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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.


Expression of osteogenic markers in explant sections detected by immunolabeling.(11a) Representative confocal micrographs showing the immunohistochemical localization of fibronectin (a1, a2), Runx2 (a3, a4), OCN (a5, a6) and TRIP-1 (a7, a8) in control scaffold & scaffold +rTRIP-1 respectively. Nuclei were stained with DAPI. Note increased expression levels of these markers with rTRIP-1 treatment. Scale bar represents 20μm for all images. 11b: Quantitation of the expression levels using Image J analysis and statistical significance obtained using Students t test. Significant increase in expression of FN, Runx2, OCN and TRIP-1 markers were observed in rTRIP-1 treated scaffolds (grey shaded box *p < 0.05) when compared to control scaffolds (white shaded box). (11c) Representative confocal micrographs showing immunohistochemical localization of Fibronectin (c1, c2, c3) Runx2 (c4, c5, c6), OCN (c7, c8, c9) and TRIP-1 (c10, c11, c12) from explant sections of MC3T3-E1 control, MC3T3-E1-TRIP-1 overexpressing and MC3T3-E1 TRIP-1 shRNA cells seeded scaffolds respectively. (11d) Graph showing the percentage of positive area calculated using Image J. Statistical significance was calculated using Students t test. Expression of Fibronectin, Runx2, OCN and TRIP-1 markers show a significant increase in MC3T3–TRIP-1 OE cells seeded scaffolds (dark grey shaded box (*p < 0.05) when compared to control (white shaded box). Scaffolds preseeded with TRIP-1 knocked down cells show a significant reduction in expression (light grey shaded box, #p < 0.05). (11e) Secondary antibody negative controls. Scale bar represents 10–20 μm.
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f11: Expression of osteogenic markers in explant sections detected by immunolabeling.(11a) Representative confocal micrographs showing the immunohistochemical localization of fibronectin (a1, a2), Runx2 (a3, a4), OCN (a5, a6) and TRIP-1 (a7, a8) in control scaffold & scaffold +rTRIP-1 respectively. Nuclei were stained with DAPI. Note increased expression levels of these markers with rTRIP-1 treatment. Scale bar represents 20μm for all images. 11b: Quantitation of the expression levels using Image J analysis and statistical significance obtained using Students t test. Significant increase in expression of FN, Runx2, OCN and TRIP-1 markers were observed in rTRIP-1 treated scaffolds (grey shaded box *p < 0.05) when compared to control scaffolds (white shaded box). (11c) Representative confocal micrographs showing immunohistochemical localization of Fibronectin (c1, c2, c3) Runx2 (c4, c5, c6), OCN (c7, c8, c9) and TRIP-1 (c10, c11, c12) from explant sections of MC3T3-E1 control, MC3T3-E1-TRIP-1 overexpressing and MC3T3-E1 TRIP-1 shRNA cells seeded scaffolds respectively. (11d) Graph showing the percentage of positive area calculated using Image J. Statistical significance was calculated using Students t test. Expression of Fibronectin, Runx2, OCN and TRIP-1 markers show a significant increase in MC3T3–TRIP-1 OE cells seeded scaffolds (dark grey shaded box (*p < 0.05) when compared to control (white shaded box). Scaffolds preseeded with TRIP-1 knocked down cells show a significant reduction in expression (light grey shaded box, #p < 0.05). (11e) Secondary antibody negative controls. Scale bar represents 10–20 μm.

Mentions: To further elucidate whether TRIP-1 can influence osteogenesis at the cellular level in-vivo, the subcutaneously implanted hydrogel scaffolds pretreated with rTRIP-1 were subjected to immunocytochemical analysis. Results show an increase in the expression of fibronectin (FN), Runx2, Osteocalcin (OCN) and TRIP-1 (Fig. 11a–a2, a4, a6 and a8) when compared with the control scaffold (Fig. 11–a1, a3, a5 and a7).


TGF beta receptor II interacting protein-1, an intracellular protein has an extracellular role as a modulator of matrix mineralization
Expression of osteogenic markers in explant sections detected by immunolabeling.(11a) Representative confocal micrographs showing the immunohistochemical localization of fibronectin (a1, a2), Runx2 (a3, a4), OCN (a5, a6) and TRIP-1 (a7, a8) in control scaffold & scaffold +rTRIP-1 respectively. Nuclei were stained with DAPI. Note increased expression levels of these markers with rTRIP-1 treatment. Scale bar represents 20μm for all images. 11b: Quantitation of the expression levels using Image J analysis and statistical significance obtained using Students t test. Significant increase in expression of FN, Runx2, OCN and TRIP-1 markers were observed in rTRIP-1 treated scaffolds (grey shaded box *p < 0.05) when compared to control scaffolds (white shaded box). (11c) Representative confocal micrographs showing immunohistochemical localization of Fibronectin (c1, c2, c3) Runx2 (c4, c5, c6), OCN (c7, c8, c9) and TRIP-1 (c10, c11, c12) from explant sections of MC3T3-E1 control, MC3T3-E1-TRIP-1 overexpressing and MC3T3-E1 TRIP-1 shRNA cells seeded scaffolds respectively. (11d) Graph showing the percentage of positive area calculated using Image J. Statistical significance was calculated using Students t test. Expression of Fibronectin, Runx2, OCN and TRIP-1 markers show a significant increase in MC3T3–TRIP-1 OE cells seeded scaffolds (dark grey shaded box (*p < 0.05) when compared to control (white shaded box). Scaffolds preseeded with TRIP-1 knocked down cells show a significant reduction in expression (light grey shaded box, #p < 0.05). (11e) Secondary antibody negative controls. Scale bar represents 10–20 μm.
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f11: Expression of osteogenic markers in explant sections detected by immunolabeling.(11a) Representative confocal micrographs showing the immunohistochemical localization of fibronectin (a1, a2), Runx2 (a3, a4), OCN (a5, a6) and TRIP-1 (a7, a8) in control scaffold & scaffold +rTRIP-1 respectively. Nuclei were stained with DAPI. Note increased expression levels of these markers with rTRIP-1 treatment. Scale bar represents 20μm for all images. 11b: Quantitation of the expression levels using Image J analysis and statistical significance obtained using Students t test. Significant increase in expression of FN, Runx2, OCN and TRIP-1 markers were observed in rTRIP-1 treated scaffolds (grey shaded box *p < 0.05) when compared to control scaffolds (white shaded box). (11c) Representative confocal micrographs showing immunohistochemical localization of Fibronectin (c1, c2, c3) Runx2 (c4, c5, c6), OCN (c7, c8, c9) and TRIP-1 (c10, c11, c12) from explant sections of MC3T3-E1 control, MC3T3-E1-TRIP-1 overexpressing and MC3T3-E1 TRIP-1 shRNA cells seeded scaffolds respectively. (11d) Graph showing the percentage of positive area calculated using Image J. Statistical significance was calculated using Students t test. Expression of Fibronectin, Runx2, OCN and TRIP-1 markers show a significant increase in MC3T3–TRIP-1 OE cells seeded scaffolds (dark grey shaded box (*p < 0.05) when compared to control (white shaded box). Scaffolds preseeded with TRIP-1 knocked down cells show a significant reduction in expression (light grey shaded box, #p < 0.05). (11e) Secondary antibody negative controls. Scale bar represents 10–20 μm.
Mentions: To further elucidate whether TRIP-1 can influence osteogenesis at the cellular level in-vivo, the subcutaneously implanted hydrogel scaffolds pretreated with rTRIP-1 were subjected to immunocytochemical analysis. Results show an increase in the expression of fibronectin (FN), Runx2, Osteocalcin (OCN) and TRIP-1 (Fig. 11a–a2, a4, a6 and a8) when compared with the control scaffold (Fig. 11–a1, a3, a5 and a7).

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.