DEC1通过激活NFATc-1促进破骨细胞分化的作用及机制

Recently, osteoporosis (OP) is one of the most common diseases which cause huge societal and family burdens. Based on the following data we had gotten: (1) NFATc-1 and targets TRAP, CTSK and MPP9 of proximal tibia at different ages (4, 24, 52 weeks) significantly decreased in DEC1 KO (DEC1-/-) mice compared with those in WT (DEC1+/+) mice, especially at 24 weeks old mice. (2) In osteoclast differentiation of bone marrow macrophages (BMMs) from DEC1+/+ and DEC1-/- mice, NFATc-1, CTSK and MPP9 significantly decreased along with decrease of osteoclast formation from KO mice compared with those from WT mice. Therefore, it is hypothesized that DEC1 promotes osteoclast differentiation through enhancing NFATc-1. Making two types of osteoporosis model and LPS-induced calvarial model with WT and KO (DEC1+/+, DEC1-/-) mice, and making LPS-induced model with the primary cultured bone marrow mononuclear cells(BMMs), we will, on the systemic and the cellular levels, investigate the regulation and mechanism of DEC1 on promoting the osteclast differentiation through enhancing NFATc-1, which plays a role in the developing of OP, through using molecular cloning technology and modern molecular biology methods. Expected outcome will reveal the regulation and mechanism of DEC1 on promoting the osteclast differentiation through enhancing NFATc-1. This study not only deepens understanding the etiology of OP, but also provides academic evidence on developing a DEC1 antagonist for treatment of OP.

骨质疏松（OP）已成为目前造成家庭和社会沉重负担的常见病。申请者基于前期发现DEC1 敲除（KO）各年龄（4、24、52周龄）小鼠骨组织NFATc-1及下游靶基因表达较同年龄野生（WT）小鼠明显降低，尤以24周龄小鼠表现最为明显；52周龄KO小鼠出现骨量增加；在KO小鼠骨髓巨噬细胞诱导破骨细胞分化过程中NFATc-1及下游靶基因表达和破骨细胞形成均较WT小鼠明显降低的基础上，提出DEC1通过NFATc-1促进破骨细胞分化的假设。本项目拟用WT、KO小鼠制备OP和LPS诱导颅骨溶骨模型、两种基因型小鼠骨髓巨噬细胞制备LPS诱导模型，应用分子克隆技术、现代分子生物学等方法，研究并阐明DEC1通过激活NFATc-1对破骨细胞分化调控作用和机制以及在OP发生发展中的作用，揭示以DEC1为靶标药物实验治疗学意义，本项目不仅有助于深化对OP病因学的认识，也为研发DEC1拮抗剂治疗OP积累学术基础。

基于前一项目发现DEC1 敲除小鼠成骨能力明显下降而破骨能力明显增强，提出DEC1不仅影响了成骨代谢，而且也影响破骨代谢的假设。本项目应用两种基因型（DEC1+/+、DEC1-/-）小鼠、源于两种基因型（DEC1+/+、DEC1-/-）小鼠原代培养骨髓单核细胞（BMMs）诱导破骨细胞为研究对象，研究并阐明DEC1对破骨细胞破骨分化中的调控和机制。结果发现：（1）DEC1对破骨分化直接作用：5月龄DEC1-/-小鼠骨组织中TRAP（破骨细胞）数量较同龄同窝DEC1+/+小鼠明显增加，破骨分化相关因子及骨吸收相关因子明显增加，尤其是破骨分化关键因子NFATc1明显增加；源于两种基因型（DEC1+/+、DEC1-/-）小鼠的骨髓单核细胞（BMMs）进行诱导分化，源于野生型小鼠BMMs比较，源于DEC1-/-小鼠的BMMs破骨数目明显较源于野生型小鼠的多，体积更大，核融合更多，其骨吸收的功能也明显增强；体内和体外实验证实DEC1敲除促进破骨分化是通过增加Ca2+/calcineurin信号通路，最终导致破骨分化关键因子的增加。（2）由于DEC1与炎症密切相关，因此DEC1对破骨作用存在双重作用。（A）用两种基因型（DEC1+/+、DEC1-/-）小鼠制备LPS-诱导颅骨溶骨模型，结果发现：DEC1缺失可明显减轻由LPS-诱导的颅骨溶骨作用，体内和体外实验证实其机制为DEC1缺失通过转录水平抑制TLR4的表达，抑制炎症小体NLRP3的表达，从而抑制炎症因子分泌，达到减少溶骨的作用；（B）用两种基因型（DEC1+/+、DEC1-/-）小鼠制备CIA-诱导类风湿关节炎（RA）模型，结果发现DEC1缺失明显减轻CIA诱导RA表型以及软骨和骨破坏，抑制滑膜组织中多种炎性因子表达以及破骨与骨吸收相关蛋白的表达。人RA滑膜细胞（MH7A）敲减DEC1可显著降低细胞迁移和侵袭以及与RA表型相关的基因及炎性因子的表达，体内和体外实验证实DEC1缺失减轻CIA诱导RA表型的机制是通过PI3K/β-catenin/NFATc1信号通路减少炎性因子而发挥作用。研究提示：DEC1对破骨分化的调节存在双重作用（直接和间接作用），为治疗骨相关的疾病提供潜在靶点和思路。