Osteocalcin secretion by the human osteosarcoma cell line MG-63

Abstract

The human osteosarcoma cell line MG-63 has been used to study the production of the bone-specific protein, osteocalcin. In the absence of any stimuli, MG-63 cells secreted very low levels of osteocalcin. The secretion of osteocalcin started after a lag time of 10-12 h upon 1,25-(OH)₂D₃ treatment. Osteocalcin secretion was measured at doses as low as 0.03 nM (fourfold increase, p < 0.05), and this activity increased further with higher doses of 1,25-(OH)₂D₃ to reach a plateau at 50 nM. The secretion increased transiently from very low levels in sparse cell cultures to peak values in subconfluent cultures (+ 40%), two- to threefold above values obtained for confluent cells. Values for confluent cells average 55.9 + 2.0 ng/ml protein per 48 h. A similar behavior is observed for 1,25-(OH)₂D₃ receptor concentration under similar experimental conditions. B_max increased transiently from sparse to subconfluent cell cultures (40 -60% confluent) and reached values 50% lower in confluent cells. However, the receptor affinity was not affected by cell density. MG-63 cells also possessed an alkaline phosphatase isoenzyme of the bone-liver-kidney type that was stimulated by 1,25-(OH)₂D₃ treatment (two- to threefold) and inhibited by parathyroid hormone (40 nM, -25%, p < 0.025). PTH and PGE₂ increased cAMP production in a dose-dependent manner, but the cells were irresponsive to salmon calcitonin. Basal and PTH-responsive cyclic AMP production were also modulated by cell density. Dexamethasone pretreatment (100 nM, 48 h) stimulated the PTH-dependent cAMP production but failed to influence the response to PGE₂. Vitamin D₃-induced osteocalcin secretion was inhibited by 40 nM PTH (-20%, p < 0.01) and 5 nM PGE₂ (-36%, p < 0.005), a situation that could be related to the ability of these hormones to stimulate cAMP in these cells. These results show that the MG-63 cell line is a good human osteoblastlike cell model in which bone-specific protein synthesis (osteocalcin and alkaline phosphatase) is modulated in response to 1,25-(OH)₂D₃ and PTH.