The equine luteinizing hormone beta-subunit promoter contains two functional steroidogenic factor-1 response elements.

The requirements for basal expression of the LH beta-subunit promoter in pituitary gonadotropes are largely unknown. We have used the equine (e) LHbeta subunit promoter as a model to unravel the combinatorial code required for gonadotrope expression. Through the use of 5'-deletion mutagenesis, a region between -185 and -100 of the eLHbeta promoter was shown to play a critical role in maintaining basal promoter activity in alphaT3-1 and LbetaT2 cells. This region encompasses the steroidogenic factor-1 (SF-1) binding site that has been reported to have a functional role in expression of the LHbeta promoter in other species. We have also identified an additional SF-1 site at -55 to -48. Binding of SF-1 to both sites was confirmed by electrophoretic mobility shift assays. Mutations within these sites, either individually or in combination, did not attenuate basal activity of the eLHbeta promoter in alphaT3-1 cells, but did diminish promoter activity in LbetaT2 cells. Interestingly, cotransfection with an expression vector encoding SF-1 induced eLHbeta promoter activity, and this induction was abrogated by mutations within the SF-1 sites in alphaT3-1 cells. Block replacement mutagenesis was performed on the -185/-100 region of the eLHbeta promoter to identify DNA response elements responsible for maintaining basal promoter activity. From this analysis, two regions emerged as being important: a distal 31-bp segment (-181 to -150) and an element located immediately 3' to the distal SF-1 site (-119 to -106). It is hypothesized that these two regions as well as the SF-1 sites represent regulatory elements that contribute to a combinatorial code involved in targeting expression of the eLHbeta promoter to gonadotropes.     

Activation of luteinizing hormone beta gene by gonadotropin-releasing hormone requires the synergy of early growth response-1 and steroidogenic factor-1.

We have previously shown that early growth response (Egr) 1-deficient mice exhibit female infertility, reflecting a luteinizing hormone (LH) beta deficiency. Egr-1 activates the LHbeta gene in vitro through synergy with steroidogenic factor-1 (SF-1), a protein required for gonadotrope function. To test if this synergy is essential for gonadotropin-releasing hormone (GnRH) stimulation of LHbeta, we examined the activity of the LHbeta promoter in the gonadotrope cell line LbetaT2. GnRH markedly stimulated the LHbeta promoter (15-fold). Mutation of either Egr-1 or SF-1 elements within the LHbeta promoter attenuated this stimulation, whereas mutation of both promoter elements abrogated GnRH induction of the LHbeta promoter. Furthermore, GnRH stimulated Egr-1 but not SF-1 expression in LbetaT2 cells. Importantly, overexpression of Egr-1 alone was sufficient to enhance LHbeta expression. Although other Egr proteins are expressed in LbetaT2 cells and are capable of interacting with SF-1, GnRH stimulation of Egr-1 was the most robust. We also found that the nuclear receptor DAX-1, a repressor of SF-1 activity, reduced Egr-1-SF-1 synergy and diminished GnRH stimulation of the LHbeta promoter. We conclude that the synergy between Egr-1 and SF-1 is essential for GnRH stimulation of the LHbeta gene and plays a central role in the dynamic regulation of LHbeta expression.     

Pitx2 deletion in pituitary gonadotropes is compatible with gonadal development, puberty, and fertility

This report introduces a gonadotrope-specific cre transgenic mouse capable of ablating floxed genes in mature pituitary gonadotropes. Initial analysis of this transgenic line, Tg(Lhb-cre)1Sac, reveals that expression is limited to the pituitary cells that produce luteinizing hormone beta, beginning appropriately at e17.5. Cre activity is detectable by a reporter gene in nearly every LHbeta-producing cell, but the remaining hormone-producing cell types and other organs exhibit little to no activity. We used the Tg(Lhb-cre)1Sac strain to assess the role Pitx2 in gonadotrope function. The gonadotrope-specific Pitx2 knockout mice exhibit normal expression of LHbeta, sexual maturation, and fertility, suggesting that Pitx2 is not required for gonadotrope maintenance or for regulated production of gonadotropins.     

Gonadotropin-releasing hormone (GnRH) positively regulates corticotropin-releasing hormone-binding protein expression via multiple intracellular signaling pathways and a multipartite GnRH response element in alphaT3-1 cells

CRH-binding protein (CRH-BP) binds CRH with high affinity and inhibits CRH-mediated ACTH release from anterior pituitary corticotrope-like cells in vitro. In female mouse pituitary, CRH-BP is localized not only in corticotropes, but is also expressed in gonadotropes and lactotropes. To investigate the functional significance of gonadotrope CRH-BP, we examined the molecular mechanisms underlying GnRH-regulated CRH-BP expression in alphaT3-1 gonadotrope-like cells. CRH-BP is endogenously expressed in alphaT3-1 cells, and quantitative real-time RT-PCR and ribonuclease protection assays demonstrate that GnRH induces a 3.7-fold increase in CRH-BP mRNA levels. GnRH also induces intracellular CRH-BP (2.0-fold) and secreted CRH-BP (5.3-fold) levels, as measured by [125I]CRH:CRH-BP chemical cross-linking. Transient transfection assays using CRH-BP promoter-luciferase constructs indicate that GnRH regulation involves protein kinase C-, ERK- and calcium-dependent signaling pathways and is mediated via a multipartite GnRH response element that includes activator protein 1 and cAMP response element (CRE) sites. The CRE site significantly contributes to GnRH responsiveness, independent of protein kinase A, representing a unique form of multipartite GnRH regulation in alphaT3-1 cells. Furthermore, EMSAs indicate that alphaT3-1 nuclear proteins specifically bind at activator protein 1 and CRE sites. These data demonstrate novel regulation of pituitary CRH-BP, highlighting the importance of the pituitary gonadotrope as a potential interface between the stress and reproductive axes.     

Activation of translation in pituitary gonadotrope cells by gonadotropin-releasing hormone

The neuropeptide GnRH is a central regulator of mammalian reproductive function produced by a dispersed population of hypothalamic neurosecretory neurons. The principal action of GnRH is to regulate release of the gonadotropins, LH and FSH, by the gonadotrope cells of the anterior pituitary. Using a cultured cell model of mouse pituitary gonadotrope cells, alphaT3-1 cells, we present evidence that GnRH stimulation of alphaT3-1 cells results in an increase in cap-dependent mRNA translation. GnRH receptor activation results in increased protein synthesis through a regulator of mRNA translation initiation, eukaryotic translation initiation factor 4E-binding protein, known as 4EBP or PHAS (protein, heat, and acid stable). Although the GnRH receptor is a member of the rhodopsin-like family of G protein-linked receptors, we show that activation of translation proceeds through a signaling pathway previously described for receptor tyrosine kinases. Stimulation of translation by GnRH is protein kinase C and Ras dependent and sensitive to rapamycin. Furthermore, GnRH may also regulate the cell cycle in alphaT3-1 cells. The activation of a signaling pathway that regulates both protein synthesis and cell cycle suggests that GnRH may have a significant role in the maintenance of the pituitary gonadotrope population in addition to directing the release of gonadotropins.     

GnRH activates ERK1/2 leading to the induction of c-fos and LHbeta protein expression in LbetaT2 cells

GnRH acts on pituitary gonadotropes to stimulate the synthesis and release of LH and FSH. However, the signaling pathways downstream of the GnRH receptor that mediate these effects are not fully understood. In this paper, we demonstrate that GnRH activates ERK, c-Jun N-terminal kinase, and p38MAPK in the LbetaT2 gonadotrope cell line. Phosphorylation of both ERK and p38MAPK are stimulated rapidly, 30- to 50-fold in 5 min, but activation of c-Jun N-terminal kinase has slower kinetics, reaching only 10-fold after 30 min. Activation of ERK by GnRH is blocked by inhibition of MAPK kinase (MEK) and partially blocked by inhibition of PKC and calcium, but not PI3K or p38MAPK signaling. We demonstrate that phosphorylated ERK accumulates in the nucleus in a PKC-dependent manner. We also show that GnRH induces c-fos and LHbeta subunit protein expression in LbetaT2 cells via MEK. Experiments with EGTA or calcium channel antagonists indicated that calcium influx is important for the induction of both genes by GnRH. In conclusion, these results show that GnRH activates all three MAPK subfamilies in LbetaT2 cells and induces c-fos and LHbeta protein expression through calcium and MEK-dependent mechanisms. These results also demonstrate that the nuclear translocation of ERK by GnRH requires PKC signaling.     

Complementary expression of IGF-II and IGFBP-5 during anterior pituitary development

The specification of the five individual hormone-secreting cell types in the anterior pituitary requires a series of sequential cell fate decisions. We have immortalized cells at several stages along this pathway of pituitary differentiation. Here, we present analysis of differences in gene expression between an anterior pituitary precursor cell line, alphaT1-1, and an immature gonadotrope cell line, alphaT3-1, identified by using cDNA subtraction. Messenger RNA expression of members of the insulin-like growth factor signaling system, IGF-II and IGFBP-5, was found in the alphaT1-1 precursor cell line, but not in the more differentiated cell line, alphaT3-1. This inferred stage specificity was confirmed in the mouse embryo by using in situ hybridization on embryonic days e10.5 through e18.5. Expression of IGF-II and IGFBP-5 mRNAs was both temporally and spatially regulated during pituitary development. IGF-II was highly expressed in the epithelium surrounding Rathke's pouch at e10.5, while IGFBP-5 expression was restricted to the adjacent oral epithelium. At e11.5 (represented by alphaT1-1), IGF-II was expressed throughout the pouch, but was coexpressed with IGFBP-5 and alpha-subunit in the ventral portion of the pouch epithelium. On e12.5, the two mRNAs were expressed in opposing dorsoventral (IGF-II) and ventrodorsal (IGFBP-5) patterns, with IGF-II excluded from the rostral, alpha-subunit-expressing region. A decrease of both mRNAs was observed at e14.5 (equivalent to alphaT3-1), with IGF-II levels low and IGFBP-5 concentrated in the anterior pituitary rostral tip. These findings suggest that the timing of IGF-II expression and regulation of its accessibility by IGFBP-5 may play a role in anterior pituitary differentiation, survival, and/or proliferation.     

Activation of the luteinizing hormone beta promoter by gonadotropin-releasing hormone requires c-Jun NH2-terminal protein kinase

Regulation of the mitogen-activated protein kinase (MAPK) family by gonadotropin-releasing hormone (GnRH) in the gonadotrope cell line LbetaT2 was investigated. Treatment with gonadotropin-releasing hormone agonist (GnRHa) activates extracellular signal-regulated kinase (ERK) and c-Jun NH(2)-terminal kinase (JNK). Activation of ERK by GnRHa occurred within 5 min, and declined thereafter, whereas activation of JNK by GnRHa occurred with a different time frame, i.e. it was detectable at 5 min, reached a plateau at 30 min, and declined thereafter. GnRHa-induced ERK activation was dependent on protein kinase C or extracellular and intracellular Ca(2+), whereas GnRHa-induced JNK activation was not dependent on protein kinase C or on extracellular or intracellular Ca(2+). To determine whether a mitogen-activated protein kinase family cascade regulates rat luteinizing hormone beta (LHbeta) promoter activity, we transfected the rat LHbeta (-156 to +7)-luciferase construct into LbetaT2 cells. GnRH activated the rat LHbeta promoter activity in a time-dependent manner. Neither treatment with a mitogen-activated protein kinase/ERK kinase (MEK) inhibitor, PD98059, nor cotransfection with a catalytically inactive form of a mitogen-activated protein kinase construct inhibited the induction of the rat LHbeta promoter by GnRH. Furthermore, cotransfection with a dominant negative Ets had no effect on the response of the rat LHbeta promoter to GnRH. On the other hand, cotransfection with either dominant negative JNK or dominant negative c-Jun significantly inhibited the induction of the rat LHbeta promoter by GnRH. In addition, GnRH did not induce either the rat LHbeta promoter activity in LbetaT2 cells transfected stably with dominant negative c-Jun. These results suggest that GnRHa differentially activates ERK and JNK, and a JNK cascade is necessary to elicit the rat LHbeta promoter activity in a c-Jun-dependent mechanism in LbetaT2 cells.     

Androgens, progestins, and glucocorticoids induce follicle-stimulating hormone beta-subunit gene expression at the level of the gonadotrope

FSH is produced by the pituitary gonadotrope to regulate gametogenesis. Steroid hormones, including androgens, progestins, and glucocorticoids, have all been shown to stimulate expression of the FSHbeta subunit in primary pituitary cells and rodent models. Understanding the molecular mechanisms of steroid induction of FSHbeta has been difficult due to the heterogeneity of the anterior pituitary. Immortalized LbetaT2 cells are a model of a mature gonadotrope cell and express the endogenous steroid receptor for each of the three hormones. Transient transfection of each receptor, along with ligand treatment, stimulates the mouse FSHbeta promoter, but induction is severely diminished using receptors that lack the ability to bind DNA, indicating that induction is likely through direct DNA binding. All three steroid hormones act within the first 500 bp of the FSHbeta promoter where six putative hormone response elements exist. The -381 site is critical for FSHbeta induction by all three steroid hormones, whereas the -197 and -139 sites contribute to maximal induction. Interestingly, the -273 and -230 sites are also necessary for androgen and progestin induction of FSHbeta, but not for glucocorticoid induction. Additionally, we find that all three receptors bind the endogenous FSHbeta promoter, in vivo, and specifically bind the -381 site in vitro, suggesting that the binding of the receptors to this element is critical for the induction of FSHbeta by these 3-keto steroid hormones. Our data indicate that androgens, glucocorticoids, and progestins act via their receptors to directly activate FSHbeta gene expression in the pituitary gonadotrope.     

Gonadotrope and thyrotrope development in the human and mouse anterior pituitary gland

Genes and orthologous intrinsic and extrinsic factors critical for embryonic pituitary gonadotrope and thyrotrope cell differentiation have been identified mainly in rodents, but data on the human are very limited. In human fetal pituitaries examined between 14 and 19 weeks of gestation using immunofluorescent confocal microscopy, we found that most fetal gonadotropes expressed alpha-GSU, LHbeta, and FSHbeta gonadotropin subunits while almost no cells expressed alpha-GSU and LHbeta alone. Gonadotropes expressing alpha-GSU and FSHbeta only were detected in both male and female pituitaries, increasing in proportion to total gonadotropes in both males and females from 14 (approximately 4.5%) to 19 weeks (approximately 16.5%) with a peak in males of 45.5% compared with females of 16.5% at 17 weeks of gestation. When FSHbeta or LHbeta genes were expressed, gonadotropes were non-dividing. This profile of human fetal gonadotrope development differs from the current mouse model. Furthermore, while expression of alpha-GSU appears to be the lead protein in gonadotropes, in thyrotropes which ultimately express alpha-GSU with TSHbeta, we observed that most if not all thyrotropes were TSHbeta-positive but alpha-GSU-negative until around 19 weeks in human, and e15 in mouse, fetal pituitaries. Furthermore, the TSHbeta-only thyrotropes were dividing, and TSHbeta rather than alpha-GSU was the lead protein in thyrotrope development. Thus, while biologically active dimeric FSH and LH can be produced by the human fetal pituitary by 14 weeks, dimeric biologically active TSH will only be produced from around 17 weeks of gestation. The mechanism(s) responsible for the different molecular regulation of alpha-GSU gene expression in gonadotropes and thyrotropes in the developing human fetal pituitary now requires investigation.     

Activin signaling pathways in ovine pituitary and LbetaT2 gonadotrope cells

In the pituitary, activin stimulates the synthesis and release of FSH. However, the activin receptor signaling pathways that mediate these effects are poorly known. We investigated these mechanisms in primary ovine pituitary cells (POP) and in the murine LbetaT2 gonadotrope cell line. POP cells and LbetaT2 cells express the different activin receptors (types IA, IB, IIA, and IIB) and the Smad proteins (Smad-2, -3, -4, and -7). In both POP and LbetaT2 cells, activin activated several signaling pathways: Smad-2, extracellular regulated kinase-1/2 (ERK1/2), p38, and phosphatidylinositol 3'-kinase (PI3K)/Akt. Phosphorylation of ERK1/2 and p38 were stimulated (3- to 6-fold) rapidly in 5 min, whereas activation of both Smad-2 and Akt (3- to 5-fold) occurred later, in 60 min. Activin also increased the association of activin receptor IIB with PI3K. Using specific inhibitors, we demonstrated that the activation of Smad-2 was partially blocked by the inhibition of PI3K but not by the inhibition of ERK1/2 or p38, suggesting a cross-talk between the Smad and PI3K/Akt pathways. In both POP and LbetaT2 cells, FSH expression and secretion in response to activin were not altered by the inhibition of PI3K/Akt, ERK1/2, or p38 pathways, whereas they were reduced by about 2-fold by expression of a dominant negative of Smad-2 or the natural inhibitory Smad-7 in LbetaT2 cells. These results indicate that activin activates several signaling pathways with different time courses in both POP and LbetaT2 cells, but only the Smad-2 pathway appears to be directly implicated in FSH expression and release in LbetaT2 cells.