Plasma Membrane Factor XIIIA Transglutaminase Activity Regulates Osteoblast Matrix Secretion and Deposition by Affecting Microtubule Dynamics

Transglutaminase activity, arising potentially from transglutaminase 2 (TG2) and Factor XIIIA (FXIIIA), has been linked to osteoblast differentiation where it is required for type I collagen and fibronectin matrix deposition. In this study we have used an irreversible TG-inhibitor to ‘block –and-track’ enzyme(s) targeted during osteoblast differentiation. We show that the irreversible TG-inhibitor is highly potent in inhibiting osteoblast differentiation and mineralization and reduces secretion of both fibronectin and type I collagen and their release from the cell surface. Tracking of the dansyl probe by Western blotting and immunofluorescence microscopy demonstrated that the inhibitor targets plasma membrane-associated FXIIIA. TG2 appears not to contribute to crosslinking activity on the osteoblast surface. Inhibition of FXIIIA with NC9 resulted in defective secretory vesicle delivery to the plasma membrane which was attributable to a disorganized microtubule network and decreased microtubule association with the plasma membrane. NC9 inhibition of FXIIIA resulted in destabilization of microtubules as assessed by cellular Glu-tubulin levels. Furthermore, NC9 blocked modification of Glu-tubulin into 150 kDa high-molecular weight Glu-tubulin form which was specifically localized to the plasma membrane. FXIIIA enzyme and its crosslinking activity were colocalized with plasma membrane-associated tubulin, and thus, it appears that FXIIIA crosslinking activity is directed towards stabilizing the interaction of microtubules with the plasma membrane. Our work provides the first mechanistic cues as to how transglutaminase activity could affect protein secretion and matrix deposition in osteoblasts and suggests a novel function for plasma membrane FXIIIA in microtubule dynamics

Role of transglutaminase enzymes in osteoblast differentiation and matrix deposition

Bone formation is an osteoblast-mediated process that is controlled by systemic factors such as hormones, growth factors and local cues that arise from the extracellular matrix (ECM). Bone ECM is elaborated by osteoblasts and therefore they can control their own activity. The ultimate goal of bone matrix formation is to elaborate an extracellular network, consisting mainly of fibronectin and collagen type I, that is capable of mineralizing and forming a strong tissue with appropriate tensile and elastic properties. This thesis describes studies that link transglutaminases (TGs), the protein cross-linking enzymes to type I collagen matrix deposition, osteoblast differentiation and bone formation. Findings here show that MC3T3-E1 osteoblasts require TG-activity for differentiation and proper production of collagenous matrices. We also show that osteoblasts produce two transglutaminase enzymes, transglutaminase 2 (TG2) and Factor XIIIA (FXIIIA), which are both expressed during osteoblast differentiation. The work further defines the roles of the two TGs in osteoblasts and shows that FXIIIA is the main TG-enzyme with transamidating activity in osteoblasts' ECM. Production of FXIIIA is induced during osteoblast differentiation and is externalized to the cell surface, then secreted to the ECM. TG2 was mainly found on the cell surface of osteoblasts with no transamidating activity; however, it is co-localized with FXIIIA on the cell surface. Studies conducted with chemical inhibitors, TG-substrates and activity probes suggest that TG-activity is required for osteoblast differentiation at three different levels. First, by positively affecting microtubule dynamics, delivery and fusion of secretory vesicles carrying cellular collagen type I to the plasma membrane. Second, by promoting fibronectin matrix deposition and collagen type I secretion. And third, by stabilizing the interaction between fibronectin and collagen type I in the ECM. Furthermore, we demonstrated that tubulin and fibronectin are candidate substrates for FXIIIA in osteoblasts. In summary, our studies are the first to describe FXIIIA transglutaminase expression in osteoblasts in vitro and in vivo, and first to link it to collagen secretion and osteoblast differentiation. Furthermore, these studies were the first to suggest a role for cellular FXIIIA in microtubule dynamics. We conclude that transglutaminase activity arising from FXIIIA can regulate osteoblast differentiation affecting extracellular matrix deposition.La formation et le développement de l'os est un processus complexe dirigé par les ostéoblastes. Contrôlés par des hormones systémiques, des cytokines et d'autres facteurs locaux, les ostéoblastes sécrètent et assemblent la matrice extracellulaire (MEC) des tissus osseux. L'aboutissement de ce processus sera la génération d'un réseau extracellulaire constitué notamment de la fibronectine et du collagene de type I qui va se minéraliser en formant le tissu dur de l'os avec d'excellent propriétés mécaniques. Cette thèse présente des études liées aux transglutaminases (TGs) – une classe des enzymes responsables de la polymérisation (cross-linking) des protéines et d'autres composée biomacromoléculaires - en relation avec le collagène de type I secrété pendant l'élaboration de la MEC, la différentiation des ostéoblastes et l'élaboration du tissu osseux. Les principaux résultats de ces études portent sur l'observation que l'activité polymérisante de la TG est un facteur crucial pour la différentiation des cellules ostéoblastiques MC3T3-E1 et pour la production normale de la matrice collagénique. Un résultat essentiel de la présente recherche porte sur la découverte que les ostéoblastes synthétisent deux types d'enzymes TG, i.e. la transglutaminase 2 (TG2) et le facteur XIIIA (FXIIIA), qui sont tous les deux secrétés pendant la différentiation des ostéoblastes. Les résultats suivants éclaircirent les rôles des deux enzymes TG (TG2 et FXIIIA) dans l'activité des ostéoblastes en montrant que c'est le FXIIIA qui est l'enzyme TG dominante avec une activité de transamidation importante dans la MEC des ostéoblastes. FXIIIA est produit pendant la différentiation des ostéoblastes en s'externalisant vers la surface des cellules pour être par la suite sécrété dans la MEC. L'enzyme TG2 a été localisé seulement à la surface des cellules osteoblastiques. Même si le TG2 a été trouvé colocalisé avec le FXIIIA à la surface des cellules, aucune activité de transamidation n'est identifiée pour le TG2. Des études comportant des inhibiteurs chimiques, de substrats TG et de sondes d'activité TG suggèrent que l'activité TG est nécessaire pour la différentiation des ostéoblastes sur trois plans distincts, à savoir : (i) par une action bénéfique sur la dynamique des microtubules, l'acheminement et la fusion des vésicules sécrétoires qui transportent le collagène I cellulaire vers la membrane plasmatique; (ii) par l'accélération du dépôt de la matrice de fibronectine et la sécrétion du collagène de type I; (iii) par la stabilisation de l'interaction de la fibronectine avec le collagène I dans la MEC. De plus, nous avons démontré que la tubuline and la fibronectine ce sont de candidats substrat pour le facteur FXIIIA dans les ostéoblastes. En résumé, notre recherche décrit pour la première fois l'expression de l'enzyme transglutaminase FXIIIA dans les ostéoblastes, tant in vitro qu'in vivo, en corrélant l'expression du FXIIIA à la sécrétion et la différentiation des ostéoblastes. De plus, notre étude est la première en attribuant un rôle au facteur FXIIIA relative à la dynamique des microtubules. On conclut de notre étude que l'activité transglutaminase du facteur FXIIIA exerce une influence décisive dans les processus de différentiation des ostéoblastes avec un effet régulateur crucial à la sécrétion et au dépôt de la matrice extracellulaire

Comprehensive Review of Adipose Stem Cells and Their Implication in Distraction Osteogenesis and Bone Regeneration

Bone is one of the most dynamic tissues in the human body that can heal following injury without leaving a scar. However, in instances of extensive bone loss, this intrinsic capacity of bone to heal may not be sufficient and external intervention becomes necessary. Several techniques are available to address this problem, including autogenous bone grafts and allografts. However, all these techniques have their own limitations. An alternative method is the technique of distraction osteogenesis, where gradual and controlled distraction of two bony segments after osteotomy leads to induction of new bone formation. Although distraction osteogenesis usually gives satisfactory results, its major limitation is the prolonged duration of time required before the external fixator is removed, which may lead to numerous complications. Numerous methods to accelerate bone formation in the context of distraction osteogenesis have been reported. A viable alternative to autogenous bone grafts for a source of osteogenic cells is mesenchymal stem cells from bone marrow. However, there are certain problems with bone marrow aspirate. Hence, scientists have investigated other sources for mesenchymal stem cells, specifically adipose tissue, which has been shown to be an excellent source of mesenchymal stem cells. In this paper, the potential use of adipose stem cells to stimulate bone formation is discussed

Recent advances in bone regeneration: The role of adipose tissue-derived stromal vascular fraction and mesenchymal stem cells

The management of large bone defects, atrophic nonunions, and other conditions with poor bone formation presents a formidable challenge to the treating physician, as all available techniques of bone reconstruction have drawbacks. Recent advances in stem cell biology, specifically adipose tissue-derived mesenchymal stem cells (ASCs) and adipose tissue stromal vascular fraction (SVF), have opened up new horizons by providing a reliable and abundant source of stem cells with osteogenic potential that can be used in various bone tissue engineering techniques. In this review, several aspects related to the use of ASCs are addressed, such as harvesting and processing of adipose tissue, advantages of ASCs over bone marrow-derived mesenchymal stem cells, mechanism of action and safety of ASCs, and factors affecting the differentiation of ASCs. Published reports on the use of ASCs in critical size defects, nonunions, and distraction osteogenesis are also reviewed. Innovative trends in stem cell research on musculoskeletal pathologies are highlighted, with special emphasis on the increasing evidence that the direct application of freshly prepared SVF processed from adipose tissue into the bone defect to be treated without a prior differentiation or an ex vivo expansion and culture is possible. This highly promising approach may lead to the development of a one-step intraoperative cell therapy