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

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

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

The percentage of lactotropes and somatotropes expressing mERα varies during the estrous cycle.

<p>Dispersed anterior pituitary cells from rats euthanized at diestrus I or proestrus were immunostained for both mERα and PRL or GH and analyzed by flow cytometry. Each column represents the mean ± SE of anterior pituitary cells (A), lactotropes (B) or somatotropes (C) expressing mERα (n = 3–4 animals per group). *p<0.05, **p<0.01 vs diestrus I, Student’s <i>t</i> test.</p

An ERα-antagonist (MPP) blocks E2-BSA-induced apoptosis of lactotropes.

<p>Anterior pituitary cells in culture were pre-incubated with MPP (100 nM) for 30 min prior to the addition of E2-BSA (1 nM) or VEH for 120 min. Apoptosis was analyzed by TUNEL and lactotropes and somatotropes were identified by immunocytochemistry. Each column represents the percentage ± CI (95%) of TUNEL-positive cells (n≥2600) (A), lactotropes (n≥1500 cells/group) (B) and somatotropes (n≥1100 cells/group) (C). *p<0.05 vs respective control without MPP; ∧p<0.05, <0.01 vs respective control without E2-BSA, χ² test.</p

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 apoptosisFil: Radl, Daniela Betiana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Biología Celular e Histología. Centro de Investigación en Reproducción; ArgentinaFil: Zarate, Sandra Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Biología Celular e Histología. Centro de Investigación en Reproducción; ArgentinaFil: Jaita, Gabriela Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Biología Celular e Histología. Centro de Investigación en Reproducción; ArgentinaFil: Ferraris, Maria Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Biología Celular e Histología. Centro de Investigación en Reproducción; ArgentinaFil: Zaldivar, Verónica. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Biología Celular e Histología. Centro de Investigación en Reproducción; ArgentinaFil: Eijo Alvarenga, Stella Guadalupe. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Biología Celular e Histología. Centro de Investigación en Reproducción; ArgentinaFil: Seilicovich, Adriana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Biología Celular e Histología. Centro de Investigación en Reproducción; ArgentinaFil: Pisera, Daniel Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Biología Celular e Histología. Centro de Investigación en Reproducción; Argentin

Estradiol up-regulates c-FLIPlong expression in anterior pituitary cells

Anterior pituitary cell turnover depends on a tight balance between proliferation and apoptosis. We have previously shown that estrogens sensitize anterior pituitary cells to pro-apoptotic stimuli. c-FLIP (cellular-FLICE-inhibitory-protein) isoforms are regulatory proteins of apoptosis triggered by death receptors. c-FLIPshort isoform competes with procaspase-8 inhibiting its activation. However, c-FLIPlong isoform may have a pro- or anti-apoptotic function depending on its expression level. In the present study, we explored whether estrogens modulate c-FLIP expression in anterior pituitary cells from ovariectomized (OVX) rats and in GH3 cells, a somatolactotrope cell line. Acute administration of 17β-estradiol to OVX rats increased c-FLIPlong expression in the anterior pituitary gland without changing c-FLIPshort expression as assessed by Western blot. Estradiol in vitro also increased c-FLIPlong expression in anterior pituitary cells but not in GH3 cells. As determined by flow cytometry, the percentage of anterior pituitary cells expressing c-FLIP was higher than in GH3 cells. However, c-FLIP fluorescence intensity in GH3 cells was higher than in anterior pituitary cells. FasL increased the percentage of TUNEL-positive GH3 cells incubated either with or without estradiol suggesting that the pro-apoptotic action of Fas activation is estrogen-independent. Our results show that unlike what happens in nontumoral pituitary cells, estrogens do not modulate either c-FLIPlong expression or FasL-induced apoptosis in GH3 cells. The stimulatory effect of estradiol on c-FLIPlong expression could be involved in the sensitizing effect of this steroid to apoptosis in anterior pituitary cells. The absence of this estrogenic action in tumor pituitary cells could be involved in their tumor-like behavior.Fil: Jaita, Gabriela Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina; ArgentinaFil: Zarate, Sandra Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina; ArgentinaFil: Ferraris, Maria Jimena. Universidad de Buenos Aires. Facultad de Medicina; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; ArgentinaFil: Gottardo, María Florencia. Universidad de Buenos Aires. Facultad de Medicina; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; ArgentinaFil: Eijo Alvarenga, Stella Guadalupe. Universidad de Buenos Aires. Facultad de Medicina; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; ArgentinaFil: Magri, María Laura. Universidad de Buenos Aires. Facultad de Medicina; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; ArgentinaFil: Pisera, Daniel Alberto. Universidad de Buenos Aires. Facultad de Medicina; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; ArgentinaFil: Seilicovich, Adriana. Universidad de Buenos Aires. Facultad de Medicina; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; Argentin

Cell surface biotinylation of anterior pituitary cells.

<p>Cultured anterior pituitary cells from cycling rats were processed for cell surface biotinylation as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041299#s2" target="_blank"><i>Material and Methods.</i></a> Western blots from biotinylated (membrane) and non-biotinylated (intracellular) protein fractions were probed with anti-rat ERα and β-actin antibodies (A). PVDF membranes from gels loaded with membrane protein fractions were stained with reversible Ponceau S to ensure equal loading of proteins (B). M: MW marker.</p