Li-Na Wei - US grants

Vitamin A, the collective name for a group of related retinoids, is essential for normal development and growth in animals. Too little of too much results in a wide variety of biological defects, and in addition, increases toxicity caused by alcohol and various environmental toxins. The biologically active retinoid is retinoic acid (RA), and its concentration within cells is critical to health. Previous studies have indicated that a cellular retinoic acid-binding protein (CRABP) plays a major role in maintaining intracellular RA at a constant concentration. However, because CRABP is so vital in this regard, mutation and other manipulation that greatly reduce or eliminate this protein are generally lethal to the organism, and hence cannot be used to study CRABP function. The major goal of this proposal is to understand the positive and negative regulatory elements of CRABP gene and to create specific changes in this gene so its expression is altered but not eliminated in animals. Specifically, we will 1. determine the region of the gene essential for its regulation, by systematically deleting portions of the 5' region of the gene fused to a LacZ reporter, and 2. create transgenic mice in which CRABP is expressed in tissues normally not expressing this protein, as well as animals in which normal CRABP expression is blocked. Taken together, these studies should illustrate the interaction of RA and CRABP and the mechanisms that regulate intracellular RA concentration, and ultimately lead to better preventive/therapeutic application of vitamin A in humans.

DESCRIPTION: (Applicant's Abstract) It has been well documented that the mu, opioid, and kappa opioid receptors are differentially localized in distinct areas of the nervous system, and they can be detected beginning in early developmental stage, as early as mid-gestation stages in prenatal development. It is also known that the opioid receptors respond to environmental, physiological or pharmacological stimuli, which is manifested by alteration in neuronal and behavioral responses. Since the recent successful cloning of all the major opioid receptor cDNAs and the genes encoding the three receptors, it becomes clear that these genes evolved are from a common ancestral gene, but unique regulatory mechanisms are adopted for these gene expression as they evolve. Among the three genes, the mouse KOR gene is most interesting in its 5' untranslated region where two alternative promoters are used and differential splicing occurs in intron 1. Two questions are asked: 1. What are the biological functions of the duo-promoters of mouse KOR gene, and 2. How are they regulated? This proposal will focus on specific regulatory mechanisms underlying specific KOR gene expression in three aims. The first aim will address the specific activity of the dual promoters, the second aim will address the regulatory activity (cell-type and developmental specificity) of these promoters and the upstream region, including a potential silencer element, and the third aim will use transgenic mouse models to confirm genetic information generated from the first two aims and to examine novel regulatory mechanisms for KOR expression in the whole animals.

The Molecular, Cellular and Genetic Core (MCGC) facility will provide four categories of services to investigators in this Center Program. Four specific service areas are: 1) Establish a facility for the provision of oligonucleotides for numerous aspects of molecularly-oriented research, 2) Establish 'banks' of reagents commonly used in molecular and cellular biology to prevent technical redundancy and material waster, including expression vectors, libraries, bacterial strains and cell lines, 3) Provide expertise in the areas of molecular and cellular biology to investigators participating in this center and foster interaction among investigators, including the service for sequencing, expression of recombinant proteins in bacteria or years, and 4) Establish a bank of mutant mouse models created by transgenic and gene targeting techniques, for studying pharmacological questions. The main purpose of this core is to provide the investigators with reliable and cost-effective reagents and technical services as well as to foster scientific communications among investigators. Most uniquely, this core will provide valuable mutant animal models that were generated in several investigators' laboratories within this Center, to researchers who are also interested in using these animal models for their research.

The potent analgesic and psychological effects of opiates in animals have been recognized for centuries. The effects of both chronic and acute administration with opiates can occur in animals during their early life; therefore, prenatal exposure to opiates in addicted pregnant women has been a major social problem in this society. It is known that opioid receptors mediate the biological effects of both endogenous opioid peptides and administered opiates, and it is well documented that opiates have profound effects in many biological systems including early animal development. However, it is not known when and which opioid receptors begin to appear during developmental stages and what type of opioid receptors are responsible for the biological sequelae of opiates in animals. Recent cloning of several opioid receptors as well as the advancement in molecular biological techniques makes it possible to examine the molecular basis of the ontogenesis of opioid receptors and the mechanism of tolerance induction and withdrawal in utero as proposed here. Specifically, we will 1. determine the ontogenic profiles of mu, delta and kappa opioid receptors; 2. determine the effects of pharmacological and physiological (endocrine) agents on opioid receptor ontogenesis, as well as the association of opioid receptor gene expression with tolerance induction and development of withdrawal in prenatal stages. These studies will be conducted by first generating transgenic receptor mice carrying an beta-galactosidase (lacZ) or a green fluorescent protein (GFP) marker. Breeding of these transgenic mouse lines will generate double transgenic mice for parallel analysis for reporter gene expression on the same specimens, allowing the changing patterns of multiple opioid receptor genes to be examined simultaneously. This will provide unique information as to if and how the development of opioid receptor gene expression change their profile in the course of prenatal exposure to drugs or other agents. In addition, the molecular genetic basis for these changing profiles will be revealed.

Nuclear receptors function to transduce hormonal signals into transcription responses. Many of these receptors require dimerization with the retinoid receptor X (RXR), suggesting extensive interaction of vitamin A with many hormonal signals. However, for receptors without known ligands (orphan receptors), critical questions are what kind of signaling pathways do orphan receptors represent and what roles do they play in vitamin A biology. This lab has recently characterized an orphan receptor family TR2/TR4 which are specifically elevated in developing germ cells and able to modulate retinoic acid (RA) signals in several experimental systems. Based upon our preliminary studies, it is hypothesized that this orphan receptor family members function to modulate vitamin A signaling pathways by both active and passive mechanisms. Additionally, it is hypothesized that the active silencing mechanism is mediated by its ligand binding domain which encodes a trans-repressive activity and involves an interacting protein RIP140 that cross-talks with retinoic acid receptors RARs and RXRs. The passive mechanism is mediated by forming their own homo- or hetero-dimers within this family, which competes with RAR/RXR for DNA target sites. This proposal will test these hypotheses by asking three questions in three aims. 1) What is the molecular basis of the active silencing activity of TR2 that involves the interaction with RIP140? 2) What underlies the ability of TR2 and TR4 (but not RXR) hetero-dimerization? 3) Does TR2 and TR4 dimers (homo- and hetero-) efficiently compete for DNA binding sites with receptor dimers involving RARs or RXRs. Molecular and biochemical approaches will be taken to address these questions. We hope to extend our understanding of nuclear receptor functions in general, to provide a molecular explanation for the functions of orphan receptors without known hormonal signals, and to understand specifically how TR2 family members cross-talk with vitamin A for specific gene regulation. Ultimately, these studies will provide a model to address how vitamin A interacts with specific cellular factors and exerts its functions, and how the efficacy of vitamin A, as a therapeutic agent or a nutrient, may be modulated by specific cellular factors.

Vitamin A (theretinoids) exerts a wide variety of effects on biological processes, and are commonly used for preventive and therapeutic purposes. Their action is mediated primarily by two families of nuclear receptors, retinoic acid receptors (RARs) and retinoid receptors (RXRs), which regulate gene expression by recruiting coactivators in the presence of retinoic acid (RA)and corepressors in its absence. Nuclear Receptor Interacting Protein 1 (NRIP1), previously named RIP140, is a novel corepressor that represses gene expression in the presence of hormones. It is hypothesized that NRIP1 serves as a ligand-dependent, negative coregulator for hormone-elicited gene regulatory circuits to silence specific gene expression in the presence of hormones. Three aims are proposed to test this hypothesis by using the RAR/RXR system. The first aim is to vigorously examine the molecular basis of NRIP1 interaction with holo-RAR/RXR, which involves a novel C-terminal motif of NRIP1 (PRLTKTNPILYYMLQK) that diverts from a typical coactivator motif, LXXLL, or a corepressor motif, CoRNR box.The second aim is to compare NRIP 1 complex to a typical ligand-dependent coactivator complex, SRC-1, with regards to the efficiency of their interaction with receptorsand their specific associate proteins. The third aim is to address the physiological relevance of RA- dependent corepressor activity of NRIP1 by a) testing the effects of NRIP1 on Oct-3/4 gene that is directly suppressed by RA through RAR/RXR binding to an RA response element (RARE) RAREoct, and b) comparing the effects of coactivator SRC-1 on RAR(32 gene that is directly induced by RA through a typical DR5 type RARE. Studies propsed in the three aims will advance our understanding of the diversity of molecular mechanisms mediatingthe effects of vitamin A hormones. Additionally, we will test whether the current working model of hormone nuclear receptor-coregulator can be modified to accomodate complicated actions of hormones in different biologicalsystems. Finally, it is our ultimate goal to determine if the unique property of NRIP1 bears a physiological relevance in terms of the specificity of vitamin A action on a particular gene promoter.

DESCRIPTION (provided by applicant): The purpose of this proposal is to continue our ongoing investigation on the regulatory mechanisms of the mouse kappa opioid receptor (KOR) gene expression which is currently funded by an R01. Following the same direction as proposed in the original project, this renewal application further serves to understand the fundamental problems in the control of opioid receptor expression, by using both tissue culture and transgenic mouse models. It is known that opiates exert extensive pharmacological and physiological effects in animals. Like many other drugs, opioids act through specific receptors on target cells. Their very restricted expression in the adult nervous system and during developmental stages suggests that the specificity and the level of opioid receptor expression must be tightly regulated, and raises an important question of the control for their expression in a homeostatic state. In the previous award period, we have selected the KOR gene as a model to address this question, and our data indicate that KOR gene is under a multi-level regulatory circuit that involves positive and negative transcriptional regulatory mechanisms and several post-transcriptional events such as alternative splicing and translation. The negative transcriptional control is mediated through the action of vitamin A hormone (the retinoic acid), whereas alternative splicing generates KOR mRNA isoforms that have distinct RNA stability, and translation efficiency, and can be differentially transported to neuron cell bodies and fibers. In the same direction as the original project, we will continue to address our principle hypothesis that KOR gene is maintained at a homeostatic state by integrating different levels of regulatory events. At the transcriptional level, KOR is regulated in two phases during stem cell differentiation, with an initial negative effect exerted by an endocrine factor, the retinoic acid, followed by unknown positive mechanisms to reactivate KOR gene in neurons. Ultimately, we will learn: a) the molecular/genetic basis of KOR gene transcriptional regulation, particularly in the context of chromatin, b) KOR gene post-transcriptional control with regard to differential mRNA stability and transport, and c) KOR gene regulatory mechanisms extended from cell cultures to animals (transgenic mice). One important feature of this renewal proposal is that we will test our hypotheses in a more physiologically relevant condition by also studying the regulatory events at the chromatin level. Additionally, the application of transgenic mouse models is essential for understanding the genetic basis of pharmacological problems that are related to the opioid receptor system.

DESCRIPTION (provided by applicant): This application is for a renewal of a NIDA sponsored K02 (DA13926) Independent Scientist Award. The current K02 award (2002-2007) allowed the PI to develop her independent research career that focuses on genetic programming for the expression of drug receptors, in particular, the opioid receptors. It is known that the amounts of opioid receptors are crucial for the analgesic effects of opioid compounds. The control is through genetic programming that instructs the cells to express these genes properly and specifically (transcriptional control) and to produce these proteins from mRNAs (post-transcriptionally) according to the needs. In the previous studies, the major task was to dissect the basic elements responsible for transcriptional and post-transcriptional regulation of opioid receptor genes, that include determining DMA sequences and protein factors required for transcriptional control, identification of post-transcriptional events that modulate the rate, the place and the time of opioid receptor protein production in neurons, and dissecting fundamentally important mechanisms in their regulation. Over the period of 2002-2005 supported by this K02, the Pi's works were published in 35 papers. The renewal proposal will continue systemic investigation into the mechanistic basis of these biological processes and the integration in the animals for understanding the homeostatic control of opioid receptor expression. Thus, the overall direction of the research project remains the same. Specific aims are: 1) to extend studies of transcriptional regulation of KOR gene by focusing on how transcription factors interact with chromatin of the KOR gene, leading to chromatin remodeling of the promoter regions;2) to identify post-transcriptional events that regulate KOR protein production in neurons by focusing on the control of mRNA transport and local translation in neurons regulated by the 5'- and the 3'-untranslated regions;and 3) to produce transgenic mouse models to answer whether and how these regulatory events affect the manifestation of pharmacological effects and problems of opioids, such as addiction and tolerance. These studies are clinically important for the identification of crucial steps in genetic programming of opioid receptor expression that may shed light on potential targets of interventions for drug abuse problems, and are fundamentally applicable for the understanding of critical biological processes required for neuronal compartment-specific expression of proteins that are of significant pharmacological and neurological consequences. This award is also essential for the Pi's continual career development as an independent scientist in the field of drug abuse research.

The long-term goal of this project is to understandhow vitamin A signaling is modulated by orphannuclear receptors belonged to the TR2 and TR4 family. Previous studiesfocused on the repressive mechanisms of TR2 that include i) directly recruitingco-repressors like histone deacetylases (HDACs), receptor interacting protein 140 (RIP140) and SMRT (an active mechanism), and ii) competing with RA receptors (RARs) and retinoid X receptor (RXR) for DNA binding (a passive mechanism).By extending from these conclusions and based upon recent studies, this renewal proposal focuses on novel ligand-independent signaling pathways that can modify the property and activity of TR2 and TR4 for the regulation of RAR|32, cyclin D1 and apoE genes. Three hypotheses will be tested: i) the biological activity of TR2 and TR4 can be modulated by protein modification (biochemical factors) and their interaction with coregulators (kinetic factors), ii) the physiologically relevant receptor activity is manifested through their interaction with, or recruitment of, specific coregulators onto the regulatory region of the target gene (dynamic factors), and iii)the ligand- independently activated receptor complex can contribute to chromatin remodeling of target gene to activate transcription. Aim I will address the first and second hypotheses by examining the mechanisms of ligand- independent modulation of receptor activity elicited through protein modifications (using a proteomic approach) that affect: i) the biochemical nature of receptors, ii) the general property of receptors, iii) receptor- coregulator interaction kinetics and iv) dynamics of TR2 and TR4 coregularory complex on target genes. Aim II will address the third hypothesis in physiologically relevant cell cultures by manipulatingTR2 and TR4, and determiningthe effects of their modification on target genes. The biological effects to be examined include the formation of coregulatory complexes and alteration in chromatin conformation (remodeling) or histone modification on the target gene promoters (RAR|32, cyclin Dl and apoE) and theirbiological activities in P19 cell cycle progression as well as transcription efficiency of target genes. Results from both in vitro (aim 1) and in vivo (aim 2) systems will be integrated to construct a comprehensive overview of the mechanisms underlyingthe modulation of vitamin A signalingpathways by these orphan receptors, specifically with respect to signals generated from protein modifications of receptors.

This component will continue elucidating the molecular mechanisms underlying "ontogenesis" of kappa opioid receptor (KOR), i.e. production of KOR protein during developmental stages and differentiating neurons. Studies in the previous funding cycles have delineated several regulatory pathways for the control of KOR mRNA production during developmental stages, principally transcriptional control. This concludes the first phase of studies focusing on the regulation of KOR mRNA synthesis, a hall markd of KOR neurogenesis involving signaling pathways of retinoic acid (vitamin A) and nitric oxide and requires chromatin remodeling of KOR gene regulatory regions, as well as more recent findings in epigenetic control. Importantly, ontogenesis of KOR appears to be controlled, most crucially, by post-transcriptional mechanisms such as mRNA stability, transport and translation. This renewal component will focus on translational mechanism by extending from our preliminary studies that have identified Netrin-1 as a translational stimulator for KOR, and Grb7 as a translational represser of KOR. Two specfiic aims are: 1. To elucidate the mechanism that activates KOR translation via Netrin-1/Grb7 pathway. We will focus on i) regulation of translational initiation of KOR by Grb7, including studies of its molecular and biochemical features and funcitonal domains, and KOR RNA sequences bound by Grb7 which can be activated by Netrin-1, and ii) specific translational initiation step that is targeted by Grb7 including initiation factors and subcellular distribution and possible circularization of KOR mRNA. 2. Pharmacological and physiological relevance of Grb7/Netrin-1 signaling to KOR ontogenesis. We will i) perform gain- and loss-of-function studies to validate the relevance of Netrin-1/Grb7 in KOR ontogenesis using stem cells and primary neurons, and ii) examine the physiological relevance of KOR synthesis in neuronal activity such as in a specific pain circiitry and during nerve injury or as a stress response.

Retinoic acid (RA), the biologically active ingredient of vitamin A, is essential for a variety of biological processes. RA acts, primarily, by binding to nuclear RA receptor (RAR) and retinoid receptor X (RXR) to regulate gene expression. But the activities of RAR and RXR ultimately depend on the recruitment of coregulators. This project was initiated by the identification of an RA-dependent RAR coregulator named Receptor Interacting Protein 140 (RIP140), also known as Nuclear Receptor Interacting Protein 1 (Nrip1), which regulates a wide spectrum of transcription factors including all nuclear receptors. RIP140 is unique for i) wide spectrum activity in chromatin remodeling, and ii) extensive post-translational modifications (PTMs) that regulate its properties and relevance to diseases. Previous progress (2013-2017) related to this renewal has been published in 16 papers, which report: i) RIP140's activity in metabolism and innate immunity (macrophage M1/M2 polarization), ii) a robust activity of RA in activating M2 gene Arg1, iii) signals triggering RIP140's PTMs, and iv) new therapeutic potential of RA and RIP140 in managing chronic inflammatory conditions such as wound healing, via modulating macrophage M1-M2 polarization and boosting M2 gene Arg1. Based upon these findings, it is hypothesized that dampening the RIP140 level and adding RA can synergistically (additively) enhance anti-inflammation by facilitating innate immune cycle completion (M1-M2 polarization) and boosting a critical effecter gene for M2 in tissue repair, Arg1. The two aims are: i) to advance translational studies determining whether and how dampening RIP140 and applying RA can synergistically boost anti-inflammation to improve wound healing, and ii) to pursue in-depth and holistic studies answering how RIP140 modulates macrophage polarization potential at the single cell resolution and how RA orchestrates multiple gene regulatory events (chromatin remodeling, transcription and coupled RNA processing) to promote Arg1 expression for effective wound healing. Completing these studies will provide further insight for designing more efficient strategies in managing diseases related to the endogenous retinoid/RA status and inflammatory state, and for developing RA as a more effective therapeutic agent.

DESCRIPTION (provided by applicant): This application requests partial support for the 17th Federation of American Societies for Experimental Biology (FASEB) Summer Research Conference on Retinoids to be held at Itasca, Illinois on June 1-6, 2014. This bi- annual research conference has been very well attended (ranging from 100 to 200 attendees over the past 30 years) and remains the sole major national and international conference that addresses the full spectrum of retinoid biology. While the mechanisms of action and the biological functions of retinoids have been intensively studied, there is an increasing need in understanding and applying retinoids in human nutrition and diseases. Currently, a great deal of ongoing research activity is supported by the NIH (the NIH RePORT database indicates that there are currently 1,460 NIH funded individual research grants involving retinoid/vitamin A biology). The 2014 conference will start with a new Round Table panel providing a holistic and historical view by several senior investigators in the field. This will be followed by 8 symposia, 2 traditional poste sessions, and one new Poster Award in conjunction with a new Meet the Expert session to close the meeting. The goal is to foster the exchange of new research findings. In particular, we aim to encourage new/young investigators meeting and networking with senior investigators in this field, and facilitate the inclusion of new insights/ideas/techniques from scientists and clinicians in other related fields. The main topics to be addressed at the conference include both basic and translational fronts of retinoid biology: (1) retinoid metabolism; (2) retinoid enzymology; (3) retinoid signaling; (4) retinoids in development and stem cell biology; (5) retinoids in disease I - cancers; (6) retinoids in disease II - immunity and metabolism; (7) retinoids in disease III - eyes and the nervous systems; (8) retinoids and carotenoid chemical biology. The overall goal is to bring together a group of investigators (senior scientists, mid-career scientists, junior scientists, postdoctoral fellows, and graduate students) who work in diverse disciplines (as biochemists, molecular biologists, nutritional scientists, cancer researchers, vision researchers, stem cell biologists, biophysicists, structural biologists, immunologists, chemists, and clinicians) to present and discuss their newest research findings that share a common focus on the retinoids. This conference will provide a forum for critical review of recent research and incorporation of newly developed ideas and technologies into translational fronts of this classical topic. This will allow new investigations to provide needed insight into the applications of retinoids in preventing disease and maintaining health and in developing new therapeutics, and facilitate collaborations among basic scientists and clinicians.