The Role and Regulation of TRP53 Activity in Oocytes and Granulosa Cells After Radiation-induced Damage

辐射损伤后卵母细胞和颗粒细胞中 TRP53 活性的作用和调节

基本信息

批准号：
10605195
负责人：
Monique L MIlls
金额：
$ 3.4万
依托单位：
JACKSON LABORATORY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-04-01 至 2024-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10605195
关键词：
Ablation Adverse effects Affect Apoptosis Apoptotic Bone Marrow CHEK2 gene Cancer Patient Cells Chemotherapy and/or radiation DNA Damage DNA Double Strand Break Dose Environment Exposure to Female Gap Junctions Genotoxic Stress Goals Hematopoietic Stem Cell Transplantation Immunoprecipitation Infertility Ionizing radiation Life Malignant Neoplasms Mammals Mass Spectrum Analysis Methods Mus Mutagens Oocytes Ovarian Ovarian Follicle Ovary Patients Population Post-Translational Protein Processing Prediction of Response to Therapy Predisposition Premature Ovarian Failure Primordial Follicle Protein Isoforms Proteins Proteomics Radiation Radiation Dose Unit Radiation induced damage Radiation therapy Reactive Oxygen Species Regulation Reproductive Health Research Role Signal Transduction Somatic Cell Survival Rate Survivors TP53 gene TYRP1 gene Therapeutic Vitamin K 3 cancer cell cancer therapy cell type chemotherapy drug preservation egg genetic approach genotoxicity granulosa cell improved intercellular communication mutant new therapeutic target novel ovarian damage ovarian failure prevent primary ovarian insufficiency pro-apoptotic protein protein expression protein protein interaction pup reconstitution response targeted treatment therapy development therapy resistant tumorigenesis

项目摘要

Project Summary Ionizing radiation (IR) is a treatment used against cancer and to ablate patient's bone marrow prior to hematopoietic stem cell transplantation. IR induces damage (double strand breaks or reactive oxygen species) in target cells but can also kill healthy bystander cells. Primordial follicles (PFs) are a limited population of ovarian follicles that contain immature oocytes and are highly susceptible to damage. Damage induced by IR or other genotoxic chemotherapies can deplete the ovary of PFs resulting in premature ovarian insufficiency (POI) and infertility. Therefore, there is a need to understand how damage affects ovarian cells and leads to POI before less toxic therapeutic or predictive treatments can be developed. The goal of this project is to determine the mechanism of radiation-induced oocyte elimination in mammals. How PF's response to damage is largely unknown beyond a few major players. Checkpoint kinase 2 (CHEK2) primarily activates the pro-apoptotic TRP63 TA isoform (TAp63) in response to damage. In contrast, somatic cells predominantly use another CHEK2 target TRP53. Our lab showed Chek2-/- female mice receiving IR retained their PF reserve and produced healthy pups. TAp63-/- females receiving low dose IR maintained PFs while Trp53-/- mice lost their PF reserve. Suggesting that TRP53 is dispensable for apoptosis in oocytes. In treatments with higher IR dose or chemotherapies, TAp63-/- females lose PF reserve while Chek2-/- females retain PF reserve. We predicted higher doses of IR and chemotherapies cause more damage, which activates a TAp63-independent mechanism. Indeed, TAp63-/- Trp53-/- double mutant females exposed to higher dose IR retained PF reserve suggesting TRP53 triggers oocyte elimination when a certain threshold of damage is reached. Analysis of TRP53 protein expression after high dose IR identified a unique form of TRP53 in purified oocytes and the absence of the typical ~53kDa protein, detected in somatic cells. Based on these observations we hypothesize TRP53 activity in oocytes is regulated by an oocyte-specific mechanism which either activates TRP53 or restricts its action until a specific threshold of damage occurs. This proposal will utilize genetic approaches (chimeric reconstituted ovaries) and proteomic approaches (mass spectrometry) to determine TRP53 regulation in response to ovarian damage. The aims of this proposed research are to (1) determine whether TRP53-dependent PF loss is triggered intrinsically in the oocyte or is due to damage signals from somatic granulosa cells to the oocyte and 2) determine how TRP53 activity is regulated in oocytes by identifying unique post-translational modifications and/or protein interactions associated with TRP53-dependent PF loss. Defining oocyte-specific mechanisms regulating pro-apoptotic TRP53 activity in response to damage will improve our understanding of the PF response to genotoxic agents and provide targets for therapeutics to prevent oocyte loss, infertility, and POI in patients receiving genotoxic treatments.

项目摘要电离辐射（IR）是针对癌症的治疗方法造血干细胞移植。 IR诱导损伤（双链断裂或活性氧）在靶细胞中，但也可以杀死健康的旁观细胞。原始卵泡（PFS）是有限的卵巢包含未成熟卵母细胞并且高度易受损害的卵泡。 IR或其他造成的损害遗传毒性化学疗法会耗尽PF的卵巢，导致卵巢过早不足（POI）和不育。因此，有必要了解损害如何影响卵巢细胞并导致POI 可以开发毒性较小的治疗或预测性治疗方法。该项目的目的是确定辐射诱导的哺乳动物消除卵母细胞的机制。 PF对损害的反应如何在很大程度上是超越了几个主要球员。检查点激酶2（CHEK2）主要激活pro凋亡TRP63 TA同工型（TAP63）响应损坏。相反，体细胞主要使用另一个CHEK2靶 TRP53。我们的实验室显示，接受IR的CHEK2 - / - 雌性小鼠保留了PF储备并产生了健康的幼犬。 TAP63 - / - 女性接受低剂量IR维持的PF，而TRP53 - / - 小鼠失去了PF储备。暗示这一点 TRP53对于卵母细胞的凋亡是可分配的。在较高的IR剂量或化学疗法的治疗中，TAP63 - / - 女性失去PF储备，而CHEK2 - / - 女性保留PF储备。我们预测了更高剂量的IR和化学疗法会造成更多的损害，从而激活TAP63独立的机制。确实，TAP63 - / - TRP53 - / - 双重突变女性暴露于较高剂量的IR保留的PF储备建议TRP53触发器当达到一定的损害阈值时，消除卵母细胞。分析TRP53蛋白表达后高剂量IR在纯化的卵母细胞中鉴定了一种独特的TRP53形式，并且没有典型的〜53KDA蛋白，在体细胞中检测到。基于这些观察结果，我们假设卵母细胞中的TRP53活性受到调节通过卵母细胞特异性机制，该机制激活TRP53或限制其作用，直到特定的阈值发生损害。该建议将利用遗传方法（嵌合重构卵巢）和蛋白质组学方法（质谱法）确定响应卵巢损伤的TRP53调节。目的这项拟议的研究是（1）确定在本质上本质上触发了TRP53依赖性PF损失。卵母细胞或由于从体细胞颗粒细胞到卵母细胞的损伤信号引起的，2）确定TRP53的方式通过识别独特的翻译后修饰和/或蛋白质相互作用来调节卵母细胞的活性与TRP53依赖性PF损失相关。定义卵母细胞特异性机制，调节促凋亡 TRP53响应损害的活性将提高我们对PF对遗传毒性剂反应的理解并为接受遗传毒性的患者提供治疗剂，以防止卵母细胞丧失，不育和POI 治疗。