ABSTRACT
Tissue transglutaminase (TG2) is a widely distributed, protein-crosslinking enzyme having a prominent role in cell adhesion as a ß1 integrin co-receptor for fibronectin. In bone and teeth, its substrates include the matricellular proteins osteopontin (OPN) and bone sialoprotein (BSP). The aim of this study was to examine effects of TG2-mediated crosslinking and oligomerization of OPN and BSP on osteoblast cell adhesion. We show that surfaces coated with oligomerized OPN and BSP promote MC3T3-E1/C4 osteoblastic cell adhesion significantly better than surfaces coated with the monomeric form of the proteins. Both OPN and BSP oligomer-adherent cells showed more cytoplasmic extensions than those cells grown on the monomer-coated surfaces indicative of increased cell connectivity. Our study suggests a role for TG2 in promoting the cell adhesion function of two matricellular substrate proteins prominent in bone, tooth cementum and certain tumors.
Subject(s)
GTP-Binding Proteins/metabolism , Integrin-Binding Sialoprotein/genetics , Integrin-Binding Sialoprotein/metabolism , Osteoblasts/cytology , Osteopontin/genetics , Osteopontin/metabolism , Transglutaminases/metabolism , Animals , Bone and Bones/metabolism , Cell Adhesion/physiology , Cell Line , Dental Cementum/metabolism , Fibronectins/metabolism , Integrin beta1/metabolism , Mice , Osteoblasts/metabolism , Protein Binding , Protein Glutamine gamma Glutamyltransferase 2 , Tooth/metabolismABSTRACT
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.
