Sex-specific regulation of prolactin secretion by pituitary activins in postnatal development.

Serum prolactin increases from birth to adulthood in rats, being higher in females from birth. The maturation of hypothalamic/gonadal prolactin-releasing and -inhibiting factors does not explain some sex differences observed. During the first weeks of life, prolactin secretion increases, even when lactotrophs are isolated in vitro, in the absence of those controls, suggesting the participation of intra-pituitary factors in this control. The present work aimed to study the involvement of pituitary activins in the regulation of prolactin secretion during post-natal development. Sex differences were also highlighted. Female and male Sprague-Dawley rats at 11, 23 and 45postnatal days were used. Pituitary expression of activin subunits and activin receptors was maximum in p11 female pituitaries, being even higher than that observed in males. Those expressions decrease with age in females, and then the gender differences disappear at p23. Inhbb expression strongly increases at p45 in males, being the predominant subunit in this sex in adulthood. Activin inhibition of prolactin is mediated by the inhibition of Pit-1 expression. This action involves not only the canonical pSMAD pathway but also the phosphorylation of p38MAPK. At p11, almost all lactotrophs express p-p38MAPK in females, and its expression decreases with age with a concomitant increase in Pit-1. Our findings suggest that the inhibitory regulation of pituitary activins on prolactin secretion is sex specific; this regulation is more relevant in females during the first week of life and decreases with age; this intra-pituitary regulation is involved in the sex differences observed in serum prolactin levels during postnatal development.

PI(4,5)P2-dependent and -independent roles of PI4P in the control of hormone secretion by pituitary cells.

Plasma membrane and organelle membranes are home to seven phosphoinositides, an important class of low-abundance anionic signaling lipids that contribute to cellular functions by recruiting cytoplasmic proteins or interacting with the cytoplasmic domains of membrane proteins. Here, we briefly review the functions of three phosphoinositides, PI4P, PI(4,5)P2, and PI(3,4,5)P3, in cellular signaling and exocytosis, focusing on hormone-producing pituitary cells. PI(4,5)P2, acting as a substrate for phospholipase C, plays a key role in the control of pituitary cell functions, including hormone synthesis and secretion. PI(4,5)P2 also acts as a substrate for class I PI3-kinases, leading to the generation of two intracellular messengers, PI(3,4,5)P3 and PI(3,4)P2, which act through their intracellular effectors, including Akt. PI(4,5)P2 can also influence the release of pituitary hormones acting as an intact lipid to regulate ion channel gating and concomitant calcium signaling, as well as the exocytic pathway. Recent findings also show that PI4P is not only a precursor of PI(4,5)P2, but also a key signaling molecule in many cell types, including pituitary cells, where it controls hormone secretion in a PI(4,5)P2-independent manner.

Sex-specific Regulation of Prolactin Secretion by Pituitary Bradykinin Receptors.

Sex differences in the control of prolactin secretion are well documented. Sex-related differences in intrapituitary factors regulating lactotroph function have recently attracted attention. Sex differences in prolactinoma development are well documented in clinic, prolactinomas being more frequent in women but more aggressive in men, for poorly understood reasons. Kallikrein, the enzyme releasing kinins has been found in the pituitary, but there is no information on pituitary kinin receptors and their function. In the present work, we characterized pituitary bradykinin receptors (BRs) at the messenger RNA and protein levels in 2 mouse models of prolactinoma, Drd2 receptor gene inactivation and hCGß gene overexpression, in both males and females, wild type or genomically altered. BR B2 (B2R) accounted for 97% or more of total pituitary BRs in both models, regardless of genotype, and was present in lactotrophs, somatotrophs, and gonadotrophs. Male pituitaries displayed higher level of B2R than females, regardless of genotype. Pituitary B2R gene expression was downregulated by estrogen in both males and females but only in females by dopamine. Activation of B1R or B2R by selective pharmacological agonists induced prolactin release in male pituitaries but inhibited prolactin secretion in female pituitaries. Increased B2R content was observed in pituitaries of mutated animals developing prolactinomas, compared to their respective wild-type controls. The present study documents a novel sex-related difference in the control of prolactin secretion and suggests that kinins are involved, through B2R activation, in lactotroph function and prolactinoma development.

Differentiation of stem progenitor CD9/SOX2-positive cells is promoted with increased prolactin-producing and endothelial cells in the pituitary.

Sex-determining region Y-box 2 (SOX2)-positive cells are stem/progenitor cells in the adenohypophysis, comprising the anterior and intermediate lobes (AL and IL, respectively). The cells are located in the marginal cell layer (MCL) facing Rathke's cleft (primary niche) and the parenchyma of the AL (secondary niche). We previously demonstrated in vitro that the tetraspanin superfamily CD9 and SOX2 double-positive (CD9/SOX2-positive) cells in the IL-side MCL migrate to the AL side and differentiate into hormone-producing and endothelial cells in the AL parenchyma. Here, we performed in vivo studies to evaluate the role of IL-side CD9/SOX2-positive cells in pregnancy, lactation, and treatment with diethylstilbestrol (DES; an estrogen analog) when an increased population of prolactin (PRL) cells was observed in the AL of the rat pituitary. The proportions of CD9/SOX2-, CD9/Ki67-, and PRL/TUNEL-positive cells decreased in the primary and secondary niches during pregnancy and DES treatment. In contrast, the number of CD9/PRL-positive cells increased in the AL-side MCL and AL parenchyma during pregnancy and during DES treatment. The proportion of PRL/Ki67-positive cells increased in the AL-side MCL and AL parenchyma in response to DES treatment. Next, we isolated CD9-positive cells from the IL-side MCL using an anti-CD9 antibody. During cell culture, the cells formed free-floating three-dimensional clusters (pituispheres). Furthermore, CD9-positive cells in the pituisphere differentiated into PRL cells, and their differentiation potential was promoted by DES. These findings suggest that CD9/SOX2-positive cells in the IL-side MCL may act as adult stem cells in the AL parenchyma that supply PRL cells under the influence of estrogen.

Impact of Chronic Prenatal Stress on Maternal Neuroendocrine Function and Embryo and Placenta Development During Early-to-Mid-Pregnancy in Mice.

Psychological stress, both leading up to and during pregnancy, is associated with increased risk for negative pregnancy outcomes. Although the neuroendocrine circuits that link the stress response to reduced sexual motivation and mating are well-described, the specific pathways by which stress negatively impacts gestational outcomes remain unclear. Using a mouse model of chronic psychological stress during pregnancy, we investigated 1) how chronic exposure to stress during gestation impacts maternal reproductive neuroendocrine circuitry, and 2) whether stress alters developmental outcomes for the fetus or placenta by mid-pregnancy. Focusing on the stress-responsive neuropeptide RFRP-3, we identified novel contacts between RFRP-3-immunoreactive (RFRP-3-ir) cells and tuberoinfundibular dopaminergic neurons in the arcuate nucleus, thus providing a potential pathway linking the neuroendocrine stress response directly to pituitary prolactin production and release. However, neither of these cell populations nor circulating levels of pituitary hormones were affected by chronic stress. Conversely, circulating levels of steroid hormones relevant to gestational outcomes (progesterone and corticosterone) were altered in chronically-stressed dams across gestation, and those dams were qualitatively more likely to experience delays in fetal development. Together, these findings suggest that, up until at least mid-pregnancy, mothers appear to be relatively resilient to the effects of elevated glucocorticoids on reproductive neuroendocrine system function. We conclude that understanding how chronic psychological stress impacts reproductive outcomes will require understanding individual susceptibility and identifying reliable neuroendocrine changes resulting from gestational stress.

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.

Expression of Rasd1 in mouse endocrine pituitary cells and its response to dexamethasone.

Dexamethasone-induced Ras-related protein 1 (Rasd1) is a member of the Ras superfamily of monomeric G proteins that have a regulatory function in signal transduction. Rasd1, also known as Dexras1 or AGS1, is rapidly induced by dexamethasone (Dex). While prior data indicates that Rasd1 is highly expressed in the pituitary and that the gene may function in regulation of corticotroph activity, its exact cellular localization in this tissue has not been delineated. Nor has it been determined which endocrine pituitary cell type(s) are responsive to Dex-induced expression of Rasd1. We hypothesized that Rasd1 is primarily localized in corticotrophs and furthermore, that its expression in these cells would be upregulated in response to exogenous Dex administration. Rasd1 expression in each pituitary cell type both under basal conditions and 1-hour post Dex treatment were examined in adult male mice. While a proportion of all endocrine pituitary cell types expressed Rasd1, a majority of corticotrophs and thyrotrophs expressed Rasd1 under basal condition. In vehicle treated animals, approximately 50-60% of corticotrophs and thyrotrophs cells expressed Rasd1 while the gene was detected in only 15-30% of lactotrophs, somatotrophs, and gonadotrophs. In Dex treated animals, Rasd1 expression was significantly increased in corticotrophs, somatotrophs, lactotrophs, and gonadotrophs but not thyrotrophs. In Dex treated animals, Rasd1 was detected in 80-95% of gonadotrophs and corticotrophs. In contrast, Dex treatment increased Rasd1 expression to a lesser extent (55-60%) in somatotrophs and lactotrophs. Corticotrophs of the pars intermedia, which lack glucocorticoid receptors, failed to display increased Rasd1 expression in Dex treated animals. Rasd1 is highly expressed in corticotrophs under basal conditions and is further increased after Dex treatment, further supporting its role in glucocorticoid negative feedback. In addition, the presence and Dex-induced expression of Rasd1 in endocrine pituitary cell types, other than corticotrophs, may implicate Rasd1 in novel pituitary functions.

Calcium-Prolactin Secretion Coupling in Rat Pituitary Lactotrophs Is Controlled by PI4-Kinase Alpha.

The role of calcium, but not of other intracellular signaling molecules, in the release of pituitary hormones by exocytosis is well established. Here, we analyzed the contribution of phosphatidylinositol kinases (PIKs) to calcium-driven prolactin (PRL) release in pituitary lactotrophs: PI4Ks - which control PI4P production, PIP5Ks - which synthesize PI(4, 5)P2 by phosphorylating the D-5 position of the inositol ring of PI4P, and PI3KCs - which phosphorylate PI(4, 5)P2 to generate PI(3, 4, 5)P3. We used common and PIK-specific inhibitors to evaluate the strength of calcium-secretion coupling in rat lactotrophs. Gene expression was analyzed by single-cell RNA sequencing and qRT-PCR analysis; intracellular and released hormones were assessed by radioimmunoassay and ELISA; and single-cell calcium signaling was recorded by Fura 2 imaging. Single-cell RNA sequencing revealed the expression of Pi4ka, Pi4kb, Pi4k2a, Pi4k2b, Pip5k1a, Pip5k1c, and Pik3ca, as well as Pikfyve and Pip4k2c, in lactotrophs. Wortmannin, a PI3K and PI4K inhibitor, but not LY294002, a PI3K inhibitor, blocked spontaneous action potential driven PRL release with a half-time of ~20 min when applied in 10 µM concentration, leading to accumulation of intracellular PRL content. Wortmannin also inhibited increase in PRL release by high potassium, the calcium channel agonist Bay K8644, and calcium mobilizing thyrotropin-releasing hormone without affecting accompanying calcium signaling. GSK-A1, a specific inhibitor of PI4KA, also inhibited calcium-driven PRL secretion without affecting calcium signaling and Prl expression. In contrast, PIK93, a specific inhibitor of PI4KB, and ISA2011B and UNC3230, specific inhibitors of PIP5K1A and PIP5K1C, respectively, did not affect PRL release. These experiments revealed a key role of PI4KA in calcium-secretion coupling in pituitary lactotrophs downstream of voltage-gated and PI(4, 5)P2-dependent calcium signaling.

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.

New insights into progesterone actions on prolactin secretion and prolactinoma development.

Progesterone (P4) has controversial physiological effects on the regulation of the lactotroph population. While some studies have shown a negative role for P4 in prolactin secretion and lactotroph proliferation, antagonizing estradiol effects, others demonstrated a proliferative role of P4 at the pituitary level. Usually, progesterone actions in the pituitary gland were studied through their classical, genomic pathways triggered by nuclear progesterone receptors (nPRs). However, in 2003, the scene became more complex with the discovery of another group of progesterone receptors involved in rapid, non-genomic P4 effects: the membrane progesterone receptors (mPRs), which are members of the progesterone and adipoQ receptor (PAQR) family. This review examines the historical background and current data on the study of progesterone actions on PRL secretion providing new evidence of P4 effects at the hypothalamic and at the pituitary level through non-classic P4-receptors. In addition, we explore the role of progesterone in the development of experimental prolactinomas, a controversial topic in the literature.

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.

Elevated Prolactin during Pregnancy Drives a Phenotypic Switch in Mouse Hypothalamic Dopaminergic Neurons.

Altered physiological states require neuronal adaptation. In late pregnancy and lactation, a sub-population of the mouse hypothalamic tuberoinfundibular dopaminergic (TIDA) neurons alters their behavior to synthesize and release met-enkephalin rather than dopamine. These neurons normally release dopamine to inhibit prolactin secretion and are activated by prolactin in a short-loop feedback manner. In lactation, dopamine synthesis is suppressed in an opioid-dependent (naloxone-reversible) manner, meaning that prolactin secretion is disinhibited. Conditional deletion of the prolactin receptor in neurons reveals that this change in phenotype appears to be driven by prolactin itself, apparently through an alteration in intracellular signaling downstream of the prolactin receptor that favors enkephalin production instead of dopamine. Thus, prolactin effectively facilitates its own secretion, which is essential for lactation and maternal behavior. These studies provide evidence of a physiologically important, reversible alteration in the behavior of a specific population of hypothalamic neurons in the adult brain.

Role of GPER in the anterior pituitary gland focusing on lactotroph function.

Ovarian steroids control a variety of physiological functions. They exert actions through classical nuclear steroid receptors, but rapid non-genomic actions through specific membrane steroid receptors have been also described. In this study, we demonstrate that the G-protein-coupled estrogen receptor (GPER) is expressed in the rat pituitary gland and, at a high level, in the lactotroph population. Our results revealed that ~40% of the anterior pituitary cells are GPER positive and ~35% of the lactotrophs are GPER positive. By immunocytochemical and immuno-electron-microscopy studies, we demonstrated that GPER is localized in the plasmatic membrane but is also associated to the endoplasmic reticulum in rat lactotrophs. Moreover, we found that local Gper expression is regulated negatively by 17ß-estradiol (E2) and progesterone (P4) and fluctuates during the estrus cycle, being minimal in proestrus. Interestingly, lack of ovarian steroids after an ovariectomy (OVX) significantly increased pituitary GPER expression specifically in the three morphologically different subtypes of lactotrophs. We found a rapid estradiol stimulatory effect on PRL secretion mediated by GPER, both in vitro and ex vivo, using a GPER agonist G1, and this effect was prevented by the GPER antagonist G36, demonstrating a novel role for this receptor. Then, the increased pituitary GPER expression after OVX could lead to alterations in the pituitary function as all three lactotroph subtypes are target of GPER ligand and could be involved in the PRL secretion mediated by GPER. Therefore, it should be taken into consideration in the response of the gland to an eventual hormone replacement therapy.

Participation of membrane progesterone receptor α in the inhibitory effect of progesterone on prolactin secretion.

The membrane progesterone receptors (mPRα, mPRß, mPRγ, mPRδ and mPRε) are known to mediate rapid nongenomic progesterone functions in different cell types. However, the functions of these receptors in the pituitary have not been reported to date. In the present study, we show that the expression of mPRα was the highest among the mPRs in the rat anterior pituitary gland. Immunostaining of mPRα was detected in somatotrophs, gonadotrophs and lactotrophs. Interestingly, 63% of mPRα-positive cells within the pituitary were lactotrophs, suggesting that mPRα is involved in controlling prolactin (PRL) secretion in the pituitary. To test this hypothesis, rat pituitaries were incubated (1 hour) with either progesterone (P4) or the mPRα-specific agonist Org OD 02-0. PRL secretion was then measured by radioimmunoassay. The results of this experiment revealed that both P4 and Org OD 02-0 decreased PRL secretion. Moreover, the results from the GH3 cell line (CCL-82.1) showed that P4 and Org OD 02-0 inhibited PRL release, although the nuclear PR agonist R5020 was ineffective. Our investigation of the cellular mechanisms behind mPRα activity indicated that both P4 and Org OD 02-0 decreased cAMP accumulation, whereas R5020 was ineffective. In addition, the Org OD 02-0-effect on PRL release was blocked by pretreatment with pertussis toxin, an inhibitor of Go/Gi proteins. Because transforming growth factor (TGF)ß1 is a potent inhibitor of PRL secretion in lactotrophs, we lastly evaluated whether TGFß1 was activated by progesterone and whether this effect was mediated by mPRα. Our results showed that P4 and Org OD 02-0, but not R5020, increased active TGFß1 levels. This effect was not observed when cells were transfected with mPRα-small interfering RNA. Taken together, these data provide new evidence suggesting that mPRα mediates the progesterone inhibitory effect on PRL secretion through both decreases in cAMP levels and activation of TGFß1 in the lactotroph population.

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.

Local electrostatic interactions determine the diameter of fusion pores.

In regulated exocytosis vesicular and plasma membranes merge to form a fusion pore in response to stimulation. The nonselective cation HCN channels are involved in the regulation of unitary exocytotic events by at least 2 mechanisms. They can affect SNARE-dependent exocytotic activity indirectly, via the modulation of free intracellular calcium; and/or directly, by altering local cation concentration, which affects fusion pore geometry likely via electrostatic interactions. By monitoring membrane capacitance, we investigated how extracellular cation concentration affects fusion pore diameter in pituitary cells and astrocytes. At low extracellular divalent cation levels predominantly transient fusion events with widely open fusion pores were detected. However, fusion events with predominately narrow fusion pores were present at elevated levels of extracellular trivalent cations. These results show that electrostatic interactions likely help determine the stability of discrete fusion pore states by affecting fusion pore membrane composition.

Hyperpolarization-activated cyclic nucleotide-gated channels and cAMP-dependent modulation of exocytosis in cultured rat lactotrophs.

Hormone and neurotransmitter release from vesicles is mediated by regulated exocytosis, where an aqueous channel-like structure, termed a fusion pore, is formed. It was recently shown that second messenger cAMP modulates the fusion pore, but the detailed mechanisms remain elusive. In this study, we asked whether the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which are activated by cAMP, are involved in the regulation of unitary exocytic events. By using the Western blot technique, a real-time PCR, immunocytochemistry in combination with confocal microscopy, and voltage-clamp measurements of hyperpolarizing currents, we show that HCN channels are present in the plasma membrane and in the membrane of secretory vesicles of isolated rat lactotrophs. Single vesicle membrane capacitance measurements of lactotrophs, where HCN channels were either augmented by transfection or blocked with an HCN channel blocker (ZD7288), show modulated fusion pore properties. We suggest that the changes in local cation concentration, mediated through HCN channels, which are located on or near secretory vesicles, have an important role in modulating exocytosis.

Prolactin prevents hepatocellular carcinoma by restricting innate immune activation of c-Myc in mice.

Women are more resistant to hepatocellular carcinoma (HCC) than men despite equal exposure to major risk factors, such as hepatitis B or C virus infection. Female resistance is hormone-dependent, as evidenced by the sharp increase in HCC incidence in postmenopausal women who do not take hormone replacement therapy. In rodent models sex-dimorphic HCC phenotypes are pituitary-dependent, suggesting that sex hormones act via the gonadal-hypophyseal axis. We found that the estrogen-responsive pituitary hormone prolactin (PRL), signaling through hepatocyte-predominant short-form prolactin receptors (PRLR-S), constrained TNF receptor-associated factor (TRAF)-dependent innate immune responses invoked by IL-1ß, TNF-α, and LPS/Toll-like receptor 4 (TLR4), but not TRIF-dependent poly(I:C)/TLR3. PRL ubiquitinated and accelerated poststimulatory decay of a "trafasome" comprised of IRAK1, TRAF6, and MAP3K proteins, abrogating downstream activation of c-Myc-interacting pathways, including PI3K/AKT, mTORC1, p38 MAPK, and NF-κB. Consistent with this finding, we documented exaggerated male liver responses to immune stimuli in mice and humans. Tumor promotion through, but regulation above, the level of c-Myc was demonstrated by sex-independent HCC eruption in Alb-Myc transgenic mice. PRL deficiency accelerated liver carcinogenesis in Prl(-/-) mice of both sexes. Conversely, pharmacologic PRL mobilization using the dopamine D2 receptor antagonist domperidone prevented HCC in tumor-prone C3H/HeN males. Viewed together, our results demonstrate that PRL constrains tumor-promoting liver inflammation by inhibiting MAP3K-dependent activation of c-Myc at the level of the trafasome. PRL-targeted therapy may hold promise for reducing the burden of liver cancer in high-risk men and women.

The role of cyclic nucleotides in pituitary lactotroph functions.

Lactotrophs are one of the five secretory anterior pituitary cell types specialized to synthesize and release prolactin. In vitro, these cells fire action potentials (APs) spontaneously and the accompanied Ca(2+) transients are of sufficient amplitude to keep the exocytotic pathway, the transcription of prolactin gene, and de novo hormone synthesis continuously active. Basal cyclic nucleotide production is also substantial in cultured cells but not critical for the APs secretion/transcription coupling in lactotrophs. However, elevated intracellular cAMP levels enhance the excitability of lactotrophs by stimulating the depolarizing non-selective cationic hyperpolarization-activated cyclic nucleotide-regulated and background channels, whereas cGMP inhibits it by activating Ca(2+)-controlled K(+) channels. Elevated cAMP also modulates prolactin release downstream of Ca(2+) influx by changing the kinetic of secretory pores: stimulate at low and inhibit at high concentrations. Induction of prolactin gene and lactotroph proliferation is also stimulated by elevated cAMP through protein kinase A. Together, these observations suggest that in lactotrophs cAMP exhibits complex regulatory effects on voltage-gated Ca(2+) influx and Ca(2+)-dependent cellular processes.