Physiological Roles of Bone Morphogenetic Proteins (BMPs) in Skeletal

项目1。BMP4 （bone morphogenetic protein 4，骨形态发生蛋白4）信号在胚胎发生过程中对造血组织的诱导具有重要作用。然而，评估BMP4在骨髓造血中的重要性是复杂的，因为缺乏BMP4的小鼠的早期胚胎致死，以及成骨BMPs信号通路的保存。为了克服这些限制并确定BMP4在骨髓造血中的作用，我们建立了BMP4条件敲除小鼠，在这些小鼠中，我们灭活了成人骨髓中的BMP4 （Bmp4Mx1cre）。在这里，我们报道骨髓细胞中BMP4的缺失显著减少了造血干细胞的数量。6周龄Bmp4Mx1cre小鼠腹腔注射聚I:C敲除BMP4基因。注射后6 ~ 8周（诱导基因组重组后），从股骨和胫骨中分离总骨髓细胞，用造血细胞表面标记染色，流式细胞术（BD FACS Calibur）分析。我们发现，在缺乏BMP4的情况下，红细胞（Ter119+）、t细胞（CD3+）、b细胞（CD19+）和单核/巨噬细胞（CD11b+）等分化的血细胞群体没有显著改变。然而，Bmp4Mx1cre小鼠的造血干细胞组分（Lin-, c-Kit+, Sca-1+）显著降低。这些结果是针对BMP4缺失的，因为我们没有观察到BMP7缺失时造血细胞群的任何改变（Bmp7Mx1cre）。我们也没有观察到BMP4和BMP7 （BMP4 / 7mx1cre）共同缺失时的任何协同效应。这些结果表明BMP4在成人骨髓造血干细胞生态位大小的调控中具有独特而重要的作用。项目2。内源性BMP7活性是出生后关节稳态的先决条件，虽然重组骨形态发生蛋白7 （BMP7）的骨和软骨诱导活性已经确立，但内源性BMP7在骨和软骨稳态中的作用的评估一直受到BMP7敲除小鼠围产期死亡的阻碍。为了克服这些问题，我们在骨骼形成开始之前从胚胎肢体中有条件地删除BMP7，以创建缺乏BMP7的肢体骨骼。我们已经报道了缺乏局部产生的BMP7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，发现在缺乏内源性BMP7的情况下关节软骨的加速退变。24周龄小鼠膝关节矢状面红素O染色显示，在内源性BMP7缺失的情况下，关节软骨基质中蛋白多糖含量明显减少。我们在生长中的BMP7条件敲除小鼠（8周龄）中观察到不那么严重但类似的结果。我们在8周龄及以上的小鼠中观察到广泛的关节软骨退行性变可能不是由于软骨形成缺陷，因为在幼年BMP7基因敲除小鼠（4周龄）中关节结构和蛋白聚糖含量没有明显改变。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（如透明质酸酶和MMP-13）的表达。关节软骨TUNEL染色显示BMP7在8周龄时不影响软骨细胞存活。组织学评估显示，在8周龄BMP7基因敲除小鼠中观察到广泛的滑膜增生。由于大量滑膜细胞F4/80（小鼠巨噬细胞的表面标记物）呈阳性，敲除小鼠似乎发生了严重的滑膜炎。相比之下，在4周龄时，对照组和BMP7基因敲除小鼠的滑膜外观非常相似。BMP7基因敲除小鼠关节组织的基因表达分析显示，在24周龄时，MMP-13的表达增加，而透明质酸酶的表达没有增加。这些数据表明BMP7通过负性调节成年小鼠滑膜炎症和MMP表达来维持关节软骨。少

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