Overview
Regulation of gene transcription and mRNA stability in vertebrates; estrogen receptor action; tamoxifen-induced apoptosis of cancer cells; RNA interference (RNAi) and RNA binding proteins in the control of mRNA degradation.
Research Summary
The research of our laboratory involves molecular and biochemical studies of a spectrum of regulatory processes. Several models that we employ involve the ability of the steroid hormone estrogen, and estrogen antagonists, bound to its regulatory protein, the estrogen receptor (ER), to regulate gene transcription and messenger RNA stability. Our identification of proteinase inhibitor 9 (PI-9) as a hormone-regulated gene has led us to undertake diverse studies of the role of human PI-9 in mediating inflammatory processes, in fetal implantation and early development, and in the ability of cells of the immune system to destroy cancer cells. Our interest in the mechanisms by which hormones regulate mRNA stability led to our identification of the novel mRNA binding protein, vigilin. Current studies are focused on identifying the diverse biological roles that make vigilin essentially for viability in human cells and on its role in mRNA metabolism. RNA interference and other pathways for mRNA breakdown are currently the focus of intense interest. We are working to develop new applications of RNAi that will both exploit and enhance our understanding of the mechanisms of RNA interference.
Introduction to Estrogen Action
The effects of steroid hormones such as estrogens and testosterone, thyroid hormone and many other hormones are mediated through specific receptor proteins called steroid/nuclear receptors. We are analyzing the molecular mechanisms by which estrogen receptor (ER) carries out its diverse biological activities. Estrogens, acting via the estrogen receptor, regulate the growth and differentiation of cells of the female and male reproductive systems, the growth and metatastic potential of breast cancer cells and have important effects on brain development, autoimmune diseases and inflammatory processes, and bone metabolism. We are studying the actions of estrogen receptor in normal cells and in cancer cells at all three of its major sites of action; the nucleus, the cytoplasm and the cell membrane.
Estrogen Receptor Regulation of Gene Transcription
In our studies of estrogen receptor action in the nucleus, we are analyzing the role played by the DNA sequence to which the estrogen receptor binds in the ability of genes to recruit estrogen receptor bound to different ligands, and in the formation of multi-protein complexes containing activator and repressor proteins that regulate transcription. We are using powerful, recently developed, chromatin immunoprecipitation (ChIP) assays to identify the proteins in transcription complexes associated with a specific, individual, estrogen-regulated gene in living cells. One important outcome of these studies from our own and other laboratories is the demonstration that the sequence of the DNA binding site for estrogen receptor acts as a kind of allosteric effector influences the receptor's shape and works to together with different estrogenic and antiestrogenic ligands to control estrogen receptor interaction with different genes.
Tamoxifen-induced Apoptosis
Estrogens cause breast and uterine cancer. Tamoxifen is widely used to treat breast cancer. We showed that tamoxifen bound to estrogen receptor does not just prevent estrogen from binding to the receptor, but actively induces programmed cell death (apoptosis) of cancer cells. Our identification of distinct pathways by which high and low concentrations of tamoxifen induce programmed cell death led us to focus on the pathways by which low concentrations of tamoxifen bound to the estrogen receptor induce apoptosis. These studies derived in part from a new dimension in studies of steroid hormone action based on recently described non-genomic actions of estrogens mediated by activation of plasma membrane-based signal transduction pathways. Our recent studies demonstrate that tamoxifen-ER induced cell death involves both the action of signal transduction pathways and tamoxifen-ER mediated transcription. These observations also provide an important new insight into how estrogens bound to ER activate gene transcription. Microarray techniques and other methods are being used to identify target proteins involved in tamoxifen-ER mediated apoptosis.
Regulation of Inflammation and Immune Surveillance by Control of Proteinase Inhibitor 9 (PI-9)
We reported in 2000 that proteinase inhibitor 9 (PI-9) is powerfully and directly induced by estrogen in human liver cells and in human liver. PI-9 was subsequently shown by others to be the first human inhibitor of caspase 1. Caspase 1 regulates the production of pro-inflammatory proteins called cytokines. These cytokines attract and stimulate the proliferation and differentiation of cells of the immune system, which attack malignant or virally infected cells (and sometimes cause autoimmune-based diseases, such atheroschlerosis, osteoporosis and multiple sclerosis). PI-9 is also a potent inhibitor of the protease granzyme B. Granzyme B is present at high levels in cells of the immune system (T cells and natural killer [NK] cells). These cells use granules containing granzyme B to induce apoptosis of target cancer cells, or virally infected cells. PI-9 inihibits granzyme B and granzyme B-induced apoptosis. Underproduction of PI-9 is implicated in athreosclerosis while overproduction of PI-9 in many tumors is suggested to protect the cancers from immune system-mediated killing. Our regulatory studies suggest a novel feedback loop by which pro-inflammatory cytokines induce PI-9, and the induced PI-9 inhibits caspase 1, blocking further production of the pro-inflammatory cytokines. The induction of PI-9 by estrogens and by glucocorticoids provides a potential biochemical mechanism to explain the ability of these steroid hormones to protect against inflammatory diseases. Current work in the PI-9 system involves work in our laboratory and collaborative studies on regulation of PI-9 gene expression, biochemical and structural work on its mechanism of action as a protease inhibitor, identification of new intracellular proteases that are targets of PI-9 inhibition and functional studies to address the role of PI-9 and its regulation in inflammation, immune surveillance, and fetal implantation and early development.
Estrogen Regulation of mRNA Degradation
To investigate hormone control of mRNA stability, we developed a model system based on our finding that estrogen induces a 30 fold increase in the cytoplasmic stability of vitellogenin mRNA. We identified vigilin, as an estrogen-inducible protein, which binds to a segment of the mRNA important in estrogen?mediated mRNA stabilization. Remarkably, vigilin contains 15 related, but non-identical, RNA binding domains. In vitro binding of vigilin to the vitellogenin mRNA 3'-untranslatd region protects this segment of the mRNA from specific cleavage by the sequence-selective mRNA degrading enzyme, polysomal mRNAse 1 (PMR-1). To analyze mRNA degradation in living cells, we recently developed a new method for determining rates of mRNA degradation. Current research centers on the pathway for regulated vitellogenin mRNA degradation in living cells and on new models for estrogen regulation of mRNA degradation.
RNAi Applications and Vigilin in RNA Metabolism
To degrade invading RNA viruses and other double-stranded RNAs, multicelluar organisms from plants to humans, use an ancient defense mechanism called RNA interference. Recent studies enable researchers to use this degradation system to selectively destroy any desired mRNA in a vertebrate cell. RNAi allows researchers to selectively eliminate any protein of interest from a vertebrate cell. RNAi and the tiny RNAs that mediate this and related processes have generated enormous interest and were recently named "Breakthrough of the Year" by Science (Dec. 20th 2002 issue). Our interest in mRNA degradation led to both widespread use of RNAi in our research and to efforts to both expand its applications and in so doing further delineate its mechanisms. A major problem of RNAi was that the effect wears off after a few days and the mRNA is no longer degaded. We developed a simple technique for long-term RNAi. This work and recent studies in other laboratories demonstrate that short interfering siRNAs are long-lived and act catalytically. Current work focuses on the development of other new applications of RNAi.
We identified vigilin as important in the regulation of mRNA stability. With its 15 RNA binding domains vigilin is both ubiquitous in eukaryotic cells and highly conserved. This naturally focused attention on vigilin's functions. Disruption of the vigilin gene in yeast in not lethal. Researchers have proposed several functions for vigilin including, regulation of mRNA degradation, control of chromosome portioning at mitosis and regulation of translation. We used RNAi to analyze the functions of vigilin in human cells. Knockdown of vigilin using vigilin-specific siRNAs is rapidly lethal, even in non-dividing, non-mitotic, human cells. Our RNAi studies support the view that vigilin's essential functions relate to its role as an RNA binding protein, functioning in processes that involve regulation of RNA metabolism.
Last Updated: 08/12/03