| Bone remodeling includes bone formation and bone resorption. People maintain their bone metabolism in all their lifetime. Bone formation is regulated by osteoblasts, bone resorption is regulated by osteoclasts. Bone metabolism depends on the coupling balance between these two cells and precise regulation. Once the balance is broken osteoporosis and other related bone diseases would occur.NRAGE (neurotrophin receptor-interacting MAGE homolog) is a member of MAGE family, also known as MAGE-D1 or Dlxin-1. Recently, studies on the function of this gene are more concentrated on cell apoptosis, cell cycle and cell differentiation. To date, no report is found about NRAGE funtion's study on the bone development and bone metabolism. We speculate that there maybe exist a correlation between NRAGE and bone development and bone metabolism regulatory from the previous reports. NRAGE gene expression patterns indicated that the gene were expressed abundantly in limb bones and skull. In situ hybridization also showed that a strongest signal in the cell layers surrounding cartilaginous elements in bone rudiment during embryonic digit formation. Moreover, NRAGE can interact with Dlx5, Msx2, Ror2, RORαand so on. These receptors or transcription factors have been proven to play an important role in bone development or bone metabolism. In addition to that, NRAGE can affect the activity of NF-κB. Mice deficient in both the p50 and p52 subunits of NF-kB fail to generate mature osteoclasts and develop severe osteoporosis. Given the above analysis, we use NRAGE knockout mouse model with in vivo and in vitro experiments to explore the effect of NRAGE on bone metabolism. The main content includes two parts:1. To explore the role of NRAGE on bone metabolism; 2. To explore the role of NRAGE on the differentiation and bone resorption of osteoclasts.Main contents and methods:The first part:To explore the role of NRAGE on bone metabolism based on NRAGE knockout mice1) To analyse the effect of NRAGE on bone morphogenetical alteration using X-ray;2) To detect the bone BMD of the femur from 16-month-old NRAGE knockout mice and wild-type mice using the dual-energy X-ray absorptiometry (DXA);3) To detect the changes of ultrastructural of femur from 16-month-old NRAGE knockout mice and wild-type mice using Micro-CT;4) To examine the changes of biomechanical properties on NRAGE knockout mice by three-point bending test.The second part:To explore the role of NRAGE on the differentiation and bone resorption activity of osteoclasts1) The paraffin slices of the ferum from 10-week-old NRAGE knockout mice and wild-type mice were performed on TRAP staining to detect the numbers and activity of osteoclasts.2) M-CSF and RANKL induced bone marrow mononuclear cells from 10-week-old NRAGE knockout mice and wild-type mice were used to detect the effect of NRAGE on osteoclast differentiation and bone resorption activity. Total RNA were extracted from the primary osteoclasts induced by M-CSF and RANKL for 8 days, and the expression of TRAP, CTR, and cathepsink in osteoclasts were detected by Real-time PCR.3) Mouse bone marrow monocytes were isolated from 10-week-old NRAGE knockout mice and wild-type mice. M-CSF and RANKL induced at Omin,5min,15min and 30min. IKK, IκB and NF-κB phosphorylation level of NF-κB signaling pathway was detected from the primary osteoclasts.Main results:The first part:NRAGE knockout mice led to the occurrence of osteoporosis.Bone morphological characteristic of NRAGE knockout mice was not only abnormal by X-ray analysis. But the BMD of NRAGE knockout mice was significantly lower than wild-type mice. Micro-CT analysis showed that the femoral trabecular bone mineral density and bone mineral content of NRAGE knockout mice were significantly decreased.The number of trabecular bone and the trabecular thickness were reduced. All the above data suggested NRAGE knockout mice obtained osteoporosis phenotype.The second part:NRAGE gene knockout promoted the differentiation and bone resorption activity of osteoclasts1) Tissue section TRAP staining showed that the number of osteoclasts and its activity were significantly increased in NRAGE knockout mice. In vitro M-CSF and RANKL inducing osteoclasts differentiation showed NRAGE gene knockout promoted that the the differentiation of osteoclasts including the significantly increased number and remarkably increased volume. The expression of TRAP, CTR and cathepsink was also significantly increased.2) Detected the phosphorylation level of IKK, IκB and NF-κB by western blot suggested the phosphorylation level of IKK, IκB and NF-κB reached a peak at 5min after stimulated by M-CSF and RANKL.Major conclusions:1. NRAGE knockout mice obtained osteoporosis phenotype;2. NRAGE gene knockout promotes the differentiation and bone resorption activity of osteoclasts... |
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