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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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Related in: MedlinePlus

Scanning Electron microscopy analysis of the rTRIP-1 coated dentin wafer subjected to in vitro nucleation and the corresponding EDS analysis.(7a and 7c) depict the representative SEM image of 100 μg rTRIP-1 coated demineralized and deproteinized dentin wafer subjected to in-vitro nucleation for 7 days (7a) and 14 days (7c). (7b and 7d) represent the corresponding EDS analysis. (7e) Representative SEM image of 100 μg BSA coated on demineralized and deproteinized dentin wafer subjected to in-vitro nucleation for 14 days under physiological conditions. (7f) EDS spectra of BSA coated dentin wafers. (7g and 7h) Representative SEM images of native dentin. (7h) is the higher magnification of boxed area in (7g). Black arrows show mineral deposits and black arrowheads points to interfibrillar space.
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f7: Scanning Electron microscopy analysis of the rTRIP-1 coated dentin wafer subjected to in vitro nucleation and the corresponding EDS analysis.(7a and 7c) depict the representative SEM image of 100 μg rTRIP-1 coated demineralized and deproteinized dentin wafer subjected to in-vitro nucleation for 7 days (7a) and 14 days (7c). (7b and 7d) represent the corresponding EDS analysis. (7e) Representative SEM image of 100 μg BSA coated on demineralized and deproteinized dentin wafer subjected to in-vitro nucleation for 14 days under physiological conditions. (7f) EDS spectra of BSA coated dentin wafers. (7g and 7h) Representative SEM images of native dentin. (7h) is the higher magnification of boxed area in (7g). Black arrows show mineral deposits and black arrowheads points to interfibrillar space.

Mentions: To investigate the role of rTRIP-1 in biomineralization, we examined if rTRIP1 had the ability to nucleate calcium phosphate on the collagenous matrix of demineralized and deproteinized dentin wafer. SEM results showed that indeed rTRIP-1 could nucleate calcium phosphate polymorphs at 7 and 14 days respectively (Fig. 7a,c). EDX analysis of the mineral deposits showed the presence of calcium phosphate deposits and the Ca/P ratio was determined to be 1.75 and 1.85 at 7 & 14 days respectively (Fig. 7b,d). BSA coated dentin wafer also shows the presence of mineral crystals (Fig. 7e). EDX analysis (Fig. 7f) detected the presence of phosphate and calcium albeit in lesser amounts. SEM of native dentin wafer showing the mineral surface is shown in Fig. 7g,h.


TGF beta receptor II interacting protein-1, an intracellular protein has an extracellular role as a modulator of matrix mineralization
Scanning Electron microscopy analysis of the rTRIP-1 coated dentin wafer subjected to in vitro nucleation and the corresponding EDS analysis.(7a and 7c) depict the representative SEM image of 100 μg rTRIP-1 coated demineralized and deproteinized dentin wafer subjected to in-vitro nucleation for 7 days (7a) and 14 days (7c). (7b and 7d) represent the corresponding EDS analysis. (7e) Representative SEM image of 100 μg BSA coated on demineralized and deproteinized dentin wafer subjected to in-vitro nucleation for 14 days under physiological conditions. (7f) EDS spectra of BSA coated dentin wafers. (7g and 7h) Representative SEM images of native dentin. (7h) is the higher magnification of boxed area in (7g). Black arrows show mineral deposits and black arrowheads points to interfibrillar space.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f7: Scanning Electron microscopy analysis of the rTRIP-1 coated dentin wafer subjected to in vitro nucleation and the corresponding EDS analysis.(7a and 7c) depict the representative SEM image of 100 μg rTRIP-1 coated demineralized and deproteinized dentin wafer subjected to in-vitro nucleation for 7 days (7a) and 14 days (7c). (7b and 7d) represent the corresponding EDS analysis. (7e) Representative SEM image of 100 μg BSA coated on demineralized and deproteinized dentin wafer subjected to in-vitro nucleation for 14 days under physiological conditions. (7f) EDS spectra of BSA coated dentin wafers. (7g and 7h) Representative SEM images of native dentin. (7h) is the higher magnification of boxed area in (7g). Black arrows show mineral deposits and black arrowheads points to interfibrillar space.
Mentions: To investigate the role of rTRIP-1 in biomineralization, we examined if rTRIP1 had the ability to nucleate calcium phosphate on the collagenous matrix of demineralized and deproteinized dentin wafer. SEM results showed that indeed rTRIP-1 could nucleate calcium phosphate polymorphs at 7 and 14 days respectively (Fig. 7a,c). EDX analysis of the mineral deposits showed the presence of calcium phosphate deposits and the Ca/P ratio was determined to be 1.75 and 1.85 at 7 & 14 days respectively (Fig. 7b,d). BSA coated dentin wafer also shows the presence of mineral crystals (Fig. 7e). EDX analysis (Fig. 7f) detected the presence of phosphate and calcium albeit in lesser amounts. SEM of native dentin wafer showing the mineral surface is shown in Fig. 7g,h.

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