Nesfatin-1 and Nesfatin-1-like peptide suppress basal and TRH-Induced expression of prolactin and prolactin regulatory element-binding protein mRNAs in rat GH3 somatolactotrophs.

Prolactin (PRL), mainly synthesized and secreted by the lactotrophs and somatolactotrophs of the anterior pituitary, is a pleiotropic hormone that regulates lactation. In the last decade, nesfatin-1 (NESF) and NESF-like peptide (NLP), encoded in nucleobindin 1 and 2 (NUCB1 and NUCB2), respectively, were characterized as metabolic factors with a potential role in the control of pituitary hormones. We hypothesized that NUCBs and their encoded peptides (NESF and NLP) suppress PRL transcription in the pituitary. The main objective of this research was to determine whether exogenous NESF and NLP, and/or endogenous NUCB1 and NUCB2 regulate the expression of prl and preb mRNAs. Using immortalized rat somatolactotrophs (GH3 cells), dose-response studies were performed to test whether NESF and NLP affect prl and preb. Moreover, the ability of these peptides to modulate the effects of the PRL stimulator thyrotropin releasing hormone (TRH) was studied. Besides, the effects of siRNA-mediated knockdown of endogenous NUCBs on prl and preb mRNAs were determined. NESF and NLP reduced the transcription of prl and preb in GH3 cells. Both NESF and NLP also prevented the stimulatory effects of TRH prl and preb expression. The knockdown of endogenous NUCB1 attenuates both basal prl and TRH-induced expression of prl and preb, while the silencing of NUCBs did not affect the actions of exogenous NESF or NLP. Overall, this work reveals that NUCBs and encoded-peptides are novel regulators of PRL. Future research should test whether the effects observed here in GH3 cells are preserved both in vivo and at the post-transcriptional level.

The Transcription Factor NR4A2 Plays an Essential Role in Driving Prolactin Expression in Female Pituitary Lactotropes.

Differentiation of the hormone-producing cells of the pituitary represents an informative model of cell fate determination. The generation and maintenance of 2 pituitary lineages, the growth hormone (GH)- producing somatotropes and the prolactin (PRL)- producing lactotropes, are dependent on the pituitary-specific transcription factor, POU1F1. While POU1F1 is expressed in both cell types, and plays a role in activation of both the Gh and Prl genes, expression of Gh and Prl is restricted to somatotropes and lactotropes, respectively. These observations imply the existence of additional factors that contribute to the somatotrope and lactotrope identities and their hormone expressions. Prior transcriptome analysis of primary somatotropes and lactotropes isolated from the mouse pituitary identified enrichment of a transcription factor, Nr4a2, in the lactotropes. Nr4a2 was shown in a cell culture model to bind the Prl promoter at a position adjacent to Pou1f1 and to synergize with Pou1f1 in driving Prl transcription. Here we demonstrate in vivo the role of Nr4a2 as an enhancer of Prl expression by conditional gene inactivation of the Nr4a2 gene in mouse lactotropes. We demonstrate that nuclear orphan receptor transcription factor (NR4A2) binding at the Prl promoter is dependent on actions of POU1F1; while POU1F1 is essential to loading polymerase (Pol) II on the Prl promoter, Nr4a2 plays a role in enhancing Pol II release into the Prl gene body. These studies establish an in vivo role of Nr4a2 in enhancing Prl expression in mouse lactotropes, explore its mechanism of action, and establish a system for further study of the lactotrope lineage in the pituitary.

The Functional Significance of Endocrine-immune Interactions in Health and Disease.

Hormones are known to influence various body systems that include skeletal, cardiac, digestive, excretory, and immune systems. Emerging investigations suggest the key role played by secretions of endocrine glands in immune cell differentiation, proliferation, activation, and memory attributes of the immune system. The link between steroid hormones such as glucocorticoids and inflammation is widely known. However, the role of peptide hormones and amino acid derivatives such as growth and thyroid hormones, prolactin, dopamine, and thymopoietin in regulating the functioning of the immune system remains unclear. Here, we reviewed the findings pertinent to the functional role of hormone-immune interactions in health and disease and proposed perspective directions for translational research in the field.

Histone deacetylase inhibitors suppress transdifferentiation of gonadotrophs to prolactin cells and proliferation of prolactin cells induced by diethylstilbestrol in male mouse pituitary.

Diethylstilbestrol (DES), an estrogen agonist, increases prolactin (PRL) cells through transdifferentiation of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) cells to PRL cells as well as proliferation of PRL cells in adult male mouse pituitary. Since hyperacetylation of histone H3 is implicated in the regulation of activation of various genes, we examined the effect of DES on the state of histone H3 acetylation. DES significantly reduced the immunohistochemical signal for acetylated histone H3 at lysine 9 (H3K9ac) in PRL, LH and FSH cells, but not for H3K18ac or H3K23ac. DES-treated mice were injected intraperitoneally with HDAC inhibitors (HDACi), sodium phenylbutyrate (NaPB) or valproic acid (VPA), to mimic the acetylation level of histone H3. As expected, HDACi treatment restored the level of H3K9ac expression in these cells, and also inhibited DES-induced increase in PRL cells. Furthermore, NaPB and VPA also abrogated the effects of DES on the population density of both LH and FSH cells. Similarly, the numbers of proliferating and apoptotic cells in the pituitary in NaPB- or VPA-treated mice were comparable to those of the control mice. Considered together, these results indicated that the acetylation level of histone H3 plays an important role in DES-induced transdifferentiation of LH to PRL cells as well as proliferation of PRL cells.

Regulation of Pituitary Progenitor Differentiation by ß-Catenin.

The pituitary gland is a critical organ that is necessary for many physiological processes, including growth, reproduction, and stress response. The secretion of pituitary hormones from specific cell types regulates these essential processes. Pituitary hormone cell types arise from a common pool of pituitary progenitors, and mutations that disrupt the formation and differentiation of pituitary progenitors result in hypopituitarism. Canonical WNT signaling through CTNNB1 (ß-catenin) is known to regulate the formation of the POU1F1 lineage of pituitary cell types. When ß-catenin is deleted during the initial formation of the pituitary progenitors, Pou1f1 is not transcribed, which leads to the loss of the POU1F1 lineage. However, when ß-catenin is deleted after lineage specification, there is no observable effect. Similarly, the generation of a ß-catenin gain-of-function allele in early pituitary progenitors or stem cells results in the formation of craniopharyngiomas, whereas stimulating ß-catenin in differentiated cell types has no effect. PROP1 is a pituitary-specific transcription factor, and the peak of PROP1 expression coincides with a critical time point in pituitary organogenesis-that is, after pituitary progenitor formation but before lineage specification. We used a Prop1-cre to conduct both loss- and gain-of-function studies on ß-catenin during this critical time point. Our results demonstrate that pituitary progenitors remain sensitive to both loss and gain of ß-catenin at this time point, and that either manipulation results in hypopituitarism.

Histone Citrullination Represses MicroRNA Expression, Resulting in Increased Oncogene mRNAs in Somatolactotrope Cells.

Peptidylarginine deiminase (PAD) enzymes convert histone arginine residues into citrulline to modulate chromatin organization and gene expression. Although PADs are expressed in anterior pituitary gland cells, their functional role and expression in pituitary adenomas are unknown. To begin to address these issues, we first examined normal human pituitaries and pituitary adenomas and found that PAD2, PAD4, and citrullinated histones are highest in prolactinomas and somatoprolactinomas. In the somatoprolactinoma-derived GH3 cell line, PADs citrullinate histone H3, which is attenuated by a pan-PAD inhibitor. RNA sequencing and chromatin immunoprecipitation (ChIP) studies show that the expression of microRNAs (miRNAs) let-7c-2, 23b, and 29c is suppressed by histone citrullination. Our studies demonstrate that these miRNAs directly target the mRNA of the oncogenes encoding HMGA, insulin-like growth factor 1 (IGF-1), and N-MYC, which are highly implicated in human prolactinoma/somatoprolactinoma pathogenesis. Our results are the first to define a direct role for PAD-catalyzed histone citrullination in miRNA expression, which may underlie the etiology of prolactinoma and somatoprolactinoma tumors through regulation of oncogene expression.

ZBTB20 is required for anterior pituitary development and lactotrope specification.

The anterior pituitary harbours five distinct hormone-producing cell types, and their cellular differentiation is a highly regulated and coordinated process. Here we show that ZBTB20 is essential for anterior pituitary development and lactotrope specification in mice. In anterior pituitary, ZBTB20 is highly expressed by all the mature endocrine cell types, and to some less extent by somatolactotropes, the precursors of prolactin (PRL)-producing lactotropes. Disruption of Zbtb20 leads to anterior pituitary hypoplasia, hypopituitary dwarfism and a complete loss of mature lactotropes. In ZBTB20-null mice, although lactotrope lineage commitment is normally initiated, somatolactotropes exhibit profound defects in lineage specification and expansion. Furthermore, endogenous ZBTB20 protein binds to Prl promoter, and its knockdown decreases PRL expression and secretion in a lactotrope cell line MMQ. In addition, ZBTB20 overexpression enhances the transcriptional activity of Prl promoter in vitro. In conclusion, our findings point to ZBTB20 as a critical regulator of anterior pituitary development and lactotrope specification.

Bovine lactotroph cultures for the study of prolactin synthesis functions.

The aim of this study was to establish a bovine anterior pituitary-derived lactotroph (BAPDL) line that expresses prolactin (PRL) in vitro to study the mechanisms of bovine PRL synthesis and secretion. Immunohistochemistry assay of PRL in the newborn calves' anterior pituitary glands showed that most lactotrophs were located within the superior border of the lateral wings of the anterior pituitary. Tissues of the superior border of the lateral wings of the anterior pituitary were dispersed and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). The limiting dilution method was used to establish BAPDL from single cell clone. BAPDL cells constantly expressed mRNAs for PRL and pituitary-specific transcription factor 1 (Pit-1) gene and grew steadily and rapidly in the DMEM supplemented with 10% FBS. PRL immunoreactivity was present in BAPDL at passage 20. The concentration of bovine PRL in BAPDL at passage 20 culture supernatant was decreased to below 35% compared with that in BAPDL at passage 1. The effects of human epidermal growth factor (hEGF) and dopamine (DA) on the expression and secretion of PRL in BAPDL at passage 4 were also investigated. The results are consistent with those of previous studies. Thus, it can be used successfully for studying the mechanisms of stimuli regulating PRL synthesis and release.

Dose-dependent dual role of PIT-1 (POU1F1) in somatolactotroph cell proliferation and apoptosis.

To test the role of wtPIT-1 (PITWT) or PIT-1 (R271W) (PIT271) in somatolactotroph cells, we established, using inducible lentiviral vectors, sublines of GH4C1 somatotroph cells that allow the blockade of the expression of endogenous PIT-1 and/or the expression of PITWT or PIT271, a dominant negative mutant of PIT-1 responsible for Combined Pituitary Hormone Deficiency in patients. Blocking expression of endogenous PIT-1 induced a marked decrease of cell proliferation. Overexpressing PITWT twofold led also to a dose-dependent decrease of cell proliferation that was accompanied by cell death. Expression of PIT271 induced a strong dose-dependent decrease of cell proliferation accompanied by a very pronounced cell death. These actions of PIT271 are independent of its interaction/competition with endogenous PIT-1, as they were unchanged when expression of endogenous PIT-1 was blocked. All these actions are specific for somatolactotroph cells, and could not be observed in heterologous cells. Cell death induced by PITWT or by PIT271 was accompanied by DNA fragmentation, but was not inhibited by inhibitors of caspases, autophagy or necrosis, suggesting that this cell death is a caspase-independent apoptosis. Altogether, our results indicate that under normal conditions PIT-1 is important for the maintenance of cell proliferation, while when expressed at supra-normal levels it induces cell death. Through this dual action, PIT-1 may play a role in the expansion/regression cycles of pituitary lactotroph population during and after lactation. Our results also demonstrate that the so-called "dominant-negative" action of PIT271 is independent of its competition with PIT-1 or a blockade of the actions of the latter, and are actions specific to this mutant variant of PIT-1.

Ultrastructural changes in lactotrophs and folliculo-stellate cells in the ovine pituitary during the annual reproductive cycle.

In seasonal mammals living in temperate zones, photoperiod regulates prolactin secretion, such that prolactin plasma concentrations peak during the summer months and are lowest during the winter. In sheep, a short-day breeder, circulating prolactin has important modulatory effects on the reproductive system via inhibitory actions on pituitary gonadotrophs and hypothalamic gonadotrophin-releasing hormone release. The exact cellular mechanisms that account for the chronic hypersecretion of prolactin during the summer is not known, although evidence supports an intrapituitary mechanism regulated by melatonin. Folliculo-stellate (FS) cells are non-endocrine cells that play a crucial role in paracrine communication within the pituitary and produce factors controlling prolactin and gonadotrophin release. The present study examined the morphology of the FS and lactotroph cell populations and their distribution in the sheep pituitary during the annual reproductive cycle. Ovine pituitary glands were collected in the winter (breeding season; BS) and summer (nonbreeding season; NBS) and were prepared for quantitative electron microscopy to assess the effects of season on FS and lactotroph cell density, morphology and distribution, as well as on junctional contacts between cells. It was found that lactotrophs in the NBS are larger in size and contain more numerous PRL granules than lactotrophs in the BS. FS cells were also larger in the NBS compared to BS and showed altered morphology such that, in the BS, long cell processes surrounded clusters of adjacent secretory cells. Although no significant change in the number of junctions was observed between lactotrophs and FS cells, or lactotrophs and gonadotrophs, there was a significant increase in the number of adherens junctions between lactotrophs and between FS cells. These findings demonstrate seasonal plasticity in the morphology of lactotrophs and FS cells that reflect changes in PRL secretion.

Prolactin induces apoptosis of lactotropes in female rodents.

Anterior pituitary cell turnover occurring during female sexual cycle is a poorly understood process that involves complex regulation of cell proliferation and apoptosis by multiple hormones. In rats, the prolactin (PRL) surge that occurs at proestrus coincides with the highest apoptotic rate. Since anterior pituitary cells express the prolactin receptor (PRLR), we aimed to address the actual role of PRL in the regulation of pituitary cell turnover in cycling females. We showed that acute hyperprolactinemia induced in ovariectomized rats using PRL injection or dopamine antagonist treatment rapidly increased apoptosis and decreased proliferation specifically of PRL producing cells (lactotropes), suggesting a direct regulation of these cell responses by PRL. To demonstrate that apoptosis naturally occurring at proestrus was regulated by transient elevation of endogenous PRL levels, we used PRLR-deficient female mice (PRLRKO) in which PRL signaling is totally abolished. According to our hypothesis, no increase in lactotrope apoptotic rate was observed at proestrus, which likely contributes to pituitary tumorigenesis observed in these animals. To decipher the molecular mechanisms underlying PRL effects, we explored the isoform-specific pattern of PRLR expression in cycling wild type females. This analysis revealed dramatic changes of long versus short PRLR ratio during the estrous cycle, which is particularly relevant since these isoforms exhibit distinct signaling properties. This pattern was markedly altered in a model of chronic PRLR signaling blockade involving transgenic mice expressing a pure PRLR antagonist (TGΔ1-9-G129R-hPRL), providing evidence that PRL regulates the expression of its own receptor in an isoform-specific manner. Taken together, these results demonstrate that i) the PRL surge occurring during proestrus is a major proapoptotic signal for lactotropes, and ii) partial or total deficiencies in PRLR signaling in the anterior pituitary may result in pituitary hyperplasia and eventual prolactinoma development, as observed in TGΔ1-9-G129R-hPRL and PRLRKO mice, respectively.

Evidence for an internal and functional circadian clock in rat pituitary cells.

In primary cultures of rat pituitary cells and in a pituitary sommatolactotroph cell line (GH4C1), endogenous core-clock- as well as hormone-genes such as prolactin displayed a rhythmic expression pattern, fitted by a sinusoidal equation in which the period value was close to the circadian one. This is consistent with the presence of a functional circadian oscillator in pituitary cells whose importance was ascertained in GH4C1 cell lines stably expressing a dominant negative mutant of BMAL1. In these cells, both endogenous core-clock- and prolactin-genes no more displayed a circadian pattern. Some genes we recently identified as mouse pituitary BMAL1-regulated genes in a DNA-microarray study, lost their circadian pattern in these cells, suggesting that BMAL1 controlled these genes locally in the pituitary. The intra-pituitary circadian oscillator could then play a role in the physiology of the gland that would not be seen anymore as a structure only driven by hypothalamic rhythmic control.

Differences between rat strains in the development of PRL-secreting pituitary tumors with long-term estrogen treatment: In vitro insulin-like growth factor-1-induced lactotroph proliferation and gene expression are affected in Wistar-Kyoto rats with low estrogen-susceptibility.

There are differences in the susceptibility of rat strains to pituitary growth and lactotroph proliferation caused by long-term treatment with estrogens. To investigate the pituitary mechanism for this strain difference in estrogen-induced lactotroph proliferation, we compared the abilities of 17-ß estradiol (E2) and insulin-like growth factor-1 (IGF-1) to modulate lactotroph proliferation and gene expression in vitro in Wistar and Wistar-Kyoto (WKY) rats. These two strains of rats have a high and very low susceptibility to estrogen, respectively. Long-term in vivo treatment with E2 was confirmed to markedly increase pituitary weight and lactotroph proliferation in ovariectomized Wistar, but not in WKY rats. Pituitary lactotrophs in primary cultures showed similar proliferative responsiveness to the culture condition-dependent, stimulatory and inhibitory actions of E2 in both strains. The only difference in lactotroph proliferation in vitro was a lower response to IGF-1 in WKY cells compared with Wistar cells. This difference in proliferation was associated with strain differences in IGF-1-induced gene expression in Wistar and WKY cultured cells. Of the genes tested, IGF-1-induced expression of the Wnt4, Stc1, Mybl1, and Myc genes was attenuated or abolished in WKY cells. These results suggest that the proliferative response to estrogen in lactotrophs in primary culture does not reflect the proliferative response to long-term estrogen treatment observed in vivo in Wistar and WKY rats. The strain difference in proliferation and gene expression to IGF-1 may be implicated in the variable degree of susceptibility for lactotroph proliferation observed in different strains of rats following long-term estrogen treatment.

Vesicle size determines unitary exocytic properties and their sensitivity to sphingosine.

Neuroendocrine cells contain small and large vesicles, but the functional significance of vesicle diameter is unclear. We studied unitary exocytic events of prolactin-containing vesicles in lactotrophs by monitoring discrete steps in membrane capacitance. In the presence of sphingosine, which recruits VAMP2 for SNARE complex formation, the frequency of transient and full fusion events increased. Vesicles with larger diameters proceeded to full fusion, but smaller vesicles remained entrapped in transient exocytosis. The diameter of vesicle dense cores released by full fusion exocytosis into the extracellular space was larger than the diameter of the remaining intracellular vesicles beneath the plasma membrane. Labeling with prolactin- and VAMP2-antibodies revealed a correlation between the diameters of colocalized prolactin- and VAMP2-positive structures. It is proposed that sphingosine-mediated facilitation of regulated exocytosis is not only related to the number of SNARE complexes per vesicle but also depends on the vesicle size, which may determine the transition between transient and full fusion exocytosis.

Cooperative effect of E2 and FGF2 on lactotroph proliferation triggered by signaling initiated at the plasma membrane.

In the present work, we investigated the effect of 17ß-estradiol (E2) and basic fibroblast growth factor 2 (FGF2) on the lactotroph cell-proliferative response and the related membrane-initiated signaling pathway. Anterior pituitary mixed-cell cultures of random, cycling 3-mo-old female rats were treated with 10 nM E2, E2 membrane-impermeable conjugated BSA (E2-BSA), PPT (ERα agonist), and DPN (ERß agonist) alone or combined with FGF2 (10 ng/ml) for 30 min or 4 h. Although our results showed that the uptake of BrdU into the nucleus of lactotrophs was not modified by E2 or FGF2 alone, a significant increase in the lactotroph uptake of BrdU was observed after E2/FGF2 coincubation, with this effect being mimicked by PPT/FGF2. These proliferative effects were blocked by ICI 182,780 or PD-98059. The involvement of membrane ER in the proliferative response of prolactin cells induced by the steroid and FGF2 coincubation was confirmed using E2-BSA, and the association between ERα and FGF receptor was observed after E2/FGF2 treatment by immunoprecipitation. A significant increase in the ERK1/2 expression was noted after E2, E2-BSA, PPT, and FGF2 alone, which was more noticeable after E2-BSA/FGF2, E2/FGF2, or PPT/FGF2 treatments. This study provides evidence that E2 and FGF2 exert a cooperative effect on the lactotroph proliferation principally by signaling initiated at the plasma membrane triggering a genomic effect mediated by MEK/ERK1/2, a common signaling pathway, that finally regulates the lactotroph population, thus contributing to pituitary plasticity.

Roles of dopamine 2 receptor isoforms and g proteins in ethanol regulated prolactin synthesis and lactotropic cell proliferation.

Alcohol consumption has been shown to increase prolactin (PRL) production and cell proliferation of pituitary lactotropes. It also causes a reduction in the lactotrope's response to dopaminergic agents and a differential expression of dopamine 2 receptor short (D2S) and long (D2L) isoforms in the pituitary. However, the role of each of these D2 receptor isoforms and its coupled G protein in mediation of ethanol actions on lactotropes is not known. We have addressed this issue by comparing ethanol effects on the level of PRL production gene transcription rate cellular protein, G proteins and cell proliferation in enriched lactotropes and lactotrope-derived PR1 cells containing various D2 receptor isoforms. Additionally, we determined the effects of G protein blockade on ethanol-induced PRL production and cell proliferation in these cells. We show here that the D2 receptor, primarily the D2S isoform, is critically involved in the regulation of ethanol actions on PRL production and cell proliferation in lactotropes. We also present data to elucidate that the presence of the pertussis toxin (PTX)-sensitive D2S receptor is critical to mediate the ethanol stimulatory action on Gs and the ethanol's inhibitory action on Gi3 protein in lactotropes. Additionally, we provide evidence for the existence of an inhibitory action of Gi3 on Gs that is under the control of the D2S receptor and is inhibited by ethanol. These results suggest that ethanol via the inhibitory action on D2S receptor activity suppresses Gi3 repression of Gs expression resulting in stimulation of PRL synthesis and cell proliferation in lactotropes.

Immunohistochemical localization and morphometry of somatotrophs and lactotrophs in protein, probiotic and symbiotic supplemented molted layers.

Two-hundred Single Comb White Leg-Horn spent hens at the age of 70 weeks were purchased from a commercial layer farm. The birds were shifted to the Poultry Research Station, Department of Physiology and Pharmacology, University of Agriculture, Faisalabad, Pakistan. High dietary zinc (3g/kg feed/day) was used to induce molting in all the birds after one week of acclimatization. Thereafter, birds were divided in groups of 50 birds each, with the following treatments: G1 [control; crude protein (CP)16%, no other supplement], G2 (CP18%, no other supplement), G3 (CP16%, Symbiotic, 85 mg/L drinking water) and G4 (CP16%, Probiotic, 85mg/L in drinking water). Fifteen birds were slaughtered at 5% of peak of production each to collect their pituitary glands. The better egg production was seen in all the supplemented groups as compared to the control. Especially an earlier post molt production recovery and delayed decline was seen in the G2 as compared to all other groups. The immunohistochemistry of the pituitary gland reveals the decrease (P≤0.01) in the cell and nucleus size as well as area of somatotrophs in G2 and G4 as compared to G1. The cell and nucleus size as well as area of lactotrophs decreased (P≤0.01) in G2, G3 and G4 as compared to G1. The better and earlier post molt production in G2 highlights the potential role of protein supplementation in connection with the decreased lactotroph size and area in molted birds.

Estrogens induce expression of membrane-associated estrogen receptor α isoforms in lactotropes.

Estrogens are key to anterior pituitary function, stimulating hormone release and controlling cell fate to achieve pituitary dynamic adaptation to changing physiological conditions. In addition to their classical mechanism of action through intracellular estrogen receptors (ERs), estrogens exert rapid actions via cell membrane-localized ERs (mERs). We previously showed that E2 exerts a rapid pro-apoptotic action in anterior pituitary cells, especially in lactotropes and somatotropes, through activation of mERs. In the present study, we examined the involvement of mERα in the rapid pro-apoptotic action of estradiol by TUNEL in primary cultures of anterior pituitary cells from ovariectomized rats using a cell-impermeable E2 conjugate (E2-BSA) and an ERα selective antagonist (MPP dihydrochloride). We studied mERα expression during the estrous cycle and its regulation by gonadal steroids in vivo by flow cytometry. We identified ERα variants in the plasma membrane of anterior pituitary cells during the estrous cycle and studied E2 regulation of these mERα variants in vitro by surface biotinylation and Western Blot. E2-BSA-induced apoptosis was abrogated by MPP in total anterior pituitary cells and lactotropes. In cycling rats, we detected a higher number of lactotropes and a lower number of somatotropes expressing mERα at proestrus than at diestrus. Acute E2 treatment increased the percentage of mERα-expressing lactotropes whereas it decreased the percentage of mERα-expressing somatotropes. We detected three mERα isoforms of 66, 39 and 22 kDa. Expression of mERα66 and mERα39 was higher at proestrus than at diestrus, and short-term E2 incubation increased expression of these two mERα variants. Our results indicate that the rapid apoptotic action exerted by E2 in lactotropes depends on mERα, probably full-length ERα and/or a 39 kDa ERα variant. Expression and activation of mERα variants in lactotropes could be one of the mechanisms through which E2 participates in anterior pituitary cell renewal during the estrous cycle.

Cholesterol and regulated exocytosis: a requirement for unitary exocytotic events.

Since the 1970s, much effort was been expended researching mechanisms of regulated exocytosis. Early work focused mainly on the role of proteins. Most notably the discovery of SNARE proteins in the 1980s and the zippering hypothesis brought us much closer to understanding the complex interactions in membrane fusion between vesicle and plasma membranes, a pivotal component of regulated exocytosis. However, most likely due to the predictions of the Singer-Nicholson fluid mosaic membrane model, the lipid components of the exocytotic machinery remained largely overlooked. Lipids were considered passive constituents of cellular membranes, not contributing much, if anything, to the process of exocytosis and membrane fusion. Since the 1990s, this so-called proteocentric view has been gradually giving way to the new perspective best described with the term proteolipidic. Many lipids were found to be of great importance in the regulation of exocytosis. Here we highlight the role of cholesterol. Furthermore, by using high-resolution cell-attached membrane capacitance measurements, we have monitored unitary exocytotic events in cholesterol-depleted membranes. We show that the frequency of these events is attenuated, providing evidence at the single vesicle level that cholesterol directly influences the merger of the vesicle and the plasma membranes.

Curcumin suppresses HIF1A synthesis and VEGFA release in pituitary adenomas.

Curcumin (diferuloylmethane), a polyphenolic compound derived from the spice plant Curcuma longa, displays multiple actions on solid tumours including anti-angiogenic effects. Here we have studied in rodent and human pituitary tumour cells the influence of curcumin on the production of hypoxia inducible factor 1α (HIF1A) and vascular endothelial growth factor A (VEGFA), two key components involved in tumour neovascularisation through angiogenesis. Curcumin dose-dependently inhibited basal VEGFA secretion in corticotroph AtT20 mouse and lactosomatotroph GH3 rat pituitary tumour cells as well as in all human pituitary adenoma cell cultures (n=32) studied. Under hypoxia-mimicking conditions (CoCl(2) treatment) in AtT20 and GH3 cells as well as in all human pituitary adenoma cell cultures (n=8) studied, curcumin strongly suppressed the induction of mRNA synthesis and protein production of HIF1A, the regulated subunit of the hypoxia-induced transcription factor HIF1. Curcumin also blocked hypoxia-induced mRNA synthesis and secretion of VEGFA in GH3 cells and in all human pituitary adenoma cell cultures investigated (n=18). Thus, curcumin may inhibit pituitary adenoma progression not only through previously demonstrated anti-proliferative and pro-apoptotic actions but also by its suppressive effects on pituitary tumour neovascularisation.