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TGF beta receptor II interacting protein-1, an intracellular protein has an extracellular role as a modulator of matrix mineralization

View Article: PubMed Central - PubMed

ABSTRACT

Transforming growth factor beta receptor II interacting protein 1 (TRIP-1), a predominantly intracellular protein is localized in the ECM of bone. TRIP-1 lacks a signal peptide, therefore, in this study, we provide evidence that intracellular TRIP-1 can be packaged and exported to the ECM via exosomes. Overexpression of TRIP-1 in MC3T3-E1 cells resulted in increased matrix mineralization during differentiation and knockdown resulted in reduced effects. In vivo function of TRIP-1 was studied by an implantation assay performed using TRIP-1 overexpressing and knockdown cells cultured in a 3-dimmensional scaffold. After 4 weeks, the subcutaneous tissues from TRIP-1 overexpressing cells showed higher calcium and phosphate deposits, arranged collagen fibrils and increased expression of Runx2 and alkaline phosphatase. Nucleation studies on demineralized and deproteinized dentin wafer is a powerful tool to determine the functional role of noncollagenous proteins in matrix mineralization. Using this system, we provide evidence that TRIP-1 binds to Type-I collagen and can promote mineralization. Surface plasmon resonance analysis demonstrated that TRIP-1 binds to collagen with KD = 48 μM. SEM and TEM analysis showed that TRIP-1 promoted the nucleation and growth of calcium phosphate mineral aggregates. Taken together, we provide mechanistic insights of this intracellular protein in matrix mineralization.

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Transmission Electron microscopy analysis and corresponding selective area electron diffraction pattern(SAED) of rTRIP-1 nucleated mineral deposits.(8a) Representative unstained TEM image of mineral crystal nucleated by 20 μg of TRIP-1 on nickel grids subjected to mineralization for 1 h in the presence of 1 M Ca2+ and phosphate buffer. (8b) is the higher magnification of the boxed area in 8a. Lattice image shows the presence of nanocrystalline arrays (black arrows) (8c) Digitally magnified images of 8b showing lattice fringes. (8d) Corresponding SAED image showing oriented crystals with strong reflections in the (002), (004) and (211) planes of hydroxyapatite. (8e) Representative TEM image of control protein (20 μg BSA) and corresponding SAED image (8f).
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f8: Transmission Electron microscopy analysis and corresponding selective area electron diffraction pattern(SAED) of rTRIP-1 nucleated mineral deposits.(8a) Representative unstained TEM image of mineral crystal nucleated by 20 μg of TRIP-1 on nickel grids subjected to mineralization for 1 h in the presence of 1 M Ca2+ and phosphate buffer. (8b) is the higher magnification of the boxed area in 8a. Lattice image shows the presence of nanocrystalline arrays (black arrows) (8c) Digitally magnified images of 8b showing lattice fringes. (8d) Corresponding SAED image showing oriented crystals with strong reflections in the (002), (004) and (211) planes of hydroxyapatite. (8e) Representative TEM image of control protein (20 μg BSA) and corresponding SAED image (8f).

Mentions: Transmission electron microscopy analysis of mineral nucleation initiated directly on EM grids showed that the mineral deposits on the rTRIP-1 coated surface was hydroxyapatite (Fig. 8a,b), based on the characteristic selected-area electron-diffraction (SAED) patterns with distinct (002), (004) and (211) reflections (Fig. 8d). The lattice fringes showed that the deposited mineral particles possessed long range crystallographic order (Fig. 8b,c). Figure 8e,f depicts the TEM image of BSA coated grid which was used as a control and its corresponding diffused diffraction pattern.


TGF beta receptor II interacting protein-1, an intracellular protein has an extracellular role as a modulator of matrix mineralization
Transmission Electron microscopy analysis and corresponding selective area electron diffraction pattern(SAED) of rTRIP-1 nucleated mineral deposits.(8a) Representative unstained TEM image of mineral crystal nucleated by 20 μg of TRIP-1 on nickel grids subjected to mineralization for 1 h in the presence of 1 M Ca2+ and phosphate buffer. (8b) is the higher magnification of the boxed area in 8a. Lattice image shows the presence of nanocrystalline arrays (black arrows) (8c) Digitally magnified images of 8b showing lattice fringes. (8d) Corresponding SAED image showing oriented crystals with strong reflections in the (002), (004) and (211) planes of hydroxyapatite. (8e) Representative TEM image of control protein (20 μg BSA) and corresponding SAED image (8f).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5121659&req=5

f8: Transmission Electron microscopy analysis and corresponding selective area electron diffraction pattern(SAED) of rTRIP-1 nucleated mineral deposits.(8a) Representative unstained TEM image of mineral crystal nucleated by 20 μg of TRIP-1 on nickel grids subjected to mineralization for 1 h in the presence of 1 M Ca2+ and phosphate buffer. (8b) is the higher magnification of the boxed area in 8a. Lattice image shows the presence of nanocrystalline arrays (black arrows) (8c) Digitally magnified images of 8b showing lattice fringes. (8d) Corresponding SAED image showing oriented crystals with strong reflections in the (002), (004) and (211) planes of hydroxyapatite. (8e) Representative TEM image of control protein (20 μg BSA) and corresponding SAED image (8f).
Mentions: Transmission electron microscopy analysis of mineral nucleation initiated directly on EM grids showed that the mineral deposits on the rTRIP-1 coated surface was hydroxyapatite (Fig. 8a,b), based on the characteristic selected-area electron-diffraction (SAED) patterns with distinct (002), (004) and (211) reflections (Fig. 8d). The lattice fringes showed that the deposited mineral particles possessed long range crystallographic order (Fig. 8b,c). Figure 8e,f depicts the TEM image of BSA coated grid which was used as a control and its corresponding diffused diffraction pattern.

View Article: PubMed Central - PubMed

ABSTRACT

Transforming growth factor beta receptor II interacting protein 1 (TRIP-1), a predominantly intracellular protein is localized in the ECM of bone. TRIP-1 lacks a signal peptide, therefore, in this study, we provide evidence that intracellular TRIP-1 can be packaged and exported to the ECM via exosomes. Overexpression of TRIP-1 in MC3T3-E1 cells resulted in increased matrix mineralization during differentiation and knockdown resulted in reduced effects. In vivo function of TRIP-1 was studied by an implantation assay performed using TRIP-1 overexpressing and knockdown cells cultured in a 3-dimmensional scaffold. After 4 weeks, the subcutaneous tissues from TRIP-1 overexpressing cells showed higher calcium and phosphate deposits, arranged collagen fibrils and increased expression of Runx2 and alkaline phosphatase. Nucleation studies on demineralized and deproteinized dentin wafer is a powerful tool to determine the functional role of noncollagenous proteins in matrix mineralization. Using this system, we provide evidence that TRIP-1 binds to Type-I collagen and can promote mineralization. Surface plasmon resonance analysis demonstrated that TRIP-1 binds to collagen with KD = 48 μM. SEM and TEM analysis showed that TRIP-1 promoted the nucleation and growth of calcium phosphate mineral aggregates. Taken together, we provide mechanistic insights of this intracellular protein in matrix mineralization.

No MeSH data available.


Related in: MedlinePlus