Ovarian steroids control a variety of physiological functions. They exert actions through classical nuclear steroid receptors, but rapid non-genomic actions through specific membrane steroid receptors have been also described. In this study, we demonstrate that the G-protein-coupled estrogen receptor (GPER) is expressed in the rat pituitary gland and, at a high level, in the lactotroph population. Our results revealed that ~40% of the anterior pituitary cells are GPER positive and ~35% of the lactotrophs are GPER positive. By immunocytochemical and immuno-electron-microscopy studies, we demonstrated that GPER is localized in the plasmatic membrane but is also associated to the endoplasmic reticulum in rat lactotrophs. Moreover, we found that local Gper expression is regulated negatively by 17β-estradiol (E2) and progesterone (P4) and fluctuates during the estrus cycle, being minimal in proestrus. Interestingly, lack of ovarian steroids after an ovariectomy (OVX) significantly increased pituitary GPER expression specifically in the three morphologically different subtypes of lactotrophs. We found a rapid estradiol stimulatory effect on PRL secretion mediated by GPER, both in vitro and ex vivo, using a GPER agonist G1, and this effect was prevented by the GPER antagonist G36, demonstrating a novel role for this receptor. Then, the increased pituitary GPER expression after OVX could lead to alterations in the pituitary function as all three lactotroph subtypes are target of GPER ligand and could be involved in the PRL secretion mediated by GPER. Therefore, it should be taken into consideration in the response of the gland to an eventual hormone replacement therapy.
María Andrea Camilletti, Alejandra Abeledo-Machado, Jimena Ferraris, Pablo A Pérez, Erika Y Faraoni, Daniel Pisera, Silvina Gutierrez and Graciela Díaz-Torga
Liliana del V Sosa, Juan P Petiti, Florencia Picech, Sabrina Chumpen, Juan P Nicola, Pablo Perez, Ana De Paul, Javier Valdez-Taubas, Silvina Gutierrez and Alicia I Torres
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.
Alejandra Abeledo-Machado, Pablo Anibal Pérez, María Andrea Camilletti, Erika Yanil Faraoni, Florencia Picech, Juan Pablo Petiti, Silvina Gutiérrez and Graciela Diaz-Torga
Serum prolactin levels gradually increase from birth to puberty in both male and female rats, with higher levels observed in female since the first days of life. The increase in lactotroph secretion was attributed to the maturation of prolactin-inhibiting and prolactin-releasing factors; however, those mechanisms could not fully explain the gender differences observed. Prolactin secretion from isolated lactotrophs, in the absence of hypothalamic control, also increases during the first weeks of life, suggesting the involvement of intra-pituitary factors. We postulate that pituitary transforming growth factor beta 1 (TGFβ1) is involved in the regulation of prolactin secretion as well as in the gender differences observed at early postnatal age. Several components of the local TGFβ1 system were evaluated during postnatal development (11, 23, and 45 days) in female and male Sprague–Dawley rats. In vivo assays were performed to study local TGFβ1 activation and its impact on prolactin secretion. At day 11, female pituitaries present high levels of active TGFβ1, concomitant with the highest expression of TGFβ1 target genes and the phospho-Smad3 immunostaining in lactotrophs. The steady increase in prolactin secretion inversely correlates with active TGFβ1 levels only in females. Dopamine and estradiol induce TGFβ1 activation at day 11, in both genders, but its activation induces the inhibition of prolactin secretion only in females. Our findings demonstrate that: (1) TGFβ1 activation is regulated by dopamine and estradiol; (2) the inhibitory regulation of local TGFβ1 on prolactin secretion is gender specific; and (3) this mechanism is responsible, at least partially, for the gender differences observed being relevant during postnatal development.
Bethania Mongi-Bragato, Ezequiel Grondona, Liliana del Valle Sosa, Natacha Zlocowski, Ana Clara Venier, Alicia Inés Torres, Alexandra Latini, Rodrigo Bainy Leal, Silvina Gutiérrez and Ana Lucía De Paul
The molecular mechanisms underlying the capability of pituitary tumours to avoid unregulated cell proliferation are still not well understood. However, the NF-κB transcription factor, which is able to modulate not only cellular senescence but also tumour progression, has emerged as a targeted candidate. This work was focused on the NF-κB role in cellular senescence during the progression of experimental pituitary tumours. Also, the contribution of the signalling pathways in senescence-associated NF-κB activation and the senescence-associated secretory phenotype (SASP) and pro-survival-NF-κB target genes transcription were analysed. A robust NF-κB activation was seen at E20–E40 of tumour development accompanied by a marked SA-β-Gal co-reactivity in the tumour pituitary parenchyma. The induction of TNFα and IL1-β as specific SASP-related NF-κB target genes as well as Bcl-2 and Bcl-xl pro-survival genes was shown to be accompanied by increases in the p-p38 MAPK protein levels, starting at the E20 stage and strengthening from 40 to 60 days of tumour growth. It is noteworthy that p-JNK displayed a similar pattern of activation during pituitary tumour development, while p-AKT and p-ERK1/2 were downregulated. By employing a pharmacological strategy to abrogate NF-κB activity, we demonstrated a marked reduction in SA-β-Gal activity and a slight decrease in Ki67 immunopositive cells after NF-κB blockade. These results suggest a central role for NF-κB in the regulation of the cellular senescence programme, leading to the strikingly benign intrinsic nature of pituitary adenomas.