title: The Paracrine Effect of Degenerated Disc Cells on Healthy Human Nucleus Pulposus Cells Is Mediated by MAPK and NF-κB Pathways and Can Be Reduced by TGF-β1.
abstract: Inflammation is thought to have a major role in the pathogenesis of disc degeneration. Studies have shown that nucleus pulposus cells (NPCs) respond to one or two specific cytokines by regulating cell proliferation or matrix synthesis. However, the effects of a cocktail of factors secreted by degenerated disc cells on transplanted exogenous healthy NPCs remain unknown. Concentrations of multiple cytokines in degenerated disc tissue-conditioned medium (dCM) were measured using enzyme-linked immunosorbent assay (ELISA). 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and Ki67 immunofluorescence staining were used to evaluate the proliferation of cells in dCM. The function of exogenous NPCs cultured in dCM was evaluated by examining catabolic markers (ADAMTS-4, ADAMTS-5, MMP-1, MMP-3, and MMP-13), anabolic markers (TIMP-1, TIMP-2, and TIMP-3), and the extracellular matrix protein-aggrecan (ACAN) and collagen II (COL2)-expression with real time polymerase chain reaction (RT-PCR). Mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) pathway activation was observed using Western blotting. Finally, we examined the role of transforming growth factor (TGF)-β1 in reducing dCM-mediated exogenous NPC dysfunction. Levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-1α, IL-2, IL-4, IL-6, IL-8, IL-10, IL-17, interferon-γ (IFN-γ), and prostaglandin E2 (PGE2) were higher and TGF-β1 levels were lower in dCM compared with the control medium. Treatment with dCM increased the proliferation of healthy NPCs. NPCs exhibited significantly higher expression of ADAMTS-4, ADAMTS-5, MMP-1, MMP-3, and MMP-13 and decreased TIMP-2, ACAN, and COL2 expression in the dCM group in a dose- and time-dependent manner. Treatment with dCM moderately increased TIMP-1 expression and had no effect on TIMP-3 mRNA levels. The MAPK and NF-κB pathways were implicated in dCM-mediated responses of healthy NPCs. TGF-β1 partially reversed the dCM-mediated NPC dysfunction. Increased levels of inflammatory factors and decreased TGF-β1 levels in dCM suggest an inflammatory environment in degenerated disc tissue. The catabolic effect of dCM on human healthy NPCs is mediated by MAPK and NF-κB pathways and can be reduced by TGF-β1.
authors.affiliation: 1 Department of Spine Surgery, Zhongda Hospital, School of Medicine, Southeast University , Nanjing, China .|2 Department of Orthopedics, The First Affiliated Hospital of Soochow University , Suzhou, China .
authors.forename: Feng
authors.initials: F
authors.lastname: Cai
authors.affiliation: 1 Department of Spine Surgery, Zhongda Hospital, School of Medicine, Southeast University , Nanjing, China .
authors.forename: Lei
authors.initials: L
authors.lastname: Zhu
authors.affiliation: 1 Department of Spine Surgery, Zhongda Hospital, School of Medicine, Southeast University , Nanjing, China .
authors.forename: Feng
authors.initials: F
authors.lastname: Wang
authors.affiliation: 1 Department of Spine Surgery, Zhongda Hospital, School of Medicine, Southeast University , Nanjing, China .
authors.forename: Rui
authors.initials: R
authors.lastname: Shi
authors.affiliation: 1 Department of Spine Surgery, Zhongda Hospital, School of Medicine, Southeast University , Nanjing, China .
authors.forename: Xin-Hui
authors.initials: XH
authors.lastname: Xie
authors.affiliation: 1 Department of Spine Surgery, Zhongda Hospital, School of Medicine, Southeast University , Nanjing, China .
authors.forename: Xin
authors.initials: X
authors.lastname: Hong
authors.affiliation: 1 Department of Spine Surgery, Zhongda Hospital, School of Medicine, Southeast University , Nanjing, China .
authors.forename: Xiao-Hu
authors.initials: XH
authors.lastname: Wang
authors.affiliation: 1 Department of Spine Surgery, Zhongda Hospital, School of Medicine, Southeast University , Nanjing, China .
authors.forename: Xiao-Tao
authors.initials: XT
authors.lastname: Wu
mesh_terms: D000071096:ADAMTS Proteins / Q000235:genetics; D000328:Adult; D000368:Aged; D016022:Case-Control Studies; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D016326:Extracellular Matrix Proteins / Q000235:genetics; D005260:Female; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006801:Humans; D018836:Inflammation Mediators / Q000378:metabolism; D055959:Intervertebral Disc Degeneration / Q000473:pathology*; D008297:Male; D020782:Matrix Metalloproteinases / Q000235:genetics; D008875:Middle Aged; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism*; D000070614:Nucleus Pulposus / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D019899:Paracrine Communication / Q000187:drug effects*; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology*
chemical_list: D016326:Extracellular Matrix Proteins; D018836:Inflammation Mediators; D016328:NF-kappa B; D053773:Transforming Growth Factor beta1; D020928:Mitogen-Activated Protein Kinases; D000071096:ADAMTS Proteins; D020782:Matrix Metalloproteinases
keywords: MAPK; NF-κB; TGF-β1; intervertebral disc; paracrine