Ronald J. Koenig - US grants

The long term goal of this project is to elucidate the mechanism of action of thyroid hormone (T3). A beta-type c-erbA cDNA cone (erb62) has been isolated from a GH3 cell library. The erb62 protein binds T3 with high affinity, and the T3-erb62 complex stimulates gene expression directed by eh rat growth hormone (rGH) promoter. The isolation of other c-erbA cDNA clones suggests these all may encode T3 receptors with distinct functions. The objective of this proposal is to use the erb62 cDNA to rest the hypothesis that the organ specificity and developmental specificity of many T3 actions may be due to: a) differential expression of functionally distinct classes of T3 receptor molecules, or b) the requirement of other, tissue specific, factors in addition to the T3 receptor to generate a T3 response. Transient transfection studies will be used to study whether the T3-erb62 complex can stimulate rGH promoter directed gene activity in a broad range of cell lines, or whether host factors limit the response. The ability of other endogenously expressed erbA molecules to support T3 inducible rGH promoter activity also will be assessed. Stable transfectants will be used to assess other T3-erb62 effects, e.g. induction of enzyme activities and mRNA levels. The results will help define the functional role of erb62 as a T3 receptor, and assess the importance of other factors in determining the tissue specific nature of many T3 effects. This research is relevant to the diagnosis and treatment of both hypothyroidism and hyperthyroidism. It is relevant to understanding the role of thyroid hormones in brain development.

The overall goal is to increase our understanding of how thyroid hormone (T3) regulates gene expression. T3 binds to receptors (TRs), which bind to T3 response elements (TREs) in specific target genes. TREs generally consist of two (or more) binding sites (half sites) arranged as a direct repeat, inverted repeat, or everted repeat. TRs can bind to TREs as homodimers or as heterodimers with retinoid X receptors (RXRs); the relative biological importance of each of these dimer forms is uncertain. TRs regulate transcription via two domains, AF-1 and AF-2. The function of AF-1 is poorly understood. AF-2 functions by interacting with other proteins, generally known as coactivators and corepressors. T3 alters the conformation of the TR, thereby affecting which proteins interact with this receptor. Our data suggest that certain genes are regulated by TR homodimers and others by RXR-TR heterodimers, and that this is determined by the sequence of the TRE. In addition, our data suggest that TR homodimers and RXR-TR heterodimers have different coactivator requirements, and that half site orientation further influences coactivator requirements. These issues will be studied in yeast and in mammalian cells. Yeast are uniquely valuable because they lack the above proteins. Hence, TR, RXR, and various coactivators can be added back in defined ways to determine their effects on gene expression. Additionally, yeast are amenable to genetic manipulations that are essentially impossible in mammalian cells. However, confirmation of the findings in yeast must be made in mammalian cells, to demonstrate biological relevance. Three specific aims will be addressed: 1) Assess the mechanism of coactivator-independent (AF-1) TR function in yeast; 2) Assess the role of TRE structure and homodimers versus heterodimers in defining coactivator requirements in yeast; 3) Determine whether the key findings in the above aims apply to mammalian cells. The results should further our understanding of how T3 affects a broad range of metabolic processes in health and disease states such as hyperthyroidism and hypothyroidism.

[unreadable] DESCRIPTION (provided by applicant): The goal of the University of Michigan Medical Scientist Training Program is to train physician scientists primarily for careers in academic medicine with a focus on basic biomedical research. The U of Michigan Medical Center is one of the world's largest one-site complexes devoted to health education, research and patient care. There are over 1.8 million sq ft of space dedicated to education and laboratory research. In addition, a new research building is just starting to be occupied, and another new research building will begin occupancy in 2005. The Michigan MSTP provides an integrated curriculum of M.D./Ph.D. training. Matriculants have a strong history of academic success and research experience, and are graduates of outstanding colleges from all parts of the U.S. There are currently 84 trainees and 97 graduates. The curriculum begins with the two basic science years of medical school. A graduate level biochemistry course is taken as part of the 1st year of medical school. Trainees undertake a research rotation after the 1st year of medical school and one or two rotations after the 2nd year. Trainees select a Ph.D. field during the 2nd year of medical school; core participating departments include bioinformatics, biological chemistry, biophysics, cell & developmental biology, cellular & molecular biology, chemical biology, human genetics, immunology, medicinal chemistry, microbiology & immunology, molecular & integrative physiology, neuroscience, pathology, and pharmacology. Other fields are possible, e.g., biomedical engineering. Trainees take a leave of absence from medical school typically after the 2nd year to continue graduate studies full time. Upon successful completion of the thesis defense, trainees complete their clinical training. The 3rd year of medical school can be entered any month within the first half of the academic year to accommodate variations in thesis defense dates, and the 4th year of medical school is truncated to 6 months. Total training is typically 7 to 8 years. There are monthly program activities including seminars, social events and an annual scientific retreat. [unreadable] [unreadable]

In anticipation of the revolutionary impact that the techniques of molecular biology would have on diabetes research, the Michigan Diabetes Research and Training Center (MDRTC) proposed a Molecular Biology Core (MBC) to introduce these techniques to its investigators as part of its 1986 Competing Renewal Application. Because the relevant research base was just being established, a joint core with the Center for Reproductive and Developmental Biology was proposed. Weakness of the research base, and administrative concerns about joint supervision led to disapproval of the core, with a strong admonition for a subsequent reformulated supplemental application. Such an application was submitted and approved for funding by the NIDDKD Council in early 1988, but the institution of a funding "cap" for the Diabetes Center led to administrative withdrawal of the proposal. A scaled-down version of the Core was subsequently equipped and initiated with institutional funds, facilitating the entry of several Center investigators into the molecular realm. Dramatic infusion of molecular techniques into the Diabetes Center research base over the last five years, plus the recent recruitment of Jack Dixon, PhD, an outstanding diabetes-related molecular biologist experienced in directing a Diabetes Center Molecular Biology Core, full justify the initiation of a full-scale Molecular Biology Core to enhance diabetes and endocrine- related molecular research at the Center by providing cost-effective methods for enhancing research activities among investigators, by introducing and exploiting modern molecular techniques, by fostering collaboration and providing state-of-the-art methods and training to investigators in the area of molecular biology, by providing the foundation for analysis of molecular structures and function as well as generating the tools necessary to study gene regulation, and by providing disease markers to further the understanding and treatment of diabetes, its complications and related endocrine and metabolic disorders.

Rubinstein-Taybi syndrome is a genetic syndrome characterized by facial abnormalities, broad thumbs, broad toes, mental retardation and other abnormalities. It is due to mutations in the gene encoding CREB Binding Protein (CBP), although the link between the mutations and the clinical phenotype is not understood. CBP is a protein that is thought to play a critical role in thyroid hormone action--it is a co-activator protein that forms a bridge between the thyroid hormone receptor and the transcription machinery, thus transmitting the signal from thyroid hormone (T3) to allow regulation of gene expression. The CBP mutations that encompass Rubinstein-Taybi syndrome are diverse, and hence there is genetic heterogeneity in this condition. The hypothesis to be tested is that at least some of these CBP mutations disrupt the ability of CBP to function as a T3 receptor co-activator, resulting in a state of thyroid hormone resistance.

The overall goal of this proposal is to establish an NIDDK Biotechnology Center at the University of Michigan. The Center will provide access to state-of-the-art technology, equipment and advice for measuring broad patterns of gene expression. The Center will be accessible to investigators funded by the NIDDK or doing research clearly related to the mission of the NIDDK. This application will focus on DNA microarray technology, an approach that fits well with the existing strengths, resources and commitments of the University of Michigan. Resources connected with this application consist of physical space containing appropriate equipment for storing and replicating ESTs, equipment for producing and hybridizing microarray chips, and tools for data analysis. Personnel resources include trained staff for performing the above steps, including preparation of labeled probes and experts to provide guidance and advice to investigators in terms of RNA preparation and data analysis. In addition to such individualized instruction, the NIDDK Biotechnology Center will provide education to the broader community through a series of seminars. The Center will be governed by an Internal Advisory Committee that includes representatives from three existing NIDDK-funded Centers (Diabetes Research and Training Center, Renal Center, and Gut/'Peptide Center) whose members have needs for access to technologies for expression analysis. These three Centers will provide supplemental financial support for the NIDDK Biotechnology Center, as will the Medical School. The Internal Advisory Committee will oversee all aspects of the Center and will review applications submitted by individual investigators for access to microarray resources. Two external advisors with national reputations at the forefront of microarray technology will serve as consultants. This proposal will benefit from outstanding University support - the University has committed close to $2 million to acquire and distribute all sets of human, rat and mouse ESTs (sequence verified) available which will be replicated and distributed at a central facility to a number of targeted research programs including the NIDDK Biotechnology Center. These research programs share knowledge and experience in microarray technologies and develop innovative applications including novel oligonucleotide based arraying and means to integrate microarray analysis with proteomic analysis. Thus a fertile environment will ensure that advances made that are relevant to expression analysis will be promulgated efficiently by the NIDDK Biotechnology Center.

DESCRIPTION (provided by applicant): The long term goal is to understand how retinoic acid (RA; the major biologically active metabolite of vitamin A) and bone morphogenetic protein 4 (BMP4) interact to regulate development. In general terms, RA regulates gene transcription by binding to its nuclear hormone receptors, the RARs; and BMP4 is a secreted protein that binds to cell surface receptors to initiate a signaling cascade that involves SMAD activation. Although the major BMP4 promoter in bone (1A promoter) is induced by RA, we have found that a previously described minor promoter (1B) and a novel promoter in intron 2 (i2) are repressed by PA. The i2 promoter is highly expressed in developing inner ear, whereas the 1A promoter is not. BMP4 and PA are both known to be essential for normal inner ear development. Antagonism of BMP4 inhibits development of the semicircular canals. Embryonic exposure to exogenous RA results in a similar phenotype, suggesting that PA may repress BMP4 in the inner ear, helping to restrict BMP4 expression appropriately in space and time. Indeed, repression of the novel i2 promoter by RA occurs both in an inner ear derived cell line and in vivo in mouse embryonic inner ear. Furthermore, the ability of RA to inhibit semicircular canal development is overcome by recombinant BMP4 (in chicks). This proposal seeks to address three Specific Aims: 1) Determine the mechanism by which PA down-regulates BMP4 transcription; 2) Characterize expression from the BMP4 1B and i2 promoters in space and time in developing mouse embryos; and 3) Determine the in vivo effects (in mice) of mutations that destroy the BMP4 1B and i2 promoters. The first aim is relevant to RA action in general in that the mechanism(s) of repression of target genes by PA is not well understood (in contrast to gene induction), and is of interest because PA up-regulates BMP4 in bone via a different promoter, the 1A promoter. The 2nd and 3rd Aims will establish the relative roles of the IB and i2 promoters in vivo in inner ear development, and will examine whether these promoters may play important roles outside the inner ear. Accomplishing these goals will rely on classical molecular tools to study gene regulation in cell culture and in cell free systems, as well as transgenic and gene-targeted mice to investigate the in vivo functions of the BMP4 1B and i2 promoters. The studies are relevant to understanding 1) how states of vitamin A excess or deficiency affect development; 2) molecular events that lead to normal or abnormal inner ear development; and 3) the molecular pathway of gene repression by PA.

DESCRIPTION (provided by applicant): The nonthyroidal illness syndrome (NTIS), also called the sick euthyroid syndrome, is the state of a low serum thyroid hormone (T3) concentration associated with any acute or chronic illness, without intrinsic disease of the hypothalamic-pituitary-thyroid axis. The severity of the NTIS correlates directly with the severity of illness, and multiple studies show that the severity of the NTIS is an independent and powerful predictor of mortality. The long term objectives of these studies are to understand the mechanisms underlying the NTIS and to address whether it should ever be treated, and if so, what the treatment should be. The first Specific Aim will use cell culture and in vivo mouse models to address the mechanisms underlying the decreased conversion of thyroxine to T3 that characterizes the NTIS. The activity and expression of type 1 iodothyronine deiodinase (D1) are known to be decreased by illness. This effect is due at least in part to a defective ability of thyroid hormone receptors to induce transcription of the D1 gene, Dio1. The relationship between defective D1 expression, the low serum T3, and thyroid hormone receptor coactivator function will be investigated. Specific Aim 2 will use two in vivo mouse models of NTIS, endotoxin administration and sepsis, to test whether a specific therapy (forced expression of a thyroid hormone receptor coactivator) can ameliorate the NTIS and decrease mortality rate. Specific Aim 3 will evaluate the basis for impaired thyroid hormone receptor coactivator function in the NTIS. Cytokine or illness-associated potential mechanisms to be explored include induction of corepressors that compete with the coactivators, redistribution of coactivators to other genes, redistribution of coactivators to other regions of the cell, and abnormalities in post-translational modifications of coactivators. The severity of the nonthyroidal illness syndrome (NTIS) correlates directly with the severity of illness, and multiple studies show that the severity of the NTIS is an independent and powerful predictor of mortality. These studies will address whether treatment of the NTIS improves recovery from serious medical illnesses.

DESCRIPTION (provided by applicant): The long term objective is to reveal the mechanisms underlying the transcriptional activity, metabolic effects and tumorigenicity of a unique nuclear receptor transcription factor, PAX8-PPARg Fusion Protein (PPFP) that is produced as a consequence of a chromosomal translocation in follicular thyroid carcinomas. PPFP contains nearly the full sequence of the transcription factor paired box 8 (PAX8) plus the entirety of the nuclear receptor peroxisome proliferator-activated receptor g1 (PPARg1). PPFP can activate the promoters of PPAR-responsive genes, although the activity of PPFP is clearly distinct from that of PPARg. Gene expression profiling of PPFP thyroid cancers (compared to all other benign and malignant thyroid neoplasms) resulted in the identification of a PPFP cancer gene signature. The metabolic pathway most enriched in PPFP signature genes is fatty acid 2 oxidation, which is physiologically regulated by PPARs. Stably transfected clones of the non-transformed thyroid cell line PCCL3 that express PPFP in a doxycycline-dependent manner (DoxyPPFP cells) will be used to understand the importance of fatty acid metabolism in the biology of PPFP cancers, the mechanism of oncogenesis, and potential approaches to therapy. In these cells, PPFP induces many genes that are induced in PPFP thyroid cancers, and these cells form xenograft tumors and lung metastases in NOD-SCID mice. In the first Specific Aim, chromatin immunoprecipitation - deep sequencing (ChIP-Seq) will be used to identify PPFP binding sites on a genome wide level. Additional studies will evaluate whether binding to specific DNA sites requires PPFP's PAX8 DNA binding domain, its PPARg DNA binding domain, or both. Affymetrix microarray gene expression profiling will be performed on DoxyPPFP cells to allow a comparison of gene expression with genome wide DNA binding. These data also will be compared with gene expression data already obtained from PPFP cancers. Coregulatory protein recruitment by PPFP to its target genes will be evaluated. The hypothesis is that the PAX8 portion of PPFP recruits inappropriate coregulators to PPAR responsive genes, and vice versa. These studies will rely primarily on chromatin immunoprecipitation - real time PCR. Specific Aim 2 will test the hypothesis that PPFP-expressing cells are highly dependent on fatty acid 2 oxidation for cell growth; i.e., that PPFP induction of 2 oxidation creates a growth advantage. A Seahorse XF24 bioanalyzer will be used to evaluate how much energy metabolism is via glycolysis versus the TCA cycle. The use of glucose versus fatty acids as energy sources will be evaluated. The effects of PPFP on de novo fatty acid synthesis, cell proliferation and apoptosis will be assessed, to better understand the putative growth advantage brought about by PPFP. In Specific Aim 3, the effects of PPARg ligands (thiazolidinediones) on xenograft tumor and metastasis formation will be evaluated in NOD-SCID mice. The importance of PPFP functional domains on xenograft tumor formation and metastases will be assessed with DoxyPPFP cell lines expressing mutant PPFPs (for example, mutation of either the PAX8 or PPARg DNA binding domain). PUBLIC HEALTH RELEVANCE: This project is relevant to the public health because it will reveal how PAX8-PPARg Fusion Protein (PPFP) causes thyroid cancer and will suggest new approaches to therapy. In addition, many patients with diabetes take a class of drugs called thiazolidinediones that activates PPARg, but these drugs also activate PPFP and the consequences of this are not known. This study will help determine whether this diabetes therapy is beneficial or harmful to patients who also have PPFP thyroid cancers.

DESCRIPTION (provided by applicant): Approximately 35% of follicular thyroid carcinomas harbor a chromosomal translocation that fuses paired box gene 8 (PAX8) with the peroxisome proliferator-activated receptor gamma gene (PPARG), resulting in production of a PAX8-PPARG fusion protein denoted PPFP. PPFP contains the full sequence of the nuclear receptor PPARG1, and hence PPFP binds to PPARGresponsive genes and to PPARG ligands. We have created the first transgenic mouse model of this cancer. The cancer is locally invasive and forms lung metastases. Treatment with the PPARG agonist pioglitazone (Pio) shrinks the thyroid almost to control size and eliminates metastatic disease. Most remarkably, this therapeutic response is characterized by a trans differentiation-type process whereby the remaining thyroid cells develop large lipid droplets and express a wide array of PPARG- inducible adipocyte genes. Since PPARG is the master regulator of adipogenesis, these results indicate that, in the presence of Pio, PPFP is strongly PPARG-like. We postulate that the anti-tumor action of Pio in PPFP cancers is tied to this adipocyte trans differentiation-like effect; i.e., the more the thyroi cancer cells acquire a mature adipocyte phenotype, the less they retain of their malignant phenotype. In Aim 1, we will evaluate the oncogenic action of PPFP in the mouse model. There is evidence that, in the absence of Pio, PPFP can inhibit PPARG induction of some target genes, and that inhibition of endogenous PPARG may underlie the oncogenic nature of PPFP. Therefore, we will test whether the genetic deletion of PPARG mimics the expression of PPFP in terms of the development of thyroid cancer. We also will assess whether both the PAX8 and PPARG DNA binding domains within PPFP are important by studying mice in which PPFP has appropriate mutations. Analyses of histology, gene expression and DNA binding will provide insight into the genes regulated by PPFP that contribute to the development and progression of thyroid cancer. We will use a non-adipogenic PPARg ligand to test whether the adipogenic nature of the Pio response is critical to its therapeutic effect. We also will test whether arsenic trioxide is therapeutic, especially in combination with Pio. This hypothesis derives from the observation that PPFP + Pio strongly induces AQP7, a channel protein through which arsenic enters cells. In Aim 2, we will perform a phase II clinical trial to determine whether Pio is therapeutic in patients with metastatic PPFP thyroid cancer not treatable by standard therapies. The primary endpoint will be a decrease in the size of metastases. A secondary endpoint will be measurement of lipid content of metastases that do not completely resolve, based upon the observation that treatment of our mouse model of this cancer with Pio results in an adipogenic response in the surviving thyroid cells. Other secondary endpoints include changes in serum thyroglobulin and testing the ability of Pio to induce radioiodine uptake in the cancer, followed b radioiodine therapy if indicated. Overall, these studies will help elucidate mechanisms through which PPFP contributes to thyroid cancer and will help identify novel therapies both in the transgenic mouse model and in patients.

Regional Pilot and Feasibility Study Grants Program The specific aim of the Michigan Diabetes Research Center (MDRC) expanded Pilot/Feasibility Study (P/FS) Grants Program is to stimulate new research and collaboration in the areas of diabetes, its complications and related endocrine and metabolic disorders in the region. This research may be in areas of basic biomedical science or clinical research. This program will fund at least 2 P/FS grants submitted by investigators from our three regional partners per year. A minimum of $80,000 per year will be provided by the MDRC and an additional $20,000 per year will be provided through cost-sharing agreements with the University of Toledo, Michigan State University, and Wayne State University. Each year, the MDRC will solicit applications for grants from full-time instructional or research faculty at these three institutions. Those eligible include: 1) new investigators without current or past NIH research support who are beginning careers in diabetes research, 2) established investigators new to diabetes research who wish to focus their expertise on diabetes, and 3) established investigators who propose innovative research in diabetes that represents a clear departure from their ongoing research. Applications are peer-reviewed by at least two extramural investigators with expertise in the area of the application. Those with merit, as judged by this review process and the Grants Program Advisory Council which includes representatives from the three regional partner universities, will receive P/FS awards. The ultimate goal of the grant program is to enhance communication and collaboration among regional diabetes researchers, accelerate the pace of research, and enable awardees to generate sufficient preliminary data for successful applications for major research funding from the NIH or other national granting agencies.