THE ROLE OF DNA-SYNTHESIS IN THE RESPONSES OF FETAL-RAT CALVARIAE TO CORTISOL

To determine the extent to which the effects of cortisol on collagen synthesis in 21 day fetal rat calvariae are linked to its effects on cell replication, calvariae were cultured for 24-72 h with 0.1 and 1-mu-M cortisol in the presence or absence of 1 mM hydroxyurea (HU) or 30-mu-M aphidicolin (APC), inhibitors of DNA synthesis. The incorporation of [H-3]proline into collagenase-digestible protein (CDP) and [H-3]thymidine into DNA were measured during the last 2 h of culture. At 24 h HU and APC decreased thymidine incorporation by > 90% and this remained low for the duration of culture. In contrast, cortisol reduced thymidine incorporation by only 44% at 72 h. Although cortisol caused a 24 h stimulatory effect and a 48 and 72 h inhibitory effect on CDP labeling and the percentage of collagen being synthesized (PCS), HU, and APC had no effect on basal CDP labeling or PCS over the 72 h culture period. Cortisol caused parallel alterations in the steady-state levels of alpha-1(I) procollagen mRNA, suggesting that its effects occur at the pretranslational level. At 24 h HU and APC did not prevent the stimulatory effect of cortisol on CDP labeling and PCS. At 48 h the inhibitory effects of cortisol on CDP labeling and PCS were observed in the presence of APC but not in the presence of HU. At 72 h the inhibitory effects of cortisol on CDP labeling and PCS were still observed in the presence of HU and APC. The inhibitory effect of cortisol on CDP labeling was only partially reversed 48 h after removing cortisol from calvariae treated with hormone for 48 h. These data show that DNA synthesis inhibitors by themselves do not mimic the effects of cortisol on collagen synthesis nor do they prevent the early stimulation and the late inhibition of collagen synthesis by cortisol. Thus we conclude that the long-term inhibitory effect of cortisol on collagen synthesis in calvariae is largely independent of cell replication. The component of collagen synthesis that is reversible may represent the direct inhibitory effect of cortisol on differentiated osteoblasts. The nonreversible component of inhibition may be due to a more permanent effect of cortisol on cell populations within the calvariae. One possibility is that cortisol inhibits differentiation of osteoprogenitor cells to the osteoblast lineage leading to decreased osteoblast number and a subsequent inhibition of collagen production.