Regulation of hematopoiesis by sclerostin in specific niche cells in vivo

 描述（由申请人提供）：骨微环境的细胞和造血细胞之间的串扰影响彼此的行为，但在成人中骨系细胞和造血干细胞（HSC）之间的相互作用和通信的基础生物学方面存在显著的知识差距。填补这一知识空白很重要，因为它对于了解健康的HSC在骨骼疾病和衰老中如何维持或改变具有潜在意义。该项目的总体目标是确定 控制骨髓中HSC维持和分化的机制，并确定这些机制中哪些受到骨中细胞外在变化的影响。硬化蛋白（基因缩写为Sost，蛋白质缩写为SOST）是骨骼和造血系统之间细胞通讯的一种新介质。SOST是一种分泌性蛋白，主要由骨细胞（OCYs）产生，骨细胞是嵌入骨中的矿化骨细胞。SOST是一种Wnt信号传导拮抗剂，对正常骨稳态至关重要。在Sost-knockout（SostKO）小鼠中，从成骨细胞向OCYs的转变是不受控制的，导致骨量大量增加。先前的研究将其他分泌的Wnt拮抗剂的错误表达与骨稳态失调和长期HSC（LT-HSC）的维持不良联系起来。然而，SOST对LT-HSC的维持、细胞周期和分化的作用仍然是一个悬而未决的问题。成人骨髓含有支持HSC的非造血“小生境”细胞，例如间充质干细胞（MSC）、成骨细胞（OB）和内皮细胞（EC）。MSC和OB作为OCYs的祖细胞和作为HSC的小生境细胞发挥双重作用。SostKO小鼠表现出MSC、OB和EC数量增加，但Sost缺失对其维持LT-HSC功能能力的影响尚不清楚。拟议的研究将测试这样的假设，即由Sost耗竭引起的骨稳态失调以直接影响LT-HSC维持的方式改变骨微环境。SostKO小鼠模型将在系列LT-HSC移植试验中用作受体，以区分暴露于SostKO微环境后LT-HSC行为的永久或暂时变化。将评价由于BM生态位中Sost的丢失而改变的LT-HSC的细胞内在特征，例如改变的LT-HSC细胞分裂速率、Wnt信号传导激活水平和LT-HSC基因组表观遗传变化的证据。与此同时，流式细胞术分选将用于解剖不同的细胞， SostKO MSC、OB和EC在调节LT-HSC命运中的作用。短期体外共培养试验将区分LT-HSC与MSC、EC和/或OB的相互作用是否影响LT-HSC的命运，并且在分选的小生境细胞群中的基因表达分析将鉴定参与HSC维持的差异表达基因。总之，拟议的研究将产生重要的新信息，Sost缺陷如何影响LT-HSC的维护和定义的分子和细胞的球员，参与HSC/生态位串扰。

 DESCRIPTION (provided by applicant): Crosstalk between cells of the bone microenvironment and hematopoietic cells affects each other's behavior, but a significant knowledge gap exists in the basic biology of the interactions and communication between osteolineage cells and hematopoietic stem cells (HSCs) in the adult. Filling this knowledge gap is important, as it has potential significance for understanding how healthy HSCs are maintained or altered in diseases of the bone and with ageing. The overall goal of this project is to identify the mechanisms that control HSC maintenance and differentiation in the bone marrow, and define which of these mechanisms are affected by cell-extrinsic changes in the bone. Sclerostin (abbreviated Sost for the gene, SOST for the protein) is a novel mediator of cellular communication between the skeletal and hematopoietic systems. SOST is a secreted protein that is produced primarily by osteocytes (OCYs), the mineralized bone cells embedded within the bone. SOST is a Wnt signaling antagonist that is critical for normal bone homeostasis. In Sost-knockout (SostKO) mice, the transition from osteoblasts to OCYs is uncontrolled, leading to massive increases in bone mass. Previous studies connected the misexpression of other secreted Wnt antagonists with dysregulated bone homeostasis and poor maintenance of long-term HSCs (LT-HSCs). However, the role of SOST on LT-HSC maintenance, cell cycling and differentiation still remains an open question. The adult bone marrow contains non-hematopoietic "niche" cells that support HSCs, such as mesenchymal stem cells (MSCs), osteoblasts (OBs) and endothelial cells (ECs). MSCs and OBs play dual roles as progenitors of OCYs and as niche cells for HSCs. SostKO mice display increased numbers of MSCs, OBs and ECs, but the effects the loss of Sost on their functional ability of maintain LT-HSCs are not known. The proposed studies will test the hypothesis that dysregulated bone homeostasis caused by Sost-depletion alters bone microenvironments in ways that directly affect the maintenance of LT-HSCs. The SostKO mouse model will be utilized as recipients in serial LT-HSC transplantation assays to discriminate between permanent or temporary changes of LT-HSC behavior after exposure to SostKO microenvironments. Cell-intrinsic characteristics of LT-HSCs that change due to the loss of Sost in the BM niche, such as altered LT-HSC cell division rates, levels of Wnt signaling activation, and evidence of epigenetic changes to the LT-HSC genome will be evaluated. In parallel, flow cytometric sorting will be used to dissect the distinct roles of SostKO MSCs, OBs, and ECs in the regulation of LT-HSC fate. Short-term in vitro co-culture assays will distinguish whether it is the interactions of LT-HSCs with MSCs, ECs, and/or OBs that influences LT-HSC fate, and gene expression analysis in sorted niche cell populations will identify differentially expressed genes that are involved in HSC maintenance. Taken together, the proposed studies will generate important new information on how Sost-deficiency affects LT-HSC maintenance and define the molecular and cellular players that are involved in HSC/niche crosstalk.