Glucocorticoids Stimulate Protein Degradation in Lymphocytes: A Possible Mechanism of Steroid-Induced Cell Death*

Abstract

The influence of glucocorticoids on protein degradation was studied in rat splenic lymphocytes in vitro using a pulse-chase method. Degradation curves for [³H]leucine-labeled protein in control cells were biphasic, having fractional protein degradative rates (k_p) of 0.0359 h^-1 during the first 3 h and 0.0089 h^-1 during the last 5 h of an 8-h incubation period. Cells treated with 1 μM dexamethasone [9α-fluoro-11β,17,21-trihydroxy-16αmethyl- pregna-l,4-diene-3,20-dione (DEX)] degraded labeled protein at a nearly constant rate during the 8-h period, with k_p values of 0.0398 h^-1 from 0–3 h and 0.0329 h^-1 during the last 5 h of hormone exposure. This glucocorticoid-induced increase in the protein-degradative rate begins after about 3 h of hormone treatment and represents a 270% increase over the proteolytic rate in control cells. Lymphocytes labeled in vitro with [³H]phenylalanine or in vivo with [³H]leucine also respond to DEX in vitro through comparable increases in degradation of labeled protein. Concomitant with this stimulation of cellular proteolysis, glucocorticoids increase the rate of cell death in splenic lymphocytes in vitro. This hormone effect also begins after 2–3 h of incubation with 1 μM DEX, and results in a 4-fold faster rate of cell death in hormone-treated cells than in controls. Glucocorticoid-induced increases in the rates of cell death and protein degradation are both dependent on the concentration of hormone employed, with half-maximal responses observed at DEX concentrations of 0.5 and 0.2 nM, respectively. Both effects are specific only for active glucocorticoids. To investigate further the relationship between these two glucocorticoid effects, viable and nonviable subpopulations of DEX-treated and control cells were separated from each other during the course of a 12-hincubation. These studies produced the rather surprising discovery that there was no detectable difference in the quantity of radioactive protein remaining in glucocorticoid-treated and control viable cells. In contrast, glucocorticoid-treated nonviable cells contained only 60% of the labeled protein present in controlnonviable cells at every time period studied (4, 8, and 12 h of hormone exposure). These data show that the cells which were killed by DEX treatment were also those that responded to the hormone through a stimulation in protein degradation. Our findings also suggest that glucocorticoid-induced proteolysis occurs just before cell death in splenic lymphocytes, since glucocorticoid- killed cells and those which died in the absence of hormone do not degrade labeled protein. The implications of these observations are discussed in relation to the mechanism of glucocorticoid-induced lymphocytolysis.