Study of the Roles of SDF1 and CXCR4 in Hematopoiesis

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。 中性粒细胞成熟和释放的改变与各种形式的中性粒细胞减少症有关，这可能先于髓系白血病的发展并在发病机制上与之相关。 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。 遗传学研究和其他研究已确定转录因子 Gfi1 是干细胞和骨髓细胞功能的关键贡献者。 因此，Gfi1缺失的小鼠无法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在中性粒细胞成熟及其从骨髓中释放中的关系，发现Gfi1调节G-CSF信号传导。 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 髓样细胞中 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。 我们发现体外和体内 G-CSF 促进 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，抑制骨髓谱系细胞中 CXCR4 的表达。 结果，骨髓细胞对 CXCR4 独特配体 SDF1 的反应减弱。 因此，Gfi1不仅调节造血干细胞功能和骨髓细胞发育，还可能通过降低CXCR4表达和功能促进粒细胞谱系细胞从骨髓释放到外周血。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 无法正确招募 β 抑制蛋白 2，但不能将 β 抑制蛋白 1 招募到受体复合物中。结果，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。由于转录因子 Gfi1 和 G-CSF/G-CSFR 各自都是骨髓中常见骨髓细胞/单核细胞前体细胞分化的关键贡献者，因此我们研究了它们的关系。 我们发现了 Gfi1 作为 G-CSF/G-CSFR 信号传导和功能调节剂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，RasGRP1 是一种激活 Ras 的交换因子，并且 RasGRP1 是通过 Ras/丝裂原激活蛋白/细胞外信号调节激酶 (MEK/Erk) 途径的 G-CSF 信号传导所必需的。 Gfi1缺失小鼠的胸腺、脾脏和骨髓中RasGRP1 mRNA和蛋白水平降低，并且骨髓细胞中的Gfi1转导促进RasGRP1表达。当用 G-CSF 刺激时，Gfi1 缺失的骨髓细胞在激活 Erk1/2 方面存在选择性缺陷，但在激活信号转导子和转录激活子 1 (STAT1) 或 STAT3 方面没有缺陷，并且无法分化为中性粒细胞。 RasGRP1 在 Gfi1 缺陷细胞中的表达部分挽救了 G-CSF 激活的 Erk1/2，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少数，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通过促进骨髓细胞外区室中含有microRNA-126（miR126）的微泡/外泌体的积累，间接降低骨髓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掺入这些外泌体，并且 miR126 抑制骨髓 HPC、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。 由于成熟的中性粒细胞是含有 miR126 的骨髓微泡的主要来源，因此缺乏成熟中性粒细胞的 Gfi1 缺失小鼠在 HPC 的动员方面存在缺陷。此外，Gfi1缺陷小鼠的骨髓中miR126的水平异常降低，并且在HPC中表达异常高水平的VCAM1。 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。

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.