Stimulation of plasma membrane and matrix vesicle enzyme activity by transforming growth factor‐β in osteosarcoma cell cultures

Abstract

Transforming growth factor‐β (TGFβ) serves an important role in extracellular matrix formation by stimulating the production of numerous extracellular matrix proteins by connective tissue cells and by osteoblasts or bone‐forming cells. TGFβ has been shown to stimulate alkaline phosphatase (ALPase) activity in the rat osteoblast‐like osteosarcoma cell line ROS 17/2.8. Previous studies have shown that this enzyme is elevated during calcification of bone and that it is enriched in matrix vesicles, an extracellular organelle associated with initial hydroxyapatite formation. To test the hypothesis that TGFβ plays a role in regulating mineral deposition in the matrix, the effects of TGFβ on ALPase and phospholipase A₂, two enzymes associated with mineralization, were examined. ROS 17/2.8 cells were cultured at high and low density with recombinant human TGFβ (0.1–10 ng/ml) to examine the influence of cell maturation on response to TGFβ. Maximal stimulation of ALPase activity in the low density cultures was seen at 5 ng/ml; in high‐density cultures, there was further stimulation at 10 ng/ml. There was a dose‐dependent increase in ALPase activity seen in the matrix vesicles and plasma membranes in both types of cultures. Matrix vesicle ALPase exhibited a greater response to factor than did the plasma membrane enzyme. However, in low density cultures, the two membrane fractions exhibited a parallel response with greatest activity consistently in the matrix vesicles. There was a dose‐dependent increase in phospholipase A₂‐specific activity in the plasma membranes and matrix vesicles of both high‐ and low‐density cultures. In agreement with previous studies, TGFβ inhibited cellular proliferation 50%. The results show that addition of TGFβ stimulates the activity of enzymes associated with calcification. The effect of TGFβ is dependent on the stage of maturation of the cell. This study indicates that TGFβ may play an important role in induced bone formation, calcification, and fracture repair in addition to its role in promoting chondrogenesis.