Role of Vwc2-assisted activin signaling in bone formation

描述（由申请人提供）：最近，半胱氨酸结蛋白（CKP）家族由于其强大的生物学效应和功能调节而受到了极大的关注，这似乎是一种很有前途的治疗方法。通过生物信息学方法，我们确定了CKP家族的新成员“Vwc2 （von Willebrand factor C domain containing 2）”，并探索了其潜在功能。我们的初步数据表明，Vwc2基因表达与早期成骨细胞分化有关，并且Vwc2蛋白存在于骨基质中并优先定位于骨表面。当Vwc2在MC3T3-E1 （MC）成骨细胞系中稳定过表达时，体外矿化可能通过其与转化生长因子(TGF)-ss超家族成员激活素a的2A亚基异构体的特异性结合而明显受到抑制。此外，当Vwc2与激活素a蛋白一起添加时，与单独添加激活素a或Vwc2相比，Smad2磷酸化在MC细胞中协同增强，表明Vwc2促进了激活素信号传导。最近，一些证据表明，激活素信号通路抑制成骨细胞分化和矿化，而其抑制该信号通路导致体外和体内骨形成和骨强度增强。作为获得这一目标的初步步骤，我们刚刚培育了小鼠，其中Vwc2基因在感兴趣的组织中选择性地过度表达。因此，我们假设一种新的CKP成员Vwc2通过协助成骨细胞激活信号来调节骨矿化。本探索性提案的目的是在体外建立Vwc2在激活素信号传导中的功能，并确定Vwc2调节对体内生物矿化的影响。为了检验这一假设，提出了以下具体目标。1. 探讨体外Vwc2调控成骨细胞功能的潜在信号通路。2. 探讨Vwc2在体内骨形成中的作用。从这项研究中获得的数据可能为这种新型CKP成员在骨形成中的生物学功能提供见解，并有助于为骨形成和愈合的治疗提供新的分子设计。公共卫生相关性：尽管目前的骨质疏松药物市场主要由抗骨吸收药物主导，但它们不能促进骨质流失的替代。我们的研究不仅促进了我们对CKP新成员Vwc2在激活素信号传导中的认识，而且有助于确定该信号通路导致骨形成抑制的分子机制。通过完成我们提出的研究，本研究的逻辑延伸将进一步允许控制成骨细胞中的Vwc2功能，从而允许对骨形成的潜在合成代谢作用，并为降低骨折风险的努力提供骨形成的新知识。

DESCRIPTION (provided by applicant): Recently, significant attention has been paid to the cysteine knot protein (CKP) family due to their potent biological effects and modulation of their functions appears to be a promising therapeutic approach. By a bioinformatics approach, we have identified a novel member of CKP family, "Vwc2 (von Willebrand factor C domain containing 2)", and explored its potential functions. Our preliminary data indicated that Vwc2 gene expression is associated with early osteoblast differentiation, and that the Vwc2 protein is present in bone matrix and preferentially localized at the bone surface. When Vwc2 was stably overexpressed in MC3T3-E1 (MC) osteoblastic cell line, in vitro mineralization was markedly inhibited possibly via its specific binding to a transforming growth factor (TGF)-ss superfamily member, 2A subunit isoform of activin A. Moreover, when Vwc2 was added together with activin A protein, Smad2 phosphorylation was synergistically enhanced compared to either activin A or Vwc2 addition alone in MC cells indicating that Vwc2 facilitates activin signaling. Recently, several lines of evidence suggests that activin signaling inhibits osteoblastic differentiation and mineralization, whereas its inhibition of this signaling pathway leads to enhance bone formation and bone strength in vitro and in vivo. As a preliminary step of obtaining such objectives, we have just generated mice, in which the Vwc2 gene has been selectively overexpressed in tissues of interest. Thus, we hypothesize that a novel CKP member, Vwc2, modulates mineralization in bone by assisting activin signaling in osteoblasts. The objectives of this exploratory proposal are to establish the function of Vwc2 in activin signaling in vitro and to determine the effects of Vwc2 modulation on biomineralization in vivo. In order to test the hypothesis, the following specific aims are proposed. 1. To investigate the potential signaling pathway by which Vwc2 modulates osteoblast function in vitro. 2. To investigate roles of Vwc2 in bone formation in vivo. The data obtained from this study may provide insights into the biological functions of this novel CKP member in bone formation and help a new molecular design for therapies of bone formation and healing. PUBLIC HEALTH RELEVANCE: Although current osteoporosis drug market is heavily dominated by anti-resorptive agents, they fail to promote the replacement of bone loss. Our studies not only advance our knowledge of a new CKP member, Vwc2 in Activin signaling but also help define the molecular mechanisms of this signaling pathway leading to the inhibition of bone formation. By accomplishing our studies proposed, the logical extension of this research will further allow controlling the Vwc2 function in osteoblasts, thereby allowing the potential anabolic effects on bone formation, as well as provide new knowledge of bone formation critical for efforts directed at decreasing the risk of bone fracture.