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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., (© Endocrine Society 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The differentiation of the hormone-producing cell lineages of the anterior pituitary represents an informative model of mammalian cell fate determination. The generation and maintenance of two of these lineages, the GH-producing somatotropes and prolactin (PRL)-producing lactotropes, are dependent on the pituitary-specific transcription factor POU1F1. Whereas POU1F1 is expressed in both cell types, and plays a direct role in the activation of both the Gh and Prl genes, GH expression is restricted to somatotropes and PRL expression is restricted to lactotropes. These observations imply the existence of additional, cell type-enriched factors that contribute to the somatotrope and lactotrope cell identities. In this study, we use transgenic mouse models to facilitate sorting of somatotrope and lactotrope populations based on the expression of fluorescent markers expressed under Gh and Prl gene transcriptional controls. The transcriptomic analyses reveal a concordance of gene expression profiles in the two populations. The limited number of divergent mRNAs between the two populations includes a set of transcription factors that may have roles in pituitary lineage divergence and/or in regulating expression of cell type-specific genes after differentiation. Four of these factors were validated for lineage enrichment at the level of protein expression, two somatotrope enriched and two lactotrope enriched. Three of these four factors were shown to have corresponding activities in appropriate enhancement or repression of landmark genes in a cell culture model system. These studies identify novel regulators of the somatotropes and lactotropes, and they establish a useful database for further study of these lineages in the anterior pituitary.

Prolactin (PRL) is a vertebrate hormone with diverse actions in osmoregulation, metabolism, reproduction, and in growth and development. Osmoregulation is fundamental to maintaining the functional structure of the macromolecules that conduct the business of life. In teleost fish, PRL plays a critical role in osmoregulation in fresh water. Appropriately, PRL cells of the tilapia are directly osmosensitive, with PRL secretion increasing as extracellular osmolality falls. Using a model system that employs dispersed PRL cells from the euryhaline teleost fish, Oreochromis mossambicus, we investigated the autocrine regulation of PRL cell function. Unknown was whether these PRL cells might also be sensitive to autocrine feedback and whether possible autocrine regulation might interact with the well-established regulation by physiologically relevant changes in extracellular osmolality. In the cell-perfusion system, ovine PRL and two isoforms of tilapia PRL (tPRL), tPRL177 and tPRL188, stimulated the release of tPRLs from the dispersed PRL cells. These effects were significant within 5-10 minutes and lasted the entire course of exposure, ceasing within 5-10 minutes of removal of tested PRLs from the perifusion medium. The magnitude of response varied between tPRL177 and tPRL188 and was modulated by extracellular osmolality. On the other hand, the gene expression of tPRLs was mainly unchanged or suppressed by static incubations of PRL cells with added PRLs. By demonstrating the regulatory complexity driven by positive autocrine feedback and its interaction with osmotic stimuli, these findings expand upon the knowledge that pituitary PRL cells are regulated complexly through multiple factors and interactions.

The pattern of prolactin (PRL) secretion depends on the physiological state. Due to insufficient detection sensitivity of existing assays, the precise description of these patterns in mice is lacking. We described an ultrasensitive ELISA assay that can detect mouse PRL in small fractions of whole blood, allowing longitudinal studies of PRL secretion profiles in freely moving mice. Over a 24-hour period, males displayed no oscillation in PRL levels, whereas virgin and lactating females showed large pulses. Peaks of PRL secretion reached 30-40 ng/mL in lactating female mice and rarely exceeded 10 ng/mL in virgin females. These pulses of PRL in lactating females were associated with suckling. The return of pups after an experimental 12-hour weaning induced a pulse of PRL release, reaching 100 ng/mL. This approach also enabled us to assess the inhibitory tone from hypothalamic dopamine neurons on PRL secretion. We used a dopamine D2 receptor antagonist to relieve pituitary lactotrophs from the tuberoinfundibular dopaminergic inhibitory tone and demonstrate a D2-induced PRL rise that can be used to evaluate both the secretory capacity of lactotrophs and the magnitude of the inhibitory tone on pituitary PRL release. We demonstrate that, although lactotroph function is altered to enhance chronic PRL output, their secretory response to acute stimulus is not modified during lactation and that chronic hyperprolactinemia is linked to a lower inhibitory tone. The combination of a sensitive PRL ELISA and administration of D2 receptor antagonist provide a unique opportunity to investigate the function and plasticity of the lactotroph axis in freely moving mice.

Liver sexual gene dimorphism, which depends mainly on specific patterns of GH secretion, may underlie differential susceptibility to some liver diseases. Because GH and prolactin secretion are regulated by dopaminergic pathways, we studied the participation of brain and lactotrope dopamine 2 receptors (D2Rs) on liver gene sexual dimorphism, to explore a link between the brain and liver gene expression. We used global D2R knockout mice (Drd2(-/-)) and conducted a functional dissection strategy based on cell-specific Drd2 inactivation in neurons (neuroDrd2KO) or pituitary lactotropes. Disruption of neuronal D2Rs (which impaired the GH axis) decreased most of male or female-predominant class I liver genes and increased female-predominant class II genes in males, consistent with the positive (class I) or negative (class II) regulation of these genes by GH. Notably, sexual dimorphism was lost for class I and II genes in neuroDrd2KO mice. Disruption of lactotrope D2Rs did not modify class I or II genes in either sex, because GH axis was preserved. But surprisingly, 1 class II gene (Prlr) and female-predominant class I genes were markedly up-regulated in lacDrd2KO females, pointing to direct or indirect effects of prolactin in the regulation of selected female-predominant liver genes. This suggestion was strengthened in the hyperprolactinemic Drd2(-/-) female mouse, in which increased expression of the same 4 liver genes was observed, despite a decreased GH axis. We hereby demonstrate endocrine-mediated D2R actions on sexual dimorphic liver gene expression, which may be relevant during chronic dopaminergic medications in psychiatric disease.

Context: Dopamine agonists (DA) are the first choice treatment of prolactinomas. However, a subset of patients is resistant to DA, due to undefined dopamine D2 receptor (D2R) alterations. Recently, D2R was found to associate with filamin-A (FLNA), a widely expressed cytoskeleton protein with scaffolding properties, in melanoma and neuronal cells., Objective: The aim of the study was to investigate the role of FLNA in D2R expression and signaling in human tumorous lactotrophs and rat MMQ and GH3 cells., Design: We analyzed FLNA expression in a series of prolactinomas by immunohistochemistry and Western blotting. We performed FLNA silencing or transfection experiments in cultured cells from DA-sensitive or -resistant prolactinomas and in MMQ and GH3 cells, followed by analysis of D2R expression and signaling., Results: We demonstrated reduced FLNA and D2R expression in DA-resistant tumors. The crucial role of FLNA on D2R was demonstrated by experiments showing that: 1) FLNA silencing in DA-sensitive prolactinomas resulted in 60% reduction of D2R expression and abrogation of DA-induced inhibition of prolactin release and antiproliferative signals, these results being replicated in MMQ cells that endogenously express FLNA and D2R; and 2) FLNA overexpression in DA-resistant prolactinomas restored D2R expression and prolactin responsiveness to DA, whereas this manipulation was ineffective in GH3 cells that express FLNA but not D2R. No alteration in FLNA promoter methylation was detected, ruling out the occurrence of epigenetic FLNA silencing in DA-resistant prolactinomas., Conclusions: These data indicate that FLNA is crucial for D2R expression and signaling in lactotrophs, suggesting that the impaired response to DA may be related to the reduction of FLNA expression in DA-resistant prolactinomas.

Dopamine, acting through the dopamine type 2 receptor (Drd2), is the main inhibitor of pituitary prolactin (PRL) secretion and lactotroph proliferation. TGF-β1 is involved, at least in part, in mediating these actions. It was described that TGF-β1 synthesis in rat pituitary lactotrophs is up-regulated by dopamine and down-regulated by estradiol. TGF-β1 is secreted as a large latent complex. The local regulation of cytokine activation in the pituitary has not yet been explored. In this work, we studied pituitary active and total TGF-β1 content, as well as TGF-β1 mRNA, and the in vivo role of dopamine and estradiol on pituitary TGF-β1 levels. Adult female mice (wild type), and female mice with a null mutation in the Drd2 (Drd2(-/-)), were used. The loss of dopaminergic tone induced a decrease in TGF-β1 mRNA expression, in active and total cytokine content, and in TGF-β type II receptor expression. Dopamine regulation of pituitary TGF-β1 activation process was inferred by the inhibition of active cytokine by in vivo sulpiride treatment. Interestingly, in the absence of dopaminergic tone, estradiol induced a strong increase in active TGF-β1. PRL secretion correlated with active, but not total cytokine. TGF-β1 inhibitory action on lactotroph proliferation and PRL secretion was decreased in Drd2(-/-) pituitary cells, in correlation with decreased TGF-β type II receptor. The study of the TGF-β1 activation process and its regulation is essential to understand the cytokine activity. As an intermediary of dopamine inhibition of lactotroph function, TGF-β1 and local activators may be important targets in the treatment of dopamine agonist-resistant prolactinomas.

During gestation, parturition, and lactation, the endocrine axis of the dam must continually adapt to ensure the continual and healthy development of offspring. The anterior pituitary gland, which serves as the endocrine interface between the brain and periphery, undergoes adaptations that contribute to regulation of the reproductive axis. Growth factors and their receptors are potential candidates for intrapituitary and paracrine factors to participate in the functional and anatomical plasticity of the gland. We examined the involvement of the growth factor glial cell-derived neurotrophic factor (GDNF) and its receptor tyrosine kinase rearranged during transfection (Ret) in the physiological functional and anatomical plasticity of the anterior pituitary gland. We found that variations in both expression and subcellular localization of Ret during gestation and lactation are temporally correlated with changes in pituitary gland function. We showed that Ret/GDNF signaling could endorse two different functional roles depending on the physiological status. At the end of lactation and after weaning, Ret was colocalized with markers of apoptosis. We found that Ret could therefore act as a physiological dependence receptor capable of inducing apoptosis in the absence of GDNF. In addition, we identified the follicullostellate cell as a probable source for intrapituitary GDNF and proposed GDNF as a potential physiological modulator of endocrine cell function. During all stages studied, we showed that acute application of GDNF to pituitary slices was able to modulate both positively and negatively intracellular calcium activity. Altogether our results implicate Ret/GDNF as a potent pleiotropic factor able to influence pituitary physiology during a period of high plasticity.

Although removal of dopamine inhibition is established as a major factor in prolactin (PRL) release, a large body of evidence suggests that hypothalamic oxytocin (OT) may serve as a PRL-releasing hormone in the rat. PRL release is modulated by estradiol (E2), which rises between diestrus and proestrus of the estrous cycle, causing a PRL surge in the afternoon of proestrus. Given that E2 strongly modulates OT actions in both central and peripheral tissues, OT action on lactotrophs might also be modulated by the stage of the estrous cycle. To test this hypothesis, we have monitored PRL release and intracellular calcium levels ([Ca(2+)](i)) induced by OT in pituitary lactotrophs obtained from female rats in either diestrus 1 or proestrus. We found that both secretory and [Ca(2+)](i) responses to OT are significantly increased in lactotrophs obtained on proestrus. Moreover, we show that these differences are due to an increase in both the number of OT-responding lactotrophs and the magnitude of their individual [Ca(2+)](i) responses. Both secretory and [Ca(2+)](i) responses were abolished by a specific OT antagonist. Finally, dose-dependent studies show that the increased PRL-releasing effect of OT on proestrus is significant over a wide range of concentrations, particularly those observed in hypophyseal portal plasma. These results suggest that the rising E2 titers that culminate on proestrus facilitate the stimulatory action of OT on lactotrophs and support the notion that OT is a PRL-releasing hormone with an important role in the production of the proestrous surge of PRL.

Epidermal growth factor (EGF) is a potent regulator of cell function in many cell types. EGF-receptor (EGFR/ErbB1)-activated Erk1/2 has been reported to activate estrogen receptor (ER) in an estrogen (E2)-independent manner. In the pituitary lactotrophs, both EGF and E2 stimulate prolactin (PRL) release, but the nature of interactions between ErbB and ERalpha signaling is unknown. Our objectives were to 1) characterize EGF-induced PRL release, 2) determine whether this effect requires ERalpha, and 3) determine the molecular basis for cross talk between ErbB and ERalpha signaling pathways. Using GH3 cells, a rat lactotroph cell line, we report that EGF stimulates PRL gene expression and release in a dose- and time-dependent manner. EGF caused a rapid and robust activation of Erk1/2 via ErbB1 and induced phosphorylation of S118 on ERalpha in an Erk1/2-dependent manner. The global antiestrogen ICI 182780 and the ERalpha-specific antagonist 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylet hoxy)phenol]-1H-pyrazole dihydrochloride (MPP), but not the ERbeta-specific antagonist 4-[2-Phenyl-5,7-bis(trifluoromethyl) pyrazolo[1,5-a]pyrimidin-3-yl]phenol (PHTPP), blocked the EGF-induced PRL release, indicating an ERalpha requirement. This was further supported by using ERalpha knockdown by small interfering RNA. Because the antiestrogens did not block EGF-induced Mek-1 or Erk1/2 phosphorylation, ERalpha is placed downstream from the ErbB1-activated Erk1/2. These results provide the first evidence that ErbB1-induced PRL release is ERalpha dependent.

In neuroendocrine cells, discharge of hormones follows the fusion of exocytotic vesicles with the plasma membrane at confined sites; however, the molecular nature of these distinct sites remains poorly understood. We studied intact pituitary lactotrophs and plasma membrane lawns by confocal microscopy in conjunction with antibodies against rat prolactin (rPRL), soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE) proteins (syntaxin-1 and synaptobrevin-2,) and fluorescent cholera toxin subunit B (CT-B), a marker of ganglioside monosialic acid (GM1) lipid rafts, to examine 1) whether rPRL vesicles discharge cargo at GM1 rafts, 2) whether discharging rPRL vesicles interact with SNAREs, and 3) to examine the overlap of GM1 rafts, rPRL, and syntaxin-1 sites in plasma membrane lawns. In intact cells, immunofluorescently labeled rPRL poorly colocalized (<6%) with CT-B. In conditions favoring endocytotic trafficking, vesicle SNARE synaptobrevin-2 modestly colocalized (35%) with CT-B, whereas it highly colocalized (58%) with retrieved rPRL. Although partial mixing between rPRL and CT-B intracellular trafficking pathways is likely, our results indicated that rPRL discharge involves interactions with plasma membrane SNAREs, but not with GM1 rafts. In support of this, the plasma membrane SNARE syntaxin-1 poorly colocalized with CT-B (<5%), whereas it highly colocalized (75%) with rPRL in inside-out plasma membrane lawns. Spontaneous and stimulated rPRL discharge in live lactotrophs is thus associated with plasma membrane sites enriched with SNARE proteins, however, spatially confined to plasma membrane areas other than GM1 rafts.

Previous studies have provided evidence for a paracrine interaction between pituitary gonadotrophs and lactotrophs. Here, we show that GnRH is able to stimulate prolactin (PRL) release in ovine primary pituitary cultures. This effect was observed during the breeding season (BS), but not during the nonbreeding season (NBS), and was abolished by the application of bromocriptine, a specific dopamine agonist. Interestingly, GnRH gained the ability to stimulate PRL release in NBS cultures following treatment with bromocriptine. In contrast, thyrotropin-releasing hormone, a potent secretagogue of PRL, stimulated PRL release during both the BS and NBS and significantly enhanced the PRL response to GnRH during the BS. These results provide evidence for a photoperiodically modulated functional interaction between the GnRH/gonadotropic and prolactin axes in the pituitary gland of a short day breeder. Moreover, the stimulation of PRL release by GnRH was shown not to be mediated by the gonadotropins, since immunocytochemical, Western blotting, and PCR studies failed to detect pituitary LH or FSH receptor protein and mRNA expressions. Similarly, no gonadotropin receptor expression was observed in the pituitary gland of the horse, a long day breeder. In contrast, S100 protein, a marker of folliculostellate cells, which are known to participate in paracrine mechanisms within this tissue, was detected throughout the pituitaries of both these seasonal breeders. Therefore, an alternative gonadotroph secretory product, a direct effect of GnRH on the lactotroph, or another cell type, such as the folliculostellate cell, may be involved in the PRL response to GnRH in these species.

Cervical stimulation induces two daily rhythmic prolactin surges, nocturnal and diurnal, which persist for several days. We have shown that a bolus injection of oxytocin initiates a similar prolactin rhythm, which persists despite low levels of oxytocin after injection. This suggests that oxytocin may trigger the cervical stimulation-induced rhythmic prolactin surges. To investigate this hypothesis, we infused an oxytocin antagonist that does not cross the blood-brain barrier for 24 h before and after cervical stimulation and measured serum prolactin. We also measured dopaminergic neuronal activity because mathematical modeling predicted that this activity would be low in the presence of the oxytocin antagonist. We thus tested this hypothesis by measuring dopaminergic neuronal activity in the tuberoinfundibular, periventricular hypophyseal, and tuberohypophyseal dopaminergic neurons. Infusion of oxytocin antagonist before cervical stimulation abolished prolactin surges, and infusion of oxytocin antagonist after cervical stimulation abolished the diurnal and significantly decreased the nocturnal surges of prolactin. The rhythmic prolactin surges returned after the clearance of the oxytocin antagonist. Hypothalamic dopaminergic activity was elevated in antiphase with prolactin surges, and the antiphase elevation was abolished by the oxytocin antagonist in the tuberoinfundibular and tuberohypophyseal dopaminergic neurons, consistent with the mathematical model. These findings suggest that oxytocin is a physiologically relevant prolactin-releasing factor. However, the cervical stimulation-induced prolactin surges are maintained even in the absence of oxytocin actions at the lactotroph, which strongly suggests the maintenance of prolactin surges are not dependent upon oxytocin actions at the pituitary gland.

The mitogenic action of estrogen on estrogen-responsive tissues is suggested to be mediated by paracrine growth factors secreted from neighboring estrogen receptor-positive cells. Using pituitary lactotrophs in primary culture, on which estrogen exerts both mitogenic and antimitogenic actions in a cell context-dependent manner, we investigated whether a paracrine cell-to-cell interaction with other pituitary cell types was required for estrogen action. In pituitary cells, enriched for lactotrophs by 85% using differential sedimentation on a discontinuous Percoll gradient, 17beta-estradiol (E2) showed an antimitogenic action on lactotrophs in the presence of IGF-I, which was similar to that in control unenriched cells. Mitogenic actions were also seen in lactotroph-enriched cells when E2 was administered alone, in combination with serum, or in combination with the adenylate cyclase activator forskolin. Similar results were obtained in 90% lactotroph-enriched cells collected by fluorescence-activated cell sorting from transgenic rats expressing enhanced green fluorescent protein under the control of the prolactin promoter. The putative role of basic fibroblast growth factor (bFGF) as a paracrine factor mediating the mitogenic action of estrogen was not supported by the results that: 1) bFGF inhibited lactotroph proliferation; 2) immunoneutralization of bFGF failed to block E2-induced proliferation; and 3) cellular bFGF levels were not altered by E2 treatment. These results suggest that the antimitogenic and mitogenic actions of estrogen on lactotrophs do not require paracrine signals from other pituitary cell types and that estrogen directly influences lactotroph proliferation.

Cessation of lactation causes a massive loss of surplus lactotrophs in the rat pituitary gland. The factors and mechanisms involved in this phenomenon have not yet been elucidated. Besides its inhibitory control on prolactin secretion and lactotroph proliferation, evidence suggests that dopamine (DA) may be a proapoptotic factor for lactotrophs. We therefore tested the proapoptotic effect of DA on pituitary glands from virgin, lactating, and postlactating rats. By measuring mitochondrial membrane potential loss, caspase-3 activation, and nuclear fragmentation, we show that DA induces apoptosis specifically in lactotrophs from postlactating rats. We then determined that this effect was partly mediated by the DA transporter (DAT) rather than the D(2) receptor, as corroborated by the detection of DAT expression exclusively in lactotrophs from postlactating rats. We also observed tyrosine hydroxylase (TH) expression in postlactating lactotrophs that was accompanied by an increase in DA content in the anterior pituitary gland of postlactating compared with virgin rats. Finally, we observed that cells expressing TH coexpressed DAT and cleaved caspase-3. These findings show that DA may play a role in lactotroph regression during the postlactation period by inducing apoptosis. The fact that this process requires DAT and TH expression by lactotrophs themselves suggests that it may be "autocrine" in nature.