Analysis of the extracellular matrix vesicle proteome in mineralizing osteoblasts

Abstract

Many key processes central to bone formation and homeostasis require the involvement of osteoblasts, cells responsible for accumulation and mineralization of the extracellular matrix (ECM). During this complex and only partially understood process, osteoblasts generate and secrete matrix vesicles (MVs) into the ECM to initiate mineralization. Although they are considered an important component of mineralization process, MVs still remain a mystery. To better understand their function and biogenesis, a proteomic analysis of MVs has been conducted. MVs were harvested by two sample preparation approaches and mass spectrometry was utilized for protein identification. A total of 133 proteins were identified in common from the two MV preparations, among which were previously known proteins, such as annexins and peptidases, along with many novel proteins including a variety of enzymes, osteoblast-specific factors, ion channels, and signal transduction molecules, such as 14-3-3 family members and Rab-related proteins. To compare the proteome of MV with that of the ECM we conducted a large-scale proteomic analysis of collagenase digested mineralizing osteoblast matrix. This analysis resulted in the identification of 1,327 unique proteins. A comparison of the proteins identified from the two MV preparations with the ECM analysis revealed 83 unique, non-redundant proteins identified in all three samples. This investigation represents the first systematic proteomic analysis of MVs and provides insights into both the function and origin of these important mineralization-regulating vesicles.

Inflammatory Cytokines Induce a Unique Mineralizing Phenotype in Mesenchymal Stem Cells Derived from Human Bone Marrow

“…To release matrix-associated MVs, cell layers were incubated with Liberase DH Research Grade (0.14 Wünsch units/ml, Roche Diagnostics) for 30 min at 37°C. Cell supernatants and Liberase-digested samples were cleared of cells and cellular debris by centrifugation at 20,000 ϫ g for 30 min at 4°C (26). MVs from cleared supernatants were then pelleted by ultracentrifugation at 100,000 ϫ g for 60 min at 4°C (Optima TM TLX Ultracentrifuge; Beckman Coulter Inc., Fullerton, CA).…”

Proteomic Analysis of Human Osteoblastic Cells: Relevant Proteins and Functional Categories for Differentiation

“…8 Several proteomic studies have been conducted to unravel the mechanisms underlying osteogenesis. [10][11][12][13][14] Yet only a portion of the osteoblast proteome has been unveiled and additional, quantitative proteomic analyses are needed to reach the goal of capturing the full osteoblast proteome.…”

Biophysical aspects of biomineralization

“…Nascent HA crystals exposed to the extravesicular fluid have been found to bind OPN, a collagen-binding molecule (Chen et al 1992;Liu et al 2007;Martin et al 2004). Given that MVs display collagen-binding molecules on their membranes (TNAP, Annexin A5 (AnxA5)) (Balcerzak et al 2008;Wu et al 1991;Xiao et al 2007), it is conceivable that a specific binding of MVs to collagen mediates the physical transfer of HA crystals to the exterior of collagen fibrils. This model does not negate, nor is it incompatible with, data indicating that crystals are able to form in the gaps and holes of the collagen fibrils, as has been well demonstrated by the work of many investigators, but if proven correct it would help explain how crystals formed within MVs can contribute to ECM mineralization.…”