Phosphate induces formation of matrix vesicles during odontoblast-initiated mineralization in vitro

Highlights

• Novel function of phosphate in mineralization was identified.

• Phosphate induces secretion of matrix vesicles.

• Molecular characteristics of matrix vesicles distinguish them from exosomes.

Abstract

Mineralization is a process of deposition of calcium phosphate crystals within a fibrous extracellular matrix (ECM). In mineralizing tissues, such as dentin, bone and hypertrophic cartilage, this process is initiated by a specific population of extracellular vesicles (EV), called matrix vesicles (MV). Although it has been proposed that MV are formed by shedding of the plasma membrane, the cellular and molecular mechanisms regulating formation of mineralization-competent MV are not fully elucidated. In these studies, 17IIA11, ST2, and MC3T3-E1 osteogenic cell lines were used to determine how formation of MV is regulated during initiation of the mineralization process. In addition, the molecular composition of MV secreted by 17IIA11 cells and exosomes from blood and B16-F10 melanoma cell line was compared to identify the molecular characteristics distinguishing MV from other EV. Western blot analyses demonstrated that MV released from 17IIA11 cells are characterized by high levels of proteins engaged in calcium and phosphate regulation, but do not express the exosomal markers CD81 and HSP70. Furthermore, we uncovered that the molecular composition of MV released by 17IIA11 cells changes upon exposure to the classical inducers of osteogenic differentiation, namely ascorbic acid and phosphate. Specifically, lysosomal proteins Lamp1 and Lamp2a were only detected in MV secreted by cells stimulated with osteogenic factors. Quantitative nanoparticle tracking analyses of MV secreted by osteogenic cells determined that standard osteogenic factors stimulate MV secretion and that phosphate is the main driver of their secretion. On the molecular level, phosphate-induced MV secretion is mediated through activation of extracellular signal-regulated kinases Erk1/2 and is accompanied by re-organization of filamentous actin. In summary, we determined that mineralization-competent MV are distinct from exosomes, and we identified a new role of phosphate in the process of ECM mineralization. These data provide novel insights into the mechanisms of MV formation during initiation of the mineralization process.

Introduction

Extracellular vesicles (EV) is a broad term describing sub-micron size, spherical, membrane-enclosed particles released from cells to the extracellular milieu. EV are secreted from cells in various physiological and pathological conditions and can be detected in virtually all biological fluids [1], [2]. They express cell surface receptors and carry biologically active proteins, lipids, and nucleic acids. It has been shown that EV have the capacity to modulate the function of target cells in an autocrine or paracrine manner, therefore they have been recognized as an integral component of the intercellular communication [2], [3]. EV are highly heterogeneous and dynamic in nature. Their molecular composition reflects their cell-type of origin, pathophysiological cell state, biogenesis pathway, and biological function [2], [3].

Mineralization is a biological process by which crystals of calcium phosphate (hydroxyapatite, HA) are laid down within the fibrous extracellular matrix (ECM). Physiological mineralization occurs in skeletal and dental tissues (bone, terminal hypertrophic cartilage, dentin, cementum, and enamel). Mineralization can also occur ectopically (pathologic mineralization) in soft tissues, for example in the blood vessels (arterial calcification) or in joints during the late stages of osteoarthritis. The progression and extent of both physiologic and pathologic mineralization are regulated locally and systemically. Mineralization depends upon the availability of Ca^2 + and PO₄^3 − (P_i), concentration of mineralization inhibitors, and ECM composition [4], [5], [6], [7], [8], [9], [10], [11], [12], [13].

P_i participates in the mineralization process in multiple ways. First, P_i is a structural component of the inorganic phase of the mineralized ECM, thus its local availability affects the rate of HA formation. Second, addition or removal of P_i to/from various ECM proteins regulates their function in mineralization [14], [15]. Finally, it has been shown that P_i regulates expression of multiple genes involved in osteogenic differentiation and the mineralization process [16], [17], [18], [19], [20]. The P_i-induced signaling pathway is not well delineated and its mediators are largely unknown. However, it has been demonstrated that P_i signaling in mineralizing cells depends on the activity of P_i transporters and is mediated by Erk1/2, but not p38 or c-jun kinases [21], [22], [23]. P_i-induced activation of Erk1/2 is bi-phasic with the first activation happening quickly, within 15–30 min of P_i treatment, and the second activation occurring approximately 6–8 h later [23], [24].

Initiation of physiologic mineralization of cartilage, mantle dentin, and woven bone is facilitated by a specific population of EV, called matrix vesicles (MV). There is evidence suggesting that ectopic mineralization of arteries is also associated with increased secretion of EV from vascular smooth muscle cells with a presumptive role in supporting pathologic vascular mineralization [25]. Electron microscopy demonstrated that mineralizing cells, such as hypertrophic chondrocytes and newly differentiated odontoblasts and osteoblasts, shed numerous sub-micron size (80–200 nm) vesicles from their plasma membrane [26], [27], [28], [29], [30]. Therefore, it has been proposed that MV are formed by budding off the plasma membrane. The plasma membrane origin of MV has been further supported by comparative analyses of lipids and proteins, in which it was demonstrated that there are significant similarities in the molecular composition of MV and the plasma membrane of the cell of origin [31], [32]. However, in two more recent studies, vesicles containing electron-dense material composed of calcium and phosphorus were detected in the cytoplasm of mouse calvarial osteoblasts, suggesting that intracellular processes may play a role in initial HA formation [33], [34].

MV have a discrete intravesicular environment as well as protein and lipid composition that together support the accumulation of high concentrations of P_i and Ca^2 +, and subsequent HA formation. In particular, MV are enriched in tissue-nonspecific alkaline phosphatase (TNAP) and phosphoethanolamine/phosphocholine phosphatase (PHOSPHO1), whose catalytic activities provide P_i for HA formation, but have non-redundant functions in skeletal mineralization [4], [35], [36], [37], [38], [39]. Proteomic analyses of vesicles produced by chondrocytes, osteoblast cell lines, and bone marrow stromal cells undergoing osteogenic differentiation consistently detect numerous proteins that are involved in the mineralization process and matrix remodeling. This agrees with the notion that the biological function of MV is to support mineralization [32], [40], [41], [42], [43]. Of note, MV are also enriched in Ca^2 + transporters (annexins) which are commonly detected in many different types of EV [26], [38], [40], [43], [44], [45], [46], [47].

It is now recognized that cells utilize various cellular mechanisms to secrete EV of diverse biological functions. With the rising interest in using EV as diagnostic markers and therapeutic targets, it is critical to understand the differences between various populations of EV and the mechanisms regulating their secretion. In this study, we use cellular models of mineralization to gain mechanistic insights into the regulation of secretion of a specific group of EV with the biological function to promote mineralization. The goals of our study are to increase our understanding of how the mineralization process is initiated and to delineate characteristic features of mineralization-competent MV.