Prolactinomas are the most frequently observed pituitary adenomas and most of them respond well to conventional treatment with dopamine agonists (DAs). However, a subset of prolactinomas fails to respond to such therapies and is considered as DA-resistant prolactinomas (DARPs). New therapeutic approaches are necessary for these tumors. Transforming growth factor β1 (TGFβ1) is a known inhibitor of lactotroph cell proliferation and prolactin secretion, and it partly mediates dopamine inhibitory action. TGFβ1 is secreted to the extracellular matrix as an inactive latent complex, and its bioavailability is tightly regulated by different components of the TGFβ1 system including latent binding proteins, local activators (thrombospondin-1, matrix metalloproteases, integrins, among others), and TGFβ receptors. Pituitary TGFβ1 activity and the expression of different components of the TGFβ1 system are regulated by dopamine and estradiol. Prolactinomas (animal models and humans) present reduced TGFβ1 activity as well as reduced expression of several components of the TGFβ1 system. Therefore, restoration of TGFβ1 inhibitory activity represents a novel therapeutic approach to bypass dopamine action in DARPs. The aim of this review is to summarize the large literature supporting TGFβ1 important role as a local modulator of pituitary lactotroph function and to provide recent evidence of the restoration of TGFβ1 activity as an effective treatment in experimental prolactinomas.
M Victoria Recouvreux, M Andrea Camilletti, Daniel B Rifkin and Graciela Díaz-Torga
Isabel García-Tornadú, Marcelo Rubinstein, Bruce D Gaylinn, David Hill, Edith Arany, Malcolm J Low, Graciela Díaz-Torga and Damasia Becu-Villalobos
Recently, the importance of the dopaminergic D2 receptor (D2R) subtype in normal body growth and neonatal GH secretion has been highlighted. Disruption of D2R alters the GHRH–GH–IGF-I axis and impairs body growth in adult male mice. The D2R knockout (KO) dwarf mouse has not been well characterized; we therefore sought to determine somatotrope function in the adult pituitary. Using immunohistochemistry and confocal microscopy, we found a significant decrease in the somatotrope population in pituitaries from KO mice (P=0.043), which was paralleled by a decreased GH output from pituitary cells cultured in vitro. In cells from adult mice the response amplitude to GHRH differed between genotypes (lower in KO), but this difference was less dramatic after taking into account the lower basal release and hormone content in the KO cells. Furthermore, there were no significant differences in cAMP generation in response to GHRH between genotypes. By Western blot, GHRH-receptor in pituitary membranes from KO mice was reduced to 46% of the level found in wildtype (WT) mice (P=0.016). Somatostatin induced a concentration-dependent decrease in GH and prolactin (PRL) secretion in both genotypes, and 1×10−7 M ghrelin released GH in cells from both genotypes (P=0.017) in a proportionate manner to basal levels. These results suggest that KO somatotropes maintain a regulated secretory function. Finally, we tested the direct effect of dopamine on GH and PRL secretion in cells from both genotypes at 20 days and 6 months of life. As expected, we found that dopamine could reduce PRL levels at both ages in WT mice but not in KO mice, but there was no consistent effect of the neurotransmitter on GH release in either genotype at the ages studied. The present study demonstrates that in the adult male D2R KO mouse, there is a reduction in pituitary GH content and secretory activity. Our results point to an involvement of D2R signaling at the hypothalamic level as dopamine did not release GH acting at the pituitary level either in 1-month-old or adult mice. The similarity of the pituitary defect in the D2R KO mouse to that of GHRH-deficient models suggests a probable mechanism. A loss of dopamine signaling via hypothalamic D2Rs at a critical age causes the reduced release of GHRH from hypophyseotropic neurons leading to inadequate clonal expansion of the somatotrope population. Our data also reveal that somatotrope cell number is much more sensitive to changes in neonatal GHRH input than their capacity to develop properly regulated GH-secretory function.
Erika Y Faraoni, María Andrea Camilletti, Alejandra Abeledo-Machado, Laura D Ratner, Fernanda De Fino, Ilpo Huhtaniemi, Susana B Rulli and Graciela Díaz-Torga
Female transgenic mice that overexpress the human chorionic gonadotrophin β subunit (hCGβ+) develop prolactinomas, whereas hCGβ+ males do not. The high levels of circulating hCG induce massive luteinization in the ovary of hCGβ+ females, and progesterone becomes the primary steroid hormone produced, but estradiol remains at physiological level. The involvement of high levels of progesterone in lactotroph proliferation is not clearly understood; hence, the pathogenesis of prolactinomas in hCGβ+ females remains unclear. TGFβ1 is an inhibitor of lactotroph function, and the reduced TGFβ1 activity found in prolactinomas has been proposed to be involved in tumor development. The aim of the present work was to study the role of TGFβ1 in the gender-specific development of prolactinomas in hCGβ+ mice. We compared the expression of different components of the pituitary TGFβ1 system in males and females in this model. We found reduced TGFβ1 levels, reduced expression of TGFβ1 target genes, TGFβ1 receptors, Ltbp1, Smad4 and Smad7 in hCGβ+ female pituitaries. However, no differences were found between the transgenic and wild-type male pituitaries. We postulate that decreased pituitary TGFβ1 activity in hCGβ+ females is involved in the development of prolactinomas. In fact, we demonstrated that an in vivo treatment carried out for increasing pituitary TGFβ1 activity, was successful in reducing the prolactinoma development, and the hyperprolactinemia in hCGβ+ females. Moreover, the stronger TGFβ1 system found in males could protect them from excessive lactotroph proliferation. Sex differences in the regulation of the pituitary TGFβ1 system could explain gender differences in the incidence of prolactinoma.
María Andrea Camilletti, Alejandra Abeledo-Machado, Jimena Ferraris, Pablo A Pérez, Erika Y Faraoni, Daniel Pisera, Silvina Gutierrez and Graciela Díaz-Torga
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 17β-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.
Alejandra Abeledo-Machado, Pablo Anibal Pérez, María Andrea Camilletti, Erika Yanil Faraoni, Florencia Picech, Juan Pablo Petiti, Silvina Gutiérrez and Graciela Diaz-Torga
Serum prolactin levels gradually increase from birth to puberty in both male and female rats, with higher levels observed in female since the first days of life. The increase in lactotroph secretion was attributed to the maturation of prolactin-inhibiting and prolactin-releasing factors; however, those mechanisms could not fully explain the gender differences observed. Prolactin secretion from isolated lactotrophs, in the absence of hypothalamic control, also increases during the first weeks of life, suggesting the involvement of intra-pituitary factors. We postulate that pituitary transforming growth factor beta 1 (TGFβ1) is involved in the regulation of prolactin secretion as well as in the gender differences observed at early postnatal age. Several components of the local TGFβ1 system were evaluated during postnatal development (11, 23, and 45 days) in female and male Sprague–Dawley rats. In vivo assays were performed to study local TGFβ1 activation and its impact on prolactin secretion. At day 11, female pituitaries present high levels of active TGFβ1, concomitant with the highest expression of TGFβ1 target genes and the phospho-Smad3 immunostaining in lactotrophs. The steady increase in prolactin secretion inversely correlates with active TGFβ1 levels only in females. Dopamine and estradiol induce TGFβ1 activation at day 11, in both genders, but its activation induces the inhibition of prolactin secretion only in females. Our findings demonstrate that: (1) TGFβ1 activation is regulated by dopamine and estradiol; (2) the inhibitory regulation of local TGFβ1 on prolactin secretion is gender specific; and (3) this mechanism is responsible, at least partially, for the gender differences observed being relevant during postnatal development.