| Bone is one of the most important organs in our body, with its function of movement, supporting and protection, hematopoiesis, reservoir for minerals. However, aging, fracture, osteoporosis, bone defects caused by bone tumors, inflammation, bone will lose the above functions. Thus it is very important to have a healthy bone. Bone maintains its quantity by a process called bone remodeling, consisting of bone formation by osteoblasts and bone resorption by osteoclasts. Thus, to maintain a healthy bone, it is of great significance to clarify the molecular mechanisms and the regulation during bone remodeling. Bone morphogenetic proteins(BMPs) were originally discovered by their ability to induce bone and cartilage formation when implanted at ectopic sites. Although US Food and Drug Administration has approved the clinical use of recombinant human BMP-2(rh BMP-2) and rh BMP-7 for the treatment of long bone open fractures, non-union fractures and spine fusion. However, it often caused bone resorption, pseudoarthrosis, local inflammation, and its effectiveness is not obvious. Therefore, elucidating the roles and molecular mechanisms of BMP signaling in bone formation and resorption would provide new insights to improve the healthy condition of bones; the effectiveness of BMPs on bone regeneration could be enhanced by regulation of the downstream targets of BMP signaling, which would be of potential significance to treat bone diseases.In Chapter 1, we reviewed physiology and pathology of bone remodeling, introduced BMP ligands and receptors as well as BMP signaling, summarized BMP signaling in bone remodeling. To investigate the roles of BMP signaling in bone remodeling and the molecular mechanisms, we targeted on type I BMP receptors. We used gene modified animal models in our study, including conventional and VIII conditional gene knockout. The knowledge of gene knockout, especially the conditional knockout mouse model was reviewed.In Chapter 2, based on the fact that bone quantity and bone quality are important in determining the biomechanical properties of bones, we investigated the effects of BMP signaling mediated by ACVR1 or BMPR1 A in bone quantity and bone quality. We disrupted type I BMP receptors, Acvr1 and Bmpr1 a, respectively in early differentiating osteoblasts. Bone mass was assessed by micro-CT, changes in trabecular bone, osteoblasts and osteoclasts were evaluated by histomorphometry, changes in bone components was measured by Raman spectroscopy. We found that osteoblast-specific disruption of Acvr1 or Bmpr1 a increased bone mass, especially in trabecular compartments. Disruption of Bmpr1 a further increased bone mass in trabecular compartments, compared with disruption of Acvr1. In addition, in trabecular compartments, mineral crystallinity and collagen order/disorder were increased, while in cortical compartments, bone tissue mineral density and mineral-matrix ratio were decreased. This study indicated that BMP signaling mediated by ACVR1 or BMPR1 A in osteoblasts is important in determining bone quantity and bone quality.In Chapter 3, based on the results of Chapter 2 that BMP signaling mediated by ACVR1 or BMPR1 A negatively regulates bone mass, we investigated the role of BMPR1 B, another type I BMP receptor, on bone remodeling. We used a Bmpr1 b gene knockout mouse model, and studied its effects on osteoblasts and osteoclasts. We found that deletion of Bmpr1 b decreased bone mass in 8-week-old male mice, and this phenotype is transient and gender-specific. However, the decreased bone mass was neither due to the changes in osteoblastic bone formation nor osteoclastic bone resorption. In vitro study showed that differentiation of mutant osteoclasts was increased but resorption activity was decreased. There was no change in the differentiation of calvarial pre-osteoblasts, but BMP-SMAD signaling was increased upon BMP stimulation. Different from calvarial pre-osteoblasts, the differentiation of bone marrow mesenchymal progenitors was compromised, which may be a reason for the decreased bone mass in mutant mice. The different roles of BMPR1 B in different cell types may be due to the expression patterns of Bmpr1 b in different tissues. Based on the previous studies about type I BMP receptors and our current study, we proposed a working model for BMP signaling mediated by type I BMP receptors on the differentiation of osteoblasts, suggesting that different type I BMP receptors play different roles at different stages of osteoblast differentiation. This study indicated that BMPR1 B plays distinct roles in maintaining bone mass and in transducing BMP singaling, compared with BMPR1 A or ACVR1.In Chapter 4, based on the working model we proposed in Chapter 3 that BMP signaling mediated by type I BMP receptors on the differentiation of osteoblasts, and based on the fact that osteoclast-osteoblast communication plays an important role during bone remodeling and the necessary role of BMPR1 A in transducing BMP signaling, we investigated the effect of BMP signaling in osteoclasts mediated by BMPR1 A in communication with osteoblasts. It is reported that osteoclast-specific disruption of Bmpr1 a increased osteoblastic bone formation in mice. We hypothesized that BMP signaling in osteoclasts mediated by BMPR1 A regulated the production of either membrane-bound proteins or secreted factors, which then regulated osteoblast differentiation. To test this hypothesis, we co-cultured osteoblasts and osteoclasts in vitro, and found that disruption of Bmpr1 a in osteoclasts promoted osteoblast mineralization. Besides, we found that the expression of Cx43/Gja1 in osteoclasts was increased, which encodes for one of the gap junction protein connexin43/gap junction protein α1. Further, we knocked-down the expression of Cx43/Gja1 in mutant osteoclasts, and found that the mineralization of osteoblasts was decreased. This study indicated that gap junction protein α1 may be one of the downstream targets of BMP signaling in osteoclasts, which mediated osteoclast-osteoblast communication during bone remodeling. |
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