The regulation of thrombopoietin levels

血小板生成素水平的调节

基本信息

批准号：
10457889
负责人：
Rami Khoriaty
金额：
$ 53.93万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-08-01 至 2025-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10457889
关键词：
Animals Autophagocytosis Binding Biological Birth Blood Platelets Bone Marrow Cell Line Cell Maintenance Cell Maturation Cell Proliferation Cell Surface Receptors Cells Clustered Regularly Interspaced Short Palindromic Repeats Co-Immunoprecipitations Complex Defect Development Disease Dose Endoplasmic Reticulum Exhibits Failure Flow Cytometry Foundations Genetic Genetic Models Genetic Transcription Glycoproteins Hematopoietic Hematopoietic stem cells HepG2 Hepatocyte Histology Human Immunofluorescence Immunologic Impairment Interleukin-6 Knock-out Knowledge Liver Lysine Mediating Megakaryocytes Membrane Messenger RNA Mus Physiological Plasma Platelet Count measurement Play Production Proteins Regulation Ribosomes Role Testing Therapeutic Thrombocytopenia Thrombopoietin Transcriptional Regulation Ubiquitination Validation design experimental study extracellular gain of function mutation genome-wide loss of function mutation mouse genetics multicatalytic endopeptidase complex novel novel strategies overexpression progenitor receptor response secretory protein stem cell survival thrombocytosis trafficking ubiquitin-protein ligase validation studies

项目摘要

Project Summary Thrombopoietin (TPO), a plasma glycoprotein made in hepatocytes, binds to its cell surface receptor MPL expressed on megakaryocytes and megakaryocyte progenitors, promoting cell proliferation and maturation. In addition to its role in megakaryocyte development and platelet production, TPO also plays a critical role in hematopoietic stem cell survival and maintenance. In humans, gain-of-function mutations in TPO result in autosomal dominant thrombocytosis (high platelet counts), while loss-of-function mutations in TPO (or in its receptor MPL) result in congenital amegakaryocytic thrombocytopenia, a disease characterized by low platelet counts and absence of bone marrow megakaryocytes at birth with subsequent bone marrow aplasia and failure. The transcriptional regulation and plasma clearance of TPO have been well studied. In contrast, the mechanism by which TPO is secreted from hepatocytes and by which TPO is degraded intracellularly remain unknown. We have shown in preliminary results that mice deficient in LMAN1 exhibit thrombocytopenia and that the thrombocytopenia is recapitulated in mice with hepatocyte-specific LMAN1 deletion but not in mice with hematopoietic-specific LMAN1 deletion. We have additionally shown that the plasma TPO level is low in LMAN1 deficient mice (~50% of normal). Taken together with LMAN1’s known function as an endoplasmic reticulum cargo receptor, these results strongly suggest that the thrombocytopenia observed in LMAN1 deficient mice is due to impaired secretion of TPO from hepatocytes. Consistent with these findings in mice, we have shown that LMAN1 deficiency results in intracellular accumulation of TPO in human cells. Therefore, the role of LMAN1 in regulating TPO appears to be conserved in mice and humans. In this proposal, we aim to define the role of LMAN1 in regulating the plasma TPO levels in mice and to dissect the mechanism by which LMAN1 regulates TPO secretion, both under a normal physiological state and in the setting of enhanced TPO production. In additional preliminary results, we have performed an unbiased genome-scale CRISPR knock-out screen to identify novel regulators of the intracellular TPO level. This screen, performed in biological triplicates, followed by validation experiments, demonstrated that deletion of UBE3C results in intracellular accumulation of TPO but no intracellular accumulation of any of the 3 control secretory proteins tested. UBE3C is an E3 ubiquitin ligase, suggesting that TPO is a substrate for UBE3C and that in the absence of UBE3C, TPO ubiquitination is impaired, resulting in reduced degradation. Therefore, an additional aim of this proposal is to define the role of UBE3C in regulating the intracellular TPO level, both under steady state TPO production and in a state of high TPO production. This proposal has important implications for understanding the mechanisms of TPO regulation at the level of its intracellular trafficking/secretion and at the level of its intracellular degradation. Knowledge gained from this proposal may lay the foundation for the development of novel strategies to therapeutically regulate the plasma TPO level.

项目概要血小板生成素 (TPO) 是一种在肝细胞中产生的血浆糖蛋白，与其细胞表面受体 MPL 结合在巨核细胞和巨核细胞祖细胞上表达，促进细胞增殖和成熟。除了在巨核细胞发育和血小板生成中发挥作用外，TPO 还在在人类中，TPO 的功能获得性突变会导致造血干细胞的存活和维持。常染色体显性血小板增多症（高血小板计数），而 TPO（或其受体MPL）导致先天性无巨核细胞性血小板减少症，一种以低血小板为特征的疾病出生时骨髓巨核细胞计数和缺失，随后出现骨髓再生障碍相比之下，TPO 的转录调控和血浆清除已得到充分研究。 TPO从肝细胞分泌以及TPO在细胞内降解的机制仍然存在我们在初步结果中表明，LMAN1 缺陷的小鼠表现出血小板减少症和血小板减少症。肝细胞特异性 LMAN1 缺失的小鼠会出现血小板减少症，但小鼠不会出现血小板减少症我们还发现，在造血特异性 LMAN1 缺失的情况下，血浆 TPO 水平较低。 LMAN1 缺陷小鼠（约为正常小鼠的 50%）与 LMAN1 已知的内质功能结合起来。网状货物受体，这些结果强烈表明在 LMAN1 中观察到的血小板减少症小鼠的缺陷是由于肝细胞分泌 TPO 受损所致，与小鼠的这些发现一致。研究表明，LMAN1 缺陷会导致人体细胞内 TPO 的积累。 LMAN1 在调节 TPO 中的作用似乎在小鼠和人类中是保守的。定义 LMAN1 在调节小鼠血浆 TPO 水平中的作用并剖析其机制 LMAN1 在正常生理状态和 TPO 增强的情况下调节 TPO 分泌在其他初步结果中，我们进行了无偏基因组规模的 CRISPR 敲除。筛选以确定细胞内 TPO 水平的新型调节剂该筛选以生物学重复进行三次。随后的验证实验证明，UBE3C 的缺失会导致细胞内积累 TPO，但所测试的 3 种对照分泌蛋白中的任何一种都没有细胞内积累，这是 E3。泛素连接酶，表明 TPO 是 UBE3C 的底物，并且在没有 UBE3C 的情况下，TPO 泛素化受到损害，导致降解减少。因此，该提案的另一个目的是定义了 UBE3C 在调节细胞内 TPO 水平中的作用，无论是在稳态 TPO 生产还是在在 TPO 产量较高的情况下，该提案对于理解其机制具有重要意义。 TPO 调节在其细胞内运输/分泌水平和细胞内水平从该提案中获得的知识可能为新颖的开发奠定基础。治疗性调节血浆 TPO 水平的策略。