ROBO4-Mediated Vascular Integrity Regulates the Directionality of Hematopoietic Stem Cell Trafficking

摘要

class="head no_bottom_margin" id="sec1title">IntroductionHematopoietic stem cells (HSCs) reside primarily in the bone marrow (BM). This selective location results in part from the unique ability of BM niches to support HSC self-renewal and long-term maintenance. Intense interest in the complex regulation of HSC self-renewal has led to significant progress in understanding the cellular and molecular composition of BM niches (reviewed in ). Because osteoblasts are only present in bone, they may provide an environment that helps to regulate the selective location of HSCs to BM. Several lines of evidence support this notion (reviewed in ). Recent evidence also points to the vascular endothelium and associated cells as important regulators of HSC maintenance and location (), and most HSCs localize near sinusoidal endothelial cells (SECs) (). Thus, accumulating evidence indicates that vascular structures within the BM are necessary for optimal HSC function.Another mechanism that is likely involved in specifying HSC location to the BM is regulated trafficking between the BM and vasculature. HSC residence in BM niches is far from static, with circulation in the blood stream occurring under steady-state physiological conditions (), between different hematopoietic organs during development, and as an essential requirement for successful hematopoietic transplantation therapies. During trafficking to and from the BM, HSCs have to traverse the vascular endothelium. Differential vascular structures of different organs that either prevent or allow HSC entry likely play important roles in guiding HSCs specifically to the BM. Here, we show that the integrity of the vascular endothelium and its ability to regulate directional HSC trafficking to the BM depend on the single transmembrane cell-surface receptor ROBO4.We recently reported that ROBO4, expressed by HSCs, promotes HSC localization to BM niches at steady state and upon transplantation (). ROBO4 is a member of the ROBO family of guidance receptors that respond to Slits, secreted proteins that are essential for neuronal development (). ROBO4 was previously identified as an EC-selective protein () and its support of vascular integrity seems to be particularly important in dynamic situations such as vascular stress, inflammation, and pregnancy (). ROBO4 was found by our group and others to also be expressed by HSCs, but not hematopoietic progenitor or mature cells (). We previously reported that hematopoietic ROBO4 acts as an HSC-selective adhesion molecule that promotes HSC location to BM niches (). ROBO4 deletion led to increased numbers of HSCs in the peripheral blood (PB) at steady state and reduced engraftment upon competitive transplantation into wild-type (WT) mice. We also found that CXCR4, a G protein-coupled receptor and well-established regulator of HSC location (), was upregulated on ROBO4-deficient HSCs, mitigating the effects of ROBO4 loss. Consequently, ROBO4-deficient HSCs displayed heightened responsiveness to mobilization with the CXCR4 inhibitor AMD3100. Functional differences in the hematopoietic system upon ROBO4 deletion were highly selective for HSCs and did not involve alterations in the number or function of hematopoietic progenitors or mature cells. We also did not detect a defect in cell-cycle status or proliferation of either HSCs or their progeny upon ROBO4 loss or in response to Slits. Similar results were reported independently by others (). Collectively, these data demonstrated that ROBO4 on HSCs promotes HSC localization to the BM. Here, we report that in addition to ROBO4 expressed by HSCs, endothelial ROBO4 is essential for efficient HSC engraftment. Using a combination of in vitro and in vivo assays, we identify the cellular and molecular mechanisms by which endothelial ROBO4 promotes HSC location to the BM, and reveal strategies for manipulating HSC location.