Biomedical subjects

John H Nilson

Publications and source records attributed to John H Nilson.

13 recordsLinked to original sources

PubMed2006

Follicle-stimulating hormone/cAMP regulation of aromatase gene expression requires beta-catenin.

Estrogens profoundly influence the physiology and pathology of reproductive and other tissues. Consequently, emphasis has been placed on delineating the mechanisms underlying regulation of estrogen levels. Circulating levels of estradiol in women are controlled by follicle-stimulating hormone (FSH), which regulates transcription of the aromatase gene (CYP19A1) in ovarian granulosa cells. Previous studies have focused on two downstream effectors of the FSH signal, cAMP and the orphan nuclear receptor steroidogenic factor-1 (NR5A1). In this report, we present evidence for beta-catenin (CTNNB1) as an essential transcriptional regulator of CYP19A1. FSH induction of select steroidogenic enzyme mRNAs, including Cyp19a1, is enhanced by beta-catenin. Additionally, beta-catenin is present in transcription complexes assembled on the endogenous gonad-specific CYP19A1 promoter, as evidenced by chromatin immunoprecipitation assays. Transient expression and RNAi studies demonstrate that FSH- and cAMP-dependent regulation of this promoter is sensitive to alterations in the level of beta-catenin. The stimulatory effect of beta-catenin is mediated through functional interactions with steroidogenic factor-1 that involve four acidic residues within its ligand-binding domain, mutation of which attenuates FSH/cAMP-induced Cyp19a1 mRNA accumulation. Together, these data demonstrate that beta-catenin is essential for FSH/cAMP-regulated gene expression in the ovary, identifying a central and previously unappreciated role for beta-catenin in estrogen biosynthesis, and a potential broader role in other aspects of follicular maturation.

Animals↗

PubMed2004

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.

Animals↗

PubMed2004

Formation of cystic ovarian follicles associated with elevated luteinizing hormone requires estrogen receptor-beta.

Stringent regulation of LH secretion from the pituitary is vital to ovarian function in mammals. Two rodent models of LH hypersecretion are the transgenic LHbeta-C-terminal peptide (LHbetaCTP) and estrogen receptor-alpha (ERalpha)-null (alphaERKO) mice. Both exhibit ovarian phenotypes of chronic anovulation, cystic and hemorrhagic follicles, lack of corpora lutea, interstitial/stromal hyperplasia, and elevated plasma estradiol and testosterone. Because ERbeta is highly expressed in granulosa cells of the ovary, we hypothesized the intraovarian actions of ERbeta may be necessary for full manifestation of phenotypes associated with LH hyperstimulation. To address this question, we generated female mice that possess elevated LH, but lack ERbeta, by breeding the LHbetaCTP and ERbeta-null (betaERKO) mice. A comparison of LHbetaCTP, alphaERKO, and betaERKO(LHCTP) females has allowed us to elucidate the contribution of each ER form to the pathologies and endocrinopathies that occur during chronic LH stimulation of the ovary. alphaERKO ovaries respond to elevated LH by exhibiting an amplified steroidogenic pathway characteristic of the follicular stage of the ovarian cycle, whereas wild-type(LHCTP) and betaERKO(LHCTP) females exhibit a steroidogenic profile more characteristic of the luteal stage. In addition, the hemorrhagic and cystic follicles of the LHbetaCTP and alphaERKO ovaries require the intraovarian actions of ERbeta for manifestation, because they were lacking in the betaERKO(LHCTP) ovary. In turn, ectopic expression of the Leydig cell-specific enzyme, Hsd17b3, and male-like testosterone synthesis in the alphaERKO ovary are unique to this genotype and are therefore the culmination of elevated LH and the loss of functional ERalpha within the ovary.

Animals↗

PubMed2004

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.

Animals↗

PubMed2003

Targeted overexpression of luteinizing hormone causes ovary-dependent functional adenomas restricted to cells of the Pit-1 lineage.

The majority of pituitary adenomas in humans are nonmetastasizing, monoclonal neoplasms that occur in approximately 20% of the general population. Their development has been linked to a combination of extrinsic factors and intrinsic defects. We now demonstrate with transgenic mice that targeted and chronic overexpression of LH causes ovarian hyperstimulation and subsequent hyperproliferation of Pit-1-positive cells that culminates in the appearance of functional pituitary adenomas ranging from focal to multifocal expansion of lactotropes, somatotropes, and thyrotropes. Tumors fail to develop in ovariectomized mice, indicating that contributions from the ovary are necessary for adenoma development. Although the link between chronic ovarian hyperstimulation and PRL-secreting adenomas was expected, the involvement of somatotropes and thyrotropes was surprising and suggests that multiple ovarian hormones may contribute to this unusual pathological consequence. In support of this idea, we have found that ovariectomy followed by estrogen replacement results in the expansion of lactotropes selectively in LH overexpressing mice, but not somatotropes and thyrotropes. Collectively, these data indicate that estrogen is sufficient for the formation of lactotrope adenomas only in animals with a hyperstimulated ovary, whereas the appearance of GH- and TSH-secreting adenomas depends on multiple ovarian hormones. Together, our data expand current models of pituitary tumorigenesis by suggesting that chronic ovarian hyperstimulation may underlie the formation of a subset of pituitary adenomas containing lactotropes, somatotropes, and thyrotropes.

Adenoma↗

PubMed2003

Expression profiling analyses of gonadotropin responses and tumor development in the absence of inhibins.

Transgenic mice with engineered disruptions in bidirectional endocrine signaling between the pituitary and gonad have shed light on the specific effects of the loss of function of gonadotropins and inhibins. These models are valuable tools for studying ovarian biology because they phenocopy specific pathological states and have variations in ovarian tissue composition that allow us to identify genes expressed in specific cell types. We have used emerging mRNA expression profiling technologies to gain a more comprehensive view of genes that are expressed in the mammalian ovary and adrenal gland in the FSHbeta and inhibin alpha knockout mouse models. Oligonucleotide array hybridization experiments using Affymetrix GeneChip technology and NIA 15K murine cDNA microarray studies identified hundreds of transcripts differentially expressed compared with wild type, over 30 of which were selected for further characterization by Northern blot analyses. Additionally, we performed in situ hybridization studies to localize 10 mRNAs, melanocyte-specific gene 1, amino acid transporter SN2, overexpressed and amplified in teratocarcinoma (Bcat1), Forkhead box protein FOXO1, 24p3, vascular cell adhesion molecule, epiregulin, Bcl2-like10, PC3B, and retinoblastoma binding protein 7. These 10 genes have expression patterns and postulated functions suggesting that they mediate important processes in the physiology and pathology of ovarian and adrenal tissue.

Adrenal Cortex Neoplasms↗

PubMed2003

Obesity in transgenic female mice with constitutively elevated luteinizing hormone secretion.

Transgenic (TG) female mice, expressing a chimeric bovine luteinizing hormone (LH) beta-subunit/human chorionic gonadotropin beta-subunit COOH-terminal extension (bLHbeta-CTP) gene, produce high levels of circulating LH and serve as a model for functional ovarian hyperandrogenism and follicular cysts. We report here that obesity is a typical feature of these female mice. The mean body weight of the bLHbeta-CTP females was significantly higher than in controls at, and beyond 5 wk of age, and at 5 mo, it was 32% increased. At this age, the amount of white adipose tissue in the bLHbeta-CTP females was significantly increased, as reflected by the weight difference of the retroperitoneal fat pad. In addition, the expression of leptin mRNA in white adipose tissue of the TG females was elevated about twofold. Serum leptin and insulin levels, and food intake, were also increased significantly in the TG females. Brown adipose tissue (BAT) thermogenic activity, as measured by GDP binding to BAT mitochondria, was reduced (P < 0.05). Ovariectomy at the age of 3 wk totally prevented the development of obesity. In summary, the present results show that intact female bLHbeta-CTP mice are obese, have increased food consumption, and reduced BAT thermogenic activity. The weight gain can be explained partly by elevated androgens but is probably also contributed to the increased adrenal steroidogenesis. Hence, the bLHbeta-CTP mice provide a useful model for studying obesity related to elevated LH secretion, with consequent alterations in ovarian and adrenal function.

Animals↗

PubMed2003

High levels of luteinizing hormone analog stimulate gonadal and adrenal tumorigenesis in mice transgenic for the mouse inhibin-alpha-subunit promoter/Simian virus 40 T-antigen fusion gene.

Transgenic (TG) mice expressing the Simian virus 40 T-antigen under the control of the murine inhibin-alpha promoter (Inhalpha/Tag) develop granulosa and Leydig cell tumors at the age of 5-6 months, with 100% penetrance. When these mice are gonadectomized, they develop adrenocortical tumors. Suppression of gonadotropin secretion inhibits the tumorigenesis in the gonads of intact animals and in the adrenals after gonadectomy. To study further the role of luteinizing hormone (LH) in gonadal and adrenal tumorigenesis, a double TG mouse model was generated by crossing the Inhalpha/Tag mice with mice producing constitutively elevated levels of LH (bLHbeta-CTP mice). Our results show that in double TG mice (bLHbeta-CTP/Inhalpha/Tag), gonadal tumorigenesis starts earlier and progresses faster than in Inhalpha/Tag mice. Both ovarian and testicular tumors were histologically comparable with the tumors found in Inhalpha/Tag mice. In addition, adrenal tumorigenesis was found in intact double TG females, but not in Inhalpha/Tag females. Inhibin-alpha and LH receptor (LHR) were highly expressed in tumorigenic gonadal tissues, and the elevated LH levels were shown to be associated with ectopic LHR and high inhibin-alpha expression in the female adrenals. We conclude that in the Inhalpha/Tag tumor mouse model, elevated LH levels act as a tumor promoter, advancing gonadal and adrenal tumorigenesis.

Adrenal Cortex Neoplasms↗

PubMed2003

Activin induces x-zone apoptosis that inhibits luteinizing hormone-dependent adrenocortical tumor formation in inhibin-deficient mice.

Inhibin and activin are members of the transforming growth factor beta (TGF-beta) family of ligands produced and secreted primarily by the gonads and adrenals. Inhibin-null (INH(-/-)) mice develop gonadal tumors and-when gonadectomized-adrenocortical carcinoma. The mechanisms leading to adrenal tumorigenesis have been proposed to involve the lack of a gonadal factor and/or a compensatory increase in gonadotropins. In order to achieve elevation of gonadotropins without the concomitant loss of a gonadal hormone, we crossed INH(-/-) mice with a transgenic mouse strain that has chronically elevated luteinizing hormone (LH) levels (LH-CTP). Compound INH(-/-)-LH-CTP mice die within 6 weeks of age from severe cancer cachexia induced by large, activin-secreting ovarian tumors. Unexpectedly, INH(-/-)-LH-CTP mice not only fail to develop adrenal tumors but have smaller adrenals, with a regressed x zone, indicating that elevated LH levels are not sufficient to induce adrenal tumor formation. However, following gonadectomy, INH(-/-)-LH-CTP mice develop large, sex steroid-producing adrenal tumors that arise from the x zone, indicating a growth-promoting effect of high levels of LH on the adrenal cortex in the absence of ovarian tumors. In addition, in vivo and in vitro data indicate that activin induces apoptosis specifically in the adrenal x zone. The restricted expression of activin receptor subunits and Smad2 in cells of the adrenal x zone, together with the elevated activin levels in INH(-/-)-LH-CTP mice, supports the conclusion that activin inhibits adrenal tumor growth by inducing x-zone regression.

Activins↗

PubMed2003

Consequences of elevated luteinizing hormone on diverse physiological systems: use of the LHbetaCTP transgenic mouse as a model of ovarian hyperstimulation-induced pathophysiology.

Chronically elevated luteinizing hormone (LH) induces significant pathology in the LHbetaCTP transgenic mouse model, which uses the bovine gonadotropin alpha (alpha)-subunit promoter to direct transgene expression specifically to gonadotropes in the anterior pituitary. Previously, it was shown that female LHbetaCTP mice are infertile due to anovulation, develop granulosa cell tumors, and undergo precocious puberty from elevated LH and steroid hormones that fail to completely repress the alpha-subunit promoter. This chapter will discuss recent studies that further elucidate the impact of chronically elevated LH on diverse physiological systems. Granulosa cell tumors induced by elevated LH are strain dependent and prevented when transgenics are treated with human chorionic gonadotropin (hCG) surges. A granulosa cell tumor-associated transcriptome is generated, revealing several possible gene candidates for ovarian granulosa cell tumorigenesis. Primordial follicles in LHbetaCTP transgenics become depleted and oocytes exhibit increased rates of meiotic segregation defects, although meiotic competency is acquired normally. Anovulation can be rescued in transgenics by superovulation, though pregnancy fails at midgestation due to maternal factors. Uterine receptivity defects prevent implantation of normal embryos following induction of pseuodpregnancy. Transgenics develop Cushing-like adrenocortical hyperfunction with increased corticosterone production following induction of adrenal LH receptor expression. Elevated LH acts as a tumor promoter in the gonads and the adrenal gland, when expressed in conjunction with the inhibin-alpha SV40 transgene. Finally, chronic elevated LH promotes mammary tumorigenesis. The understanding of multiple clinical pathologies--including ovarian cancer, perimenopausal reproductive aging, premature ovarian failure, polycystic ovarian syndrome, Cushing's syndrome, and breast cancer--may be enhanced through further study of this useful transgenic mouse model.

Aging↗

PubMed2002

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.

Cell Line↗

PubMed2002

Characterization of integrin expression in the mouse ovary.

Integrin alpha:beta heterodimers mediate cell contacts to the extracellular matrix and initiate intracellular signaling cascades in response to a variety of factors. Integrins interact with many determinants of cellular phenotypes and play roles in controlling the development, structural integrity, and function of every type of tissue. Despite their importance, little is known about the regulation of integrin subunits in the mammalian ovary and how they function in folliculogenesis. To determine their relevance to ovarian physiology, we have studied the expression of integrin subunit mRNAs by Northern blot analysis and in situ hybridization in ovaries of wild-type, growth differentiation factor 9 (Gdf 9) knockout, FSHbeta (Fshb) knockout, and inhibin alpha (Inha) knockout mice. Integrin alpha6 mRNA is expressed in oocytes and granulosa cells of single-layer follicles and in oocytes and theca cells of multilayer follicles. Integrin alpha6 is highly expressed in Gdf 9 knockout ovaries, which are enriched in oocytes and primary (single layer) follicles because of a block at this stage of follicular development. Integrin alpha(v) mRNA is most highly expressed in the granulosa cells of multilayer growing follicles, and therefore only low levels of expression are detectable in the Gdf 9 knockout ovaries. Integrin beta1 mRNA exhibits a broad expression pattern in ovaries, including oocytes, granulosa cells, theca cells, and corpora lutea. Integrin beta3 mRNA is expressed in theca and interstitial cells and is upregulated in corpora lutea. It is nearly undetectable in ovaries of Fshb knockout mice, which develop preantral follicles but have no luteal cells. Integrin beta5 mRNA is predominantly expressed in granulosa cells of multilayer follicles. It is expressed at high levels in the Fshb knockout mice and in a compartmentalized manner in the granulosa cell/Sertoli cell tumors that develop in the Inha knockout mice. Specific integrins are associated with ovarian cellular phenotypes in mice, which raises intriguing possibilities as to integrin functions in oocyte competence, follicular development, luteinization, and granulosa cell proliferation.

Animals↗

PubMed2002

Ovulatory surges of human CG prevent hormone-induced granulosa cell tumor formation leading to the identification of tumor-associated changes in the transcriptome.

Granulosa cell tumors comprise approximately 10% of ovarian tumors and, although rare, are clinically important due to their potential for malignancy and recurrence. Although their morphological features have been carefully described, the global changes in gene expression associated with their formation remain undetermined. To initiate this characterization, we used a transgenic mouse model in which granulosa cell tumors occur with 100% penetrance in CF-1 mice that harbor a novel transgene encoding a chimeric LHbeta subunit. When this transgene is expressed in other strains of mice, including (C57BL/6 female symbol x CF-1 male symbol,Tg) F1 hybrids, luteomas develop even though levels of LH remain high. This dichotomous response permits a longitudinal comparison of global changes in transcriptomes uniquely associated with either granulosa cell tumors or luteomas. Herein we report numerous changes in the transcriptome, including a decrease in LH receptor mRNA and increases in several mRNAs that encode secreted proteins previously associated with granulosa cell tumors. Furthermore, we identified a constellation of mRNAs that encode proteins that may serve as new markers for this tumor phenotype. Additional experiments indicated that periodic treatment with human CG prevented formation of granulosa cell tumors in mice genetically predisposed to tumor development and, instead, led to the appearance of luteomas. More importantly, ovarian transcriptomes from the luteomas induced by ovulatory doses of human CG permitted refined confirmation of gene expression changes that were uniquely associated with either granulosa cell tumors in the permissive CF-1 genetic background or in luteomas in the F1 hybrids. Together, these dynamic changes in the ovarian transcriptome indict various signaling pathways potentially involved in mediating the actions of LH over time and, depending on genetic background, the formation of either a luteoma or a granulosa cell tumor.

Animals↗