No abstract

Fibronectin (FN) is a cell adhesion protein that binds integrins in a process also involving the protein-crosslinking enzyme transglutaminase 2 (TG2) as a co-receptor. The cell-adhesive property of TG2 has been linked to a complex formation with FN and to its ability to crosslink and polymerize FN on the cell surface. We tested here the effects of extracellular FN, before and after in vitro crosslinking and polymerization by TG2, on MC3T3-E1 osteoblast adhesion. We show that TG2-mediated crosslinking creates large, compacted chain-like protein clusters that include both TG2 and FN molecules as analyzed by Western blotting and atomic force microscopy. Crosslinking of FN significantly promotes osteoblast adhesion as measured by crystal violet staining, and enhances beta(1)-integrin clustering on the cell surface as visualized by immunofluorescence microscopy. We hypothesize that TG2-mediated crosslinking enhances the cell-adhesive properties of FN by increasing the molecular rigidity of FN in the extracellular matrix.

A pro-mineralization function for transglutaminase 2 (TG2) has been suggested in numerous studies related to bone, cartilage, and vascular calcification. TG2 is an enzyme which can perform protein crosslinking functions, or act as a GTPase/ATPase depending upon different stimuli. We have previously demonstrated that TG2 can act as an ATPase in a Ca(2+)-rich environment and that it can regulate phosphate levels in osteoblast cultures. In this study, we investigate the role MT1-MMP in regulating the ATPase activity of TG2. We report that proteolytic cleavage of TG2 by MT1-MMP in vitro results in nearly a 3-fold increase in the ATPase activity of TG2 with a concomitant reduction in its protein-crosslinking activity. We show that MC3T3-E1 osteoblasts secreted full-length TG2 and major smaller fragments of 66 and 56 kDa, the latter having ATP-binding abilities. MT1-MMP inhibition by a neutralizing antibody suppressed mineralization of osteoblast cultures to 35% of control, and significantly reduced phosphate levels in conditioned medium (CM). Furthermore, MT1-MMP inhibition abolished two of TG2 fragments in the cultures, one of which, the 56-kDa fragment, has ATPase activity. Neutralization of MT1-MMP at early phases of mineralization significantly reduced mineral deposition, but had no effect in later phases implying MT1-MMP and TG2 might contribute to the initiation of mineralization. The cleavage of TG2 by MT1-MMP likely occurs on the cell surface/pericellular matrix where MT1-MMP and TG2 were co-localized. Based on these data, we propose that MT1-MMP modulates the extracellular function TG2 as part of a regulatory mechanism activates the pro-mineralization function of TG2.