Prenylation inhibitors stimulate both estrogen receptor alpha transcriptional activity through AF-1 and AF-2 and estrogen receptor beta transcriptional activity.
BACKGROUND
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.
Cestac
Philippe
P
Département Innovation Thérapeutique et Oncologie Moléculaire, Centre de Physiopathologie de Toulouse Purpan, INSERM U563 and Institut Claudius Regaud, Toulouse, France. cestac.p@chu-toulouse.fr
Sarrabayrouse
Guillaume
G
Médale-Giamarchi
Claire
C
Rochaix
Philippe
P
Balaguer
Patrick
P
Favre
Gilles
G
Faye
Jean-Charles
JC
Doisneau-Sixou
Sophie
S
D001549:Benzamides / Q000494:pharmacology; D001943:Breast Neoplasms / Q000473:pathology; D004122:Dimethylallyltranstransferase / Q000037:antagonists & inhibitors*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D047628:Estrogen Receptor alpha / Q000096:biosynthesis* / Q000235:genetics; D047629:Estrogen Receptor beta / Q000096:biosynthesis* / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D008156:Luciferases / Q000037:antagonists & inhibitors / Q000378:metabolism; D008715:Methionine / Q000031:analogs & derivatives / Q000494:pharmacology; D009154:Mutation; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured; D020741:rho GTP-Binding Proteins / Q000502:physiology*; D020742:rhoA GTP-Binding Protein / Q000502:physiology*; D020743:rhoB GTP-Binding Protein / Q000502:physiology*; D000075024:rhoC GTP-Binding Protein
D001549:Benzamides; D004791:Enzyme Inhibitors; D047628:Estrogen Receptor alpha; D047629:Estrogen Receptor beta; C096856:FTI 277; C102521:GGTI 298; D008715:Methionine; D008156:Luciferases; D004122:Dimethylallyltranstransferase; C066690:RHOC protein, human; D020741:rho GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein; D020743:rhoB GTP-Binding Protein; D000075024:rhoC GTP-Binding Protein