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

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.

Extracellular Matrix Metalloproteinase 2 Levels Are Regulated by the Low Density Lipoprotein-related Scavenger Receptor and Thrombospondin 2

“…Pro-MMP2 by APMA-We recently reported that TSP2-null fibroblasts have a marked adhesive defect when plated on a number of protein substrates, and we showed that this defect resulted from a 2-fold increase in MMP2 protein in the conditioned media of the cells (6). The adhesive defect was corrected by inhibitors of MMP2, by transfection of a full-length TSP2 cDNA gene into the cells, or by long term preincubation with recombinant TSP2 but not by adsorption of TSP2 to tissue culture plastic.…”

“…Dermal fibroblasts, isolated from adult animals, also show an adhesive defect in vitro that is most evident when cells are plated on a variety of pure protein substrates in the absence of serum. Adhesion was restored by prolonged (48 h) incubation of TSP2-null cells with recombinant mouse TSP2 or by transfection with a TSP2 cDNA gene (6). The basis for this adhesive defect was recently investigated and was shown by zymography to result from an increase in matrix metalloproteinase 2 (MMP2) levels in both the conditioned media and cell layers of cultured cells (6).…”

“…The basis for this adhesive defect was recently investigated and was shown by zymography to result from an increase in matrix metalloproteinase 2 (MMP2) levels in both the conditioned media and cell layers of cultured cells (6). Although virtually all of the enzyme that was analyzed was in the zymogen or pro-MMP2 form, an increase in active MMP2 in TSP2-null cells was inferred from the observation that inhibitors of MMP2, including TIMP2 and a neutralizing anti-MMP2 antibody, corrected the adhesive defect (6).…”

“…Aliquots of the resulting lysates were counted for radioactivity in a liquid scintillation counter to measure the internalization of 125 I-MMP2 by fibroblasts. Cell Attachment Assays and Zymography-Analyses for attachment of fibroblasts on fibronectin-coated 96-well plates were performed as described previously (6). Fibronectin was coated at a concentration of 5 g/ml in phosphate-buffered saline.…”

“…Normal rabbit IgG was purchased from Vector Laboratories (Burlingame, CA) and was used as a control. Zymography of conditioned media was performed as described by Yang et al (6).…”