RU-486 (RU 38486; mifepristone) is a steroid that antagonizes many of the documented in vitro and in vivo effects of GCs such as DEX, by competing for the ligand binding site on the GR and inducing a modification of the three-dimensional structure of the bound complex that prevents transactivation.
56,57 RU-486 inhibits DEX-induced myocilin expression in cultured human TM cells,
58 and we endeavored to replicate this result for myocilin protein release by cultured monkey TM, for both DEX and BOL-303242-X, and to establish that both of these compounds' effects in our model were mediated via the GR. Using one of the monkey TM cell strains used in the previous studies, and with DEX and BOL-303242-X each at 30 nM, i.e., within a log
10 unit of their EC
50s with respect to myocilin expression (
Table 2), co-incubation with RU-486 in a concentration range from 10 to 1000 nM resulted in dose-dependent abrogation of both DEX- and BOL-303242-X–induced elevations in myocilin protein detected in CM (
Fig. 6, left, middle). Myocilin protein values were reduced to vehicle control levels at the highest RU-486 concentration tested, 1000 nM (
Fig. 6, left, middle). In the context of the concentration of either agonist compound tested, the estimated EC
50s for RU-486 were not significantly different (15.48 ± 6.03 nM, with 95% confidence limits of 7.21 and 33.22 nM in the presence of DEX; 11.74 ± 4.37 nM, with 95% confidence limits of 5.66 and 24.37 nM for BOL-303242-X). Similar to previous findings by Shepard et al.,
58 at a few concentrations intermediate in the dose range, RU-486 alone caused some slight but statistically significant increases in myocilin protein expression (
Fig. 6, right), indicating low level partial agonism. These data demonstrated that RU-486 was a highly effective antagonist, with equivalent potency for inhibiting induction of myocilin protein by both BOL-303242-X and DEX, and suggested that these latter two compounds both exerted their effects in monkey TM cells via the classic GR.