Human osteosarcoma cells spontaneously release matrix-vesicle-like structures with the capacity to mineralize

Abstract

Nearly all models of skeleton, cartilage, and dentin mineralization evoke a specific role for matrix vesicles (MV) and alkaline phosphatase (ALP). Nevertheless, the mechanism underlying MV production, mineralization, and the pivotal role of ALP is largely unknown. Previous studies in this laboratory demonstrated that ALP in a human osteosarcoma cell line (SAOS-2) is of the tissue nonspecific ('bone') isoenzyme and lipid-anchored to the plasma membrane in ecto-orientation [1], thus reminiscent of osteoblasts in vivo [2]. Herein, we show that these cells spontaneously release ALP-rich structures (MVs) with the capacity to mineralize. MVs from SAOS-2 cells are 100-200 nm in diameter with characteristic trilaminar membranes. ALP in these vesicles is hydrophobic and lipid-anchored in ecto-orientation in a manner similar to the ALP in the parent SAOS-2 cells. 5'-Nucleotidase, another plasma membrane enzyme, is also abundant in MVs; adenylate cyclase is relatively deficient. Analysis of plasma membrane and MV proteins by 2-D gel electrophoresis reveals many common constituents; nevertheless, MVs contain several unique (or greatly enriched) proteins indicating that SAOS-2 MVs originate from specialized regions of the plasma membrane and are released in the same orientation as the plasma membrane. MVs, unlike plasma membrane vesicles, can cause the formation of insoluble calcium and phosphate in a manner that i) requires ALP substrates; ii) is blocked by ALP inhibition or inactivation; and iii) is not dependent on intact MVs. SAOS-2 derived MVs contain at least 3 protein kinases and their substrates. ALP does not, however, have a major role in regulating the phosphorylation state of these phosphoproteins.