Prenylation inhibitors stimulate both estrogen receptor alpha transcriptional activity through AF-1 and AF-2 and estrogen receptor beta transcriptional activity

Abstract

Introduction: We showed in a previous study that prenylated proteins play a role in estradiol stimulation of proliferation. However, these proteins antagonize the ability of estrogen receptor (ER) alpha to stimulate estrogen response element (ERE)-dependent transcriptional activity, potentially through the formation of a co-regulator complex. The present study investigates, in further detail, how prenylated proteins modulate the transcriptional activities mediated by ERalpha and by ERbeta.

Methods: The ERE-beta-globin-Luc-SV-Neo plasmid was either stably transfected into MCF-7 cells or HeLa cells (MELN cells and HELN cells, respectively) or transiently transfected into MCF-7 cells using polyethylenimine. Cells deprived of estradiol were analyzed for ERE-dependent luciferase activity 16 hours after estradiol stimulation and treatment with FTI-277 (a farnesyltransferase inhibitor) or with GGTI-298 (a geranylgeranyltransferase I inhibitor). In HELN cells, the effect of prenyltransferase inhibitors on luciferase activity was compared after transient transfection of plasmids coding either the full-length ERalpha, the full-length ERbeta, the AF-1-deleted ERalpha or the AF-2-deleted ERalpha. The presence of ERalpha was then detected by immunocytochemistry in either the nuclei or the cytoplasms of MCF-7 cells. Finally, Clostridium botulinum C3 exoenzyme treatment was used to determine the involvement of Rho proteins in ERE-dependent luciferase activity.

Results: FTI-277 and GGTI-298 only stimulate ERE-dependent luciferase activity in stably transfected MCF-7 cells. They stimulate both ERalpha-mediated and ERbeta-mediated ERE-dependent luciferase activity in HELN cells, in the presence of and in the absence of estradiol. The roles of both AF-1 and AF-2 are significant in this effect. Nuclear ERalpha is decreased in the presence of prenyltransferase inhibitors in MCF-7 cells, again in the presence of and in the absence of estradiol. By contrast, cytoplasmic ERalpha is mainly decreased after treatment with FTI-277, in the presence of and in the absence of estradiol. The involvement of Rho proteins in ERE-dependent luciferase activity in MELN cells is clearly established.

Conclusions: Together, these results demonstrate that prenylated proteins (at least RhoA, RhoB and/or RhoC) antagonize the ability of ERalpha and ERbeta to stimulate ERE-dependent transcriptional activity, potentially acting through both AF-1 and AF-2 transcriptional activities.

Figure 1

Effects of prenyltransferase inhibitors on estrogen response element (ERE)-dependent luciferase activity (a) in stably transfected MELN cells and (b) in transiently transfected MCF-7 cells. Both MELN cells and MCF-7 cells are deprived of estradiol (E2) for 4 days, and the MCF-7 cells are transiently transfected with the ERE-β-glob-Luciferase and the Renilla luciferase plasmids. Three hours after transfection, cells were treated or not with FTI-277 (10 μM or dithiothreitol/dimethylsulfoxide vehicle). Twenty-four hours later, cells were stimulated with E2 (5 nM) or ethanol and were treated or not with FTI-277 or GGTI-298 (10 μM or 5 μM, respectively, or dithiothreitol/dimethylsulfoxide vehicle). Luciferase activity was quantified 16 hours after E2 addition, as described in Materials and methods. Results are expressed in arbitrary units after normalization. Error bars indicate the mean values ± standard deviation from triplicate experiments, and the results are representative of two independent experiments. Results obtained show that prenylation inhibitors statistically increase the luciferase activity induced by E2 compared with the activity induced by E2 alone in MELN cells only (* P < 0.02).

Figure 2

Effects of prenyltransferase inhibitors on estrogen response element-dependent luciferase activity in HELN cells transfected with estrogen receptor (ER) α or ERβ. Cells, deprived of estradiol (E2) for 4 days, were co-transfected with the Renilla luciferase plasmid and either HEG0 (full-length ERα), pSG5-REβ (full-length ERβ) or pSG5 (empty vector). Five hours after transfection, cells were treated or not with FTI-277 (10 μM or dithiothreitol/dimethylsulfoxide vehicle), and 24 hours later they were stimulated with E2 (5 nM) or ethanol and were treated or not with FTI-277 or GGTI-298 (10 μM or 5 μM, respectively, or dithiothreitol/dimethylsulfoxide vehicle). Luciferase activity was quantified 16 hours after E2 addition, as described in Materials and methods. Results are expressed in arbitrary units after normalization. Error bars indicate the mean values ± standard deviation from triplicate experiments, and the results are representative of three independent experiments. Results obtained show that prenylation inhibitors statistically increase the luciferase activity on their own compared with control cells (white bars) and statistically increase the luciferase activity induced by E2 compared with the activity induced by E2 alone (grey bars) in MELN cells transfected with ERα or ERβ (* P < 0.05).

Figure 3

Effects of prenyltransferase inhibitors on estrogen response element-dependent luciferase activity in HELN cells, transfected with estrogen receptor (ER) α or its mutants. Cells, deprived of estradiol (E2) for 4 days, were co-transfected with the Renilla luciferase plasmid and either HEG0 (full-length ERα), HEG19 (AF-1-deleted ERα), HEG7 (AF-2-deleted ERα) or pSG5 (empty vector). Five hours after transfection, cells were treated or not with FTI-277 (10 μM or dithiothreitol/dimethylsulfoxide vehicle), and 24 hours later they were stimulated with E2 (5 nM) or ethanol and treated or not with FTI-277 or GGTI-298 (10 μM or 5 μM, respectively, or dithiothreitol/dimethylsulfoxide vehicle). Luciferase activity was quantified 16 hours after E2 addition, as described in Materials and methods. Results are expressed in arbitrary units after normalization. Error bars indicate the mean values ± standard deviation from triplicate experiments, and the results are representative of three independent experiments. Results obtained show that prenylation inhibitors statistically increase or do not statistically increase the luciferase activity on their own compared with control cells (white bars) and statistically increase the luciferase activity induced by E2 compared with the activity induced by E2 alone (grey bars) in HELN cells transfected with ERα or its mutants (* P < 0.02).

Figure 4

Immunocytochemical analysis of the effects of prenyltransferase inhibitors on estrogen receptor (ER) α distribution in MCF-7 cells. (a) Cells, deprived of estradiol (E2) for 7 days, were treated or not with FTI-277 (10 μM or dithiothreitol/dimethylsulfoxide vehicle), and 24 hours later they were stimulated with E2 (5 nM) or ethanol and treated or not with FTI-277 or GGTI-298 (10 μM or 5 μM, respectively, or vehicle). After 24 hours, cells were fixed and stained. One randomly selected field is presented for each treatment. (b) Data were quantified by determining the grey value of both the nucleus and the cytoplasm for each cell counted, as described in Materials and methods. For each experimental condition, six randomly selected fields were analyzed. The total number of cells present in the fields ranged from 250 to 400. Error bars indicate the mean values ± standard error of the mean, and the results are representative of two independent experiments.

Figure 5

Effect of C3 exoenzyme on estrogen response element-dependent luciferase activity in MCF-7 cells. Cells, deprived of estradiol (E2) for 4 days, were treated with C3 exoenzyme as described in Materials and methods. Twenty-four hours later the cells were stimulated with E2 (5 nM) or ethanol. Luciferase activities were quantified 16 hours after E2 addition, as described in Material and methods. Results are expressed in arbitrary units after protein normalization. Error bars indicate the mean values ± standard deviation from triplicate experiments, and the results are representative of two independent experiments.