP, CIP4 (Cdc42 interacting protein 4), in a yeast two hybrid screen with the Src kinase Lyn as bait. CIP4 is a member of the F-BAR family of proteins, which remodel the plasma membrane and promote actin polymerization. To determine CIP4's function, we created a CIP4-null mouse. We found that CIP4-/- mice display thrombocytopenia, similar to that observed in WASP-/- mice. The mechanism for thrombocytopenia in WAS is not known. Some studies demonstrate an autoimmune basis; others a defect in proplatelet production. Because our mice do not display signs of autoimmune disease, we favor the hypothesis ("defective proplatelet formation hypothesis") that CIP4-WASP forms a signaling pathway that promotes platelet production and that a deficiency of either CIP4 or WASP perturbs actin polymerization in megakaryocytes, which results in defective proplatelet formation. An alternative "immune destruction hypothesis" states that a defect in this CIP4-WASP pathway promotes autoimmunity and immune-mediated destruction of platelets. We propose to address these hypotheses through the following two specific aims: 1) the defective proplatelet hypothesis by culturing megakaryocyte precursors and megakaryocytes from wild-type, CIP4-/-, WASP-/-, and CIP4-/-WASP-/- mice and analyzing their proplatelet formation. We will use these unique mice strains to analyze proplatelet formation, the interaction of CIP4 with microtubules in megakaryocytes, and ultrastructural features of platelets; and 2) Test the immune destruction hypothesis by labeling platelets and measuring their clearance rates in wild-type, CIP4-/-, WASP-/-, and CIP4-/-WASP-/- mice and measuring their T, B, and NK cell functions. We will use these unique mice strains to measure platelet survival and to correlate with altered immune function. PUBLIC HEALTH RELEVANCE: Platelet production is critical for normal control of bleeding. Excessive bleeding and thrombocytopenia characterizes Wiskott - Aldrich syndrome (WAS). We have discovered a protein CIP4 that interacts physically and functionally with WAS Protein (WASP). The mechanism for the thrombocytopenia in WAS is not known. We will study platelet production in CIP4 knockout and CIP4/WASP double knockout mice to determine the precise mechanism that results in thrombocytopenia. Since platelets are involved in cardiovascular, stroke, and inflammatory diseases, our work has the potential to impact more broadly on human health.