Transglutaminase expression and activity in MC3T3-E1 osteoblast cultures

Transglutaminases are enzymes that stabilize extracellular matrices by catalyzing the formation of protease-resistant isopeptide crosslinks between and within their substrate proteins. Two transglutaminase isoforms, tissue transglutaminase (tTG) and factor XIIIa (FXIIIa), are expressed in skeletal tissues. Tissue transglutaminase has been previously localized in bone to osteoblasts, the osteoid layer and the pericellular matrix of osteocytes. Factor XIIIa has been localized to mineralizing chondrocytes, however, its expression in osteoblasts has not been reported yet. In order to understand the role of transglutaminases in hard tissue formation, we have investigated tTG and FXIIIa expression and activity during osteoblast differentiation and mineralization using the well-characterized MC3T3-E1 preosteoblast cell line. We show that tTG and FXIIIa are expressed at all time points examined in the cell cultures, and that their expression and activity does not appear to be significantly changed by the differentiation or mineralization state of the cells. We show that tTG localizes homogeneously throughout the cells and the extracellular matrix, whereas FXIIIa distribution is polarized to one side of the cells at earlier time points, but becomes incorporated into the matrix produced by differentiating cells in mature cultures. These expression profiles suggest a dual role, initially in cell adhesion and attachment and, later, in protein assembly, where the transglutaminases likely stabilize the matrix and assist in the formation of an extracellular matrix competent for subsequent mineralization. The identification and characterization of two transglutaminase isoforms in osteoblasts suggest a potentially overlapping roles for these two enzymes in the MC3T3-E1 cell culture system