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Record Count: 29
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DESCRIPTION (provided by applicant): The heme moiety of denatured hemoproteins (heme-proteins) is degraded by HO-1 & HO-2, to CO and biliverdin (BV), an HO activity inhibitor; BV is reduced by its reductase (BVR) to the antioxidant, bilirubin. CO has anti-inflammatory and vasodilatory activities. Stimuli that cause oxidative stress and hemoprotein denaturation, such as surgical interventions, inherited and transmitted hemolytic diseases, and certain drugs, industrial and environmental agents, induce ho-1. The kidney tubules are the target of potent pro-oxidant activity of heme-compounds. To date, no effective strategy has been described to counter heme-protein renal toxicity; however, increase in HO-1 activity by activation of stress-activated response elements, e.g., AP-1/CRE, AREs (antioxidant response elements) is considered cytoprotective. The activation involves cell-line independent binding of basic leucine zipper (bZip) transcription factors: c-Jun, ATF- 2/CREB, and Nrf2. The MAPK and PI3-K pathways transduce signals for activation of bZip factors and "cross talk" using PKCs. bZip factor activity is subject to the identity of its dimeric partner. Phosphorylation of the ultimate target gene product, e.g., HO-1, alters its activity and turnover. In vitro and in cultured cells, we have discovered that: the human (h) BVR is one of the rare kinases that control MAPK and PI3-K signaling; is a bZip factor; activated by ho-1 inducers; and, traffics between the cytosol and nucleus. hBVR binds to AP-l/CRE and ARE elements and also enhances ATF-2 and Nrf2 binding to AP-1 and/or ARE; promotes induction and activation of ATF-2, c-Jun and c-Fos; activates kinase mediators of ho-1 response, i.e. PKCs and PKB/Akt; and, causes cell differentiation. BVR regulates ho-1 oxidative stress response and its anti-apoptotic effect. The overall objective of this application is to further investigate regulation of HO-1 activity by BVR at the molecular and cellular levels and to extend the investigation to the intact animal. Specific aims are to examine: i) function of BVR as kinase:kinase in phosphorylation, activity, and turnover of HO-1; 2) the role of hBVR in transcriptional activity of ATF-2 and Nrf2; and, 3) whether increased levels of BVR protect against heme-mediated injury. Mice expressing hBVR in renal tubules and treated with the hemolytic agent, phenylhydrazine, will be analyzed for pathophysiology, antioxidant status, kinase activities and HO-1 levels. Liver will serve as the non-target organ as control for the expression of the transgene.
DESCRIPTION (provided by applicant): The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor and one of a family of proteins containing the bHLH-PAS domain structure. Members of this family are involved in responding to signals in the tissue environment and serve regulatory roles in development and cellular differentiation. Although the AhR has been conserved throughout evolution and mice lacking the AhR show many defects in several organ systems, its normal function is not known. Recent work has identified an endogenous ligand, 2-(1'H-indole-3'- carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), from porcine lung and demonstrated this to be a potent AhR agonist. However, unlike toxic AhR ligands like 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), ITE produces no toxicity in mice even at very high doses. It is hypothesized that ITE differentially interacts with the AhR as compared to toxic ligands such that exposure results in different molecular and biological consequences. It is also hypothesized that, based on work indicating a role of the AhR in the regulation of inflammation, that ITE may have a novel role as a regulatory molecule for inflammatory processes in the lung. Using primary mouse and human lung fibroblasts, we will rigorously determine, by microarray analyses, differences and/or similarities in the gene profile induced by ITE and TCDD. Using both isolated lung cells and a pre-clinical mouse model of lung inflammation, we will also determine whether the inflammatory response is similar or different in the presence of ITE, TCDD, or known AhR antagonists, and will begin to characterize the cellular and signaling pathways that may determine these similarities or differences. The goals of these studies are of particular significance given the wealth of data indicating a role of inflammation in the pathogenesis of several diseases including chronic obstructive pulmonary disease, hypertension, cardiovascular disease, allergic diseases such as asthma, and cancer. Understanding the role of ITE and the AhR in modulating inflammatory processes may lead to possible therapeutic interventions.
DESCRIPTION (provided by applicant): The Ah receptor (AhR) has been shown to be largely responsible for the toxic and tumor promotional properties of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), especially in rodents. In addition, the AhR is a key regulator of xenobiotic metabolism. The human population is exposed to low levels of TCDD and related compounds (e.g. planar PCBs), mostly through the diet. However, the actual long-term health effects of human exposure to TCDD remain to be fully elucidated. Little is known about the biochemical processes involved in the activation and regulation of this ligand-activated helix-loop-helix/basic region transcription factor. In this application the ability of the human and mouse AhR to regulate gene transcription through protein-protein interactions will be examined in three specific aims; 1) Use transgenic mouse models to compare the ability of the human versus the mouse Ah receptor to activate gene expression and mediate liver tumor promotion, 2) Determine the ability of the AhR to modulate transcriptional activity other than through binding to dioxin response elements (DRE), and 3) Determine whether the AhR can alter gene regulation as a monomer. To achieve these goals, a combination of AhR-null cell lines and null-AhR mice with a mutant form of mAhR, WT-mAhR or human AhR "knocked-in" will be used. An emphasis will be placed on determining the multiple mechanisms that lead to direct AhR-mediated changes in gene expression and the target genes that are affected, as well as examine liver toxicity upon exposure to TCDD. Significant preliminary data is provided to support each aim, including the production of the hAhR and mAhR-A78D transgenic mouse models. The combined results from these studies will establish how the AhR/ARNT heterocomplex mediates transcription through a DRE and a non-DRE driven mechanism, and whether differences in the structure of the human AhR results in altered modulation of gene transcription. Lay Relevance: Liver insufficiency can result from environmental and dietary chemical exposure and lead to death in humans. Results from these studies will determine the mechanisms used by the Ah receptor to alter gene regulation that can lead to liver toxicity or tumor formation.
Crisp Terms/Key Words: protein protein interaction, aromatic hydrocarbon receptor, neoplastic process, human genetic material tag, active site, environmental toxicology, tissue /cell culture, receptor binding, toxicant interaction, dioxin, liver, gene induction /repression, transcription factor, genetic transcription, molecular cloning, transfection, site directed mutagenesis, biological signal transduction, genetically modified animal, laboratory mouse
DESCRIPTION (provided by applicant): The major long-term objectives of the proposed research are [1] to test the hypothesis that lead interacts with calcium binding proteins of the C2 and annexin families, and [2] to identify genes and proteins of these calcium-binding families that are regulated following lead exposure of rats. Lead poisoning remains a pervasive problem in the United States, affecting at least 5% of all children. The proposed research is intended to elucidate molecular mechanisms underlying lead toxicity. Previous studies have demonstrated potent interactions between lead and proteins of the C2 domain family (e.g. protein kinase C and synaptotagmin) and annexins. Furthermore, lead exposure of cells has been shown to regulate the expression of genes encoding calcium-binding annexins. The four specific aims of this proposal are [1] to measure the interactions of lead with proteins of the C2 domain and annexin families, in order to determine the possible targets of lead. [2] To measure gene expression in the brain, kidney and liver of lead-exposed rats. This in vivo model may reveal whether lead exposure differentially regulates the expression of genes encoding calcium-binding proteins. [3] To extend gene expression studies to well characterized cell lines (astrocytes, PC12 cells, fibroblasts and normal rat kidney cells). These studies will complement gene expression measurements from the in vivo model. [4] To deposit gene expression data into a publicly accessible database. Together these studies may reveal which calcium binding proteins interact with lead, and which genes encoding calcium-binding proteins are regulated by lead exposure.
Crisp Terms/Key Words: microarray technology, molecular biology information system, synaptotagmin, neurotoxicology, PC12 cell, cell line, northern blotting, annexin, calcium binding protein, in situ hybridization, astrocyte, intermolecular interaction, lead poisoning, liver, kidney, western blotting, gene expression, fibroblast, polymerase chain reaction, hippocampus, frontal lobe /cortex, laboratory rat
DESCRIPTION (provided by applicant):
The goal of this project is to elucidate redox-responsive developmental pathways and gene regulatory networks that mediate susceptibility to environmental redox stressors. Redox chemistry is at the core of biology, providing the energy that fuels life but also producing toxic byproducts in the form of reactive oxygen species (ROS). ROS production can lead to oxidative stress, a hallmark of many human diseases (such as diabetes) and environmentally-induced pathologies (such as those associated with alcohol abuse). Biological signaling systems are therefore often responsive to redox chemistry. While many environmental redox stressors are also known to cause developmental malformations in humans, particularly in the developing nervous system, the redox-sensitive regulatory networks that mediate this susceptibility are largely unknown. The sea urchin embryo provides a useful comparative model for addressing this problem, as its genome has been sequenced and annotated, and because of the fact that it is a deuterostome and hence developmentally more similar to humans than other invertebrate model organisms. A number of findings indicate that ectodermal cell fate along the oral-aboral axis of the sea urchin embryo is specified via a redox-sensitive regulatory network, and can be specifically perturbed (radialized) by redox stressors such as metal ions and hypoxia. Ectodermal cell fate specification is mediated by Nodal signaling, which in turn is dependent on p38 mitogen activated protein kinase (MARK). The specific aims of this project are to (1) test the hypothesis that p38 mitogen activated protein kinase (MARK) activity is regulated by redox signaling in the developing ectoderm; (2) identify redox-responsive cis-elements and transcription factors that regulate Nodal activity; and (3) identify pathways through which redox stressors perturb ectodermal patterning and affect human development and disease. To achieve these aims, the project will make use of highly specific molecular reagents including mitochondrially-targeted enzymatic anti-oxidants, morpholino-antisense mediated knockdown, and cis-regulatory analysis of the Nodal gene. In addition, a microarray approach will be used to identify the redox-sensitive transcriptome. Finally, the Comparative Toxicogenomics Database (CTD) at MDIBL will be used to determine the relevance of the pathways discovered in sea urchins to human health, and to generate hypotheses that might explain specific human diseases.
DESCRIPTION (provided by applicant): The long-term goal of this study is to understand the role that the mitogen-activated protein kinase (MAPK) signaling pathways play in the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Dioxin is a widely spread environmental contaminant that exerts diverse species-specific toxic effects in animals and humans, including immune, reproductive and developmental toxicity, cancer, wasting syndrome and death. Dioxin toxicity is mediated by the activation of a cytosolic aromatic hydrocarbon receptor (AHR) that functions as a ligand-activated transcription factor and whose homozygous ablation protects mice from dioxin toxicity. Dioxin is non-genotoxic and, like other tumor promoters, is believed to exert its effects by promoting signaling pathways ultimately responsible for cell proliferation and cell survival. The (MAPKs) are the primary effectors of many of those signal transduction pathways. However, the molecular connections between dioxin-activated signaling pathways and AHR function have yet to be established. Dioxin-induced MAPKs play an important role in Ah receptor activation, because their suppression causes impaired AHR function. In addition, specific MAP kinase modules regulate Ah receptor function in a tissue specific manner. This proposal will test the hypothesis that dioxin-induced MAP kinase pathways regulate the activity and function of Ah receptor as a transcription factor. We will focus on the molecular identification of the signaling factors involved in dioxin action on MAPKs, the characterization of the mechanism of MAP kinase-mediated Ah receptor activation and the analysis of dioxin-induced AHR functions regulated by MAPKs in culture cells and in mice deficient in signaling factors of the MAPK pathways. To achieve these aims, we will use novel approaches that bring together an understanding of signal transduction pathways with the analysis of the molecular biology of the toxic response. Results from this work will further our understanding of cross-talks between dioxin-elicited biological pathways, will identify molecular factors critical for the diverse toxic effects of dioxin and will help characterize primary candidate targets for its prevention. Understanding the signaling mechanisms responsible for AHR activation by dioxin will provide a wealth of information immediately applicable to the study of the toxicity of the more than 400 environmental toxicants and Ah receptor agonists of which dioxin is the prototype.
Crisp Terms/Key Words: environmental exposure, cell proliferation, aromatic hydrocarbon receptor, mitogen activated protein kinase, gel mobility shift assay, SDS polyacrylamide gel electrophoresis, tissue /cell culture, autoradiography, protein structure function, phosphorylation, dioxin, ligand, western blotting, transcription factor, gene expression, biological signal transduction, genetically modified animal, laboratory mouse
DESCRIPTION (provided by applicant): Peroxisome proliferators are an important class of tumor promoters that bind to members of the nuclear receptor superfamily named PPARs (Peroxisome proliferator-activated receptors). Several subtypes of PPAR have been discovered (alpha, beta, gamma), although the predominant liver subtype (PPARalpha) is our primary interest due to the critical role it plays in carcinogenesis. Little is known about the biochemical properties or post-translational mechanisms that regulate this important receptor. Thus, the central hypothesis to be tested is that PPAR( transcriptional activity is regulated by site-specific phosphorylation and entry into a dynamic chaperone complex containing hsp90 and/or XAP2 The central hypothesis and proposed specific aims are firmly supported by preliminary findings from our laboratories. Four specific aims are proposed: 1). Determine the sites of phosphorylation of PPAR( and examine their role in modulating receptor activity; 2) Examine the role of the co-chaperone protein XAP2 in the regulation of PPAR function; 3). Determine the mechanisms that regulate XAP2 expression; and 4). Examine the role of PPAR chaperones (hsp90, XAP2, hsp70, p23) on transcriptional regulatory complexes. The phosphoprotein PPARalpha is found in cytosolic extracts as a complex with hsp90 and XAP2; both proteins repress PPARalpha activity. This would suggest that regulation of PPAR( activity is quite complex. A series of in vitro and in vivo studies are proposed utilizing a wide variety of techniques including transgenic and knockout mice, immortalized cell lines, transient and stable transfections and protein biochemistry. Taken together the proposed studies will greatly enhance our knowledge of PPARalpha's mechanism of action and hence our understanding of an important class of carcinogen.
DESCRIPTION (provided by applicant): Toxins, pathogenic infection (viral and bacterial), and physical injury to the liver results in a loss of hepatic tissue, triggering a regenerative response to restore liver cell mass. Dysregulation in the repair process can lead to liver failure or liver cancer. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor functionally identified with proliferative processes. The AhR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) the prototype for a class of compounds responsible for a range of toxic or adaptive endpoints, inhibits liver regeneration following tissue injury thus implicating the AhR in liver repair. Our long-term goal is to understand mechanistically how the AhR contributes to liver homeostasis by regulating cell proliferation, and thereby identify the molecular basis for TCDD-induced disruption of normal biological processes. We hypothesize that the AhR plays an important role in liver homeostasis, in part by regulating progress through G1 phase of the cell cycle in proliferating hepatocytes. The goal of this proposal is to examine the functional relationship between AhR activity and TCDD responsiveness proteins known to regulate hepatocyte proliferation. Specifically, the planned studies will examine the molecular basis and relative contribution of cyclin-dependent kinase 2 (CDK2) inhibition and plasminogen activator inhibitor-1 (PAI-1) expression on liver regeneration. TCDD alters CDK2 activity and PAI-1 expression during liver regeneration, representing distinct intracellular and extracellular (autocrine or paracrine) mechanisms of action that conspire to inhibit the restorative response to injury. Preliminary results also demonstrate that AhR regulation of the PAI-1 gene involves a novel (non-XRE) DNA element that will be characterized in detail. The proposed studies are an extension of currently funded research looking at the AhR in regulating hepatic proliferation in cell culture models, and seeks to build on previous findings using a physiologically relevant (non-transformed) model of cell proliferation. Parallels between humans and mice in the regenerative response, instills confidence that findings generated during these studies will be directly applicable to the human condition.
Crisp Terms/Key Words: protein protein interaction, aromatic hydrocarbon receptor, retinoblastoma protein, gel mobility shift assay, toxin metabolism, environmental toxicology, receptor expression, receptor binding, DNA binding protein, protein structure function, protein sequence, toxicant interaction, dioxin, complementary DNA, liver regeneration, immunoprecipitation, western blotting, immunocytochemistry, molecular cloning, carbopolycyclic compound, polymerase chain reaction, cell growth regulation, laboratory rat, laboratory rabbit
DESCRIPTION (provided by applicant): Dioxin and related halogenated aromatic hydrocarbons are ubiquitous, persistent environmental contaminants causing adverse responses to human and wildlife. Most of the toxic responses induced by dioxin are mediated by the aryl hydrocarbon receptor (AhR). Therefore, central to our understanding of dioxin-induced toxicity is to elucidate the mechanism of the AhR-regulated gene expressions. In earlier studies, we have found a physical association and functional reciprocal repression between the AhR and NF-kB pathways (J. Biol. Chem. 274,510). Because NF-kB is a pleiotropic transcription factor involved in many physiological functions that are known to be adversely affected by dioxin, the AhR-mediated suppression of NF-kB offers a mechanism for some aspects of hitherto poorly understood dioxin-induced toxic responses, such as the immune suppression and abnormal skin proliferation. Reciprocally, suppression of AhR by NF-kB activation has also offered an underlying mechanism for the long-standing observation that inflammatory cytokines and lipopolysaccharide suppress AhR-regulated cytochrome P450 1A1/1A2 and decrease capacity of xenobiotic (including clinical drugs) metabolism (J. Biol. Chem. 276,39638). In recent studies, by using chromatin immunoprecipitation (CHIP) assay, we have obtained new results revealing that the AhR/NF-kB interaction converges at level of transcription involving (1) control of transcription elongation and (2) chromatin modifications. In AIM I of this proposal we will investigate a mechanism in which AhR/NF-kB interaction regulates cyp1a1 transcription elongation by directly interacting with p-TEFb (positive transcription elongation factor b), which plays a critical role in elongation control. In AIM II, we will investigate histone modifications (histone acetylation and methylation) in response to the diametrically opposing actions of AhR and NF-kB and to establish the residue-specific and combinatorial patterns of histone modifications (histone code) associated with "on and off" states of cyp1a1. We will also investigate a novel AhR interactive protein (identified by CytoTrap yeast two hybrid screening) SUV39H2 methyltransferase for its role in AhR-mediated gene silencing, which may be important for male imprinting. The proposed studies will help us gain mechanistic understandings of the functions of AhR and NF-kB in normal physiology as well as pathogenesis induced by dioxin and related compounds.
Crisp Terms/Key Words: protein protein interaction, yeast two hybrid system, aromatic hydrocarbon receptor, nuclear factor kappa beta, SDS polyacrylamide gel electrophoresis, methyltransferase, environmental toxicology, histone, lipopolysaccharide, phosphorylation, dioxin, immunoprecipitation, cytokine, gene induction /repression, transcription factor, gene expression, polymerase chain reaction, biological signal transduction
DESCRIPTION (provided by applicant): The broad, long term goals of this proposal are to use a novel class of antagonists of the aryl hydrocarbon receptor (AHR), AHR-Protacs, developed herein as 1) a research tool to delineate the mechanisms by which activation of the AHR pathway by environmental agents leads to carcinogenesis in the human population and identify roles of the AHR in other disease processes and 2) a therapeutic agent to treat cancers, diabetes and cardiovascular diseases. Exposures to environmental factors, such as benzo[a]pyrene and 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) are thought to play important roles in the development of human cancers. While these agents are known to exert many of their carcinogenic actions via activation of the AHR, the mechanisms by which the AHR elicits the key events are unknown. Further, it has not yet been established whether targeting the AHR would be an effective chemopreventive approach. Finally, emerging evidence implicates a role for the AHR in not only cancer, but also diabetes and cardiovascular diseases. The immediate goals of the current proposal are to optimize and characterize novel AHR antagonists, some of which have already been developed in our laboratories, as appropriate AHR antagonists. Specific Aims: (1) Synthesis of optimum AHR Protacs. (2) Determine that the optimum AHR Protac molecule(s) inhibits AHR signaling in cultured cells with high efficacy. (3) Determine whether the optimum AHR Protac(s) inhibits the ability of TCDD to alter cell cycle, apoptosis and senescence. (4) Determine whether the optimum AHR Protac(s) alters the AHR pathway with high specificity. The work proposed herein will develop a novel research tool that can be used to identify how inappropriate activation of the AHR by environmental carcinogens leads to human cancers and to understand the role of the AHR in normal biological processes. In addition, this work will set the stage for the development of drugs that target the AHR and may be clinically useful not only as agents that may prevent cancers, but may also be effective in the treatment of diabetes and cardiovascular diseases.
DESCRIPTION: (Adapted from the Investigator's Abstract) This is a
continuation application for a long-term project on effects of trace metal
ions on gene expression in human cells. During the last period, we began to
investigate the effects of toxic metal ions on signal transduction pathways
used for global regulation of gene expression, cell proliferation and
survival. The tumor promoter arsenite (As+3) was found to be a potent
activator of two mitogen activated protein kinase (MAPK) cascades that
stimulate the activity of transcription factor AP-1. This effect of As+3 is
due to inhibition of a dual-specificity protein phosphatase, JNK
phosphatase, whose normal function is to keep the MAPKs, JNK and p38 in a
low activity state. As induction of AP-1 activity is closely linked to
tumor promotion, the JNK phosphatase is probably an important mediator of
As+3 cocarcinogenesis. It may also be involved in As+3 induced inflammatory
disease. To examine the physiological role of the JNK phosphatase we will
characterize and molecularly identify it using a combination of biochemical
and molecular biological approaches. Dominant-negative mutants will be
transfected into cultured cell lines to inhibit endogenous JNK phosphatase
activity and thus assess its function in cell physiology. We will also
examine the susceptibility of mouse strains and cell lines deficient in JNK
or JNK phosphatase to As+3 induced cocarcinogenesis and toxicity. As+3 and
other trace metals were also proposed to act through non-specific induction
of oxidant stress. We therefore started to investigate the regulation of
transcription factor NF-KB, which was proposed to be a major sensor of
oxidant stress. We recently purified and cloned a key component in the
pathway leading to NF-KB activation, the protein kinase responsible for
phosphorylation and eventual degradation of the inhibitors of NF-KB, the
IKBs. We now propose to study the molecular mechanism by which oxidants
lead to activation of this IKB kinase (IKK). As NF-KB plays a key role in
inflammation, understanding the regulation of IKK activity by oxidants, such
as ozone, will provide a molecular basis for oxidant induced inflammatory
disease.
Crisp Terms/Key Words: mitogen activated protein kinase, nuclear factor kappa beta, oxidative stress, enzyme activity, phosphoprotein phosphatase, human genetic material tag, environmental toxicology, tissue /cell culture, protein tyrosine kinase, tumor promoter, cocarcinogen, molecular oncology, metal poisoning, heavy metal, metal complex, gene mutation, gene induction /repression, transcription factor, protooncogene, gene expression
DESCRIPTION (provided by applicant): Our long-term objective is to determine the mechanisms by which developmental exposure to dioxins and other ubiquitous arylhydrocarbon receptor (AhR) ligands alters reproductive functions in adulthood. We recently showed that a single developmental exposure to the potent AhR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), interferes with estrogen-dependent sexual differentiation of the male anteroventral periventricular nucleus (AVPV). The AVPV controls the female pattern of luteinizing hormone (LH) release; therefore, TCDD-exposed males have a female-like AVPV structure and they show the female, cyclic pattern of LH release in adulthood. Considering that estradiol (E2) derived from fetal production of testosterone is responsible for the sexual differentiation process, we hypothesize that TCDD interferes with E2 regulation of a set of genes during development. To test this hypothesis, we will use Affymetrix microarrays and cluster analysis to identify sex-specific genes that are regulated by E2 and TCDD. We will use a "redundancy model", testing different animal treatments predicted by our hypothesis to target the same set of genes. We will validate our microarray findings using RT-QPCR, in situ hybridization histochemistry, developmental ontogeny studies, dose response studies and promoter analysis. Our findings will be important for identifying genes underlying effects of dioxins and other ubiquitous AhR ligands on reproductive toxicity, as well as on other neural functions disrupted by perinatal TCDD exposure in a sex-specific manner. Such findings will be important for mechanism-based risk assessment, as well as for development of pharmaceutical interventions to prevent neurotoxicity.
DESCRIPTION (provided by applicant): The long-term objective of this proposal is to understand the molecular mechanisms underlying the biological responses to dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD) exposure. TCDD, the prototypic dioxin and a model for many other organochlorinated compounds, produces many apparently unrelated biological effects, ranging from chloracne in humans to developmental teratogenesis, tumor promotion, thymic atrophy, wasting syndrome and death in laboratory animals. In addition, TCDD, a rodent carcinogen, is strongly suspected of being carcinogenic also in humans. The molecular basis of the biological effects of TCDD is largely unknown. Dioxin is a ligand for the aromatic hydrocarbon (Ah) receptor (AHR), which, as a dimer with the Ah receptor nuclear translocator protein ARNT, mediates the transcriptional activation of genes in the CYP 1 family of cytochrome P450 monooxygenases. However, activation of the CYP1A1, CYP1A2 and CYP1 B1 genes, although one of the best characterized effects of Ah receptor activation by TCDD, does not adequately explain the diversity of TCDD effects. Our recent global expression profiling analyses of human hepatoma cells shows that exposure to dioxin induces or represses a total of more than 300 genes, with repression being the more frequent. Induction may readily be explained by the transactivating potential of the AHR, but gene repression is a novel effect of the activated AHR that is uncharacterized at the molecular level. The goal of the experiments proposed here is to define and characterize the regulatory interactions between the activated AHR and other transcription factors, co-regulators and chromatin remodeling factors responsible for the effects of dioxin on gene expression. The major objectives of this work are, (1) to define the role of discrete domains of the AHR in gene regulation; (2) to use proteomic analyses to identify AHR coregulatory partners in gene induction and repression; and (3) to clone the promoters of AHR regulated genes and characterize their response to dioxin exposure. Results from these experiments will be crucial for our understanding of the long-range biological consequences of exposure to dioxin and to other organochlorinated compounds and will help formulate an adequate rationale to deal with health problems arising from an ever-increasing exposure to these environmental agents.
DESCRIPTION (provided by applicant): The long-term goal of our laboratory is to understand, in molecular detail, how protein kinase signaling pathways together with phosphoserine/threonine-binding domains regulate multiple aspects of cell proliferation, including cell cycle progression and the cellular response to DNA damage. In the present proposal, we explore the function of MAPKAP Kinase-2, a stress-responsive protein kinase activated by p38 MAPK, as a critical regulator of S-phase and mitotic progression in response to environmental and endogenous types of DNA damage. We use a combination of extensive biochemistry and molecular cell biology to explore the signal transduction mechanisms involved in MAPKAP Kinase-2 activation after DNA damage induced by chemicals and UV-C irradiation, and examine how MAPKAP Kinase-2 functions together with other checkpoint kinases such as Chk1, to control cell cycle progression after genotoxic stress in cells in culture. We go on to develop a conditional MAPKAP Kinase-2 knock-out mouse to explore whether MAPKAP Kinase-2 functions as a tumor suppressor in genetically defined models of sarcoma and lung cancer, and in environmental carcinogen-induced models of colorectal and skin cancer. Finally, we explore whether down-regulation of MAPKAP Kinase-2 facilitates cell death after intentional chemically-induced DNA damage such as chemotherapy. These studies should clarify how signals from the p38 MAPK-MAPKAP Kinase-2 pathway, a global stress-responsive network that is activated by a wide variety of toxic insults, integrate with those from dedicated DNA damage response pathways, to regulate the cellular response to genotoxic stress. The results of the proposed experiments should reveal whether MAPKAP Kinase-2 functions as a tumor suppressor gene that modulates the risk of cancer after exposure to environmental agents, and whether specific targeting of MAPKAP Kinase-2 would be of therapeutic value for sensitizing tumors to the cytotoxic effects of conventional chemotherapy.
Crisp Terms/Key Words: phosphoserine, laboratory mouse, genetically modified animal, biochemistry, biological signal transduction, cell death, cell cycle, enzyme structure, disease /disorder model, molecular biology, sarcoma, lung neoplasm, protein kinase, protein structure function, DNA damage, mitogen activated protein kinase, cell proliferation, tumor suppressor protein, binding site, serine threonine protein kinase
DESCRIPTION (provided by applicant): Malignant melanoma is on the rise in industrialized nations. Indeed, the incidence of mortality related to melanoma has increased by 34% in the United States from 1973-1992. However, there are few effective treatments developed for melanoma. We propose that the aryl hydrocarbon receptor (AhR) pathway plays a role in melanoma progression through the activation of matrix remodeling enzymes. We have chosen to use 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as the agent to activate the AhR- pathway. TCDD and related polycyclic and halogenated aromatic hydrocarbons (PAH/HAH) are ubiquitous environmental contaminants that are the unintentional by-products of industrial combustion. TCDD is ideal for examination of the AhR pathway in melanoma for three reasons: (1.) skin is a target tissue for TCDD-activation of the AhR pathway; (2.) and TCDD is not metabolized; and (3) TCDD is non-mutagenic, and therefore the effects we observe should be directly related to activation of the AhR pathway. We hypothesize that TCDD-activation of the AhR/Arnt pathway stimulates melanoma progression by altering expression of the genes involved in matrix remodeling, specifically the matrix metalloproteinases (MMPs). MMP activity is necessary for cell migration through matrix barriers, and expression of these enzymes correlates with aggressive and invasive tumors. Our preliminary data in melanoma cells demonstrate that both non-invasive and invasive melanoma lines are responsive to TCDD, and invasive melanoma cells express MMPs in response to TCDD exposure. Further, we also show increased invasiveness of melanoma cells when treated with TCDD. This suggests that TCDD directly increases melanoma invasion by activating matrix degradation. Therefore, the specific aims for this proposal are: (1.) Elucidate the molecular mechanism mediating TCDD-induced expression of MMPs in melanoma cell lines. (2.) Elucidate the role of the AhR in TCDD-induced changes in MMP expression in melanoma. (3.) Demonstrate the role of the AhR pathway in melanoma migration and invasion using an in vitro invasion assay. (4.) Using a three-dimensional skin model system, determine the role of cell-cell interaction on AhR-activation of MMP expression.
DESCRIPTION (provided by applicant): Environmental exposures to polychlorinated biphenyls (PCBs) are known in humans as well as lab animals to cause immunosuppression, thyroid disease, endocrine disruption, and damage to the central nervous system. Not all humans or laboratory animals respond similarly to the same dose -indicating interindividual genetic differences. In rodents to elicit these pathologies, planar PCBs must bind to, and activate, the aryl hydrocarbon receptor (AHR). Despite this overriding role for planar-PCB-mediated AHR activation in toxicity, the AHR up-regulates CYP1A2, which in liver sequesters and protects distant tissues against planar PCBs. Both the AHR and CYP1A2 are polymorphic in humans: the AHR exhibits >12-fold differences in ligand-binding affinity; liver basal CYP1A2 shows >60-fold differences in subjects having no known exposure to inducers. With regard to fetal exposure, our studies in mice demonstrate that risk of birth defects by planar TCDD depends on the high-affinity AHR and is also greatly increased in fetuses carried by dams that lack CYP1A2. PCBs represent mixtures having many dozens of different congeners; which congener is toxic, and the rates of uptake, metabolism and excretion are difficult to determine in humans, and most studies in lab animals look at a single congener. We have studied mice with the high- (Ahrb, B6) vs low- (B6.D2-Ahrd) affinity AHR, and with or without the Cyp1a2 gene. Using these mice, we hypothesize that Ahrb fetuses carried by Cyp1a2(-/-) dams will be most susceptible, and Ahrd fetuses carried by Cyp1a2(+/+) dams most resistant, to deficits in learning, memory, and other behaviors caused by planar PCBs. For the funding period, we propose to: [1] determine tissue distribution of each of eight PCB congeners (most relevant to humans) given as a mixture -comparing B6 vs B6.D2-Ahrd, and Cyp1a2(+/+) vs Cyp1a2(-/-) dams and their offspring; and [2] evaluate the in utero and lactational effects of this orally administered PCB mixture on learning, memory, and other behaviors in offspring of these treated dams, starting at postnatal day 60. These studies will define the impact of a fetal basis for adult disease. The Ahr and Cyp1a2 genotypes in these mice represent the extremes for variability of these two genes in the human population. There exist genetic differences in mouse (and human) populations, which represent a gradient of at-risk individuals. Project Narrative: Polychlorinated biphenyls (PCBs) are widespread persistent organic pollutants linked to numerous human health problems, including learning and memory deficits in children of exposed mothers. The Ahr and Cyp1a2 genotypes in our mouse models represent the extremes for variability in these two genes in the human population, and both genes likely play a role in susceptibility following PCB exposure. These studies will define the fetal basis for adult disease and help to identify individuals at greatest risk of PCB-induced neurotoxicity.
DESCRIPTION (provided by applicant): Dioxins, generated both commercially and naturally, are chlorinated polycyclic aromatic hydrocarbons that are highly toxic environmental contaminants. These agents are known to be potent rodent carcinogens and suspected human carcinogens. The best known prototype of this group of agents is 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD). It has been well-documented that most, if not all, of the TCDD effects are mediated through the Ah receptor (AhR). In an effort to better understand the mechanism of TCDD action, we will investigate the molecular mechanism of the AhR signaling pathway. The working hypothesis is as follows: Upon TCDD binding, nuclear translocation of the receptor occurs and the AhR forms the AhR/Arnt/DRE complex in the nucleus, leading to activation of gene transcription. We discovered that p23 and CyP40 potentiate the formation of this AhR ternary complex in vitro (Refs: 1. Shetty, P. V., Wang, X., and Chan, W. K. (2004) Arch. Biochem. Biophys. 429, 42-9; 2. Shetty, P. V., Bhagwat, B. Y., and Chan, W. K. (2003) Biochem. Pharmacol. 65, 941-8) and these proteins appear to affect the AhR signaling in cell culture studies. This proposal focuses on the endogenous roles of p23 and CyP40 in the AhR signaling. Four specific aims have been proposed as follows: We will use p23 and CyP40 knockdown and overexpressed cells to determine whether (1) the heterodimerization of AhR and Arnt and the binding of the heterodimer to the DRE are affected by p23 and CyP40 in intact cells (Aim 1); the assembly of the AhR complex to the enhancer region prior to activation of gene transcription is affected by p23 and CyP40 in intact cells (Aim 2) and (3) the fate of the nuclear AhR is affected by p23 and CyP40. We will also examine the requirements of p23 and CyP40 in the AhR signaling (Aim 4). Deletion and mutation studies will be performed to map out the minimal structural requirement of p23 and CyP40 for the AhR function. Interactions between CyP40 and AhR will be examined and characterized. CyP40-interacting proteins that are essential for the full CyP40 effect on the AhR function will be identified and characterized.
DESCRIPTION (provided by applicant): The Ah-receptor (AHR) is a ligand activated transcription factor that belongs to the family of basic-helix-loop-helix/PER-ARNT-SIM (bHLH/PAS) proteins. AHR-mediated signaling has been extensively investigated in the C57BL/6J mouse model system in an attempt to define the components of the pathway, understand protein and DNA interactions and define specific changes in gene expression that likely impact human health. There has been less emphasis placed on the degradation of AHR protein especially as it relates turning the AHR signaling pathway off. Unfortunately, the proteins that modulate the degradation of the AHR and the domains of the AHR that are necessary for degradation remain to be identified. Thus, multifaceted approaches must be initiated to define candidate target enzymes involved in the degradation process and obtain "gain of function" mutants of the AHR. Therefore, the central hypothesis of this proposal is that defects in protein degradation will be manifest as "gain of function" mutants of the AHR. Three sets of studies are proposed to address this hypothesis and isolate the first ligand- activated gain of function mutants in the AHR pathway. First, studies will be carried out that utilize a bacterial two-hybrid screen to identify proteins interacting with the COOH-terminal domain of the AHR. Second, cell lines expressing a stably integrated GFP tagged AHR will be used to carry out a genetic screen for cells that do not degrade the AHR. Finally, yeast strains will be generated that express a functional AHR signaling pathway. Specific genes involved in protein degradation will be deleted from these strains to assess their role in AHR degradation. Thus, the proposed experiments utilize candidate gene approaches as well as genetic screens and employ mammalian cells, yeast and bacteria to address this hypothesis. The mechanism involved in turning off AHR-mediated signaling and the regulation of this pathway are especially critical with respect to halogenated aromatic compounds that are not readily metabolized or cleared from an organism. In addition, since modeling of AHR signaling and risk assessment in humans relies heavily on understanding the complete signaling pathway, it is essential to determine how the AHR pathway is attenuated. Statement of Relevance: Human health can be adversely affected by exposure to environmental contaminants. For chemicals that act through association with endogenous proteins and modulation of gene regulation, it is essential to understand how the pathways are regulated. Understanding how the Ah receptor protein is degraded is critical to risk assessment of chemicals typified by 2,3,7,8, tetrachlorodibezo-p-dioxin (Dioxin), and will be applicable to other toxicologically relevant receptors (i.e. steroid hormone receptors).
DESCRIPTION (PROVIDED BY APPLICANT): The Ah receptor (AhR) is a ligand-dependent transcription factor that mediates the toxic and biological effects of a variety of chemicals and it is believed that these effects result from receptor-dependent alterations in gene expression in susceptible cells. While the best characterized and highest affinity ligands for the AhR include a variety of toxic halogenated aromatic hydrocarbons, recent studies have revealed that the AhR can be activated by a wide variety of structurally dissimilar chemicals. This observation strongly suggest that the AhR has a promiscuous ligand binding domain (LED) and raises questions regarding the actual spectrum of chemicals which can bind to and/or activate the AhR. Although it's clear that that the AhR plays the key pivotal role in mediating the response of a cell to AhR ligands, there is little information with respect to the specific binding interactions of these ligands within the AhR LED or how these interactions lead to ligand-dependent activation of the AhR and AhR signal transduction. We hypothesize that specific binding interactions between AhR ligands and key amino acids contained within the AhR LBD are responsible for differential ligand affinity and specificity as well as ligand-induced changes in the AhR LBD structure that leads to AhR activation. Testing of this hypothesis has not been previously possible due to the lack of a 3- dimensional model of the AhR LBD. However, our recent development of a homology model of the PAS domain of the AhR LBD provides an avenue by which to carry out a mechanistically-directed structural analysis of these hitherto undefined events. Accordingly, we propose to develop, optimize and validate of homology model of the structure of the AhR LBD based on existing crystal structures for PAS domains from related proteins. Validation of the model will be confirmed using site-directed mutagenesis and characterization of AhR functional activities of mutant AhRs. QSAR and molecular docking approaches will be used to model the specific binding of ligands within the modeled AhR LBDs and will utilize binding data obtained by functional analysis of a library of novel flavonoids and indirubins. Site-directed mutagenesis and AhR functional studies will also be used in combination with modeling approaches to identify and characterize the regions of the AhR and AhR LBD responsible for interactions with hsp90 and the effect of ligand binding on these interactions. Overall, the studies proposed here will not only allow provide the first detailed analysis of the mechanisms by which ligands bind to and activate the AhR, but they will provide new insights into observed differences in species- and ligand-specific binding and activation of the AhR.
Crisp Terms/Key Words: aromatic hydrocarbon receptor, animal genetic material tag, toxicant screening, environmental toxicology, tissue /cell culture, receptor expression, receptor binding, DNA binding protein, protein structure function, gene expression, transfection, eicosanoid, species difference, indole
The overall goal of our research is to understand the molecular mechanism by which halogenated aromatic hydrocarbons (HAHs), polycyclic aromatic hydrocarbons (PAHs) and related chemicals interact with the Ah receptor (AhR) to alter gene expression and responses of cells and animals to these inducers. The AhR is a ligand-dependent transcription factor that mediates the majority of the biological and toxicological actions of HAHs and PAHs. Significant species, tissue- and ligand-specific differences have been reported in the spectrum of toxic and biological responses observed following exposure to HAHs and PAHs but also in the concentration of chemicals needed to produce these responses, with HAHs being significantly more potent than PAHs. Although differential responsiveness to HAHs and PAHs can result from a variety of biochemical and physiological characteristics in target cells, it is generally accepted that the greater toxicological and biological potency of HAHs results from their significantly higher AhR binding affinity and resistance to metabolism. Recent evidence has demonstrated that differences exist in the potency and efficacy of HAHs and PAHs as activators of AhR-dependent gene expression that are separate from those directly related to their persistence and metabolic stability in the cell. We hypothesize that some of the differences in the potency and biological responses produced by PAHs and HAHs are directly related to ligand-specific differences in the structure of the AhR protein and/or AhR protein complex that alters the functionality of the AhR and its relative affinity/specificity for DNA/chromatin. Accordingly, here we propose to conduct detailed comparative studies to characterize the similarities and differences in the activation and persistence of ligand and DNA/chromatin binding of mouse AhR occupied by selected HAHs and PAHs in vitro and in cells in culture and to identify and characterize ligand-dependent changes in AhR structure. The DREnucleotide
specificity for transcriptional activation of gene expression by AhR complexes bound by HAHs
and PAHs and similarities and differences in HAH- and PAH-induction of gene expression assessed in cells culture and CYP null mice using microarrays. Finally, transgenic animals expressing a consitutively active (e.g., ligand-independent) AhR complex will be generated to examine contributions of the AhR and the ligand to the adverse effects associated with this persistent AhR activation. Overall, these studies will provide insights into the species- and ligand-specific differences in the ability of HAHs and PAHs to activate the AhR, the mechanisms responsible for the persistence of this activation and the role that it plays in the toxic and biological effects of HAHs and PAHs.
DESCRIPTION (provided by applicant): Our long-term goal is to understand the molecular mechanisms that govern the cellular stress response, thereby exploring the potential of targeting the cellular stress signaling circuitry for prevention and treatment of human diseases. In this proposal, we will study the integration of the stress signaling network; i.e. "wiring the stress signaling circuitry", using the regulation of UV signaling by the crosstalk between NF-kappaB and JNK1 as a model system.
Using both genetic and biochemical approaches, we recently found that the transcription factor NF-kappaB, which is known as a key survival factor in cells, surprisingly functions as a pro-death factor in UV-induced apoptosis by promoting activation of c-Jun N-terminal protein kinase 1 (JNK1). Specifically, RelA/p65, which is a major transactivating subunit of the NF-kappaB family, in its pre-existing nuclear form controls expression of protein kinase c delta (PKCdelta) in non-stimulated cells. This "priming" effect allows UV to quickly activate PKCdelta, which is required for rapid and robust activation of JNK1 and cell death. We hypothesize that the novel crosstalk between NF-kappaB and JNK1 is critical in "wiring" the UV signaling circuitry in programmed cell death and tumorigenesis.
This proposal is novel, as it will determine the molecular mechanism by which the NF-kappaB-PKCdelta axis regulates UV-induced JNK1 activation and cell death, to elucidate the molecular mechanism by which PKCdelta participates in the integration of the UV signaling circuitry, and to determine the pathophysiological relevance of the novel crosstalk between NF-kappaB and JNK1 in response to physical stress in vivo.
This study will put forward a novel paradigm regarding the molecular mechanism by which the UV signaling circuitry is integrated and will also provide the rationale in developing novel strategies for prevention and treatment of physical stress-related human diseases and cancer. PUBLIC HEALTH RELEVANCE: Ultraviolet (UV) is a major physical stress and is also a complete carcinogen in skin cancer. This research is designed to determine how the information of UV-irradiation is "wired" by the crosstalk between two major cell signaling regulators, NF-:B and JNK1 in physiological and/or pathological events such as programmed cell death apoptosis and tumorigenesis. This study will test a novel paradigm regarding the molecular mechanism by which the UV signaling circuitry is integrated and will also provide the rationale in developing novel strategies for prevention and treatment of physical stress-related human diseases and cancer.
DESCRIPTION (provided by applicant): Background: We have recently discovered a new toxic effect of the aryl hydrocarbon receptor (AhR) ligand 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD or dioxin). Specifically, exposure during pregnancy impairs mammary gland development and suppresses the coordinated induction of milk proteins, resulting in impaired lactation and neonatal mortality.
Objectives/Hypothesis: The objectives of the proposed studies are (1) to further characterize this novel finding and (2) to identify the lactogenic regulatory pathways adversely affected by exposure to dioxin. The hypothesis for these studies is that AhR activation during pregnancy disrupts the normal signaling that directs pregnancy-associated mammary development and milk protein gene expression, resulting in impaired epithelial cell differentiation and lactation. Specific Aims: 1).To determine whether defects in lactogenesis result from direct effects on mammary tissue, we will cross-transplant mammary tissue from wild-type and AhR-null mice. 2) To identify the mechanism underlying impaired milk production, we will determine whether exposure to TCDD deregulates the activation of NF-kappaB-, STATSa- and C/EBbeta-mediated signalling pathways in mammary epithelial cells. 3) To determine whether stunted glandular development during pregnancy results from deregulation of proliferation, differentiation, apoptosis or defects in multiple pathways, we will further characterize the effects of exposure to TCDD on these processes in mammary cells during pregnancy. 4) To identify additional molecular pathways that are deregulated following exposure to TCDD, we will compare the expression of factors known to regulate to glandular differentiation and lactogenesis in glands derived from vehicle- and TCDD-treated pregnant mice using a combination of gene expression profiling and immunocytochemical methods. Mammary tissue from AhR-null mice will be used to distinguish defects that are directly AhR-mediated from defects that arise due to an upstream lesion. Significance: The proposed studies address an area that is clinically-relevant but has received very little attention. An estimated 3-6 million mothers of live infants annually are either unable to or have significant difficulty initiating breastfeeding. The causes of this problem are not clear, and very little is known about the effects of exposure to environmental contaminants on lactogenesis. Furthermore, since the mechanisms that control lactogenesis also regulate proliferation and differentiation in other organs, and exposure to AhR ligands disrupts the proliferation and differentiation of epithelial cells in other tissues, findings from these studies will have broad biological significance, and will help us better understand the mechanisms by which dioxin-like chemicals adversely affect epithelial cells throughout the body.
DESCRIPTION (provided by applicant): Breast cancer is one of the leading causes of premature death in women and development of chemoprevention and chemotherapeutic interventions wilt require understanding of the important steps that lead to tumor formation, growth and metastasis. Estrogen receptor (ER)-positive and -negative breast cancer cells have been extensively used as models for studying signaling pathways that are important for breast tumor growth. Both 17beta-estradiol (E2) and polypeptide growth factors (GFs) have been identified as important mitogens for breast cancer cell growth. We hypothesize that ERalpha-dependent activation of nongenomic phosphatidyl inositol 3-kinase (PI3-K) and mitogen-activated protein kinase (MAPK) pathways by E2 are important for breast cancer cell proliferation, and the proposed studies will investigate the mechanisms of non-genomic activation by E2. Aim 1 will focus on the role of ERalpha in activation of PI3-K and MAPK using ER-positive breast cancer cells (MCF-7 and ZR-75) and ER-negative (CHO and COS) cells as models. The domains of ERalpha required for activation of kinases and the importance of cell context will be determined in transient transfection studies. The mechanisms of non-genomic action of E2 and the role of specific kinases in mediating activation of PI3-K and MAPK will be investigated in Aim 2. The studies will also determine the biological significance of direct ERalpha interactions with Src-SH2, G proteins, the p85 regulatory subunit of PI3-K and the IGF-1 receptor. Aim 3 will focus on downstream mechanisms of ERalpha-dependent activation of c-fos by kinases. Dominant negative p85, MAPKK and Src expression plasmids transiently or stably transfected into breast cancer cells will be used to investigate the contribution of the MAPK and PI3-K pathways to the mitogenic activity of E2 in breast cancer cells (in vitro) and mammary tumors in athymic nude mice bearing breast cancer cell xenografts. The in vitro mechanistic studies will also be complemented by research on the contributions of kinase activation by E2 on cell proliferation and tumor growth and thereby define potential therapeutic targets for treating breast cancer.
Crisp Terms/Key Words: athymic mouse, biological signal transduction, cell growth regulation, estradiol, transfection, gene expression, protooncogene, growth factor, insulinlike growth factor, breast neoplasm, neoplastic cell, neoplastic growth, protein structure function, G protein, growth factor receptor, estrogen receptor, tissue /cell culture, enzyme activity, MCF7 cell, mitogen activated protein kinase, phosphatidylinositol 3 kinase, cell proliferation, fos protein, protein protein interaction
DESCRIPTION (provided by applicant):
Children born small for gestational age (SGA) have a significantly elevated risk of cardiovascular and metabolic diseases in adulthood; however, data is limited on how SGA may impact gonadal development and reproductive health. Epidemiological studies and registry surveys demonstrate that altered intrauterine growth increases the risks of congenital hypospadias, cryptorchidism and testicular cancer approximately 2- to 3-fold. Evidence for these outcomes points towards alterations in the normal functions of Sertoli and Leydig cells. Both animal and human studies suggest that impaired peri-pubertal growth can affect testis size and function into adulthood. A study by Main et al., examined testis growth from birth to 3 months of age in healthy Finnish and Danish newborns and found a significant correlation between weight for gestational age and testis size. Therefore, elucidating signaling networks which modulate peri-pubertal testis growth and identifying environmental toxicants which impinge upon these pathways will significantly impact environmental health. The Akt gene family effects testis growth. Akt1 -deficient mice are born small for gestational age and have significantly smaller testis throughout their lifetime. Preliminary data indicate that the Akt1 signaling pathway plays a significant role in maintaining peri-pubertal testicular homeostasis. A striking sensitivity is observed in vivo for germ cell apoptosis in testis of Akt1-deficient mice exposed to MEHP, a peri-pubertal Sertoli cell toxicant, and a reduction in testis and reproductive potential in mice exposed to 6-N-propylthiouracil (PTU), a thyroid toxicant which targets Sertoli cell proliferation and differentiation. Based on these findings, it is hypothesized that the Akt gene family plays a critical role in peri- pubertal testis development and that toxicants which target the Sertoli cell at this developmental window provide a crucial link between the environment and the etiology of male reproductive disease. The hypothesis will be tested through the following Specific Aims: 1.) Identify the mechanisms by which Akt1 suppresses Sertoli cell mediated germ cell apoptosis following MEHP exposure. 2.) Identify the mechanisms by which Akt1 promotes testis growth and development following PTU exposure, and 3.) Identify relevant Sertoli cell toxicant-induced stress response networks. Relevance: This research will define critical signaling networks targeted by peri-pubertal reproductive toxicants and delineate how they impact male reproductive health.
DESCRIPTION (provided by applicant): We propose that pattern recognition receptors, whose normal function is in innate immune responses, are also activated by stress-generated ligands, such as molecules released by dying cells, normal and oxidized lipids and a variety of so called "danger" signals in addition to environmental toxins and pathogen associated molecular patterns. Activation of such receptors either by endogenous- (i.e. host-generated) or pathogen- generated ligands turns on stress-activated protein kinases, such as JNK, p38 MAPK and IKK, that serve as molecular transducers that contribute to development of chronic inflammatory diseases. Importantly, these pathogenic mechanisms allow the integration of environmental factors and genetic susceptibility loci that together contribute to the development of some of the most common chronic diseases, including type 2 diabetes, asthma, inflammatory bowel disease and chronic liver disease. In the previous grant period we have generated strong evidence in support of this hypothesis by focusing on the pathogenic functions of the JNK and IKK signaling pathways. We also generated a mouse model expressing the equivalent of the most common susceptibility allele for Crohn's disease, an inflammatory bowl disease whose pathogenesis is affected by genetic and environmental factors. In the present period we will focus our main effort on the pathogenic function of different classes of pattern recognition receptors as targets for stress - and injury- generated stimuli, as well as continue with our studies on the role of p38 MAPK in liver inflammation and toxicity. More specifically we will examine: 1) the role of protein kinase C isozymes and Toll like receptors (TLRs) in obesity-induced JNK activation and insulin resistance; 2) the role of TLRs in toxin-induced liver injury, liver inflammation and liver cancer; 3) examine the role of p38 MAPK in toxin-induced liver injury, liver inflammation and liver cancer; 4) examine the mechanism by which the intracellular NOD-like receptor NOD2 leads to activation of caspase 1 and IL-lbeta secretion; 5) construct a conditional mouse mutant that allows constitutive NOD2 activation and use it along with our previously generated Nod2delta33 knockin mutant to examine effects of tobacco smoke, microparticles and bacterial products on development of NOD2- modulated colonic and airway inflammation. To accomplish these aims we will use a combination of cellular biochemistry, molecular genetics and experimental pathology, an approach that has been proven effective during the previous project period.
DESCRIPTION (provided by applicant):
It is widely appreciated that environmental stresses, such as exposure to toxins or pathogens, can have profound impact on individual health and well-being. How these "stressors" exert their influence is less well understood. While specific targets of environmental stresses have been identified for some toxins and pathogens, we propose that the impact of environmental stresses is mediated through the pathway(s) and/or networks in which the target molecule participates. Furthermore, even where a causal association between an environmental stress and a biological target has been identified, the biological insight that must precede a strategy for therapeutic intervention has generally been slow in coming. We suggest that the phenotypic effects of environmental stresses are mediated by alterations in a dynamic network of gene products and metabolites, and such networks, normal and perturbed, exhibit emergent properties that cannot be understood one gene at a time. Our central hypothesis is that one must understand changes in complex cellular networks to fully understand the link between genotype, environment, and phenotype. The Center for Cancer Systems Biology (CCSB) at the Dana-Farber Cancer Institute, together with a growing number of laboratories, is mapping and modeling cellular pathways and networks both "locally" at the scale of "molecular circuits" made of a few interacting molecules, and "globally" at the scale of the whole proteome; fundamental biological insights have already emerged from this body of work. We propose to use a model system as a surrogate for generalized environmental stresses to begin understanding how cellular pathways and networks are altered or modified as a consequence of environmental insult. We have chosen a defined, model system with a variety of disease outcomes: viral infection. Here we explore the concept that environmental stresses, as exemplified by viruses, influence local and global properties of networks to induce "disease states". Our plans to achieve these goals are summarized in the following specific aims: 1) Profile all binary viral-host protein-protein interactions for a group of viruses with related biological properties 2) Systematically test each viral protein for its ability to disrupt or alter host-host protein interactions, 3) Integrate the resulting interaction and perturbation data with diverse genomic data sources to derive dynamic cellular network models.
DESCRIPTION (provided by applicant): The aryl hydrocarbon receptor (AhR) is an orphan nuclear receptor and a central mediator of the effects of an entire class of environmental toxicants. The AhR has an undefined role in normal and disrupted embryogenesis as well. Reproductive senescence is accelerated by 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD, dioxin), the specific and potent AhR ligand, at environmentally relevant exposures and one consequence is a decline in oocyte and subsequent embryo quality. As women increasingly delay attempts at pregnancy into their 30s and beyond, any acceleration of reproductive aging has dire consequences for the possibility of successful pregnancy and the risk of birth defects. Furthermore, early reproductive senescence in women is associated with decreased lifespan and quality of life. Although the effects of AhR ligands on oocyte numbers have been studied extensively, little is known about the AhR, oocyte quality and subsequent organization of the early embryo. In our preliminary work, we have identified early chromosomal and cytoskeletal defects that may be pathognomonic for exposure of oocytes and embryos to AhR ligands such as dioxins; this can explain the decreased fertility resulting from chronic exposure with age. The objective of this project is to identify checkpoints of oocyte development and early embryogenesis that are regulated by AhR ligands. Our central hypothesis is that environmental exposure to AhR ligands reinforces the decline in oocyte and embryo quality with age. This work, once completed, promises new insights to prevent the loss of fertility in polluted environments and slow the loss of fertility with age. The research team will achieve these experimental goals through the following specific aims: Specific Aim 1: Determine the effect of defined AhR activation on critical periods of oocyte maturation and early embryogenesis in vitro. This aim encompasses the most detailed investigation into the impact of AhR activation or antagonism on the loss of oocyte and embryo quality to date. Endpoints include embryonic morphology, cytoskeleton and chromatin and its epigenetic modulation in oocytes and embryo. Specific Aim 2: Determine the impact of chronic, environmentally relevant AhR activation and antagonism on oocyte and embryo quality during normal and accelerated reproductive senescence. Oocytes and embryos from young and middle aged rats will be assessed for morphological and cytoskeletal conformation and chromatin remodeling and epigenetic modification and imprinting from oogenesis to early embryogenesis. This will provide insight into decreased oocyte and embryo quality and fertility with age and the importance of the AhR pathway in the aging ovary and oocyte. This work is novel through use of realistic exposures to AhR agonists and a focus on subtle measures oocyte and embryo quality and epigenetics. These studies explore a novel diagnostic indicator of compromised oocyte and embryo quality due to exposure to toxic AhR ligands. This project will provide crucial knowledge for the identification and prevention of infertility and birth defects due to the AhR pathway in older mothers. Hundreds of manmade compounds including dioxins, polychlorinated biphenyls and carcinogens in cigarette smoke act on cells through the aryl hydrocarbon receptor (AhR) pathway. We have recently reported detrimental effects of AhR activation on aging of the ovary and a number of defects in eggs and embryos from females exposed to AhR ligands. This project will provide crucial knowledge for the identification and prevention of infertility and birth defects due to the AhR pathway in older mothers.
DESCRIPTION (provided by applicant): Understanding the molecular details of
dioxin/Ah receptor (AHR) signal transduction will yield a number of benefits.
First, identifying modifiers of AHR signaling will help explain the tissue and
species specificity of dioxin toxicity and thus will aid attempts to predict
the human and environmental risk that results from exposure to these
pollutants. Second, the AHR is a prototype of a large super family of
environmental sensors. What we learn here will shed significant light on a
variety of environment-gene interactions. Third, it is likely that the AHR will
be involved in a variety of human disorders. Detailed information about
signaling can guide the pharmacology necessary to develop related therapeutics.
Finally, identifying endogenous activators of the AHR will provide one of the
most significant clues as to how and why this protein signals in development
and begin to explain the conservation of this protein throughout evolution. In
an effort to answer these questions, we propose the following specific aims:
Aim 1: Identify/understand modifiers of AHR signaling through the use of
genetic screens in the yeast, S cerevisiae. We will employ two screening
approaches in yeast. First we will use a library of deletions to determine the
role of each yeast gene product on AHR signal transduction. Second, we will
expand upon these screens by using mammalian cDNAs to perform high copy
modifier screens in yeast. Identified modifiers will then be characterized
using both statistical clustering of their pharmacological action, as well as
biochemical approaches to elucidate mechanism. Aim 2: Understand the mechanism
of AHR modifiers through the use of signaling kinetics in mammalian cell
culture. We propose to use fluorescently tagged molecules and reporters to
elucidate the cellular mechanisms of AHR modifiers using a mammalian culture
system. Once developed, this system will also serve as the basis for an
activator screen described in Aim 3. Aim 3: Determine how the AHR signals
during development. The phenotype of Ah null mice suggests the existence of a
developmental activator of the AHR. To identify this factor, we propose to: 1)
identify those developmental sites where the activator is most likely to be
expressed; 2) screen for cDNAs encoding protein factors that activate the AHR
using an activator trap protocol in cell culture; and 3) characterize the
soluble factor derived from murine heart that activates the AHR in cell
culture. Aim 4: Complete the high-resolution domain mapping studies of the AHR.
DESCRIPTION (provided by applicant): Elucidation of the specific steps involved in signal transduction pathways activated in cellular responses to DNA damage and other cellular stresses can provide critical insights into the genesis of malignancies and suggest new therapeutic approaches. Inhibitors of topoisomerase I have become an important component of our armamentarium in treating human cancers, however, little is known about the signal transduction pathways that participate in cellular responses to these agents. Experiments are proposed here that will elucidate the signaling pathways that are activated in cells following exposure to topoisomerase I inhibitors, including the roles of ATM and/or ATR and the numerous proteins that have been found to be downstream of these protein kinases in other cellular responses. Hypoxia is a cellular stress that plays a critical role in tumor development and appears to influence tumor prognosis. Studies are proposed that will also elucidate the cellular signaling pathways that determine cellular outcome in the presence of hypoxic stress. Since many of the steps in these pathways participate in controlling specific stages of the cell cycle, these characterizations should provide new insights into the nature of the cellular lesions that are present following exposure to these cellular stresses. P53 protein is one of the critical modulators of cellular responses to stress and a series of proposed experiments specifically address the regulation of p53 protein levels and function following DNA damage and hypoxia. In particular, continued or enhanced translation of p53 protein in the face of these stresses suggests a unique mechanism of translational regulation and the role of the 5'-UTR of p53 in this process will be investigated. In addition, experiments are proposed that will elucidate the importance of post-translational modifications of p53 protein and cellular redox status in controlling the ability of p53 protein to bind to specific DNA promoter sequences following stress. Finally, one of the major goals of understanding the molecular basis of these responses is to improve interventions in the treatment of cancer. Towards this end, responses of tumor xenografts with specific lesions in one or more of the signaling steps in these pathways will be examined. In tow, these studies should provide significant new information that has implications for both tumor development and therapy.
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