FBXO6泛素化降解MMP-14保护软骨终板退变的分子机制研究

Cartilage endplate (CEP) degeneration is considered as an important cause of lumbar intervertebral disc (IVD) degeneration, while the underlying mechanism of CEP degeneration is still unclear. F-box protein 6 (FBXO6) is a component of the Skp1-cullin-Fbox ubiquitin E3 ligases. In our previous studies, we found that (1) TGF-β1 and FBXO6 were decreased while MMP-14 was significantly upregulated in human degenerated CEP; (2) the binding of MMP-14 and FBXO6 was observed by Co-IP and immunofluorescence co-localization analysis in murine ATDC5 chondrocyte cells; overexpression of FBXO6 degenerated MMP-14 by the ubiquitin-proteasome protein-degradation pathway, while the knockdown of FBXO6 caused the elevation of MMP-14, which degenerated the extracellular matrix by MMP-14 itself and activation proMMP-13. (3) Meanwhile we found that TGF-β1 could enhance FBXO6 expression in vitro and there were three highly probable SMAD2/3/4 binding sites on the promoter of FBXO6 predicted by JASPAR database. We hypothesize that the decline of FBXO6 expression in the endplate chondrocyte is resulted from the decreasing activation of SMAD2/3 signaling pathway, which results in decline of MMP-14 ubiquitylation and increases the content of active MMP-13, causing CEP degeneration at last. This view has not been reported in the literature yet. In this study, we will further (1) focus on the role of FBXO6 in the protection of CEP degeneration and the underlying molecular mechanism by using COL2 cre FBXO6-/- mice; (2) elucidate the interaction protein domains of FBXO6 and MMP-14 that binding to MMP-14 and causing MMP-14 degradation; (3) verify that SMAD2/3 can bind to FBXO6 promoter by SMAD2-/- mice and chromatin immunoprecipitation; (4) upregulate FBXO6 as a potential molecule target for biological treatment of CEP and IVD degeneration. Overall, we hope to illuminate the function of TGFβ1-SMAD2/3-FBXO6-MMP14 pathway in CEP degeneration and provide a new target for endplate cartilage degeneration therapy.

下腰痛中软骨终板退变发生机制仍不明确。我们前期研究发现：（1）人体退变软骨终板中TGFβ1和FBXO6表达降低，MMP14增高；（2）ATDC5细胞系中FBXO6和MMP14存在蛋白互作；FBXO6过表达可泛素化降解MMP14，抑制MMP13激活，而突变或沉默则相反；（3）TGFβ1促进FBXO6表达。我们假设TGFβ1通过SMAD2/3促进FBXO6表达导致泛素化降解MMP14，从而抑制MMP13激活来延缓终板退变。本课题（1）用COL2 cre FBXO6敲除小鼠研究软骨终板细胞外基质降解的分子机制；（2）研究FBXO6与MMP14相互结合的分子结构域；（3）研究SMAD2/3在FBXO6启动子上可能的结合位点;（4）探索FBXO6过表达对软骨终板退变的防治。综上我们希望阐明TGFβ1-SMAD2/3-FBXO6-MMP14对软骨终板细胞外基质的调控机制，为防治软骨终板退变提供新靶点。

下腰痛中椎间盘退变发生机制仍不明确。我们前期研究发现：（1）人体退变椎间盘中TGFβ1和FBXO6表达降低，MMP14增高；（2）ATDC5细胞系中FBXO6和MMP14存在蛋白互作；FBXO6过表达可泛素化降解MMP14，抑制MMP13激活，而突变或沉默则相反；（3）TGFβ1促进FBXO6表达。我们假设TGFβ1通过SMAD2/3促进FBXO6表达导致泛素化降解MMP14，从而抑制MMP13激活来延缓椎间盘退变。本课题（1）用COL2 cre FBXO6敲除小鼠研究椎间盘细胞外基质降解的分子机制；（2）研究FBXO6与MMP14相互结合的分子结构域；（3）研究SMAD2/3在FBXO6启动子上可能的结合位点;（4）探索FBXO6过表达对椎间盘退变的防治。我们通过分析 FBXO6 和 MMP14 在退化的人类 NP 组织和老化诱导的自发退化 IVD 样本中的表达，确定了 FBXO6 和 MMP14 在 NP 退化中的作用。 使用 FBXO6 条件敲除小鼠，我们证实NP 细胞中 FBXO6 的条件性敲除增加了 MMP14 的表达并加速了退化过程。此外，体外实验表明，FBXO6 通过调节 MMP14 泛素化和降解以及退化 NP 中的炎症环境来抑制细胞外基质中MMP13 的激活组织可以通过促进 FOXO1 磷酸化来下调 FBXO6。退化的 NP 细胞中 FBXO6 表达的下降是由于IL-1β/TNF-α/IL-6 的表达增加和 FOXO1 的磷酸化增强，导致其细胞质易位。 FBXO6 下调导致 MMP14 泛素化减少和 pro-MMP13 激活增加，导致 NP 细胞外基质变性。这些研究将为椎间盘退变的防治提供新的靶点，具有重大的理论和应用价值。