ROLE OF PTPU AND SHPTP2 IN ENDOTHELIAL CELLS

许多重要的生理过程都受到蛋白质-酪氨酰的调控 磷酸化，其由蛋白质酪氨酰激酶（PTK）控制， 蛋白质酪氨酰磷酸酶（PTPs）。内皮细胞增殖， 迁移和分化的启动、维持和调节， 生长因子，其中许多通过受体PTKs（RTK）信号。两 内皮细胞特异性RTK，如VEGF和Tie/Tek成员 亚家族，以及更广泛表达的PTK，如FGFR 1， PDGFR和HGFR对内皮细胞有重要作用。障碍 RTK活动可能在人类疾病中发挥作用;例如，Tie家族 RTK与血管瘤综合征有关。干扰酪氨酰 磷酸化途径可能产生抗血管生成疗法;增强 这些通路可有助于诸如伤口 愈合/血运重建。像其他细胞一样，内皮细胞对 抑制信号，如细胞接触。尽管我们已经了解到 关于PTKs如何调节内皮细胞，几乎一无所知， 特异性PTP的作用和对抑制性信号的了解很少 m内皮细胞。本研究的目的是定义功能 PTPmu和SHPTP 2在培养的内皮细胞和小鼠体内的表达。 在其他实验室的工作已经确定，PTPmu，一种跨膜PTP， 参与嗜同性相互作用并与钙粘蛋白相关， 组织培养细胞中的连环蛋白复合物。我们发现，在体内， HPTPmu几乎仅在内皮细胞中表达。 免疫细胞化学研究表明PTPmu定位于细胞-细胞间 连接并与体内的连环蛋白共定位。此外，初步 数据表明，针对HPTPmu胞外域的抗体可以抑制 在细胞培养“创伤”测定中的再内皮化。SHPTP 2是一种非 广泛表达的跨膜PTP，包括在内皮细胞 细胞对成纤维细胞和上皮细胞系的研究表明， SHPTP 2是增殖所必需的，以响应一些但不是所有的RTK 并且在一些但不是所有的细胞类型中。这些RTK和细胞类型特定 SHPTP 2要求的差异表明，SHPTP 2在 内皮细胞不能简单地从其它细胞类型推断出来。我们将 测试假设：（i）PTPmu是一个重要的负调节因子， 内皮细胞增殖，和（ii）SHPTP 2是所需的阳性 内皮RTK通路中的元件。抗PTPmu抗体的作用， 可溶性和颗粒结合的胞外域构建体，和显性阴性 PTPmu突变体对主动脉和毛细血管内皮细胞增殖的影响， 迁移和分化将被确定。的机制 将测定从细胞表面去除PTPmu的量，同样将测定从细胞表面去除PTPmu的量。 其与钙粘蛋白/连环蛋白复合物的结合性质。候选人PTPmu 将通过生物化学和遗传学相结合的方法来确定目标。 SHPTP 2是否被酪氨酰磷酸化和/或与RTKs相关， 将评估内皮细胞。瞬时和/或稳定转染 显性阴性SHPTP 2突变体，沿着显微注射抗 SHPTP 2融合蛋白将用于确定功能， SHPTP 2在内皮SHPTP 2抗体中的位置和细胞RTK信号传导 途径。最后，内皮细胞特异性启动子和一种新的“敲- 在”策略将被用来检查显性否定的影响， PTPmu和SHPTP 2突变体在转基因小鼠体内的表达。这些研究 应该产生新的见解如何PTPs有助于维持 血管内稳态，并可能有助于疾病状态。

Many important physiological processes are regulated by protein-tyrosyl phosphorylation, which is controlled by protein-tyrosyl kinases (PTKs) and protein-tyrosyl phosphatases (PTPs). Endothelial cell proliferation, migration, and differentiation are initiated, maintained, and regulated by growth factors, many of which signal through receptor PTKs (RTKs). Both endothelial cell-specific RTKs, such as VEGFRs and members of the Tie/Tek subfamily, as well as more widely expressed PTKs, such as FGFR1, the PDGFR, and the HGFR have important actions on endothelial cells. Disorders in RTK activity may play a role in human diseases; for example, Tie family RTKs are implicated in hemangioma syndromes. Interfering with tyrosyl phosphorylation pathways may yield anti-angiogenic therapies; enhancing such pathways may aid in processes such as wound healing/revascularization. Like other cells, endothelial cells respond to inhibitory signals such as cell contact. Although much has been learned about how PTKs regulate endothelial cells, almost nothing is known about the roles of specific PTPs and little is known about inhibitory signaling m endothelial cells. The goal of this research is to define the function of PTPmu and SHPTP2 in cultured endothelial cells and in vivo in mice. Work in other labs has established that PTPmu, a transmembrane PTP, participates in homophilic interactions and associates with cadherin- catenin complexes in tissue culture cells. We have found that, in vivo, HPTPmu is expressed almost exclusively in endothelial cells. Immunocytochemical studies indicate that PTPmu is located at cell-cell junctions and co-localizes with catenins in vivo. Furthermore, preliminary data indicate that antibodies against the ectodomain of HPTPmu may inhibit re-endothelialization in a cell culture "wounding" assay. SHPTP2 is a non- transmembrane PTP that is expressed widely, including in endothelial cells. Studies in fibroblast and epithelial cell lines indicate that SHPTP2 is required for proliferation in response to some, but not all RTKs and in some, but not all cell types. These RTK- and cell type-specific differences in SHPTP2 requirements indicate that SHPTP2's role in endothelial cells cannot simply be inferred from other cell types. We will test the hypotheses that: (i) PTPmu is an important negative regulator of endothelial cell proliferation, and (ii) SHPTP2 is a required positive element in endothelial RTK pathways. The effects of anti-PTPmu antibodies, soluble and particle bound ectodomain constructs, and dominant negative PTPmu mutants on aortic and capillary endothelial cell proliferation, migration, and differentiation will be determined. The mechanism by which PTPmu is removed from the cell surface will be determined, as will the nature of its association with cadherin/catenin complexes. Candidate PTPmu targets will be identified by a combined biochemical and genetic approach. Whether SHPTP2 is tyrosyl phosphorylated and/or associated with RTKs in endothelial cells will be assessed. Transient and/or stable transfections of dominant negative SHPTP2 mutants, along with microinjection of anti- SHPTP2 fusion proteins will be used to determine the function(s) and position of SHPTP2 in endothelial SHPTP2 antibodies an cell RTK signaling pathways. Finally, endothelial cell specific promoters and a novel "knock- in" strategy will be used to examine the effects of dominant negative mutants of PTPmu and SHPTP2 in vivo in transgenic mice. These studies should yield new insights into how PTPs contribute to the maintenance of vascular homeostasis and may contribute to disease states.