Abstract：
BACKGROUND: Matrix metallopeptidases (MMPs) are critical matrix-degrading molecules and they are frequently overexpressed in degenerative discs. This study aimed to investigate the mechanism for MMP upregulation.
METHODS: Immunoblot and RT-qPCR were used for detecting protein and gene expression levels. 4-month-old and 24-month-old C57BL/6 mice were used for evaluating intervertebral disc degeneration (IDD). An ubiquitination assay was used to determine protein modification. Immunoprecipitation and mass spectrometry were used for identifying protein complex members.
RESULTS: We identified the elevation of 14 MMPs among 23 members in aged mice with IDD. Eleven of these 14 MMP gene promoters contained a Runx2 (runt-related transcription factor 2) binding site. Biochemical analyses revealed that Runx2 recruited a histone acetyltransferase p300 and a coactivator NCOA1 (nuclear receptor coactivator 1) to assemble a complex, transactivating MMP expression. The deficiency of an E3 ligase called HERC3 (HECT and RLD domain containing E3 ubiquitin-protein ligase 3) resulted in the accumulation of NCOA1 in the inflammatory microenvironment. High throughput screening of small molecules that specifically target the NCOA1-p300 interaction identified a compound SMTNP-191, which showed an inhibitory effect on suppressing MMP expression and attenuating the IDD process in aged mice.
CONCLUSIONS: Our data support a model in which deficiency of HERC3 fails to ubiquitinate NCOA1, leading to the assembly of NCOA1-p300-Runx2 and causing the transactivation of MMPs. These findings offer new insight into inflammation-mediated MMP accumulation and also provide a new therapeutic strategy to retard the IDD process.
METHODS: Immunoblot and RT-qPCR were used for detecting protein and gene expression levels. 4-month-old and 24-month-old C57BL/6 mice were used for evaluating intervertebral disc degeneration (IDD). An ubiquitination assay was used to determine protein modification. Immunoprecipitation and mass spectrometry were used for identifying protein complex members.
RESULTS: We identified the elevation of 14 MMPs among 23 members in aged mice with IDD. Eleven of these 14 MMP gene promoters contained a Runx2 (runt-related transcription factor 2) binding site. Biochemical analyses revealed that Runx2 recruited a histone acetyltransferase p300 and a coactivator NCOA1 (nuclear receptor coactivator 1) to assemble a complex, transactivating MMP expression. The deficiency of an E3 ligase called HERC3 (HECT and RLD domain containing E3 ubiquitin-protein ligase 3) resulted in the accumulation of NCOA1 in the inflammatory microenvironment. High throughput screening of small molecules that specifically target the NCOA1-p300 interaction identified a compound SMTNP-191, which showed an inhibitory effect on suppressing MMP expression and attenuating the IDD process in aged mice.
CONCLUSIONS: Our data support a model in which deficiency of HERC3 fails to ubiquitinate NCOA1, leading to the assembly of NCOA1-p300-Runx2 and causing the transactivation of MMPs. These findings offer new insight into inflammation-mediated MMP accumulation and also provide a new therapeutic strategy to retard the IDD process.
摘要：
背景：基质金属肽酶（MMP）是关键的基质降解分子，它们经常在退行性椎间盘中过度表达。本研究旨在探讨MMP上调的机制。
方法：使用免疫印迹和RT-qPCR检测蛋白质和基因表达水平。4月龄和24月龄C57BL/6小鼠用于评价椎间盘退变(IDD)。使用泛素化测定法来确定蛋白质修饰。免疫沉淀和质谱用于鉴定蛋白质复合物成员。
结果：我们在患有IDD的老年小鼠中确定了23个成员中14个MMP的升高。这14种MMP基因启动子中有11种含有Runx2（runt相关转录因子2）结合位点。生化分析表明，Runx2招募了组蛋白乙酰转移酶p300和共激活剂NCOA1（核受体共激活剂1）来组装一个复合物，反式激活MMP表达。称为HERC3的E3连接酶（含有E3泛素蛋白连接酶3的HECT和RLD结构域）的缺乏导致炎症微环境中NCOA1的积累。特异性靶向NCOA1-p300相互作用的小分子的高通量筛选鉴定了化合物SMTNP-191,其在老年小鼠中显示出对抑制MMP表达和减弱IDD过程的抑制作用。
结论：我们的数据支持一个模型，其中HERC3缺乏未能使NCOA1泛素化，导致NCOA1-p300-Runx2组装并引起MMP的反式激活。这些发现为炎症介导的MMP积累提供了新的见解，也为延缓IDD过程提供了新的治疗策略。
方法：使用免疫印迹和RT-qPCR检测蛋白质和基因表达水平。4月龄和24月龄C57BL/6小鼠用于评价椎间盘退变(IDD)。使用泛素化测定法来确定蛋白质修饰。免疫沉淀和质谱用于鉴定蛋白质复合物成员。
结果：我们在患有IDD的老年小鼠中确定了23个成员中14个MMP的升高。这14种MMP基因启动子中有11种含有Runx2（runt相关转录因子2）结合位点。生化分析表明，Runx2招募了组蛋白乙酰转移酶p300和共激活剂NCOA1（核受体共激活剂1）来组装一个复合物，反式激活MMP表达。称为HERC3的E3连接酶（含有E3泛素蛋白连接酶3的HECT和RLD结构域）的缺乏导致炎症微环境中NCOA1的积累。特异性靶向NCOA1-p300相互作用的小分子的高通量筛选鉴定了化合物SMTNP-191,其在老年小鼠中显示出对抑制MMP表达和减弱IDD过程的抑制作用。
结论：我们的数据支持一个模型，其中HERC3缺乏未能使NCOA1泛素化，导致NCOA1-p300-Runx2组装并引起MMP的反式激活。这些发现为炎症介导的MMP积累提供了新的见解，也为延缓IDD过程提供了新的治疗策略。
