Author

Anna Malyala

Date

May 2007

Document Type

Dissertation

Degree Name

Ph.D.

Department

Dept. of Physiology and Pharmacology

Institution

Oregon Health & Science University

Abstract

The goal of this dissertation is to define the underlying molecular mechanisms of estrogen-mediated effects that influence homeostasis. The rapid and enduring effects of estrogen in the central nervous system are complex and overlap with multiple signaling pathways such that defining its mechanism of action has been difficult. By elucidating hypothalamic genes and signaling pathways regulated by estrogen, we will develop a better understanding of how multiple physiological processes are affected and interconnected. Furthermore, revealing estrogen's molecular effects will enable development of specific and targeted therapies for disorders associated with fertility and homeostasis. The background for this dissertation is presented in chapter 1. It includes the fundamental role of estrogen in reproduction and its link to energy balance; illustration of the hypothalamus as a key mediator of estrogen action; approaches taken to understand the multifaceted signaling of estrogen; review of phosphatidylinositol 3-kinase signaling; and finally, the putative roles of other regulated estrogen-regulated genes such as gec1 in hypothalamic signaling. Chapter 2 explains the high throughput approach to identify estrogen-regulated genes in the hypothalamus. We describe the method used to create a guinea pig brainspecific eDNA library that was subsequently used for printing eDNA microarray chips. Microarray analysis of estrogen- and vehicle-treated hypothalamic tissue reveals that many regulated genes are involved in signal transduction and neurotransmission. Futhermore, upon examination of the promoter and enhancer sequences of several regulated genes, we find that estrogen affects transcription directly via estrogen response elements (ERE) but also via non-classical mechanisms independent of EREs. Further analysis of the microarray data show that PI3K p55y and many transcripts encoding pleckstrin homology domain-containing proteins are differentially regulated, suggesting that PI3K signaling is critical for some estrogen-mediated effects. In chapter 3, we present findings on the expression of PI3K regulatory subunits in the central nervous system. We propose a model in which estrogen alters the sensitivity of molecules that signal through the PI3K pathway by changing the regulatory subunit levels. Furthermore, electrophysiology data reveal that PI3K signaling may mediate some of the rapid estrogen effects at the membrane. These data suggest estrogen acts at multiple levels of the PI3K pathway to mediate rapid and sustained effects. Chapter 4 highlights other genes regulated by estrogen in the hypothalamus. Gec1, a known estrogen-regulated gene in the periphery, is highly homologous to the GABAA receptor associated protein (GABARAP) and we find that gec1 expression increases with estrogen in the arcuate nucleus. GABARAP facilitates GABAA receptor trafficking and insertion into the plasma membrane, and evidence suggests that gec1 may also function similarly. Interestingly, PI3K signaling facilitates the insertion of GABAA receptors to the neuronal membrane and implies that estrogen could act in multiple ways to affect rapid signaling and gene transcriptional events. Finally in Chapter 5, a general discussion on these research findings is presented. Future experiments building upon the research presented here are listed and thus will provide more insight on the neuronal effects of estrogen to modulate homeostasis.

Identifier

doi:10.6083/M46M34TF

School

School of Medicine

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