Daniel Pisera

Estrogens exert a rapid apoptotic action in anterior pituitary cells

It is now accepted that estrogens not only stimulate lactotrope proliferation but also sensitize anterior pituitary cells to proapoptotic stimuli. In addition to their classical mechanism of action through binding to intracellular estrogen receptors (ERs), there is increasing evidence that estrogens exert rapid actions mediated by cell membrane-localized ERs (mERs). In the present study, we examined the involvement of membrane-initiated steroid signaling in the proapoptotic action of estradiol in primary cultures of anterior pituitary cells from ovariectomized rats by using estren, a synthetic estrogen with no effect on classical transcription and a cell-impermeable 17beta-estradiol conjugate (E2-BSA). Both compounds induced cell death of anterior pituitary cells after 60 min of incubation as assessed by flow cytometry and the [3-(4,5-dimethylthiazol-2-yl)]-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay. Estren, E2, and E2-BSA induced apoptosis of lactotropes and somatotropes as evaluated by the deoxynucleotidyltransferase-mediated dUTP nick end-labeling assay and immunodetection of prolactin (PRL) and growth hormone (GH). The proapoptotic effect of E2-BSA was abrogated by ICI-182,780, an antagonist of ERs. The expression of membrane-associated ERalpha was observed in PRL- and GH-bearing cells. Our results indicate that estradiol is able to exert a rapid apoptotic action in anterior pituitary cells, especially lactotropes and somatotropes, by a mechanism triggered by mERs. This mechanism could be involved in anterior pituitary cell turnover.

Lipopolysaccharide- and Tumor Necrosis Factor-α-Induced Changes in Prolactin Secretion and Dopaminergic Activity in the Hypothalamic-Pituitary Axis

Bacterial lipopolysaccharide (LPS) affects pituitary hormone secretion, including prolactin release, by inducing synthesis and release of cytokines such as tumor necrosis factor-α (TNF-α). Since prolactin is mainly under tonic inhibitory control of dopamine, we investigated the effect of LPS and TNF-α on the hypothalamic-pituitary dopaminergic system. LPS (100–250 µg/rat, i.p.) decreased serum prolactin levels after 1 or 3 h. Sulpiride, a dopaminergic antagonist, increased serum prolactin and blocked the inhibitory effect of LPS. LPS increased hypothalamic dopamine and DOPAC concentrations and the DOPAC/dopamine ratio both in mediobasal hypothalamus and the posterior pituitary. LPS also enhanced dopamine and DOPAC concentration in the anterior pituitary. LPS elevated plasma levels of epinephrine, norepinephrine and dopamine but it did not modify the concentration of epinephrine or norepinephrine in the tissues studied. The administration of TNF-α (i.c.v., 1 h, 100 ng/rat) decreased serum prolactin but did not affect plasma catecholamine levels. TNF-α did not modify the DOPAC/dopamine ratio in hypothalamus or posterior pituitary but increased dopamine and DOPAC concentrations in the anterior pituitary. Incubations of hypothalamic explants showed that TNF-α did not modify in vitro basal dopamine release and reduced K+-evoked dopamine release. On the contrary, incubations of posterior pituitaries showed that TNF-α significantly increased basal and K+-evoked dopamine release. These results indicate that LPS and TNF-α increase dopamine turnover in the hypothalamic-pituitary axis. This increase in dopaminergic activity could mediate the inhibitory effect of LPS and TNF-α on prolactin release. Furthermore, the increase in dopaminergic activity elicited by LPS could be mediated by an increase in hypothalamic TNF-α during endotoxemia.

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 17β-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.

Tumor Necrosis Factor-Alpha-Induced Nitric Oxide Restrains the Apoptotic Response of Anterior Pituitary Cells

We previously reported that tumor necrosis factor-α (TNF-α) inhibits cell proliferation whereas it stimulates apoptosis of anterior pituitary cells in an estrogen-dependent manner. Also, we showed that nitric oxide (NO) mediates the inhibitory effect of TNF-α on prolactin release. Here, we studied the effect of TNF-α on nitric oxide synthase (NOS) activity and expression in anterior pituitary cells from cycling and ovariectomized (OVX) rats, and the role of NO in TNF-α induced apoptosis of anterior pituitary cells. NOS activity was higher in anterior pituitary cells from rats in proestrus than in diestrus and was stimulated by 17β-estradiol (10–9 M, E2). TNF-α (50 ng/ml) stimulated NOS activity in anterior pituitary cells from rats at both stages of the estrous cycle and in cells from OVX rats cultured either with or without E2. Inducible NOS (iNOS) gene expression was higher in anterior pituitary cells from rats in proestrus than in diestrus and its expression was enhanced by TNF-α. Acute administration of E2 to OVX rats increased endothelial NOS (eNOS) expression in the anterior pituitary gland. Also, E2 increased eNOS mRNA in dispersed anterior pituitary cells from OVX rats, and this effect was blocked by TNF-α. nNOS expression in the anterior pituitary gland was higer at proestrus than at diestrus but eNOS expression was similar at both stages. TNF-α decreased eNOS mRNA in anterior pituitary cells from rats at proestrus or diestrus. In anterior pituitary cells from OVX rats, TNF-α failed to induce apoptosis but was able to induce it when cells were incubated with NAME or NMMA, NOS inhibitors that did not affect cell viability per se. In the presence of E2, NAME induced apoptosis and enhanced the proapoptotic effect of TNF-α. In conclusion, our study shows that TNF-α upregulates iNOS gene expression whereas it downregulates estrogen-induced eNOS expression in anterior pituitary cells. Endogenous NO may restrain rather than mediate the proapoptotic effect of TNF-α in anterior pituitary cells.

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.

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.

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.

Effect of Interleukin-6 and Tumor Necrosis Factor-α on GABA Release from Mediobasal Hypothalamus and Posterior Pituitary

The release of cytokines during infection, inflammation and stress induces brain-mediated responses, including alterations of neuroendocrine functions. We examined the effect of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) on release of γ-aminobutyric acid (GABA) from mediobasal hypothalamic (MBH) explants and posterior pituitaries (PP) of male rats. IL-6 (10 ng/ml) did not modify basal GABA release from MBH and PP, but significantly increased GABA release under depolarizing conditions (40 mM K+). This effect was abolished by incubation of the tissue with indomethacin, an inhibitor of cyclooxygenase activity, indicating that prostaglandins could mediate the stimulation of GABA release induced by IL-6. On the contrary, TNF-α (50 ng/ml) significantly decreased K+-evoked GABA release from both MBH and PP. This inhibitory effect was not modified by indomethacin. Neither IL-6 nor TNF-α affected nitric oxide synthesis, as measured by [14C]citrulline production. The current results indicate that IL-6 stimulates GABA release from both hypothalamus and posterior pituitary by a mechanism mediated by prostaglandins. On the contrary, TNF-α inhibits GABA release from both tissues. These results suggest the possibility that GABAergic activity in the hypothalamic-pituitary axis could be involved in neuroendocrine responses to cytokines.

Effect of lipopolysaccharide on tumor necrosis factor and prolactin release from rat anterior pituitary cells.

TNF-α plays a critical role in the cascade of neuroendocrine events during inflammation and septic shock. It also affects the release of pituitary hormones and acts as a growth factor in immune and nonimmune cells. The aim of the present study was to investigate the release of TNF-α from rat anterior pituitary cells and the effect of the steroid medium on its release. Cultured anterior pituitary cells from lactating rats spontaneously released TNF-α. The presence of lipopolysaccharide (LPS, 0.1 µg/mL) in the culture medium significantly increased TNF-α release and inhibited prolactin release. Chronic estrogenization of ovariectomized rats or the presence of 17 β-estradiol in the culture medium also increased TNF-α release. LPS significantly stimulated TNF-α release in all groups and abrogated the estrogen-induced prolactin release. We also investigated the effect of TNF-α on prolactin release. The presence of TNF-α (50 ng/mL) in the culture medium inhibited prolactin release from anterior pituitary cells. These data show that anterior pituitary cells in culture release TNF-α and that this release is stimulated by estrogens. Our results also indicate that LPS inhibits prolactin release in an estrogenic environment, suggesting that TNF-α could affect pituitary hormone release during endotoxemia.

Estrogens modulate the inhibitory effect of tumor necrosis factor-α on anterior pituitary cell proliferation and prolactin release

Considering that tumor necrosis factor-α (TNF-α) is involved in normal tissue homeostasis and that its receptors are expressed in the anterior pituitary, we examined the expressed in the anterior pituitary, we examined the effect of this cytokine on pituitary cell growth. Because anterior pituitary function depends on hormonal environment, we also investigated the influence of gonadal steroids in the effects of TNF-α on cell proliferation and the release of PRL from anterior pituitary cells. In addition, the release of TNF-α and its action on the release of PRL from anterior pituitary cells of rats at different stages of the estrous cycle was evaluated. In minimum essential medium D-valine, a medium that restricts fibroblastic proliferation, TNF-α (10 and 50 ng/mL) reduced 3H-Thymidine incorporation, DNA content, and active cell number. TNF-α failed to affect proliferation of cells from ovariectomized (OVX) rats. However, it significantly inhibited growth of cells from OVX rats cultured with 17ß-estradiol (E2) (10−9M) and from chronically estrogenized rats. TNF-α decreased the release of PRL from cells of intact rats, especially in proestrous, OVX rats cultured with E2 and chronically estrogenized rats. The release of anterior pituitary TNF-α was higher in proestrous rats. These results indicate that TNF-α plays and inhibitory role in anterior pituitary cell growth and the release of PRL in an estrogen-dependent manner.