Normal reproductive function in mammals requires precise control of LH synthesis and secretion by gonadotropes of the anterior pituitary. Synthesis of LH requires expression of two genes [alpha-glycoprotein subunit (alphaGSU) and LHbeta] located on different chromosomes. Hormones from the hypothalamus and gonads modulate transcription of both genes as well as secretion of the biologically active LH heterodimer. In males and females, the transcriptional tone of the genes encoding alphaGSU and LHbeta reflects dynamic integration of a positive signal provided by GnRH from hypothalamic neurons and negative signals emanating from gonadal steroids. Although alphaGSU and LHbeta genes respond transcriptionally in the same manner to changes in hormonal input, different combinations of regulatory elements orchestrate their response. These hormone-responsive regulatory elements are also integral members of much larger combinatorial codes responsible for targeting expression of alphaGSU and LHbeta genes to gonadotropes. In this review, we will profile the genomic landscape of the promoter-regulatory region of both genes, depicting elements and factors that contribute to gonadotrope-specific expression and hormonal regulation. Within this context, we will highlight the different combinatorial codes that control transcriptional responses, particularly those that mediate the opposing effects of GnRH and one of the sex steroids, androgens. We will use this framework to suggest that GnRH and androgens attain the same transcriptional endpoint through combinatorial codes unique to alphaGSU and LHbeta. This parallelism permits the dynamic and coordinate regulation of two genes that encode a single hormone.

Targeted overexpression of LH in transgenic mice causes hyperproliferation of Pit-1-positive pituitary cells and development of functional adenomas. To characterize gene expression changes associated with pituitary tumorigenesis, we performed microarray studies using Affymetrix GeneChips comparing expression profiles from pituitary tumors in LH-overexpressing mice to wild-type control pituitaries. We identified a number of candidate genes with altered expression in pituitary tumors. One of these, p8 (candidate of metastasis-1), encodes a native high-mobility group-like transcription factor previously shown to be necessary for ras-mediated transformation of mouse embryonic fibroblasts and also implicated in breast cancer progression. Herein, we show that expression of p8, normally quiescent in adult pituitary, localizes to tumor foci containing lactotropes, suggesting a linkage with their transformation. To further establish the functional significance of p8 in pituitary tumorigenesis, we constructed several clonal cell lines with reduced expression of p8 from a parent GH3 somatolactotrope cell line. These clonal derivates, along with the parent cell line, were tested for tumorigenicity by injection into athymic mice. When compared with wild-type GH3 with higher levels of p8, GH3 cells with reduced expression of p8 displayed attenuated tumor development or failed to develop tumors at all. Similar results were obtained with gonadotrope-derived cell lines that displayed reduced expression of p8. Together, these data suggest that maintenance of the transformed phenotype of pituitary GH3 cells requires expression of p8 and that it may play a similar role when reexpressed in a subset of lactotropes that form prolactinomas in vivo.

A comparison between two pituitary-derived cell lines (␣T3-1 and L␤T2) that represent gonadotropes at early and late stages of development, respectively, was performed to further elucidate the genomic repertoire required for gonadotrope specification and luteinizing hormone ␤ (LH␤) gene expression. One isolated clone that displayed higher expression levels in L␤T2 cells encodes p8, a high mobility group-like protein with mitogenic potential that is up-regulated in response to proapoptotic stimuli and in some developing tissues. To test the functional significance of this factor in developing gonadotropes, a knockdown of p8 in L␤T2 cells was generated. The loss of p8 mRNA correlated with loss of endogenous LH␤ mRNA and the loss of activity of a transfected LH␤ promoter-driven reporter, even upon treatment with gonadotropin-releasing hormone. In addition, expression of p8 mRNA in developing mouse pituitary glands mirrored its expression in the gonadotrope-derived cell lines and coincided with the first detectable appearance of LH␤ mRNA. In contrast, p8 mRNA was undetectable in the pituitary glands of normal adults. Taken together, our data indicate that p8 is a stage-specific component of the gonadotrope transcriptome that may play a functional role in the initiation of LH␤ gene expression during embryonic cellular differentiation.With its ability to stimulate gonadal steroidogenesis and gametogenesis, luteinizing hormone (LH) 1 is essential for normal reproductive function in mammals (1, 2). Luteinizing hormone is a heterodimeric protein composed of an ␣ glycoprotein hormone subunit (␣GSU) common to all members of the glycoprotein family of hormones that is non-covalently linked to a unique LH␤ subunit that confers its biological specificity (1, 2). Biosynthesis of LH depends on the coordinated expression of both the ␣GSU and LH␤ subunit genes. In humans, the single copy ␣GSU gene resides on chromosome 6q12-q21 (Locus ID 1081) while the LH␤ gene resides amid a cluster of six chorionic gonadotropin-␤ genes on chromosome 19q13.32 (Locus ID 1082). In addition to their different locations within the human genome, the pattern of expression of the genes encoding ␣GSU and LH␤ are temporally and spatially distinct.During development ␣GSU is seen throughout Rathke's pouch as early as embryonic day (e) 9.5 in the mouse (3). By later stages of pituitary development and in adult mammals, ␣GSU expression is limited to thyrotropes and gonadotropes (4). Gonadotrope-specific expression of the ␣GSU gene is controlled by an array of regulatory elements, including the pituitary glycoprotein hormone basal element (5, 6), ␣ basal elements (6), gonadotrope-specific element (7), and tandemly repeated cAMP response elements (8 -10), as well as the intricate interplay between their cognate binding proteins (6).In a fashion similar to the ␣GSU promoter, the LH␤ gene is regulated by a combinatorial array of transcription factors and regulatory elements. In this case, however, the elements consist of those that bind early growth response ...

Reproduction depends on regulated expression of the LHbeta gene. Tandem copies of regulatory elements that bind early growth response protein 1 (Egr-1) and steroidogenic factor 1 (SF-1) are located in the proximal region of the LHbeta promoter and make essential contributions to its activity as well as mediate responsiveness to GNRH: Located between these tandem elements is a single site capable of binding the homeodomain protein Pitx1. From studies that employ overexpression paradigms performed in heterologous cell lines, it appears that Egr-1, SF-1, and Pitx1 interact cooperatively through a mechanism that does not require the binding of Pitx1 to its site. Since the physiological ramifications of these overexpression studies remain unclear, we reassessed the requirement for a Pitx1 element in the promoter of the LHbeta gene using homologous cell lines and transgenic mice, both of which obviate the need for overexpression of transcription factors. Our analysis indicated a striking requirement for the Pitx1 regulatory element. When assayed by transient transfection using a gonadotrope-derived cell line (LbetaT2), an LHbeta promoter construct harboring a mutant Pitx1 element displayed attenuated transcriptional activity but retained responsiveness to GNRH: In contrast, analysis of wild-type and mutant expression vectors in transgenic mice indicated that LHbeta promoter activity is completely dependent on the presence of a functional Pitx1 binding site. Indeed, the dependence on an intact Pitx1 binding site in transgenic mice is so strict that responsiveness to GnRH is also lost, suggesting that the mutant promoter is inactive. Collectively, our data reinforce the concept that activity of the LHbeta promoter is determined, in part, through highly cooperative interactions between SF-1, Egr-1, and Pitx1. While Egr-1 can be regarded as a key downstream effector of GnRH, and Pitx1 as a critical partner that activates SF-1, our data firmly establish that the Pitx1 element plays a vital role in permitting these functions to occur in vivo.

Regulated synthesis of luteinizing hormone (LH) requires coordinated transcriptional control of the ␣ and LH␤ subunits in pituitary gonadotropes. Several cis-acting elements and trans-acting factors have been defined for control of the LH␤ promoter through heterologous cell culture models. In this report, we describe the identification of bipartite NF-Y (CBF/CP1) binding sites within the proximal bovine LH␤ promoter. When multimerized, one of these sites activates the heterologous, minimal HSV thymidine kinase promoter in the gonadotrope-derived cell line ␣T3-1. The functional role of the promoter-distal site in regulating the full-length bovine LH␤ promoter was assessed in vivo using transgenic mice harboring a mutant promoter linked to the chloramphenicol acetyltransferase reporter gene. While this element is important for conferring high level activity of the LH␤ promoter in pituitary, it does not appear to be essential for mediating gonadotropin-releasing hormone (GnRH) regulation. This is the first characterization of a cis-acting element within this GnRH-dependent promoter that is restricted to regulating basal expression and not GnRH-induced activity.Stimulation of gametogenesis and synthesis of sex steroids by luteinizing hormone (LH) 1 is essential for reproduction in all mammalian species. Thus, understanding the regulated synthesis and secretion of this hormone from the gonadotropes in the pituitary is paramount to understanding the reproductive process.LH is a member of the glycoprotein hormone family whose members are composed of a shared ␣ subunit that combines with unique, hormone-defining ␤ subunits. Synthesis and secretion of LH requires coordinated, gonadotrope-specific expression of the genes encoding both the ␣ and LH␤ subunits (1). Gonadotrope-specific expression of the ␣ subunit gene requires a number of cis-acting elements located within the promoterproximal 400 bp of the 5Ј-flanking region of the gene (2-7).Discernment of the essential cis-acting elements involved in expression of the LH␤ subunit has lagged behind that made for the ␣ subunit due to the lack of readily accessible, appropriate model systems.Five models have been used to uncover regulatory elements within the LH␤ promoter. These include cell lines corresponding to kidney fibroblasts (CV-1) (8, 9), modified somatotropes (GGH 3 -1Ј) (10, 11), primordial gonadotropes (␣T3-1) (12-14), and differentiated gonadotropes (L␤T2) (15, 16) as well as transgenic mice (17-21). Using these approaches, four transcription factors have been identified that regulate expression of the LH␤ subunit gene through direct binding to its promoter (see Fig. 1A). These include the orphan nuclear receptor, SF-1 (8, 15, 21), an early growth response protein, Egr-1 (22, 23), a bicoid-related homeodomain protein, Pitx1 (9), and the ubiquitous transcription factor, Sp1 (11). There are two Egr-1 and SF-1 binding sites that exist in a pairwise conformation interrupted by a Pitx1 binding site within the promoter-proximal 150 bp (24). This configuration is con...

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Although the ability of estradiol to enhance pituitary sensitivity to GnRH is established, the underlying mechanism(s) remain undefined. Herein, we find that approximately 9,100 bp of 5' flanking region from the ovine GnRH receptor (oGnRHR) gene is devoid of transcriptional activity in gonadotrope-derived cell lines and is not responsive to either estradiol or GnRH. In stark contrast, this same 9,100 bp promoter fragment directed tissue-specific expression of luciferase in multiple lines of transgenic mice. To test for hormonal regulation of the 9,100-bp promoter, ovariectomized transgenic females were treated with a GnRH antiserum alone or in combination with estradiol. Treatment with antiserum alone reduced pituitary expression of luciferase by 80%. Pituitary expression of luciferase in animals receiving both antiserum and estradiol was approximately 50-fold higher than animals receiving antiserum alone. The estradiol response of the -9,100-bp promoter was equally demonstrable in males. In addition, a GnRH analog (D-Ala-6-GnRH) that does not cross-react with the GnRH antiserum restored pituitary expression of luciferase in males passively immunized against GnRH to levels not different from castrate controls. Finally, treatment with both estradiol and D-Ala-6-GnRH increased pituitary expression of luciferase to a level greater than the sum of the individual treatments suggesting synergistic activation of the transgene by these two hormones. Thus, despite the complete absence of transcriptional activity and hormonal responsiveness in vitro, 9,100 bp of proximal promoter from the oGnRHR gene is capable of directing tissue-specific expression and is robustly responsive to both GnRH and estradiol in transgenic mice. To begin to refine the functional boundaries of the critical cis-acting elements, we next constructed transgenic mice harboring a transgene consisting of 2,700 bp of 5' flanking region from the oGnRHR gene fused to luciferase. As with the -9,100 bp promoter, expression of luciferase in the -2,700 lines was primarily confined to the pituitary gland, brain and testes. Furthermore, the passive immunization-hormonal replacement paradigms described above revealed both GnRH and estradiol responsiveness of the -2,700-bp promoter. Thus, 2,700 bp of proximal promoter from the oGnRHR gene is sufficient for tissue-specific expression as well as GnRH and estradiol responsiveness. Given the inability to recapitulate estradiol regulation of GnRHR gene expression in vitro, transgenic mice may represent one of the few viable avenues for ultimately defining the molecular mechanisms underlying estradiol regulation of GnRHR gene expression.

A line of transgenic mice harboring a fusion gene consisting of 1900 bp of proximal 5'-flanking region from the murine GnRH receptor gene linked to the complementary DNA encoding luciferase was established to determine whether this promoter can direct tissue-specific expression in vivo and serve as a model for identifying the molecular mechanisms underlying hormonal regulation of this gene. Of 10 tissues screened, luciferase was detected predominantly in pituitary gland, but also in brain and testis. To assess hormonal regulation, luciferase activity was measured in intact males and ovariectomized females treated with an anti-GnRH serum alone, and in combination with testosterone or 17beta-estradiol. No effect of steroid treatment on transgene expression was detected. However, immunoneutralization of GnRH resulted in decreased serum LH concentrations and suppressed pituitary expression of luciferase. Furthermore, the effects of GnRH antiserum could be prevented by the administration of a noncross-reactive GnRH agonist. Thus, 1900 bp of 5'-flanking DNA from the murine GnRH receptor gene are sufficient to target luciferase expression in transgenic mice to established sites of GnRH receptor gene expression. Furthermore, we suggest that GnRH regulation of GnRH receptor gene expression is mediated by regulatory elements residing within 1900 bp of the 5'-flanking region.