Xu Kang, Wang Xiaofei, Zhang Qianshi, Liang Anjing, Zhu Haipeng, Huang Dongsheng, Li Chunhai, Ye Wei
Experimental Center of Surgery, Sun Yat‑sen Memorial Hospital, Sun Yat‑sen University, Guangzhou, Guangdong 510120, P.R. China.
Department of Spine Surgery, Sun Yat‑sen Memorial Hospital, Sun Yat‑sen University, Guangzhou, Guangdong 510120, P.R. China.
Mol Med Rep. 2016 Oct;14(4):3961-8. doi: 10.3892/mmr.2016.5730. Epub 2016 Sep 6.
During the pathogenesis of intervertebral disc degeneration, pro‑inflammatory cytokines, including tumor necrosis factor‑α (TNF‑α), stimulate the degradation of the extracellular matrix (ECM) of intervertebral discs via the activity of catabolic enzymes including matrix metalloproteinases (MMPs), disintegrins and metalloproteinases with thrombospondin motifs (ADAMTSs), and cyclooxygenase 2 (Cox2). The transcriptional promoters of the human catabolic enzymes MMPs, ADAMTS, Cox2 and Syndecan 4 contain at least one specificity protein‑1 (Sp1) transcription factor‑binding site. The present study investigated the role of Sp1 in the regulation of the mRNA and protein expression of the aforementioned catabolic enzyme genes in nucleus pulposus cells, using reverse transcription‑quantitative polymerase chain reaction, western blot, transfection and RNA interference. The data demonstrated that Sp1 transcription factor protein expression is induced by TNF‑α and interleukin‑1β. Specific inhibitors of Sp1 DNA binding to its GC‑rich consensus site, WP631 and mithramycin A, partially suppressed TNF‑α‑induced catabolic enzyme expression and activity. Genetic inhibition of Sp1 by small interfering RNA‑mediated Sp1 knockdown partially inhibited catabolic enzyme induction by TNF‑α. In addition, Sp1 transcription factor inhibitors decreased the activity of MMP3, ADAMTS4 and ADAMTS5 promoters. Furthermore, chromatin immunoprecipitation revealed functional Sp1 binding sites at ‑577/‑567 bp within the ADAMTS4 promoter and ‑718/‑708 bp within the ADAMTS5 promoter. These results provide pharmacological and genetic evidence of the importance of Sp1 in catabolic enzyme gene regulation during TNF‑α stimulation. Thus, Sp1 may represent an effective target in reducing intervertebral disc‑associated ECM loss.
在椎间盘退变的发病机制中，包括肿瘤坏死因子-α（TNF-α）在内的促炎细胞因子通过分解代谢酶的活性刺激椎间盘细胞外基质（ECM）的降解，这些分解代谢酶包括基质金属蛋白酶（MMPs）、含血小板反应蛋白基序的解聚素和金属蛋白酶（ADAMTSs）以及环氧合酶2（Cox2）。人类分解代谢酶MMPs、ADAMTS、Cox2和Syndecan 4的转录启动子至少包含一个特异性蛋白-1（Sp1）转录因子结合位点。本研究采用逆转录-定量聚合酶链反应、蛋白质印迹、转染和RNA干扰等方法，研究Sp1在调节髓核细胞中上述分解代谢酶基因的mRNA和蛋白表达中的作用。数据表明，Sp1转录因子蛋白表达由TNF-α和白细胞介素-1β诱导。Sp1与富含GC的共有位点结合的特异性抑制剂WP631和光神霉素A可部分抑制TNF-α诱导的分解代谢酶表达和活性。通过小干扰RNA介导的Sp1敲低对Sp1进行基因抑制可部分抑制TNF-α诱导的分解代谢酶表达。此外，Sp1转录因子抑制剂降低了MMP3、ADAMTS4和ADAMTS5启动子的活性。此外，染色质免疫沉淀显示在ADAMTS4启动子内-577 / -567 bp处和ADAMTS5启动子内-718 / -708 bp处存在功能性Sp1结合位点。这些结果提供了药理学和遗传学证据，证明Sp1在TNF-α刺激期间对分解代谢酶基因调控的重要性。因此，Sp1可能是减少椎间盘相关ECM损失的有效靶点。
