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.

造血前体细胞产生中性粒细胞及其释放到外周循环是受到高度调控的过程，可确保血液中中性粒细胞稳态的维持及其响应细菌感染和其他信号而升高。中性粒细胞成熟/释放缺陷与各种形式的中性粒细胞减少症相关，这可能先于髓系白血病的发展并在发病机制上与髓系白血病的发展相关。 G-CSF 已成为粒细胞生成的重要生理调节剂，因为携带集落刺激因子 (G-CSF) 或其受体纯合缺失的小鼠严重中性粒细胞减少，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的施用诱导骨髓中髓系细胞的扩增，并促进中性粒细胞和造血祖细胞从骨髓释放到外周血。基于这些特性，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。最近，CXCR4 竞争性抑制剂 AMD3100/Plerixaflu 已获得 FDA 批准，可作为与 G-CSF 联合使用的造血前体细胞动员剂。 G-CSF 的生物活性仅由其激活在骨髓谱系祖细胞上表达的 G-CSF 受体 (R) 介导。来自遗传学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至外周血。 WHIM 是一种与 CXCR4 胞内结构域突变相关的遗传性疾病，导致 CXCR4 功能增强，导致未成熟中性粒细胞滞留在骨髓中，并导致严重的外周中性粒细胞减少症。 AMD3100 是 SDF-1 与其受体结合的竞争性抑制剂，也是 SDF-1 的突变形式，可诱导 CXCR4 表面受体的长期下调，促进中性粒细胞和造血细胞动员至外周血。在用 G-CSF 动员干细胞期间，骨髓中的 SDF-1 和 CXCR4 蛋白水平降低。我们已经研究了导致 CXCR4 表达减少的机制。最初，我们发现 G-CSF 降低表达 G-CSFR 的骨髓 Gr1+ 骨髓细胞中 CXCR4 的表达。在其他研究中，我们获得的证据表明转录抑制因子 Gfi-1 参与 G-CSF 诱导的粒细胞谱系细胞从骨髓到外周血的动员：G-CSF 促进 Gfi-1 的表达，从而降低 CXCR4表达和功能。在相关实验中，我们生成了 CXCR4 突变体，模仿 WHIM 综合征患者 C 端结构域的突变。我们检查了野生型 CXCR4 的信号传导机制，并与突变型 CXCR4 受体的信号传导进行了比较。我们的结果表明，与正常受体不同，突变体CXCR4无法适当地将β抑制蛋白2招募到受体复合物中。因此，突变型 CXCR4 受体从细胞表面到细胞质区室的内化被延迟，降解被延迟，并且来自突变型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，并且同时携带正常和突变等位基因，因此最终结果是 CXCR4 信号传导时间延长，因为它是正常和突变受体激活的结果。因此，WHIM 患者具有超功能的 CXCR4 受体，并且可能由于配体 SDF1 的持续信号传导而无法将中性粒细胞从骨髓释放到外周血，配体 SDF1 将成熟的中性粒细胞保留在骨髓室中。除了促进成熟骨髓细胞的释放外，G-CSF还促进HSPC（造血干/祖细胞）从骨髓释放到外周血。通过 G-CSF 将造血祖细胞 (HPC) 从骨髓动员到外周血是获得干细胞移植物进行造血细胞移植的主要手段，避免侵入性骨髓采集。由于 HPC 代表 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。我们现在发现，G-CSF通过促进含有microRNA-126（miR126）的微泡/外泌体在骨髓HSPC、基质细胞和内皮细胞上的积累，间接降低表面血管细胞粘附分子1（VCAM-1）的表达。骨髓细胞外室。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 抑制骨髓 HSPC、基质细胞和内皮细胞上的 VCAM-1 表达。与此相符，miR126 缺失的小鼠对 G-CSF 的动员反应减弱。总而言之，我们的结果表明 miR126 参与了骨髓和外周部位之间 HPC 运输的调节，阐明了 VCAM-1 在 G-CSF 介导的动员中的作用，并对改进选择性动员 HPC 的方法具有重要意义。正在进行的旨在进一步了解 HSPC 动员的研究已检测到受体/配体对 EphrinB2/EphB4 的重要作用。因此，用阻断肽阻断这种相互作用可以防止小鼠干细胞从骨髓动员到血液。骨髓切片的免疫组织化学分析显示，骨髓窦内皮选择性表达EphB4，而在造血细胞中未检测到EphB4。肝窦内皮不表达肝配蛋白B2，而是在骨髓造血细胞中表达。迁移实验表明 EphrinB2/EphB4 对 HSPC 跨内皮迁移至关重要，但对其他成熟造血细胞的迁移则不然。因此，ephrinB2 的激动剂激活应促进选择性 HSPC 从骨髓中排出。相反，抑制 EphrinB2/EphB4 相互作用（阻止 HSPC 从骨髓中排出）可用于减少 HSPC 对肿瘤进展的贡献。其他正在进行的研究主要集中在主动脉内皮生成造血细胞，以及这一关键发育步骤背后的生化要求的特征。我们发现 EphrinB2 的表达对于小鼠 ES 细胞造血的发育至关重要，并且 EphrinB2 关键性地调节协调发育造血的转录因子的表达。我们目前正在探索分化早期阶段 ES 细胞中 EphrinB2 表达的机制。迫切需要生成用于临床移植的 HSPC。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。