Age-dependent decline in bone nodule formation stimulating activity in ratserum is mainly due to the change in the corticosterone level

The replacement of fetal bovine serum with rat serum in a culture medium br ought about a marked increase in the formation of mineralized bone nodules (BN) in primary cultures of rat calvarial cells. These effects of rat serum were most prominent when added during the early phase of the culture, indi cating that the serum factor mainly acts on the cells during the growing ph ase. A significant increase in BN formation was observable at final rat ser um concentration as low as 1%, and the effect was dependent on serum concen tration, at least up to 10%. The addition of rat serum also increased alkal ine phosphatase (ALP) activity, collagen synthesis, and DNA synthesis in ca lvarial cells. BN formation stimulating activity was extractable with ethyl acetate. The ethyl acetate extract was purified by TSK-GEL OH-120 column c hromatography by monitoring the stimulation of ALP activity in ROS 17/2.8 c ells. The chromatographic behavior of the ALP activity was found to be iden tical to that of corticosterone, the major glucocorticoid in rodents and th e preincubation of the purified fraction with anticorticosterone antibody a bolished the ALP stimulating activity. These results suggest that BN format ion stimulating activity in rat serum is mainly attributable to corticoster one. The concentration of serum corticosterone decreased with age in parall el with BN formation stimulating activity, which suggests that the physiolo gical level of corticosterone may have a regulatory role in the maintenance of osteoblast function. J. Cell. Biochem. 81:547-556, 2001. (C) 2001 Wiley -Liss, Inc.