Matrix vesicles produced by osteoblast-like cells in culture become significantly enriched in proteoglycan-degrading metalloproteinases after addition of ?-Glycerophosphate and ascorbic acid

Abstract

Matrix vesicles, media vesicles, and plasma membranes from three well-characterized, osteoblast-like cells (ROS 17/2.8, MG-63, and MC-3T3-E1) were evaluated for their content of enzymes capable of processing the extracellular matrix. Matrix vesicles were enriched in alkaline phosphatase specific activity over the plasma membrane and contained fully active neutral, but not acid, metalloproteinases capable of digesting proteoglycans, potential inhibitors of matrix calcification. Matrix vesicle enrichment in neutral metalloproteinase varied with the cell line, whereas collagenase, lysozyme, hyaluronidase, and tissue inhibitor of metalloproteinases (TIMP) were not found in any of the membrane fractions examined. MC-3T3-E1 cells were cultured for 32 days in the presence of ascorbic acid (100 micrograms/ml), beta-glycerophosphate (5 mM), or a combination of the two, to assess changes in matrix vesicle enzymes during calcification. Ascorbate or beta-glycerophosphate alone had no effect, but in combination produced significant increases in both active and total neutral metalloproteinase in matrix vesicles and plasma membranes, with the change seen in matrix vesicles being the most dramatic. This correlated with an increase in the formation of von Kossa-positive nodules. The results of the present study indicate that osteoblast-like cells produce matrix vesicles enriched in proteoglycan-degrading metalloproteinases. In addition, the observation that matrix vesicles contain significantly increased metalloproteinases under conditions favorable for mineralization in vitro lends support to the hypothesis that matrix vesicles play an important role in extracellular matrix processing and calcification in bone.

Proteoglycans in Dentinogenesis

“…To emphasize this aspect, Dean et al (1994), using three differing osteoblast cell lines, reported that the matrix vesicles produced by these cells are enriched in PGdegrading MMPs. In addition, in conditions favorable for mineralization in vitro, the matrix vesicles contained significantly increased levels of metalloproteinases, lending support to the proposed role that matrix vesicles play in ECM processing and mineralization.…”

Mineralization of Decalcified Bone Occurs Under Cell Culture Conditions and Requires Bovine Serum But Not Cells

“…The majority of these studies have utilized an osteoblastic cell line in culture media that typically contain ionic calcium levels between 1.8 mM [Minimum Essential Medium (MEM), aMEM, and Dulbecco's Modified Eagle Medium (DMEM)] and 2.6 mM [Biggers, Gwatkin and Judah b (BGJ/b) Medium, Fitton Jackson Modification], ionic phosphate levels of 0.9 mM (DMEM) to 1.6 mM (BGJ/b), and 10 mM b-glycerophosphate [1][2][3][4][5][6][7][8][9][10][11][12][13][14]. The role of the bglycerophosphate is generally accepted to be as a source of phosphate for mineral growth, but it may affect mineral deposition in other ways as well [15].…”

Calcitonin gene-related peptide elevates calcium and polarizes membrane potential in MG-63 cells by both cAMP-independent and -dependent mechanisms

“…From among an assortment of osteoblast-like cell lines, clonal osteosarcoma-derived human MG-63 cells have been described as a model for early-stage immature preosteoblasts because of the nature of their response to factors like l,25(OH) 2 -vitamin D 3 and their regulated temporal development of bone-type alkaline phosphatase activity (4,9). We very recently found (22) that MG-63 cells prominently express the type-1 CGRP receptor [calcitonin receptor-like receptor (CRLR)/receptor activity-modifying protein (RAMP)1] and respond to CGRP with 35-fold increases in cAMP formation and rapid phosphorylation or dephosphorylation of several types of mitogen-activated protein kinase.…”