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

Chromosomal integrity after UV irradiation requires FANCD2-mediated repair of double strand breaks

Fanconi Anemia (FA) is a rare autosomal recessive disorder characterized by hypersensitivity to inter-strand crosslinks (ICLs). FANCD2, a central factor of the FA pathway, is essential for the repair of double strand breaks (DSBs) generated during fork collapse at ICLs. While lesions different from ICLs can also trigger fork collapse, the contribution of FANCD2 to the resolution of replication-coupled DSBs generated independently from ICLs is unknown. Intriguingly, FANCD2 is readily activated after UV irradiation, a DNA-damaging agent that generates predominantly intra-strand crosslinks but not ICLs. Hence, UV irradiation is an ideal tool to explore the contribution of FANCD2 to the DNA damage response triggered by DNA lesions other than ICL repair. Here we show that, in contrast to ICL-causing agents, UV radiation compromises cell survival independently from FANCD2. In agreement, FANCD2 depletion does not increase the amount of DSBs generated during the replication of UV-damaged DNA and is dispensable for UV-induced checkpoint activation. Remarkably however, FANCD2 protects UV-dependent, replication-coupled DSBs from aberrant processing by non-homologous end joining, preventing the accumulation of micronuclei and chromatid aberrations including non-homologous chromatid exchanges. Hence, while dispensable for cell survival, FANCD2 selectively safeguards chromosomal stability after UV-triggered replication stress.Fil: Federico, Maria Belén. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Vallerga, María. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Radl, Daniela Betiana. Autoridad Regulatoria Nuclear; ArgentinaFil: Paviolo, Natalia Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Bocco, Jose Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Di Giorgio, Marina. Autoridad Regulatoria Nuclear; ArgentinaFil: Soria, Gastón. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Gottifredi, Vanesa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentin

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

DA increases the percentage of apoptotic PR1-D2S cells incubated in the presence of E2.

<p>PR1-D2S cells were cultured with DMEM-F12-SS in the absence (VEH) or presence of E2 (1 nM) for 48 h. Then the cells were incubated with DA (1 µM) with or without E2 for 4 h. Each column represents the percentage of TUNEL-positive cells ± CI (>2,000 cells/group). Data from at least two separate experiments were analyzed by χ². ** p<0.01 vs respective control without DA. ∧∧ p<0.01 vs respective control without E2.</p

A p38 MAPK inhibitor blocks the apoptosis of anterior pituitary cells induced by D2R activation.

<p>Anterior pituitary cells were cultured with E2 (1 nM) for 48 h. Then the cells were preincubated with SB203580 (1 µM), a p38 MAPK inhibitor, for 30 min and then incubated with DA (1 µM, A and B) or CAB (1 µM, C and D) in the presence or absence of SB203580 for 4 h. Each column represents the percentage of TUNEL-positive cells ± CI (>2,000 cells/group, A, C) or the percentage of TUNEL positive lactotropes ± CI (>500 cells/group B, D). Data from at least two separate experiments were analyzed by χ². ** p<0.01 vs respective control without DA or CAB. ∧ p<0.05 vs respective control without SB203589.</p

DA increases apoptosis in PR1-D2S cells incubated in the presence of E2.

<p>PR1-V, PR1-D2L or PR1-D2S cells were cultured with DMEM-F12-SS in the absence (A) or presence of E2 (1 nM, B) for 48 h. Then the cells were incubated with DA (1 µM-100 µM) in the presence or absence of E2 for 4 h and apoptosis was detected by ELISA. Each column represents the mean ± SE expressed of OD as the percentage of control without DA (n = 5 wells/group). Data were analyzed by ANOVA followed by Dunnett's test. * p<0.05 vs control without DA.</p

p38 MAPK is involved in DA-induced apoptosis of PR1-D2S cells.

<p>A: PR1-D2S cells were cultured with E2 (1 nM) for 48 h. Then the cells were preincubated with SB203580 (1 µM), a p38 MAPK inhibitor, for 30 min and then incubated with DA (1 µM) in the presence or absence of SB203580 for 4 h. Each column represents the percentage of TUNEL-positive cells ± CI (>2,000 cells/group). Data from at least two separate experiments were analyzed by χ². ** p<0.01 vs respective control without DA. ∧∧ p<0.01 vs respective control without SB203580. B: PR1-D2S cells were cultured with or without E2 (1 nM) for 48 h. Then the cells were incubated with DA (1 µM) in the presence of E2 or VEH for 15 min, 30 min or 4 h. Proteins were extracted and p38 MAPK and phospho-p38 MAPK were detected by western blot.</p

CAB induces apoptosis of anterior pituitary cells in an E2-dependent manner.

<p>OVX and E2 treated rats were injected with CAB (1 mg/kg, ip) or vehicle (CONTROL) and euthanized 16 h later. Anterior pituitary cells were dispersed and apoptosis was detected by Annexin-V and flow cytometry. Each column represents the mean ± SE of the percentage of apoptotic cells (n = 8 rats/group). Data were analyzed by two-way ANOVA, followed by Tuckey's test. *p<0.05 vs. respective control without CAB, ∧p<0.05 vs respective control without E2.</p