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 ␤-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 g 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 (TG) 1 (EC 2.3.2.13) is a widely distributed intra-and extracellular calcium-dependent enzyme, which catalyzes the formation of high molecular mass complexes of its substrate proteins by creating isopeptide crosslinks from glutamine and lysine residues and releasing ammonia (1, 2). TG is suggested to be involved in matrix maturation and stabilize the tissue with cross-links that are resistant to normal proteolysis (1, 2). TG is closely related to wound healing which suggests a role for it in tissue remodeling and repair (3,4). Immunohistochemical data have also demonstrated the presence of TG in mineralizing cartilage and bone (5, 6) and the enzyme is thought to participate in matrix cross-linking before the tissue undergoes calcification (5, 6). The number of proteins serving as glutaminyl substrates for TG is restricted indicating the physiological importance of its functions (1). The roles of TG and the actions of its enzymatic products, meaning high molecular weight proteins, are still unclear.Osteopontin (OPN), a prominent and potentially multifunctional acidic phosphoglycoprotein (7,8), is a substrate of TG (9 -11). OPN is a major product of bone forming cells, osteoblasts, but is not specific to bone. It is also synthesized in other types of tissues and found in, e.g. inner ear, brain, kidney (7), and atherosclerotic plaques (13,14), and it is also secreted into milk (12) and urine (15). Its production is also related to immunity, infection, and cancer (8). Osteoblasts express OPN at an early developmental stage of bone formation (16,17). In bone, OPN is deposited into unmineralized matrix pri...

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 microplateand 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 ␣ 2 HS-glycoprotein (AHSG), of which the latter two are novel substrates. Crosslinking 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. (J Bone Miner Res 2002;17:2161-2173)

Microfibrils are essential elements in elastic and nonelastic tissues contributing to homeostasis and growth factor regulation. Fibrillins form the core of these multicomponent assemblies. Various human genetic disorders, the fibrillinopathies, arise from mutations in fibrillins and are frequently associated with aberrant microfibril assembly. These disorders include Marfan syndrome, Weill-Marchesani syndrome, Beals syndrome, and others. Although homotypic and heterotypic fibrillin self-interactions are considered to provide critical initial steps, the detailed mechanisms for microfibril assembly are unknown. We show here that the C-terminal recombinant half of fibrillin-1 assembles into disulfidebonded multimeric globular structures with peripheral arms and a dense core. These globules are similar to the beaded structures observed in microfibrils isolated from tissues. Only these C-terminal fibrillin-1 multimers interacted strongly with the fibrillin-1 N terminus, whereas the monomers showed very little self-interaction activity. The multimers strongly inhibited microfibril formation in cell culture, providing evidence that these recombinant assemblies can also interact with endogenous fibrillin-1. The Cterminal self-interaction site was fine-mapped to the last three calcium-binding EGF domains in fibrillin-1. These results suggest a new mechanism for microfibril formation where fibrillin-1 first oligomerizes via its C terminus before the partially or fully assembled bead-like structures can further interact with other beads via the fibrillin-1 N termini. extracellular matrix ͉ fibrillinopathies ͉ Marfan syndrome ͉ protein assembly

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Bone development and formation during embryogenesis as well as postnatally during bone remodeling is a complex process controlled systemically and locally by hormones, growth factors and matrix molecules. Transglutaminases (TGases) are the protein cross-linking enzymes, which have long been implicated in bone development and formation. Two members of TGase family, TG2 (also called tissue transglutaminase) and FXIIIA (the enzymatic A subunit of coagulation factor XIII), are expressed in chondrocytes and osteoblasts. The results of analyses in vivo and in vitro accumulated to date indicate an important role of these enzymes in promoting chondrocyte and osteoblast differentiation and matrix mineralization. These effects could be mediated by protein cross-linking activity of TGases, by GTPase activity of TG2 or via non-catalytic signaling effects. The aim of this review is to summarize the available data regarding the expression, localization and activity of TG2 and FXIIIA in mineralizing tissues and to discuss a number of mechanisms by which TGases could exert their promineralizing effects.

Bone wound healing after surgical drilling/cutting initially involves a typical inflammatory response with a leukocyte-rich cell infiltrate whose professional phagocytes (neutrophils and macrophages) clear the wound site of various bacterial (if present), particulate, and insoluble components arising from the original wounding event. As part of this process, in a surgical model of bone repair in rats, osteopontin (OPN) secreted by macrophages – with its known mineral-binding properties arising from abundant calcium-binding phosphorylations and overall net negative charge – binds to the newly exposed mineralized surfaces of particulate bone debris and the osseous wound margins created by the drilling, as shown by high-resolution immunogold labeling and transmission electron microscopy. For bone debris powder, OPN serves as an opsonin for clearance by macrophage phagocytosis, as demonstrated in vitro by phagocytosis assays using cultured J774.A1 murine macrophages and OPN-coated microbeads. Macrophage-secreted OPN binding to the bone wound margins contributes to cement line (plane) formation with subsequent OPN additions to the cement line coming from osteoblast lineage cells arriving at this site to effect bone repair upon further osteoblast differentiation, and extracellular matrix deposition and mineralization. Such interfacial OPN is thought to contribute to the cell adhesion, cell signaling, and matrix mineralization events required to effectively integrate the new bone into the preexisting bone at the margins of the drill site.