增龄性骨形成能力下降的分子机制：Plekho1对BMP信号通路的调控作用

The reduction in aging-induced bone formation leads to increased osteoporotic fracture risk in aged patients. Emerging evidences indicated that dysregulation of ubiquitylation plays an important role in aging-associated diseases. Thus far, no study has established the pathological role of ubiquitination-related molecules identified from aged human bone specimens in regulating osteoblastic bone formation during aging. Pleckstrin homology domain containing, family member 1 (PLEKHO1, also known as CKIP-1) specifically targets the linker region between the WW domains of smad ubiquitination regulatory factor 1 (Smurf1) to promote its ubiquitylation of Smad 1/5. However, the exact pathological role of Plekho1 in aging-induced bone formation reduction is still not well elaborated. In the previous study, we firstly found increased PLEKHO1 expression associated with elevated ubiquitylation of Smad 1/5, reduced BMP signaling (phosphorylation of Smad 1/5) and bone formation during aging in bone specimens from human and rodents. Thus， we raised our hypothesis that increased PLEKHO1 suppress BMP signaling to inhibit bone formation during aging. To test our hypothesis, firstly, by genetic ‘loss-of-function’ and ‘gain-of-function’ mutation approach, we created the osteoblast-specific Plekho1 knockout (Osx;Ckip-1fl/fl) / knock-in (Osx/Plekho1), and osteoblast-specific Smad1 knock-in mice (Osx/Smad1) to investigate the functional role of Plekho1 in bone formation reduction during aging. Secondly, by pharmacological approach, we specifically silencing Plekho1 in osteoblasts using Plekho1 siRNA encapsulated in (AspSerSer)6-liposome to examine the effects of silencing Plekho1 within osteoblasts on age-related decreases in bone formation in aged female ovariectomized and male rats. If the hypothesis was approved, the increased PLEKHO1 suppress BMP signaling to inhibit bone formation during aging, indicating that therapeutic silencing Plekho1 in osteoblasts may be a potential bone anabolic strategy to reverse established osteoporosis in aged patients.

增龄性骨形成下降是引起老年骨质疏松症进而导致骨质疏松性骨折发生的主要原因，然而其分子机制并没有被明确阐述。申请人团队在细胞实验证实泛素化相关蛋白PLEKHO1通过作用于泛素链接酶Smurf1的WW结构域的铰链区而促进后者对BMP信号通路Smad1/5 的泛素化，更发现老年骨质疏松症患者骨标本中PLEKHO1表达水平增龄性升高而骨形成增龄性下降。因此，申请人提出研究假设：在老年骨质疏松症的发生发展中，成骨细胞内上调的PLEKHO1通过抑制BMP信号通路而引起增龄性骨形成下降。为了检验该研究假设，申请人分别通过基因手段（成骨细胞特异性基因敲除/敲入动物模型）失活/激活突变Plekho1和药理手段（靶向成骨细胞的核酸递送系统包裹Plekho1 siRNA）静默成骨细胞内的Plekho1探索PLEKHO1调控增龄性骨形成下降的分子机制，并为治疗老年骨质疏松症提供分子靶点及有转化潜能的干预手段。

增龄性骨形成下降是引起老年骨质疏松症进而导致骨质疏松性骨折发生的主要原因，然而其分子机制并没有被明确阐述。项目组团队在细胞实验证实泛素化相关蛋白PLEKHO1通过作用于泛素链接酶Smurf1的WW结构域的铰链区而促进后者对BMP信号通路Smad1/5 的泛素化，更发现老年骨质疏松症患者骨标本中PLEKHO1表达水平增龄性升高而骨形成增龄性下降。因此，我们提出的研究假设：在老年骨质疏松症的发生发展中，成骨细胞内上调的PLEKHO1通过抑制BMP信号通路而引起增龄性骨形成下降。为了检验该研究假设，在该项目的研究开展过程中分别通过基因手段（成骨细胞特 异性基因敲除/敲入动物模型）失活/激活突变Plekho1和药理手段（靶向成骨细胞的核酸递送 系统包裹Plekho1 siRNA）静默成骨细胞内的Plekho1探索PLEKHO1调控增龄性骨形成下降的分子机制。 我们发现（1）证实Plekho1基因失活突变能够增强及BMP信号通路，进而减缓增龄性骨形成下调。(2) 明确Plekho1基因功能激活突变能够进一步抑制成骨细胞内BMP信号通路，并加速增龄性骨形成的下调。(3) 发现特异性静默老年骨质疏松症动物模型成骨细胞内Plekho1 基因能够加强成骨细胞内的BMP信号通路，并改善增龄性骨形成的下调。 通过该项目的研究，明确了Plekho1基因调控增龄性骨形成下调过程中对成骨细胞内的BMP信号通路的负调控作用，从而为治疗老年骨质疏松症提供分子靶点及有转化潜能的干预手段。