Transglutaminase activity arising from Factor XIIIA is required for stabilization and conversion of plasma fibronectin into matrix in osteoblast cultures.
Circulating plasma fibronectin (pFN), produced by hepatocytes, is a major component of the noncollagenous bone matrix where it was recently shown in vivo in mice to control the biomechanical quality as well as the mineral-to-matrix ratio in bone. FN fibrillogenesis is a process generally requiring FN binding to cellular integrins, and cellular tension to elongate and assemble the molecule. Whether soluble pFN undergoes cell-mediated assembly in bone is not fully established. FN is a well-known substrate for transglutaminases (TGs), which are protein-crosslinking enzymes capable of stabilizing macromolecular structures. The role of this modification regarding the function of FN in bone matrix has remained unknown. Osteoblasts express two TGs-transglutaminase 2 and Factor XIIIA-and we have shown that Factor XIIIA is the main TG active during osteoblast differentiation. In the present study, conducted using MC3T3-E1 osteoblast cultures and bone marrow stromal cells, we demonstrate that pFN requires a TG-mediated crosslinking step to form osteoblast matrix in vitro. This modification step is specific for pFN; cellular FN (EDA-FN) does not serve as a TG substrate. Inhibition of pFN assembly using a TG inhibitor, or depletion of pFN from cell culture serum, dramatically decreased total FN matrix assembly in the osteoblast cultures and affected both the quantity and quality of the type I collagen matrix, and decreased lysyl oxidase and alkaline phosphatase levels, resulting in decreased mineralization. Experiments with isozyme-specific substrate peptides showed that FXIIIA is responsible for the crosslinking of pFN. Addition of exogenous preactivated FXIIIA to osteoblast cultures promoted pFN assembly from the media into matrix. Exogenous TG2 had no effect. Analysis of pFN and EDA-FN fibrils by immunofluorescence microscopy demonstrated that they form distinct matrix network, albeit with minor overlap, suggesting different functions for the two FN forms. Further analysis using EDA-FN blocking antibody showed that it regulated preosteoblast proliferation whereas pFN depletion from the serum had no effect on this process. In conclusion, our study shows that pFN assembly into bone matrix in vitro requires FXIIIA transglutaminase activity making pFN assembly an active, osteoblast-mediated process.
Division of Biomedical Sciences, Faculty of Dentistry, McGill University, Montreal, QC, Canada.
Cui
C
Cui
Division of Biomedical Sciences, Faculty of Dentistry, McGill University, Montreal, QC, Canada.
Shuai
S
Wang
Division of Biomedical Sciences, Faculty of Dentistry, McGill University, Montreal, QC, Canada.
Vamsee D
VD
Myneni
Department of Applied Molecular Biosciences, Graduate School of Pharmaceutical Sciences, Nagoya University, Chikusa, Nagoya, Japan.
Kiyotaka
K
Hitomi
Division of Biomedical Sciences, Faculty of Dentistry, McGill University, Montreal, QC, Canada; Division of Experimental Medicine, Department of Medicine, Faculty of Medicine, McGill University, Montreal, QC, Canada. Electronic address: mari.kaartinen@mcgill.ca.
Mari T
MT
Kaartinen
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002454:Cell Differentiation; D002460:Cell Line; D024042:Collagen Type I / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism; D026122:Factor XIIIa / Q000378:metabolism*; D005353:Fibronectins / Q000097:blood*; D019204:GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D008954:Models, Biological; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D012995:Solubility; D013379:Substrate Specificity; D011503:Transglutaminases / Q000037:antagonists & inhibitors / Q000378:metabolism*
D024042:Collagen Type I; D003432:Cross-Linking Reagents; D005353:Fibronectins; D026122:Factor XIIIa; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D000469:Alkaline Phosphatase; D019204:GTP-Binding Proteins
Collagen type I; Extracellular matrix; Factor XIIIA; Osteoblasts; Plasma fibronectin; Transglutaminase