Loss of the protein NUPR1 (p8) leads to delayed LHB expression, delayed ovarian maturation, and testicular development of a sertoli-cell-only syndrome-like phenotype in mice.

The high mobility group factor NUPR1, also known as p8 and com1, plays a role in temporal expression of the beta subunit of luteinizing hormone, LHB, during gonadotroph development. At Embryonic Day (e) 16.5, LHB is detectable in wild-type (Nupr1(+/+)) but not Nupr1 knockout (Nupr1(-/-)) mice. LHB is initiated by e17.5 in Nupr1(-/-) mice, and expression is fully recovered by Postnatal Day (p) 2. Factors indicative of pituitary maturation, GATA2, CGA, and TSH, are not differentially expressed in Nupr1(-/-) and Nupr1(+/+) embryos at e17.5. Therefore, the delay in LHB expression does not appear to result from delayed pituitary development. In addition, the role of NUPR1 in gonadotropin expression appears specific for LHB, as no difference in FSHB is observed in Nupr1(-/-) and Nupr1(+/+) embryos. The gonads are also impacted by the absence of NUPR1. Ovaries of female Nupr1(-/-) mice lack corpora lutea (CL) at 8 wk, an age at which CL are present in all Nupr1(+/+) littermates. Sexual maturity is recovered by 11 wk in Nupr1(-/-) mice. Conversely, the testes of Nupr1(-/-) males appear normal through 8 mo of age. By 10 mo, however, these mice develop a condition in which a significant number of seminiferous tubules lack germ cells, an abnormality reminiscent of human Sertoli-cell-only syndrome. NUPR1 is undetectable in Nupr1(+/+) gonadotrophs by p2 and remains absent in adulthood, but quantitative PCR analysis indicates Nupr1(+/+) adult ovaries and testes express Nupr1 mRNA. Therefore, the ovarian and testicular phenotypes may be due to the loss of NUPR1 directly at the gonads.

The murine p8 gene promoter is activated by activating transcription factor 4 (ATF4) in the gonadotrope-derived LbetaT2 cell line.

The factor p8 is a high mobility group (HMG) A family member that is upregulated during the cellular stress response in numerous tissues. Because expression of this protein encourages cellular transformation, our goal is to characterize the mechanism by which the p8 gene is regulated. Using LbetaT2 cells as a model of a transformed cell in which p8 plays a role in tumor formation, we dissected the p8 promoter into its minimal functional units and found that activating transcription factor 4 (ATF4), a factor also upregulated during cellular stress responses, enhances p8 promoter activity in a dose-dependent manner. In addition, ATF4 binds in the highly conserved major activation domain of the p8 proximal promoter between -130 and -100 bp. Furthermore, we show that six of the nine base pairs that encompass the putative element are essential for ATF4 binding. These findings increase our knowledge of the mechanisms regulating the p8 gene in a genetically defined tumor model.

Multiple and overlapping combinatorial codes orchestrate hormonal responsiveness and dictate cell-specific expression of the genes encoding luteinizing hormone.

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.

Reexpression of p8 contributes to tumorigenic properties of pituitary cells and appears in a subset of prolactinomas in transgenic mice that hypersecrete luteinizing 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.

Embryonic expression of the luteinizing hormone beta gene appears to be coupled to the transient appearance of p8, a high mobility group-related transcription factor.

A comparison between two pituitary-derived cell lines (alpha T3-1 and L beta 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 beta (LH beta) gene expression. One isolated clone that displayed higher expression levels in L beta 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 beta T2 cells was generated. The loss of p8 mRNA correlated with loss of endogenous LH beta mRNA and the loss of activity of a transfected LH beta 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 beta 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 beta gene expression during embryonic cellular differentiation.