Role and Mechanism of Claudin-11 Action and Signaling in Bone

项目摘要/摘要 骨质疏松症在美国是一个严重的公共卫生问题，并构成巨大的经济负担。这个 众所周知，骨质疏松症的发病机制是骨破坏增加，而不是平行补偿。 增加新组织的合成。唯一被批准的治疗骨质疏松症的合成代谢药物，PTH，也 增加骨吸收，从而限制其长期使用。因此，迫切需要发展 治疗骨质疏松症的新合成代谢疗法。在我们努力识别控制分子和它们的 有助于调节成骨的信号通路，我们发现了一个新的作用 Claudin(Cldn)家族的紧密连接蛋白。在这个RO1应用中，我们的重点是阐明角色和 基于我们令人兴奋的新数据，Cldn11在调节成骨细胞(OBS)功能中的作用机制 靶向破坏Cldn11基因的小鼠表现出严重的骨小梁体积缺陷(40%)。 此外，在骨折愈合过程中，Cldn11的表达增加了几倍。虽然传统的角色是 Cldns是为了调节细胞外小分子的运输，我们有了新的令人兴奋的初步数据 提示Cldn11通过与跨膜蛋白Tetraspanin3相互作用而非典型地作用于OBS。 (Tspan3)，调节ADAM10介导的Notch信号转导。我们将按如下方式测试此Cldn11操作模型： 1)为了验证在OBS中表达的Cldn11调节骨小梁形成的假设，我们将 用显微CT、组织学、骨质学等方法研究OB特异性Cldn11转基因(TG)小鼠的骨骼表型 力学测试和基因表达，并确定Cldn11 KO小鼠骨形成减少是否可以 通过在OBS中转基因表达Cldn11而获救。2)检验Cldn11对OBS影响的假设 通过其与Tspan3的相互作用，我们将确定Cldn11/Tspan3的功能结果 体外评价Tspan3基因敲除是否取消Cldn11介导的OBS分化的相互作用 在活体内。3)验证Cldn11/Tspan3对OBS的作用是通过ADAM10介导的假设 Notch信号的调节，我们将评估Cldn11/Tspan3相互作用中断的后果 编码ADAM10的显性负性突变体Cldn11的过表达对ADAM10成熟度和活性的影响 Tspan3结合结构域，并确定Cldn11/Tspan3相互作用是否调节Notch信号和OB 通过调节ADAM10活性实现分化。4)检验Cldn11促进骨折的假设 通过调节Notch信号进行愈合，我们将确定Cldn11 Tg和/或Tspan3的骨折表型 KO和对照组小鼠采用稳定的闭合性股骨骨折模型。我们将使用Notch信号报告器 评估Cldn11转基因小鼠骨折处Notch信号是否激活，并确定 用ADAM10抑制剂治疗可阻断激活的Notch信号。成功完成我们建议的 研究将提供有关Cldn11调节OB功能的途径的重要信息，并可能 提供新的药物靶点，促进OBS的合成代谢活性，用于治疗骨质疏松症。

Project Summary/Abstract Osteoporosis is a significant public health problem in the U.S. and poses a substantial financial burden. The pathogenesis of osteoporosis is known to involve increased destruction of bone, not compensated by parallel increases in the synthesis of new tissue. The only approved anabolic drug for osteoporosis, PTH, also increases bone resorption, thereby limiting its long term use. Thus, there is an urgent need for development of novel anabolic therapies for the treatment of osteoporosis. In our effort to identify control molecules and their signaling pathways that contribute to the regulation of osteogenesis, we have discovered a novel role for the Claudin (Cldn) family of tight junction proteins. In this RO1 application, our focus is on elucidating the role and mechanism of action of Cldn11 in regulating functions of osteoblasts (OBs) based on our exciting new data that mice with targeted disruption of the Cldn11 gene exhibit a severe deficit in trabecular bone volume (40%). Additionally, Cldn11 expression is increased several-fold during fracture healing. While the traditional role of Cldns is to regulate paracellular transport of small molecules, we have new exciting preliminary data that suggests that Cldn11 acts on OBs non-canonically via interacting with a transmembrane protein, Tetraspanin3 (Tspan3), to regulate ADAM10-mediated Notch signaling. We will test this model of Cldn11 action as follows: 1) To test the hypothesis that the Cldn11 expressed in OBs regulates trabecular bone formation, we will characterize the skeletal phenotype of OB-specific Cldn11 transgenic (Tg) mice by micro-CT, histology, mechanical testing and gene expression and determine if the reduced bone formation in Cldn11 KO mice can be rescued by transgenic expression of Cldn11 in OBs. 2) To test the hypothesis that Cldn11 effects on OBs are mediated via its interaction with Tspan3, we will determine the functional consequence of Cldn11/Tspan3 interactions by evaluating if knockdown of Tspan3 abolishes Cldn11-mediated differentiation in OBs, in vitro and in vivo. 3) To test the hypothesis that Cldn11/Tspan3 effects on OBs are mediated via ADAM10-mediated regulation of Notch signaling, we will evaluate the consequence of disruption of the Cldn11/Tspan3 interaction on ADAM10 maturation and activity via overexpression of a dominant negative mutant Cldn11 encoding the Tspan3 binding domain and determine if the Cldn11/Tspan3 interaction regulates Notch signaling and OB differentiation via modulation of ADAM10 activity. 4) To test the hypothesis that Cldn11 promotes fracture healing via regulating Notch signaling, we will determine the fracture phenotype in Cldn11 Tg and/or Tspan3 KO and control mice using a stabilized closed femoral fracture model. We will use notch signaling reporter mice to evaluate if Notch signaling is activated at the fracture site in the Cldn11 Tg mice and determine if treatment with an ADAM10 inhibitor blocks activated Notch signaling. Successful completion of our proposed studies will provide important information on the pathway by which Cldn11 regulates OB functions and could provide novel drug targets to promote anabolic activities of OBs for treatment of osteoporosis.