Matricellular proteins as modulators of cell-matrix interactions: adhesive defect in thrombospondin 2-null fibroblasts is a consequence of increased levels of matrix metalloproteinase-2

Mol Biol Cell. 2000 Oct;11(10):3353-64. doi: 10.1091/mbc.11.10.3353.

Abstract

Thrombospondin 2 (TSP2)-null mice, generated by disruption of the Thbs2 gene, display a variety of connective tissue abnormalities, including fragile skin and the presence of abnormally large collagen fibrils with irregular contours in skin and tendon. In this study we demonstrate that TSP2-null skin fibroblasts show a defect in attachment to a number of matrix proteins, and a reduction in cell spreading. To investigate the molecular mechanisms responsible for these abnormal cell-matrix interactions, we compared the levels of matrix metalloproteinases (MMPs) in wild-type and mutant fibroblasts. Isolation and analysis of gelatinases from conditioned media by gelatin-agarose affinity chromatography and gelatinolytic assays demonstrated that TSP2-null fibroblasts produce a 2-fold increase in gelatinase A (MMP2) compared with wild-type cells. The adhesive defect was corrected by treatment of TSP2-null fibroblasts with soluble TSP2, with the MMP inhibitors BB94 and tissue inhibitor of metalloproteinase-2, and with a neutralizing antibody to MMP2. Moreover, stable transfection of TSP2-null fibroblasts with mouse TSP2 cDNA corrected both the adhesive defect and the altered expression of MMP2. Finally, MMP2 was shown to interact with TSP2 in a direct-binding plate assay. We conclude that TSP2 plays an important role in cell-matrix interactions, and that a deficiency in the protein results in increased levels of MMP2 that contribute to the adhesive defect in TSP2-null fibroblasts and could play a role in the complex phenotype of TSP2-null mice.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Antibodies / pharmacology
  • Cell Adhesion / genetics
  • Cell Adhesion / physiology*
  • Cell Adhesion Molecules / physiology*
  • Cells, Cultured
  • Culture Media, Conditioned
  • Extracellular Matrix / physiology*
  • Extracellular Matrix Proteins / metabolism*
  • Fibroblasts / physiology*
  • Kinetics
  • Matrix Metalloproteinase 2 / genetics*
  • Matrix Metalloproteinase 2 / metabolism
  • Mice
  • Mice, Knockout
  • Recombinant Proteins / metabolism
  • Skin / cytology*
  • Skin Physiological Phenomena
  • Thrombospondins / deficiency
  • Thrombospondins / genetics
  • Thrombospondins / physiology*
  • Transfection

Substances

  • Antibodies
  • Cell Adhesion Molecules
  • Culture Media, Conditioned
  • Extracellular Matrix Proteins
  • Recombinant Proteins
  • Thrombospondins
  • thrombospondin 2
  • Matrix Metalloproteinase 2