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| Steroid Receptor Biology |
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Research Projects Projects available: 1. Molecular pathogenesis of granulosa cell tumours of the ovary Supervisor: Professor Peter Fuller Granulosa cell tumours of the ovary represent 10% of ovarian tumours. These tumours arise as their name implies, from the granulosa cells of the ovarian follicle. They both respond to endocrine signals and also synthesise hormones. The group at PHIMR has over the last decade been involved in their clinical characterisation, particularly the development of the use of inhibin as a tumour marker, and also in early studies to define the molecular pathogenesis. In particular, we wish to establish the events, (i.e. mutations) that give rise to these endocrine tumours. The approach involves the use of a panel of tumours obtained from women with the disease and also the use of two established cell lines. Our current focus is on the analysis of the status of signalling pathways known to be important in the regulation of growth and differentiation in granulosa cells in both the tumours and cell lines. The project would involve the use of molecular techniques for mutagenesis detection to seek to identify abnormalities in signalling pathways, particularly known oncogenes in these tumours. Where abnormalities are found, their significance would then be analysed in the cell lines. The cell lines and the tumours also express the novel oestrogen receptor, ER b . Little is known, at least in granulosa cells, of the genes regulated by this molecule and the cell lines place us in a unique position to determine the genes that are regulated by ER b . This will be identified using the microarray facility recently established in the institute. The analysis and characterisation of these genes is likely to be of significance in reproductive function.
2. Understanding the molecular mechanisms of action of aldosterone and the mineralocorticoid receptor Supervisor: Professor Peter Fuller The adrenal steroid aldosterone is a key regulator of blood pressure. Excessive aldosterone levels cause hypertension and contribute to the pathogenesis of cardiac disease. Our laboratory has a program of work focused on various aspects of aldosterone action and the diseases that may arise from this action. This project examines the receptor protein for aldosterone, the mineralocorticoid receptor (MR), which acts as a transcription factor to regulate gene expression. We are offering the following Honours projects: 2a. Regulation of target genes by the MR: This project would involve the analysis of the regulatory regions of an aldosterone-induced gene (channel-inducing factor) in the colon using molecular biology, cell culture systems, transfection and reporter assays. 2b. Interdomain interactions: The MR consists of a number of functional domains that interact with one another. The student will examine the effect of this "cross-talk" on receptor function in vitro and in cultured mammalian cells, using techniques to measure ligand binding, receptor activity and the ability of the receptor to interact with other proteins involved in transcription. 2c. Interacting proteins: Using a yeast screening method, we have identified a number of novel proteins that interact with the MR, and that may be important for the ability of the receptor to regulate gene expression. The student will examine the ability of these proteins to interact with and regulate MR function in vitro and in cultured mammalian cells, using techniques to measure MR ligand and DNA binding, and receptor activity.
3. Cellular localization of mineralocorticoid receptor-mediated vascular inflammation and cardiac fibrosis. Supervisors: Dr. Morag Young and Professor Peter Fuller We have used the Cre-Lox technique to delete MR expression in a tissue-specific manner in the cardiovascular system to identify the cells types critical for the development of vascular inflammation and cardiac fibrosis. Identification of the critical cell types will allow a focused investigation of the cellular mechanisms involved in the establishment and progression of this pathology. We have shown that MR signalling in the context of high salt leads to inflammation, fibrosis and ultimately heart failure. This CV remodelling is a direct effect of MR activation in the CV system, independent of blood pressure, hypertrophy and hypokalemia. More recently we have shown that this pathology is largely reversible following MR blockade in established disease. There are 2 specific projects in this section: 1. Identify the critical cell types responsible for the generation of mineralocorticoid (MC)/salt mediated vascular inflammation, cardiac fibrosis and renal fibrosis using transgenic mice in which the mineralocorticoid receptor (MR) has been inactivated (in vivo). 2. Determine, in those cell types, shown to be critical in the development of cardiac fibrosis, the specific response to inactivation of the MR-protective enzyme 11 b HSD2, under circumstances of normal plasma aldosterone (in vitro). Outcomes and significance: We hope to identify the cells/tissues in the heart and kidney that are the site of activation of the MR. This is an essential precursor to gaining a molecular and cellular understanding of the “pathological consequences” of MR activation and strategies to ameliorate this. Although the available MR blockers are effective their side-effect profiles severely limit their use. Cardiac specific blockade will avoid the major limitations, hyperkalemia, which is a consequence of concomitant renal MR blockade. Tissue-specificity is now well established for other steroid receptors, eg. the oestrogen receptor in breast cancer. A knowledge therefore of the target cells will enable identification of the properties needed to create cardiac-specific MR antagonists. 4. The role of the MR in macrophage function. Supervisor : Dr. Morag Young We have shown for the first time that tissue selective knockout of the MR in macrophages changes the normal physiological profile of macrophage specific markers. Given that the MR is not protected in the cells by the specificity-conferring enzyme 11 b HSD2 this receptor is probably acting as a receptor for glucocorticoids under normal circumstances. We wish to fully characterise the response of these macrophages, compared to wild type (normal) macrophages, under normal and stimulated (LPS, oxidative stress etc) conditions using primary cell culture and FACs analysis. We will further examine the differences in macrophage response to various steroid ligands that can bind the receptor. Primary cell culture, cell lines, immunohistochemistry and RT PCR are some techniques that will be involved in this work. This work may involve use of human monocytes isolated from blood.
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