Unraveling the effect of the inflammatory microenvironment in spermatogenesis progression.

Experimental autoimmune orchitis (EAO) is a chronic inflammatory disorder that causes progressive spermatogenic impairment. EAO is characterized by high intratesticular levels of nitric oxide (NO) and tumor necrosis factor alpha (TNFα) causing germ cell apoptosis and Sertoli cell dysfunction. However, the impact of this inflammatory milieu on the spermatogenic wave is unknown. Therefore, we studied the effect of inflammation on spermatogonia and preleptotene spermatocyte cell cycle progression in an EAO context and through the intratesticular DETA-NO and TNFα injection in the normal rat testes. In EAO, premeiotic germ cell proliferation is limited as a consequence of the undifferentiated spermatogonia (CD9+) cell cycle arrest in G2/M and the reduced number of differentiated spermatogonia (c-kit+) and preleptotene spermatocytes that enter in the meiotic S-phase. Although inflammation disrupts spermatogenesis in EAO, it is maintained in some seminiferous tubules at XIV and VII-VIII stages of the epithelial cell cycle, thereby guaranteeing sperm production. We found that DETA-NO (2 mM) injected in normal testes arrests spermatogonia and preleptotene spermatocyte cell cycle; this effect reduces the number of proliferative spermatogonia and the number of preleptotene spermatocytes in meiosis S-phase (36 h after). The temporal inhibition of spermatogonia clonal amplification delayed progression of the spermatogenic wave (5 days after) finally altering spermatogenesis. TNFα (0.5 and 1 µg) exposure did not affect premeiotic germ cell cycle or spermatogenic wave. Our results show that in EAO the inflammatory microenvironment altered spermatogenesis kinetics through premeiotic germ cell cycle arrest and that NO is a sufficient factor contributing to this phenomenon.

JAK2/STAT5 Pathway Mediates Prolactin-Induced Apoptosis of Lactotropes.

Prolactinomas are increasingly viewed as a "problem of signal transduction." Consequently, the identification of factors and signaling pathways that control lactotrope cell turnover is needed in order to encourage new therapeutic developments. We have previously shown that prolactin (PRL) acts as a proapoptotic and antiproliferative factor on lactotropes, maintaining anterior pituitary cell homeostasis, which contrasts with the classical antiapoptotic and/or proliferative actions exerted by PRL in most other target tissues. We aimed to investigate the PRLR-triggered signaling pathways mediating these nonclassical effects of PRL in the pituitary. Our results suggest that (i) the PRLR/Jak2/STAT5 pathway is constitutively active in GH3 cells and contributes to PRL-induced apoptosis by increasing the Bax/Bcl-2 ratio, (ii) PRL inhibits ERK1/2 and Akt phosphorylation, thereby contributing to its proapoptotic effect, and (iii) the PI3K/Akt pathway participates in the PRL-mediated control of lactotrope proliferation. We hypothesize that the alteration of PRL actions in lactotrope homeostasis due to the dysregulation of any of the mechanisms of actions described above may contribute to the pathogenesis of prolactinomas.

Use of prolactin receptor antagonist to better understand prolactin regulation of pituitary homeostasis.

The anterior pituitary is permanently regulated by processes of apoptosis and proliferation in order to maintain tissue homeostasis. Several factors have been implicated in this regulation and lately, prolactin (PRL) has been included into that list. However, since PRL is secreted by anterior pituitary lactotropes, the actual outcome of its autocrine/paracrine actions on pituitary cells has remained difficult to assess. The availability of the pure PRL receptor antagonist Del1-9-G129R-hPRL has been helpful to circumvent this problem. While PRL has been traditionally associated with increased cell proliferation, recent studies revealed that this hormone actually induces apoptosis and decreases proliferation of anterior pituitary cells, by mechanisms involving the PRL receptor. The aim of this short review is to overview our current understanding of the regulation of pituitary homeostasis by PRL. Moreover, studies involving Del1-9-G129R-hPRL have helped anticipate to what extent future treatments involving PRL receptor inhibitors may interfere with processes regulated by PRL at the central level.

Prolactin receptor antagonism in mouse anterior pituitary: effects on cell turnover and prolactin receptor expression.

Since anterior pituitary expresses prolactin receptors, prolactin secreted by lactotropes could exert autocrine or paracrine actions on anterior pituitary cells. In fact, it has been observed that prolactin inhibits its own expression by lactotropes. Our hypothesis is that prolactin participates in the control of anterior pituitary cell turnover. In the present study, we explored the action of prolactin on proliferation and apoptosis of anterior pituitary cells and its effect on the expression of the prolactin receptor. To determine the activity of endogenous prolactin, we evaluated the effect of the competitive prolactin receptor antagonist Δ1-9-G129R-hPRL in vivo, using transgenic mice that constitutively and systemically express this antagonist. The weight of the pituitary gland and the anterior pituitary proliferation index, determined by BrdU incorporation, were higher in transgenic mice expressing the antagonist than in wild-type littermates. In addition, blockade of prolactin receptor in vitro by Δ1-9-G129R-hPRL increased proliferation and inhibited apoptosis of somatolactotrope GH3 cells and of primary cultures of male rat anterior pituitary cells, including lactotropes. These results suggest that prolactin acts as an autocrine/paracrine antiproliferative and proapoptotic factor in the anterior pituitary gland. In addition, anterior pituitary expression of the long isoform of the prolactin receptor, measured by real-time PCR, increased about 10-fold in transgenic mice expressing the prolactin receptor antagonist, whereas only a modest increase in the S3 short-isoform expression was observed. These results suggest that endogenous prolactin may regulate its own biological actions in the anterior pituitary by inhibiting the expression of the long isoform of the prolactin receptor. In conclusion, our observations suggest that prolactin is involved in the maintenance of physiological cell renewal in the anterior pituitary. Alterations in this physiological role of prolactin could contribute to pituitary tumor development.

Estradiol increases the expression of TNF-α and TNF receptor 1 in lactotropes.

BACKGROUND: Estrogens are recognized modulators of pituitary cell renewal, sensitizing cells to mitogenic and apoptotic signals. Tumor necrosis factor-α (TNF-α) is a proinflammatory cytokine that plays an important role in tissue homeostasis modulating cell proliferation, differentiation and death. We previously demonstrated that TNF-α-induced apoptosis of anterior pituitary cells from female rats is estrogen-dependent and predominant in cells from rats at proestrus when estradiol levels are the highest. AIMS: Considering that one of the mechanisms involved in the apoptotic action of estrogens can result from increased expression of cytokines and/or their receptors, the aim of the present study was to evaluate the effect of estrogens on the expression of TNF-α and its receptor, TNF receptor 1 (TNFR1), in anterior pituitary cells. METHODS/RESULTS: TNFR1 expression, determined by Western blot, was higher in anterior pituitary glands from rats at proestrus than at diestrus. Incubation of anterior pituitary cells from ovariectomized rats with 17ß-estradiol enhanced TNFR1 protein expression. As determined by double immunocytochemistry, the expression of TNF-α and TNFR1 was detected in prolactin-, GH-, LH- and ACTH-bearing cells. 17ß-estradiol increased the percentage of TNF-α and TNFR1-immunoreactive lactotropes but did not modify the number of GH-bearing cells expressing TNF-α or TNFR1. CONCLUSION: Our results demonstrate that estradiol increases the expression of TNF-α and TNFR1 in anterior pituitary cells, especially in lactotropes. The sensitizing action of estrogens to proapoptotic stimuli at proestrus in the anterior pituitary gland may involve changes in the expression of the TNF-α/TNFR1 system.

Anterior pituitary gland synthesises dopamine from l-3,4-dihydroxyphenylalanine (l-dopa).

Prolactin (PRL) is a hormone principally secreted by lactotrophs of the anterior pituitary gland. Although the synthesis and exocytosis of this hormone are mainly under the regulation of hypothalamic dopamine (DA), the possibility that the anterior pituitary synthesises this catecholamine remains unclear. The present study aimed to determine if the anterior pituitary produces DA from the precursor l-3,4-dihydroxyphenylalanine (l-dopa). Accordingly, we investigated the expression of aromatic l-amino acid decarboxylase (AADC) enzyme and the transporter vesicular monoamine transporter 2 (VMAT2) in the anterior pituitary, AtT20 and GH3 cells by immunofluorescence and western blotting. Moreover, we investigated the production of DA from l-dopa and its release in vitro. Then, we explored the effects of l-dopa with respect to the secretion of PRL from anterior pituitary fragments. We observed that the anterior pituitary, AtT20 and GH3 cells express both AADC and VMAT2. Next, we detected an increase in DA content after anterior pituitary fragments were incubated with l-dopa. Also, the presence of l-dopa increased DA levels in incubation media and reduced PRL secretion. Likewise, the content of cellular DA increased after AtT20 cells were incubated with l-dopa. In addition, l-dopa reduced corticotrophin-releasing hormone-stimulated adrenocorticotrophic hormone release from these cells after AADC activity was inhibited by NSD-1015. Moreover, DA formation from l-dopa increased apoptosis and decreased proliferation. However, in the presence of NSD-1015, l-dopa decreased apoptosis and increased proliferation rates. These results suggest that the anterior pituitary synthesises DA from l-dopa by AADC and this catecholamine can be released from this gland contributing to the control of PRL secretion. In addition, our results suggest that l-dopa exerts direct actions independently from its metabolisation to DA.

Estradiol increases the Bax/Bcl-2 ratio and induces apoptosis in the anterior pituitary gland.

BACKGROUND: Estrogens are recognized as acting as modulators of pituitary cell renewal, sensitizing cells to mitogenic and apoptotic signals, thus participating in anterior pituitary homeostasis during the estrous cycle. The balance of pro- and antiapoptotic proteins of the Bcl-2 family is known to regulate cell survival and apoptosis. AIMS: In order to understand the mechanisms underlying apoptosis during the estrous cycle, we evaluated the expression of the proapoptotic protein Bax and the antiapoptotic proteins Bcl-2 and Bcl-xL in the anterior pituitary gland in cycling female rats as well as the influence of estradiol on the expression of these proteins in anterior pituitary cells of ovariectomized rats. METHODS/RESULTS: As determined by Western blot, the expression of Bax was higher in anterior pituitary glands from rats at proestrus than at diestrus I, Bcl-2 protein levels showed no difference and Bcl-xL expression was lower, thus increasing the Bax/Bcl-2 ratio at proestrus. Assessed by annexin V binding and flow cytometry, the percentage of apoptotic anterior pituitary cells was higher in rats at proestrus than at diestrus I. Chronic estrogen treatment in ovariectomized rats enhanced the Bax/Bcl-2 ratio and induced apoptosis. Moreover, incubation of cultured anterior pituitary cells from ovariectomized rats with 17beta-estradiol for 24 h increased the Bax/Bcl-2 ratio, decreased Bcl-xL expression and induced apoptosis. CONCLUSION: Our results demonstrate that estradiol increases the ratio between proapoptotic and antiapoptotic proteins of the Bcl-2 family. This effect could participate in the sensitizing action of estrogens to proapoptotic stimuli and therefore be involved in the high apoptotic rate observed at proestrus in the anterior pituitary gland.

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.

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.

Prolactin and its receptor as therapeutic targets in glioblastoma multiforme.

Although prolactin (PRL) and its receptor (PRLR) have been detected in glioblastoma multiforme (GBM), their role in its pathogenesis remains unclear. Our aim was to explore their contribution in GBM pathogenesis. We detected PRL and PRLR in all GBM cell lines tested. PRLR activation or overexpression using plasmid transfection increased proliferation, viability, clonogenicity, chemoresistance and matrix metalloproteinase activity in GBM cells, while PRLR antagonist ∆1-9-G129R-hPRL reduced their proliferation, viability, chemoresistance and migration. Meta-analysis of transcriptomic data indicated that PRLR was expressed in all grade II-III glioma (GII-III) and GBM samples. PRL was upregulated in GBM biopsies when compared to GII-III. While in the general population tumour PRL/PRLR expression did not correlate with patient survival, biological sex-stratified analyses revealed that male patients with PRL+/PRLRHIGH GBM performed worse than PRL+/PRLRLOW GBM. In contrast, all male PRL+/PRLRHIGH GII-III patients were alive whereas only 30% of PRL+/PRLRLOW GII-III patients survived after 100 months. Our study suggests that PRLR may be involved in GBM pathogenesis and could constitute a therapeutic target for its treatment. Our findings also support the notion that sexual dimorphism should be taken into account to improve the care of GBM patients.

Apoptosis of lactotrophs induced by D2 receptor activation is estrogen dependent.

BACKGROUND/AIMS: Dopamine (DA) inhibits prolactin release and reduces lactotroph proliferation by activating D2 receptors. DA and its metabolite, 6-hydroxydopamine (6-OHDA), induce apoptosis in different cell types. DA receptors and DA transporter (DAT) were implicated in this action. Considering that estradiol sensitizes anterior pituitary cells to proapoptotic stimuli, we investigated the effect of estradiol on the apoptotic action of DA and 6-OHDA in anterior pituitary cells, and the involvement of the D2 receptor and DAT in the proapoptotic effect of DA. METHODS: Viability of cultured anterior pituitary cells from ovariectomized rats was determined by MTS assay. Apoptosis was evaluated by Annexin-V/flow cytometry and TUNEL. Lactotrophs were identified by immunocytochemistry. RESULTS: DA induced apoptosis of lactotrophs in an estrogen-dependent manner. In contrast, estradiol was not required to trigger the apoptotic action of 6-OHDA. Cabergoline, a D2 receptor agonist, induced lactotroph apoptosis, while sulpiride, a D2 receptor antagonist, blocked DA-induced cell death. The blockade of DAT by GBR12909 did not affect the apoptotic action of DA, but inhibited 6-OHDA-induced apoptosis. CONCLUSION: These data show that DA, through D2 receptor activation, induces apoptosis of estrogen-sensitized anterior pituitary cells, and suggest that DA contributes to the control of lactotroph number not only by inhibiting proliferation but also by inducing apoptosis.

Humanin inhibits apoptosis in pituitary tumor cells through several signaling pathways including NF-κB activation.

Humanin (HN) and Rattin (HNr), its homologous in the rat, are peptides with cytoprotective action in several cell types such as neurons, lymphocytes and testicular germ cells. Previously, we have shown that HNr is expressed in pituitary cells and that HN inhibited the apoptotic effect of TNF-α in both normal and tumor pituitary cells. The aim of the present study was to identify signaling pathways that mediate the antiapoptotic effect of HN in anterior pituitary cells from ovariectomized rats and in GH3 cells, a somatolactotrope cell line. We assessed the role of STAT3, JNK, Akt and MAPKs as well as proteins of the Bcl-2 family, previously implicated in the antiapoptotic effect of HN. We also evaluated the participation of NF-κB in the antiapoptotic action of HN. STAT3 inhibition reversed the inhibitory effect of HN on TNF-α-induced apoptosis in normal and pituitary tumor cells, indicating that STAT3 signaling pathway mediates the antiapoptotic effect of HN on pituitary cells. Inhibition of NF-κB pathway did not affect action of HN on normal anterior pituitary cells but blocked the cytoprotective effect of HN on TNF-α-induced apoptosis of GH3 cells, suggesting that the NF-κB pathway is involved in HN action in tumor pituitary cells. HN also induced NF-κB-p65 nuclear translocation in these cells. In pituitary tumor cells, JNK and MEK inhibitors also impaired HN cytoprotective action. In addition, HN increased Bcl-2 expression and decreased Bax mitochondrial translocation. Since HN expression in GH3 cells is higher than in normal pituitary cells, we may suggest that through multiple pathways HN could be involved in pituitary tumorigenesis.

Opposite effects of dihydrotestosterone and estradiol on apoptosis in the anterior pituitary gland from male rats.

Hormones locally synthesized in the anterior pituitary gland are involved in regulation of pituitary cell renewal. In the pituitary, testosterone (T) may exert its actions per se or by conversion to dihydrotestosterone (DHT) or 17ß-estradiol (E2) by 5α-reductase and aromatase activity, which are expressed in this gland. Previous reports from our laboratory showed that estrogens modulate apoptosis of lactotropes and somatotropes from female rats. Now, we examined the in vitro and in vivo effects of gonadal steroids on apoptosis of anterior pituitary cells from adult male rats. T in vitro did not modify apoptosis in anterior pituitary cells from gonadectomized (GNX) male rats. DHT, a non-aromatizable androgen, exerted direct antiapoptotic action on total anterior pituitary cells and folliculo-stellate cells, but not on lactotropes, somatotropes, or gonadotropes. On the contrary, E2 exerted a rapid apoptotic effect on total cells as well as on lactotropes and somatotropes. Incubation of anterior pituitary cells with T in presence of Finasteride, an inhibitor of 5α-reductase, increased the percentage of TUNEL-positive cells. In vivo administration of DHT to GNX rats reduced apoptosis in the anterior pituitary whereas E2 exerted proapoptotic action and reduced cells in G2/M-phase of the cell cycle. In summary, our results indicate that DHT and E2 have opposite effects on apoptosis in the anterior pituitary gland suggesting that local metabolization of T to these steroids could be involved in pituitary cell turnover in males. Changes in expression and/or activity of 5α-reductase and aromatase may play a role in the development of anterior pituitary tumors.

Gene therapy for pituitary tumors.

Pituitary tumors are the most common primary intracranial neoplasms. Although most pituitary tumors are considered typically benign, others can cause severe and progressive disease. The principal aims of pituitary tumor treatment are the elimination or reduction of the tumor mass, normalization of hormone secretion and preservation of remaining pituitary function. In spite of major advances in the therapy of pituitary tumors, for some of the most difficult tumors, current therapies that include medical, surgical and radiotherapeutic methods are often unsatisfactory and there is a need to develop new treatment strategies. Gene therapy, which uses nucleic acids as drugs, has emerged as an attractive therapeutic option for the treatment of pituitary tumors that do not respond to classical treatment strategies if the patients become intolerant to the therapy. The development of animal models for pituitary tumors and hormone hypersecretion has proven to be critical for the implementation of novel treatment strategies and gene therapy approaches. Preclinical trials using several gene therapy approaches for the treatment of anterior pituitary diseases have been successfully implemented. Several issues need to be addressed before clinical implementation becomes a reality, including the development of more effective and safer viral vectors, uncovering novel therapeutic targets and development of targeted expression of therapeutic transgenes. With the development of efficient gene delivery vectors allowing long-term transgene expression with minimal toxicity, gene therapy will become one of the most promising approaches for treating pituitary adenomas.

Effects of lipopolysaccharide on neurokinin A content and release in the hypothalamic-pituitary axis.

The administration of bacterial lipopolysaccharide (LPS) markedly affects pituitary secretion, and its effects are probably mediated by cytokines produced by immune cells or by the hypothalamo-pituitary axis itself. Since neurokinin A (NKA) plays a role in inflammatory responses and is involved in the control of prolactin secretion, we examined the in vivo effect of LPS on the concentration of NKA in hypothalamus and pituitary (assessed by RIA) and serum prolactin levels in male rats. One hour after the intraperitoneal administration of LPS (250 microg/rat), NKA content was decreased in the posterior pituitary but not in the hypothalamus or anterior pituitary. Three hours after injection, LPS decreased NKA concentration in the hypothalamus and anterior and posterior pituitary. In all the conditions tested, LPS significantly decreased serum prolactin. We also examined the in vitro effects of LPS (10 microg/ml), interleukin-6 (IL-6, 10 ng/ml) and tumor necrosis factor alpha (TNF-alpha, 50 ng/ml) on hypothalamic NKA release. Interleukin-6 increased NKA release without modifying hypothalamic NKA concentration, whereas neither LPS nor TNF-alpha affected them. Our results suggest that IL-6 may be involved in the increase of hypothalamic NKA release induced by LPS. NKA could participate in neuroendocrine responses to endotoxin challenge.

Characterization of tachykinin NK2 receptor in the anterior pituitary gland.

Tachykinins are a family of bioactive peptides that interact with three subtypes of receptors: NK1, NK2 and NK3. Substance P has greater affinity for NK1, and neurokinin A (NKA) for NK2 receptor subtype. Although only NK1 receptor has been characterized in the anterior pituitary gland, some evidence suggests the existence of NK2 receptors in this gland. Therefore, we investigated the presence of NK2 receptors in the anterior pituitary gland of male rats by radioligand binding studies using labeled SR48968, a non peptidic specific antagonist. [3H]SR48968 specific binding to cultured anterior pituitary cells was time-dependent and saturable, but with a lower affinity than previously reported values for cells expressing NK2 receptors. Unlabeled NKA inhibited only partially [(3)H]SR48968 specific binding to whole anterior pituitary cells. Since SR48968 is a non polar molecule, we performed experiments to discriminate surface from intracellular binding sites. SR48968 exhibited both surface and intracellular specific binding. Analysis of the surface-bound ligand indicated that [3H]SR48968 binds to one class of receptor with high affinity. Neurokinin A completely displaced [3H]SR48968 surface specific binding fitting to a two-site/two-state model with high and low affinity. Additionally, immunocytochemical studies showed that the NK2 receptor is expressed at least in a subset of lactotropes. These results demonstrate the presence of NK2 receptors in the anterior pituitary gland and suggest that NKA actions in this gland are mediated, at least in part, by the NK2 receptor subtype.

Prolactin induces apoptosis of lactotropes in female rodents.

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

Antiapoptotic factor humanin is expressed in normal and tumoral pituitary cells and protects them from TNF-α-induced apoptosis.

Humanin (HN) is a 24-amino acid peptide with cytoprotective action in several cell types such as neurons and testicular germ cells. Rattin (HNr), a homologous peptide of HN expressed in several adult rat tissues, also has antiapoptotic action. In the present work, we demonstrated by immunocytochemical analysis and flow cytometry the expression of HNr in the anterior pituitary of female and male adult rats as well as in pituitary tumor GH3 cells. HNr was localized in lactotropes and somatotropes. The expression of HNr was lower in females than in males, and was inhibited by estrogens in pituitary cells from both ovariectomized female and orquidectomized male rats. However, the expression of HNr in pituitary tumor cells was not regulated by estrogens. We also evaluated HN action on the proapoptotic effect of TNF-α in anterior pituitary cells assessed by the TUNEL method. HN (0.5 µM) per se did not modify basal apoptosis of anterior pituitary cells but completely blocked the proapoptotic effect of TNF-α in total anterior pituitary cells, lactotropes and somatotropes from both female and male rats [corrected]. Also, HN inhibited the apoptotic effect of TNF-α on pituitary tumor cells. In summary, our results demonstrate that HNr is present in the anterior pituitary gland, its expression showing sexual dimorphism, which suggests that gonadal steroids may be involved in the regulation of HNr expression in this gland. Antiapoptotic action of HN in anterior pituitary cells suggests that this peptide could be involved in the homeostasis of this gland. HNr is present and functional in GH3 cells, but it lacks regulation by estrogens, suggesting that HN could participate in the pathogenesis of pituitary tumors.

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

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

Dopamine-induced apoptosis of lactotropes is mediated by the short isoform of D2 receptor.

Dopamine, through D2 receptor (D2R), is the major regulator of lactotrope function in the anterior pituitary gland. Both D2R isoforms, long (D2L) and short (D2S), are expressed in lactotropes. Although both isoforms can transduce dopamine signal, they differ in the mechanism that leads to cell response. The administration of D2R agonists, such as cabergoline, is the main pharmacological treatment for prolactinomas, but resistance to these drugs exists, which has been associated with alterations in D2R expression. We previously reported that dopamine and cabergoline induce apoptosis of lactotropes in primary culture in an estrogen-dependent manner. In this study we used an in vivo model to confirm the permissive action of estradiol in the apoptosis of anterior pituitary cells induced by D2R agonists. Administration of cabergoline to female rats induced apoptosis, measured by Annexin-V staining, in anterior pituitary gland from estradiol-treated rats but not from ovariectomized rats. To evaluate the participation of D2R isoforms in the apoptosis induced by dopamine we used lactotrope-derived PR1 cells stably transfected with expression vectors encoding D2L or D2S receptors. In the presence of estradiol, dopamine induced apoptosis, determined by ELISA and TUNEL assay, only in PR1-D2S cells. To study the role of p38 MAPK in apoptosis induced by D2R activation, anterior pituitary cells from primary culture or PR1-D2S were incubated with an inhibitor of the p38 MAPK pathway (SB203850). SB203580 blocked the apoptotic effect of D2R activation in lactotropes from primary cultures and PR1-D2S cells. Dopamine also induced p38 MAPK phosphorylation, determined by western blot, in PR1-D2S cells and estradiol enhanced this effect. These data suggest that, in the presence of estradiol, D2R agonists induce apoptosis of lactotropes by their interaction with D2S receptors and that p38 MAPK is involved in this process.