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。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调节因子，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。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SDF-1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。骨髓中成骨细胞、基质细胞和内皮细胞组成性地表达SDF-1；造血细胞表达CXCR4。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Gr-1+髓样细胞中CXCR4的表达。这与早期观察结果一致，即在g - csf诱导动员后循环中发现的中性粒细胞和CD34+造血细胞中的CXCR4水平降低。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，骨髓细胞对CXCR4独特配体SDF-1的反应降低。因此，Gfi-1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。我们的初步结果表明，与正常受体不同，突变的CXCR4不能适当地将β抑制蛋白招募到受体复合物中。最近，一种新的SDF-1受体被发现，命名为RDC-1/CXCR7。该受体在骨髓中性粒细胞释放中的作用尚未被研究。此外，还产生了GFI-1缺失小鼠，表现出严重的中性粒细胞减少症和中性粒细胞成熟缺陷。正在进行的研究主要集中在研究RDC-1/CXCR7的功能和GFI-1对髓细胞分化的调节。

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. G-CSF has emerged a critical 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. 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 SDF-1 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 mmature 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. Osteoblasts, stromal cells and endothelial cells constitutively express SDF-1 in the bone marrow; hematopoietic cells express CXCR4. 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 Gr-1+ myeloid cells, which express G-CSFR. This is consistent with earlier observations that CXCR4 levels are reduced on neutrophils and CD34+ hematopoietic cells found in the circulation after G-CSF-induced mobilization. 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 SDF-1 are reduced. Thus, Gfi-1 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 preliminary results indicate that unlike the normal receptor, mutant CXCR4 fails to appropriately recruit beta arrestin to the receptor complex. Recently, a new receptor for SDF-1 has been identified, named RDC-1/CXCR7. The role of this receptor in the release of neutrophils from the bone marrow has not been investigated. In addition, GFI-1 null mice have been generated, showing severe neutropenia and defective neutrophil maturation. Ongoing studies are focused on the study of RDC-1/CXCR7 function and on GFI-1 regulation of myeloid differentiation.