TRIP-1 Promotes the Assembly of an ECM That Contains Extracellular Vesicles and Factors That Modulate Angiogenesis

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.

Disrupted Protein Expression and Altered Proteolytic Events in Hypophosphatemic Dentin Can Be Rescued by Dentin Matrix Protein 1

“…PRS is a non-specific stain for a variety of collagen types that reveals information about a tissue's collagen content, molecular order, and organization (Lattouf et al, 2014). Our group has previously used the PRS method in conjunction with polarized light microscopy to quantify the de novo collagen synthesis in a subcutaneous implantation model (Chen and George, 2018). In this study, polarized light microscopy revealed significant differences in the absolute angles of collagen fibers between the genotypes.…”

“…Polarized images of the Sirius Red stained sections were analyzed using the opensource CT-FIRE software (Bredfeldt et al, 2014). Regions of interest (dentin, PDL, and bone) were annotated using the ROI manager tool and quantitative CT-FIRE analysis was performed on each to obtain collagen fiber length, width, and angle, as previously published (Chen and George, 2018). For WT versus Hyp comparisons in Figure 1, statistical analysis was performed using multiple t-tests with the Holm-Sidak correction for multiple comparisons, alpha = 0.05.…”

Microfluidic device with a carbonate‐rich hydroxyapatite micro‐coating

“…Matrix vesicles are observed as aggregates in some areas or scattered among collagen fibrils in others, consistent with previous studies that show aggregates of matrix vesicles on mineralizing osteoblasts. [45][46][47] This further suggests that the cells are engaged in the primary phase of mineralization.…”

Biomineralization of Enamel and Dentin Mediated by Matrix Proteins

“…Immunogold labeling also suggested that TRIP1 could bind to monomeric collagen aggregates. These observations suggest that TRIP1 in the ECM could bind both collagen and calcium ions to initiate matrix mineralization (Chen and George 2018).…”