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 LT2) 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 LT2 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 LT2 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.
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