PDGFRβ泛素化修饰和蛋白酶体抑制剂在老年性骨折愈合中的作用机制研究

Bone fractures in the elderly usually show delayed or non-union repair, which is accompanied with reduced angiogenesis and osteoblastogenesis. Ubiquitin-proteasome system (UPS)-mediated protein degradation plays a critical role in the regulation of mesenchymal stem cells (MSC) differentiation and function. However, it is still unclear whether UPS contributes to fracture repair. We performed fracture surgery on 3-month-old young and 20-month-old aged mice, and found that the aged mice showed delayed repair and less MSCs and blood vessels in their fracture calluses. In addition, more ubiquitinated platelet-derived growth factor receptor β (PDGFRβ) had been detected by ubiquitin proteomics in the fracture calluses of aged mice. PDGFRβ signaling is critical for MSC proliferation and blood vessel maintenance. Our preliminary results indicated that PDGFRβ was significantly degraded by UPS in the fracture calluses of aged mice, which accounts for the less responses in aged fractured mice to the treatment of PDGF-BB, the ligand of PDGFRβ. Strikingly, the proteasome inhibitor, Bortezomib (Btz), restored the effect of PDGF-BB in treating bone fracture in aged mice. Btz+PDGF-BB synergistically increased fracture repair in aged mice, which could be blocked by the PDGFRβ inhibitor, su16f. Furthermore, higher level of TGFβ expression was detected in aged bone tissues, which promoted UPS-mediated PDGFRβ degradation in proteasome. We hypothesize that delayed fracture repair in aged mice is associated with TGFβ-mediated ubiquitination and proteasome degradation of PDGFRβ in MSCs, resulting in reduced angiogenesis and osteoblastogenesis, which could be prevented by Btz+PDGF-BB combination therapy. Our project will elucidate the involvement of protein ubiquitination and degradation in fracture repair, and provide preclinical evidence for using Btz+PDGF-BB in the promotion of fracture repair in the elderly.

老年性骨折常延迟愈合或不愈合，并伴随血管形成和间充质干细胞（MSC）成骨能力降低。泛素化-蛋白酶体降解途径在调控MSC分化与功能过程中起重要作用，但在骨折愈合中的作用尚不明确。本项目前期研究发现老年小鼠骨折愈合延迟，骨痂中MSC和血管数目减少，泛素化蛋白增多。PDGFRβ信号通路对骨折血管生成及MSC增殖至关重要，但老年小鼠骨痂细胞中PDGFRβ泛素化和降解均显著上调，从而导致其激动剂PDGF-BB失去对老年小鼠骨折愈合的治疗作用，而蛋白酶体抑制剂Bortezomib (Btz) 能重新恢复其疗效。我们还发现老年小鼠骨组织中较高的TGFβ诱导了PDGFRβ的泛素化及降解，本项目将阐明该过程相关的分子机制。同时，我们将探讨PDGFRβ的泛素化修饰和降解在老年性骨折愈合缺陷中的作用及其分子机制，为临床联合应用Btz+PDGF-BB治疗老年性骨折提供理论依据。

老龄化被认为是影响骨折愈合的一个重要因素之一，但其机制和治疗方法的研究进展并不如人意。泛素-蛋白酶系统（UPS）是介导细胞内蛋白翻译后泛素化修饰和降解的重要细胞生命机制。研究显示大量成骨细胞调节因子受UPS调控，比如RUNX2和JUNb。骨相关研究报道蛋白酶体抑制剂，Bortezomib（Btz）可以通过抑制RUNX2的降解来预防骨质疏松。我们通过实验发现Btz不仅可以促进年轻和老年小鼠骨折愈合，还能增加骨痂中的间充质干细胞（MSC）。采用泛素化（Ub）蛋白质组学方法发现Btz在MSC中候选靶蛋白中，PDGFRb既能促进MSC增殖，且在老年小鼠骨痂中蛋白表达量降低，而Ub-PDGFRb增高。PDGF-BB治疗老年小鼠骨折失效，而Btz联合PDGF-BB能纠正PDGF-BB的作用，并起到协同促进老年骨折愈合。通过筛选，我们发现老年小鼠骨痂中TGFb的表达量显著增加，且能体外诱导PDGFRb的蛋白降解，但能被Btz所阻断。TGFb可以增加PDGFRb的E3泛素连接酶Cbl和Cbl-b的表达。本项目从组织学、生物力学和细胞学水平综合证实了Btz+PDGF-BB在协同促进老年小鼠骨折愈合中的作用，并且通过转基因动物模型短期敲除PDGFRb+细胞证明其在小鼠骨折愈合过程中的重要作用。最后我们通过一系列分子生物学方法证实TGFb既能促进PDGFRb的Tyr1021磷酸化，又能增加Cbl和Cbl-b的表达，并促进其与PDGFRb结合，从而增加PDGFRb的泛素化和降解, 进而导致了成骨和成血管减少。结合最新的科学前沿发展，我们发现老年小鼠增多的TGFb主要来自于骨痂中增多的衰老细胞，senolytic药物剔除衰老细胞能降低TGFb水平及TGFb中和抗体皆能促进老年骨折愈合。本项目通过机制研究证实多种可用于治疗老年骨折的可行性治疗手段，具有一定的临床意义。