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

• 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
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-22570135/
• 关键词: ホルモンと生理活性物質; 骨形成因子(BMP); BMP4; BMP7; 造血微小環境; 造血幹細胞; 関節軟骨; コンディショナルノックアウトマウス; BMP; 骨芽細胞; FACS

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。BMP 4调节骨髓造血干细胞小生境的大小。BMP 4（Bone morphogenetic protein 4）信号在胚胎发育过程中诱导造血组织的形成中起着关键作用。然而，评估BMP 4在骨髓造血中的重要性是复杂的，因为缺乏BMP 4的小鼠的早期胚胎致死率，以及成骨BMP的信号传导途径的保守性。为了克服这些局限性并定义BMP 4在骨髓造血中的作用，我们建立了BMP 4条件性敲除小鼠，在其中我们灭活了成人骨髓中的BMP 4（Bmp 4 Mx 1 cre）。在这里，我们报告了骨髓细胞中BMP 4的缺失显著减少了造血干细胞的数量。通过腹腔注射poly I：C将BMP 4基因敲除至6周龄的Bmp 4 Mx 1cre小鼠。注射后6 - 8周（诱导基因组重组后），从股骨和胫骨分离总骨髓细胞，用造血细胞化学染色， ...更多信息 II表面标志物，并通过流式细胞术（BD FACS Calibur）分析。我们发现，在不存在BMP 4的情况下，分化的血细胞如红细胞（Ter 119+）、T细胞（CD 3+）、B细胞（CD 19+）和单核细胞/巨噬细胞（CD 11b+）的群体没有显著改变。然而，造血干细胞分数（Lin-，c-Kit+，Sca-1+）在Bmp 4 Mx 1cre小鼠中显著降低。这些结果对于BMP 4的丢失是特异性的，因为我们在BMP 7（Bmp 7 Mx 1cre）不存在的情况下没有观察到造血细胞群体的任何改变。我们也没有观察到在BMP 4和BMP 7（Bmp 4/7 Mx 1cre）共同缺失的情况下的任何协同效应。这些结果提示BMP 4在调节成人骨髓造血干细胞龛大小中具有独特而重要的作用。内源性BMP 7活性是出生后关节稳态的先决条件虽然重组骨形态发生蛋白7（BMP 7）的骨和软骨诱导活性已被充分确立，但对内源性BMP 7在骨和软骨稳态中的作用的评估已受到BMP 7敲除小鼠围产期致死性的阻碍。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缺失BMP 7，以产生缺乏BMP 7的肢体骨骼。我们已经报道了缺乏局部产生的BMP 7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。在这项研究中，为了评估内源性BMP 7在出生后关节内稳态中的作用，我们对成年BMP 7条件性敲除小鼠的膝关节进行了详细的组织学分析，发现在缺乏内源性BMP 7的情况下关节软骨加速退化。来自24周龄小鼠的膝关节的矢状切片的番红O染色揭示了在不存在内源性BMP 7的情况下关节软骨基质中蛋白聚糖含量的显著损失。我们在生长中的BMP 7条件性敲除小鼠（8周龄）中观察到了不太严重但类似的结果。我们在8周龄及以上的小鼠中观察到的广泛的关节软骨退化可能不是由于软骨形成的缺陷，因为在幼年BMP 7敲除小鼠中关节结构和蛋白多糖含量没有显著的改变为了进一步分析BMP 7在维持关节软骨中的生理作用，我们研究了软骨细胞存活，滑膜炎症的严重程度和基质降解酶如透明质酸酶和MMP-13的表达。关节软骨的TUNEL染色显示BMP 7在8周龄时不影响软骨细胞的存活。组织学评价显示，在8周龄BMP 7敲除小鼠中观察到广泛的滑膜增生。似乎在基因敲除小鼠中发生了严重的滑膜炎，因为滑膜中大量的细胞对F4/80（小鼠巨噬细胞的表面标志物）呈阳性。相比之下，在4周龄时，对照和BMP 7敲除小鼠之间的滑膜外观非常相似。BMP 7基因敲除小鼠关节组织的基因表达分析显示，MMP-13的表达在24周龄时增加，但透明质酸酶的表达没有增加。这些数据表明，BMP 7通过负调节成年小鼠的滑膜炎症和MMP表达来维持关节软骨。少

项目成果

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
• 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

• 发表时间: 2011
• 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

Endogenous bmp7 activity is prerequisite for postnatal joint homeostasis

内源性 bmp7 活性是产后关节稳态的先决条件

• 发表时间: 2012
• 作者: 森俊輔;鈴木健夫;鈴木勉;遠藤斗志也;吉久徹;Abula K.
• 通讯作者: Abula K.

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.