Transglutaminase activity regulates osteoblast differentiation and matrix mineralization in MC3T3-E1 osteoblast cultures

Transglutaminase (TG) enzymes and protein crosslinking have long been implicated in the formation of mineralized tissues. The aim of this study was to analyze the expression, activity and function of TGs in differentiating osteoblasts to gain further insight into the role of extracellular matrix protein crosslinking in bone formation. MC3T3-E1 (subclone 14) pre-osteoblast cultures were treated with ascorbic acid and β-glycerophosphate to induce cell differentiation and matrix mineralization. Expression of TG isoforms was analyzed by RT-PCR. TG activity was assessed during osteoblast differentiation by in vitro biochemical assays and by in situ labeling of live cell cultures. We demonstrate that MC3T3-E1/C14 osteoblasts express two TG isoforms—TG2 and FXIIIA. Abundant TG activity was observed during cell differentiation which increased significantly after thrombin treatment, a result confirming the presence of FXIIIA in the cultures. Ascorbic acid treatment, which stimulated collagen secretion and assembly, also stimulated externalization of TG activity, likely from FXIIIA which was externalized upon this treatment as analyzed by immunofluoresence microscopy. Inhibition of TG activity in the cultures by cystamine resulted in complete abrogation of mineralization, attributable to decreased matrix accumulation and an arrested state of osteoblast differentiation as measured by decreased levels of bone sialoprotein, osteocalcin and alkaline phosphatase. Additional functional studies and substrate characterization showed that TG activity was required for the formation of a fibronectin–collagen network during the early stages of matrix formation and assembly. This network, in turn, appeared to be essential for further matrix production and progression of the osteoblast differentiation program, and ultimately for mineralization.