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: 9153665
• 负责人: Giovanna Tosato
• 金额: $ 51.77万
• 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/9153665
• 关键词: AMD3100; Agonist; Alleles; Area; Bacterial Infections; Binding; Biochemical; Biological; Blood; Blood Cells; Blood Circulation; Bone Marrow; Bone Marrow Cells; C-terminal; CSF3 gene; CXCL12 gene; CXCR4 Receptors; CXCR4 gene; Cell Lineage; Cell surface; Cells; Clinical; Collection; Colony-Stimulating Factor Receptors; Colony-Stimulating Factors; Complex; Defect; Derivation procedure; Development; Disease; Dominant-Negative Mutation; Down-Regulation; Endothelial Cells; Endothelium; Ensure; FDA approved; Generations; Genetic study; Goals; Granulopoiesis; Hematopoiesis; Hematopoietic; Hematopoietic Stem Cell Mobilization; Hematopoietic stem cells; Hereditary Disease; Heterozygote; In Vitro; Investigation; Knockout Mice; Ligands; Link; Maintenance; Malignant Neoplasms; Mature Bone; Mediating; Methods; MicroRNAs; Minority; Mus; Mutation; Myelogenous; Myeloid Cells; Myeloid Leukemia; Neutropenia; Patients; Peptides; Peripheral; Pharmaceutical Preparations; Physiological; Process; Production; Property; Proteins; Receptor Cell; Recruitment Activity; Regulation; Research Design; Research Project Grants; Role; Signal Transduction; Site; Staging; Stem cells; Stromal Cell-Derived Factor 1; Stromal Cells; Surface; Syndrome; Therapeutic; Time; Transcription Repressor/Corepressor; Transplantation; Vascular Cell Adhesion Molecule-1; base; chemokine; controlled release; embryonic stem cell; extracellular; hematopoietic cell transplantation; improved; inhibitor/antagonist; migration; mutant; neutrophil; peripheral blood; prevent; progenitor; receptor; reconstitution; research study; response; selective expression; stem; trafficking; transcription factor; tumor progression

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. Defective neutrophil maturation/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. 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, promotes 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: G-CSF promotes expression of Gfi-1, which reduces 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 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. In addition to promoting the release of mature myeloid cells, G-CSF promotes the release of HSPC (hematopoietic stem/progenitor cells) from the bone marrow to the peripheral blood. 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 small 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 HSPC, 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 HSPC, stromal cells and endothelial cells readily incorporate these exosomes, and that miR126 represses VCAM-1 expression on bone marrow HSPC, stromal cells and endothelial cells. In line with this, miR126-null mice display a reduced mobilization response to G-CSF. 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. Ongoing studies designed to further understanding of HSPC mobilization have detected an important role of the receptor/ligand pair EphrinB2/EphB4. Thus, blocking this interaction with blocking peptides prevents stem cell mobilization from the bone marrow to the blood in mice. Immunohistochemical analysis of bone marrow sections has revealed that bone marrow sinusoidal endothelium selectively expresses EphB4, which is not detected in hematopoietic cells. The sinusoidal endothelium does not express ephrinB2, which is instead expressed in the bone marrow hematopoietic cells. Transmigration experiments suggest that EphrinB2/EphB4 are critical for HSPC trans-endothelial migration, but not the migration of other mature hematopoietic cells. Thus, agonist activation of ephrinB2 should promote selective HSPC exit from the bone marrow. Conversely, inhibition of EphrinB2/EphB4 interaction, which blocks HSPC exit from the bone marrow, could be exploited to reduce HSPC contribution to tumor progression. Other ongoing studies have focused on the generation of hematopoietic cells from aortic endothelium, and the characterization of the biochemical requirements underlying this critical developmental step. We have discovered that EphrinB2 expression is critical to the development of hematopoiesis from murine ES cells, and that EphrinB2 critically regulates the expression of transcription factors that orchestrate developmental hematopoiesis. We are currently exploring the mechanistic aspects of EphrinB2 expression in ES cells at bhe very early stages of differentiation. There is a critical need for generation of HSPC to be used in clinical transplantation. The ultimate goal of our investigation is to develop a method for production of such cells in vitro.

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/释放有缺陷与各种形式的嗜中性粒细胞减少有关，这可能在可能之前与髓样白血病的发展有关。 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。 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，可促进中性粒细胞和造血细胞向周围血液的动员。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围血液的动员：G-CSF促进GFI-1的表达，从而促进了CXCR4表达和功能。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。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。除促进成熟的髓样细胞的释放外，G-CSF还促进了HSPC（造血干/祖细胞）从骨髓到外周血的释放。 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代表了通过G-CSF动员的所有血细胞中的一小部分，因此需要了解开发选择性药物的潜在机制。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，间接地降低了骨髓HSPC，基质细胞和内皮细胞上表面血管细胞粘附分子1（VCAM-1）的表达。我们发现HSPC，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HSPC，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。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。旨在进一步了解HSPC动员的正在进行的研究已经检测到受体/配体对Ephrinb2/ephb4的重要作用。因此，阻断与阻断肽的这种相互作用可防止干细胞从骨髓到小鼠血液的动员。骨髓切片的免疫组织化学分析表明，骨髓正弦内皮有选择地表达EPHB4，这在造血细胞中未检测到。正弦内皮不表达ephrinb2，而不是在骨髓造血细胞中表达。移民实验表明，ephrinb2/ephb4对于HSPC跨层层迁移至关重要，但不是其他成熟造血细胞的迁移。因此，Ephrinb2的激动剂激活应促进骨髓中的选择性HSPC退出。相反，可以利用阻断HSPC退出的Ephrinb2/Ephb4相互作用的抑制作用，以减少HSPC对肿瘤进展的贡献。其他正在进行的研究集中在主动脉内皮细胞中产生造血细胞，以及这一关键发育步骤的生化需求的表征。我们已经发现，以phrinb2的表达对于鼠ES细胞的造血作用至关重要，而ephrinb2严重调节了调节发育型造血的转录因子的表达。我们目前正在探索ES细胞在分化的早期阶段的ES细胞中Ephrinb2表达的机械方面。在临床移植中使用HSPC的产生至关重要。我们研究的最终目标是开发一种体外生产此类细胞的方法。