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

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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.


Overexpression and Silencing of TRIP-1 protein in MC3T3-E1 cells.CFP-TRIP-1 plasmid was used for establishing a stable cell line overexpressing TRIP-1. (3a) Light microscopy images of Control (a1) and TRIP-1 overexpressing cells (a2). Note the change in the cellular morphology (black arrows). (3a3 and 3a4) Confocal microscopy images of control and TRIP-1 overexpressing cells. Note the accumulation of TRIP-1 on the cell membrane as shown by yellow arrows (a4). Western blot analysis of total cell lysate (3b) and secretome (3d) from control, TRIP-1 overexpressed and TRIP-1 silenced cells shows the expression of TRIP-1. (3c) Quantitative analysis of TRIP-1 protein expression showing 2.5-fold increase in overexpressed cells and 70% reduction in expression in TRIP-1 silenced cells. Student’s t-test used to obtain statistical significance with respect to control. *, # Represents significance of p < 0.05.
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f3: Overexpression and Silencing of TRIP-1 protein in MC3T3-E1 cells.CFP-TRIP-1 plasmid was used for establishing a stable cell line overexpressing TRIP-1. (3a) Light microscopy images of Control (a1) and TRIP-1 overexpressing cells (a2). Note the change in the cellular morphology (black arrows). (3a3 and 3a4) Confocal microscopy images of control and TRIP-1 overexpressing cells. Note the accumulation of TRIP-1 on the cell membrane as shown by yellow arrows (a4). Western blot analysis of total cell lysate (3b) and secretome (3d) from control, TRIP-1 overexpressed and TRIP-1 silenced cells shows the expression of TRIP-1. (3c) Quantitative analysis of TRIP-1 protein expression showing 2.5-fold increase in overexpressed cells and 70% reduction in expression in TRIP-1 silenced cells. Student’s t-test used to obtain statistical significance with respect to control. *, # Represents significance of p < 0.05.

Mentions: To understand its function in osteoblasts, we overexpressed TRIP-1 in MC3T3-E1 cells and observed their morphology. Light microscopy analysis showed that with overexpression the cells changed their morphology from cobble-stone-like appearance (Fig. 3a–a1) to spindle-shaped cells, which were elongated, polarized and aligned themselves in straight parallel lines (Fig. 3a–a2). Confocal image of the cells showed accumulation of TRIP-1 on the plasma membrane (Fig. 3a–a4) when compared with the control (Fig. 3a–a3). Western blot analysis in Fig. 3b confirmed the overexpression and knocked down TRIP-1 protein levels in the total cell lysate and quantitative analysis showed a 2.5-fold increase and 70% reduction in TRIP-1 expression in overexpressed and shRNA treated cells respectively (Fig. 3c). Similar TRIP-1 expression pattern was observed in the secretome obtained from these cells. (Fig. 3d).


TGF beta receptor II interacting protein-1, an intracellular protein has an extracellular role as a modulator of matrix mineralization
Overexpression and Silencing of TRIP-1 protein in MC3T3-E1 cells.CFP-TRIP-1 plasmid was used for establishing a stable cell line overexpressing TRIP-1. (3a) Light microscopy images of Control (a1) and TRIP-1 overexpressing cells (a2). Note the change in the cellular morphology (black arrows). (3a3 and 3a4) Confocal microscopy images of control and TRIP-1 overexpressing cells. Note the accumulation of TRIP-1 on the cell membrane as shown by yellow arrows (a4). Western blot analysis of total cell lysate (3b) and secretome (3d) from control, TRIP-1 overexpressed and TRIP-1 silenced cells shows the expression of TRIP-1. (3c) Quantitative analysis of TRIP-1 protein expression showing 2.5-fold increase in overexpressed cells and 70% reduction in expression in TRIP-1 silenced cells. Student’s t-test used to obtain statistical significance with respect to control. *, # Represents significance of p < 0.05.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5121659&req=5

f3: Overexpression and Silencing of TRIP-1 protein in MC3T3-E1 cells.CFP-TRIP-1 plasmid was used for establishing a stable cell line overexpressing TRIP-1. (3a) Light microscopy images of Control (a1) and TRIP-1 overexpressing cells (a2). Note the change in the cellular morphology (black arrows). (3a3 and 3a4) Confocal microscopy images of control and TRIP-1 overexpressing cells. Note the accumulation of TRIP-1 on the cell membrane as shown by yellow arrows (a4). Western blot analysis of total cell lysate (3b) and secretome (3d) from control, TRIP-1 overexpressed and TRIP-1 silenced cells shows the expression of TRIP-1. (3c) Quantitative analysis of TRIP-1 protein expression showing 2.5-fold increase in overexpressed cells and 70% reduction in expression in TRIP-1 silenced cells. Student’s t-test used to obtain statistical significance with respect to control. *, # Represents significance of p < 0.05.
Mentions: To understand its function in osteoblasts, we overexpressed TRIP-1 in MC3T3-E1 cells and observed their morphology. Light microscopy analysis showed that with overexpression the cells changed their morphology from cobble-stone-like appearance (Fig. 3a–a1) to spindle-shaped cells, which were elongated, polarized and aligned themselves in straight parallel lines (Fig. 3a–a2). Confocal image of the cells showed accumulation of TRIP-1 on the plasma membrane (Fig. 3a–a4) when compared with the control (Fig. 3a–a3). Western blot analysis in Fig. 3b confirmed the overexpression and knocked down TRIP-1 protein levels in the total cell lysate and quantitative analysis showed a 2.5-fold increase and 70% reduction in TRIP-1 expression in overexpressed and shRNA treated cells respectively (Fig. 3c). Similar TRIP-1 expression pattern was observed in the secretome obtained from these cells. (Fig. 3d).

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&thinsp;=&thinsp;48&thinsp;&mu;M. 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.