Physiological Roles of Bone Morphogenetic Proteins (BMPs) in Skeletal

骨形态发生蛋白 (BMP) 在骨骼中的生理作用

基本信息

批准号：
22570135
负责人：
TSUJI Kunikazu
金额：
$ 3万
依托单位：
Tokyo Medical and Dental University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2010
资助国家：
日本
起止时间：
2010 至 2012
项目状态：
已结题

项目摘要

Project 1. BMP4 regulates the hematopoietic stem cell niche size in bone marrow BMP4 (Bone morphogenetic protein 4) signal has critical roles in inducing hematopoietic tissues during embryogenesis. However, evaluating the importance of BMP4 in bone marrow hematopoiesis is complicated by early embryonic lethality in mice lacking BMP4, and by conservation of signaling pathways for osteogenic BMPs. To overcome these limitations and to define the roles of BMP4 in bone marrow hematopoiesis, we established BMP4 conditional knockout mice in which we inactivated BMP4 in adult bone marrow (Bmp4Mx1cre). Here we report that the loss of BMP4 in bone marrow cells significantly reduced the hematopoietic stem cell population. BMP4 gene was knocked-out by the intraperitoneal injection of poly I:C into Bmp4Mx1cre mice at 6 weeks of age. Six to 8 weeks after the injection (after inducing the genomic recombination), total bone marrow cells were isolated from femurs and tibiae, stained by hematopoietic ce … More ll surface markers, and analyzed by flow-cytometry (BD FACS Calibur). We found that the populations of differentiated blood cells such as Erythrocytes (Ter119+), T-cells (CD3+), B-cells (CD19+), and Monocytes/Macrophages (CD11b+) were not significantly altered in the absence of BMP4. However, the hematopoietic stem cell fraction (Lin-, c-Kit+, Sca-1+) was significantly decreased in Bmp4Mx1cre mice. These results are specific for the loss of BMP4, because we did not observe any alteration in hematopoietic cell population in the absence of BMP7 (Bmp7Mx1cre). We also did not observe any synergistic effect in the co-absence of BMP4 and BMP7 (Bmp4/7Mx1cre). These results suggest the unique and important roles of BMP4 in the regulation of hematopoietic stem cell niche size in adult bone marrow.Project 2. Endogenous BMP7 activity is prerequisite for postnatal joint homeostasis While the osteo- and chondro-inductive activities of recombinant bone morphogenetic protein 7 (BMP7) are well established, evaluation of the role of endogenous BMP7 in bone and cartilage homeostasis has been hampered by perinatal lethality in BMP7 knockout mice. To overcome these problems, we employed conditional deletion of BMP7 from the embryonic limb prior to the onset of skeletogenesis to create limb skeletons lacking BMP7. We have reported that the absence of locally produced BMP7 had no effect on postnatal limb growth, articular cartilage formation, maintenance of bone mass, or fracture healing. In this study, to evaluate the roles of endogenous BMP7 in postnatal joint homeostasis, we performed detailed histological analyses of the knee joint in adult BMP7 conditional knockout mice and found the accelerated degeneration of articular cartilage in the absence of endogenous BMP7. Safranin O staining of sagittal sections of the knee joint from 24 week-old mice revealed the significant loss in proteoglycan contents in articular cartilage matrix in the absence of endogenous BMP7. We observed less severe but similar results in the growing BMP7 conditional knockout mice (8 week-old). Extensive articular cartilage degeneration we observed in the mice at 8 week-old andolder may not be due to the defect in cartilage formation since there was no significant alteration in both articular structure and proteoglycan contents in the juvenile BMP7 knockout mice (at 4 weeks of age).To further analyze the physiological roles of BMP7 in the maintenance of articular cartilage, we investigated the chondrocyte survival, severity of synovial inflammation, and expression of matrix-degrading enzymes, such as hyaluronidase and MMP-13, in the BMP7 knockout mice. TUNEL staining of articular cartilage revealed that BMP7 did not affect chondrocyte survival at 8 weeks of age. Histological evaluation revealed that extensive synovial hyperplasia was observed in 8-week old BMP7 knockout mice. It seemed that severe synovitis occurred in the knockout mice since significant numbers of the cells in synovial membrane were positive for F4/80, a surface marker for mice macrophages. In contrast, appearance of synovial membrane was quite similar between control and BMP7 knockout mice at 4 weeks of age. Gene expression analysis of joint tissue from BMP7 knockout mice revealed that the expression of MMP-13 but not hyaluronidase was increased at 24 weeks of age. These data suggest that BMP7 maintains articular cartilage by negatively regulating synovial inflammation and MMP expression in the adult mice. Less

项目 1. BMP4 调节骨髓中的造血干细胞生态位大小 BMP4（骨形态发生蛋白 4）信号在胚胎发生过程中诱导造血组织中发挥关键作用。然而，评估 BMP4 在骨髓造血中的重要性因早期胚胎致死性而变得复杂。缺乏 BMP4 的小鼠，并通过保护成骨 BMP 的信号通路来克服这些限制并确定 BMP4 在骨髓中的作用。我们建立了 BMP4 条件性敲除小鼠，其中我们灭活了成人骨髓中的 BMP4（Bmp4Mx1cre），在此我们报告，骨髓细胞中 BMP4 的丢失显着减少了通过腹膜内敲除的造血干细胞群。注射后 6 至 8 周（诱导后）将 Poly I:C 注射到 6 周龄的 Bmp4Mx1cre 小鼠中。基因组重组），从股骨和胫骨中分离出总骨髓细胞，用造血细胞表面标记物染色，并通过流式细胞术（BD FACS Calibur）进行分析，我们发现分化的血细胞群，例如红细胞（Ter119+）。 )、T 细胞 (CD3+)、B 细胞 (CD19+) 和单核细胞/巨噬细胞(CD11b+) 在 BMP4 缺失的情况下没有显着改变，但是，Bmp4Mx1cre 小鼠中的造血干细胞分数 (Lin-、c-Kit+、Sca-1+) 显着降低。因为我们没有观察到在缺乏 BMP7 (Bmp7Mx1cre) 的情况下造血细胞群有任何变化，我们也没有观察到任何协同效应。在 BMP4 和 BMP7 共同缺失的情况下 (Bmp4/7Mx1cre) 这些结果表明 BMP4 在调节成人骨髓中造血干细胞生态位大小方面具有独特而重要的作用。项目 2. 内源性 BMP7 活性是出生后关节的先决条件。体内平衡虽然重组骨形态发生蛋白 7 (BMP7) 的骨和软骨诱导活性已得到充分证实，对内源性 BMP7 在骨和软骨稳态中的作用的评估因 BMP7 敲除小鼠的围产期致死性而受到阻碍。为了克服这些问题，我们在骨骼发生开始之前从胚胎肢体中条件性删除 BMP7，以创建缺乏 BMP7 的肢体骨骼。我们报道，本地产生的 BMP7 的缺乏对出生后肢体生长、关节软骨形成、骨量维持或骨折愈合没有影响。为了评估内源性 BMP7 在出生后关节稳态中的作用，我们对成年 BMP7 条件敲除小鼠的膝关节进行了详细的组织学分析，发现在矢状切片缺乏内源性 BMP7 的情况下，关节软骨加速退化。 24 周龄小鼠的膝关节显示，在缺乏内源性 BMP7 的情况下，关节软骨基质中的蛋白多糖含量显着减少。在生长中的 BMP7 条件性敲除小鼠（8 周龄）中，我们在 8 周龄及以上的小鼠中观察到广泛的关节软骨退化，这可能不是由于软骨形成的缺陷，因为两个关节都没有明显的变化。幼年 BMP7 敲除小鼠（4 周龄）的结构和蛋白多糖含量。为了进一步分析 BMP7 在维持关节软骨中的生理作用，我们研究了 BMP7 敲除幼年小鼠的结构和蛋白多糖含量。 BMP7 敲除小鼠的软骨细胞存活率、滑膜炎症的严重程度以及基质降解酶（例如透明质酸酶和 MMP-13）的表达，关节软骨的 TUNEL 染色显示 BMP7 不影响 8 周龄时的软骨细胞存活率。评估显示，8周龄BMP7敲除小鼠出现广泛的滑膜增生，似乎发生了严重的滑膜炎。敲除小鼠的滑膜中大量细胞呈 F4/80（小鼠巨噬细胞表面标记）阳性，相比之下，4 周龄时对照小鼠和 BMP7 敲除小鼠的滑膜外观非常相似。对 BMP7 敲除小鼠关节组织的分析表明，24 周龄时，MMP-13 的表达增加，但透明质酸酶的表达没有增加。这些数据表明，BMP7 通过负作用维持关节软骨。调节成年小鼠滑膜炎症和 MMP 表达。

项目成果

期刊论文数量（0）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Conditional deletion of BMP7 from the limb skeleton does not affect bone formation or fracture repair.

DOI：
10.1002/jor.20996
发表时间：
2010-03
期刊：
Journal of orthopaedic research : official publication of the Orthopaedic Research Society
影响因子：
0
作者：
Tsuji K;Cox K;Gamer L;Graf D;Economides A;Rosen V
通讯作者：
Rosen V

Mesenchymal stem cells in synovial fluid increase after meniscus injury.

DOI：
10.1007/s11999-013-3418-4
发表时间：
2014-05
期刊：
Clinical orthopaedics and related research
影响因子：
4.2
作者：
Matsukura Y;Muneta T;Tsuji K;Koga H;Sekiya I
通讯作者：
Sekiya I

BMP4 regulates the hematopoietic stem cell niche size in bone marrow

BMP4 调节骨髓中造血干细胞生态位大小

DOI：
发表时间：
2011
期刊：
影响因子：
0
作者：
森俊輔;鈴木健夫;鈴木勉;遠藤斗志也;吉久徹;Abula K.;T. Yoshihisa;Kunikazu Tsuji
通讯作者：
Kunikazu Tsuji

Properties and usefulness of aggregates of synovial mesenchymal stem cells as a source for cartilage regeneration.

DOI：
10.1186/ar3869
发表时间：
2012-06-07
期刊：
Arthritis research & therapy
影响因子：
4.9
作者：
Suzuki S;Muneta T;Tsuji K;Ichinose S;Makino H;Umezawa A;Sekiya I
通讯作者：
Sekiya I

Differences in gene expression between the otic capsule and other bones.

DOI：
10.1016/j.heares.2010.02.006
发表时间：
2010-06-14
期刊：
HEARING RESEARCH
影响因子：
2.8
作者：
Stankovic, Konstantina M.;Adachi, Osamu;Tsuji, Kunikazu;Kristiansen, Arthur G.;Adams, Joe C.;Rosen, Vicki;McKenna, Michael J.
通讯作者：
McKenna, Michael J.