The F-BAR protein CIP4 in WAS-dependent thrombocytopenia

描述(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综合征是一种X连锁疾病，以血小板减少、免疫缺陷、湿疹、淋巴瘤和自身免疫风险增加为特征。受影响的基因编码细胞骨架蛋白WASP。黄蜂通过RhoGTP酶CDC42发生构象变化，导致肌动蛋白聚合。黄蜂是如何导致血小板减少的，这是IS最常见的表现，目前尚不清楚。CDC42、Src激酶、膜磷脂和SH3结构域控制WASP的功能。我们实验室在以Src激酶Lyn为诱饵的酵母双杂交筛选中发现了WASP的一个新的调节子CIP4(CDC42相互作用蛋白4)。CIP4是F-bar蛋白家族中的一员，它可以重塑质膜，促进肌动蛋白聚合。为了确定CIP4的S功能，我们创造了一个CIP4缺失的小鼠。我们发现，CIP4-/-小鼠表现出血小板减少症，与WASP-/-小鼠相似。WASs中的血小板减少的机制尚不清楚。一些研究证明了自身免疫的基础；另一些研究证明了原血小板生成的缺陷。由于我们的小鼠没有表现出自身免疫性疾病的迹象，我们支持这样的假说(缺陷原血小板形成假说)，即CIP4-WASP形成促进血小板产生的信号通路，并且CIP4或WASP的缺陷扰乱巨核细胞中的肌动蛋白聚合，从而导致缺陷的原血小板形成。另一种“免疫破坏假说”认为，CIP4-WASP途径的缺陷促进了自身免疫和免疫介导的血小板破坏。我们建议通过以下两个具体目标来解决这些假说：1)通过培养CIP4-/-、WASP-/-和CIP4-/-WASP-/-野生型、CIP4-/-WASP-/-和CIP4-/-WASP-/-小鼠的巨核细胞前体和巨核细胞，并分析它们的血小板原形成情况，提出有缺陷的血小板原假说。我们将使用这些独特的小鼠品系来分析前血小板的形成、CIP4与巨核细胞中微管的相互作用以及血小板的超微结构特征；以及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它们的T、B和NK细胞功能来检验免疫破坏假说。我们将使用这些独特的小鼠品系来测量血小板存活率，并与改变的免疫功能相关联。

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.