title: Fibronectin fragments and the cleaving enzyme ADAM-8 in the degenerative human intervertebral disc.
abstract: METHODS
  The presence of fibronectin fragments (FN-fs) and the cleaving enzyme, A disintegrin and metalloproteinase domain-containing protein (ADAM)-8 were examined in human intervertebral disc (IVD) tissue in vitro.
  
  
  OBJECTIVE
  To investigate the presence and pathophysiological concentration of FN-fs and their cleaving enzyme, ADAM-8, in the human IVD tissue.
  
  
  BACKGROUND
  The 29-kDa FN-f has been shown to result in extracellular matrix loss in rabbit IVDs. However, the concentration of this biologically active fragment in the degenerative human IVD tissue has previously not been determined. Furthermore, it is critical to identify the enzyme(s) responsible for FN cleavage in the IVD.
  
  
  METHODS
  Human degenerative IVD tissues were removed during spinal surgery. A normal seeming young adult and an infant human cadaveric sample were obtained as controls. Soluble proteins were extracted, and analyzed by Western blotting using antibodies specific for the human FN neoepitope VRAA²⁷¹. A purified 29-kDa FN-f was used to allow estimation of the concentration of FN-fs in the tissues. ADAM-8, a FN-cleaving enzyme, was analyzed by Western blotting and immunostaining.
  
  
  RESULTS
  All adult IVD tissues contain many FN-f species, but these species were absent from the infant disc tissue. Moderately degenerative discs contained the highest amount of FN-fs; the concentration was estimated to be in the nanomolar range per gram of tissue. ADAM-8, known to cleave FN resulting in the VRAA²⁷¹ neoepitope, was present in the human disc. ADAM-8 primarily localized in the pericellular matrix of the nucleus pulposus tissue, as determined by immunostaining.
  
  
  CONCLUSIONS
  This is the first report that N-terminal FN-fs are consistently present in IVD tissues from adult subjects. The pathophysiological concentration of these fragments is estimated to be at nanomolar range per gram of IVD tissue. Furthermore, ADAM-8, known to cleave FN, is present at the pericellular matrix of disc cells.
authors.affiliation: *Department of Orthopedic Surgery, Rush University Medical Center, Chicago, IL †Department of Orthopedic Surgery, Thomas Jefferson University, Philadelphia, PA ‡Department of Biochemistry, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA §Department of Medicine, Section of Rheumatology, Perelman School of Medicine, and Philadelphia Veterans Affairs Medical Center, University of Pennsylvania, Philadelphia, PA ¶Department of Biochemistry, Rush University Medical Center, Chicago, IL ‖Philadelphia Veterans Affairs Medical Center, and Department of Physical Medicine and Rehabilitation, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA; and **Dow Agrosciences, Indianapolis, IN.
authors.forename: Nancy
authors.initials: N
authors.lastname: Ruel
authors.forename: Dessislava Z
authors.initials: DZ
authors.lastname: Markova
authors.forename: Sherrill L
authors.initials: SL
authors.lastname: Adams
authors.forename: Carla
authors.initials: C
authors.lastname: Scanzello
authors.forename: Gabriella
authors.initials: G
authors.lastname: Cs-Szabo
authors.forename: David
authors.initials: D
authors.lastname: Gerard
authors.forename: Peng
authors.initials: P
authors.lastname: Shi
authors.forename: D Greg
authors.initials: DG
authors.lastname: Anderson
authors.forename: Marc
authors.initials: M
authors.lastname: Zack
authors.forename: Howard S
authors.initials: HS
authors.lastname: An
authors.forename: Di
authors.initials: D
authors.lastname: Chen
authors.forename: Yejia
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D051722:ADAM Proteins / Q000378:metabolism*; D000328:Adult; D015153:Blotting, Western; D002102:Cadaver; D005353:Fibronectins / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D007223:Infant; D055959:Intervertebral Disc Degeneration / Q000378:metabolism* / Q000601:surgery; D008565:Membrane Proteins / Q000378:metabolism*; D008875:Middle Aged; D010446:Peptide Fragments / Q000378:metabolism*; D059748:Proteolysis
chemical_list: D005353:Fibronectins; D008565:Membrane Proteins; D010446:Peptide Fragments; D051722:ADAM Proteins; C068490:ADAM8 protein, human