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.
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.
Yang
Z
Z
Department of Biochemistry, University of Washington, Seattle, Washington 98195, USA.
Kyriakides
T R
TR
Bornstein
P
P
D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D002448:Cell Adhesion / Q000235:genetics / Q000502:physiology*; D015815:Cell Adhesion Molecules / Q000502:physiology*; D002478:Cells, Cultured; D017077:Culture Media, Conditioned; D005109:Extracellular Matrix / Q000502:physiology*; D016326:Extracellular Matrix Proteins / Q000378:metabolism*; D005347:Fibroblasts / Q000502:physiology*; D007700:Kinetics; D020778:Matrix Metalloproteinase 2 / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D011994:Recombinant Proteins / Q000378:metabolism; D012867:Skin / Q000166:cytology*; D012879:Skin Physiological Phenomena; D019699:Thrombospondins / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D014162:Transfection; D000099262:Thrombospondin 2
D000906:Antibodies; D015815:Cell Adhesion Molecules; D017077:Culture Media, Conditioned; D016326:Extracellular Matrix Proteins; D020778:Matrix Metalloproteinase 2; D011994:Recombinant Proteins; D019699:Thrombospondins; D000099262:Thrombospondin 2