Abstract
Transforming growth factor beta receptor II interacting protein-1 (TRIP-1) was recently localized in the mineralized matrices of bone and dentin. The function of TRIP-1 in the ECM is enigmatic, as it is known to function as an intracellular endoplasmic reticulum protein during protein synthesis. Based on its localization pattern in bones and teeth, we posited that TRIP-1 must function as a regulatory protein with multiple functions during mineralization. In this study, we determined the in vivo function of TRIP-1 by an implantation assay performed using recombinant TRIP-1 and TRIP-1 overexpressing and knocked down cells embedded in a 3D biomimetic scaffold. After 4 weeks, the subcutaneous tissues from TRIP-1 overexpressing cells and scaffolds containing recombinant TRIP-1 showed higher expression levels of several ECM proteins such as fibronectin and collagen I. Picrosirius red and polarized microscopy was used to identify the birefringence of the collagen fibrils in the extracellular matrix (ECM). Interestingly, knockdown of TRIP-1 resulted in lower fibronectin and downregulation of the activation of the ERK MAP kinase. We further demonstrate that TRIP-1 overexpression leads to higher expression of pro-angiogenic marker VEGF and downregulation of anti-angiogenic factors such as pigment epithelium-derived factor and thrombospondin. Field emission scanning electron microscope results demonstrated that TRIP-1 overexpressing cells released large amount of extracellular microvesicles which were localized on the fibrillar matrix in the ECM. Overall, this study demonstrates that TRIP-1 can promote secretion of extracellular vesicles, synthesis of key osteogenic ECM matrix proteins and promote angiogenesis.
Highlights
Osteoblasts and odontoblasts are bone and dentin forming cells that synthesize and assemble a specialized extracellular matrix (Gajjeraman et al, 2007; George and Veis, 2008)
Results show an increase in the expression of the major extracellular matrix (ECM) proteins (Figure 6A) such as type I collagen (Col1) (B1–B3), alkaline phosphatase (ALP) a key enzyme that initiates mineralization (B4–B6) and Glucose regulated protein 78 (GRP-78) that we have recently identified in the matrices of bone and dentin (B10–B12)
TRIP-1 overexpression led to an increase in the expression of type-II receptor and higher TGF-β ligand localized in the ECM
Summary
Osteoblasts and odontoblasts are bone and dentin forming cells that synthesize and assemble a specialized extracellular matrix (Gajjeraman et al, 2007; George and Veis, 2008). Biological apatite deposited in the extracellular matrix (ECM) is orchestrated by an organic extracellular matrix that controls mineral nucleation and growth (He et al, 2003). This extracellular matrix is a complex network of collagens, glycoproteins such as fibronectin, SIBLING protein family members, phosphoproteins, proteoglycans, and polysaccharides There are yet unidentified proteins in the matrix that may play a regulatory role in initiating physiological processes such as cell differentiation and angiogenesis during the formation of mineralized tissues
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