Rho GTPase regulation in trophoblasts by c-Jun N-terminalkinase signaling

项目总结 胎盘发育在妊娠早期是至关重要的，因为这一过程的失调可能会导致 胎儿宫内发育受限、先兆子痫和流产。胎盘的基本步骤 发育是指绒毛外滋养层细胞侵入子宫内膜。关键的调解人 侵袭性滋养层细胞的表型是由小GTP酶Rho活性驱动的细胞骨架重塑。 细胞骨架的动态变化对于启动细胞极性和形成细胞突起是必不可少的 以及驱动细胞迁移的粘连，而对这些变化的紧密协调调节对于 胎盘发育成功。然而，调控Rho GTP酶的特定信号机制 滋养层细胞侵袭过程中的细胞骨架重塑仍不清楚。之前发布的数据已 提示丝裂原活化蛋白激酶(MAPKs)的c-Jun-N末端激酶(JNK)亚家族可能 参与滋养层细胞的侵袭，然而其实现这一目标的机制尚不清楚。 我们实验室最近的筛查已经确定了Rho GTP酶激活蛋白(Rho Gap)SYDE1和SYDE2 作为基于激酶-底物对接作用的新型JNK底物。现有文献表明， SYDE1和SYDE2在滋养层细胞迁移和细胞骨架重塑中的作用因此，SYDE1很可能 和/或SYDE2可能参与调节Rho GTP酶细胞骨架重排的JNK信号轴 以及在滋养层入侵过程中的迁移。这项建议的目的是阐明JNK是如何调节 SYDE1和SYDE2控制滋养层细胞的迁移和侵袭。这项提案将决定 JNK调节滋养层细胞SYDE1和SYDE2的功能 SYDE1基因对人滋养层细胞形态、细胞骨架组织和迁移的影响 或SYDE2缺失或过度表达。胎盘组织样本也将被分析以检查临床 JNK信号通过这些蛋白的相关性。将使用GTP酶生物传感器分析来确定 SYDE1和SYDE2的JNK磷酸化正向调节其作为Rho Gap的活性。体外GTP酶 检测将确定这一机制是否通过影响GAP酶活性来实现。为了 确定任何观察到的效应是否依赖于JNK磷酸化、JNK抑制剂和对接位置 突变的SYDE1和SYDE2将被用来阻断JNK特异性的磷酸化。最终，确定JNK和 小分子GTP酶调控因子在滋养层细胞信号转导中的作用 它们的作用机制将使它们成为治疗重度妊娠的分子靶点。 与滋养层细胞异常迁移相关的并发症。

PROJECT SUMMARY Placental development is critical in the early stages of pregnancy, as dysregulation of this process can lead to intrauterine growth restriction of the fetus, preeclampsia, and miscarriage. A fundamental step in placental development is the invasion of extravillous trophoblast cells into the endometrium of the uterus. A key mediator of the invasive trophoblast phenotype is cytoskeletal remodeling driven by the activity of the small GTPase Rho. Dynamic changes in the cytoskeleton are indispensable for initiating cell polarity and forming cell protrusions and adhesions that drive cell migration, and the tightly coordinated regulation of these changes is critical for successful placental development. However, the specific signaling mechanisms that regulate Rho GTPase cytoskeletal remodeling during trophoblast invasion remain unclear. Previously published data have suggested that the c-Jun-N-terminal kinase (JNK) subfamily of mitogen-activated protein kinases (MAPKs) may be involved in trophoblast invasion, however the mechanism by which it accomplishes this is not well understood. Recent screens from our lab have identified the Rho GTPase activating proteins (Rho GAPs) SYDE1 and SYDE2 as novel JNK substrates based on kinase-substrate docking interactions. Existing literature indicate roles of SYDE1 and SYDE2 in migration in trophoblasts and cytoskeletal remodeling. It is therefore likely that SYDE1 and/or SYDE2 may be involved in a JNK signaling axis that regulates Rho GTPase cytoskeletal rearrangement and migration during trophoblast invasion. The objective of this proposal is to elucidate how JNK modulates SYDE1 and SYDE2 to control migration and invasion of trophoblasts. This proposal will determine the functional consequences of JNK-mediated regulation of SYDE1 and SYDE2 in trophoblasts by examining the effects on cell morphology, cytoskeletal organization, and migration in human trophoblast cells harboring SYDE1 or SYDE2 deletion or overexpression. Placental tissue samples will also be analyzed to examine clinical relevance of JNK signaling through these proteins. GTPase biosensor assays will be used to determine whether JNK phosphorylation of SYDE1 and SYDE2 positively regulates their activity as Rho GAPs. In vitro GTPase assays will determine whether this mechanism is accomplished by affecting GAP enzymatic activity. In order to establish whether any observed effects are dependent on JNK phosphorylation, JNK inhibitors and docking-site mutant SYDE1 and SYDE2 will be used to ablate JNK-specific phosphorylation. Ultimately, identifying JNK and small GTPase regulators as a key molecular players in trophoblast signaling, as well as understanding the mechanisms by which they act, will allow them to be explored as molecular targets to treat severe pregnancy complications related to aberrant trophoblast migration.