Cloning retinoid and peroxisome proliferator-activated nuclear receptors of the Pacific oyster and in silico binding to environmental chemicals

Abstract

Disruption of nuclear receptors, a transcription factor superfamily regulating gene expression in animals, is one proposed mechanism through which pollution causes effects in aquatic invertebrates. Environmental pollutants have the ability to interfere with the receptor’s functions through direct binding and inducing incorrect signals. Limited knowledge of invertebrate endocrinology and molecular regulatory mechanisms, however, impede the understanding of endocrine disruptive effects in many aquatic invertebrate species. Here, we isolated three nuclear receptors of the Pacific oyster, Crassostrea gigas: two isoforms of the retinoid X receptor, CgRXR-1 and CgRXR-2, a retinoic acid receptor ortholog CgRAR, and a peroxisome proliferator-activated receptor ortholog CgPPAR. Computer modelling of the receptors based on 3D crystal structures of human proteins was used to predict each receptor’s ability to bind to different ligands in silico. CgRXR showed high potential to bind and be activated by 9-cis retinoic acid and the organotin tributyltin (TBT). Computer modelling of CgRAR revealed six residues in the ligand binding domain, which prevent the successful interaction with natural and synthetic retinoid ligands. This supports an existing theory of loss of retinoid binding in molluscan RARs. Modelling of CgPPAR was less reliable due to high discrepancies in sequence to its human ortholog. Yet, there are suggestions of binding to TBT, but not to rosiglitazone. The effect of potential receptor ligands on early oyster development was assessed after 24h of chemical exposure. TBT oxide (0.2μg/l), all-trans retinoic acid (ATRA) (0.06 mg/L) and perfluorooctanoic acid (20 mg/L) showed high effects on development (>74% abnormal developed D-shelled larvae), while rosiglitazone (40 mg/L) showed no effect. The results are discussed in relation to a putative direct (TBT) disruption effect on nuclear receptors. The inability of direct binding of ATRA to CgRAR suggests either a disruptive effect through a pathway excluding nuclear receptors or an indirect interaction. Our findings provide valuable information on potential mechanisms of molluscan nuclear receptors and the effects of environmental pollution on aquatic invertebrates.

Nuclear Receptors and Development of Marine Invertebrates

“…Contrasting the situation in annelids, mollusk RARs have lost the ability to bind RA. It has been suggested that mollusk RARs secondarily lost the capacity to bind RA by accumulating independent single mutations in different mollusk lineages [ 51 , 56 ]. Accordingly, mollusk RARs likely function as constitutive transcriptional repressors [ 50 , 51 , 102 ].…”

“…Even though it is still not clear to what extent the teratogenic effects of EDCs in invertebrates are mediated by NRs, it has been shown that NRs are involved in embryonic and post-embryonic development in these animals [ 47 , 48 , 49 , 50 , 51 , 52 , 53 ]. Yet, the mechanisms at play are not necessarily conserved with vertebrates, revealing lineage-specific adaptions in both invertebrates and vertebrates [ 1 , 10 , 17 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 ]. The aim of this review is to describe and discuss the developmental functions of NRs in invertebrates, with a special focus on marine organisms, highlighting the particular importance of comparative approaches using emerging marine invertebrate models for the field of evolutionary developmental biology.…”

Endocrine disruption and altered sexual development in aquatic organisms: an invertebrate perspective

“…Despite this variance, the potential threat of ED for aquatic species is virtually ubiquitous, as illustrated by stickleback ( Pungitius pungitius Linnaeus, 1758) from remote rivers in the Arctic (St Lawrence Island, Alaska) where expression of VTG in male fish was consistent with exposure to oestrogenic PCBs – a legacy from previous military installations. Gene expression changes and DNA methylation in gonads were also allied to PCB exposure (von Hippel et al ., 2018). Higher in the food chain, terns from PCB-contaminated locations, feeding on fish and aquatic invertebrates such as molluscs and crustaceans, exhibited skewed sex ratios and elevated intersex frequencies –supporting evidence of ED and highlighting the potential threat of bioaccumulation and trophic transfer of EDCs (Hart et al ., 2003).…”

Pharmacological evidence for the role of RAR in axon guidance and embryonic development of a protostome species

“…For example, an RAR cloned from the mollusk Thais clavigera (TcRAR), failed to show transcriptional activity in response to atRA (Urushitani et al, 2013). Likewise, computational analysis of an RAR ortholog from the Pacific oyster (C. gigas), did not support atRA binding (Vogeler, Galloway, Isupov, & Bean, 2017). It was proposed that the RAR of a common ancestor of the deuterostomes and protostomes likely possessed atRA binding capabilities, but that mutations in the ligand binding domain resulted in a loss of this ability in protostomes.…”