title: Abundance of calpain and aggrecan-cleavage products of calpain in degenerated human intervertebral discs.
abstract: OBJECTIVE
  To assess the expression of calpains and calpain-induced aggrecan fragmentation in early and advanced stages of degeneration of human intervertebral discs (IVDs).
  
  
  METHODS
  Disc tissue samples of 55 patients (mean age, 51.2 ± 22.3 years) who underwent intervertebral fusion were divided into groups with early and advanced degeneration based on the Thompson magnetic resonance imaging (MRI) scale. In advanced degeneration group, five patients (mean age, 35.5 ± 11.4 years) of lumbar disc herniation (LDH) were included. Protein levels of m- and μ-calpains and their inhibitor calpastatin were assayed, and immunohistochemical techniques were used to localize and quantify the production of the enzymes. To investigate calpain activity, we assayed purified aggrecan fragmentation in disc tissue by Western blotting and immunohistochemistry with VPGVA antibody, which recognizes the m-calpain generated neo-epitope GVA.
  
  
  RESULTS
  Discs at early stages of degeneration expressed low levels of m- and μ-calpains and calpastatin, and few cells expressed degenerative enzymes. At more advanced stages of degeneration, the expression and number of cells immunopositive for m-calpain, μ-calpain and calpastatin were significantly higher. Further finding showed that anti-GVA-reactive aggrecan fragments were significantly higher in discs at advanced compared with early stages of degeneration. Herniated disc samples showed stronger expression and more cells immunopositive for calpains, calpastatin and GVA in the nucleus pulposus than in the annulus fibrous.
  
  
  CONCLUSIONS
  The expression of calpains, together with m-calpain-induced degradation products of extracellular matrix, was correlated with the degree of disc degeneration in human IVD tissue. These findings suggest that calpains may be involved in IVD degeneration via proteoglycan (PG) cleavage.
authors.affiliation: Department of Orthopedic Surgery, Gifu University Graduate School of Medicine, Gifu City, Gifu, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Fukuta
authors.forename: K
authors.initials: K
authors.lastname: Miyamoto
authors.forename: K
authors.initials: K
authors.lastname: Suzuki
authors.forename: H
authors.initials: H
authors.lastname: Maehara
authors.forename: T
authors.initials: T
authors.lastname: Inoue
authors.forename: A
authors.initials: A
authors.lastname: Hara
authors.forename: K
authors.initials: K
authors.lastname: Kikuike
authors.forename: A
authors.initials: A
authors.lastname: Taguchi
authors.forename: K
authors.initials: K
authors.lastname: Shimizu
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D053674:Aggrecans / Q000378:metabolism*; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000502:physiology*; D002154:Calpain / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002648:Child; D005109:Extracellular Matrix / Q000473:pathology; D016326:Extracellular Matrix Proteins / Q000378:metabolism; D005260:Female; D006801:Humans; D055959:Intervertebral Disc Degeneration / Q000201:enzymology / Q000378:metabolism*; D008159:Lumbar Vertebrae / Q000201:enzymology* / Q000473:pathology; D008279:Magnetic Resonance Imaging; D008297:Male; D008875:Middle Aged; D012447:Sacrum / Q000201:enzymology / Q000473:pathology
chemical_list: D053674:Aggrecans; D002135:Calcium-Binding Proteins; D016326:Extracellular Matrix Proteins; C033668:calpastatin; D002154:Calpain; C449000:m-calpain; C449002:mu-calpain