Regulation of hematopoiesis by sclerostin in specific niche cells in vivo

 描述(申请人提供)：骨微环境细胞和造血细胞之间的串扰会影响彼此的行为，但在成人骨线细胞和造血干细胞(HSCs)之间的相互作用和交流的基础生物学方面存在显著的知识差距。填补这一知识空白是重要的，因为它对于了解健康的HSC在骨骼疾病和衰老中是如何维持或改变的具有潜在的意义。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，并确定这些机制中的哪些机制受到骨骼中细胞外源性变化的影响。Skerostin(基因缩写为Sost，蛋白质缩写为Sost)是骨骼系统和造血系统之间细胞通讯的一种新的介体。SOST是一种分泌型蛋白质，主要由骨细胞(OCys)产生，骨细胞是嵌入骨骼中的矿化骨细胞。SOST是一种Wnt信号拮抗剂，对正常的骨骼动态平衡至关重要。在SOST基因敲除(SostKO)小鼠中，从成骨细胞到OCys的转变不受控制，导致骨量大量增加。以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造血干细胞(LT-HSCs)的维护不良联系在一起。然而，SOST在LT-HSC维持、细胞周期和分化中的作用仍是一个悬而未决的问题。成人骨髓含有支持造血干细胞的非造血细胞，如间充质干细胞(MSCs)、成骨细胞(OBS)和内皮细胞(ECs)。MSCs和OBS既是OCys的祖细胞，又是HSCs的生态位细胞。SostKO小鼠表现为MSCs、OBS和ECs数量增加，但SOST丢失对其维持LT-HSCs功能的影响尚不清楚。这项拟议的研究将检验这样一种假设，即耗竭骨量引起的骨稳态失调改变了骨微环境，从而直接影响了肝移植-造血干细胞的维持。SostKO小鼠模型将被用作系列LT-HSC移植试验的受体，以区分暴露于SostKO微环境后LT-HSC行为的永久性或暂时性变化。将评估由于BM利基中Sost的丢失而改变的LT-HSC的细胞固有特征，例如改变的LT-HSC细胞分裂率、Wnt信号激活水平以及LT-HSC基因组的表观遗传学变化的证据。同时，将使用流式细胞仪分选来解剖不同的 SostKO MSCs、OBS和ECs在调节LT-HSC命运中的作用。短期的体外共培养分析将区分LT-HSC与MSCs、ECs和/或OBS的相互作用是否影响LT-HSC的命运，而分类的利基细胞群体中的基因表达分析将识别参与HSC维持的差异表达基因。综上所述，拟议的研究将产生关于SOST缺乏如何影响LT-HSC维持的重要新信息，并确定参与HSC/生态位串扰的分子和细胞角色。

 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.