The ERα membrane pool modulates the proliferation of pituitary tumours

in Journal of Endocrinology
Correspondence should be addressed to A I Torres: atorres@cmefcm.uncor.edu

*(L d V Sosa and J P Petiti contributed equally to this work)

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The molecular mechanisms underlying the ERα nuclear/cytoplasmic pool that modulates pituitary cell proliferation have been widely described, but it is still not clear how ERα is targeted to the plasma membrane. The aim of this study was to analyse ERα palmitoylation and the plasma membrane ERα (mERα) pool, and their participation in E2-triggered membrane-initiated signalling in normal and pituitary tumour cell growth. Cell cultures were prepared from anterior pituitaries of female Wistar rats and tumour GH3 cells, and treated with 10 nM of oestradiol (E2). The basal expression of ERα was higher in tumour GH3 than in normal pituitary cells. Full-length palmitoylated ERα was observed in normal and pituitary tumour cells, demonstrating that E2 stimulation increased both, ERα in plasma membrane and ERα and caveolin-1 interaction after short-term treatment. In addition, the Dhhc7 and Dhhc21 palmitoylases were negatively regulated after sustained stimulation of E2 for 3 h. Although the uptake of BrdU into the nucleus in normal pituitary cells was not modified by E2, a significant increase in the GH3 tumoural cell, as well as ERK1/2 activation, with this effect being mimicked by PPT, a selective antagonist of ERα. These proliferative effects were blocked by ICI 182780 and the global inhibitor of palmitoylation. These findings indicate that ERα palmitoylation modulated the mERα pool and consequently the ERK1/2 pathway, thereby contributing to pituitary tumour cell proliferation. These results suggest that the plasma membrane ERα pool might be related to the proliferative behaviour of prolactinoma and may be a marker of pituitary tumour growth.

 

      Society for Endocrinology

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    (A) Basal expression of total REa in normal and pituitary tumour cells and after E2 treatment for 30 min. (B) Protein extracts from pituitary cells were tested for palmitoylated ERα following the ABE assay. Biotinylated proteins were purified, separated by SDS-PAGE and stained with anti-REα antibody. ERα palmitoylation is prominent in protein extracts that were treated with hydroxylamine (H). In addition, in GH3-transfected SX8-Cher tumour cells, palmitoylated SX8 was used as a control of palmitoylated protein. The input in (A) and (B) was 10% of total protein extract and Tris: pull-down without H. Relative mRNA expression levels of Dhhc7 (C) and Dhhc21 (D) in normal pituitary and GH3 tumour cells. Dhhc7 (E), Dhhc21 (F) and Dhhc11 (G) mRNA expression levels after treatment with E2 (10 nM) at 3, 6 and 9 h. A significant decrease in the Dhhc7 and Dhhc21 mRNA levels was observed in normal and GH3 cells stimulated with E2 for 3 h compared to control. Expression levels were calculated by quantitative real-time PCR analysis. The β-actin gene was used as the internal reference gene and the ΔΔCT method was used for relative quantification and expressed as fold over control: *P < 0.05 Dhhc7 vs C and ^P < 0.05 Dhhc21 vs C.

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    Cell-surface immunostaining of ERα in non-permiabilised normal (A) and tumour GH3 (B) cell cultures treated with E2 (10 nM) for 30 min with or without pre-treatment of 2BP. Microphotograph represent the merging of light transmitted and immunofluorescence field. CLEM of normal pituitary (C) and GH3 tumour (D) rat cell cultures were treated with E2. (1) Immunofluorescence labelling for ERα, (2) TEM images for the whole section and (3) CLEM overlay of TEM on the corresponding FLM image. Arrows indicate ERα at plasma membrane. Bar: 10 µm. Biotinylation of cell-surface biotinylated proteins in normal pituitary and tumour GH3 rat cells. Surface membrane proteins were biotinylated in normal control (E) and tumour GH3 (F) pituitary cells or in cells treated with E2 (10 nM) for 30 min with or without 2BP pre-incubation. Whole-cell lysates were subjected to avidin pull-down using streptavidin-agarose beads, and the recovered cell-surface biotinylated proteins (pellet) and intracellular unbiotinylated proteins (supernatant) were analysed by western blot and stained with anti-REα antibody. In the pellet fraction, ERα expression showed an increase after E2 treatment but a decrease after pre-treatment with 2BP. The ERα expression did not change after treatment in the supernatant. The expression of the FGF and EGF receptors and β-actin confirmed equivalent total protein loading. Images correspond to a representative experiment from a total of three with similar results. Values are expressed as mean ± s.e.m. *P < 0.05 E2 vs C and ^P < 0.05 E2-2BP vs E2. A full colour version of this figure is available at https://doi.org/10.1530/JOE-18-0418.

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    Association between ERα and caveolin 1. Normal pituitary (A) and tumour GH3 (C) rat cell cultures were treated with E2 (10 nM) for 30 min with or without pre-treatment of 2BP. The total cell extracts were used for immunoprecipitation (IP) using anti-ERα and the immunoprecipitates were then probed with anti-cav-1. *P < 0.01 E2 vs C and ^P < 0.05 E2-2BP vs E2. In total anterior pituitary cell culture lysates, both antibodies recognised the antigens (input). Electron micrographs of normal pituitary (B) and tumour GH3 (D) with double immune-labelling of ERα (15 nm gold particle) and cav-1 (5 nm gold particle). The black arrows indicate the ERα-cav-1 adhered to the plasma membrane and the white arrows indicate the cytosolic ERα. g, granule; bar: 100 nm.

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    Representative micrographs of immunohistochemical staining for BrdU (red) and quantitative analysis in normal (A and B) and tumour GH3 (C and D) cell cultures. The cells were treated with E2 (10 nM) or PPT (10 nM) for 30 min with or without pre-treatment with 2BP or ICI 182780 (I, 100 nM). BrdU was added for an additional 24 h and the data represent the proportion of positive BrdU cells in the total cells. Significant differences were found in GH3 cell proliferation: *P < 0.01 E2 or PPT vs C and ^P < 0.05 I+ E2 vs E2. #P < 0.05 E2 or PPT vs E2 or PPT and ~P < 0.05 2BP-I+E2 vs E2. Bar: 100 µm. (E) Western blotting of ERK1/2 and Akt in total extract from tumour pituitaries treated with E2 (10 nM) or PPT (10 nM) for 30 min with or without pre-treatment of 2BP or ICI 182780 (I, 100 nM). The amount of protein levels was normalised by comparison with total ERK1/2 and AKT expression A representative panel of three independent experiments is shown. (F and G) Quantitative analysis of BrdU staining and western blotting of ERK1/2 in total extract from cells treated with EGF (10 ng/mL) for 30 min with or without pre-treatment with 2BP (10 µM). Significant differences were found in GH3 cell proliferation: *P < 0.01 EGF vs C, while that cell proliferation and ERK1/2 phosphorylation did not change with 2BP treatment. A full colour version of this figure is available at https://doi.org/10.1530/JOE-18-0418.

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