Regulatory effect of estradiol mediated by ERalfa palmitoylation on the proliferative activity in normal and tumoral adenohypophyseal cells

SOSA L DEL V.; PICECH, F; PETITI J P; VALDEZ-TAUBAS, J; CHUMPEN S; GUIDO, C; VACA, AM; GUTIÉRREZ, S; NICOLA, JP; TORRES, A I; DE PAUL, A L

Praga

Congreso; 12th International Congress of Cell Biology; 2016

Czech Society for Cell Biology

Previously, we have demonstrated that estradiol (E2), through membrane estrogen receptor α (mERα), modulates the functional activity in adenohypophysial cells. One requirement for ER location at the plasma membrane is the presence of a hydrophobic segment as a part of the receptor structure, being reported the addition of a palmitate molecule, commonly called palmitoylation, in steroid receptors. The aim of this study was to analyze the ERα palmitoylation and their implication in E2-triggered membrane-initiated signaling in normal and tumoral pituitary cell proliferation. Adenohypophysial cells from female Wistar rats and tumoral GH3 cell were exposed to E2 (10 nM) for 30 min. Palmitoylation of REα was determined by acyl-biotin exchange method; mREα expression was evaluated by membrane proteins biotinylation and mREα and caveolin 1 (cav1) association by immunoprecipitation. Cell proliferation was analyzed by BrdU uptake and REα, cav-1 and phosphorylated and total ERK1/2 expression by WB. The palmitoylases participation was evaluated using the inhibitor 2-bromo palmitate (2BP, 10 uM) and the DHHC-7 and DHHC-21 mRNA levels were determined by qPCR after 3, 6, 9 h of E2 stimulation. Statistics: Test-t. The ERα palmitoylated was detected in normal and GH3 adenohypophysis cells. The general inhibitor of palmitoylation induced loss of mERα location and inhibited the REα/cav1 association in both cell culture conditions. In addition, E2 treatment was able to regulate DHHC-7 and -21 expressions showing a decrease in mRNA levels at 3h, reaching similar levels to control after 6h. In primary cultures, the BrdU uptake was not modified by E2 treatment, whereas ERK1/2 phosphorylation was inhibited by 2BP. In contrast, GH3 cell proliferation was stimulated after E2 treatment, data in correlation with the significant increase of ERK1/2 phosphorylation levels, effects that were inhibited by 2BP. These findings suggest that ERα palmitoylated contribute to E2 effects initiated at the plasma membrane triggering the tumoral GH3 cells proliferation and that E2 could modulate to DHHC-7 and -21 enzymes thereby regulating mERα expression.