title: Regulation of gelatinase-A (MMP-2) production by ovine intervertebral disc nucleus pulposus cells grown in alginate bead culture by Transforming Growth Factor-beta(1)and insulin like growth factor-I.
abstract: The aim of this study was to gain information relevant to disc repair processes. Limited degradation of the collagen matrix by matrix metalloproteases (MMPs) may facilitate the loosening of cell-cell and cell-matrix interactions within the injured intervertebral disc (IVD) to favour the penetration of blood vessels and migration of fibroblasts into the defect to promote repair processes. Gelatinase A (MMP-2) has a particularly important role to play in angiogenesis, in the present study we investigated the in vitro regulation of MMP-2 by Transforming Growth Factor-beta 1 (TGF-beta 1) and Insulin-like Growth Factor-1 (beta IGF-I) in cells from the nucleus pulposus (NP) of the ovine IVD. Ovine NP cells were grown in alginate bead cultures in complete medium (10% foetal calf serum) for 7 days, established in serum-free conditions for 24 h, then stimulated with TGF-beta 1 (0.1 or 10 ng/ml) or IGF-I (2 or 50 ng/ml) +/-Concanavalin A (20 microg/ml) for an additional 48 h. Conditioned medium was examined for matrix metalloproteases using gelatin zymography, Tissue Inhibitor of Metalloproteinase 2 (TIMP-2) and Membrane Type 1 Matrix Metalloproteinase (MT1-MMP) were immunolocalised in beads. Pro (72 kDa) and active (59 kDa) MMP-2 were the major gelatinolytic MMPs detected in control cultures, the TGF-beta 1 and IGF-I treatments significantly decreased levels of the active MMP-2, inclusion of Concanavalin A resulted in a complete reversal of this trend with IGF-I, and to a lesser extent with TGF-beta 1. Cell surface levels of TIMP-2 and MT1-MMP were decreased by the TGF-beta 1 treatment while IGF-I only appeared to decrease TIMP-2 expression. The findings of this study provide some insight as to why dense avascular connective tissues such as the intervertebral disc have such a poor healing potential.
authors.affiliation: Institute of Bone and Joint Research, The Royal North Shore Hospital, St Leonards, NSW, Australia.
authors.forename: S T
authors.initials: ST
authors.lastname: Pattison
authors.forename: J
authors.initials: J
authors.lastname: Melrose
authors.forename: P
authors.initials: P
authors.lastname: Ghosh
authors.forename: T K
authors.initials: TK
authors.lastname: Taylor
mesh_terms: D000464:Alginates / Q000737:chemistry; D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002478:Cells, Cultured; D004792:Enzyme Precursors / Q000096:biosynthesis; D018093:Gelatinases / Q000096:biosynthesis; D020723:Glucuronic Acid; D006603:Hexuronic Acids; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D007403:Intervertebral Disc / Q000166:cytology* / Q000201:enzymology*; D020778:Matrix Metalloproteinase 2 / Q000096:biosynthesis*; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases / Q000096:biosynthesis / Q000378:metabolism; D008863:Microspheres; D012756:Sheep; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D053773:Transforming Growth Factor beta1; D014945:Wound Healing
chemical_list: D000464:Alginates; D004792:Enzyme Precursors; D006603:Hexuronic Acids; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D019716:Tissue Inhibitor of Metalloproteinase-2; D007334:Insulin-Like Growth Factor I; D020723:Glucuronic Acid; D018093:Gelatinases; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases; C069860:progelatinase; D020778:Matrix Metalloproteinase 2