Mari T. Kaartinen

Pyrophosphate Inhibits Mineralization of Osteoblast Cultures by Binding to Mineral, Up-regulating Osteopontin, and Inhibiting Alkaline Phosphatase Activity

Inorganic pyrophosphate (PPi) produced by cells inhibits mineralization by binding to crystals. Its ubiquitous presence is thought to prevent “soft” tissues from mineralizing, whereas its degradation to Pi in bones and teeth by tissue-nonspecific alkaline phosphatase (Tnap, Tnsalp, Alpl, Akp2) may facilitate crystal growth. Whereas the crystal binding properties of PPi are largely understood, less is known about its effects on osteoblast activity. We have used MC3T3-E1 osteoblast cultures to investigate the effect of PPi on osteoblast function and matrix mineralization. Mineralization in the cultures was dose-dependently inhibited by PPi. This inhibition could be reversed by Tnap, but not if PPi was bound to mineral. PPi also led to increased levels of osteopontin (Opn) induced via the Erk1/2 and p38 MAPK signaling pathways. Opn regulation by PPi 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 PPi, hydrolysis by Tnap reduces its mineral inhibiting potency. Using enzyme kinetic studies, we have shown that PPi inhibits Tnap-mediated Pi release from β-glycerophosphate (a commonly used source of organic phosphate for culture mineralization studies) through a mixed type of inhibition. In summary, PPi 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.

Transglutaminase Regulation of Cell Function

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.

Cross-linking of Osteopontin by Tissue Transglutaminase Increases Its Collagen Binding Properties

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 and Its Substrates in Bone

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.

Cartilage Formation and Calcification in Arteries of Mice Lacking Matrix Gla Protein

Matrix Gla protein (MGP/Mgp) is a protein expressed predominantly by vascular smooth muscle cells (VSMCs) and by chondrocytes. Transgenic mice lacking Mgp die 1-3 months after birth due to calcification of elastic fibers and rupture of large elastic arteries such as the aorta [6]. Here, we report on cartilage formation that commonly occurs in calcified arteries of Mgp m / m mice. Using histology, von Kossa staining, immunohistochemistry, and Western blotting, together with examination of cellular markers for VSMCs and extracellular matrix markers for cartilage, we provide evidence for cell transformation from VSMC to chondrocyte in the arterial media in the absence of Mgp. At 2 weeks of age in the aorta of Mgp m / m mice, VSMCs lose immunostaining for smooth muscle f -actin concomitant with the appearance of cartilage molecules as shown by immunohistochemical staining and Western blotting for aggrecan, link protein, and type II collagen. These data provide evidence that the absence of Mgp, and/or calcification of the ECM, in the arterial media can trigger chondrocyte differentiation and cartilage formation in blood vessels.

Transglutaminase-catalyzed Cross-linking of Osteopontin Is Inhibited by Osteocalcin

Osteocalcin, the most abundant noncollagenous protein of bone matrix, has been demonstrated to inhibit bone growth by gene knockout experiments (Ducy, P., Desbois, C., Boyce, B., Pinero, G., Story, B., Dunstan, C., Smith, E., Bonadio, J., Goldstein, S., Gundberg, C., Bradley, A., and Karsenty, G. (1996)Nature 382, 448–452). Its specific functional mechanism in bone metabolism is, however, largely unknown. In this study, we provide evidence that osteocalcin has an inhibitory effect on tissue transglutaminase activity, as measured by cross-linking of osteopontin, another bone matrix protein. Using a set of synthetic peptides, we found that the inhibitory activity resided within the first 13 N-terminal amino acid residues of osteocalcin. An N-terminal peptide also inhibited cross-linking of another tissue transglutaminase substrate, β-casein. The inhibitory peptide was shown to have affinity for the substrates of transglutaminase rather than for the enzyme. Since the N terminus of osteocalcin exhibits homology to the substrate recognition site sequences of two transglutaminases, we conclude that the inhibitory effect is most likely due to competition with the enzyme for the transglutaminase-binding region of the substrates, osteopontin and β-casein, which prevents access of the enzyme to them to perform its function. The interference of osteocalcin with osteopontin cross-linking gives osteocalcin a new potential function as the first protein inhibitor of tissue transglutaminase. This suggests a specific role and a plausible mechanism for it as a modulator of maturation, stabilization, and calcification of bone matrix.

Transglutaminases in mineralized tissues.

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.

Hierarchies of Extracellular Matrix and Mineral Organization in Bone of the Craniofacial Complex and Skeleton

Structural hierarchies are common in biologic systems and are particularly evident in biomineralized structures. In the craniofacial complex and skeleton of vertebrates, extracellular matrix and mineral of bone are structurally ordered at many dimensional scales from the macro level to the nano level. Indeed, the nanocomposite texture of bone, with nanocrystals of apatitic mineral embedded within a crosslinked matrix of fibrillar and nonfibrillar proteins, imparts to bone the very mechanical properties and toughness it needs to function in vital organ protection, musculoskeletal movement and mastication. This article focuses on how hierarchies of extracellular matrix protein organization influence bone cell behavior, tissue architecture and mineralization. Additional attention is given to recent work on the molecular determinants of mineral induction in bone, and how the mineralization process is subsequently regulated by inhibitory proteins.

Biogenesis of extracellular microfibrils: Multimerization of the fibrillin-1 C terminus into bead-like structures enables self-assembly

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 disulfide-bonded 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 C-terminal 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.

Osteopontin Upregulation and Polymerization by Transglutaminase 2 in Calcified Arteries of Matrix Gla Protein-deficient Mice:

Matrix Gla protein (MGP) is a potent inhibitor of soft tissue calcification, and Mgp gene deletion in mice results in arterial calcification. Our aim was to examine osteopontin (OPN) expression and localization, and posttranslational processing of OPN by the crosslinking enzyme transglutaminase 2 (TG2), in the calcified aorta of Mgp-deficient (Mgp−/−) mice. Using immunohistochemistry and light and electron microscopy, we report that following mineralization occurring in the arterial media of Mgp−/− aortas, OPN is upregulated and accumulates at the surface of the calcified elastic lamellae. Macrophages were observed in direct contact with this OPN-rich layer. Western blot analysis of extracted Mgp−/− aortas revealed that the majority of the OPN was in high molecular mass protein complexes, indicating modification by a crosslinking enzyme. Consistent with this observation, TG2 expression and γ-glutamyl-∊-lysyl crosslink levels were also increased in Mgp−/− aortas. In addition to the mineral-inhibiting actions of OPN, and based on data linking OPN and TG2 with cell adhesion in various cell types including monocytes and macrophages, we propose that TG2 interactions with OPN lead to protein polymerization that facilitates macrophage adhesion to the calcified elastic lamellae to promote clearance of the ectopic mineral deposits.