Wnt4/Fzd10/LRP6-NLK信号轴调控炎症微环境促进强直性脊柱炎异位成骨的机制研究

Axial skeleton ankylosis resulted from ectopic bone formation is a major cause of disability in patients with Ankylosing Spondylitis (AS). No targeted and effective therapy is so far available to prevent ankylosis progression in clinical practice. Previous studies have shown that low-intensity inflammation in the ethesis is essential for ectopic new bone formation in AS, while high-intensity inflammation has inhibitory effect. Previous studies have shown Wnt4 has dual function of inflammation suppression and osteogenesis induction. In our previous study, we observed Wnt4 overexpression in samples collected from AS correction surgeries. In a cell model of osteogenesis in inflammatory microenvironment, we found Wnt4 induced osteogenic differentiation by activation of NLK-JNK signaling. Wnt4 also suppressed inflammatory cytokines expression through activation of NLK and inhibition of NFκB. These Wnt4 molecular functions were highly Fzd10/LRP6 dependent. Therefore we hypothesize that Wnt4/Fzd10/LRP6-NLK signal axis enhances ectopic new bone formation in AS by regulation of enthesis inflammatory microenvironment. To prove this hypothesis, we propose the current study, including in vitro study for investigating the molecular mechanism of Wnt4/Fzd10/LRP6-NLK signal axis regulating enthesis inflammatory microenvironment and enhancing bone formation, as well in vivo study for validating its role in ankylosis. The potential findings would provide more insight of AS and help with identification of novel treatment strategy.

异位成骨导致的脊柱强直是强直性脊柱炎（AS）致残的重要原因，目前机制尚不清楚，临床上无有效的治疗方法。研究证实，附着点局部的低水平炎症刺激是异位成骨的必要条件，而高水平炎症则抑制异位成骨的发生。前期研究发现Wnt4具有抑制炎症和促进成骨的双重作用，申请人进一步发现，Wnt4在AS组织标本中高表达；Wnt4通过激活NLK进而抑制单核细胞的NFκBd通路及其下游的炎症因子表达，并通过激活NLK-JNK通路促进成骨细胞分化，且以上作用需要Wnt受体Fzd10与LRP6的参与。由此我们提出科学假设：Wnt4/Fzd10/LRP6-NLK信号轴通过抑制局部炎症水平和促进成骨分化的双重作用调控附着点区域炎症微环境，促进异位成骨的发生。本课题拟在细胞模型中探讨该信号轴上述作用的详细分子机制，并在动物模型中验证其在异位成骨病理过程中的作用。本研究有望揭示AS异位成骨的新机制，为其防治提供新的思路和靶点。

异位成骨导致的脊柱强直是强直性脊柱炎（AS）致残的重要原因，目前机制尚不清楚，临床上无有效的治疗方法。结合既往文献复习与前期预实验结果，课题组提出科学假设：Wnt4/Fzd10/LRP6-NLK信号轴通过抑制局部炎症水平和促进成骨分化的双重作用调控附着点区域炎症微环境，促进异位成骨的发生。本课题组围绕该假设按计划进行，进展顺利。.重要进展如下：.1．为明确AS中炎症相关的异位成骨的分子机制，本课题组建立模拟炎症微环境中成骨分化的细胞模型，并探讨Wnt蛋白对成骨的作用以及具体的调控机制。结果发现Wnt4通过Wnt4–Fzd1/6–LRP5/6–β-catenin和Wnt4–Fzd2–JNK-p38通路促进成骨分化。.2．课题组拓展研究，探讨CaSR在AS异位成骨中的作用。结果发现，成骨前体细胞中CaSR-PLCγ增强其成骨分化能力，介导了AS脊柱强直的形成。.3．课题组拓展研究，探究细胞外基质在AS异位成骨中的作用。结果发现，成纤维细胞分泌的细胞外基质TNC通过降低基质黏附力促进成软骨分化，进而促进AS病理性新骨的形成。.4．课题组拓展研究，结果发现BRD4通过上调lncRNA MANCR促进异位成骨的发生。.5．课题组拓展研究，结果发现HOTTIP通过与WDR5结合并激活Wnt/β-catenin信号通路促进成骨分化。.以上研究结果已整理发表SCI杂志论文5篇，其中中科院I区、IF>10论文2篇，中科院II区、IF>5论文2篇（FASEB J. 2019；EMBO Mol Med, 2020； Ann Rheum Dis, 2021；Bone Joint Res, 2021；Biochem Biophys Res Commun, 2020）。在本研究基础上，课题组主要成员相继获得国家自然科学基金面上项目2项，广东省自然科学基金杰出青年项目1项，参与培养研究生5名（2名获国家奖学金）。