Jennifer Schlezinger - US grants

Many studies have demonstrated that aryl hydrocarbon receptor (AhR) agonists, including polynuclear aromatic hydrocarbons (PAH) that are ubiquitously distributed in the environment, adversely affect the immune system. Epidemiological analysis of human populations exposed to AhR agonists revealed multi-faceted immune dysfunction in those individuals. Studies in the Sherr laboratory have shown that PAH rapidly induce apoptosis in murine bone marrow stromal cell-dependent preB cells. The preB cell "death signal" was shown to be produced by the stromal cells by an AhR-dependent mechanism. Initial studies have shown that PAH activate NF-kappaB-DNA binding in bone marrow stromal cells and suggested that NF-kappaB also may participate in the pathway leading to production of the "death signal." Therefore, the specific aims of this study were developed to address the hypothesis that AhR agonists alter the NF-kappaB signaling pathway in bone marrow stromal cells, resulting in adverse biological responses potentially including the production of the preB cell "death signal." We will: 1) determine how NF-kappaB is modulated by a model PAH/AhR agonist in bone marrow stromal cells, 2) define PAH-mediated alterations in the kinase signaling cascade upstream of NF-kappaB, and 3) define requirements for AhR-p65 association and transcriptional activity.

In keeping with the programmatic theme of developmental toxicity, the goal of these studies is to determine the molecular mechanisms by which environmental chemicals impair the development of the mammalian immune system. Our previous studies demonstrated that environmentally ubiquitous aryl hydrocarbon receptor (AhR) agonists profoundly affect immune system development by inducing bone marrow pro- and pro/pre-B lymphocyte death. In the course of defining the molecular mechanisms through which this immunotoxicity is manifest, we found that other environmental chemicals included on the list of priority chemicals designated by the Agency for Toxic Substances and Disease Registry, notably agonists of the peroxisome proliferator activated receptor/ (PPARy) such as di-(2-ethylhexyl) phthalate/ mono-(2-ethylhexyl) phthalate, deliver a potent death signal that involves intracellular signaling pathways distinct from those activated by AhR ligands. Furthermore, an endogenous bone marrow-derived PPARy agonist, 15-deoxy-Al2|I4-prostaglandin J2, or a naturally occurring RXRa agonist, 9-cw-retinoic acid, enhances the inhibition of B cell proliferation. Our working model of PPARy agonist-induced death, which is based on a considerable foundation of new information, proposes a pathway that involves the activation of and interaction between caspases, kinase signaling cascades and NF-KB. Accordingly, three specific aims are proposed: 1. Map PPARy agonist-induced caspase-dependent bone marrow B cell apoptosis signaling pathways. 2. Map the kinase activation cascade in PPARy agonist-induced death and define its relationship to caspase and NF-KB activation. 3. Define the molecular mechanisms of chemical synergy resulting in developing B cell apoptosis. These studies not only will contribute to our understanding of the molecular effects of PPARy agonists on developing B cells, but also will validate a platform that is easily applicable to the study of other immunotoxic environmental chemicals, either individually or in complex mixtures, at the molecular level.

DESCRIPTION (provided by applicant): Historically, the aryl hydrocarbon receptor (AhR) has been studied for its transcriptional regulation of genes encoding cytochrome P450 enzymes, which metabolize environmental and endogenous substrates into toxic and mutagenic intermediates. Accumulating studies support the hypothesis that the AhR also plays an important role in malignant epithelial cell growth and invasion apart from its role in formation of mutagens and in the absence of environmental chemicals. This new paradigm is based on several key observations: 1) AhR expression is increased dramatically in carcinogen-induced rat and mouse mammary tumors and in "spontaneous" human mammary tumor lines. 2) Constitutive AhR activation is indicated by nuclear AhR localization in rat, mouse, and human mammary tumors and by AhR binding to gene promoters in the absence of environmental chemicals. 3) Constitutively active AhR regulates the expression of multiple genes, including CYP1B1, CK21, and Slug, a master regulator of tumor invasion. 4) Recent studies suggest that increased AhR activity in mammary tumors also contributes to cell migration and invasiveness. 5) Molecular downregulation of the AhR suppresses breast cancer cell proliferation and reverts cells to a non-aggressive phenotype. Molecular and biologic strategies have provided significant evidence that the AhR participates, beyond mutagenesis, in multiple mechanisms that contribute to tumor formation, growth and invasion. Therefore, we can exploit our ability to examine effects of constitutively active AhR to determine how chemical antagonism of the AhR may translate into breast cancer prevention or a therapeutic approach to suppress tumor progression. Thus, we propose a new hypothesis: Targeting the constitutively active AhR with naturally occurring, non-toxic antagonists represents a feasible therapeutic approach to inhibit breast tumor growth and invasion. Three specific aims are proposed: 1. Investigate strategies to maximize antagonism of the AhR by examining the potential for synergistic interaction in mixtures of antagonists, performing a high-throughput screen for novel, potent antagonists from natural product extract libraries (NCI Natural Products Repository) and examining the "chemical knockout" approach for improving AhR inactivation. 2. Define the molecular mechanisms of chemical antagonism of the constitutively active AhR in a breast cancer model by establishing antagonist effects on AhR transactivation of endogenous gene expression and examining antagonist-mediated changes in AhR-DNA interactions. 3. Establish the functional consequences of chemically antagonizing the constitutively active AhR using optimal AhR antagonists. The translational impact of these studies lies in the ability of known and newly identified antagonists to suppress tumor growth and invasion. Here, potentially therapeutic AhR antagonists will be evaluated for their ability to block the biological outcomes of constitutive AhR activity in human mammary tumor cell lines. Collectively, these studies will provide the foundation for preclinical studies on the potential for potent AhR antagonists to prevent and/or treat breast cancer in vivo. PUBLIC HEALTH RELEVANCE: We hypothesize that the hyper-expression of a protein, called the aryl hydrocarbon receptor, and its binding to DNA contributes to the growth and progression of breast tumors. Here we propose that chemicals that impede the function of this receptor (i.e. antagonists) will be effective at downregulating this protein's activity and therefore will suppress breast tumor growth and metastasis. Screening of plant and marine natural product libraries will provide a source of novel antagonists that can be tested for their interaction with this receptor and their mechanism of interference with tumor growth, ultimately resulting in the development of therapeutic agents for the treatment of breast cancer.

DESCRIPTION (provided by applicant): Osteoporosis is the primary public health threat for the aging population. Osteoporosis has been likened to obesity of the bone because normal bone mass is lost as it is replaced with adipose tissue. A well recognized risk factor for development of osteoporosis is the decline of estrogen secretion in women at menopause. A newly recognized risk factor is a high fat diet and obesity. An underappreciated aspect of this bone health crisis is the contribution of exposure to environmental obesogens, contaminants that disrupt the homeostatic controls of adipogenesis and energy balance. A growing number of environmental contaminants, including organotins and phthalates, are being recognized for their ability to activate peroxisome proliferator activated receptor gamma (PPAR?), the master regulator of adipocyte differentiation. Activation of PPAR? in bone is associated with increased adipogenesis and decreased osteogenesis. Since PPAR? is poised at the apex of a regulatory network that controls multi-potent marrow stem cell (MSC) differentiation, it is posited that environmental obesogens are bone marrow toxicants. What is unclear is how exposure to both a high fat diet and environmental obesogens cooperate to modify bone homeostasis. The long term goal is to determine how activation of nuclear receptors in the bone marrow by environmental contaminants modifies MSC differentiation and how skewed MSC differentiation impacts bone marrow function. The objective of this proposal is to examine how exposure to a high fat diet and environmental obesogens cooperate to impair osteogenesis. It is hypothesized 1) that environmental obesogens induce adipogenesis and suppress osteogenesis through activation of PPAR? and RXR, which is a central point of regulatory control in directing MSC differentiation between osteogenic and adipogenic lineages, 2) that co-exposure to dietary fatty acids facilitates adipogenesis and 3) that exposure to a high fat diet will synergize with environmental obesogens to accelerate the progression of osteo- porosis in vivo. By pursuing the following two Specific Aims these hypotheses will be tested: 1) Determine the functional interactions of fatty acid and obesogen exposure and their effects on the mechanisms that control MSC differentiation. Alterations in central transcriptional mechanisms that control the balance between adipogenic and osteogenic differentiation that are induced by a phthalate, an organotin, and dietary fatty acids in primary MSC cultures and define the qualitative/quantitative effects of co-exposure will be delineated. 2) Examine the effect of a high fat diet on obesogen-induced osteoporosis in vivo. The effect of low dose tributyltin (TBT) exposure and diets differing in fat content in intact and ovariectomized female C57BL/6 mice on bone structure, quality and expression of mediators of adipogenesis, osteogenesis and osteoclast activity will be investigated. Given the growing aging population that is already at risk for development of osteoporosis, it is urgent that the molecular mechanisms driving diet- and contaminant-driven suppression of osteogenesis be identified so that appropriate approaches can be developed to prevent the onset/progression of disease.

Peroxisome proliferator activated receptor y (PPARy) is poised at the apex of a regulatory network that controls bone physiology, yet it remains unclear how activation of PPARy in the bone marrow may alter the microenvironment that supports life-long B cell development. This is an important problem, as a growing number of environmental contaminants, including Superfund chemicals such as phthalates and organofins, are being recognized for their ability to acfivate PPARy and its heterodimerization partners the retinoid X receptors (RXR). Our long-term goal is to understand the molecular mechanisms by which individual and complex mixtures of Superfund chemicals impair development in the mammalian immune system, a system that requires ongoing development in the face of continuing pathogen exposures. The objective here is to determine the role of PPARy acfivafion in phthalate- and organofin-induced alteration of bone marrow physiology. We hypothesize that environmental PPAR/RXR ligands suppress B lymphopoiesis by two mechanisms, directly by inducing apoptosis in eariy B cells and indirectly by altering the bone marrow microenvironment that supports lymphopoiesis, resulting in aging-like suppression of immune responses. We will investigate this hypothesis by pursuing three Specific Aims: 1) Determine the relationship between PPAR and RXR acfivation and the functional consequences for multipotent mesenchymal stromal cell differentiation by determining changes in the osteogenic transcriptome induced by a phthalate, an organotin, and contaminant mixtures, 2) Determine the mechanisms by which environmental PPAR/RXR agonists damage B lymphopoiesis, both directly and indirectly by defining mechanisms of toxicant-induced apoptosis and by testing contaminant-altered bone mamow environments for the ability to support B cell development, and 3) Determine mechanisms by which in vivo exposure to environmental PPAR/RXR agonists negatively affects bone physiology, lymphopoiesis and immune responses by examining organotin-induced defects in bone integrity, B cell development and B cell function. Critical knowledge will be gained to refine human risk assessment and to improve prevention of both bone loss and immune compromise.

Project Summary / Abstract Obesity and low bone quality (osteopenia/osteoporosis) are prevalent public health issues worldwide, contributing significantly to metabolic disease and fracture risk. A growing body of evidence suggests that environmental exposures are contributing to the incidence and severity of these pathological conditions. Both legacy Superfund chemicals (organotins, polychlorinated biphenyls (PCB)) and Superfund chemicals of emerging concern (organophosphate flame retardants) have been recognized as metabolic and bone disruptors. While a strong physiological coupling of obesity and osteoporosis may seem unlikely, the recent discoveries of fat and bone regulatory crosstalk suggest that they share common origins. Adipose and bone tissue each contain multipotent cells whose differentiation and fate are regulated by common nuclear receptors. One such nuclear receptor is the peroxisome proliferator activated receptor ? (PPAR?), an essential regulator of adipocyte differentiation and function and a negative regulator of bone homeostasis. Critical gaps in our knowledge prevent our understanding of the potential for environmental PPAR? activators to contribute to development of disease, including: 1) how environmental PPAR? ligands disrupt metabolic health, whereas therapeutic PPAR? ligands used to treat type 2 diabetes improve metabolic health; 2) whether developmental exposures program both adipose and bone dysfunction in adulthood, and 3) how environmental PPAR? activators interact with other classes of adipose- and bone-disrupting chemicals (e.g. PCBs). Our novel and compelling preliminary data show that that tributyltin (TBT) alters adipose and bone differentiation in vitro and adipose and bone homeostasis in vivo, through its interaction with PPAR? and with the retinoid X receptor (RXR). We identified a novel PPAR? ligand, the common organophosphate flame retardant triphenyl phosphate (TPhP), which coordinately enhances adipocyte differentiation and suppresses bone formation in vitro. TPhP stimulates adipose accumulation and hepatic steatosis following adult exposures, and lipodystrophy and systemic inflammation following perinatal exposures. TPhP and TBT activate PPAR?, but also induce adipocyte transcriptomes that are distinct from those induced by a therapeutic PPAR? ligand. Here, we propose to investigate the hypothesis that TPhP and TBT selectively modulate PPAR??s activation and function to compromise adipose and bone homeostasis. We will address three Specific Aims: 1) to determine how early life exposures to TPhP and TBT impact adipose and bone homeostasis in adulthood; 2) to define how selective activation of PPAR? by TPhP and TBT modifies adipocyte function, and 3) to define the roles of PPAR? and RXR in disruption of osteoblast/osteoclast function and crosstalk by TPhP and TBT. Collectively, our work will enhance our knowledge of the physiological processes driving adipose and bone dyshomeostasis and of how early life toxicant exposures exacerbate risk of developing disease, contributing to our ability to minimize/prevent chronic disability caused by exposure to Superfund chemicals.

Project Summary We are in the midst of an unprecedented, modern pandemic as a result of the evolution of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019. SARS-CoV-2 is one of three known coronaviruses that can replicate in the lower respiratory tract and cause pneumonia and acute respiratory distress syndrome, which can be fatal. However, people are not equally susceptible to development of severe SARS-CoV-2 infection disease (COVID-19). Some risk factors are known, including male sex and comorbidities related to metabolic disease. This pandemic has occurred during an endemic exposure to a class of chemicals called per- and polyfluoroalkyl substances (PFAS) in the United States. Daily exposures occur via PFAS contaminated food, drinking water, dust and air, resulting in nearly universal detection in people examined. What we do not know is how PFAS exposure may influence susceptibility to SARS-CoV-2 infection and COVID-19. SARS-CoV-2 infects airway epithelial cells, triggering a Th1-polarizing pro- inflammatory response. Resolution of the infection is driven by CD8+ T cell-mediated clearance of infected cells and inactivation of the free virus by antibody-binding. Disease severity is associated with lymphopenia and reduced IFN-? production by CD4+ T cells. PFAS are well-known immunosuppressive agents in rodent models, and PFAS are associated with reduced antibody titers following vaccinations in humans. Our data, and others, show that PFAS are agonists for nuclear receptors, including peroxisome proliferator activated receptor ? (PPAR?), constitutive androstane receptor (CAR) and pregnane X receptor (PXR) and that their respective transcriptional programs are upregulated following in vivo exposure. Intriguingly, activation of at least PPAR? and PXR in T cells results in Th2-skewing, reduced IFN-? production, and lymphopenia. Here, we propose to examine the interaction between exposure to legacy (perfluorooctanoic acid, PFOA) and replacement (perfluoro(2-methyl-3-oxahexanoic) acid, GenX) PFAS and infection with SARS-CoV-2. We will test the hypothesis that PFAS exposure enhances susceptibility to SARS-CoV-2 infection via interaction with nuclear receptors. First, there are critical, species-specific differences in proteins regulating susceptibility to SARS- CoV-2 infection (angiotensin-converting enzyme 2 (ACE2)) and the biological effects of PFAS (PPAR?). In Aim 1 we will generate a novel hACE2/hPPAR? transgenic mouse and examine the effects of SARS-CoV-2 infection in mice with human relevant steady-state body burdens of PFAS. Second, efficient CD4+ T cell function is essential for minimizing risk of developing COVID-19. PFAS activate multiple nuclear receptors known to regulate immune function and T cell function. In Aim 2, we will use adeno-associated virus-mediated transduction of PPAR?, CAR and PXR shRNA in vivo, to test the necessity for each receptor in enhancing susceptibility to SARS-CoV-2 and how PFOA?s effects are modified. The results will provide essential information on how concurrent exposures to environmental chemicals enhance the risk of severe COVID-19.