Abstract
Previous studies have shown 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3)-responsive alkaline phosphatase in cultured growth zone cartilage chondrocytes is localized in extracellular matrix vesicles (MV). Since osteoblast-like cells also have 1,25-(OH)2D3-responsive alkaline phosphatase, this study determined whether the 1,25-(OH)2D3-responsive enzyme activity is localized to MV produced by these cells as well. Osteoblast-like cells from rat (ROS 17/2.8), mouse (MC 3T3), human (MG 63), and rat growth zone cartilage were cultured in Dulbecco's modified Eagle's medium containing 10(-7)-10(-12) M 1,25-(OH)2D3. Alkaline phosphatase total activity and specific activity were measured in the cell layer, MV, and plasma membrane (PM) fractions. MV and PM purity were verified by electron microscopy and MV alkaline phosphatase specific activity compared to PM (MV versus PM: ROS 17/2.8 6 x; MG 63, 5.5 x; MC 3T3, 33 x; GC, 2 x). There was a dose-dependent stimulation of MV alkaline phosphatase (5- to 15-fold increase at 10(-7)-10(-9) M) in all cell types in response to the 1,25-(OH)2D3. The PM enzyme was stimulated in a parallel fashion in the osteoblast cultures. No effect of 1,25-(OH)2D3 was observed in growth cartilage PM. Although MV accounted for less than 20% of the total activity they contributed 50% of the increase in alkaline phosphatase activity in the cell layer in response to 1,25-(OH)2D3 and MV specific activity was enriched 10 times over that of the cell layer. These are common features of MV produced by cells which calcify their matrix and suggest that hormonal regulation of MV enzymes may be important in primary calcification.
Highlights
Cultured growth zone cartilage chondrocytes is local- Alkaline phosphatase activity appears to be regulated by ized in extracellular matrixvesicles (MV)
Alkaline phosphatase in and Matrix vesicles (MV) alkaline phosphatase specific activity com- matrix vesicles produced by resting zone cells, which do not pared to plasma membranes (PM)(MV uersus PM: ROS 1 7 / 2 36 X; MG 63, calcify their matrix i n viuo, is stimulated by 24,25-(OH)&
In calcifying tissues like growth cartilage and bone, mineralization is regulated by vitamin D and is associated with increased alkaline phosphatase activity, an enzyme that is enriched in the matrix vesicles isolated from cartilage
Summary
Cell Layer-Specific activity of alkaline phosphatase in the cell layer varied with the source of cells. Of the osteoblast cell lines, ROS 17/2.8 cells had the highest specific activity (2.7 f 0.3), followed by MC 3T3 cells (0.8 f 0.5) and MG 63 cells (0.2 -+ 0.0). 1,25-(OH)2D3-responsiveMatrix VesicleAlkaline Phosphatase of other cell and matrix components.
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