|Budget Amount *help
¥4,680,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥1,080,000)
Fiscal Year 2012: ¥1,040,000 (Direct Cost: ¥800,000、Indirect Cost: ¥240,000)
Fiscal Year 2011: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
Fiscal Year 2010: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
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
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