Margaret A. Shupnik - US grants

Lutropin (LH), a pituitary glycoprotein hormone, plays a critical role in gonadal development and function, and regulates the production of sex steroids such as estradiol (E2). LH consists of two subunits, the alpha subunit common to all glycoprotein hormones, and the unique LHBeta subunit which is under more stringent physiological regulation. LH production in vivo is under both positive and negative regulation by E2, but it is unclear whether this feedback occurs at the hypothalamic or pituitary level, or both. We have shown that the transcription rate of the kLHBeta gene is negatively regulated by E2 in vivo, but preliminary experiments indicate E2 stimulation of transcription in pituitary fragments and in transient expression assays. The first aim is to define the direct actions of E2 on the LHBeta gene by measuring E2- regulated transcription free of hypothalamic influences. The time and dose dependence of the E2 response will be assessed in isolated pituitary fragments and in cell culture in the absence and presence of gonadotropin releasing hormone (GnRH). The second aim is to identify regions of the LHBeta gene which confer an E2 response. Filter-binding assays with labeled gene fragments and purified E2 receptor complex indicate at least one putative receptor binding site in the 5'-flanking region of the LHBeta gene. This region and other potential receptor binding sites will be studied by Exonuclease III and DNAse I footprinting studies, as well as by mutational analysis, to identify the sequence for receptor binding. Gene regions necessary for the biological E2 response will be identified by transient expression assays, in which upstream LHBeta gene regions inserted next to a thymidine kinase or prolactin promoter controlling expression of a reporter gene are transfected into GH3 cells or normal pituitary cells. It is anticipated that the receptor binding region will be required for the response, but other DNA elements and binding proteins may be necessary, and will be identified. The ability of specific gene regions to confer the E2 response to the reporter gene, and the sequence and positioning of these elements will be determined by deletion analysis. The importance of specific nucleotides within these sequences will be evaluated by site-directed mutagenesis. The ability of other hormones to modulate the E2 response will be tested. The interaction of the antiestrogen tamoxifen with LHBeta gene E2 regulatory regions will be measured by tamoxifen-E2 receptor footprint analysis, and tamoxifen and progesterone modulation of the E2 effects will be investigated by transcription, and in transient expression assays. Reporter gene constructs containing the homologous LHBeta gene promoter and 5'flanking regions will be transfected into pituitary cells and tested for expression and E2 regulation in the absence or presence of GnRH. These studies will add to our understanding of the direct E2 effects on LH production and clarify some of the complex hormonal regulation in the reproductive process.

This is a Shannon Award providing partial support for research projects that fall short of the assigned institute's funding range but are in the margin of excellence. The Shannon award is intended to provide support to test the feasibility of the approach; develop further tests and refine research techniques; perform secondary analysis of available data sets; or conduct discrete projects that can demonstrate the PI's research capabilities or lend additional weight to an already meritorious application. Further scientific data for the CRISP System are unavailable at this time.

The Center for Biological Timing consists of 20 laboratories, 17 of which are at the University of Virginia. This award will renovate laboratory space for five center investigators as well as core facility research and research training space for all investigators. The renovated laboratory space will be located in the Department of Biology and in the multistory building of the Health Science Center. The total amount of renovated space is approximately 12,000 gross square feet and will be used for the latest molecular bio-technology applied to biological timing. The improvement of the laboratory space will greatly enhance the research productivity and research training opportunities in the Center laboratories.

Molecular biology techniques have revolutionized the ability of reproductive biologists to address specific physiological questions in a rapid, sensitive manner. This approach, however, often requires a large time investment to acquire appropriate expertise and equipment, additional time commitments to keep current in a rapidly advancing field, and critical attention to quality control for even the most routine and repetitive techniques. A major cost for many molecular experiments is the labor involved to provide dependable reagents and probes. A centralized, well-equipped core is the most efficient and economical way to provide recombinant DNA methodology and reagents for projects to pursue innovate molecular work in reproductive medicine. The objective of the Molecular Core is to provide services, expertise, and equipment to research projects addressing clinical and basic questions in reproduction. Three research projects within the U54, and five additional projects approved by the Reproductive Science Branch require access to the Core for routine services of probe labeling (end-label, random priming, and single-strand RNA riboprobes), DNA isolation, plasmid growths and purification, DNA sequencing, and DNA fragment isolation and subcloning procedures. The core director and personnel will provide advice and expertise on the design of probes and expression vectors, various RNA and DNA quantitation methods, and specialized techniques such as library screening. Core personnel will provide timely, quality-controlled service and reagents to approved projects on a priority basis. Approve investigators can access the core for specialized equipment to perform more advanced techniques on a availability basis. Finally, Core personnel will continue to improve and develop additional services and procedures as required by projects accessing the core, and update current services to provide the most sensitive, precise, and cost-effective reagents.

Thyrotropin (TSH) pituitary glycoprotein hormone, plays a critical role in thyroid hormone production and thus in normal metabolism. TSH consists of an alpha subunit common to all glycoprotein hormones, and the unique TSHbeta subunit which is under more stringent physiological regulation. We have previously demonstrated that the hypothalamic peptide thyrotropin- releasing hormone (TRH) stimulates TSHbeta gene transcription in rat pituitary cell cultures, and portions of the 5'-flanking region of the gene confer TRH-stimulated responses to reporter genes in transient expression assays. Our first specific aim is to identify the specific TSHbeta gene elements which confer TRH responsiveness to luciferase constructs in GH3 and cultured pituitary cells. Bal 31 digestions, linker-scanner and point mutations of the gene will be performed, and the exact positioning of relevant DNA elements identified. Transient expression experiments will determine if TRH-sensitive gene regions colocalize with calcium and kinase C responses, and the effect of calcium on basal and TRH-stimulated TSHbeta gene transcription in normal pituitary cells will be measured. The second specific aim is to identify and isolate pituitary DNA-binding proteins which confer TRH stimulation to the TSHbeta gene. Binding of nuclear proteins to the TSHbeta gene will be assessed by DNAse I or Exonuclease III footprinting studies. Specific binding between defined gene elements and transcription factors will be measured by gel mobility shift assays and Southwestern blot analysis. Affinities of DNA-protein interactions will be calculated by Scatchard analysis and by competition and equilibrium binding studies. Because a transcription factor termed Pit-1/GHF-1 is thought to play a role in TRH stimulation of prolactin, DNA binding and transfection experiments with Pit-1 expression vector and the TSHbeta gene will be performed. These studies will determine if Pit-1 binds to the TSHbeta gene and assesses its potential role in TRH stimulation of TSHbeta. Transcription factors involved in the TRH response will be cloned from expression vector libraries by screening for DNA-binding proteins with specific TSHbeta gene enhancer oligonucleotides. The potential biological role of these proteins will be evaluated by cotransfection experiments using cloned factors and TRH receptor cDNA. Alternatively, protein factors can be isolated by chromatography on DNA enhancer oligonucleotide affinity columns. The combination of these approaches will directly define the DNA and proteins necessary to implement the TRH transcriptional response, and will provide important insight into the mechanism by which membrane-acting hormones exert effects at the gene level.

GnRH is released in pulses and acts on the pituitary to stimulate the synthesis and release of LH and FSH. This pulsatility is crucial to maintain normal fertility. We previously demonstrated frequency-dependent, subunit-specific effects of GnRH pulses on rat gonadotropin gene transcription in vivo and in vitro, and now have GnRH-responsive luciferase (LUC) reporter gene constructs for the rat alpha and LHbeta genes. Frequency-dependent effects on transcription could arise from differential activation of Ca+2 and protein C(PCK)/mitogen-activated protein kinase (MAPK) cascade and/or divergent GnRH-responsive gene regions and transcription factors. The role of different intracellular signals in GnRH-stimulated endogenous gonadotropin gene transcription will be assessed by nuclear run-off assays in gonadotropes after treatment with specific activators of inhibitors of Ca+2 influx, PKC, and MAPK, in the absence or presence of GnRH. Pituitary cells from transgenic mice expressing LHbetaLUC will be similarly treated and Lhbeta promotor activity measured. Finally, alphaLUC and LHbetaLUC constructs will be transfected into clonal gonadotrope cell lines, and GnRH-stimulated activity measured in the absence or presence of inhibitors of calcium influx, MAPK, and with co-transfected dominant negative or constitutively active forms of MAPK and calcium-calmodulin kinase (CAMK). MAPK and CAMK enzyme activity will be measured directly in treated cells. GnRH- sensitive elements of the alpha and LHbeta genes will be identified by transfection analysis using deletion/mutation constructs of the reporter genes. Activity of the isolated DNA elements will be demonstrated on heterologous promoters, and DNA-nuclear protein binding performed by gels shift and footprinting assays and southwestern blot analysis. The ability of steroids to directly influence the response to GnRH, and binding of nuclear proteins to GnRH-sensitive gene regions will be determined. These studies will help define the underling mechanisms of frequency-dependent regulation of gonadotropin synthesis, and begin to identify specific cellular targets which confer the GnRH response.

Estrogen (E), acting through the estrogen receptor (ER), plays a critical role in gene expression in target tissues such as breast, bone, and the pituitary, where it controls hormone production and cell proliferation. Cell-specific responses to E are well-documented, and mechanisms include differential expression of ER isoforms, distinct pathways of ligand- independent ER stimulation, and differential expression of ER coactivator and repressor proteins. We previously identified a pituitary-specific truncated ERalpha in rat pituitary cells (TERP). TERP is transcribed from a unique promoter and dramatically regulated by E, divergent from full-length ER, such that the TERP:ER ratio varies from 0:1 to 3-4:1. We cloned the intronic TERP promoter for the rat and mouse ERalpha genes, and found it is stimulated by E in transfection assays. TERP cannot bind DNA, but modulates ER transcriptional activity biphasic manner, dependent on TERP:ER ratios. When TERP:ER ratios are high (greater than 1:1) ER activity is suppressed by the formation of TERP:ER heterodimers that cannot bind DNA. At low ratios TERP stimulates ER activity, and we hypothesize this occurs by titration of repressor proteins. One such protein is Repressor of Estrogen Receptor Activity (REA), expressed in pituitary cells, and other repressors may be identified. We will determine the biological role of TERP and its physiological regulation in four specific aims. 1) Interactions of TERP with REA and other ER repressors will be evaluated at the level of protein-protein interactions, the ability of TERP to compete with ER binding to repressors, and to rescue suppressed ER transcription. 2) Expression of the mouse TERP promoter will be analyzed in transient transfection assays for basal enhancer and E-regulated regions. Proteins binding to these sites will be identified, and the ability of TERP to autoregulate its own promoter will be determined. 3) Expression of TERP mRNA during development in male and female mice will be evaluated. 4) Transgenic mice in which TERP expression is eliminated by selective TERP promoter disruption will be produced, and physiological responses of lactotropes and gonadotropes to E will be characterized. Cell-specific ER isoforms with varying biological activities has profound physiological implications for cell function and growth. These studies will provide important information on mechanisms for positive and negative E feedback at the cellular level.

Molecular biology techniques have revolutionized the ability of reproductive biologists to address specific physiological questions in a rapid, sensitive manner. This approach, however, often requires a large time investment to acquire appropriate expertise and equipment, additional time commitments to keep current in a rapidly advancing field, and critical attention to quality control for even the most routine and repetitive techniques. A major cost for many molecular experiments is the labor involved to provide dependable reagents and probes. A centralized, well-equipped core is the most efficient and economical way to provide recombinant DNA methodology and reagents for projects to pursue innovative molecular work in reproductive medicine. The objective of the Molecular Core is to provide services, expertise, and equipment to research projects addressing clinical and basic questions in reproduction. Three research projects within the U54, and five additional projects approved by the Reproductive Sciences Branch require access to the Core for routine services of probe labeling (end-label, random priming, and single-strand RNA riboprobes), DNA isolation, plasmid growths and purification, DNA sequencing, and DNA fragment isolation and subcloning procedures. The core director and personnel will provide advice and expertise on the design of probes and expression vectors, various RNA and DNA quantitation methods, and specialized techniques such as library screening. Core personnel will provide timely, quality controlled service and reagents to approved projects on a priority basis. Approved investigators can access the core for specialized equipment to perform more advanced techniques on an availability basis. Finally, Core personnel will continue to improve and develop additional services and procedures as required by projects accessing the core, and update current services to provide the most sensitive, precise, and cost-effective reagents.

GnRH pulses and sex steroids act on the pituitary to regulate the synthesis and secretion of LH and FSH. In the past funding period, we cloned the rat alpha-subunit promoter, identified GnRH-responsive DNA elements and transcription factors for rat LHbeta and alpha-subunit promoters, and characterized intracellular signaling pathways mediating GnRH responses. The LHbeta promoter requires pulsatile GnRH stimulation in vivo, and has two cooperating complex DNA elements, a distal element with overlapping Sp1 and CArG box sites, and a proximal element with two bipartite sites for SF-1 and Egr-1. Both elements bind Sp1 family zinc finger proteins that could differentially affect transcription. Relative roles of these transcription factors in GnRH stimulation will be assessed by DNA-protein binding, chromatin immunoprecipitation (CHIP) and real-time RT-PCR, and transfection ot deletion/mutation luciferase constructs in LbetaT2 ceils. Preliminary data show that steroids both enhance (17beta- estradiol (E) or pM dihydrotestosterone, DHT) and suppress (nM DHT) the GnRH transcriptional response, without steroid receptor binding to DNA. Coactivator/integrator proteins SNURF and CBP will be tested in cotransfection and protein-protein binding studies for modulation of the GnRH response. Parallel biochemical and functional strategies will be used to investigate both stimulatory and suppressive effects of steroids in normal gonadotropes and LbetaT2 cells. These include tests of altered promoter occupancy by Sp1/Egr- 1 family members in ChIP assays, rescue of suppression by overexpression of transcription factors, and alteration of gene expression by focused microarray analysis and real-time RT-PCR. Promoter regions and transcription factors mediating steroid responses will be defined. Nuclear receptor requirements will be tested with steroid antagonists, and the Tfm androgen receptor mutant mouse. Non-genomic steroid effects on intracellular signaling pathways will be measured in the absence or presence of GnRH. These studies will further our understanding of GnRH regulation of the gonadotropins, and steroid modulation of this response. This research has important implications for understanding fertility disorders such as PCOS, in which elevated circulating androgens are accompanied by altered GnRH and LH secretion.

Program 2 -Endocrinology includes basic, translational, and clinical studies with a common theme of evaluating hormonal and growth factor regulation of cell growth and function. Major investigative efforts are focused in the areas of steroid hormone action and steroid-dependent cancers, and on how growth hormones and cytokines act to promote cell growth, tumor development and metastasis through direct, paracrine, autocrine, or novel mechanisms. The overall scientific goals of the Program are to understand how these processes are regulated in normal cells and tissues, how dysregulation of hormonally-regulated pathways result in aberrant growth of cancer cells and metastasis, and how to prevent or treat such cancers. To achieve these goals, investigators conduct studies on: Hormone regulatory processes in normal and cancer cells;Potential therapeutic targets for prevention of or treatment of cancer;and Collaborative studies on patient treatment and survival. The Program has 21 investigators from 9 clinical and basic science departments in the School of Medicine and College of Arts and Sciences, representing a wide variety of intellectual, technical and clinical expertise. Program investigators interact through dedicated Program meetings, seminar series such as those sponsored by the Cancer Center and Endocrinology, and the Endocrinology Cancer Center Program retreat. Program members also collaborate with other investigators in program projects and working groups for prostate and breast cancer, and women's oncology. These collaborations have been highly successful, as shown by joint grants and publications. For example, of 333 total Program publications, 24% have intra-programmatic, and 28% have inter-programmatic authors. These joint efforts include work on mechanisms of cross-talk between steroids and growth factors in cancer, identification of common signaling molecules for steroids and growth factors in cell proliferation, and mechanisms by which steroid hormone-dependent cancers become hormone-independent. Membership requires that an investigator have an established cancer-focused or cancer-relevant program on endocrine regulation of cell growth and function. The cancer focus has increased substantially since the last renewal, with NCI funding increasing 3-fold ($1.2M in 2005) and cancer-directed funding from the DOD increasing 2- fold. In the past year, Program faculty received over $7 million in peer-reviewed support and over $8.7 million dollars in total direct cost (peer-reviewed and non peer-reviewed).

bioimaging /biomedical imaging; tissue /cell preparation; tissue resource /registry

Temporally regulated secretion of the pituitary gonadotropins luteinizing hormone (LH) and folliclestimulating hormone (FSH) is critical for steroidogenesis and fertility. LH and FSH are tightly controlled by hypothalamic gonadotropin-releasing hormone (GnRH) pulses, and directly and indirectly by ovarian steroids. GnRH pulse amplitude and frequency are regulated physiologically by estrogen (E), progesterone (P) and testosterone (T). High-frequency GnRH pulses occur with E stimulation and favor LH secretion and LH subunit gene transcription, whereas low-frequency pulses occur with P plus E and preferentially stimulate FSH secretion and the FSHbeta gene. Polycystic ovarian syndrome (PCOS) conservatively affects between 6-8% of women of reproductive age, and is characterized by hyper-androgenism, erratic menstrual cycles, and infertility. Many women also have insulin resistance and hyper-insulinemia, with or without obesity. Between 30-90% of PCOS patients have increased LH/FSH ratios, and P is less effective in reducing GnRH pulse frequency and altering gonadotropin secretion in these women. P sensitivity can be partially restored by antiandrogens in some women. Treatment with the insulin sensitizing drug, metformin, can also restore fertility in some subjects, but sites and mechanism of action of insulin or this drug on the reproductive axis are unclear. Understanding mechanisms underlying GnRH gonadotrope regulation is critical to develop treatments for PCOS. Proposed studies will use the prenatal androgenized (PNA) mouse model that mimics many symptoms of PCOS, including elevated LH and T, erratic reproductive cycles and glucose intolerance. We will examine regulated gonadotropin expression, and responses to GnRH and P feedback in PNA and control gonadotropes. Treatment of mice with antiandrogen and metformin will test potential contributions of elevated T or insulin insensitivity to PNA responses. The role of androgen receptor CAG repeat length in influencing T responses will be evaluated in vitro and in transgenic mice. We will also examine the pulsatile GnRH stimulation of gonadotropin gene transcription requirement for proteasome activity, and if insulin or T modulate this process and may thus contribute to dysregulated LH/FSH ratios seen in PCOS.

WOMEN'S ONCOLOGY (WON) ? PROJECT SUMMARY/ABSTRACT The Women's Oncology Program (WON) aims to stimulate high quality basic, translational, and clinical research and trials in women's cancers through collaborations, program interactions, information sharing, and faculty recruitment, and to translate the research findings into cutting edge diagnostics and treatments for cancer. Leaders of the Women's Oncology program include Margaret A. Shupnik, PhD, Professor of Medicine and Physiology, an expert in molecular endocrinology and estrogen receptor action; Susan C. Modesitt. MD, Professor of Obstetrics and Gynecology, an expert in gynecology-oncology clinical trials; and Joellen M. Schildkraut, PhD, MPH Professor of Public Health Sciences, an expert in the genetics and epidemiology of women's cancers, particularly breast and ovarian cancer. WON represents a wide variety of intellectual, technical, and clinical expertise with 25 members from 10 different basic science and clinical departments in the School of Medicine and School of Engineering and Applied Science, and 3 associate members. Cancer Center support for faculty recruitment and retention, forums for information exchange such as the Cancer Center Seminar Series, Women's Oncology monthly research meetings and Program Retreat, Cancer Center pilot research funds, and support for and from our Shared Resources have enabled the success of our research programs, which have become increasingly cancer-focused and interactive. The high quality of the resulting science resulted in numerous high impact publications, including 22% inter- programmatic and 18% intra-programmatic publications over the past 5 years. Current funding is over $4.5M, including $2M in NCI funding, and patient accrual was robust for patients with Breast (17.5%) and Ovarian (45.8%) cancers in 2015. With over 300,000 new diagnoses of these cancers yearly in the United States, it remains essential to develop new treatments for cancers resistant to current therapeutic approaches, methods for early detection and prognostic indicators for responses, and methods to assess risk and prevent breast and gynecological cancers. The WON program has developed specific aims and transdisciplinary groups of investigators to tackle these critical issues in women's cancers. Aim 1: To investigate pathways of therapeutic resistance in women's cancers and identify prognostic indicators and new molecular targets. Aim 2: To understand how dysregulation of metabolism and obesity contribute to women's cancers and identify potential new therapeutic targets. Aim 3. To identify behavioral, hormonal and genetic risks for women's cancers, and improve detection methods for these cancers. Each aim includes basic, translational and clinical research and trials that cut across all women's cancers.