Prolactin Synthesis Research Articles

Loss of tumorigenicity of estrogen receptor (ER) beta-expressing breast cancer MCF-7 cells

The mammary gland is the major target tissue of prolactin (PRL) in mammals. Although this pituitary hormone has been long suspected to be involved in the progression of human breast cancer, the failure of clinical improvement by treatment with dopamine agonists (which lower circulating levels of PRL) rapidly reduced the interest of oncologists concerning a potential role of PRL in the development of breast cancer. Within the last few years, however, several studies reported first, that PRL is also synthesized by mammary epithelial cells, and second that it may exert a proliferative action in an autocrine/paracrine manner. In agreement with a recent epidemiological study, these observations have led to a reconsideration of the role of PRL as an active participant in breast cancer, and are an impetus to redefine the molecular targets of anti-prolactin strategies since dopamine analogs are assumed to be inefficient on extrapituitray PRL synthesis. In this review, we briefly summarize the current knowledge of PRL effects on both normal and tumor mammary cells, and we discuss the most relevant articles supporting the autocrine-paracrine action of PRL in the breast. With the aim of defining putative new molecular targets, we propose an overview of the main PRL receptor signaling cascades known to be triggered by PRL in mammary epithelial cells or, when not available, in other cell types. Finally, because proteolytic fragments of rat PRL have been shown to inhibit the angiogenic process, which may be relevant for preventing the progression of solid tumors such as breast tumors, we discuss the hypothesis that the enzymatic cleavage of human PRL could also represent a new molecular target in the search for alternative strategies in the treatment of breast cancer.

Effects of prolactin inhibition during late gestation on the immunity of gilts and foetuses

The goal of the present study was to determine the effects of inhibiting prolactin (PRL) synthesis for three consecutive 20-d periods during the second half of gestation, on the immune status of gilts and their foetuses. Crossbred gilts were randomly assigned as controls (n = 12) or received 10 mg of bromocriptine orally three times daily from days 50 to 69 (BR50, n = 12), days 70 to 89 (BR70, n = 12), or days 90 to 109 (BR90, n = 12) of gestation. All gilts were injected subcutaneously with ovalbumin (OVA) on days 53 and 72 of gestation. Blood samples were collected on days 50, 60, 70, 90 and 109 of gestation to evaluate the antibody response. Cellular immunity, as measured by lymphocyte proliferative response and production of interferon-γ, was characterized on controls and BR50 gilts until the end of gestation. Six foetuses from five litters per treatment were selected and their thymus and spleen were excised and weighed. Splenocytes and thymocytes were assayed to characterize lymphocyte sub-populations and to evaluate splenocyte responses to mitogenic stimulation. Average spleen weight in foetuses from BR90 gilts was significantly lower (P < 0.05) than that of foetuses from control or BR70 gilts. In all gilts treated with bromocriptine,percentages of CD4+ and CD8+ lymphocyte populations in foetus spleens were numerically reduced compared with those obtained in foetus from control gilts but the differences were not significant. In BR50 gilts, inhibition of PRL synthesis did not affect the proliferative response of lymphocytes to mitogenic stimulations compared with control group, but tended (P = 0.13) to increase the production of interferon-γ. A time effect showed that production of interferon-γ by leukocytes was reduced (P < 0.02) on days 80 and 100 compared with days 50 and 70, regardless of bromocriptine treatment. The antibody response of gilts to OVA was not affected by bromocriptine treatments. In conclusion, inhibition of PRL synthesis by bromocriptine did not significantly affect immune response of pregnant gilts. In foetuses, although the differences between treatments were not significant, data suggest that the foetal development of immune tissues seems to be impaired by bromocriptine treatment. Key words: Bromocriptine, prolactin, immunity, pregnancy, sow

Regulation of Prolactin Gene Expression by Vasoactive Intestinal Peptide and Dopamine in the Turkey: Role of Ca2+ Signalling

Our recent work has demonstrated that dopamine, acting through D2 dopamine receptors on pituitary cells, inhibits the stimulatory effects of vasoactive intestinal peptide (VIP) on prolactin release and prolactin gene transcription. It is hypothesised that the stimulatory and inhibitory roles of VIP and dopamine, respectively, on prolactin synthesis and release are mediated by their opposite effects on intracellular Ca2+ concentration ([Ca2+]i) in lactotrophs. The present study aimed: (i) to investigate the effect of VIP and dopamine on [Ca2+]i of cultured turkey anterior pituitary cells and (ii) to examine the role of Ca2+ signalling in mediating the regulatory effects of VIP and dopamine on prolactin mRNA levels and prolactin release. Changes in [Ca2+]i were measured spectrofluorometrically using Fura-2/AM as a fluorescent Ca2+ indicator. Semi-quantitative reverse transcription-polymerase chain reaction and radioimmunoassay were used to determine prolactin mRNA levels and prolactin release, respectively. VIP or the L-type Ca2+ channel activator, Bay K8644 (Bay) increased [Ca2+]i in a concentration- and time-dependent fashion, an effect abolished by preincubating the cells with R(-)-propylnorapomorphine HCl, a D2 dopamine receptor agonist (D2AG) or Verapamil (VR), a specific L-type Ca2+ channel blocker. Similarly, either VR or D2Ag diminished the VIP/Bay stimulatory effect on prolactin expression and release. On the other hand, pretreatment of pituitary cells with thapsigargin (TG) or neomycin (NEO), to deplete the intracellular Ca2+ stores, showed no effect on basal or VIP-stimulated prolactin mRNA levels; although VIP-induced prolactin release was partially inhibited by NEO but not TG. These results suggest that intracellular Ca2+ represents a common signal transduction pathway through which VIP and dopamine can exert antagonistic control on prolactin synthesis and release in avian lactotrophs.

Oestrogen Requires the Insulin‐like Growth Factor‐I Receptor for Stimulation of Prolactin Synthesis via Mitogen‐Activated Protein Kinase

Sex steroids and growth factors interact at the intracellular level in a variety of tissues to control numerous physiological functions. Oestrogen is known to stimulate prolactin synthesis and secretion, but the effect of insulin-like growth factor (IGF)-I is less clear. We used GH3 cells, a somatolactotroph cell line, to study the interaction of 17beta-oestradiol (E(2)) and IGF-I on prolactin protein levels and the intracellular mechanisms involved. Cell cultures were treated with E(2) (10 nM) and/or IGF-I (10 ng/ml) for 8 h. The real-time reverse transcriptase-polymerase chain reaction, Western blot and enzyme-immunoassay were used to determine changes in prolactin mRNA and protein levels. At this time-point, there were no significant changes in cell number, prolactin mRNA expression, or the amount of secreted prolactin. However, E(2) increased intracellular prolactin concentrations. IGF-I alone had no effect, but blocked the stimulatory effect of E(2). MAPK (ERK1/2) activation, as determined by Western blot analysis, increased with both E(2) and IGF-I, but not with the combination of these factors. The MAPK inhibitor PD98059 blocked the ability of E(2) to increase intracellular prolactin concentrations. Similarly, the IGF-I receptor antagonist, JB1, blocked the effect of E(2) on prolactin synthesis and MAPK activation, as did the oestrogen receptor antagonist ICI182 780. These results suggest that, to stimulate prolactin synthesis, E(2) activates the MAPK cascade and that this requires the presence of both oestrogen and IGF-I receptors.

Identification of Prolactin Synthesis at Early Differentiation Stage of T Cells in the Thymus

Ectopic gene expression of prolactin (PRL) has been reported in different tissues among several species. Recently, lymphocytes have also been shown to produce PRL, which is considered to be involved in the immunoregulatory system. However, when and where PRL synthesis occurs in lymphocytes remain unknown. In the present study, we attempted to examine the existence of PRL producing cells in the mouse thymus and spleen of immune tissues. We performed double fluorescence immunohistochemistry (DFIH) for mouse PRL (mPRL) and mouse CD4 that was specifically produced in T cells, and for mPRL and mouse CD25 or CD44, which specify the CD4+ precursor cells in the early stage of T cell differentiation in the thymus. Similarly, we performed DFIH for mPRL and mouse IL-4 or IFN-γ which are specifically produced in helper T cells of the mouse spleen. The immunoreactive substance for mPRL was co-localized in some CD4 or CD44 immunopositive cells in the thymus and some IL-4 or IFN-γ producing cells in the spleen. In conclusion, we found that mPRL is synthesized at the early stage of T cell differentiation.

Effect of aqueous extract of Acacia nilotica ssp adansonii on milk production and prolactin release in the rat.

In view of the traditional belief that Acacia nilotica ssp adansonii (AN) can stimulate milk production in lactating women, experiments were performed to determine the effect of an aqueous extract of AN on milk production in rats. Female rats that received oral doses of aqueous extract of this plant during their first lactation produced about 59% more milk than controls (P<0.01). Pup weight gain was also significantly higher than that in the control group. A lower dose, comparable to that used by women to improve their milk yield, led to about 33% more milk with the same growth rate for pups as that in the high-dose group. The extract of AN was found to stimulate the synthesis and release of prolactin (PRL) significantly (P<0.05). In addition, the mammary glands of oestrogen-primed rats treated with the extract showed clear lobuloalveolar development with milk secretion. This study demonstrates that the aqueous extract of AN can stimulate milk production and PRL release in the female rat and could consequently have the properties claimed for lactating women.

Immunocytochemical detection of effects of TRH and T 4 on prolactin- and TSH-producing cells in the pituitary gland of Rana ridibunda

Effects of synthetic thyrotropin-releasing hormone (TRH) and various doses of thyroxin (T 4) on prolactin (PRL)-producing cells and thyrotropic cells in the pituitary were investigated in adult male and female Rana ridibunda frogs. Animals were given 200 μg TRH once a week for 4 weeks and 0.2–0.5 mg T 4 during 3 days per week for a period of 2 weeks by injections in the groin. PRL-producing cells and thyrotropic cells were identified with light microscopical and electron microscopical immunocytochemical methods, using rabbit anti-PRL and rabbit anti-thyroid stimulating hormone (TSH) as primary antibodies. TRH caused cytological changes in both cell types, which were consistent with increased synthesis and release of both PRL and TSH. Treatment with 0.5 mg T 4 activated both cell types less than TRH treatment did, whereas 0.2 and 0.4 mg T 4 caused inactivation of both cell types. In conclusion, mammalian TRH is effective on both types of frog pituitary cells. Our study suggests that T 4 has a positive rather than a negative effect when concentrations above a certain threshold are given.

Post-Operative Evaluation of Anterior Pituitary Function in a Nigerian with Hyperprolactinaemia: A Case Report and Review of Literature

Prolactinoma is an endocrine abnormality associated with hyperprolactinaemia of which there is paucity of data in this environment probably due to under-reporting, under diagnosis and inadequate skill for management. This is a report of a 42 year old Nigerian who underwent surgery for pituitary prolactinoma in Western Germany and was referred to our hospital for assessment of anterior pituitary reserve on return to Nigeria. Basal values of glucose, cortisol, T3, T4, TSH, LH, FSH, and prolactin were measured following which a triple function test was conducted by intravenous administration of a bolus consisting of 250ug of ACTH, 200ug TRH and 100ug GnRH. Basal values demonstrated normoglycaemia and euthyroidism while responses to TRH and GnRH indicated restoration of anterior pituitary capacity for synthesis and control of TSH, LH, FSH and prolactin post-operatively. (Key words: Hyperprolactinaemia, Hypophysectomy, Triple function test, Immunoassay). Sahel Medical Journal Vol.7(1) 2004: 41-45

Identification of estrogen-responsive genes in the GH3 cell line by cDNA microarray analysis

To identify estrogen-responsive genes in somatolactotrophic cells of the pituitary gland, a rat pituitary cell line GH3 was subjected to cDNA microarray analysis. GH3 cells respond to estrogen by growth as well as prolactin synthesis. RNAs extracted from GH3 cells treated with 17β-estradiol (E2) at 10 −9 M for 24 h were compared with the control samples. The effect of an antiestrogen ICI182780 was also examined. The array analysis indicated 26 genes to be up-regulated and only seven genes down-regulated by E2. Fourteen genes were further examined by real-time RT-PCR quantification and 10 were confirmed to be regulated by the hormone in a dose-dependent manner. Expression and regulation of these genes were then examined in the anterior pituitary glands of female F344 rats ovariectomized and/or treated with E2 and 8 out of 10 were again found to be up-regulated. Interestingly, two of the most estrogen-responsive genes in GH3 cells were strongly dependent on E2 in vivo. #1 was identified as calbindin-D9k mRNA, with 80- and 118-fold induction over the ovariectomized controls at 3 and 24 h, respectively, after E2 administration. #2 was found to be parvalbumin mRNA, with 30-fold increase at 24 h. Third was c- myc mRNA, with 4.5 times induction at 24 h. The levels were maintained after one month of chronic E2 treatment. Identification of these estrogen-responsive genes should contribute to understating of estrogen actions in the pituitary gland.

Author Index of Volume 192

The dopaminergic regulation of prolactin (PRL) and growth hormone (GH) synthesis were studied in monolayer cultures of rat pituitary tumor (GH3) cells. PRL synthesis could not be inhibited by dopamine at concentrations ranging from 10−9 to 10−5 M. Bromocriptine inhibited PRL synthesis only at the very high concentration of 10−5 M, and this effect was not reversible by metoclopramide. GH synthesis was inhibited by 10−6 and 10−5 M dopamine but not by bromocriptine, and the dopamine effect was blocked by metoclopramide. These results suggest the existence of a defective dopaminergic regulation of PRL and GH synthesis in GH3 cells.

Hypoxia Inhibits Expression of Prolactin and Secretion of Cathepsin-D by the GH4C1 Pituitary Adenoma Cell Line

Diminished oxygen concentration within growing tumors may stimulate neovascularization by inducing both up-regulation of angiogenic factors and down-regulation of antiangiogenic agents. A potentially important molecule in the growth of pituitary adenomas is prolactin (PRL), which can be cleaved by cathepsin-D to yield a 16-kDa form (16K-PRL) with potent antiangiogenic effects. We examined the expression of PRL in cultured GH4C1 pituitary adenoma cells after exposure to hypoxia (0.1% oxygen) for periods of 12 to 36 hours. In contrast to increased expression of the angiogenic factor vascular endothelial growth factor in hypoxic cells, PRL mRNA and levels of intracellular and secreted PRL were significantly reduced under hypoxia. The reduction was not attributable to a general suppression of either transcription or protein synthesis. Although 16K-PRL was not evident in conditioned medium at physiologic pH, lowering the pH to mimic the acidic tumor microenvironment resulted in generation of 16K-PRL, which was sharply reduced in medium drawn from hypoxic cells. Production of 16K-PRL was blocked by the cathepsin-D inhibitor pepstatin-A, and the reduced 16K-PRL formation in hypoxic-conditioned medium correlated with a decrease in secretion of cathepsin-D and its precursor, procathepsin-D. Thus, hypoxia acts upon GH4C1 cells to increase vascular endothelial growth factor expression, decrease PRL synthesis, and suppress conversion of PRL to 16K-PRL via inhibition of cathepsin-D proteolysis. These mechanisms may act in concert to stimulate angiogenesis in prolactinomas.

P.4.035 Effects of cerebrolysin on quantitative EEG activity in elderly controls and in patients with brain damage

The control of prolactin (PRL) cell activity in Salmo gairdneri was investigated in vivo and in vitro. In some in vivo experiments treatment was followed by estimation of pituitary PRL content by gel electrophoresis or of PRL cell nuclear area by light microscopy. In the remainder, treatment was followed by incubation of the pituitary glands in drug-free medium for estimation of PRL synthesis and release. The dopamine precursor, l-dopa (20 mg/kg), reduced pituitary PRL content. Conversely, the dopamine-receptor blocker, domperidone (10 mg/kg), increased total PRL content and amount released in the subsequent incubation. The initial serotonin precursor, l-tryptophan (75 mg/kg), increased pituitary PRL content and PRL cell nuclear area. 5-HTP (20 mg/kg), the immediate serotonin precursor, increased both percentage PRL release and total PRL levels during subsequent incubation. Pargyline (25 mg/kg) treatment to inhibit serotonin catabolism elevated PRL levels in pituitary and medium during subsequent incubation. The serotonin synthesis blocker, parachlorophenylalanine (pCPA; 100 mg/kg), nonsignificantly reduced PRL cell nuclear area. When this was followed by incubation, percentage PRL release and total PRL fell significantly. During in vitro incubation, dopamine (2 μg/ml) reduced the release of PRL into the medium, while serotonin (10−5 M) increased PRL release. These results suggest that both an inhibitory dopaminergic and a stimulatory serotonergic system may be involved in PRL cell regulation in S. gairdneri. The lack of any significant effect of cortisol (1 μg/ml), somatostatin (300 ng/ml, GABA (100 ng/ml) and TRH 100 ng/ml) on PRL release in vitro suggested little or no involvement of these putative regulatory factors in PRL cell regulation.

Tyroxine hydroxylase (TH) downregulation in hyperstimulated ovaries reveals the dopamine agonist bromocriptine (Br2) as an effective and specific method to block increased vascular permeability (VP) in OHSS

Objective: Ovarian hyperstimulation syndrome (OHSS) is induced by the ovarian release of vasoactive-angiogenic substances which increase vascular permeability (VP). Vascular Endothelial Growth Factor (VEGF) plays a major role in this regard. Inhibition of VEGF action has been accompanied by decrease in VP. However, the use of such strategy as antiangiogenic tumoral drugs in women has been abandoned due to toxic effects. Aiming to find a specific-non toxic treatment for OHSS, we have looked at the expression of gene products regulated under OHSS conditions, and developed new approaches employing the D-2 dopamine agonist Bromocriptine (Br2). Design: In the first series of experiments, we compared ovarian gene expression in animals (n = 8 per group) subjected to 3 different regimens of gonadotropins: Pregnant Mare′s Serum Gonadotropin (PMSG) 10 IU/day for 4 days and hCG (30 IU) the fifth day (OHSS group); PMSG (10 IU) plus hCG (10 IU) 2 days later (MILD group); and PMSG (1 IU) plus hCG (1 IU) 48 hrs later (CONTROL group). Fourty-eight hours after hCG, VP was measured, and mRNA from ovaries extracted to perform gene expression profiles in macroarray filters containing 14,000 genes. Only genes with a 3-fold up or down regulation were considered to be significantly regulated. The hybridization results were confirmed by quantitative Fluorescence(QF)-PCR, immunostaining, and cluster analysis. In the second series of experiments, Br2 was employed in OHSS animals (40 IU PMSG+30IU hCG). Different doses of Br2: 1.6, 4, 10 and 25 mg/kg were intraperitoneally administered the day of hCG and 24 hrs later. VP was measured 48 hrs after hCG. Materials and Methods: Immature female Wistar rats were employed. In the macroarray technology, RNA was reverse transcribed and transferred to the nylon membrane filters under radioactive conditions. Clusters of 5×5 spot signals were analyzed using the AIDA software, and its expression paired to the other groups after background signal substraction. To confirm gene expression, we used the same RNA and performed QF-PCR in an ABIPRISM 7700 thermocycler to amplify 3 up-regulated and 3 down-regulated genes. To measure VP, 0.2 ml 5mM Evans blue(EB) were injected via the femoral vein. After 30 min, the peritoneal cavity was irrigated with saline. Light absorption was measured in the recovered fluid at 595 nm and the VP expressed as μg EB/100 g body weight. Results: Gene expression in the 3 groups showed 80 upregulated and 7 downregulated genes in OHSS as compared to MILD and CONTROLS. Upregulated genes were grouped in 5 families: Cholesterol synthesis,VEGF signal transduction, Prostaglandins synthesis, oxidative stress process and Prolactin synthesis. The down-regulation of Tyrosine-hydroxylase (TH, enzyme responsible for Dopamine synthesis) was considered a characteristic of OHSS too. As a result, the D2 dopamine agonist Br2 was employed. Increased VP in OHSS animals (30.7±5.8 μg EB/100 g body weight) was significantly reduced when 1.6 mg Br2 (15.3±3.3; p = 0.04), 4 mg Br2 (14.2±2.2; p = 0.04), 10 mg Br2 (10.1±2.6; p = 0.009), and 25 mg Br2 (8.0±1.9; p = 0.001) were employed. Conclusion: OHSS is associated with the up and down regulation of several families of genes in the ovaries. In addition to VEGF, the up and down regulation of genes encoding for prolactin and dopamine expression, encouraged the successful use of Br2 to prevent increased VP. These experiments provide the rationale to test Br2 as a safe and possible effective medication in the prevention/treatment of OHSS.

Heterogeneous regulation of individual lactotroph cells by photoperiod in the Syrian hamster ( Mesocricetus auratus)

In many mammals, changes in daylength (photoperiod) regulate multiple aspects of physiology, including the synthesis and secretion of the anterior pituitary hormone prolactin. Here, we tested the hypothesis that individual lactotroph cells exhibit a heterogeneous response to changes in photoperiod, by exploiting a recently developed assay for prolactin gene expression in single pars distalis (PD) cells. Male Syrian hamsters were exposed to either long (LD; 16 h light: 8 h dark) or short (SD; 8 h light: 16 h dark) photoperiods for 12 weeks. Response of the lactotrophic axis to photoperiod was confirmed by the significantly ( P<0.01) lower plasma prolactin concentrations in SD than LD hamsters. Analysis of freshly dispersed PD cells by in situ hybridisation demonstrated that photoperiod has no effect ( P>0.05) on the proportion of PD cells (approximately 25%) that expressed prolactin mRNA. Heterogeneity of prolactin mRNA expression was observed in both LD and SD. A similar proportion of cells expressed low levels of prolactin mRNA in both photoperiods, suggesting that they may be unresponsive to photoperiod change. In contrast, the remaining PD cells that expressed prolactin mRNA exhibited markedly increased gene expression in LD, consistent with the selective recruitment of a lactotroph subpopulation to a more transcriptionally active state in this photoperiod.

Dopaminergic regulation of avian prolactin gene transcription.

It is well documented that prolactin (PRL) release and PRL gene expression in birds are controlled by the tonic stimulation of hypothalamic vasoactive intestinal peptide (VIP). However, there is good evidence that dopamine (DA) exerts both stimulatory (at the hypothalamic level) and inhibitory (at the pituitary level) effects on PRL secretion. The interactions between VIP and DA in the regulation of PRL gene transcription are not known. This study was designed to examine the effects of a D(2) DA receptor agonist (D(2)AG; R(-)-propylnorapomorphine HCl) on basal and VIP-stimulated PRL gene transcription rate, PRL mRNA steady-state levels, PRL mRNA stability and PRL release from cultured turkey anterior pituitary cells. The D(2)AG (10(-)(10) M) completely inhibited the stimulatory effect of VIP (10(-)(7) M) upon nascent PRL mRNA as determined utilizing a nuclear run-on transcription assay. To examine further the effect of the D(2)AG on PRL mRNA post-transcriptional events, anterior pituitary cells were treated with different concentrations of D(2)AG (10(-)(12)-10(-)(4) M). Semi-quantitative RT-PCR and RIA were performed to determine the levels of PRL mRNA and PRL content in the medium respectively. The results show that D(2)AG inhibited VIP-stimulated PRL mRNA steady-state levels as well as basal and VIP-stimulated PRL release, effects which were diminished by the D(2) DA receptor antagonist, S(-)-eticlopride HCl (10(-)(10) M). Actinomycin D (5 microg/ml), an inhibitor of mRNA synthesis, was used to assess the effect of D(2)AG on PRL mRNA stability in response to VIP. The stimulatory effect of VIP on PRL mRNA stability was completely negated by the D(2)AG (from a half-life of 53.0+/-2.3 h in VIP-treated cells to 25.5+/-1.6 h in D(2)AG+VIP-treated cells, P<or=0.05). These results support the hypothesis that VIP and DA play a major role in the regulation of PRL gene expression in avian species, at both the transcriptional and post-transcriptional levels. In addition, these findings suggest that the DAergic system inhibits PRL release and synthesis by antagonizing VIP at the pituitary level via D(2) DA receptors.

The human neuroendocrine thyrotropin-releasing hormone receptor promoter is activated by the haematopoietic transcription factor c-Myb.

Thyrotropin-releasing hormone (TRH) receptor (TRHR) is a G-protein-coupled receptor playing a crucial role in the anterior pituitary where it controls the synthesis and secretion of thyroid-stimulating hormone and prolactin. Its widespread presence not only in the central nervous system, but also in peripheral tissues, including thymus, indicates other important, but unknown, functions. One hypothesis is that the neuropeptide TRH could play a role in the immune system. We report here that the human TRHR promoter contains 11 putative response elements for the haematopoietic transcription factor c-Myb and is highly Myb-responsive in transfection assays. Analysis of Myb binding to putative response elements revealed one preferred binding site in intron 1 of the receptor gene. Transfection studies of promoter deletions confirmed that this high-affinity element is necessary for efficient Myb-dependent transactivation of reporter plasmids in CV-1 cells. The Myb-dependent activation of the TRHR promoter was strongly suppressed by expression of a dominant negative Myb-Engrailed fusion. In line with these observations, reverse transcriptase PCR analysis of rat tissues showed that the TRHR gene is expressed both in thymocytes and bone marrow. Furthermore, specific, high-affinity TRH agonist binding to cell-surface receptors was demonstrated in thymocytes and a haematopoietic cell line. Our findings imply a novel functional link between the neuroendocrine and the immune systems at the level of promoter regulation.

Calcitonin inhibits prolactin promoter activity in rat pituitary GGH3 cells: evidence for involvement of p42/44 mitogen-activated protein kinase in calcitonin action.

Previous findings from our laboratory have shown that pituitary calcitonin-like peptide (pit-CT) is synthesized and released by gonadotrophs and inhibits prolactin (PRL) release, synthesis, and lactotroph proliferation. To investigate further the regulation of PRL gene transcription by CT, we examined the effect of CT on rat PRL (rPRL) promoter activity in rat pituitary GGH3 cells. GGH3 cells were transiently transfected with rPRL promoter- luciferase and control plasmids. Thirty-six hours later, the cells were treated with CT or other agents and their effect on luciferase activity was examined. The effect of CT and/or thyrotropin-releasing hormone (TRH) on p42/44 mitogen-activated protein kinase (MAPK) activity was also investigated. CT inhibited basal rPRL promoter activity in a dose-dependent fashion, with an approximate IC(50) of 3 nM. The maximal inhibition occurred 1 h after the CT addition, and the peptide was equipotent in inhibiting 600 and 2500 rPRL promoter constructs. CT also inhibited TRH-, Bay K 8644-, and ionomycin-induced rPRL promoter activity. CT mimicked the actions of MEK inhibitors U0126 and PD 980089. However, CT could not inhibit rPRL promoter activity in GGH3 cells expressing constitutively active ERK1 or ERK2. CT markedly attenuated phospho-MAPK immunoreactivity in untreated as well as TRH-treated GGH3 cells. These results suggest that CT inhibits rPRL promoter activity by antagonizing Ca(2+) and ERK1/2-mediated signaling events. They also demonstrate that CT is a potent inhibitor of early events associated with PRL gene activation and may play an important role in regulation of lactotroph function.

Effects of insulin-like growth factors (IGF-I and -II) on growth hormone and prolactin release and gene expression in euryhaline tilapia, Oreochromis mossambicus

We investigated in vitro effects of insulin-like growth factors (IGF-I and -II) on growth hormone (GH) and prolactin (PRL) release and gene expression in euryhaline tilapia, Oreochromis mossambicus. Pituitaries were removed from freshwater-acclimated adult males and incubated for 2–24 h in the presence of human IGF-I or -II at doses ranging from 1–1000 ng/ml (0.13–130 nM). IGF-I at concentrations higher than 10 ng/ml and IGF-II higher than 100 ng/ml significantly inhibited GH release after 8, 16, and 24 h. No effect of IGFs was seen during the first 4 h of incubation. IGFs at the same concentrations also significantly attenuated GH gene expression after 24 h, although no effect was seen at 2 h. By contrast, PRL 188 release was stimulated significantly and in a dose-related manner by IGF-I at concentrations higher than 10 ng/ml and by IGF-II at concentrations higher than 100 ng/ml within 2 h. No stimulation was observed after 4 h. Similarly, both IGFs at concentrations higher than 10 ng/ml increased PRL 177 release within 2 h. However, no significant effect of IGF-I or -II was observed on mRNA levels of both PRLs after 2 and 24 h at all concentrations examined. These results clearly indicate differential regulation of GH and PRL release and synthesis by IGFs in the tilapia pituitary, i.e., rapid-acting, stimulatory effects of IGFs on PRL release and slow-acting, inhibitory effects on GH release and synthesis.

Differential regulation of pituitary hormone secretion and gene expression by thyrotropin-releasing hormone. A role for mitogen-activated protein kinase signaling cascade in rat pituitary GH3 cells.

We examined the possible involvement of mitogen-activated protein (MAP) kinase activation in the secretory process and gene expression of prolactin and growth hormone. Thyrotropin-releasing hormone (TRH) rapidly stimulated the secretion of both prolactin and growth hormone from GH3 cells. Secretion induced by TRH was not inhibited by 50 microM PD098059, but was completely inhibited by 1 microM wortmannin and 10 microM KN93, suggesting that MAP kinase does not mediate the secretory process. Stimulation of GH3 cells with TRH significantly increased the mRNA level of prolactin, whereas expression of growth hormone mRNA was largely attenuated. The increase in prolactin mRNA stimulated by TRH was inhibited by addition of PD098059, and the decrease in growth hormone mRNA was also inhibited by PD098059. Transfection of the cells with a pFC-MEKK vector (a constitutively active MAP kinase kinase kinase), significantly increased the synthesis of prolactin and decreased the synthesis of growth hormone. These data taken together indicate that MAP kinase mediates TRH-induced regulation of prolactin and growth hormone gene expression. Reporter gene assays showed that prolactin promoter activity was increased by TRH and was completely inhibited by addition of PD098059, but that the promoter activity of growth hormone was unchanged by TRH. These results suggest that TRH stimulates both prolactin and growth hormone secretion, but that the gene expressions of prolactin and growth hormone are differentially regulated by TRH and are mediated by different mechanisms.

Influence of Photoinhibition on the Morphology and Function of Pituitary Lactotropes in Male Golden Hamsters

Inhibition of prolactin (PRL) secretion has been previously shown in pituitaries from male and female hamsters exposed to short photoperiods. The purpose of the present study was to analyze the possible quantitative immunohistochemical and ultrastructural changes of PRL cells in male golden hamsters undergoing regression and spontaneous recrudescence, correlating the morphological findings with circulating PRL levels. Thus, adult male golden hamsters were exposed for 8, 16, 22 and 28 weeks to either short photoperiods (SP: 6 h light, 18 h darkness) or long photoperiods (LP: 14 h light, 10 h darkness). Pituitaries were processed for both light and electron microscopy, and serum levels of PRL were assessed by radioimmunoassay (RIA). Volume density (VD = cell area/reference area) and cell density (CD = number of cells/reference area) of lactotropes were measured with an image analysis system (Imaging Technology, Software Optimas 5.2). One hundred lactotropes were recorded for measuring several ultrastructural parameters. When analyzing the lactrotrope cell population from animals submitted to SP, the VD and CD were found to be significantly (p < 0.05) diminished with respect to those of the groups submitted to LP at weeks 8, 16 and 22. However, at week 28 a spontaneous recrudescence appeared. The lactotropes from animals submitted to LP for 8, 16, 22 and 28 weeks exhibited numerous large electrondense secretory granules. The rough endoplasmic reticulum (RER) presented some flat cisternae and numerous free ribosomes. Animals submitted to SP for 8 weeks showed a number of medium and large secretory granules, and the RER exhibited mainly numerous free ribosomes. In those animals submitted to SP for 16 and 22 weeks, lactotropes were found smaller and showed small and medium-sized secretory granules decreased in number. The Golgi complex exhibited some immature granules and dilated cisternae, while the RER did not present differences with respect to the 8-week SP group. The hamsters submitted to SP for 28 weeks presented medium and large secretory granules, and the RER exhibited dilated irregular cisternae. The ultrastructural morphometric parameters showed a decrease (p < 0.05) in the number of secretory granules and exocytotic profiles, as well as a diminution (p < 0.05) in the areas of Golgi complex, RER, secretory granules, and individual mean area of secretory granule during the 16 and 22 weeks of SP. Serum PRL levels were severely reduced under SP. This decrease was greater at 16 weeks than at 8 weeks of exposure. When hamsters were kept for 22 weeks under SP, PRL levels started their recovery. Thus, at the end of the experiment, the PRL values returned to near those of LP group. In summary, we found quantitative immunohistochemical and ultrastructural, as well as biochemical changes that suggest an inhibition of synthesis, storage and release of PRL in male golden hamsters submitted to SP, with a spontaneous recrudescence appearing at the end of the study.