I型胶原通过Caveolin-1调节TGF-β/Smad信号在成骨不全症中的作用及机制研究

Osteogenesis imperfecta (OI), characterized by bone fragility and frequent fractures, is a genetic disorder caused by mutations of type I collagen. The impairment.of osteogenic differentiation is the pathogenesis of OI. Transforming growth factor-β (TGF-β) has been shown to inhibit osteoblast differentiation, and TGF-β/Smad signaling was found increased in OI. However, the mechanism of excessive TGF-β/Smad signaling in OI remains unclear. The degradation of TGF-β type I receptor (TβRI) by caveolin-dependent endocytosis pathway is an important way to suppress TGF-β/Smad signaling. Recently, we found that the expression of Caveolin-1, a key molecule of caveolin-dependent endocytosis pathway, was downregulated in OI, indicating that the inhibition of TGF-β signaling via caveolin-dependent endocytosis was decreased in OI. Previous evidence also suggests that type I collagen could mediate the phosphorylation of FAK, and FAK/Src signaling pathway could regulate the expression of Caveolin-1. Therefore, the objective of this study is to explore the specific mechanism of type I collagen in the regulation of Caveolin-1 expression via integrin α2β1-FAK/Src signaling pathway; to verify the effect of type I collagen on degrading TβRI and inhibiting TGF-β/Smad signaling through caveolin-dependent endocytosis in OI; and to assess the efficacy of inhibiting TβRI activity or enhancing Caveolin-1 function in the treatment of OI. Findings from this study will provide new directions and strategies for the treatment of OI.

成骨不全症（OI）是I型胶原相关基因突变所致的遗传病，表现为骨脆性增加、骨折等，其机制为成骨分化成熟障碍。TGF-β可抑制成骨分化，研究表明OI中TGF-β/Smad信号增强，但机制不明。微囊蛋白（Caveolin）依赖内吞途径降解TGF-βI型受体（TβRI）是抑制TGF-β信号的重要方式，而我们预实验发现该途径的关键分子Caveolin-1在OI中表达显著降低，提示OI中微囊蛋白依赖内吞途径抑制TGF-β作用减弱。研究表明I型胶原可激活FAK，而FAK/Src可调控Caveolin-1表达。为此，本研究拟探讨I型胶原通过整合素α2β1-FAK/Src信号通路调控Caveolin-1表达的机制以及I型胶原通过微囊蛋白依赖内吞途径降解TβRI和负调节TGF-β/Smad信号在OI中的作用；并探讨抑制TβRI活性或增强Caveolin-1功能治疗OI的疗效。以期为治疗OI提供新靶点和新思路。

成骨不全症（OI）是I型胶原相关基因突变所致的遗传病，表现为骨脆性增加、骨折等，其机制为成骨分化成熟障碍。TGF-β可抑制成骨细胞晚期阶段分化，而既往研究发现TGF-β/Smad信号增强参与OI发病过程，但具体机制尚不明确。本研究中，我们以oim鼠（OI模型鼠）及野生型鼠（wt鼠）为研究对象，发现oim鼠成骨分化能力显著低于wt鼠，而TGF-β(10 ng/ml)对oim鼠成骨细胞（OB）中Runx2、ALP、OCN的抑制作用以及Smad2/3的激活作用均显著高于wt鼠；这提示oim鼠对TGF-β的作用更为敏感。进一步实验发现oim鼠OB中TGF-β受体（TβRI和TβRII）的蛋白水平表达显著高于wt鼠，而mRNA水平两者无差异；放线菌酮实验发现oim鼠成骨细胞中TβRI和TβRII半衰期显著长于wt鼠；；这提示oim鼠OB中TGF-β受体降解出现障碍。对比研究发现oim鼠中Smad7蛋白含量以及Smad7/TβRI共表达显著高于wt鼠，而SARA蛋白含量及SARA/TβRI共表达显著低于wt鼠；相比wt鼠，oim鼠中TβRI在脂筏区域的含量显著高于非脂筏区域；这提示oim鼠中Caveolin依赖内吞降解途径减弱。机制研究发现oim鼠中Caveolin-1(Cav-1)磷酸化显著高于wt鼠；敲除Cav-1表达，可逆转I型胶原对TβRI的降解作用；预先采用整合素α2β1中和性抗体、FAK抑制剂、Src抑制剂干预OB，可逆转I型胶原对Cav-1磷酸化和TβRI降解的作用；这提示I型胶原可能通过整合素α2β1-FAK-Src信号通路诱导Cav-1的磷酸化，OI中由于I型胶原异常导致Cav-1磷酸化减弱，进而导致Caveolin依赖内吞途径降解TβRI作用减弱，促使 TGF-β/Smad 信号增强。体内研究进一步采用TβRI酶活性抑制剂SD-208治疗4周龄oim鼠和wt鼠，治疗8周显示SD-208可显著降低oim鼠的骨折发生次数，micro-CT结果显示SD-208可有效增加oim鼠骨密度和骨量，组织学结果显示SD-208可有效促进oim鼠骨形成能力和成骨分化能力，生物力学结果显示SD-208可显著改善oim鼠的生物力学特性。这提示SD-208具有改善oim异常骨结构以及降低骨折的疗效。本研就结果将为SD-208用于临床治疗成骨不全症患者提供理论依据。