Gonadotropins decrease estrogen receptor-beta messenger ribonucleic acid stability in rat granulosa cells

We have previously shown that the preovulatory LH surge down-regulates estrogen receptor-beta (ERbeta) messenger RNA (mRNA) levels selectively in the granulosa cells of preovulatory follicles. To gain insight into the underlying mechanisms, we examined whether the LH-induced loss of ERbeta mRNA expression in rat granulosa cells is attributable to the hormone-induced changes at the level of transcription and/or mRNA degradation. When the rate of ERbeta gene transcription was assessed in cultured granulosa cells, by nuclear run-off assays, we observed only a marginal effect of hCG on ERbeta gene transcription. In contrast, when ERbeta mRNA levels were estimated in granulosa cells that were cultured in the presence of 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), an RNA synthesis inhibitor, we observed a significant inhibitory effect of human CG (hCG) on ERbeta mRNA expression at a magnitude similar to that observed in the absence of DRB. Forskolin (FSK) and 2-O-tetradecanol-phorbol-13-acetate (TPA), pharmacological agents that mimic LH actions in granulosa cells, also showed similar effects. Thus, these results suggest that LH decreases ERbeta mRNA expression in the granulosa cells of preovulatory follicles, primarily by destabilizing the preexisting ERbeta mRNA. We next determined the decay rate of the ERbeta mRNA in granulosa cells that were cultured in the presence of DRB and additional hCG, FSK, or TPA for various time periods, by estimating ERbeta mRNA levels, using semiquantitative RT-PCR assays and subsequent linear regression analyses. The half-life of the ERbeta mRNA in the presence of vehicle was 17.87 +/- 1.2 h (n = 4). hCG dramatically decreased the half-life of the ERbeta mRNA (4.85 +/- 0.49 h, n = 4). Similarly, both FSK and TPA decreased the half-life of the ERbeta mRNA to 3.57 +/- 0.31 h and 4.02 +/- 0.13 h, respectively. We extended these findings by examining whether the LH-induced down-regulation of the ERbeta mRNA is cycloheximide-sensitive. When granulosa cells were cultured in the presence of cycloheximide, a protein synthesis inhibitor, the inhibitory effects of hCG, FSK, and TPA on ERbeta mRNA levels were abolished. Similar results were obtained in the presence or absence of DRB, indicating that the hormone-induced destabilization of the ERbeta mRNA is coupled with translation processes. Taken together, our results demonstrate that LH decreases ERbeta mRNA expression, predominantly at the posttranscriptional level, in a cycloheximide-sensitive manner.