NEUROFIBROMIN (Nf1) SIGNALING AND TARGET GENES IN OSTEOBLASTS

描述(由申请人提供)：我们的实验室有兴趣鉴定和阐明骨重建调节剂的分子作用机制。这种兴趣使我们研究了神经纤维连接蛋白，一种由NF1基因编码的GTP酶激活蛋白在骨重建中的作用。NF1基因突变导致1型神经纤维瘤病(NF1)，这是一种综合征，除其他表现外，其特征是致残骨骼异常，其来源不明。为了确定Nf1在骨生物学中的作用，并克服Nf1-/-小鼠的胚胎致死性，我们培育了成骨细胞中Nf1特异性缺失的小鼠(Nf1ob-/-小鼠)。在初步数据中，我们表明成骨细胞中NF1缺乏会导致骨形成增加而导致骨量增加。我们还提供了证据表明，ERK和RSK2以及成骨细胞分化所需的转录因子ATF4可能是Nf1信号转导的靶点，参与了Nf1ob-/-小鼠的高骨量。令人惊讶的是，成骨细胞中NF1缺乏还通过未知的机制增加破骨细胞分化和骨吸收。因此，这些数据以及我们最近关于ATF4在骨吸收中的作用的发现表明，在成骨细胞中，ATF4活性的增加可能是NF1骨骼表现的主要原因，包括在Nf1ob-/-小鼠和人类中观察到的骨吸收增加。 在这一应用中，我们建议使用WT和NF1-/-成骨细胞/破骨细胞共培养和细胞信号研究来表征成骨细胞中NF1调控破骨细胞生成、骨吸收、胶原合成和骨形成的信号通路。为了确认ATF4是成骨细胞中Nf1信号转导的靶点，并证明这些发现在体内的相关性，我们将试图挽救Nf1ob-/-小鼠的骨表型，从遗传学上通过在Nf1ob-/-小鼠中移除一个ATF4副本，在药理学上通过在Nf1ob-/-小鼠和成骨细胞中阻断RAS和Mark来挽救Nf1ob-/-小鼠的骨表型。 相关：神经纤维瘤病(NF1)以衰弱的骨骼异常为特征，这些异常通常是进行性的，难以治疗。该项目旨在了解这些骨骼异常的起源，并利用NF1的小鼠模型测试可以改善该综合征骨骼表现的候选药物。它还旨在表征新的信号通路和调控骨重塑的靶基因，长期目标是更好地了解影响骨骼的疾病，并提出合理的治疗方案。

DESCRIPTION (provided by applicant): Our laboratory is interested in identifying and elucidating the molecular mechanism of action of regulators of bone remodeling. This interest led us to study the role of neurofibronin, a GTPase activating protein encoded by the NF1 gene in bone remodeling. Mutation in NF1 causes Neurofibromatosis Type 1 (NF1), a syndrome characterized, among other manifestations, by disabling skeletal abnormalities, whose origin is unknown. To characterize the role of Nf1 in bone biology and to overcome the embryonic lethality of Nf1-/- mice, we generated mice deficient for Nf1 specifically in osteoblasts (Nf1ob-/- mice). In preliminary data, we show that Nf1 deficiency in osteoblasts leads to a high bone mass caused by an increase in bone formation. We also present evidence that the kinases ERK and RSK2 as well as ATF4, a transcription factor required for osteoblast differentiation, may be a target of Nf1 signaling involved in the high bone mass of Nf1ob-/- mice. Surprisingly, Nf1 deficiency in osteoblasts also increases osteoclast differentiation and bone resorption through yet unknown mechanisms. These data, along with our recent findings related to the role of ATF4 in bone resorption therefore suggest that, in osteoblasts, an increase in ATF4 activity may be responsible for most of the NF1 skeletal manifestations, including the increase in bone resorption observed in Nf1ob-/- mice and humans. In this application, we propose to use WT and Nf1-/- osteoblast/osteoclast co-cultures and cell signaling studies to characterize the signaling pathways whereby Nf1 in osteoblasts regulate osteoclastogenesis, bone resorption, collagen synthesis and bone formation. To confirm that ATF4 is a target of Nf1 signaling in osteoblasts and to demonstrate the in vivo relevance of these findings, we will attempt to rescue the bone phenotypes of Nf1ob-/- mice, genetically by removing one copy of Atf4 in Nf1ob-/- mice and pharmacologically by blocking Ras and MARK in Nf1ob-/- mice and osteoblasts. Relevance: Neurofibromatosis (NF1) is characterized by debilitating skeletal abnormalities that are often progressive and difficult to treat. This project aims at understanding the origin of these bony abnormalities and to test pharmacological candidates that could ameliorate the skeletal manifestations of the syndrome using a mouse model of NF1. It also aims at characterizing novel signaling pathways and target genes regulating bone remodeling, with the long-term goal to better understand diseases affecting the skeleton and to propose rationale therapies.