Study of the Roles of SDF1 and CXCR4 in Hematopoiesis

SDF1和CXCR4在造血中的作用研究

基本信息

批准号：
10926064
负责人：
Giovanna Tosato
金额：
$ 88.56万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10926064
关键词：
AMD3100 Adult Aging Architecture Area Binding Biochemical Blood Cells Bone Marrow Bone Marrow Cells CSF3 gene CXCR4 Receptors CXCR4 gene Cancer Model Cell Aging Cell Line Cell Lineage Cell physiology Cells Cellular Structures Cerebellum Circulation Collagen Colony-Stimulating Factor Receptors Colony-Stimulating Factors Defect Development Discontinuous Capillary Disease Dominant-Negative Mutation Endothelial Cells Endothelium EphB4 Receptor Epigenetic Process FDA approved Genetic Recombination Genome Glues Goals Granulopoiesis Hematinics Hematopoiesis Hematopoietic Hematopoietic System Hematopoietic stem cells Heterogeneity Image In Vitro Individual Infiltration K562 Cells Legal patent Ligands Ligation Link Location Malignant Neoplasms Maps Mesenchymal Modeling Movement Mus Myelogenous Myeloid Cells Nature Neurons Pathway interactions Pattern Peripheral Phenotype Phosphorylation Physiological Population Proteins RGS3 gene Regulation Reporter Research Project Grants Role Signal Transduction Site Stromal Cell-Derived Factor 1 System Tamoxifen Technology Therapeutic Tumor Promotion aged bone bone cell cadherin 5 cell motility chemokine epigenome experimental study genetic approach inducible Cre inhibitor new technology novel peripheral blood pharmacologic prevent progenitor receptor reconstitution recruit response single cell analysis single cell technology stem stem cells technology development transcriptome transcriptome sequencing transcriptomics tumor tumor growth tumor progression

项目摘要

Previous studies from many groups, including our group, have identified a critical role of the chemokine CXCL12/SDF1 and its receptor CXCR4 in the retention and mobilization of myeloid cells from the bone marrow. CXCL12 is mostly produced by the non-hematopoietic cells of bone marrow, particular in the CAR (CXCL12 abundant reticular) and in other mesenchymal cells in the bone marrow. The bone marrow hematopoietic cells generally express CXCR4, and the signaling interaction between CXCL12/CXCR4 serves to retain hematopoietic cells in the bone marrow. G-CSF is a critical physiological regulator of granulopoiesis: mice carrying homozygous deletions of colony-stimulating factor (G-CSF) or its receptor are severely neutropenic, and dominant-negative mutations of G-CSFR have been linked to severe defects of granulopoiesis. Administration of G-CSF induces an expansion of myeloid lineage cells in the bone marrow, and promotes the release of mature myeloid cells and hematopoietic progenitor cells from the bone marrow to the peripheral blood. Thus, G-CSF at pharmacologic concentrations is widely used to induce granulopoiesis and to mobilize hematopoietic progenitors to the peripheral blood. Functionally, G-CSF reduces CXCR4 and CXCL12 levels in the bone marrow through a variety of mechanisms. As a consequence, the glue that retains hematopoietic cells in the bone marrow is loosened and massive exit of mature and immature hematopoietic cells is achieved. A CXCR4 competitive inhibitor, AMD3100/Plerixafluor, has been approved by FDA as a mobilizing agent for hematopoietic precursors in conjunction with G-CSF. Recently, our studies on HSPC mobilization have detected an important role of the receptor/ligand pair EphrinB2/EphB4. We unveiled the mutually exclusive bone marrow distribution of EphB4 receptors in the sinusoids and EphrinB2 ligands in hematopoietic cells, and discovered an EphB4/EphrinB2-dependent pathway that controls HSPCs mobilization. Blockade of EphB4/EphrinB2 reduced HSPCs and other myeloid cells mobilization to the circulation. In murine cancer models, in which hematopoietic cells derived from the bone marrow promote tumor growth, EphB4/EphrinB2 blockade reduced tumor infiltration with HSPCs and tumor progression. These results identify EphB4/EphrinB2 signaling as critical to hematopoietic cells mobilization from bone marrow and provide a new strategy for reducing cancer progression by targeting the bone marrow. Other ongoing studies stem from previous observations in the neuronal system, particularly in the cerebellum, showing a biochemical link between EphrinB2 and CXCR4 that regulates cerebellar cell motility. More specifically, these studies showed that PDZ-dependent signaling by EphrinB2 recruits the PDZ-containing RGS3 protein, which in turn can bind and shut down CXCR4 signaling. In so doing, active EphrinB2 regulates CXCL12-induced motility of CXCR4-positive neuronal cells. We hypothesized that this interaction may also occur in the context of the bone marrow where CXCL12 (produced by non-hematopoietic cells), CXCR4 and EphrinB2 (expressed by hematopoietic cells) are abundant. In proof-of-principle experiments we have documented the proximal co-localization of CXCR4 and EphrinB2 in mouse hematopoietic cells. Initial mapping of sites where these molecules associate in the bone marrow showed a broad pattern, with some degree of signal concentration at sites more proximal to the spongiform bone. This mapping will be continued by bone marrow imaging of WT and genetically-modified mouse lines. In vitro experiments have clarified how EphrinB2 regulates hematopoietic cell responses to CXCL12. In brief, phosphorylated EphrinB2 reduced hematopoietic cell migration in response to CXCL12. This effect is more prominent in subsets of immature hematopoietic cells. Current studies are focused on identifying the specific bone marrow niche that includes CXCL12-producing cells, CXCR4 and EphrinB2 co-expressing cells, and EphB expressing cells. Based on our experiments in vitro, we have hypothesized that such niche would serve as a "motility unit" in the bone marrow, promoting the movement of specific hematopoietic stem or progenitor cells within the bone marrow. Other ongoing studies have identified a novel population of endothelial cells in the mouse bone marrow that bears markers of typical endothelial cells and also bears some features of mesenchymal cells. This identification is based on analysis of single-cell transcriptomic analysis of hematopoietic-cell depleted bone marrow cell populations. We have now been able to mark this population by generating a mouse line in which Col1a2 (protein Collagen Type1 Alpha 2 chain)-expressing cells are tracked by a fluorescent reporter after Tamoxifen-induced Cre recombination. We have isolated an endothelial (VE-Cadherin+) bone marrow cell population that expressed Col1a2 and have begun to analyze its function in the adult bone marrow. Since individual cell-components of this population share the markers of endothelial and mesenchymal cells, current efforts aim at the purification and characterization of these cells genetically, epigenetically, phenotypically, and functionally. Important, if preliminary observations, have made us conclude that a subset of bone-marrow resident hematopoietic cells retains the ability to undergo endothelial-to-mesenchymal transition contributing to adult hematopoiesis. Currently, we are using genetic approaches to bone marrow lineage tracking to analyze further our observations. These efforts have highlighted the importance of single-cell technologies, particularly for the purpose of lineage-tracking. Current technological limitations have prevented effective multiplex analysis of the epigenome at a single-cell level. They have also prevented single-cell analysis of the transcriptome and epigenome in individual cells. To overcome this limitation, we have developed a new technology that relies on the establishment of "re-usable" single-cells and proximity ligation-based mapping of epigenetic marks to the genome. This technology has successfully been applied to single-cell analysis of a model hematopoietic cell line (K562 cells). More recent developments of this technology have envisioned and provided proof-of-principle that single-cell analysis of transcriptome and epigenome is possible. A patent was issued both in the US and the EU and an additional patent application has been filed. Importantly, we have applied these technical developments to a fundamental question in hematopoiesis, i.e. the relationship between epigenome and transcriptome in regulation of stem and progenitor hematopoietic cells function. An extension of this project is focused on the roles of the epigenome in hematopoietic cell aging. Currently, the results of RNA sequencing from young and aged hematopoietic single-cells is being analyzed.

包括我们小组在内的许多小组之前的研究已经确定了趋化因子 CXCL12/SDF1 及其受体 CXCR4 在骨髓中髓样细胞的保留和动员中的关键作用。 CXCL12 主要由骨髓的非造血细胞产生，特别是在 CAR（CXCL12 丰富的网状细胞）和骨髓中的其他间充质细胞中。骨髓造血细胞普遍表达CXCR4，CXCL12/CXCR4之间的信号相互作用有助于将造血细胞保留在骨髓中。 G-CSF 是粒细胞生成的关键生理调节因子：携带集落刺激因子 (G-CSF) 或其受体纯合缺失的小鼠患有严重中性粒细胞减少症，并且 G-CSFR 的显性失活突变与粒细胞生成的严重缺陷有关。 G-CSF的施用诱导骨髓中骨髓谱系细胞的扩增，并促进成熟骨髓细胞和造血祖细胞从骨髓释放到外周血。因此，药理学浓度的G-CSF被广泛用于诱导粒细胞生成并将造血祖细胞动员至外周血。从功能上讲，G-CSF 通过多种机制降低骨髓中 CXCR4 和 CXCL12 的水平。结果，保留骨髓中造血细胞的胶被松动，成熟和未成熟造血细胞大量排出。 CXCR4 竞争性抑制剂 AMD3100/Plerixaflu 已被 FDA 批准作为与 G-CSF 联合使用的造血前体细胞动员剂。最近，我们对 HSPC 动员的研究发现了受体/配体对 EphrinB2/EphB4 的重要作用。我们揭示了血窦中 EphB4 受体和造血细胞中 EphrinB2 配体的相互排斥的骨髓分布，并发现了控制 HSPC 动员的 EphB4/EphrinB2 依赖性途径。阻断 EphB4/EphrinB2 会减少 HSPC 和其他骨髓细胞向循环的动员。在小鼠癌症模型中，来自骨髓的造血细胞促进肿瘤生长，EphB4/EphrinB2 阻断可减少 HSPC 的肿瘤浸润和肿瘤进展。这些结果表明 EphB4/EphrinB2 信号传导对于骨髓中造血细胞的动员至关重要，并提供了一种通过靶向骨髓来减少癌症进展的新策略。其他正在进行的研究源于之前对神经系统（尤其是小脑）的观察，显示 EphrinB2 和 CXCR4 之间调节小脑细胞运动的生化联系。更具体地说，这些研究表明 EphrinB2 的 PDZ 依赖性信号传导会招募含有 PDZ 的 RGS3 蛋白，而 RGS3 蛋白反过来又可以结合并关闭 CXCR4 信号传导。在此过程中，活性 EphrinB2 调节 CXCL12 诱导的 CXCR4 阳性神经元细胞的运动。我们假设这种相互作用也可能发生在骨髓中，其中富含 CXCL12（由非造血细胞产生）、CXCR4 和 EphrinB2（由造血细胞表达）。在原理验证实验中，我们记录了 CXCR4 和 EphrinB2 在小鼠造血细胞中的近端共定位。这些分子在骨髓中结合的位点的初步绘制显示出广泛的模式，在更接近海绵状骨的位点处有一定程度的信号集中。该图谱将通过野生型和转基因小鼠品系的骨髓成像继续进行。体外实验阐明了 EphrinB2 如何调节造血细胞对 CXCL12 的反应。简而言之，磷酸化 EphrinB2 减少了响应 CXCL12 的造血细胞迁移。这种效应在未成熟造血细胞亚群中更为突出。目前的研究重点是确定特定的骨髓生态位，其中包括 CXCL12 生成细胞、CXCR4 和 EphrinB2 共表达细胞以及 EphB 表达细胞。根据我们的体外实验，我们假设这样的生态位将充当骨髓中的“运动单元”，促进骨髓内特定造血干细胞或祖细胞的运动。其他正在进行的研究已经在小鼠骨髓中发现了一个新的内皮细胞群，它们具有典型内皮细胞的标记，并且还具有间充质细胞的一些特征。该鉴定基于对造血细胞耗尽的骨髓细胞群的单细胞转录组分析。现在，我们已经能够通过生成小鼠系来标记该群体，其中在他莫昔芬诱导的 Cre 重组后，荧光报告基因可以跟踪表达 Col1a2（1 型胶原蛋白 Alpha 2 链）的细胞。我们分离出了表达 Col1a2 的内皮 (VE-钙粘蛋白+) 骨髓细胞群，并开始分析其在成人骨髓中的功能。由于该群体的各个细胞成分共享内皮细胞和间充质细胞的标记，因此当前的努力旨在对这些细胞进行遗传、表观遗传、表型和功能的纯化和表征。重要的是，初步观察使我们得出结论，骨髓驻留造血细胞的一个子集保留了经历内皮到间质转化的能力，有助于成人造血。目前，我们正在使用遗传方法进行骨髓谱系追踪，以进一步分析我们的观察结果。这些努力凸显了单细胞技术的重要性，特别是对于谱系追踪的目的。当前的技术限制阻碍了在单细胞水平上对表观基因组进行有效的多重分析。它们还阻止了对单个细胞的转录组和表观基因组的单细胞分析。为了克服这一限制，我们开发了一种新技术，该技术依赖于“可重复使用”单细胞的建立和基于邻近连接的表观遗传标记到基因组的映射。该技术已成功应用于模型造血细胞系（K562细胞）的单细胞分析。这项技术的最新发展已经设想并证明了转录组和表观基因组的单细胞分析是可能的。该产品已在美国和欧盟获得专利，并已提交额外的专利申请。重要的是，我们已将这些技术发展应用于造血的一个基本问题，即表观基因组和转录组在干细胞和祖造血细胞功能调节中的关系。该项目的扩展重点是表观基因组在造血细胞衰老中的作用。目前，正在分析年轻和老年造血单细胞的RNA测序结果。

项目成果

期刊论文数量（5）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Contribution of viral and cellular cytokines to Kaposi's sarcoma-associated herpesvirus pathogenesis.

病毒和细胞细胞因子对卡波西肉瘤相关疱疹病毒发病机制的贡献。

DOI：
发表时间：
2008-10
期刊：
影响因子：
5.5
作者：
Gasperini, Paola;Sakakibara, Shuhei;Tosato, Giovanna
通讯作者：
Tosato, Giovanna

Ephrin ligands and Eph receptors contribution to hematopoiesis.

Ephrin 配体和 Eph 受体对造血作用的贡献。

DOI：
发表时间：
2017-09
期刊：
影响因子：
0
作者：
Tosato; Giovanna
通讯作者：
Giovanna

Inactivation of axon guidance molecule netrin-1 in human colorectal cancer by an epigenetic mechanism.

通过表观遗传机制使人结直肠癌中轴突引导分子 netrin-1 失活。

DOI：
发表时间：
2022-06-30
期刊：
影响因子：
3.1
作者：
Nakayama, Hironao;Ohnuki, Hidetaka;Nakahara, Masako;Nishida;Sakaue, Tomohisa;Fukuda, Shinji;Higashiyama, Shigeki;Doi, Yuki;Mitsuyoshi, Masahiro;Okimoto, Takashi;Tosato, Giovanna;Kusumoto, Chiaki
通讯作者：
Kusumoto, Chiaki

Investigation of the interactions between the EphB2 receptor and SNEW peptide variants.

研究 EphB2 受体和 SNEW 肽变体之间的相互作用。

DOI：
发表时间：
2014-12
期刊：
影响因子：
0
作者：
Ma, Buyong;Kolb, Stephanie;Diprima, Michael;Karna, Molleshree;Tosato, Giovanna;Yang, Qiqi;Huang, Qiang;Nussinov, Ruth
通讯作者：
Nussinov, Ruth

Impaired recruitment of Grk6 and beta-Arrestin 2 causes delayed internalization and desensitization of a WHIM syndrome-associated CXCR4 mutant receptor.

Grk6 和 beta-Arrestin 2 的募集受损会导致 WHIM 综合征相关 CXCR4 突变受体的内化延迟和脱敏。