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

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

Cellular mechanisms of estrogen-and dopamine-induced control of glandular kallikrein in the anterior pituitary of the rat

Glandular kallikrein (GK, a trypsin-like serine protease) exhibits estrogen induction and dopamine repression in rat pituitary lactotrophs. Steroid induction may reflect primary actions to increase selectively the synthesis of specific proteins, or may be part of broad cellular responses secondary to steroid-induced phenotype transitions. This study examined the cellular mechanisms underlying estrogen and dopaminergic control of lactotroph GK using a quantified immunocytochemical approach. Pituitaries from ovariectomized rats exhibited little GK staining. Estradiol treatment for 10 days produced dose-dependent increases in pituitary mass, the percentage of lactotrophs (indicating lactotroph proliferation) and the percentage of GK-positive cells. Also, GK staining intensity was dependent upon estradiol dose, increasing 4-fold between 5 g and 50 g/48 h. Dopamine receptor blockade with haloperidol (2.5 mg/kg/24 h) elicited weak GK immunostaining in 46% of the lactotrophs in the absence of estradiol, and markedly potentiated GK staining intensity elicited with low but not high doses of estradiol. The results suggest that GK induction is a primary estrogen effect, and is not secondary to a phenotype transition: the induction is enhanced by estrogen-induced lactotroph proliferation. Dopaminergic systems strongly inhibit GK induction by low estradiol levels. This dopaminergic modulation may shift the induction of lactotroph GK to physiological events associated with high estradiol levels or low dopaminergic tone.     

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

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

Apoptotic cell death of oestrogen activated lactotrophs induced by tamoxifen

Stimulation and inhibition of lactotroph cells cause remarkable morphological and functional changes. In keeping with these changes, the size of the lactotroph cell population undergoes striking alterations due to proliferation or cell death. Factors involved in the induction of apoptosis of pituitary cells are not well established. We demonstrated earlier that oestrogens prevent lactotroph cells of female rats to die by apoptosis induced by bromocryptine treatment, a fact that can be reversed in ovariectomised rats. In this study, we developed experimental models for in vivo and in vitro studies to gain further insight on the survival effect of oestrogens on lactotrophs. In rats pretreated with oestrogens, tamoxifen generates a massive cell death by apoptosis as validated by the TUNEL technique and DNA electrophoresis of pituitary gland. On electron microscope observations, numerous lactotrophs exhibited progressive morphological changes in the nuclei compatible with the apoptotic process. The cells remaining intact also exhibit signs of inhibition due to a significant transformation of regular lactotrophs in atypical subtypes. In pituitary cell cultures exposed to tamoxifen and oestrogen simultaneously, most of the lactotrophs displayed features of apoptosis in the nucleus. The present reports gathered new evidences on the apoptogenic potential of tamoxifen on lactotroph cells, and corroborates the contribution of oestrogens to sustain both a balanced population of lactotrophs and a competent secretory activity. The concept that opposed activities, such as inhibition and stimulation, can activate apoptosis is also strengthen by these observations.     

Prolactin receptor signaling mediates the osmotic response of embryonic zebrafish lactotrophs

The pituitary hormone prolactin (PRL) regulates salt and water homeostasis by altering ion retention and water uptake through peripheral osmoregulatory organs. To understand the role of osmotic homeostasis in the development of PRL-secreting lactotrophs, we generated germline transgenic zebrafish coexpressing red fluorescent protein directed by Prolactin regulatory elements (PRL-RFP) and green fluorescent protein by the Pro-opiomelanocortin promoter (POMC-GFP). Transparent embryos expressing fluorescent markers specifically targeted to lactotrophs and corticotrophs, the two pituitary lineages involved in teleost osmotic adaptation, allowed in vivo dynamic tracing of pituitary ontogeny during altered environmental salinity. Physiological osmotic changes selectively regulate lactotroph but not corticotroph proliferation during early ontogeny. These changes are not suppressed by pharmacological dopamine receptor blockade but are completely abrogated by morpholino knockdown of the PRL receptor. PRL receptor signaling exerts robust effects on lactotroph development and plays a permissive role in lactotroph osmo-responsiveness, reflecting the dual peripheral and central interactions required for early pituitary development and embryonic homeostasis.     

Somatotrophs and lactotrophs: an immunohistochemical study of Gallus domesticus pituitary gland at different stages of induced moult

The objective of this study was to determine the distribution of somatotrophs and lactotrophs and conduct a morphometrical analysis of immunoreactive somatotrophs and lactotrophs in the pituitary glands of White Leghorn Hens (Gallus domesticus) during the period of induced moult. We divided the periods of induced moulting into three phases viz. 7, 14 and 21 days. The labeled alkalinephsphatase method with anti-GH (growth hormone) and anti-PRL (prolactin) as a primary antibody was used to detect somatotrophs and lactotrophs, in the midsagital sections of chicken adenohypophysis. Immunohistochemistry showed that somatotrophs are not only confined to the cephalo-caudal axis but can also be found in the caudal lobe; while lactotrophs were distributed in both lobes of the anterior pituitary gland at all stages of moulting (7, 14 and 21 days). Lactotrophs were of different shapes but somatotrophs were oval to round in morphology. At the given stages of induced moulting, some hypertrophied lactotrophs were also present after 7 days of induced moult in the anterior pituitary gland. However, there were moulting-related changes: from 7 to 21 days of induced moulting the immunoreactive-PRL cell population decreased, while the mean lactotroph size was more than that of somatotrophs. Basic quantitative and morphological information relating to somatotrophs and lactotrophs during the period of induced moult in laying hens is reported here and the changes brought about by induced moulting are restricted to PRL positive cells rather than GH positive cells.Key words: Moult, pituitary gland, somatotrophs, lactotrophs, chicken.     

Estradiol promotes pituitary cell proliferation and gonadotroph differentiation at different doses and with different mechanisms in chick embryo

Estrogens are known to play a role in the feedback regulation of pituitary gonadotropin secretion in adults. However, it is still unknown whether estrogens are involved in promoting pituitary development. In this study, we selected chick embryo as the animal model and microinjected different doses of estradiol (E2) at stage E27–28, which was when endogenous E2 was not detected. First, the results demonstrated that E2 at different doses promoted pituitary cell proliferation and gonadotroph differentiation. Lower doses of E2 had a more significant effect on cell proliferation, while higher doses of E2 were required for luteinizing hormone (LH) secreting cell differentiation. Furthermore, the levels of early growth response protein 1 (Egr-1) and GATA2 mRNAs were also elevated with E2 treatment at a higher dose than that required to increase the level of proliferating cell nuclear antigen (PCNA) in vitro. To investigate whether estrogen receptors (ERs) mediate these effects of estradiol, the ER antagonist ICI 182,780 was added, and the results showed that ICI 182,780 did not modify the enhancing effects of E2 on cell proliferation; however, it inhibited the stimulatory effect of E2 on LH secreting cell differentiation. These results suggest that E2 at different doses promotes pituitary cell proliferation and gonadotroph differentiation with different mechanisms. Our results are important to further understanding of the physiological and pharmacological functions and related mechanisms of estrogens and their receptors, although the related mechanism need to be elucidated in future studies.     

Different Behavior of Lactotroph Cell Subpopulations in Response to Angiotensin II and Thyrotrophin-Releasing Hormone

1. In the present investigation we have extended the study of lactotroph subpopulations in primary pituitary cell cultures. Male rats with or without previous estrogenization followed by A-II or TRH treatments were selected as experimental models.2. The TRH increased up to 50% the PRL released in both whole and ORQX + EB rats (P < 0.05). In contrast, A-II treatment introduced no changes in PRL secretion from cell cultures derived from whole male rats but attained a significant augmentation (about 75%) of PRL secreted by ORQX + EB pituitary cells.3. The addition of TRH and A-II to cultures of ORQX + EB-derived lactotrophs induced cytological changes compatible with a high secretory activity. In estrogen-treated rats the prevailing lactotroph subpopulation is type I. In cell cultures from control and A-II treated whole male pituitaries, the majority of lactotrophs consists of atypical subpopulations of II and III cells, with smaller secretory granules (between 150 and 300 nm in diameter).4. Morphometry of immunostained lactotrophs performed on light microscopic preparations revealed that about 30–36% of the total cell count were lactotrophs. This percentage was fixed and did not change significantly after TRH and A-II treatments.5. The present results confirm the presence of morphological and functional subtypes of lactotroph cells in rat pituitary. Typical PRL cell population shows the highest responsiveness to angiotensin II and TRH action. This functional heterogeneity of lactotroph subtypes may reflect an important and scarcely explored factor in the regulatory process of prolactin secretion.     

Ultrastructural immunolocalization of IGF-1 and insulin receptors in rat pituitary culture: evidence of a functional interaction between gonadotroph and lactotroph cells

We have investigated the expression of receptors for insulin and insulin-like growth factor 1 (IGF-1) in rat pituitary cells in vitro and examined the morphological and proliferative changes induced in adenohypophyseal cells by insulin and IGF-1. The proliferation of lactotrophs was determined by double-immunostaining for bromodeoxyuridine and prolactin. Incubation with insulin (10, 100 or 1000 ng/ml) or IGF-1 (5, 30 or 100 ng/ml) for 48 or 72 h significantly increased the number of lactotrophs undergoing mitosis. Co-incubation of insulin or IGF-1 with genistein (25 μM), an inhibitor of the tyrosine kinase receptor, reduced the proliferation of lactotrophs elicited by the hormone and the growth factor. The receptors for insulin and IGF-1 were localized in intact pituitary cells by ultrastructural immunocytochemistry with the colloidal gold-protein A technique. Gonadotrophs expressed both receptors, specific labelling being restricted to this cell type. Electron-microscopical observations of pituitary cell cultures incubated with insulin or IGF-1 revealed gonadotroph cells exhibiting the fine-structural features of enhanced protein synthetic activity. These findings suggest that both insulin and IGF-1 are able to induce the proliferation of lactotrophs through an indirect mechanism mediated by a factor synthesized by gonadotroph cells, in addition to stimulating the biosynthetic activity of the gonadotroph in a direct manner.This work was supported by grants from the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) and the Secretaría de Ciencia y Tecnología de la Universidad Nacional de Córdoba (SECyT).     

Pituitary, pancreatic and gut neuroendocrine defects in protein tyrosine phosphatase-sigma-deficient mice.

The expression of receptor protein tyrosine phosphatase sigma (PTPfinal sigma) is developmentally regulated in neuronal and neuroendocrine tissues. We have previously shown that mice deficient in PTPfinal sigma demonstrate nervous system abnormalities, pituitary hypoplasia, increased neonatal mortality (60%), and death from a wasting syndrome at 2-3 wk of age (38%). We have now examined the role of PTPfinal sigma on pituitary, pancreas and enteroendocrine cytodifferentiation, hormone production, and development. The adenohypophyses of PTPfinal sigma(-/-) mice were small and exhibited reduced GH and PRL immunoreactivity. Cells containing TSH, LH, FSH, ACTH, pituitary-specific POU homeodomain factor (Pit-1), ER, and steroidogenic factor 1 were found in normal proportions and distributions. The diminished expression of GH and PRL was not associated with apoptosis of somatotrophs or lactotrophs. Pit-1-positive TSH-negative cells were detected, suggesting that impaired GH and PRL synthesis was not attributable to Pit-1 deficiency. In the knockout mice, pancreatic islets were hypoplastic with reduced insulin immunoreactivity, and there was also variable expression of gut hormones. Functionally, the GH deficiency was associated with hypoglycemia and death in the PTPfinal sigma(-/-) neonate and accordingly, ip administration of GH rescued the PTPfinal sigma(-/-) neonate and normalized the blood glucose. These data indicate that PTPfinal sigma plays a major role in differentiation and development of the neuroendocrine system.     

Cross-talk between the estrogen receptor-, protein kinase A-, and mitogen-activated protein kinase-mediated signaling pathways in the regulation of lactotroph proliferation in primary culture

Using pharmacological means we investigated the functional interactions between the estrogen receptor (ER)-, protein kinase A (PKA)-, and mitogen-activating protein kinase (MAPK)-mediated pathways in the regulation of lactotroph proliferation in primary culture. Treatment of lactotrophs for 28 h with the PKA inhibitor H89 or KT5720, an effective inhibitor of forskolin-induced proliferation, inhibited both insulin- and estradiol-induced proliferation. Inhibition of the MAPK activity by PD98059 or U0126 suppressed not only insulin-induced proliferation but also forskolin- and estradiol-induced proliferation. However, treatment for 28 h with the antiestrogens 4-hydroxy tamoxifen and ICI182780 failed to antagonize estradiol-induced lactotroph proliferation but instead enhanced it. Prolonging the antiestrogen treatment time from 28 to 88 h was effective in antagonizing estradiol-induced proliferation with this long-term treatment also inhibiting insulin- and forskolin-induced proliferation. There was no decrease in these mitogen-induced proliferations following treatment with a progesterone antagonist or protein kinase C inhibitor. These results suggest that cross-talk occurs between the ER-, PKA-, and MAPK-mediated signaling pathways in the regulation of lactotroph proliferation, and that antiestrogens stimulate and inhibit estradiol-induced proliferation in a time-dependent manner.     

Estrogen-modulated estrogen receptor x Pit-1 protein complex formation and prolactin gene activation require novel protein synthesis

Both estrogen receptor (ER) and Pit-1 proteins are essential for the estrogen-activated expression of the rat prolactin gene. Our results show that ER.Pit-1 protein complex formation is reduced by estrogen in GH3 and PR1 rat pituitary tumor cells. In the latter, this decrease was blocked by cycloheximide, a protein synthesis inhibitor. On the other hand, the direct addition of estrogen to PR1 cell lysates had no effect on the formation of ER.Pit-1 complexes. Estrogen-activated prolactin gene expression was also inhibited by cycloheximide, suggesting that some form of protein synthesis is involved in ER.Pit-1 complex formation and subsequent prolactin gene activation. In support of this notion, we showed that estrogen-induced regulation of ER.Pit-1 complex formation could be transferred from cell lysates prepared from estrogen-treated PR1 cells to control cell lysates. This is not true for GH3 cells; instead, direct administration of estrogen to GH3 cell lysates readily abolished ER.Pit-1 protein complex formation in a dose-dependent manner, and such estrogen-induced regulation was blocked by the antiestrogen ICI 182,780. These findings thus indicate that 1) interaction between ER and Pit-1 proteins is estrogen-regulated in ways specific to different cell types, and 2) auxiliary protein factor synthesis may be involved in this process.