Gabriela Jaita

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.

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.

Anterior Pituitary Cell Renewal during the Estrous Cycle

The anterior pituitary gland undergoes a process of cell renewal during the estrous cycle. Although the occurrence of proliferation and death of anterior pituitary cells at specific stages of the estrous cycle is well known, the underlying mechanisms that regulate these processes are still being uncovered. In spite of the recognized proliferative effects of estrogens on lactotropes, recent evidence shows that estrogens can also trigger antiproliferative and apoptotic responses in anterior pituitary cells. In the present review we analyze the actions of gonadal steroids on proliferation and death of anterior pituitary cells during the estrous cycle and the mediators involved in these actions. Estradiol sensitizes anterior pituitary cells not only to mitogenic stimuli but also to apoptotic signals and upregulates local synthesis of tropic growth factors as well as proapoptotic cytokines. Several growth factors and cytokines have been shown to induce estrogen-dependent lactotrope proliferation and death, whereas progesterone antagonizes estrogen-induced effects. These locally synthesized factors may mediate the effects of gonadal steroids in the process of anterior pituitary cell renewal during the estrous cycle.

Role of Estrogens in Anterior Pituitary Gland Remodeling during the Estrous Cycle

In this review, we analyze the action of estrogens leading to the remodeling of the anterior pituitary gland, especially during the estrous cycle. Proliferation and death of anterior pituitary cells and especially lactotropes is regulated by estrogens, which act by sensitizing these cells to both mitotic and apoptotic stimuli such as TNF-alpha, FasL and dopamine. During the estrous cycle, the changing pattern of gonadal steroids is thought to modulate both cell proliferation and death in the anterior pituitary gland, estrogens being key players in cell turnover. The mechanisms involved in estrogen-modulated cell renewal in the anterior pituitary gland during the estrous cycle could include an increase in the expression of proapoptotic cytokines as well as the increase in the Bax/Bcl-2 ratio at proestrus, when estrogen levels are highest and a peak of apoptosis, in particular of lactotropes, is evident in this gland. Estrogens exert rapid antimitogenic and proapoptotic actions in the anterior pituitary through membrane-associated estrogen receptors, a mechanism that might also be involved in remodeling of this gland during the estrous cycle.

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 17β-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. 17β-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.

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.

Inhibition of nuclear factor-kappa B sensitises anterior pituitary cells to tumour necrosis factor-α- and lipopolysaccharide-induced apoptosis.

Nuclear factor‐kappa B (NF‐κB), an important pro‐inflammatory factor, is a crucial regulator of cell survival. Both lipopolysaccharide (LPS) and tumour necrosis factor (TNF)‐α activate NF‐κB signalling. Oestrogens were shown to suppress NF‐κB activation. Oestrogens exert a sensitising action to pro‐apoptotic stimuli such as LPS and TNF‐α in anterior pituitary cells. In the present study, we show by western blotting that 17β‐oestradiol (E2) decreases TNF‐α‐induced NF‐κB/p65 and p50 nuclear translocation in primary cultures of anterior pituitary cells from ovariectomised (OVX) rats. Also, the in vivo administration of E2 decreases LPS‐induced NF‐κB/p65 and p50 nuclear translocation. To investigate whether the inhibition of NF‐κB pathway sensitises anterior pituitary cells to pro‐apoptotic stimuli, we used an inhibitor of NF‐κB activity, BAY 11‐7082 (BAY). BAY, at a concentration that fails to induce apoptosis, has permissive action on TNF‐α‐induced apoptosis of lactotrophs and somatotrophs from OVX rats, as assessed by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL). Pharmacological inhibition of NF‐κB signalling enhances E2‐sensitising effect to TNF‐α‐induced apoptosis in lactotrophs but not in somatotrophs. In vivo administration of BAY allowed LPS‐induced apoptosis in anterior pituitary cells from OVX rats (determined by fluorescence activated cell sorting). Furthermore, LPS‐induced expression of Bcl‐xL in pituitaries of OVX rats is decreased by E2 administration. Our results show that inhibition of the NF‐κB signalling pathway sensitises anterior pituitary cells to the pro‐apoptotic action of LPS and TNF‐α. Because E2 inhibits LPS‐ and TNF‐α‐activated NF‐κB nuclear translocation, the present study suggests that E2 sensitises anterior pituitary cells to TNF‐α‐ and LPS‐induced apoptosis by inhibiting NF‐κB activity.

N-terminal prolactin-derived fragments, vasoinhibins, are proapoptoptic and antiproliferative in the anterior pituitary.

The anterior pituitary is under a constant cell turnover modulated by gonadal steroids. In the rat, an increase in the rate of apoptosis occurs at proestrus whereas a peak of proliferation takes place at estrus. At proestrus, concomitant with the maximum rate of apoptosis, a peak in circulating levels of prolactin is observed. Prolactin can be cleaved to different N-terminal fragments, vasoinhibins, which are proapoptotic and antiproliferative factors for endothelial cells. It was reported that a 16 kDa vasoinhibin is produced in the rat anterior pituitary by cathepsin D. In the present study we investigated the anterior pituitary production of N-terminal prolactin-derived fragments along the estrous cycle and the involvement of estrogens in this process. In addition, we studied the effects of a recombinant vasoinhibin, 16 kDa prolactin, on anterior pituitary apoptosis and proliferation. We observed by Western Blot that N-terminal prolactin-derived fragments production in the anterior pituitary was higher at proestrus with respect to diestrus and that the content and release of these prolactin forms from anterior pituitary cells in culture were increased by estradiol. A recombinant preparation of 16 kDa prolactin induced apoptosis (determined by TUNEL assay and flow cytometry) of cultured anterior pituitary cells and lactotropes from ovariectomized rats only in the presence of estradiol, as previously reported for other proapoptotic factors in the anterior pituitary. In addition, 16 kDa prolactin decreased forskolin-induced proliferation (evaluated by BrdU incorporation) of rat total anterior pituitary cells and lactotropes in culture and decreased the proportion of cells in S-phase of the cell cycle (determined by flow cytometry). In conclusion, our study indicates that the anterior pituitary production of 16 kDa prolactin is variable along the estrous cycle and increased by estrogens. The antiproliferative and estradiol-dependent proapoptotic actions of this vasoinhibin may be involved in the control of anterior pituitary cell renewal.

Gonadal steroids modulate Fas-induced apoptosis of lactotropes and somatotropes

We have previously reported that Fas activation induces apoptosis of anterior pituitary cells from rats at proestrus but not at diestrus and in an estrogen-dependent manner. In this study, we evaluated the effect of Fas activation on apoptosis of lactotropes and somatotropes during the estrous cycle and explored the action of gonadal steroids on Fas-induced apoptosis. Also, we studied whether changes in Fas expression are involved in the apoptotic response of anterior pituitary cells. Fas activation increased the percentage of TUNEL-positive lactotropes and somatotropes at proestrus but not at diestrus. FasL triggered apoptosis of somatotropes only when cells from ovariectomized rats were cultured in the presence of 17 β-estradiol (E2). Progesterone (P4) blocked the apoptotic action of the Fas/FasL system in lactotropes and somatotropes incubated with E2. Both E2 and P4 increased the percentage of cells expressing Fas at the cell membrane. Our results show that Fas activation induces apoptosis of lactotropes and somatotropes at proestrus but not at diestrus. Gonadal steroids may be involved in the apoptotic response of lactotropes and somatotropes, suggesting that Fas activation is implicated in the renewal of these pituitary subpopulations during the estrous cycle. The effect of gonadal steroids on Fas expression may be only partially involved in regulation of the Fas/FasL apoptotic pathway in the anterior pituitary gland.