title: Expression of matrix metalloproteinase-2 and -9 in human ligamentum flavum cells treated with tumor necrosis factor-α and interleukin-1β.
abstract: OBJECTIVE
  An in vitro study was performed to understand the potential roles of matrix metalloproteinase (MMP)-2 and MMP-9 in the elastin degradation of human ligamentum flavum (LF) cells via treatment with tumor necrosis factor-α (TNFα) and interleukin-1β (IL-1β). Previous studies have identified a decreased elastin to collagen ratio in hypertrophic LF. Among the extracellular matrix remodeling endopeptidases, MMP-2 and MMP-9 are known to have elastolytic activity. The hypothesis that activated LF cells exposed to inflammation would secrete MMP-2 and MMP-9, thereby resulting in elastin degradation, was examined.
  
  
  METHODS
  To examine MMP-2 and MMP-9 expression in human LF, cells were isolated and cultured from LF tissues that were obtained during lumbar disc surgery. Isolated LF cells were equally divided into 3 flasks and subcultured. Upon cellular confluency, the LF cells were treated with TNFα, IL-1β, or none (as a control) and incubated for 48 hours. The conditioned media were collected and assayed for MMP-2 and MMP-9 using gelatin zymography and Western blot analysis. The electrophoresis bands were compared on densitometric scans using ImageJ software.
  
  
  RESULTS
  The conditioned media from the isolated human LF cells naturally expressed 72-kD and 92-kD gelatinolytic activities on gelatin zymography. The IL-1β-treated LF cells presented sustained increases in the proenzyme/zymogen forms of MMP-2 and -9 (proMMP-2 and proMMP-9), and activeMMP-9 expression (p = 0.001, 0.022, and 0.036, respectively); the TNFα-treated LF cells showed the most elevated proMMP9 secretion (p = 0.006), as determined by Western blot analyses. ActiveMMP-2 expression was not observed on zymography or the Western blot analysis.
  
  
  CONCLUSIONS
  TNFα and IL-1β promote proMMP-2 and proMMP-9 secretion. IL-1β appears to activate proMMP-9 in human LF cells. Based on these findings, selective MMP-9 blockers or antiinflammatory drugs could be potential treatment options for LF hypertrophy.
authors.affiliation: Department of Neurosurgery, Korea University College of Medicine; and.
authors.forename: Bum-Joon
authors.initials: BJ
authors.lastname: Kim
authors.affiliation: Department of Neurosurgery, Korea University College of Medicine; and.
authors.forename: Junseok W
authors.initials: JW
authors.lastname: Hur
authors.affiliation: Department of Neurosurgery, Thejoeun Hospital, Seoul, Korea.
authors.forename: Jong Soo
authors.initials: JS
authors.lastname: Park
authors.affiliation: Department of Neurosurgery, Korea University College of Medicine; and.
authors.forename: Joo Han
authors.initials: JH
authors.lastname: Kim
authors.affiliation: Department of Neurosurgery, Korea University College of Medicine; and.
authors.forename: Taek-Hyun
authors.initials: TH
authors.lastname: Kwon
authors.affiliation: Department of Neurosurgery, Korea University College of Medicine; and.
authors.forename: Youn-Kwan
authors.initials: YK
authors.lastname: Park
authors.affiliation: Department of Neurosurgery, Korea University College of Medicine; and.
authors.forename: Hong Joo
authors.initials: HJ
authors.lastname: Moon
mesh_terms: D015153:Blotting, Western; D003094:Collagen / Q000378:metabolism*; D004549:Elastin / Q000378:metabolism*; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D053583:Interleukin-1beta / Q000494:pharmacology*; D017843:Ligamentum Flavum / Q000166:cytology*; D008297:Male; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism*; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D053583:Interleukin-1beta; D014409:Tumor Necrosis Factor-alpha; D003094:Collagen; D004549:Elastin; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9
keywords: ECM = extracellular matrix; EDTA = ethylenediaminetetraacetic acid; FBS = fetal bovine serum; HBSS = Hank’s balanced saline solution; IL-1β = interleukin-1β; LF = ligamentum flavum; MMP = matrix metalloproteinase; P/S = penicillin/streptomycin; SDS-PAGE = sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TIMP = tissue inhibitor of the metalloproteinase; TNFα = tumor necrosis factor−α; elastin degradation; interleukin-1β; ligamentum flavum cells; matrix metalloproteinase−2; matrix metalloproteinase−9; proMMP = proenzyme/zymogen form of MMP; tumor necrosis factor-α