BRD4调控破骨细胞分化参与骨性关节炎软骨下骨重塑的机制研究

Subchondral bone remodeling is an important feature of osteoarthritis (OA). As the only cells type that are capable of bone resorption, the function of osteoclasts (OCs) are closely related to the remodeling of subchondral bone. Our previous study found that the expression level of BRD4 gene, a member of bromodomain and extra-terminal (BET) family proteins, was increased gradually in RANKL induced OCs differentiation and maturation. Furthermore, BRD4 inhibitor JQ1 could reduce the differentiation and maturation of Ocs. To further construct osteoclast specific BRD4 gene conditional knockout (cKO) mice, We found that the knee joint subchondral bone of BRD4 cKO mice exhibit an increased bone mass with characteristics most consistent with osteopetrosis phenotype. Those results suggesting that BRD4 may plays an important role in OCs-mediated subchondral bone remodeling. In addition, we previously demonstrated that HMGB1 is a direct down stream target of BRD4, while the recombination HMGB1 protein promoted the differentiation and maturation of OCs. Based on these novel observations, this studies we aim to: 1. Analysis the skeletal phenotype of BRD4 cKO mice comprehensively and confirm the physiological role of BRD4 in OC-mediated bone resorption in vitro; 2. Determine the regulation mechanism between BRD4 and HMGB1 during OCs maturation; 3. Investigate the role of BRD4 in subchondral bone remodeling of knee joint in an ACLT-induced OA model; 4. Explore the potential rescue effects of BRD4 inhibitor CPI-203 on subchondral bone remodeling in ACLT OA mice. The further investigation of this project will potentially identify novel molecular targets for OC mediated subchondral bone remodeling in osteoarthritis.

软骨下骨重塑是骨性关节炎的重要特征。破骨细胞作为人体内唯一具有骨吸收功能的细胞，功能状态与软骨下骨重塑密切相关。项目组研究发现BRD4在RANKL诱导的破骨细胞分化过程中表达逐渐增高，BRD4抑制剂JQ1显著抑制破骨细胞分化，破骨细胞特异性BRD4条件性敲除小鼠软骨下骨表现为骨硬化表型，提示BRD4在软骨下骨重塑过程中发挥重要功能。此外，项目组发现HMGB1是BRD4的直接作用靶点，而HMGB1重组蛋白可以显著促进破骨细胞分化成熟。结合上述工作基础，本研究将系统分析：1.BRD4条件性基因敲除的骨骼表型及BRD4对破骨细胞分化功能的影响；2.破骨分化过程中BRD4对HMGB1的调控作用；3.研究BRD4对于骨性关节炎小鼠膝关节软骨下骨重塑的调控作用；4.研究BRD4抑制剂CPI-203对骨性关节炎小鼠软骨下骨重塑的挽救作用。本研究为临床治疗破骨细胞介导的软骨下骨重塑提供了新的分子靶点。

本项目前期围绕BRD4在OA发生发展中的重要作用展开研究，深入剖析了BRD4作为一个重要的OA的软骨损伤相关标记物，靶向抑制BRD4可以显著抑制软骨细胞炎症反应和分解代谢反应，同时，我们发现HMGB1作为BRD4的直接下游靶点，且HMGB1的表达和出核激活均依赖BRD4的调控。同时BRD4在调控NF-κB信号通路的激活中具有重要作用。本项目进一步从BRD4对于破骨细胞分化成熟的调控作用展开系列研究。课题组在本项目的前期研究中发现:(1)BRD4基因在成熟破骨细胞内高表达；(2)构建破骨细胞内特异性的BRD4条件性基因敲除小鼠，分析发现在破骨细胞内敲除BRD4基因后，小鼠膝关节软骨下骨小梁数量增多，骨量增加，而骨小梁间隙变小，表现为骨硬化表型;(3)HMGB1是BRD4下游的重要调控基因:进一步分析发现，BRD4一方面可以通过招募P-TEFb，另一方面还可直接结合于HMGB1蛋白，介导HMGB1的的表达和激活。项目主要研究了BRD4条件性基因敲除小鼠膝关节软骨下骨组织超微结构分析、组织形态学分析和破骨细胞活跃程度分析，并在体外进行了BRD4下游转录因子CDK9的抑制剂LDC067对破骨细胞分化的影响研究。本课题组也积极探索小分子中药单体对骨关节炎的影响，争取找到治疗新方法。我们发现EsculentosideA通过减轻炎症和抑制破骨细胞生成来预防骨关节炎；Nangibotide通过抑制软骨下骨骨丢失性重塑，调节成骨分化，进而干预骨性关节炎发生发展；Toddalolactone通过减轻软骨侵蚀以及抑制软骨下骨骨丢失性重塑进而干预骨性关节炎进程。此外，我们聚焦软管下骨重塑对骨关节炎的影响，并基于软骨靶向的可视化干预策略研究，制备了具有软骨靶向和光热响应性的双药纳米微球用于骨关节炎治疗，为骨关节炎提供治疗新策略；我们通过CoNPs磨损颗粒探索研究假体周围磨损颗粒影响成骨细胞功能的相关机制，具有进一步探索的研究意义。