Study of the Roles of SDF1 and CXCR4 in Hematopoiesis

SDF1和CXCR4在造血中的作用研究

• 批准号: 8552822
• 负责人: Giovanna Tosato
• 金额: $ 48.82万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 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/8552822
• 关键词: AMD3100; Alleles; Area; Bacterial Infections; Binding; Biological; Blood; Blood Cells; Blood Circulation; Bone Marrow; Bone Marrow Cells; C-terminal; CSF3 gene; CXCL12 gene; CXCR4 Receptors; CXCR4 gene; Cell Differentiation process; Cell Lineage; Cell physiology; Cell surface; Cells; Collection; Colony-Stimulating Factor Receptors; Colony-Stimulating Factors; Commit; Complex; DNA Sequence; Defect; Derivation procedure; Development; Disease; Dominant-Negative Mutation; Down-Regulation; Endothelial Cells; Endothelium; Ensure; FDA approved; Generations; Genetic; Goals; Granulopoiesis; Guanine Nucleotide Exchange Factors; Guanine Nucleotides; Hematopoiesis; Hematopoietic; Hematopoietic Stem Cell Mobilization; Hematopoietic stem cells; Hereditary Disease; Heterozygote; In Vitro; Investigation; Knockout Mice; Ligands; Link; MEKs; Maintenance; Malignant Neoplasms; Mediating; Messenger RNA; MicroRNAs; Minority; Mitogen-Activated Protein Kinases; Mitogens; Mus; Mutation; Myelogenous; Myeloid Cells; Myeloid Leukemia; Neutropenia; Pathway interactions; Patients; Peripheral; Pharmaceutical Preparations; Physiological; Process; Property; Proteins; Receptor Cell; Recruitment Activity; Regulation; Research Project Grants; Role; STAT1 protein; STAT3 gene; Signal Transduction; Site; Source; Spleen; Staging; Stem cells; Stromal Cell-Derived Factor 1; Stromal Cells; Surface; Syndrome; Therapeutic; Thymus Gland; Time; Transcription Repressor/Corepressor; Vascular Cell Adhesion Molecule-1; base; chemokine; controlled release; extracellular; granulocyte; hematopoietic cell transplantation; improved; in vivo; inhibitor/antagonist; monocyte; mutant; neutrophil; peripheral blood; progenitor; receptor; reconstitution; research study; response; stem; trafficking; transcription factor

The generation of neutrophils from hematopoietic precursors and their release to the peripheral circulation are highly regulated processes that ensure the maintenance of homeostatic neutrophil levels in the blood and their rise in response to bacterial infections and other signals. Altered neutrophil maturation and release are associated with various forms of neutropenia, which may precede and be pathogenetically linked to the development of myeloid leukemias. G-CSF has emerged a critical physiological regulator of granulopoiesis since 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 neutrophils and hematopoietic progenitor cells from the bone marrow to the peripheral blood. Based on these properties, G-CSF is widely used to induce granulopoiesis and to mobilize hematopoietic progenitors to the peripheral blood. More recently, a CXCR4 competitive inhibitor, AMD3100/Plerixafluor, has been approved by FDA and a mobilizing agent for hematopoitic precursors in conjunction with G-CSF. Genetic studies and other studies have identified the transcription factor Gfi1 as a critical contributor to stem cells and myeloid cell function. Thus, mice null for Gfi1 fail to produce mature neutrophils, which has been attributed to a block in neutrophil maturation at the stage of common granulocyte/monocyte precursors. We have investigated the relationship between Gfi1 and G-CSF/G-CSFR in neutrophil maturation and their release from the bone marrow, and found that Gfi1 regulates G-CSF signaling. The biological activities of G-CSF are solely mediated by its activation of the G-CSF-receptor (R) that is expressed on myeloid lineage progenitor cells. Compelling evidence from genetic studies and other studies demonstrated that G-CSF indirectly promotes hematopoietic cell and neutrophil mobilization to the peripheral blood by modulating the activities of the chemokine SDF1 and/or its receptor CXCR4. WHIM, a genetic disorder associated with mutations in the intracellular domain of CXCR4 leading to increased CXCR4 function causes a retention of immature neutrophils into the bone marrow and severe peripheral neutropenia. AMD3100, a competitive inhibitor of SDF-1 binding to its receptor and a mutant form of SDF-1, which induces prolonged downregulation of the CXCR4 surface receptor, promote the mobilization of neutrophils and hematopoietic cells to the peripheral blood. During stem cell mobilization with G-CSF, SDF-1 and CXCR4 protein levels decrease in the bone marrow. We have examined the mechanisms responsible for reduced CXCR4 expression. Initially, we found that G-CSF reduces CXCR4 expression in bone marrow Gr1+ myeloid cells, which express G-CSFR. In additional studies, we have obtained evidence that the transcriptional repressor Gfi-1 is involved in G-CSF-induced mobilization of granulocytic lineage cells from the bone marrow to the peripheral blood. We found that in vitro and in vivo G-CSF promotes expression of Gfi-1 and down-regulates expression of CXCR4. Gfi-1 binds to DNA sequences upstream of the CXCR4 gene and represses CXCR4 expression in myeloid lineage cells. As a consequence, myeloid cell responses to the CXCR4 unique ligand SDF1 are reduced. Thus, Gfi1 not only regulates hematopoietic stem cell function and myeloid cell development but also likely promotes the release of granulocytic lineage cells from the bone marrow to the peripheral blood by reducing CXCR4 expression and function. In related experiments, we have generated mutants of CXCR4 that mimic mutations in the C-terminal domain found in patients with WHIM syndrome. We have examined the signaling mechanisms from wild-type CXCR4 and compared with signaling from mutants CXCR4 receptors. Our results indicate that unlike the normal receptor, mutant CXCR4 fails to appropriately recruit beta arrestin2, bur not beta arrestin1 to the receptor complex. As a consequence internalization of the mutant CXCR4 receptor from the cell surface to the cytoplasmic compartment is delayed, degradation is delayed, and signaling from the mutant receptor is also delayed. Since WHIM patients are heterozygotes for the mutant CXCR4 receptor and carry both the normal and the mutant allele, the net result is that CXCR4 signaling is extended in time, as it is the result of activation of both the normal and the mutant receptor. Thus, patients with WHIM have a super-functional CXCR4 receptor and presumably fail to release neutrophils from the bone marrow to the peripheral blood due to continuous signaling by the ligand SDF1, which holds the mature neutrophils in the bone marrow compartment. Since both the transcription factor Gfi1 and G-CSF/G-CSFR individually are critical contributors of myeloid cell differentiation from common myeloid/monocyte precursors in the bone marrow, we have investigated their relationship. We have uncovered a previously unrecognized function of Gfi1 as a regulator of G-CSF/G-CSFR signaling and function. Specifically, we found that Gfi1 regulates the expression of Ras guanine nucleotide releasing protein 1 (RasGRP1), an exchange factor that activates Ras, and that RasGRP1 is required for G-CSF signaling through the Ras/mitogen-activated protein/extracellular signal-regulated kinase (MEK/Erk) pathway. Gfi1-null mice have reduced levels of RasGRP1 mRNA and protein in thymus, spleen, and bone marrow, and Gfi1 transduction in myeloid cells promotes RasGRP1 expression. When stimulated with G-CSF, Gfi1-null myeloid cells are selectively defective at activating Erk1/2, but not signal transducer and activator of transcription 1 (STAT1) or STAT3, and fail to differentiate into neutrophils. Expression of RasGRP1 in Gfi1-deficient cells partially rescues Erk1/2 activation by G-CSF and allows neutrophil maturation by G-CSF. Mobilization of hematopoietic progenitor cells (HPC) from the bone marrow to the peripheral blood by G-CSF is the primary means to acquire stem cell grafts for hematopoietic cell transplantation avoiding invasive bone marrow collection. Since HPC represent a minority of all blood cells mobilized by G-CSF, there is a need for understanding the underlying mechanisms to develop selective drugs. We now found that G-CSF indirectly reduces expression of surface vascular cell adhesion molecule 1 (VCAM-1) on bone marrow HPC, stromal cells and endothelial cells by promoting the accumulation of microRNA-126 (miR126)-containing microvescicles/exosomes in the bone marrow extracellular compartment. We find that HPC, stromal cells and endothelial cells readily incorporate these exosomes, and that miR126 represses VCAM-1 expression on bone marrow HPC, stromal cells and endothelial cells. In line with this, miR126-null mice display a reduced mobilization response to G-CSF. Since mature neutrophils represent the main source of bone marrow microvesicles containing miR126, Gfi1-null mice that lack of mature neutrophils are defective in the mobilization of HPC. In addition, bone marrows of Gfi1-deficient mice have abnormally reduced levels of miR126, and express abnormally high levels of VCAM1 in the HPC. Altogether, our results implicate miR126 in the regulation of HPC trafficking between the bone marrow and peripheral sites, clarify the role of VCAM-1 in G-CSF-mediated mobilization, and have important implications for improved approaches to selective mobilization of HPC.

造血前体的中性粒细胞及其释放到周围循环的产生是高度调节的过程，可确保血液中稳态中性粒细胞水平的维持以及它们对细菌感染和其他信号的响应。 中性粒细胞成熟和释放的改变与各种形式的嗜中性粒细胞减少有关，这可能是在与髓样白血病的发展有关的。 G-CSF已经出现了粒状植物的关键生理调节剂，因为携带纯合子刺激因子（G-CSF）或其受体的小鼠或其受体严重中性粒细胞减少，而G-CSFR的显性阴性突变已与严重的颗粒状缺陷联系在一起。 G-CSF的给药诱导骨髓中髓样细胞的扩张，并促进中性粒细胞和造血祖细胞从骨髓到外周血的释放。 基于这些特性，G-CSF被广泛用于诱导粒状并动员造血祖细胞向外周血液。最近，FDA批准了CXCR4竞争性抑制剂AMD3100/Plerixafluor，并与G-CSF一起批准了造血前体的动员剂。 遗传研究和其他研究已将转录因子GFI1确定为干细胞和髓样细胞功能的关键因素。 因此，用于GFI1的小鼠未能产生成熟的中性粒细胞，这归因于中性粒细胞成熟的块在公共粒细胞/单核细胞前体的阶段。 我们已经研究了GFI1和G-CSF/G-CSFR在中性粒细胞成熟中的关系，并从骨髓中释放出来，发现GFI1调节G-CSF信号传导。 G-CSF的生物学活性仅通过其在髓样谱系祖细胞上表达的G-CSF受体（R）的激活来介导。 来自遗传研究和其他研究的令人信服的证据表明，G-CSF通过调节趋化因子SDF1和/或其受体CXCR4的活性，间接促进造血细胞和中性粒细胞动员到外周血。 WIM是一种与CXCR4细胞内结构域突变相关的遗传疾病，导致CXCR4功能增加会导致未成熟的中性粒细胞保留到骨髓和严重的外周中性粒细胞减少症中。 AMD3100是SDF-1与其受体结合的竞争性抑制剂，SDF-1的突变体形式诱导了CXCR4表面受体的延长下调，促进了中性粒细胞和造血细胞向外周血液的动员。在用G-CSF动员干细胞的过程中，骨髓中的SDF-1和CXCR4蛋白水平降低。 我们已经检查了负责降低CXCR4表达的机制。 最初，我们发现G-CSF降低了表达G-CSFR的骨髓GR1+髓样细胞中的CXCR4表达。在其他研究中，我们获得了证据表明，转录阻遏物GFI-1参与G-CSF诱导的动员粒细胞谱系细胞从骨髓到外周血的动员。 我们发现体外和体内G-CSF促进了GFI-1的表达并下调CXCR4的表达。 GFI-1与CXCR4基因上游的DNA序列结合，并抑制髓样谱系细胞中的CXCR4表达。 结果，髓样细胞对CXCR4独特的配体SDF1的反应减少了。 因此，GFI1不仅调节造血干细胞功能和髓样细胞发育，而且还可能通过减少CXCR4表达和功能来促进粒细胞谱系细胞从骨髓到外周血液的释放。在相关的实验中，我们已经产生了CXCR4的突变体，即在综合症患者中模仿C末端结构域中的突变。我们已经检查了野生型CXCR4的信号传导机制，并将其与突变体CXCR4受体的信号传导进行了比较。 我们的结果表明，与正常的受体CXCR4不同，突变体CXCR4无法适当募集beta抑制素2，而不是bur beta beta抑制素1。因此，从细胞表面到细胞质室的突变体CXCR4受体的内在化延迟，降解被延迟，并且来自突变体受体的信号传导也被延迟。 由于WHIM患者是突变CXCR4受体的杂合子，并同时携带正常和突变等位基因，因此净结果是CXCR4信号的时间扩大，因为这是正常受体和突变体受体激活的结果。 因此，由于配体SDF1的连续信号传导而导致的嗜中性粒细胞的患者具有超功能的CXCR4受体，并且大概无法从骨髓到外周血液释放中性粒细胞，这使配体SDF1的连续信号传导，该信号使骨髓隔室中的成熟中性粒细胞保持。由于转录因子GFI1和G-CSF/G-CSFR单独是骨髓中常见的髓样/单核细胞前体的髓样细胞分化的关键因素，因此我们研究了它们的关系。 我们已经发现了GFI1先前未识别的功能，作为G-CSF/G-CSFR信号传导和功能的调节剂。具体而言，我们发现GFI1调节Ras鸟嘌呤核苷酸释放蛋白1（RASGRP1）的表达，这是一种激活RAS的交换因子，RASGRP1是通过RAS/MITITOGON激活的蛋白质/外细胞外蛋白/外细胞信号受调控的激酶（Mek/Erk）path的G-CSF信号传导所必需的。 GFI1-NULL小鼠在胸腺，脾和骨髓中降低了RASGRP1 mRNA和蛋白质水平，而髓样细胞中的GFI1转导可促进RASGRP1的表达。当用G-CSF刺激时，GFI1-NULL髓样细胞在激活ERK1/2时有选择性有缺陷，但没有信号转录器和转录1（STAT1）或STAT3的激活因子，并且未能区分为中性粒细胞。 RASGRP1在GFI1缺陷型细胞中的表达部分通过G-CSF挽救ERK1/2的激活，并允许通过G-CSF成熟中性粒细胞。 G-CSF从骨髓到外周血的造血祖细胞（HPC）动员是获取造血细胞移植的主要手段，避免了造成侵入性的骨髓骨髓收集。 由于HPC代表通过G-CSF动员的所有血细胞中的少数血细胞，因此需要了解开发选择性药物的基本机制。 现在，我们发现，G-CSF通过促进MicroRNA-126（MiR126）的积累 - 骨髓-126（MiR126）的积累 - 连接微伏菌丝/exosomes/exosomes，在骨髓HPC，基质细胞和内皮细胞上间接降低表面血管细胞粘附分子1（VCAM-1）的表达。 我们发现HPC，基质细胞和内皮细胞很容易掺入这些外泌体，并且MiR126在骨髓HPC，基质细胞和内皮细胞上抑制VCAM-1的表达。 与此相一致，miR126-null小鼠显示了对G-CSF的动员响应降低。 由于成熟的嗜中性粒细胞代表了含有miR126的骨髓微囊泡的主要来源，因此缺乏成熟中性粒细胞的GFI1-NULL小鼠在动员HPC时有缺陷。另外，GFI1缺陷小鼠的骨髓具有异常降低的miR126水平，并且在HPC中表达异常高的VCAM1。 总而言之，我们的结果暗示了MiR126在骨髓和外围部位之间的HPC运输调节中，阐明了VCAM-1在G-CSF介导的动员中的作用，并且对改善HPC选择性动员的方法具有重要意义。