Sp1 downregulates proinflammatory cytokine‑induced catabolic gene expression in nucleus pulposus cells

Xu,Kang; Wang,Xiaofei; Zhang,Qianshi; Liang,Anjing; Zhu,Haipeng; Huang,Dongsheng; Li,Chunhai; Ye,Wei

doi:10.3892/mmr.2016.5730

Sp1 downregulates proinflammatory cytokine‑induced catabolic gene expression in nucleus pulposus cells

Authors:
- Kang Xu
- Xiaofei Wang
- Qianshi Zhang
- Anjing Liang
- Haipeng Zhu
- Dongsheng Huang
- Chunhai Li
- Wei Ye
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Affiliations: 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, Department of Spine Surgery, The Second Xiangya Hospital of Central South University, Changsha, Hunan 410011, P.R. China
Published online on: September 6, 2016 https://doi.org/10.3892/mmr.2016.5730
Pages: 3961-3968

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Abstract

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.

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