Impaired Accumulation of Ribonucleic Acid Precursors and Depletion of Ribonucleic Acid in Glucocorticoid-treated Bone Cells

We have examined the effects of cortisol in vitro on RNA metabolism in isolated, cultured bone cells. Treatment for as little as 2 hours decreased the incorporation of radioactively labeled nucleosides (uridine, adenosine, or guanosine) into RNA. This appears to result from reduced influx or phosphorylation (or both) of free nucleosides, since the accumulation of radioactivity in intracellular nucleotide pools (UMP, UDP, UTP) was also inhibited, but efflux of uridine nucleotides was not increased. Inhibition persisted after refeeding treated cells with fresh, cortisol-free medium, and was evident at a high (1 mm) as well as a low (13 µm) starting concentration of uridine in the incubation medium, suggesting that it was not caused by enhanced dilution of radioactive uridine in expanded intra- or extracellular pools of nonradioactive uridine. The possibility that the accumulation of radioactivity in nucleotide pools was inhibited by a feedback mechanism caused by prior reduction in RNA synthesis seems unlikely, in view of the simultaneous and equivalent effects of cortisol on the labeling of nucleotide pools and RNA. Treatment of bone cells with actinomycin D was accompanied by a decrease in the accumulation of RNA precursors only after inhibition of RNA synthesis was well established. Protein synthesis may be required for this response to cortisol since cortisol was ineffective when cells were treated simultaneously with either puromycin or cycloheximide, inhibitors of protein synthesis which did not in themselves block nucleoside incorporation. In addition to inhibiting the accumulation of RNA precursors and their incorporation into RNA, cortisol treatment gradually reduced the total content of RNA per culture (5 to 10% in 5 hours, 20 to 30% in 15 hours) and the amount of 18 S and 28 S ribosomal RNA per culture. Two observations suggest that this loss of RNA results in part from an acceleration of RNA breakdown: (a) the breakdown of endogenous, previously labeled RNA was increased in isolated microsomes derived from cortisol-treated cells and (b) addition of cortisol to cells which had been continuously labeled with radioactive uridine from initial seeding until treatment significantly enhanced the decline in RNA total radioactivity during the chase period. In addition, the rate of RNA synthesis, as estimated from the change in RNA specific radioactivity during the chase period, was decreased by treatment with cortisol. Cortisol effects were related to its glucocorticoid activity, and were directly proportional to its starting concentration, minimal changes appearing at near physiological concentrations (10 to 100 mµm).