The regulation of thrombopoietin levels

血小板生成素水平的调节

• 批准号: 10590666
• 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
• 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/10590666
• 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; autosome; cell type; 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

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.

项目摘要 血小板生成素（TPO）是肝细胞产生的一种血浆糖蛋白，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。在 TPO除了在巨核细胞发育和血小板生成中起作用外，还在 造血干细胞存活和维持。在人类中，TPO的功能获得性突变导致 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，一种以血小板减少为特征的疾病 出生时骨髓巨核细胞计数和缺失，随后出现骨髓发育不全， 失败TPO的转录调控和血浆清除已被充分研究。而反观 TPO从肝细胞分泌和TPO在细胞内降解的机制仍然存在 未知我们已经在初步结果中表明，LMAN 1缺陷的小鼠表现出血小板减少症， 血小板减少症在肝细胞特异性LMAN 1缺失的小鼠中重现，但在小鼠中没有 造血特异性LMAN 1缺失我们还表明，血浆TPO水平低， LMAN 1缺陷小鼠（约为正常小鼠的50%）。结合LMAN 1作为内质网的已知功能， 网状货物受体，这些结果强烈表明，在LMAN 1中观察到的血小板减少症 缺陷型小鼠是由于肝细胞TPO分泌受损。与小鼠的这些发现一致，我们 已经表明LMAN 1缺陷导致TPO在人细胞中的细胞内积累。因此 LMAN 1在调节TPO中的作用似乎在小鼠和人中是保守的。在本建议中，我们的目标是 确定LMAN 1在调节小鼠血浆TPO水平中的作用，并分析其机制， LMAN 1调节TPO分泌，在正常生理状态下和在增强的TPO设置下都是如此 生产在额外的初步结果中，我们进行了无偏的基因组规模CRISPR敲除， 筛选以鉴定细胞内TPO水平的新调节剂。该筛选以生物学一式三份进行， 随后的验证实验表明，UBE 3C的缺失导致细胞内积累 的TPO，但没有细胞内积累的任何3个对照分泌蛋白测试。UBE 3C是E3 泛素连接酶，这表明TPO是UBE 3C的底物，并且在不存在UBE 3C的情况下，TPO 泛素化受损，导致降解减少。因此，本提案的另一个目的是 确定UBE 3C在稳态TPO产生和细胞内TPO水平调节中的作用。 在高TPO生产状态下。这一建议对于理解这些机制具有重要意义 TPO调节在其细胞内运输/分泌水平和在其细胞内运输/分泌水平， 降解从这一建议中获得的知识可能为小说的发展奠定基础 治疗性调节血浆TPO水平的策略。