FLNA expression modulates pathological markers of pituitary neuroendocrine tumours.

Due to the current limited knowledge about the role of filamin A (FLNA) in pituitary tumour progression, we aimed to analyse FLNA expression levels and its impact on aggressive markers of pituitary neuroendocrine tumours (PitNETs), using an integrative approach of in vivo and in vitro models and human samples. An increase in the expression levels of FLNA was observed in the advanced tumoural stages of the hyperplastic adenomatous pituitary model, concomitant with a decrease in cell proliferation and with a modification in the subcellular localisation of this protein. Similarly, overexpression of FLNA in the somatolactotropic GH3 cell line induced a decrease in the cell proliferation, promoted a migratory phenotype, increased invasion activity, and decreased the prolactin secretion. Cyclin D1 (CCND1) and cyclin-dependent kinase 4 (CDK4) expression increased in both models in correlation with the increase observed in FLNA levels. When human tissues were analysed a significant increase of FLNA was observed in PitNETs compared to normal pituitary gland, with heterogeneous intracellular localisation. Higher levels of FLNA expression were observed in tumours with invasive characteristics. These results underline the crucial roles of FLNA as a modulator of pathological markers and as a potential prognostic marker in pituitary tumours.

Unraveling swine hepatitis E in the central region of Argentina through ELISA development and epidemiological insights.

Hepatitis E virus (HEV) is a public health concern globally, causing acute viral hepatitis in humans. Genotype-3 HEV (HEV-3), the most frequently genotype detected in South America, is zoonotic and the main reservoirs are the domestic pig and wild boar. Circulation of HEV-3 in Argentina has been confirmed in humans as well as in pig herds, wild boar and environmental waters. However, data are scarce mainly due to the inaccessibility of serological assays in this country. In order to provide insights in the epidemiology of HEV in swine in Argentina, we developed an indirect ELISA based on the native recombinant protein ORF2 and conducted a serological survey to determine the prevalence of seropositive swine in small-scale pig farms in the central region of Argentina. The method was evaluated in a panel of 157 serum samples, resulting in relative sensitivity of 98.6 % (95 % CI 95 %-100 %) and relative specificity of 97.7 % (95 % CI 94 %-100 %) compared to a commercial test. An almost perfect agreement was obtained between the two tests (Kappa index of 0.961). A survey on 294 samples from 49 small-scale farms resulted in a seropositivity rate of 54 %. Seropositive animals were found in 34 out of 49 (69.4 %) farms. Most of the farms (70.6 %) had over 50 % of seropositive animals. The wide spreading of HEV in the swine population of Tandil, Argentina, underscore the need to better understand the epidemiology of HEV in the region, enabling the implementation of targeted interventions to mitigate the impact of this virus on public health.

Exploring hepatitis E virus seroprevalence and associated risk factors among the human population in Tandil, Buenos Aires, Argentina.

Background: Hepatitis E virus (HEV) infection is a common cause of acute clinical hepatitis worldwide and is emerging as a disease in Argentina. It is primarily transmitted through contaminated water and food, following the fecal-oral route. Furthermore, is a zoonotic disease with swine as the primary reservoir. Prevalence of HEV infection in humans in several regions of Argentina remains unknown. Objectives: (i) Determine the seroprevalence of HEV among the human population in Tandil, Buenos Aires, Argentina; (ii) Evaluate its association with demographic, socioeconomic and other risk exposures variables, and (iii) Describe and analyze spatial patterns related to HEV infection. Methods: From August 2020 to July 2021, serum samples were collected from 969 individuals aged 1-80 years. Seroprevalence and 95% Confidence Interval was determined. To assess the factors associated with the presence of anti-HEV antibodies, associations between the variables and seropositivity were evaluated through bivariate and multivariate analysis. Spatial scanning for clusters of positivity was carried out. Factors associated with these clusters were also assessed. Results: Anti-HEV antibodies were detected in 4.64% (IC 95% 3.27-6.02) of samples. Dark urine was associated with seropositivity (p = 0.02). Seropositivity was linked with the presence of natural water courses near their households (p = 0.02); the age (p = 0.04); and previous travel to Europe (p = 0.04). A spatial cluster of low rates of HEV seropositivity was detected, with greater distance of the households to water courses associated to the cluster, and male sex inversely associated to it. Discussion and conclusion: This study is the first study to investigate the prevalence of HEV in the population from Tandil, Buenos Aires, Argentina. Considering HEV infection in the differential diagnosis in individuals presenting acute hepatitis is highlighted. The incorporation of HEV testing into blood screening policies should be mandatory. Factors related to the infection and spatial patterns of high and low risk were determined, and should be considered when implementing specific preventive measures.

A multispecies competitive nanobody-based ELISA for the detection of antibodies against hepatitis E virus.

The hepatitis E virus (HEV) is an emergent zoonotic virus causing viral hepatitis worldwide. Clinically, hepatitis E is not easily distinguished from other types of acute viral hepatitis. There is a need for HEV diagnostic assays to detect and prevent interspecies transmission among susceptible populations. Nanobodies (Nbs) are expressed recombinantly in different systems, produced with high yields, and have superior physicochemical properties compared with conventional antibodies (Ab). Several Nbs against ORF2, the capsid protein and main antigen, were selected and produced in E. coli. Nb39 and Nb74 specifically recognized HEV ORF2 (genotypes 3 and 4). A competitive ELISA (cELISA) was developed and validated using a reference panel of human (n = 86) and swine sera (n = 116) tested in comparison with a commercial kit. The optimal cutoff values determined by ROC analysis were 69.16% (human) and 58.76% (swine); the sensitivity and specificity were high: 97.4% (95% CI 86.5-99.5%) and 95.8% (95% CI 86.0-98.8%) for human vs. 100% (95% CI 93.5-100%) and 98.3% (95% CI 91.0-99.7%) for swine. Further, the cELISA detected total anti-HEV antibodies in wild boar, deer, and mice. To our knowledge, this is the first report of production of Nbs against HEV-3 ORF2 for diagnostic purposes.

Environmentally relevant DEHP exposure during gestational and lactational period inhibits filamin a testicular expression.

Toxicological studies have revealed that DEHP exposure during pregnancy may induce developmental disorders, especially in male offspring, leading to morphological and functional alterations in the reproductive system by mechanisms that should be investigated. Thus, the aim of this work was to analyze the testicular toxicity induced by an environmentally relevant DEHP dose during development and its impact on FLNA, a protein that participates in the blood-testis barrier assembly. We used male Wistar rats exposed to DEHP during pregnancy and lactation. The results showed that DEHP exposure during development and lactation increased body weight, decreased gonadal weight and shortened anogenital distance. This phthalate induced morphological changes in the testis, suggestive of hypospermatogenesis. DEHP exposure decreased the number of FLNA positive cells and the expression of FLNA and claudin-1 in prepubertal testes. Furthermore, DEHP inhibited FLNA and claudin-1 protein expression in adult male rats. These results indicated that exposure to DEHP during gestation and lactation perturbed testis development and suggested that FLNA is a target protein of DEHP, possibly contributing to the phthalate-induced damage on BTB.

Regulation of FGF2-induced proliferation by inhibitory GPCR in normal pituitary cells.

Introduction: Intracellular communication is essential for the maintenance of the anterior pituitary gland plasticity. The aim of this study was to evaluate whether GPCR-Gαi modulates basic fibroblast growth factor (FGF2)-induced proliferative activity in normal pituitary cell populations. Methods: Anterior pituitary primary cell cultures from Wistar female rats were treated with FGF2 (10ng/mL) or somatostatin analog (SSTa, 100nM) alone or co-incubated with or without the inhibitors of GPCR-Gαi, pertussis toxin (PTX, 500nM), MEK inhibitor (U0126, 100µM) or PI3K inhibitor (LY 294002, 10 µM). Results: FGF2 increased and SSTa decreased the lactotroph and somatotroph BrdU uptak2e (p<0.05) whereas the FGF2-induced S-phase entry was prevented by SSTa co-incubation in both cell types, with these effects being reverted by PTX, U0126 or LY294002 pre-incubation. The inhibition of lactotroph and somatotroph mitosis was associated with a downregulation of c-Jun expression, a decrease of phosphorylated (p) ERK and pAKT. Furthermore, SSTa was observed to inhibit the S-phase entry induced by FGF2, resulting in a further increase in the number of cells in the G1 phase and a concomitant reduction in the number of cells in the S phases (p< 0.05), effects related to a decrease of cyclin D1 expression and an increase in the expression of the cell cycle inhibitors p27 and p21. Discussion: In summary, the GPCR-Gαi activated by SSTa blocked the pro-proliferative effect of FGF2 in normal pituitary cells via a MEK-dependent mechanism, which acts as a mediator of both anti and pro-mitogenic signals, that may regulate the principal effectors of the G1 to S-phase transition.

Ultrastructural and Molecular Evidence of Macroautophagy in Functioning PitNETs and Experimental Pituitary Tumors.

INTRODUCTION: Macroautophagy is a lysosome-mediated degradation process that controls the quality of cytoplasmic components and organelles, with its regulation depending on autophagy-related proteins (Atg) and with Beclin1/Atg6 and microtubule-associated protein light chain 3 (LC3/Atg8) being key players in the mammalian autophagy. As reports on this mechanism in the field of pituitary neuropathology and neuroendocrinology are scarce, our study analyzed the ultrastructural signs of macroautophagy and the expression of Beclin1 and LC3 proteins in human functioning PitNETs and in experimental pituitary tumors. METHODS: A group of humans functioning PitNETs and an experimental lactotroph model in rats of the F344 strain stimulated with estradiol benzoate (BE) were used. Ultrastructural and molecular evidence of the macroautophagic process was evaluated using different techniques. RESULTS: In functioning PitNETs cohort, 60% exhibited evidence of macroautophagy, with a significant difference found for Beclin1 and LC3 between macro- and micro-PitNETs (p < 0.05). In the experimental model, the expression of both Beclin1 and LC3 proteins was immunopositive in normal and tumoral glands when analyzed by immunofluorescence, Western blot, and immunohistochemistry. In the experimental model, protein expression was associated with increased glandular size and weight. CONCLUSIONS: Our study revealed evidence of macroautophagy at the pituitary level and the important role of Beclin1 and LC3 in the progression of functioning PitNETs, implying that this mechanism participate in regulating pituitary cell growth.

Cationic polymer contributes to broaden the spectrum of vancomycin activity achieving eradication of Pseudomonas aeruginosa.

Vancomycin (VAN) is unable to penetrate the outer membrane of Gram-negative bacteria and reach the target site. One approach to overcome this limitation is to associate it with compounds with permeabilizing or antimicrobial properties. Eudragit E100® (Eu) is a cationic polymer insufficiently characterized for its potential antimicrobial action. Eu-VAN combinations were characterized, the antimicrobial efficacy against Pseudomonas aeruginosa was evaluated and previous studies on the effects of Eu on bacterial envelopes were extended. Time-kill assays showed eradication of P. aeruginosa within 3-6 h exposure to Eu-VAN, whilst VAN was ineffective. Eu showed regrowth in 24 h and delayed colony pigmentation. Although permeabilization of bacterial envelopes or morphological alterations observed by TEM and flow cytometry after exposure to Eu were insufficient to cause bacterial death, they allowed access of VAN to the target site, since Eu-VAN/Van-FL-treated cultures showed fluorescent staining in all bacterial cells, indicating Van-FL internalization. Consequently, Eu potentiated the activity of an otherwise inactive antibiotic against P. aeruginosa. Moreover, Eu-VAN combinations exhibited improved physicochemical properties and could be used in the development of therapeutic alternatives in the treatment of bacterial keratitis.

Oophorectomy improves pituitary activin inhibitory function preventing lactotroph hyperplasia development.

Among pituitary adenomas, prolactinomas are the most frequently diagnosed (about 50%). Dopamine agonists are generally effective in the treatment of prolactinomas. However, a subset of about 25% of patients does not respond to these agents. The management of drug-resistant prolactinomas remains a challenge for endocrinologists and new inhibitory treatments are needed. Pituitary activins inhibit lactotroph function. Its expression and action were found reduced in animal models of lactotroph hyperplasia (female mice overexpressing the B subunit of the human chorionic gonadotrophin and female mice knockout for dopamine receptor type 2). In these models, an oophorectomy avoids prolactinoma development. Hormonal replacement with oestradiol and/or progesterone is not enough to reach the tumor size observed in transgenic females. We postulated that the loss of gonadal inhibins after an oophorectomy contributes to prevent hyperplasia development. Here, we demonstrated that an oophorectomy at 2 months age recovers the following in adulthood: (i) pituitary activin expression, (ii) activin receptor expression specifically in lactotroph population, (iii) activin biological activity in lactotrophs with a concomitant reduction of Pit-1 expression. To summarize, when an oophorectomy is performed, inhibins are lost and the inhibitory action of pituitary activins on lactotroph population is recovered, helping to prevent lactotroph hyperplasia development. These results emphasize the importance of the inhibitory action of activins on lactotroph function, positioning activins as a good therapeutic target for the treatment of resistant prolactinomas.

Perinatal DEHP exposure modulates pituitary estrogen receptor α and ß expression altering lactotroph and somatotroph cell growth in prepuberal and adult male rats.

Phthalates are synthetic chemicals widely used to make polyvinylchloride (PVC) soft and flexible. Of these, Di-(2-ethylhexyl) phthalate (DEHP) is the most commonly used, with high human exposure occurring as early as the fetal developmental stage and affecting the endocrine system. We focused on the perinatal DEHP effects on pituitary estrogen receptor (ER) expression in male rats, explored their impact on lactotroph and somatotroph cell growth, and evaluated the direct effects of this phthalate on pituitary cell cultures. Our results showed that DEHP perinatal exposure was unable to modify the ERα+ pituitary cell number from prepuberal rats, but increased ERß+ cells. In adulthood, the pituitary ERα+ cells underwent a slight decrease with ERß showing the greatest changes, and with a significant increase observed in somatotroph cells. Also, in vitro, DEHP reduced the ERα+ cells, increased the percentage of ERß+ pituitary cells and modified the Ki67 index, as well as decreasing the lactotrophs and increasing the somatotroph cells. In conclusion, the present study showed that DEHP induced ER expression changes in normal pituitary glands from male rats in in vivo and in vitro conditions, suggesting that DEHP could differentially modulate lactotroph and somatotroph cell growth, possibly as a consequence of ER imbalance.

Changes of stem cell niche during experimental pituitary tumor development.

To investigate the putative stem cell/tumor stem cell (SC/TSC) niche contribution to hyperplasic/adenomatous pituitary lesions, we analyzed variation in the pituitary stem cell population during the development of experimental pituitary tumors. Pituitary tumors were induced in female F344 rats with estradiol benzoate for 5, 10, 20 and 30 days. Cells positive for GFRa2, Sox2, Sox9, Nestin, CD133 and CD44 were identified in the marginal zone and in the adenoparenchyma in both control and 30D groups, with predominant adenoparenchyma localization of GRFa2 and SOX9 found in tumoral pituitaries. GFRa2, Nestin, CD133 and CD44 were upregulated at the initial stages of tumor growth, whereas Sox9 significantly decreased at 5D, with Sox2 remaining invariable during the hyperplasic/adenomatous development. In addition, isolated pituispheres from normal and tumoral pituitary glands enriched in SC/TSC were characterized. Pituispheres from the 30D glands were positive for the above-mentioned markers and showed a significant increase in the proliferation. In conclusion, our data revealed pituitary SC pool fluctuations during hyperplastic/adenomatous development, with differential localization of the SC/TSC niche in this process. These findings may help to provide a better understanding of these cell populations, which is crucial for achieving advancements in the field of pituitary tumor biology.

TGF-ß1/Smad2/3 signaling pathway modulates octreotide antisecretory and antiproliferative effects in pituitary somatotroph tumor cells.

Octreotide (OCT) is used to inhibit hormone secretion and growth in somatotroph tumors, although a significant percentage of patients are resistant. It has also been tested in nonfunctioning (NF) tumors but with poor results, with these outcomes having been associated with SSTR2 levels and impaired signaling. We investigated whether OCT inhibitory effects can be improved by TGF-ß1 in functioning and nonfunctioning somatotroph tumor cells. OCT effects on hormone secretion and proliferation were analyzed in the presence of TGF-ß1 in WT and SSTR2-overexpressing secreting GH3 and silent somatotroph tumor cells. The mechanism underlying these effects was assessed by studying SSTR and TGFßR signaling pathways mediators. In addition, we analyzed the effects of OCT/TGF-ß1 treatment on tumor growth and cell proliferation in vivo. The inhibitory effects of OCT on GH- and PRL-secretion and proliferation were improved in the presence of TGF-ß1, as well as by SSTR2 overexpression. The OCT/TGF-ß1 treatment induced downregulation of pERK1/2 and pAkt, upregulation of pSmad3, and inhibition of cyclin D1. In vivo experiments showed that OCT in the presence of TGF-ß1 blocked tumor volume growth, decreased cell proliferation, and increased tumor necrosis. These results indicate that SSTR2 levels and the stimulation of TGF-ß1/TGFßR/Smad2/3 pathway are important for strengthening the antiproliferative and antisecretory effects of OCT.

Association of PTP4A3 expression and tumour size in functioning pituitary adenoma: a descriptive study.

BACKGROUND: PTP4A3 is a subclass of a protein tyrosine phosphatase super family and is expressed in a range of epithelial neoplasms. We evaluated PTP4A3 expression and its association with clinicopathological parameters in different types of functioning pituitary adenomas. METHODS: A total of 34 functioning pituitary adenomas samples were evaluated in this observational study. PTP4A3 expression was examined by immunohistochemical staining, and, possible correlations between PTP4A3 and some clinicopathological variables were investigated. RESULTS: PTP4A3 was expressed in 19 out of 34 tumours (55%), at the cytoplasmic level of tumorous cells. Moreover, there was significant association (p=0.042) between PTP4A3 expression and tumorous size. CONCLUSIONS: PTP4A3 was expressed in more than half of the tumours analysed, with there being a significant association with the tumorous size of functioning adenomas. This allows to speculate that PTP4A3 may regulate tumour growth, although further investigations are necessary to determine if this phosphatase can serve as a biomarker or used as a therapeutic target in pituitary macroadenomas.

The phthalate DEHP modulates the estrogen receptors α and ß increasing lactotroph cell population in female pituitary glands.

Humans are exposed to numerous endocrine disruptors on a daily basis, which may interfere with endogenous estrogens, with Di-(2-ethylhexyl) phthalate (DEHP) being one of the most employed. The anterior pituitary gland is a target of 17ß-estradiol (E2) through the specific estrogen receptors (ERs) α and ß, whose expression levels fluctuate in the gland under different contexts, and the ERα/ß index is responsible for the final E2 effect. The aim of the present study was to evaluate in vivo and in vitro the DEHP effects on ERα and ß expression in the pituitary cell population, and also its impact on lactotroph and somatotroph cell growth. Our results revealed that perinatal exposure to DEHP altered the ERα and ß expression pattern in pituitary glands from prepubertal and adult female rats and increased the percentage of lactotroph cells in adulthood. In the in vitro system, DEHP down-regulated ERα and ß expression, and as a result increased the ERα/ß ratio and decreased the percentages of lactotrophs and somatotrophs expressing ERα and ß. In addition, DEHP increased the S + G2M phases, Ki67 index and cyclin D1 in vitro, leading to a rise in the lactotroph and somatotroph cell populations. These results showed that DEHP modified the pituitary ERα and ß expression in lactotrophs and somatotrophs from female rats and had an impact on the pituitary cell growth. These changes in ER expression may be a mechanism underlying DEHP exposure in the pituitary gland, leading to cell growth deregulation.

TGFß1 regulates prolactin secretion during postnatal development: gender differences.

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 (TGFß1) 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 TGFß1 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 TGFß1 activation and its impact on prolactin secretion. At day 11, female pituitaries present high levels of active TGFß1, concomitant with the highest expression of TGFß1 target genes and the phospho-Smad3 immunostaining in lactotrophs. The steady increase in prolactin secretion inversely correlates with active TGFß1 levels only in females. Dopamine and estradiol induce TGFß1 activation at day 11, in both genders, but its activation induces the inhibition of prolactin secretion only in females. Our findings demonstrate that: (1) TGFß1 activation is regulated by dopamine and estradiol; (2) the inhibitory regulation of local TGFß1 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.

Pivotal role of NF-κB in cellular senescence of experimental pituitary tumours.

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-ß-Gal co-reactivity in the tumour pituitary parenchyma. The induction of TNFα and IL1-ß 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-ß-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.

Early events following bovine leukaemia virus infection in calves with different alleles of the major histocompatibility complex DRB3 gene.

Cattle maintaining a low proviral load (LPL) status after bovine leukaemia virus (BLV) infection have been recognized as BLV controllers and non-transmitters to uninfected cattle in experimental and natural conditions. LPL has been associated with host genetics, mainly with the BoLA class II DRB3 gene. The aim of this work was to study the kinetics of BLV and the host response in Holstein calves carrying different BoLA-DRB3 alleles. Twenty BLV-free calves were inoculated with infected lymphocytes. Two calves were maintained uninfected as controls. Proviral load, total leukocyte and lymphocyte counts, anti-BLVgp51 titres and BLVp24 expression levels were determined in blood samples at various times post-inoculation. The viral load peaked at 30 days post-inoculation (dpi) in all animals. The viral load decreased steadily from seroconversion (38 dpi) to the end of the study (178 dpi) in calves carrying a resistance-associated allele (*0902), while it was maintained at elevated levels in calves with *1501 or neutral alleles after seroconversion. Leukocyte and lymphocyte counts and BLVp24 expression did not significantly differ between genetic groups. Animals with < 20 proviral copies/30 ng of DNA at 178 dpi or < 200 proviral copies at 88 dpi were classified as LPL, while calves with levels above these limits were considered to have high proviral load (HPL) profiles. All six calves with the *1501 allele progressed to HPL, while LPL was attained by 6/7 (86%) and 2/6 (33%) of the calves with the *0902 and neutral alleles, respectively. One calf with both *0902 and *1501 developed LPL. This is the first report of experimental induction of the LPL profile in cattle.

mPRs represent a novel target for PRL inhibition in experimental prolactinomas.

Membrane progesterone receptors are known to mediate rapid nongenomic progesterone effects in different cell types. Recent evidence revealed that mPRα is highly expressed in the rat pituitary, being primarily localized in lactotrophs, acting as an intermediary of P4-inhibitory actions on prolactin secretion. The role of mPRs in prolactinoma development remains unclear. We hypothesize that mPR agonists represent a novel tool for hyperprolactinemia treatment. To this end, pituitary expression of mPRs was studied in three animal models of prolactinoma. Expression of mPRs and nuclear receptor was significantly decreased in tumoral pituitaries compared to normal ones. However, the relative proportion of mPRα and mPRß was highly increased in prolactinomas. Interestingly, the selective mPR agonist (Org OD 02-0) significantly inhibited PRL release in both normal and tumoral pituitary explants, displaying a more pronounced effect in tumoral tissues. As P4 also regulates PRL secretion indirectly, by acting on dopaminergic neurons, we studied mPR involvement in this effect. We found that the hypothalamus has a high expression of mPRs. Interestingly, both P4 and OrgOD 02-0 increased dopamine release in hypothalamus explants. Moreover, in an in vivo treatment, that allows both, pituitary and hypothalamus actions, the mPR agonist strongly reduced the hyperprolactinemia in transgenic females carrying prolactinoma. Finally, we also found and interesting gender difference: males express higher levels of pituitary mPRα/ß, a sex that does not develop prolactinoma in these mice models. Taken together, these findings suggest mPRs activation could represent a novel tool for hyperprolactinemic patients, especially those that present resistance to dopaminergic drugs.

The ERα membrane pool modulates the proliferation of pituitary tumours.

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.