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

c-Jun N 末端激酶信号传导对滋养细胞中 Rho GTPase 的调节

• 批准号: 10749994
• 负责人: Claire Song
• 金额: $ 4.77万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10749994
• 关键词: Ablation; Adhesions; Affect; Biological Assay; Biosensor; Blood Vessels; CRISPR/Cas technology; Cell Polarity; Cells; Cellular Morphology; Chorionic villi; Cultured Cells; Cytoskeletal Modeling; Cytoskeleton; Data; Docking; Endometrium; Family; Fetal Growth Retardation; Fetus; Genes; Guanosine Triphosphate Phosphohydrolases; Human; In Vitro; Invaded; JUN gene; Literature; MAPK8 gene; Mediating; Mediator; Mitogen-Activated Protein Kinases; Molecular; Molecular Target; Monomeric GTP-Binding Proteins; Mothers; N-terminal; Outcome; Phenotype; Phosphorylation; Phosphotransferases; Placentation; Pre-Eclampsia; Pregnancy; Pregnancy Complications; Process; Proteins; Publishing; Regulation; Role; Signal Transduction; Site; Spontaneous abortion; Tissue Sample; Tissues; Uterus; Vascular blood supply; Villous; cell motility; clinically relevant; cytotrophoblast; inhibitor; kinase inhibitor; knock-down; migration; mutant; novel; overexpression; rho; rho GTP-Binding Proteins; rho GTPase-activating protein; trophoblast; wound healing

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.

项目概要 胎盘发育在怀孕早期至关重要，因为这一过程的失调可能会导致 胎儿宫内生长受限、先兆子痫和流产。胎盘的基本步骤 发育是绒毛外滋养层细胞侵入子宫内膜。关键调解人 侵袭性滋养层表型的一个重要特征是由小 GTP 酶 Rho 的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 和驱动细胞迁移的粘附，以及这些变化的紧密协调调节对于 胎盘发育成功。然而，调节 Rho GTPase 的特定信号机制 滋养层侵袭期间的细胞骨架重塑仍不清楚。此前公布的数据有 表明丝裂原激活蛋白激酶 (MAPK) 的 c-Jun-N 末端激酶 (JNK) 亚家族可能 参与滋养层侵袭，然而其实现这一点的机制尚不清楚。 我们实验室最近的筛选已鉴定出 Rho GTP 酶激活蛋白 (Rho GAP) SYDE1 和 SYDE2 作为基于激酶-底物对接相互作用的新型 JNK 底物。现有文献表明的作用 SYDE1 和 SYDE2 在滋养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 GTPase 细胞骨架重排的 JNK 信号轴 和滋养层侵袭期间的迁移。该提案的目的是阐明 JNK 如何调节 SYDE1和SYDE2控制滋养细胞的迁移和侵袭。该提案将决定 通过检查滋养层中 JNK 介导的 SYDE1 和 SYDE2 调节的功能后果 对携带 SYDE1 的人滋养层细胞的细胞形态、细胞骨架组织和迁移的影响 或 SYDE2 缺失或过度表达。还将分析胎盘组织样本以检查临床 JNK 信号传导通过这些蛋白质的相关性。 GTPase 生物传感器检测将用于确定是否 SYDE1 和 SYDE2 的 JNK 磷酸化正向调节它们作为 Rho GAP 的活性。体外GTP酶 测定将确定该机制是否是通过影响 GAP 酶活性来实现的。为了 确定任何观察到的效应是否依赖于 JNK 磷酸化、JNK 抑制剂和对接位点 突变体 SYDE1 和 SYDE2 将用于消除 JNK 特异性磷酸化。最终，识别出 JNK 并 小 GTP 酶调节剂作为滋养层信号传导的关键分子参与者，以及了解 它们的作用机制将使它们能够作为治疗严重妊娠的分子靶标进行探索 与异常滋养层迁移相关的并发症。