The regulation of thrombopoietin levels

血小板生成素水平的调节

• 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
• 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

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 促血小板生成素(TPO)是一种产生于肝细胞的血浆糖蛋白，与其细胞表面受体MPL结合 在巨核细胞和巨核祖细胞上表达，促进细胞增殖和成熟。在……里面 除了在巨核细胞发育和血小板生成中的作用外，TPO还在 造血干细胞的存活和维持。在人类中，TPO功能获得性突变导致 常染色体显性性血小板增多症(高血小板计数)，而TPO(或ITS)功能丧失突变 受体MPL)导致先天性无核细胞血小板减少症，这是一种以血小板减少为特征的疾病 出生时骨髓巨核细胞的计数和缺失，随后的骨髓再生障碍性贫血和 失败了。TPO的转录调控和血浆清除已经得到了很好的研究。相比之下， 肝细胞分泌TPO和细胞内降解TPO的机制仍然存在 未知。我们已经在初步结果中表明，LMAN1基因缺陷的小鼠表现出血小板减少和 在肝细胞特异性LMAN1缺失的小鼠中，血小板减少症重新出现，但在小鼠中没有 具有造血特异性的LMAN1缺失。我们还发现血浆中TPO水平较低。 LMAN1缺陷小鼠(约正常的50%)。与Lman1‘S共同发挥内质网功能 这些结果强烈提示在LMAN1中观察到的血小板减少 缺陷小鼠是由于肝细胞分泌TPO受损所致。与在老鼠身上的这些发现一致，我们 已有研究表明，LMAN1缺乏会导致TPO在细胞内积聚。因此， LMAN1在调节TPO中的作用在小鼠和人类中似乎是保守的。在这项建议中，我们的目标是 明确LMAN1在调节小鼠血浆TPO水平中的作用，并剖析其作用机制 LMAN1调节TPO的分泌，无论是在正常生理状态下，还是在TPO增强的情况下 制作。在更多的初步结果中，我们进行了无偏见的基因组规模的CRISPR敲除 筛选以确定细胞内TPO水平的新调节因子。这个筛查，以生物三联的形式进行， 随后的验证实验证明，UBE3C的缺失会导致细胞内积累 TPO，但未见3种对照分泌蛋白在细胞内蓄积。UBE3C是E3 泛素连接酶，提示TPO是UBE3C的底物，在没有UBE3C的情况下，TPO 泛素化受到损害，导致降解减少。因此，这项提议的另一个目的是 确定UBE3C在调节细胞内TPO水平中的作用，包括在稳定状态下TPO的产生和 处于TPO产量高的状态。这一建议对理解这些机制具有重要意义。 TPO在细胞内转运/分泌水平和细胞内水平的调节 退化。从这一提议中获得的知识可能会为小说的发展奠定基础 治疗调节血浆TPO水平的策略。