Mark Lawson - US grants

DESCRIPTION (Taken from the applicant's Abstract) The neuroendocrine cells of the hypothalamus and the multiple endocrine cell lineages of the anterior pituitary are central to coordinating endocrine function. Defining the relationships and functional integration of these endocrine cell types is essential for understanding the complex interplay of widely differing tissues and organ systems regulated by endocrine effectors. The aims of this proposal investigate the developmental relationships of the pituitary endocrine cells and their relationship to the neuroendocrine hypothalamus. Recent data have demonstrated the role of the transcription factor GATA-4, a member of a family of developmentally regulated transcription factors, in the regulation of the alpha-glycoprotein hormone subunit gene in pituitary gonadotropes and of the gonadotropin-releasing hormone (GnRH) gene in hypothalamus. Studies of GATA-4 throughout development have identified this factor in embryonic progenitors to both the GnRH neurons and the pituitary gonadotropes. This proposal seeks to describe the extent of GATA factor expression in the hypothalamus and pituitary and to examine the expression of GATA-4 during development. In the first aim, we propose to define the extent of GATA-4 gene expression in cultured cell lines derived from the various pituitary endocrine lineages and in the mature and developing mouse pituitary. We will also investigate the transcriptional determinants specifying expression of GATA-4 in pituitary cells as well as examine potential regulatory pathways affecting GATA-4 expression. In the second aim, we propose parallel studies examining the extent of the expression of GATA-4 in the mature and developing hypothalamus. We also seek to determine the regulatory elements directing cell type-specific expression of GATA-4 in GnRH neurons, as well as possible regulation of the GATA-4 gene by known regulators of GnRH neurons. The identification of common elements regulating GATA-4 expression in neuroendocrine and pituitary cells will provide a basis for investigating the molecular mechanisms regulating the differentiation of these differing but functionally highly integrated cells. Further, the third aim proposes the development of a transgenic mouse model system expressing a reporter gene under the regulation of the GATA-4 promoter. This model system will facilitate studies of the regulation of GATA-4 in the adult and embryonic mouse. Overall, the proposed studies seek to develop a model for the differentiation of important endocrine tissues by examination of shared regulatory factors that are essential for their development.

DESCRIPTION (provided by applicant): Activation of the gonadotropin-releasing hormone (GnRH) receptor initiates a number of complex signaling cascades in pituitary gonadotropes. The rhythmic stimulation of gonadotropes by pulsatile GnRH underlies the differential control of luteinizing hormone and follicle-stimulating hormone production, and ultimately the control of reproduction. GnRH receptor signaling cascades regulate gonadotropin secretion, gene transcription and cell differentiation. We have shown that GnRH regulates protein synthesis and activates the unfolded protein response (UPR), a protective response to modulate stressful demands of protein synthesis. The UPR also activated by inflammatory and metabolic stress signals. The regulatory schemes utilized by the GnRH receptor are essential to maintain cell homeostasis and sensitivity to recurring GnRH pulses. To maintain pulse sensitivity and hormone synthesis, gonadotropes must resolve the intracellular alterations induced by secretion, increase protein synthesis to continue to meet these demands, and resolve the signaling events activated by the previous pulse to allow full response to the next. We have identified the UPR and control of translation as central elements maintaining gonadotrope sensitivity to GnRH and biosynthetic capacity. We propose to examine these components in vitro to determine their mechanism of regulation and in experimental mouse models to determine their physiological role in reproduction. The UPR may provide a direct link between physiological stress and reproductive function. Specific Aim 1: The Unfolded Protein Response in gonadotrope function. We will determine the role of the three main regulators of the unfolded protein response, EIF2AK3 and ERN1, in normal gonadotrope cell function Specific Aim 2: Mechanisms of mRNA redistribution in response to GnRH We have demonstrated that GnRH causes a redistribution of mRNA in gonadotropes. We will determine the specificity of redistribution and the factors determining susceptibility to redistribution. Specific Aim 3: Physiological consequences of an impaired stress response in mice We will examine the role of the unfolded protein response in normal gonadotrope cell function by examining the reproductive impact of pituitary-specific knockout of critical UPR regulatory factors. PUBLIC HEALTH RELEVANCE: To preserve normal reproductive hormone production and to respond correctly to environmental cues, pituitary cells must minimize cellular stress and adapt to the demand of continued hormone production. We are investigating the mechanisms cells use to adapt to stress and preserve normal cell function. These regulatory pathways are essential to assure proper reproductive hormone synthesis and reproductive success.

DESCRIPTION (provided by applicant): The candidate has a broad training background in molecular biology and in animal model systems for the study of gene expression and endocrinology, including transgenic mice, rats, and hamsters. The candidate has training in all molecular biology techniques and has published manuscripts utilizing them. The training background includes virology, cellular and molecular biology, analysis of DNA-protein and protein- protein interactions, gene transcription, mRNA translation, and signal transduction. In vivo techniques include the creation and analysis of transgenic mice, tissue and organ analysis of mice, rats and hamsters, hormone replacement, dose-response assay paradigms, minor surgical procedures, immunohistochemistry, organotypic culture. Past awards include an independent R03 award and three prestigious postdoctoral fellowships. The environment provides an exceptional atmosphere for career development and further training. The candidate is a member of a U54 center, serving as a project co- investigator, and was recently awarded an R0l. He interacts with both basic science and clinical research faculty in his and other departments. His department has committed laboratory space in a modern, well equipped six year old facility, and he will move to a new expanded facility, that will open in 2003. The candidate's immediate goals are to increase laboratory personnel, to meet the scientific goals set out in the current R0l in support of renewal of that award, to initiate new research projects with the aim of supporting a successful application for a second R0l, and to achieve promotion to a tenured Associate Professor in the approximate time frame of this award. Although the candidate seeks relief from formal teaching and administrative duties normally required for progression to a tenured appointment, the candidate has a strong desire to provide an excellent training environment for graduate and postdoctoral level students. The candidate also seeks relief of salary burden on his current funding to support new laboratory personnel. The candidate proposes to initiate studies on the effects of pulsatile gonadotropin- releasing hormone on the translational and transcriptional regulation of gene expression. The candidate presents the hypothesis that releasing hormone modulates both transcriptional and non-transcriptional changes in gene expression and cell metabolism, and therefore regulates both short term and long term changes in gene transcription and mRNA utilization. Expanded aims are directed at understanding the modulation of pulsatile hormone stimulation by other endocrine effectors, such as insulin, growth factors, and gonadal steroids. Techniques to be applied to this problem include the use of the gonadotrope cell lines and primary pituitary cells for in a perifusion system, allowing the manipulation of the pulsatile regime. Isolation of actively translated and total mRNA from treated and control cells will allow identification and quantification of global changes in gene expression.

DESCRIPTION (provided by applicant): Evidence from studies in humans, animal models, and in vitro cell model systems suggest the tight metabolic control of reproductive fitness. In particular, insulin receptor signaling has been shown to play an important role in fertility in systems ranging from C. elegans to mice. Clinical studies in humans and observations in knockout mouse models suggest a direct link between insulin action and the neuroendocrine control of reproduction. Studies in animal model systems ranging from Syrian hamsters, rats, ewes and some clinical observations indicate that insulin can suppress pituitary reproductive hormone synthesis. Studies in primary pituitary culture indicate that insulin is a positivie regulator of hormone synthesis. Thus the role of insulin as a positive or negative regulator of reproductive function is still controversial. We will utilize an in vitro cell model of the pituitary gonadotropes and mouse primary pituitary culture to investigate the role of insulin signaling in the modulation of gonadotropin output and to address the mechanistic basis for this action. We will identify the point in gonadotropin synthesis and release that is regulated by insulin. We will also identify the mechanism of convergent GnRH and insulin signaling. It is our overall goal to understand how insulin serves as a modulator of gonadotropin synthesis, and ultimately of reproductive function. Aim 1: Insulin regulation of gonadotropin production. Circulating insulin level inversely correlates with gonadotropin production. Using the gonadotrope cell line, LbetaT2, we will determine the point of insulin action by assessing the effects on hormone secretion, protein synthesis, and transcription of gonadotropins. Aim 2: Cross receptor signaling of GnRH and insulin in a gonadotrope cell model. Preliminary observations indicate that insulin attenuates the response to GnRH stimulation. We hypothesize that this is due .to modification of signaling components targeted by the GnRH receptor. We will determine the points of convergent intracellular signaling by GnRH and insulin. We will investigate PIS kinase, MAP kinase, and G protein signalling cascades and negative feedback regulators of receptor signaling to determine which cascades and intermediate proteins mediate insulin effects on GnRH signaling in gonadotropes.

PROJECT SUMMARY Androgens and obesity-related metabolic factors, such as free fatty acids (FFAs), are critical regulators of the reproductive axis. Along with the emerging epidemic of obesity, negative impacts of high adiposity on the female reproductive axis are becoming evident. In addition, women affected with Polycystic Ovary Syndrome (PCOS), the most common reproductive disorder in reproductive-aged women, experience hyperandrogenism and metabolic features including obesity. Evidence indicates that the gonadotropin luteinizing hormone (LH) is suppressed in high body mass individuals relative to lean women, and this is true in both normal women and women with PCOS. Recent work has also revealed, in both normal and PCOS women, a paradox in the association of elevated LH levels with elevated testosterone, despite the well-recognized role of testosterone in suppressing the reproductive neuroendocrine axis. Interestingly, androgen appears to influence both the hypothalamic reproductive circuits, dictating the pace of LH secretion, as well as pituitary gonadotrope responsiveness to incoming GnRH pulses, suggesting a complex regulation of LH secretion patterns. The overall goal of Project II is to investigate the impact of FFA and androgen on the female reproductive neuroendocrine axis, either individually or in combination, in both normal and PCOS-like conditions. In Aim 1, we will elucidate how FFAs alter in vivo LH pulse dynamics as well as the pituitary LH response to GnRH pulses. We hypothesize that FFA induce cell stress in gonadotropes and alter hypothalamic pulsatility and gonadotrope responses to GnRH. We will test the impact of acute and chronic exposure to a panel of FFAs on GnRH pulsatility and pituitary sensitivity to GnRH in vivo and in vitro, using new models of GnRH challenge and perifusion pituitary culture. In Aim 2, we will test how androgens alter LH pulse generation and the in vivo pituitary response to GnRH in females. We will assess the effects of in vivo DHT treatment on both pulsatile LH secretion and pituitary responsiveness to GnRH pulses, and use Cre-lox technology to determine in what specific neuroendocrine cell- types androgens act to alter LH pulses in vivo. In Aim 3, we will examine the combined interaction of FFA and androgens in normal females and in a mouse model of PCOS. We will determine how LH pulsatility and the gonadotrope response to GnRH pulses are altered in the face of both elevated androgens and FFAs. Because PCOS women have elevated androgens and FFAs, but paradoxically also exhibit rapid, high LH pulses, we will use a novel mouse model of PCOS to investigate the interaction of androgens and obesity on LH pulse pattern and secretion in PCOS-like conditions. We will then assess gene expression changes specifically in Kiss1 and gonadotrope cells in a PCOS mouse model and compare these changes with those induced individually by androgen, FFA, or high BMI alone. Together, these Aims will delineate the mechanisms by which androgens and FFAs regulate LH pulsatile secretion and contribute to PCOS, providing novel insight for diagnosis and treatment of female infertility.

PROJECT SUMMARY A diverse educational and scientific community is a vital component for developing a robust work force that can address the technological and social challenges of the U.S. in a new global economy. However, the access of students from disadvantaged social and economic backgrounds to graduate education (Ph.D. level) is still low in comparison with individuals from more privileged socioeconomic groups. This disparity is amplified during academic life, resulting in a lack of faculty diversity at most U.S. universities. To address this problem, the Initiative for Maximizing Student Development (IMSD) program was established in 2008 at the University of California San Diego (UCSD), which focuses on motivating, mentoring, and facilitating the transition of students from disadvantaged economic and social backgrounds and students with disabilities from college to graduate school. The UCSD-IMSD program is composed of two consecutive phases directed at mentoring students from their initial college education to enrolling into a Ph.D. program in biomedical sciences. Phase 1 is directed at introducing freshmen students with no or limited prior research experience to scientific work via fundamental experimental instruction within the Basic Methodology Training Laboratory (BMTL). In this setting, students learn the essential research principles and skills (laboratory safety, basic techniques, data collection, and analysis) that will prepare them to participate in organized research projects. During Phase 2, students participate in hands-on, bench research projects under the mentorship of established, well-funded investigators, where they develop an appetite for science. Students are trained in several aspects of science, including experimental design, execution, data analysis and presentations, increasing their competitiveness as graduate school candidates. In addition, students are motivated to enroll into Ph.D. programs and are assisted during the process.