Nuclear Receptors: Connecting Human Health to the Environment

STEFANO LORENZETTI AND LAURA NARCISO

Istituto Superiore di Sanità – ISS, Department of Food Safety and Veterinary Public Health, Food and Veterinary Toxicology Unit, viale Regina Elena 299, 00161 Rome, Italy

1.1 Introducing Nuclear Receptors

Nuclear receptors (NRs) are evolutionary conserved proteins whose encoding genes are expressed in the animal kingdom (metazoans); they are also present in animals that do not have any endocrine system. NRs function as transcription factors activated by small (<1000 Da) lipophilic compounds able to cross the plasma membrane and, owing to their discovery as mediators of the sex steroid hormones, they were initially defined as endocrine receptors, although recently some of them have been suggested to act as sensors of their environment interacting with ligands external to the host organism (xenobiotics). Indeed, such ligand-activated transcription factors control levels of both xenobiotics (i.e., man-made chemicals such as pesticides and plasticizers) and endobiotics (i.e., endogenous chemicals such as sex steroid and thyroid hormones, vitamins) since the xenosensing activity of the NRs developed evolutionarily to support the spread of metazoans during the Cambrian age through the setting up of a whole endocrine system. The survival of all organisms (i.e., metazoans) relies on energy maintenance (via dietary intake, storage and utilization) and self-propagation (via reproduction), two physiological activities completely controlled by the central nervous system (CNS) through the signaling to the peripheral effector tissues/organs: NRs allow different, multiple signals to be integrated between central and peripheral organs acting as xenosensors and orchestrating hormone-dependent signaling.

1.2 Linking the Environment to the Human Organism: Nuclear Receptors as Mediators of the Action of Endocrine-active Compounds (EACs)

Ligands of NRs are usually defined as endocrine-active compounds (EACs) or endocrine-disrupting chemicals (EDCs), substances able to interfere with the function of hormonal systems affecting human and wild-life health, for example, contributing to developmental, reproductive and metabolic diseases. Although many EACs are xenobiotics – man-made chemicals manufactured by industry and released into the environment (e.g., pesticides, plasticizers, flame retardants, organotins, alkylphenols dioxins, polychlorinated biphenyls) – many others are naturally occurring (e.g., phyto- and mycoestrogens), being present in plants or fungi as part of their defensive mechanism against physical and biological stresses. Either via the environment or via the food chain, exposure to EACs is usually persistent and common to a wide range of compounds whose role might be potentially both harmful (e.g., xenobiotics) and/or beneficial (e.g., phytoestrogens). Indeed, industrialized and agricultural areas are typically polluted with a wide range of chemicals spread into the air, soil and groundwater, hence people working with (or in some cases living near sources of) pesticides, fungicides and other man-made chemicals are particularly exposed to these toxic compounds and thus have a higher risk of developing reproductive and/or endocrine dysfunction. With dietary and environmental exposure, EACs can affect the endocrine system by (i) mimicking natural hormones, (ii) antagonizing their action or (iii) modifying their synthesis, metabolism and transport. Most of the reported harmful effects of EACs are attributed to their interference with hormone-like, NR-mediated signaling.

1.3 How Nuclear Receptors Work

As mentioned above, human NRs (Table 1.1) constitute a superfamily of 48 ligand-activated transcription factors able to regulate cognate gene networks involved in key physiological functions such as cell growth and differentiation, development, homeostasis or metabolism. NRs have a fairly simple and conserved general structure and, as depicted in Figure 1.1a, is constituted by five distinct domains characterized by subdomains having specific functions. The modulatory A/B domain, at the amino terminus of each NR, contains the transcriptional activation function (AF-1) that, together with the AF-2 domain, takes part in receptor dimerization, nuclear localization and binding to co-activators and co-repressors (Figure 1.1b). The C domain is highly conserved since overlaps with the DNA-binding domain (DBD), a region recognizing NR-specific response elements (NR-RE) in the promoter sequences of targeted genes. The DBD contains two zinc fingers and TA boxes: the first zinc finger has a highly conserved sequence, termed P-box, involved in NR–DNA helix binding, whereas the second zinc finger contains a D-box necessary for protein–protein interactions and also for NR binding to the NR-RE. Finally, the TA boxes within the DBD are essential in NRs acting as monomers. NR binding specificity relies on the orientation of the binding sites (the single consensus binding site being the sequence AGGTCA) that can be formed by AGGTCA inverted, reverted or direct repeats, and also on their separation (from one to eight nucleotides) between the two single consensus binding sites. Indeed, whereas some NRs bind with high stringency to the consensus binding site, others display greater flexibility. The D domain is a short hinge region allowing each NR to undergo conformational changes upon ligand binding. The E–F domain, close to the carboxy terminus, contains the ligand-binding domain (LBD), a region containing the AF-2 domain that, as mentioned above, participates with the AF-1 domain in receptor dimerization, nuclear localization and binding to co-activators and co-repressors. The LBD is structured in α-helices that provide conformational flexibility to this region, allowing the DBD–LBD interaction and thus contributing also to the recruitment of...