Transglutaminases (TGs) are multifunctional proteins having enzymatic and scaffolding functions that participate in regulation of cell fate in a wide range of cellular systems and are implicated to have roles in development of disease. This review highlights the mechanism of action of these proteins with respect to their structure, impact on cell differentiation and survival, role in cancer development and progression, and function in signal transduction. We also discuss the mechanisms whereby TG level is controlled and how TGs control downstream targets. The studies described herein begin to clarify the physiological roles of TGs in both normal biology and disease states.

Inorganic pyrophosphate (PP_i) produced by cells inhibits mineralization by binding to crystals. Its ubiquitous presence is thought to prevent “soft” tissues from mineralizing, whereas its degradation to P_i in bones and teeth by tissue-nonspecific alkaline phosphatase (Tnap, Tnsalp, Alpl, Akp2) may facilitate crystal growth. Whereas the crystal binding properties of PP_i are largely understood, less is known about its effects on osteoblast activity. We have used MC3T3-E1 osteoblast cultures to investigate the effect of PP_i on osteoblast function and matrix mineralization. Mineralization in the cultures was dose-dependently inhibited by PP_i. This inhibition could be reversed by Tnap, but not if PP_i was bound to mineral. PP_i also led to increased levels of osteopontin (Opn) induced via the Erk1/2 and p38 MAPK signaling pathways. Opn regulation by PP_i was also insensitive to foscarnet (an inhibitor of phosphate uptake) and levamisole (an inhibitor of Tnap enzymatic activity), suggesting that increased Opn levels did not result from changes in phosphate. Exogenous OPN inhibited mineralization, but dephosphorylation by Tnap reversed this effect, suggesting that OPN inhibits mineralization via its negatively charged phosphate residues and that like PP_i, hydrolysis by Tnap reduces its mineral inhibiting potency. Using enzyme kinetic studies, we have shown that PP_i inhibits Tnap-mediated P_i release from β-glycerophosphate (a commonly used source of organic phosphate for culture mineralization studies) through a mixed type of inhibition. In summary, PP_i prevents mineralization in MC3T3-E1 osteoblast cultures by at least three different mechanisms that include direct binding to growing crystals, induction of Opn expression, and inhibition of Tnap activity.

Osteopontin, a major noncollagenous bone protein, is an in vitro and in vivo substrate of tissue transglutaminase, which catalyzes formation of cross-linked protein aggregates. The roles of the enzyme and the polymeric osteopontin are presently not fully understood. In this study we provide evidence that transglutaminase treatment significantly increases the binding of osteopontin to collagen. This was tested with an enzyme-linked immunosorbent assay. The results also show that this increased interaction is clearly calcium-dependent and specific to osteopontin. In dot blot overlay assay 1 μg of collagen type I was able to bind 420 ng of in vitro prepared and purified polymeric osteopontin and only 83 ng of monomeric osteopontin, indicating that the transglutaminase treatment introduces a 5-fold amount of osteopontin onto collagen. Assays using a reversed situation showed that the collagen binding of the polymeric form of osteopontin appears to be dependent on its conformation in solution. Circular dichroism analysis of monomeric and polymeric osteopontin indicated that transglutaminase treatment induces a conformational change in osteopontin, probably exposing motives relevant to its interactions with other extracellular molecules. This altered collagen binding property of osteopontin may have relevance to its biological functions in tissue repair, bone remodeling, and collagen fibrillogenesis.

Tissue transglutaminase (tTG) is an intra- and extracellular, protein-cross-linking enzyme that has been implicated in apoptosis, matrix stabilization, and cell attachment in a variety of tissues. This study provides in vivo evidence in bone of TG activity, its tissue localization, and identification of its substrates. In microplate- and blotting-based activity assays using biotinylated primary amine as a probe, we show TG activity in protein extracts from the mineralized compartment of intramembranous rat bone. Avidin affinity purification of bone extract labeled with biotinylated primary amine in the presence of tTG, in conjunction with Western blotting, permitted identification of three major noncollagenous TG substrates in bone: osteopontin (OPN), bone sialoprotein (BSP), and α2 HS-glycoprotein (AHSG), of which the latter two are novel substrates. Cross-linking and labeling of purified proteins confirmed their ability to serve as TG substrates, because they readily incorporated biotinylated primary amine and formed large protein aggregates in the presence of tTG. All three proteins were also identified in the high molecular weight complexes extractable from the mineralized compartment of bone. Two-dimensional (2D) gel electrophoretic analysis combined with Western blotting indicated that the proteins are not cross-linked to each other, but form distinct homotypic polymers. In the extracellular matrix of bone, tTG and isopeptide bonds were localized by immunohistochemistry in the osteoid and in the pericellular matrix surrounding osteocytes. At the cellular level, osteoblasts and osteocytes were immunostained for tTG. Collectively, these data suggest a role for tTG and its covalently cross-linked substrates in cell adhesion and possibly also in bone matrix maturation and calcification.