Cell-specificity of transforming growth factor-beta response is dictated by receptor bioavailability.
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Members of the transforming growth factor-beta (TGFbeta) family control diverse cellular responses including differentiation, proliferation, controlled cell death and migration. The response of a cell to an individual ligand is highly restricted yet the signaling pathways for TGFbeta, activin and bone morphogenic proteins share a limited number of receptors and activate similar intracellular cytoplasmic co-regulators, Smads. A central question in the study of this family of ligands is how cells titrate and integrate each TGFbeta-like signal in order to respond in a cell- and ligand-specific manner. This study uses the pituitary gonadotrope cell line, LbetaT2, as a model to delineate the relative contribution of TGFbeta and activin ligands to follicle-stimulating hormone (FSH) biosynthesis. It was found that pituitary gonadotrope cells do not express the TGFbeta type II (TbetaRII) receptor and are therefore not responsive to the TGFbeta ligand. Transfection of the receptor restores TGFbeta signaling capabilities and the TGFbeta-mediated stimulation of FSHbeta gene transcription in LbetaT2 cells. Consequently, we evaluated the presence of the TbetaRII in the adult mouse pituitary. TbetaRII does not co-localize with FSH-producing cells; however it is detected on the cell surface of prolactin- and growth hormone-positive cells. Taken together, these results suggest that the bioavailability of the TGFbeta-specific receptor rather than TGFbeta dictates pituitary gonadotrope selectivity to activin, which is necessary to maintain normal reproductive function. It is likely that the ligand-restricted mechanisms employed by the gonadotrope are present in other cells, which could explain the distinct control of many cellular processes by members of the TGFbeta superfamily. (+info)
Activin modulates the transcriptional response of LbetaT2 cells to gonadotropin-releasing hormone and alters cellular proliferation.
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Both GnRH and activin are crucial for the correct function of pituitary gonadotrope cells. GnRH regulates LH and FSH synthesis and secretion and gonadotrope proliferation, whereas activin is essential for expression of FSH. Little is known, however, about the interplay of signaling downstream of these two hormones. In this study, we undertook expression profiling to determine how activin pretreatment alters the transcriptional response of LbetaT2 gonadotrope cells to GnRH stimulation. Activin treatment alone altered the transcriptional profile of 303 genes including inducing that of the 17beta-hydroxysteroid dehydrogenase B1 gene that converts estrone to 17beta-estradiol, altering the sensitivity of the cells to estrone. Furthermore, activin had a dramatic effect on the response of LbetaT2 cells to GnRH. Hierarchical clustering of 2453 GnRH-responsive genes identified groups of genes the response of which to GnRH was either enhanced or blunted after activin treatment. Mapping of these genes to gene ontology classifications or signaling pathways highlighted significant differences in the classes of altered genes. In the presence of activin, GnRH regulates genes in pathways controlling cell energetics, cytoskeletal rearrangements, organelle organization, and mitosis in the absence of activin, but genes controlling protein processing, cell differentiation, and secretion. Therefore, we demonstrated that activin enhanced GnRH induction of p38MAPK activity, caused GnRH-dependent phosphorylation of p53, and reduced the ability of GnRH to cause G1 arrest. Thus, although activin alone changes a modest number of transcripts, activin pretreatment dramatically alters the response to GnRH from an antiproliferative response to a more differentiated, synthetic response appropriate for a secretory cell. (+info)
Differential effects of GnRH and androgens on Cres mRNA and protein in male mouse anterior pituitary gonadotropes.
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The Cres gene defines a new subgroup in the family 2 cystatins of cysteine protease inhibitors. However, unlike typical cystatins, CRES does not inhibit cysteine proteases but rather inhibits the serine protease prohormone convertase 2, an enzyme with roles in proprotein processing in the neuroendocrine system. Cres is expressed in the gonadotropes and colocalizes with LHbeta, suggesting a role in the regulation of gonadotrope secretion. Our present studies were carried out to examine the regulation of Cres mRNA and protein expression by GnRH and steroid hormones, thus providing clues regarding its role in gonadotropes. Castration profoundly reduced Cres mRNA, while replacement with estradiol (E(2)), testosterone (T), or dihydrotestosterone (DHT) further decreased Cres, suggesting negative regulation by GnRH or steroid hormones. The administration of Antide, a GnRH antagonist, resulted in a 3-fold increase in Cres mRNA, supporting a negative regulation by GnRH. Because all hormonal manipulations in vivo resulted in alterations in steroid hormones, organ culture was used to assess the effects of GnRH independent of steroids. Mouse pituitaries cultured in the absence of GnRH or steroids showed high Cres mRNA levels, while DHT or E(2) resulted in decreases of 25% and 68%, respectively. However, an 85% decrease in Cres mRNA occurred following the administration of GnRH, demonstrating that GnRH, and to a lesser degree E(2), negatively regulate Cres mRNA in gonadotropes. Examination of CRES protein by immunohistochemistry showed that levels were profoundly reduced following castration, while DHT and in part T, but not E(2), restored CRES levels. Castrated mice treated with Antide showed little effect. However, castrated mice treated with Antide + DHT showed a dramatic recovery of CRES, suggesting that androgens act directly at the level of the gonadotrope to regulate CRES protein. Together, our studies suggest that Cres mRNA and protein are low at peak gonadotrope secretory activity, possibly as a means to allow proprotein processing events to occur that are integral to gonadotrope function. (+info)
A novel technique for temporally regulated cell type-specific Cre expression and recombination in the pituitary gonadotroph.
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Inducing tissue-specific genetic alterations under temporal control allows for the analysis of gene function in particular cell types at specified points in time. We have generated a system for tetracycline-controlled expression of Cre recombinase in mice using the unique CreTeR vector. The gonadotroph-specific bovine alpha-subunit (Balpha) promoter fragment was subcloned into the CreTeR vector, creating a technique for highly regulated expression of Cre recombinase exclusively in pituitary gonadotrophs. Control of Cre recombinase in the CreTeR vector was demonstrated in LbetaT2 pituitary cell lines, where Cre protein was detected in cells treated with doxycycline, but not in untreated cells. In transgenic mice, Cre was expressed in pituitary gonadotrophs of mice treated with doxycycline, but not in non-pituitary tissues or in transgenic mice not treated with doxycycline. We demonstrated Cre expression in the gonadotroph by immunostaining showing co-localization of Cre recombinase with the beta-subunit of LH (LH-beta). Furthermore, by crossing Balpha/CreTeR with R26R mice, we were able to demonstrate functional recombination within pituitary gonadotrophs, detected by lacZ expression. The Balpha/CreTeR mice described here can be used to study the function of virtually any gene in the gonadotroph; in particular, this will be useful in studying genes, which may have distinct roles in development and in the adult. (+info)
Activin B can signal through both ALK4 and ALK7 in gonadotrope cells.
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BACKGROUND: Activins stimulate pituitary FSH synthesis via transcriptional regulation of the FSHbeta subunit gene (Fshb). Like other members of the TGFbeta superfamily, these ligands signal through complexes of type I and type II receptor serine/threonine kinases. The type I receptors, or activin receptor-like kinases (ALKs), propagate intracellular signals upon ligand binding and phosphorylation by associated type II receptors. ALK4 is generally regarded as the type I receptor for activins; however, recent data suggested that activin B and AB might also signal through ALK7. Here, we examined a role for ALK7 in activin B-regulated Fshb transcription. METHODS: We analyzed ALK7 mRNA expression in immortalized gonadotrope cells, LbetaT2, and adult murine pituitary by RT-PCR. We next transfected LbetaT2 cells with wild-type and kinase-deficient (Lys to Arg, KR) forms of ALK4 and ALK7 and examined the effects of these receptors on activin A and B stimulated Fshb promoter-reporter activity. Cells were also transfected with constitutively active (Thr to Asp, TD) forms of the receptors and their effects on endogenous Fshb mRNA levels and phosphorylation of transfected Smad2/3 were measured by RT-PCR and Western blot, respectively. Finally, we measured ALK4(TD) and ALK7(TD) stimulation of Fshb transcription when endogenous Smad3 levels were depleted using short hairpin RNAs. RESULTS: ALK7 mRNA was expressed in LbetaT2 cells and pituitary gland. Transfection of ALK4 cDNA potentiated the effects of both activin A and activin B on Fshb promoter-reporter activity in LbetaT2 cells. In contrast, ALK7 transfection selectively potentiated activin B's effects. Transfection of ALK4(KR) and ALK7(KR) partly inhibited basal and activin B-stimulated reporter activity, whereas ALK4(TD) and ALK7(TD) potently stimulated the Fshb promoter and endogenous mRNA levels. Transfection of both ALK4(TD) and ALK7(TD) stimulated Smad2/3 phosphorylation, and the effects of both receptors on Fshb promoter activity were inhibited by depletion of endogenous Smad3 protein levels. CONCLUSION: These data suggest that immortalized gonadotropes express ALK7 and that activin B can signal through this receptor to stimulate Fshb transcription. The relative roles of endogenous ALK4 and ALK7 receptors in mediating activin B's effects in these cells have yet to be determined. (+info)
Activin and glucocorticoids synergistically activate follicle-stimulating hormone beta-subunit gene expression in the immortalized LbetaT2 gonadotrope cell line.
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FSH is produced by the pituitary gonadotrope to regulate gametogenesis. Production of the beta-subunit of FSH is the rate-limiting step in FSH synthesis, and a number of peptide and steroid hormones within the reproductive axis have been found to regulate transcription of the FSH beta-subunit gene. Although both activin and glucocorticoids are notable regulators of FSHbeta by themselves, we find that cotreatment results in a synergistic interaction on the mouse FSHbeta promoter at the level of the gonadotrope using transient transfection of a reporter gene into the LbetaT2 immortalized gonadotrope-derived cell line. This synergistic interaction is specific to FSHbeta, because only additive effects of these two hormones are observed on LH beta-subunit, GnRH receptor, and mouse mammary tumor virus gene expression. Components of both activin and glucocorticoid signaling are found to be necessary for synergy, and there are specific cis elements on the mouse FSHbeta promoter that contribute to the synergistic response as well. We also identify novel activin-responsive regions in the mouse FSHbeta promoter and find that the -120 site can bind Smad2/3 in vitro. In addition, the glucocorticoid receptor and Smad3 are sufficient to confer a striking synergy with glucocorticoids on the mouse FSHbeta promoter. Our studies provide the first evidence of a synergistic interaction between activin and glucocorticoids within the gonadotrope cell and demonstrate that this synergy can occur directly at the level of the mouse FSHbeta promoter. (+info)
Cell-specific expression of X-linked inhibitor of apoptosis in the anterior pituitary of streptozotocin-induced diabetic rats.
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Cell death is increased in the anterior pituitary of poorly controlled diabetic rats, but anti-apoptotic mechanisms are also activated. We hypothesized that specific cell types are selectively protected against diabetes-induced cell death. To determine when anti-apoptotic mechanisms are activated, streptozotocin-induced diabetic rats were killed after 1, 4, 6 and 8 weeks of evolution. Anterior pituitaries were processed for western blot analysis to determine changes in the intrinsic cell death pathway and upstream kinases involved in cell protection mechanisms. An increase in cell death was detected by ELISA at 4 weeks of diabetes. TUNEL labelling demonstrated that this corresponded to death of primarily lactotrophs, a few somatotrophs, and no thyrotrophs, corticotrophs or gonadotrophs. Levels of phosphorylated (p) Akt were increased at 1 week of diabetes, while pERK1/2 levels increased at 4 weeks and pJNK at 6 weeks. Activation of caspase 3 decreased and anti-apoptotic members of the Bcl-2 protein family increased as early as 1 week after diabetes onset. These changes were coincident with increased IGF-I receptor levels. Levels of X-linked inhibitor of apoptosis protein (XIAP) increased significantly after 6 weeks of diabetes, as did activation of nuclear factor (NF)kappaB. Double immunohistochemistry indicated that XIAP was expressed in less than 1% of lactotrophs and gonadotrophs, approximately 50% of somatotrophs and more than 90% of corticotrophs and thyrotrophs. These results suggest that some cell survival mechanisms are rapidly activated in the anterior pituitary, even before increased cell death can be detected, while others are more delayed. Furthermore, both pituitary cell death and expression of protective mechanisms such as XIAP are cell-type specific. (+info)
Over the past decade, substantial advances have been made in our understanding of the transcription factors which regulate gene expression in gonadotropes. One of the most important of these factors, steroidogenic factor-1 (SF-1; NR5A1) is critical for gonadotropin and GnRH-receptor expression. Interestingly, a closely related nuclear hormone receptor, liver receptor homologue-1 (LRH-1; NR5A2) has recently been detected in the anterior pituitary gland; however, its functional significance in this tissue has not been investigated. For the experiments reported here, we hypothesized that LRH-1 plays a previously unrecognized role in gonadotrope physiology. Towards this end, we first demonstrate LRH-1 mRNA and protein expression in both primary pituitary cells and gonadotrope-derived cell lines. We next show that LRH-1 stimulates promoter activity of the GnRH-receptor and gonadotropin subunit genes. Within the LHbeta gene, this response appears to be mediated by DNA-binding and transactivation through previously characterized SF-1 cis-elements. To our knowledge, this is the first report demonstrating a functional role for LRH-1 in the gonadotrope population of the anterior pituitary gland. (+info)