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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.

No MeSH data available.


Overexpression and Silencing of TRIP-1 in MC3T3-E1 cells influences osteogenic differentiation.Real-Time PCR analysis depicting the expression levels of TRIP-1 in MC3T3-E1-TRIP-1 overexpressing cells (4a) and TRIP-1 knocked-down cells (4b). A 3-fold increase in expression was observed in TRIP-1 OE cells when compared to untransfected cells. TRIP-1 expression decreased 70% in TRIP-1 knocked-down cells. No significant change in TRIP-1 expression was observed in mock vector transfected cells and non-target control shRNA treated cells. (4c) Real-Time PCR analysis showing osteogenic gene expression normalized to the control cells. Note increase in the expression levels of osteogenic differentiation markers such as ALP, Runx2, and Collagen type 1 in TRIP-1 OE cells. TRIP-1 silencing showed a downregulation in expression of ALP, Runx2 and OCN. Data are represented as mean fold change obtained from triplicate experiments with standard deviation as error. Student’s t-test was used to obtain statistical significance with respect to control. *, @ Represents significance of p < 0.05.
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f4: Overexpression and Silencing of TRIP-1 in MC3T3-E1 cells influences osteogenic differentiation.Real-Time PCR analysis depicting the expression levels of TRIP-1 in MC3T3-E1-TRIP-1 overexpressing cells (4a) and TRIP-1 knocked-down cells (4b). A 3-fold increase in expression was observed in TRIP-1 OE cells when compared to untransfected cells. TRIP-1 expression decreased 70% in TRIP-1 knocked-down cells. No significant change in TRIP-1 expression was observed in mock vector transfected cells and non-target control shRNA treated cells. (4c) Real-Time PCR analysis showing osteogenic gene expression normalized to the control cells. Note increase in the expression levels of osteogenic differentiation markers such as ALP, Runx2, and Collagen type 1 in TRIP-1 OE cells. TRIP-1 silencing showed a downregulation in expression of ALP, Runx2 and OCN. Data are represented as mean fold change obtained from triplicate experiments with standard deviation as error. Student’s t-test was used to obtain statistical significance with respect to control. *, @ Represents significance of p < 0.05.

Mentions: RT-PCR quantitation showed a 3-fold increase of TRIP-1 in overexpressed cells (Fig. 4a) while efficient knockdown was observed in TRIP-1 shRNA cells (Fig. 4b). In each case, the expression levels was normalized to untransfected cells. Mock vector and non-target control shRNA were used as negative controls. We then analyzed the osteogenic gene expression profile in overexpressing and TRIP-1 knocked down MC3T3-E1 cells. Osteogenic gene expression and quantitation analysis showed upregulation of alkaline phosphatase (ALP), Runx2 Type I collagen, Osteocalcin (OCN) and Osterix (OSX). The expression of these genes were downregulated in TRIP-1 silenced cells (Fig. 4c). Protein level of Runx2 was confirmed by Western blot analysis in the control and transgenic cell lines (Supplementary Figure S4).


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 in MC3T3-E1 cells influences osteogenic differentiation.Real-Time PCR analysis depicting the expression levels of TRIP-1 in MC3T3-E1-TRIP-1 overexpressing cells (4a) and TRIP-1 knocked-down cells (4b). A 3-fold increase in expression was observed in TRIP-1 OE cells when compared to untransfected cells. TRIP-1 expression decreased 70% in TRIP-1 knocked-down cells. No significant change in TRIP-1 expression was observed in mock vector transfected cells and non-target control shRNA treated cells. (4c) Real-Time PCR analysis showing osteogenic gene expression normalized to the control cells. Note increase in the expression levels of osteogenic differentiation markers such as ALP, Runx2, and Collagen type 1 in TRIP-1 OE cells. TRIP-1 silencing showed a downregulation in expression of ALP, Runx2 and OCN. Data are represented as mean fold change obtained from triplicate experiments with standard deviation as error. Student’s t-test was used to obtain statistical significance with respect to control. *, @ Represents significance of p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f4: Overexpression and Silencing of TRIP-1 in MC3T3-E1 cells influences osteogenic differentiation.Real-Time PCR analysis depicting the expression levels of TRIP-1 in MC3T3-E1-TRIP-1 overexpressing cells (4a) and TRIP-1 knocked-down cells (4b). A 3-fold increase in expression was observed in TRIP-1 OE cells when compared to untransfected cells. TRIP-1 expression decreased 70% in TRIP-1 knocked-down cells. No significant change in TRIP-1 expression was observed in mock vector transfected cells and non-target control shRNA treated cells. (4c) Real-Time PCR analysis showing osteogenic gene expression normalized to the control cells. Note increase in the expression levels of osteogenic differentiation markers such as ALP, Runx2, and Collagen type 1 in TRIP-1 OE cells. TRIP-1 silencing showed a downregulation in expression of ALP, Runx2 and OCN. Data are represented as mean fold change obtained from triplicate experiments with standard deviation as error. Student’s t-test was used to obtain statistical significance with respect to control. *, @ Represents significance of p < 0.05.
Mentions: RT-PCR quantitation showed a 3-fold increase of TRIP-1 in overexpressed cells (Fig. 4a) while efficient knockdown was observed in TRIP-1 shRNA cells (Fig. 4b). In each case, the expression levels was normalized to untransfected cells. Mock vector and non-target control shRNA were used as negative controls. We then analyzed the osteogenic gene expression profile in overexpressing and TRIP-1 knocked down MC3T3-E1 cells. Osteogenic gene expression and quantitation analysis showed upregulation of alkaline phosphatase (ALP), Runx2 Type I collagen, Osteocalcin (OCN) and Osterix (OSX). The expression of these genes were downregulated in TRIP-1 silenced cells (Fig. 4c). Protein level of Runx2 was confirmed by Western blot analysis in the control and transgenic cell lines (Supplementary Figure S4).

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.