Study of the Roles of SDF1 and CXCR4 in Hematopoiesis

从造血前体产生中性粒细胞并释放到外周循环是一个高度调控的过程，它确保维持血液中中性粒细胞水平的稳态，并在细菌感染和其他信号的反应中升高。中性粒细胞成熟和释放的改变与各种形式的中性粒细胞减少有关，这可能先于骨髓性白血病的发展，并在病理上与之相关。由于携带集落刺激因子（G-CSF）或其受体纯合缺失的小鼠严重中性粒细胞减少，G-CSF已成为颗粒生成的关键生理调节剂，并且G-CSF的显性阴性突变与颗粒生成的严重缺陷有关。给药G-CSF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向外周血的释放。基于这些特性，G-CSF被广泛用于诱导粒细胞生成和动员造血祖细胞进入外周血。最近，一种CXCR4竞争性抑制剂AMD3100/ plerixfluor已被FDA批准，并作为造血前体动员剂与G-CSF联合使用。遗传研究和其他研究已经确定转录因子Gfi1是干细胞和髓细胞功能的关键贡献者。因此，Gfi1缺失的小鼠不能产生成熟的中性粒细胞，这归因于中性粒细胞在普通粒细胞/单核细胞前体阶段的成熟受阻。我们研究了Gfi1和G-CSF/G-CSFR在中性粒细胞成熟和骨髓释放中的关系，发现Gfi1调节G-CSF信号。G-CSF的生物活性仅通过激活在髓系祖细胞上表达的G-CSF受体(R)来介导。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向外周血的动员。WHIM是一种遗传性疾病，与CXCR4细胞内结构域突变相关，导致CXCR4功能增加，导致未成熟中性粒细胞滞留骨髓和严重的外周中性粒细胞减少。AMD3100是一种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向外周血的动员。在干细胞动员G-CSF过程中，骨髓中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可降低表达G-CSFR的骨髓Gr1+髓样细胞中CXCR4的表达。在其他研究中，我们已经获得证据表明转录抑制因子Gfi-1参与了g - csf诱导的粒细胞谱系细胞从骨髓到外周血的动员。我们发现G-CSF在体外和体内均能促进Gfi-1的表达，下调CXCR4的表达。Gfi-1与CXCR4基因上游的DNA序列结合，抑制髓系细胞中CXCR4的表达。因此，髓细胞对CXCR4独特配体SDF1的反应降低。因此，Gfi1不仅调节造血干细胞功能和髓系细胞发育，还可能通过降低CXCR4的表达和功能，促进粒细胞系细胞从骨髓向外周血的释放。在相关实验中，我们已经生成了CXCR4的突变体，该突变体模拟了WHIM综合征患者c端结构域的突变。我们研究了野生型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。我们的研究结果表明，与正常受体不同，突变的CXCR4不能适当地招募β - arrestin2，但不能招募β - arrestin1到受体复合物中。因此，突变的CXCR4受体从细胞表面到细胞质室的内化被延迟，降解被延迟，突变受体的信号传导也被延迟。由于WHIM患者是突变的CXCR4受体的杂合子，并且同时携带正常和突变的等位基因，因此最终结果是CXCR4信号在时间上延长，因为它是正常和突变受体同时激活的结果。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中，该配体将成熟的中性粒细胞保存在骨髓腔室中。由于转录因子Gfi1和G-CSF/G-CSFR都是骨髓中髓细胞从普通髓细胞/单核细胞前体分化的关键因素，我们研究了它们之间的关系。我们已经发现了Gfi1作为G-CSF/G-CSFR信号和功能的调节因子的先前未被认识的功能。具体来说，我们发现Gfi1调节Ras鸟嘌呤核苷酸释放蛋白1 （RasGRP1）的表达，Ras鸟嘌呤核苷酸释放蛋白1是一种激活Ras的交换因子，并且RasGRP1是通过Ras/丝裂原活化蛋白/细胞外信号调节激酶（MEK/Erk）途径进行G-CSF信号传导所必需的。Gfi1缺失小鼠胸腺、脾脏和骨髓中RasGRP1 mRNA和蛋白水平降低，骨髓细胞中Gfi1的转导促进了RasGRP1的表达。当G-CSF刺激时，gfi1缺失的髓细胞选择性地激活Erk1/2，但不激活信号换能器和转录激活因子1 （STAT1）或STAT3，并且不能分化为中性粒细胞。RasGRP1在gfi1缺陷细胞中的表达部分地挽救了G-CSF对Erk1/2的激活，并允许G-CSF使中性粒细胞成熟。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用于造血细胞移植的主要手段，避免了侵入性骨髓采集。由于HPC只占G-CSF动员的所有血细胞的一小部分，因此有必要了解其潜在机制以开发选择性药物。我们现在发现G-CSF通过促进骨髓细胞外室中含有microRNA-126 （miR126）的微囊/外泌体的积累，间接降低了骨髓HPC、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HPC、基质细胞和内皮细胞很容易结合这些外泌体，miR126抑制骨髓HPC、基质细胞和内皮细胞中VCAM-1的表达。与此一致，mir126缺失的小鼠对G-CSF的动员反应降低。由于成熟中性粒细胞是含有miR126的骨髓微泡的主要来源，缺乏成熟中性粒细胞的gfi1缺失小鼠在动员HPC方面存在缺陷。此外，gfi1缺陷小鼠的骨髓miR126水平异常降低，HPC中VCAM1表达异常高水平。总之，我们的研究结果表明miR126参与了HPC在骨髓和外周部位之间运输的调节，阐明了VCAM-1在g - csf介导的动员中的作用，并对改进HPC的选择性动员方法具有重要意义。

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.