RESUMO
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 less than 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. Bmax 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 less than 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.(ABSTRACT TRUNCATED AT 250 WORDS)
