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.

Cuprous oxide nanoparticles incorporated into a polymeric matrix embedded in fabrics to prevent spread of SARS-CoV-2.

This paper describes the development of a coating for cotton and polypropylene (PP) fabrics based on a polymeric matrix embedded with cuprous oxide nanoparticles (Cu2O@SDS NPs) in order to inactivate SARS-CoV-2 and manufactured by a simple process using a dip-assisted layer-by-layer technology, at low curing temperature and without the need for expensive equipment, capable of achieving disinfection rates of up to 99%. The polymeric bilayer coating makes the surface of the fabrics hydrophilic, enabling the transportation of the virus-infected droplets to achieve the rapid inactivation of SARS-CoV-2 by contact with the Cu2O@SDS NPs incorporated in the coated fabrics.

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.

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.

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.

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.

Trastuzumab inhibits pituitary tumor cell growth modulating the TGFB/SMAD2/3 pathway.

In pituitary adenomas, early recurrences and resistance to conventional pharmacotherapies are common, but the mechanisms involved are still not understood. The high expression of epidermal growth factor receptor 2 (HER2)/extracellular signal-regulated kinase (ERK1/2) signal observed in human pituitary adenomas, together with the low levels of the antimitogenic transforming growth factor beta receptor 2 (TBR2), encouraged us to evaluate the effect of the specific HER2 inhibition with trastuzumab on experimental pituitary tumor cell growth and its effect on the antiproliferative response to TGFB1. Trastuzumab decreased the pituitary tumor growth as well as the expression of ERK1/2 and the cell cycle regulators CCND1 and CDK4. The HER2/ERK1/2 pathway is an attractive therapeutic target, but its intricate relations with other signaling modulators still need to be unraveled. Thus, we investigated possible cross-talk with TGFB signaling, which has not yet been studied in pituitary tumors. In tumoral GH3 cells, co-incubation with trastuzumab and TGFB1 significantly decreased cell proliferation, an effect accompanied by a reduction in ERK1/2 phosphorylation, an increase of SMAD2/3 activation. In addition, through immunoprecipitation assays, a diminution of SMAD2/3-ERK1/2 and an increase SMAD2/3-TGFBR1 interactions were observed when cells were co-incubated with trastuzumab and TGFB1. These findings indicate that blocking HER2 by trastuzumab inhibited pituitary tumor growth and modulated HER2/ERK1/2 signaling and consequently the anti-mitogenic TGFB1/TBRs/SMADs cascade. The imbalance between HER2 and TGFBRs expression observed in human adenomas and the response to trastuzumab on experimental tumor growth may make the HER2/ERK1/2 pathway an attractive target for future pituitary adenoma therapy.

Oxidative stress and mitochondrial adaptive shift during pituitary tumoral growth.

The cellular transformation of normal functional cells to neoplastic ones implies alterations in the cellular metabolism and mitochondrial function in order to provide the bioenergetics and growth requirements for tumour growth progression. Currently, the mitochondrial physiology and dynamic shift during pituitary tumour development are not well understood. Pituitary tumours present endocrine neoplastic benign growth which, in previous reports, we had shown that in addition to increased proliferation, these tumours were also characterized by cellular senescence signs with no indication of apoptosis. Here, we show clear evidence of oxidative stress in pituitary cells, accompanied by bigger and round mitochondria during tumour development, associated with augmented biogenesis and an increased fusion process. An activation of the Nrf2 stress response pathway together with the attenuation of the oxidative damage signs occurring during tumour development were also observed which will probably provide survival advantages to the pituitary cells. These neoplasms also presented a progressive increase in lactate production, suggesting a metabolic shift towards glycolysis metabolism. These findings might imply an oxidative stress state that could impact on the pathogenesis of pituitary tumours. These data may also reflect that pituitary cells can modulate their metabolism to adapt to different energy requirements and signalling events in a pathophysiological situation to obtain protection from damage and enhance their survival chances. Thus, we suggest that mitochondria function, oxidative stress or damage might play a critical role in pituitary tumour progression.

The expansion of adult stem/progenitor cells and their marker expression fluctuations are linked with pituitary plastic adaptation during gestation and lactancy.

Extensive evidence has revealed variations in the number of hormone-producing cells in the pituitary gland, which occur under physiological conditions such as gestation and lactancy. It has been proposed that new hormone-producing cells differentiate from stem cells. However, exactly how and when this takes place is not clear. In this work, we used immunoelectron microscopy to identify adult pituitary stem/progenitor cells (SC/P) localized in the marginal zone (MZ), and additionally, we detected GFRa2-, Sox2-, and Sox9-positive cells in the adenoparenchyma (AP) by fluorescence microscopy. Then, we evaluated fluctuations of SC/P mRNA and protein level markers in MZ and AP during gestation and lactancy. An upregulation in stemness markers was shown at term of gestation (AT) in MZ, whereas there were more progenitor cell markers in the middle of gestation and active lactancy. Concerning committed cell markers, we detected a rise in AP at beginning of lactancy (d1L). We performed a BrdU uptake analysis in MZ and AP cells. The highest level of BrdU uptake was observed in MZ AT cells, whereas in AP this was detected in d1L, followed by a decrease in both the MZ and AP. Finally, we detected double immunostaining for BrdU-GFRa2 in MZ AT cells and BrdU-Sox9 in the AP d1L cells. Taken together, we hypothesize that the expansion of the SC/P niche took place mainly in MZ from pituitary rats in AT and d1L. These results suggest that the SC niche actively participates in pituitary plasticity during these reproductive states, contributing to the origin of hormone cell populations.

Evidence of cellular senescence during the development of estrogen-induced pituitary tumors.

Although pituitary adenomas represent 25% of intracranial tumors, they are usually benign, with the mechanisms by which these tumors usually avoid an invasive profile and metastatic growth development still remaining unclear. In this context, cellular senescence might constitute a plausible explanation for the benign nature of pituitary adenomas. In this study, we investigated the emergence of cellular senescence as a growth control mechanism during the progression of estrogen-induced pituitary tumors. The quantification of Ki67-immunopositive cells in the pituitaries of estrogenized male rats after 10, 20, 40, and 60 days revealed that the mitogenic potential rate was not sustained for the whole period analyzed and successively decreased after 10 days of estrogen exposure. In addition, the expression of cellular senescence features, such as the progressive rise in the enzymatic senescence-associated b-galactosidase (SA-b-gal) activity, IL6, IL1b, and TGFb expression, was observed throughout pituitary tumor development. Furthermore, tumoral pituitary cells also displayed nuclear pATM expression, indicating activated DNA damage signaling, with a significant increase in p21 expression also being detected. The associations among DNA damage signaling activation, SA-b-gal expression, and p21 may provide a reliable combination of senescence-associated markers for in vivo pituitary senescence detection. These results suggest a role for this cellular process in the regulation of pituitary cell growth. Thus, cellular senescence should be conceived as a contributing component to the benign nature of pituitary adenomas, thereby influencing the capability of the pituitary gland to avoid unregulated cell proliferation.

Involvement of MEK/ERK1/2 and PI3K/Akt pathways in the refractory behavior of GH3B6 pituitary tumor cells to the inhibitory effect of TGFß1.

Pituitary tumor cells have a poor response to the growth inhibitory effect of TGFß1, possibly resulting from the cross talk of TGFß/Smads signal with other signaling pathways, an undescribed mechanism in these tumoral cells. To address this hypothesis, we investigated whether the mitogen-activated extracellular signal-regulated kinase (MEK)/ERK1/2 and phosphoinositide-3 kinase/protein kinase B (PI3K/Akt) pathways were able to regulate the antimitogenic effect of TGFß1 on GH3B6 cells. TGFß1 treatment decreased the cell proliferation and induced an activation of mothers against decapentaplegic homolog 2/3 (Smad2/3), effects that were potentiated by MEK and PI3K inhibitors, thus indicating the existence of a cross talk between TGFß1/Smad with the MEK/ERK1/2 or PI3K/Akt pathways. In addition, through immunoprecipitation assays, a direct interaction was observed between Smad2/3-ERK1/2 and Smad2/3-Akt, which decreased when the GH3B6 cells were incubated with TGFß1 in the presence of MEK or PI3K inhibitors, thereby suggesting that the ERK1/2- and Akt-activated states were involved. These Smad2/3-ERK1/2 and Smad2/3-Akt associations were also confirmed by confocal and transmission electron microscopy. These findings indicate that the TGFß1-antimitogenic effect in GH3B6 cells was attenuated by the MEK/ERK1/2 and PI3K/Akt pathways via modulating Smad2/3 phosphorylation. This molecular mechanism could explain in part the refractory behavior of pituitary tumor cells to the inhibitory effect of TGFß1.

Functional Toll-like receptor 4 expressed in lactotrophs mediates LPS-induced proliferation in experimental pituitary hyperplasia.

Toll like receptor 4 (TLR4) has been characterized for its ability to recognize bacterial endotoxin lipopolysaccharide (LPS). Considering that infections or inflammatory processes might contribute to the progression of pituitary tumors, we analyzed the TLR4 functional role by evaluating the LPS effect on lactotroph proliferation in primary cultures from experimental pituitary tumors, and examined the involvement of PI3K-Akt and NF-κB activation in this effect. In addition, the role of 17ß-estradiol as a possible modulator of LPS-induced PRL cell proliferation was further investigated. In estrogen-induced hyperplasic pituitaries, LPS triggered lactotroph cell proliferation. However, endotoxin failed to increase the number of lactotrophs taking up BrdU in normal pituitaries. Moreover, incubation with anti-TLR4 antibody significantly reduced LPS-induced lactotroph proliferation, suggesting a functional role of this receptor. As a sign of TLR4 activation, an LPS challenge increased IL-6 release in normal and tumoral cells. By flow cytometry, TLR4 baseline expression was revealed at the plasma membrane of tumoral lactotrophs, without changes noted in the percentage of double PRL/TLR4 positive cells after LPS stimulus. Increases in TLR4 intracellular expression were detected as well as rises in CD14, p-Akt and NF-κB after an LPS challenge, as assessed by western blotting. The TLR4/PRL and PRL/NF-κB co-localization was also corroborated by immunofluorescence and the involvement of PI3K/Akt signaling in lactotroph proliferation and IL-6 release was revealed through the PI3K inhibitor Ly-294002. In addition, 17ß-estradiol attenuated the LPS-evoked increase in tumoral lactotroph proliferation and IL-6 release. Collectively these results demonstrate the presence of functional TLR4 in lactotrophs from estrogen-induced hyperplasic pituitaries, which responded to the proliferative stimulation and IL-6 release induced by LPS through TLR4/CD14, with a contribution of the PI3K-Akt and NF-κB signaling pathways.

Testosterone abrogates TLR4 activation in prostate smooth muscle cells contributing to the preservation of a differentiated phenotype.

Prostate smooth muscle cells (pSMCs) are capable of responding to inflammatory stimuli by secreting proinflammatory products, which causes pSMCs to undergo dedifferentiation. Although it has been proposed that androgens decrease proinflammatory molecules in many cells and under various conditions, the role of testosterone in the prostate inflammatory microenvironment is still unclear. Therefore, our aim was to evaluate if testosterone was able to modulate the pSMCs response to bacterial LPS by stimulating primary pSMC cultures, containing testosterone or vehicle, with LPS (1 or 10 µg/ml) for 24-48 h. The LPS challenge induced pSMCs dedifferentiation as evidenced by a decrease of calponin and alpha smooth muscle actin along with an increase of vimentin in a dose-dependent manner, whereas testosterone abrogated these alterations. Additionally, an ultrastructural analysis showed that pSMCs acquired a secretory profile after LPS and developed proteinopoietic organelles, while pSMCs preincubated with testosterone maintained a more differentiated phenotype. Testosterone downregulated the expression of surface TLR4 in control cells and inhibited any increase after LPS treatment. Moreover, testosterone prevented IκB-α degradation and the LPS-induced NF-κB nuclear translocation. Testosterone also decreased TNF-α and IL6 production by pSMCs after LPS as quantified by ELISA. Finally, we observed that testosterone inhibited the induction of pSMCs proliferation incited by LPS. Taken together, these results indicate that testosterone reduced the proinflammatory pSMCs response to LPS, with these cells being less reactive in the presence of androgens. In this context, testosterone might have a homeostatic role by contributing to preserve a contractile phenotype on pSMCs under inflammatory conditions.

GH3B6 pituitary tumor cell proliferation is mediated by PKCalpha and PKCepsilon via ERK 1/2-dependent pathway.

BACKGROUND: In this report, we explored the role of PKCalpha and PKCe as mediators of phorbol 12-myristate13-acetate (PMA)-induced proliferation in pituitary tumor GH3B6 cells, and determined if the ERK1/2 and Akt pathways were activated. METHODS: The GH3B6 cell proliferation was estimated by BrdU incorporation and the cell cycle progression by flow cytometric cell cycle analysis. We determined the expression of PKCalpha and PKCe in membrane and cytosolic fractions by western blotting. The subcellular redistribution of both PKC isozymes was analyzed by confocal microscopy. RESULTS: Incubation with PMA for 15 min stimulated PKCalpha and PKCe activation, which was correlated with the phosphorylation of ERK1/2 but not Akt. The activation of both these PKC isozymes was closely associated with the stimulation of proliferation and the cell cycle progression induced by PMA in GH3B6 cells, an effect that was blocked by the inhibitors of PKCalpha (Gö6976) and PKCe (eV1-2). In addition, the pretreatment with the inhibitor of ERK1/2 (PD98059) prevented the mitogenic activity induced by treatment with PMA for 15 min. CONCLUSION: We demonstrated that the activation of PKCalpha and PKCe by phorbol ester in tumor pituitary GH3B6 cells led to cell proliferation and cell cycle progression, effects that involved ERK1/2 activation.

Bromocriptine induces parapoptosis as the main type of cell death responsible for experimental pituitary tumor shrinkage.

Bromocriptine (Bc) produces pituitary tumoral mass regression which induces the cellular death that was classically described as apoptosis. However, recent works have related that other mechanisms of cell death could also be involved in the maintenance of physiological and pathological pituitary homeostasis. The aim of this study was to evaluate and characterize the different types of cell death in the involution induced by Bc in experimental rat pituitary tumors. The current study demonstrated that Bc induced an effective regression of estrogen induced pituitary tumors by a mechanism identified as parapoptosis. This alternative cell death was ultrastructurally recognized by extensive cytoplasmic vacuolization and an increased cell electron density, represented around 25% of the total pituitary cells counted. Furthermore, the results obtained from biochemical assays did not correspond to the criteria of apoptosis or necrosis. We also investigated the participation of p38, ERK1/2 and PKC delta in the parapoptotic pathway. An important observation was the significant increase in phosphorylated forms of these MAPKs, the holoenzyme and catalytic fragments of PKC delta in nuclear fractions after Bc administration compared to control and estrogen treated rats. Furthermore, the immunolocalization at ultrastructural level of these kinases showed a similar distribution pattern, with a prevalent localization at nuclear level in lactotrophs from Bc treated rats. In summary, we determined that parapoptosis is the predominant cell death type involved in the regression of pituitary tumors in response to Bc treatment, and may cause the activation of PKC delta, ERK1/2 and p38.

Specific subcellular targeting of PKCalpha and PKCepsilon in normal and tumoral lactotroph cells by PMA-mitogenic stimulus.

The variations of the intracellular localization of the individual protein kinase C (PKC) isoforms are related with their different biological functions. In this study, we have investigated the precise intracellular translocation of endogenous PKCalpha and PKCepsilon in PMA-stimulated normal and tumoral lactotroph cells by using confocal and immunogold electron microscopy, which was correlated with the rate of cell proliferation of both pituitary cell phenotypes. The present results showed that the short phorbol ester incubation stimulated the proliferation of normal and tumoral lactotroph cells, as determined by the measurement of the BrdU-labelling index. The translocation of PKCalpha to plasma and nuclear membranes induced by PMA was more marked than that observed for PKCepsilon in normal and tumoral lactotroph cells. Our results showed that PKCs translocation to the plasma and nuclear membranes varied from isozyme to isozyme emphasizing that PKCalpha could be related with the mitogenic stimulus exerted by phorbol ester. These data support the notion that specific PKC isozymes may exert spatially defined effects by virtue of their directed translocation to distinct intracellular sites.

Activation of PKC epsilon induces lactotroph proliferation through ERK1/2 in response to phorbol ester.

The aim of this investigation was to contribute to current knowledge about intracellular mechanisms that are involved in lactotroph cell proliferation, by evaluating the role of PKCalpha, PKCepsilon and extracellular-signal regulated kinase (ERK) 1/2 in response to phorbol 12-myristate13-acetate (PMA). In primary pituitary cultures, the activation of protein kinase C (PKC) by PMA for 15 min stimulated lactotroph proliferation; whereas a prolonged activation for 3-8h diminished this proliferative effect. The use of PMA for 15 min-activated PKCepsilon and ERK1/2, whereas incubation with PMA for 3 h induced PKCalpha activation and attenuated the PMA-triggered phosphorylation of ERK1/2. The following inhibitors: PKCs (bisindolylmaleimide I), PKCepsilon (epsilonV1 peptide) and ERK1/2 (PD98059) prevented the mitogenic activity induced by PMA for 15 min. Lactotroph cells stimulated with PMA for 15 min showed a translocation of PKCepsilon to membrane compartment and nucleus. These results thus establish that PKCepsilon plays an essential role in the lactotroph proliferation induced by PMA by triggering signals that involve ERK1/2 activation.

Estradiol interacts with insulin through membrane receptors to induce an antimitogenic effect on lactotroph cells.

The signaling mechanisms of estrogens interact with those of growth factors to control the pituitary gland functions. The contribution of the membrane bound estrogen receptor in these actions is not fully understood. In this study, we focused on the regulatory action of estradiol in interaction with insulin on the secretory and proliferative lactotroph cell activities from primary pituitary cell cultures. Furthermore, we studied the involvement of ERK1/2, PKC epsilon and Pit-1 in these actions. In serum free conditions, estradiol and estradiol-BSA promoted a differential secretory activity on PRL cells but were unable to induce lactotroph cell proliferation. However, both free and conjugated estradiol were competent arresting the mitogenic activity promoted by insulin. Estradiol, estradiol-BSA and insulin stimuli increased the PKC epsilon, phosphorylated ERK 1/2 and Pit-1 expression, although combined treatments with estradiol/insulin or estradiol-BSA/insulin induced a significant reduction in these levels, in close correlation with the decrease of lactotroph cell proliferation. The pre-treatment with PKC inhibitor BIM significantly inhibited the ERK activation promoted by insulin without modifying the ERK expression levels induced by estradiol or estradiol-BSA. By immuno-electron-microscopy the alpha nuclear estrogen receptor was localized in the plasma membrane of lactotroph cells. These findings suggest that the membrane bound ER participates modulating lactotroph cells proliferation via PKC epsilon, ERK1/2 and Pit-1. The interactions between estradiol and growth factors, inducing both mitogenic and antimitogenic effects, could provide glandular plasticity preventing an over-proliferation induced by growth factors.