The retinoic acid receptor (RAR) in molluscs: Function, evolution and endocrine disruption insights

Aquatic Toxicology 208 (2019) 80–89

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The retinoic acid receptor (RAR) in molluscs: Function, evolution and endocrine disruption insights ⁎

Ana Andréa,b,c, ,1, Raquel Ruivoa, ⁎ Miguel M. Santosa,b, ,2

⁎,1

, Elza Fonsecaa,b, Elsa Froufea, L. Filipe C. Castroa,b,

⁎,2

T

,

a CIMAR/CIIMAR, Interdisciplinary Centre of Marine and Environmental Research, University of Porto, Avenida General Norton de Matos s/n, 4450-208, Matosinhos, Portugal b FCUP – Department of Biology, Faculty of Sciences, University of Porto, Rua do Campo Alegre, 4169-007, Porto, Portugal c ICBAS - Institute of biomedical Sciences Abel Salazar, University of Porto, Rua Jorge de Viterbo Ferreira 228, 4050-313, Porto, Portugal

A R T I C LE I N FO

A B S T R A C T

Keywords: Retinoic acid receptor Retinoid X receptor Retinoids Endocrine disrupting chemicals Molluscs

Retinoid acid receptor (RAR)-dependent signalling pathways are essential for the regulation and maintenance of essential biological functions and are recognized targets of disruptive anthropogenic compounds. Recent studies put forward the inability of mollusc RARs to bind and respond to the canonical vertebrate ligand, retinoic acid: a feature that seems to have been lost during evolution. Yet, these studies were carried out in a limited number of molluscs. Therefore, using an in vitro transactivation assay, the present work aimed to characterize phylogenetically relevant mollusc RARs, as monomers or as functional units with RXR, not only in the presence of vertebrate bone fine ligands but also known endocrine disruptors, described to modulate retinoid-dependent pathways. In general, none of the tested mollusc RARs were able to activate reporter gene transcription when exposed to retinoic acid isomers, suggesting that the ability to respond to retinoic acid was lost across molluscs. Similarly, the analysed mollusc RAR were unresponsive towards organochloride pesticides. In contrast, transcriptional repressions were observed with the RAR/RXR unit upon exposure to retinoids or RXR-specific ligands. Loss-of-function and gain-of-function mutations further corroborate the obtained results and suggest that the repressive behaviour, observed with mollusc and human RAR/RXR heterodimers, is possibly mediated by ligand biding to RXR.

1. Introduction Retinoids are derived metabolites of vitamin A, essential for the regulation of multiple biological processes including development, tissue maintenance, vision and reproduction (Maden and Hind, 2003). Retinoic acid (RA) isomers, namely all-trans- and 9-cis-RA, are the main active form of retinoids acting through binding and modulation of two nuclear receptors (NRs), the retinoic acid receptor (RARs) and/or retinoid X receptors (RXR) (Rochette-Egly and Germain, 2009). RAR and RXR are members of the NR superfamily, a class of mostly ligand-dependent transcription factors that regulate the expression of specific gene subsets (Rochette-Egly and Germain, 2009). NRs share a welldefined domain structure which includes a highly conserved DNAbinding domain (DBD) and a moderately conserved ligand-binding

domain (LBD) (Aranda and Pascual, 2001; Germain et al., 2003, 2006; Rochette-Egly and Germain, 2009). To mediate RA effects, RARs form heterodimers with RXRs to efficiently bind to specific RA responsive elements (RAREs) in the regulatory regions of target genes (Germain et al., 2003). In vitro formation of RAR homodimers has also been suggested, as well as heterodimerization with the thyroid hormone receptor, yet, the physiological role of these receptor couples is still unclear (Lee and Privalsky, 2005; Osz et al., 2012). Contrary to RXRs, the RARs is among the NRs family members that were initially perceived as chordate novelty (Albalat, 2009; Bertrand et al., 2004; Canestro et al., 2006). Yet, with the increasing number of genome sequencing projects, of both model and non-model invertebrate taxa, it became clear that RAR appeared earlier in Metazoa evolution and was present in the last common ancestor of the bilateral clade, and

⁎ Corresponding authors at: CIMAR/CIIMAR, Interdisciplinary Centre of Marine and Environmental Research, University of Porto, Avenida General Norton de Matos s/n, 4450-208, Matosinhos, Portugal. E-mail addresses: [email protected] (A. André), [email protected] (R. Ruivo), fi[email protected] (L.F.C. Castro), [email protected] (M.M. Santos). 1 These authors contributed equally to this work (as first author). 2 These authors contributed equally to this work (as last author).

https://doi.org/10.1016/j.aquatox.2019.01.002 Received 17 July 2018; Received in revised form 4 January 2019; Accepted 4 January 2019 Available online 07 January 2019 0166-445X/ © 2019 Elsevier B.V. All rights reserved.

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Polyplacophora class. From Conchifera, the sister clade of Aculifera, we selected two Gastropoda species, Patella vulgata (Patellogastropoda) and Nucella lapillus (Caenogastropoda); and one Bivalvia, Crassostrea gigas (Ostreoida). The selected clades share a common ancestor, with Polyplacophora as the most basal and closely related to the common ancestor (Kocot et al., 2011). Regarding N. lapillus, P. vulgata and C. gigas receptor sequences were already available (Gesto et al., 2016; Gutierrez-Mazariegos et al., 2014a; Vogeler et al., 2014).

secondarily lost in some groups (i.e. Arthropods) (Albalat and Canestro, 2009; Andre et al., 2014; Bertrand et al., 2004; Campo-Paysaa et al., 2008; Canestro et al., 2006). Still, information regarding RARs isolation and functional characterization, as well as information concerning its role on RA signalling remains very sparse for most invertebrate taxa (Gutierrez-Mazariegos et al., 2014b; Urushitani et al., 2013; Vogeler et al., 2017). To date, advances have been mostly made for chordate invertebrates, such as the cephalochordate Branchiostoma lanceolatum or the urochordates Polyandrocarpa misakiensis and Botrylloides leachi (Escriva et al., 2006, 2002; Hisata et al., 1998; Kamimura et al., 2001; Rinkevich et al., 2007). In B. lanceolatum and P. misakiensis, RARs were shown to bind putative natural ligands (RA isomers), activate target gene transcription, and form heterodimers with RXRs, similarly to vertebrate orthologues (Escriva et al., 2006; Kamimura et al., 2001). Regarding protostomes, despite the seemingly high degree of amino acids conservation generally found within the LBDs, when compared to chordate RARs, recent studies with two molluscs species (Thais clavigera and Nucella lapillus) demonstrated the receptors’ inability to activate target gene transcription and/or bind to retinoids (Campo-Paysaa et al., 2008; Gutierrez-Mazariegos et al., 2014a; Urushitani et al., 2013). These observations were linked to a subset of amino acid substitutions, suggested to be required for receptor/ligand interaction (GutierrezMazariegos et al., 2014a; Urushitani et al., 2013). In silico analysis with the pacific oyster (Crassostrea gigas) receptor further supported this view (Vogeler et al., 2017). In contrast, RAR from the annelid Platynereis dumerilii was shown to have conserved the ability to bind and respond to RA, although at higher concentrations than the chordate orthologue(s) (Handberg-Thorsager et al., 2018). Taken together, these evidences suggest that during lophotrochozoans evolution molluscs RARs may have lost the ability to activate gene transcription in the presence of RA; yet, given the limited number of Mollusca species addressed, it remains unclear if this trait is conserved along the whole phylum (Gutierrez-Mazariegos et al., 2014a; Urushitani et al., 2013). RA-signalling is also a target of endocrine disruptions (Novák et al., 2009; Santos et al., 2012). Several studies have demonstrated that several anthropogenic compounds, such as organochloride pesticides, have the ability to disrupt mammalian RAR-signalling pathways (Inoue et al., 2010; Kamata et al., 2008; Lemaire et al., 2005). Yet, given the strong focus on vertebrate RA-signalling pathway disruption, comprehensive knowledge is still lacking for invertebrate receptors (Castro and Santos, 2014; Novák et al., 2009; Santos et al., 2012). Additionally, RXR ligands could also modulate heterodimeric signalling cascades. The organotin TBT has been shown to be a high affinity ligand for both vertebrate and lophotrochozoan RXRs (André et al., 2017; Bouton et al., 2005; Castro et al., 2007; Urushitani et al., 2011), and has been linked to perturbations of lipid metabolism and gastropods imposex development (Castro et al., 2007; Grün et al., 2006; Kanayama et al., 2005; le Maire et al., 2009; Lyssimachou et al., 2015; Nishikawa et al., 2004). Yet, it remains to be clarified if this compound modulates the heterodimeric response of retinoid receptors in molluscs. In the present work we aimed to gain insights into RAR-signalling pathway evolution and endocrine disruption in molluscs. Therefore, we selected four mollusc species, taking in consideration their phylogenetic position, and characterized mollusc RAR and RXR orthologues as monomer and in heterodimer using an in vitro luciferase reporter assay approach in the presence of putative natural ligands, synthetic agonist and antagonists, as well as TBT and two organochlorine pesticides.

2.2. RNA extraction Total RNA from adult mature mollusc gonads were extracted using a combination of methods. Initially, tissues were homogenized with PureZOL RNA Isolation Reagent® (Bio-Rad) and nucleic acids extracted with chloroform, according to the manufacturer’s instructions. Then, the resulting aqueous phase was used to isolate the total RNA using the Kit illustra RNAspin Mini RNA Isolation (GE Healthcare) with on column DNAse I digestion, starting from the ethanol step. The cDNA synthesis was performed with the iScript™cDNA Synthesis Kit (Bio-Rad) according to the manufacturer’s instructions. 2.3. RAR and RXR isolation in A. crinita First, degenerate oligonucleotide primers were designed based on conserved NR domains of phylogenetically related species (Supplementary Figs. 1 and 2, and Table 1). To obtain the remaining sequence, the initial products were extended using the SMARTer™ RACE cDNA Amplification Kit (Clontech) following the manufacturer instructions. RACE PCR primers were designed using the initial degenerated sequences and subsequent partial fragments were obtained combining RACE, nested, hemi-nested and/or degenerate PCR approaches (Supplementary Figs. 1 and 2, and Supplementary Tables 1 and 2). All PCR amplification reactions were carried out with Phusion Flash High-Fidelity PCR Master Mix (ThermoFisher); the obtained fragments were purified using NZYGelpure (Nzytech), cloned into pGEM-T Easy Vector System (Promega) and further transformed using Nzy5α competent cells (Nzytech). Purified fragments and whole coding sequences were verified by automated sequencing (GATC). 2.4. Sequence and phylogenetic analysis Nuclear receptor amino acid sequences were retrieved from GenBank and Ensembl via tBLASTn and BLASTp searches (Benson et al., 2013; Geer et al., 2010) (Supplementary Table 3). Sequences were aligned using MAFFT alignment software (Katoh and Toh, 2010) using default parameters and visualized and edited in Geneious ®v7.1.7. Conserved amino acid residues, relevant for interaction with all-transRA, were identified according to previous works (Escriva et al., 2006; Gesto et al., 2016; Gutierrez-Mazariegos et al., 2014a; Hisata et al., 1998; Renaud et al., 1995). Amino acid sequences were aligned with MAFFT (Katoh et al., 2002) alignment software using default parameters and visualized and edited in Geneious® v7.1.7. The alignment was stripped from columns with at least 80% of gaps resulting in an alignment with 61 sequences and 534 positions. Maximum Likelihood phylogenetic analysis was performed using PhyML 3.0 server with smart model selection: the inferred amino acid substitution model JTT + G+I + F was selected for phylogenetic analysis. (Guindon et al., 2010; Guindon and Gascuel, 2003). Bayesian-like transformation of aLRT was selected to assess branch support (Anisimova et al., 2011). Trees were visualized with FigTree v1.4.3. The Retinoid X Receptor (RXR) was used as outgroup to root the tree.

2. Materials and methods 2.1. Species selection Four mollusc species were selected, from different classes, according to their phylogenetic position. From the Aculifera clade we selected Acanthochitona crinita (Neoloricata; for identification we used CO1 barcoding approach - see Supplementary material 1) that belongs to the

2.5. Construction of plasmid vectors and mutagenesis The Ligand binding domain (LBD) and Hinge regions of selected RARs and RXRs were amplified using specific primers and cloned into 81

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Fig. 1. Partial alignment of human and mollusk RAR protein sequences. In the DBD the P-box is presented in purple box, D-box in green box and the T-box in red box. Key amino acid that interact with all-trans-RA in the human RARγ ligand-binding pocket are highlighted; direct or indirect hydrogen bonds, between the substrate and LBP residues, are highlighted in grey; in blue, hydrophobic and Van der Waals interactions. The positions of the twelve helices from the LBD are shown (H1-12) in a delimited brown line. Domain motifs and residues inferred from: Renaud et al., (1995); Gutierrez-Mazariegos et al. (2014a) and Gesto et al. (2016). H. sapiens (Hs), T. clavigera (Tc), N. Lapillus (Nl), P. vulgata (Pv), C. gigas (Cg), L. stagnalis (Ls), A. crinita (Ac).

(Promega). Firefly Luciferase (pGL4.31) and Renilla Luciferase (pBIND) luminescent activities were assessed using the Dual luciferase assay system according to manufacturer’s instructions (Promega) and measured with a Synergy HT Multi-Mode Microplate Reader (Biotek). Renilla Luciferase, co-expressed with the LBD hybrid proteins, served as an internal control for transfection efficiency (Schagat et al., 2007). Experiments were repeated at least three times.

pBIND and/or pACT vectors (Promega, accession numbers AF264722 and AF264723.1), to produce fusion proteins with the yeast transcriptional activator GAL4 (LBD-GAL4) or the viral enhancer, VP16 (LBDVP16), which acts on proximal downstream promoters, respectively (Duffy, 2002; Hagmann et al., 1997). Primers and restriction sites are listed in Supplementary Table 4. Cloned sequences were verified by automated sequencing (GATC). N. lapillus RAR gain of function double mutant (Ala230Ser and Val353Ile) was produced according to previous reports (Gutierrez-Mazariegos et al., 2014a). Human RXRα and N. lapillus RXRa, loss-of-function mutants, towards TBT, were also produced (Cys432Ala and Cys409Ala, respectively). Mutagenesis procedures were outsourced to NZYTech (Portugal).

2.7. Data analysis and statistics Transactivation results were expressed as fold-induction, resulting from the ratio between Firefly Luciferase (pGL4.31) and Renilla Luciferase (pBIND) luminescent activities followed by normalization with solvent control. Results are presented as average of the normalized values, and the bars corresponds to the standard error values. For statistical analysis, data from Luciferase and Renilla ratio was used. Data were analysed using SigmaPlot 11 software. Significant difference from DMSO control were estimated using one-way ANOVA followed by Fisher LSD Test or ANOVA on Ranks followed by Dunn’s test if assumptions were not met. Differences were considered significant when p < 0.05.

2.6. Transfection and transactivation assays COS-1 cells were maintained in DMEM with phenol red (Invitrogen) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Invitrogen) at 37 °C with a humidified atmosphere and 5% CO2. Prior to transfection, cells were seeded on 24-well culture plates at a density of 2 × 105 live cells/ well. The following day, cells were transfected using 2 μl Lipofectamine 2000 (Invitrogen) in Opti-MEM reduced serum medium (Gibco, Thermo Fisher), and construct plasmids, to a final volume of 350 μl. For single NR LBD transfections, cells were incubated with 0.5 μg of pBIND-LBD-GAL4 and 1 μg of pGAL4.31 reporter vector (Promega, accession number DQ487213). For double NR LBD transfections, cells were incubated with 0.5 μg of pBIND-LBDGAL4, 0.75 μg of pACT-LBD-VP16 and 1 μg of pGAL4.31 (CheckMate™ Mammalian Two-Hybrid System, Promega). After 5 h of incubation, cells were washed with Phosphate Buffer Saline (PAA Biotech) and exposed to the test compounds in phenol red-free DMEM supplemented with 10% dextran-coated charcoal-treated serum (Invitrogen) and 1% penicillin/streptomycin (Invitrogen). Final concentration were as follows: 1 μM 9-cis-RA, 1 μM all-trans-RA, 0.1 μM 4-oxo-RA, 1 μM synthetic RAR agonist and antagonists (LE135, CD2665, TTNPB), 0.5 μM RXR agonist HX630, 0.1 μM TBT, and 10 μM organochlorine pesticides (Endrin, Dieldrin). DMSO was used as solvent to prepare test compound solutions and was used as solvent control; the final solvent concentration per well did not exceed 0.1%. Each test condition was assayed in duplicate. The following day, cells were washed and gently lysed, for 15 min at 37 °C and 90 rpm, using 100 μL of Passive Lysis Buffer

3. Results and discussion 3.1. Mollusc RAR phylogenetic and sequence analysis Genome research studies predict the presence of retinoid metabolic and signalling key players in molluscs (Campo-Paysaa et al., 2008; Theodosiou et al., 2010). Endogenous retinoids have been detected in gastropods species, mainly in gonads (Dmetrichuk et al., 2008, 2006; Gesto et al., 2012, 2013; Gesto et al., 2016). In addition, it has been demonstrated that N. lapillus and Osilinus lineatus are capable of metabolizing retinoid precursors into RA isomers (Gesto et al., 2012, 2013). These findings clearly indicate the presence of an active pathway for the biosynthesis and signalling of RA in molluscs. In support of this, several biological effects of retinoids have been described and considered conserved between molluscs and vertebrates (Campo-Paysaa et al., 2008; Créton et al., 1993; Gesto et al., 2016). Through a combination of PCR strategies, we were able to isolate the full-length sequence of a cDNA encoding for a RAR orthologue from 82

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Fig. 2. Phylogenetic analysis of the RAR gene family. Tree was constructed using Maximum likelihood phylogenetic analysis with a bayesian-like transformation of aLRT selected to assess branch support; Thyroid hormone NRs were used to root the tree. As highlighted in the figure, the isolated A. crinita sequence robustly clusters with mollusc RARs. A. californica (Aca), A. carolinensis (Acar), A. crinita (Ac), A. crinita (Ac), B. floridae (Bf), B. glabrata (Bg), C. teleta (Ct), C. intestinalis (Ci), C. gigas (Cg), D.rerio (Dr), D. magna (Dma), G. gallus (Gg), H. sapiens (Hs), L. stagnalis (Ls), L. migratoria (Lm), L. gigantea (Lg), M. musculus (Mm), N. Lapillus (Nl), P. vulgata (Pv), P. dumerilli (Pd), P. misakiensis (Pm), S. kowalevskii (Sk), T. clavigera (Tc), X. laevis (Xl), X. tropicalis (Xt). Accession numbers can be found in Supplementary Table 4.

human RAR homologs (Fig. 1). In the case of the D-box of N. lapillus RAR three amino acid substitutions were found, whereas in P. vulgata, A. crinita and C. gigas RARs only one amino acid substitution is observed when compared to human RARs. Regarding the T-box only P. vulgata RAR displays an amino acid substitution (Fig. 1). Nonetheless, the identified substitutions are most likely non-disruptive given that amino acid properties are conserved (i.e. size, charge). Previous crystallographic analysis revealed that the human RARs LBP is formed by 25 key amino acid residues that are important for establishing a direct contact to RA (Fig. 1): localized in helixes (H) 1, H3, H5, the β-turn, loop 6–7, H11, loop 11–12 and H12 (Renaud et al., 1995). These amino acids are highlighted in the alignment presented in Fig. 1, whose residues positions are presented with respect to human RARγ. This amino acid signature is not entirely conserved in mollusc RAR LBDs (Fig. 1). The N. lapillus RAR have 15 out of the 25 residues key amino acid residues conserved, whereas C. gigas, A. crinita and P. vulgata have 16 (Fig. 1). These amino acid residues substitutions are not necessarily shared among the molluscs. Regarding C. gigas RAR, a recent analysis highlighted six of these residue mutations as possibly disabling with respect to the correct binding of retinoids: Ser234, Met308, Asn289, Val230, Gly237 and Leu408 (Vogeler et al., 2017); these residues correspond to Ala234, Leu271, Phe230, Ser289, Gly237 and Met408, in human RARγ, respectively. With human RARγ, all-trans-RA forms a stable hydrogen bond network between its carboxyl group and Lys236, Leu233, Arg278, and Ser289 residues (Renaud et al., 1995). Only in A. crinita RAR these key amino acids are conserved, whereas the other mollusc RARs show at least one substitution, with the Arg278 being the only residues conserved in all (Fig. 1). In addition, residues Phe230, Ala397 and Met408 were suggested to be essential since mutations lead to loss of both ligand binding and transactivation abilities (Renaud et al., 1995). In molluscs, Phe230 is substituted by a Val or Ile, and Met408 is replaced by Leu (Fig. 1), whereas, Ala234 shows substitutions similar to those observed in human RARα and β. Residues Met408 and Ile412, were also

Table 1 Amino acid identity (%) of the DNA-binding and Ligand Binding Domains between mollusc and H. sapiens RAR isoforms. Percentage determined by sequence alignment in NCBI protein blastp tool. NlRAR

HsRARα HsRARβ HsRARγ TcRA NlRAR CgRAR PvRAR AcRAR

CgRAR

PvRAR

AcRAR

DBD

LBD

DBD

LBD

DBD

LBD

DBD

LBD

81% 81% 85% 95% – 91% 88% 89%

53% 50% 50% 68% – 60% 63% 62%

86% 85% 89% 96% 91% – 95% 92%

49% 51% 58% 62% 60% – 65% 69%

86% 86% 89% 93% 88% 95% – 93%

55% 56% 53% 66% 63% 65% – 64%

85% 85% 89% 93% 89% 92% 93% –

51% 52% 49% 67% 62% 69% 64% –

A. crinita, which encodes a protein with 432 amino acids. Sequence alignment of isolated and retrieved mollusc RARs, together with the human RARs homologs, is depicted in Fig. 1. Full-length amino acid sequences of RAR from selected species were used to conduct molecular phylogenetic analysis. The isolated receptor robustly clustered with other lophotrochozoan species (Fig. 2). Amino acid sequence alignment comparisons revealed that the selected study mollusc RAR orthologues exhibit the NRs typical modular structure contain the two main domains, the DBD and the LBD (Aranda and Pascual, 2001). Overall, among the selected mollusc species, sequence identity ranged from 88 to 95% and from 50 to 69%, for the DBD and LBD, respectively (Table 1). Between mollusc and human RARs sequence identity ranged from 81 to 89% and from 49 to 58%, for the DBD and LBD, respectively (Table 1). NR DBDs have three highly-conserved sequence elements, termed as P-, D-, and T-box, essential for the recognition and binding to response elements and serving as dimerization interfaces between the dimeric DBDs (Germain et al., 2003). Within the DBD molluscs RARs P-box (CEGCKG) sequence residues are fully conserved when compared to 83

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suggested to play an important role in 9-cis-RA binding in human RARα, with mutations of this two residues yielding loss of binding ability (Tate and Grippo, 1995). In our set of mollusc species these amino acids are not conserved being replaced by a Leu and a Val, respectively (Fig. 1). Overall, sequence alignment analysis suggests that mollusc RARs exhibit a range of amino acid substitution within their LBP which might modulate their affinities towards classical RAR ligands.

3.2. Transcriptional response of mollusc RARs in the presence of all-transRA, 9-cis-RA and 4-oxo-RA In order to investigate if the previously observed loss of RAR’s ability to activate target gene transcription was conserved in the entire phyla, we performed transient transactivation assays with LBD-GAL4 fusion proteins, and a luciferase reporter as readout, with RAR LBDs from representative mollusc species: A. crinita, C. gigas, P. vulgata and N. lapillus. Human RARγ was used as control. The selected test ligands included RA isomers 9-cis- and all-trans-RA considered as the main natural high affinity ligands of human RARs (Allenby et al., 1993; Idres et al., 2002), as well as 4-oxo-RA, a metabolite resulting from RA catabolism. Previous studies reported that, in mammals, several RA catabolic metabolites are able to bind and induce RAR-mediated transcription, yielding biological activity: notably in embryogenesis and cell growth (Idres et al., 2002; Pijnappel et al., 1993; van der Leede et al., 1997; Van heusden et al., 1998). Fig. 3A displays the results from the reporter gene transactivation assay. Tested mollusc RARs did not yield significant activations when exposed to the examined retinoids, while the human orthologue was significantly responsive to all three compounds, in agreement with previous findings (Idres et al., 2002; Lemaire et al., 2005). Interestingly, for P. depressa RAR exposure to 4oxo-RA induced a significant transactivation repression (Fig. 3A). The obtained results are in agreement with previous reports for N. lapillus RAR in the presence of retinoids (Gutierrez-Mazariegos et al., 2014a). Also, a ligand binding modelling study predicted the inability of C. gigas RAR to interact with 9-cis-RA and all-trans-RA, due to significant amino acid substitutions in the LBP (Vogeler et al., 2017). Overall, our findings suggest that the loss of RARs ability to mediate gene transcription in response to retinoids might be globally conserved through the entire molluscan phyla. In addition, RAR from the annelid P. dumerilii was shown to retain the capacity to bind and respond to RA (Handberg-Thorsager et al., 2018). Thus, it can be hypothesized that the ability of RAR to promote gene expression in response to retinoids was already present in the last common ancestor of all lophotrochozoans and was subsequently lost in molluscs, after the mollusc/ annelid split. This loss of activation might be due to changes in the LBD of mollusc RARs: as illustrated by the described substitutions of some of the key 25 amino acid residues identified by crystallography (Renaud et al., 1995). Yet, theses substitutions are not fully shared among the studied molluscan RARs, which can denote some plasticity and account for the repressive behaviour observed in P. vulgata upon exposure to 4oxo-RA. Nonetheless, the ability of molluscan RARs to induce retinoiddependent gene expression was hampered; yet, we cannot exclude that other residues, beyond this set of 25, may also determine retinoid binding and receptor activation. Besides molluscan RARs, other invertebrate NRs have been suggested to exhibit distinct ligand-binding affinities or activation mechanisms when compared to vertebrate NRs. For example, estrogen receptor (ER) orthologues, from molluscs and from the cephalochordate B. lanceolatum, were shown to be unable to bind estradiol and other vertebrate steroid ligands (Paris et al., 2008; Thornton et al., 2003). Furthermore, the Octopus vulgaris ER was proposed to act as a constitutive activator of gene transcription (Keay et al., 2006).

Fig. 3. NR-mediated luciferase activity in response to 1 μM all-trans-RA, 1 μM 9cis-RA, 0.1 μM 4-oxo-RA, 0.5 μM HX630 and 0.1 μM TBT: (A) RAR, (B) RXR and (C) RAR/RXR heterodimer. Results are expressed as average fold induction with respect to vehicle control DMSO (means ± SEM); * denotes significant differences from vehicle control DMSO, calculated using one-way ANOVA followed by FISHER LSD Test.

3.3. Transcriptional activity of mollusc RAR/RXR heterodimers In vertebrates, RAR dimerizes with RXR, yielding a functional unit with the ability to bind to specific DNA elements and modulate gene transcription (Chambon, 2005; Glass, 1994; Kurokawa et al., 1995). This heterodimeric protein complex composed by RAR/RXR is generally classified as non-permissive, since it is only activated when a ligand binds to RAR. Yet, the roles of the individual receptors within this non-permissive heterodimers are still under debate. In fact, as a heterodimer with RAR, RXR was shown to retain the ability to bind ligands and even recruit coactivators in vitro; however, when the RAR/ RXR heterodimer is complexed with corepressors, ligand-binding to RXR not only seems insufficient to release corepressors but also increases the corepressor-heterodimer interaction (Germain et al., 2002; Kersten et al., 1996; Lammi et al., 2008; Minucci et al., 1997). In agreement, some studies demonstrate that mammalian RAR/RXR 84

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that mammalian RXRs bind with high affinity to 9-cis-RA and have negligible binding affinity towards all-trans-RA (Allenby et al., 1993; Heyman et al., 1992; Levin et al., 1992). Yet, in the arthropod Locusta migratoria, RXR was shown to bind similarly to both 9-cis-RA and alltrans-RA (Nowickyj et al., 2008). Nonetheless, all-trans-RA can spontaneously isomerize to 9-cis-RA (Levin et al., 1992). Thus, isomerization of all-trans-RA to 9-cis-RA could have occurred during our exposure assay leading to the observed results. The metabolite 4-oxo-RA did not yield significant RXR-dependent transactivation signals in the tested species (Fig. 3B). This is in agreement with previous reports suggesting that this catabolic metabolite binds weakly to human RXRα (Pijnappel et al., 1993). Taken together, these results suggest that in molluscs, the repressive behaviour of the RAR/RXR heterodimer is possibly mediated by ligand biding to RXR. In fact, all-trans-RA, TBT and HX630 ligands, inducing a negative regulation of luciferase transcription, were able to promote RXR-dependent transactivation, but not RAR, in the individual receptor assays. The inability of mollusc RAR to bind and/or mediate gene transcription in the presence of retinoids further corroborates this hypothesis (Gutierrez-Mazariegos et al., 2014a; Urushitani et al., 2013). Mechanistically, this could reflect the capacity of RXR to stabilize corepressor interaction in the context of the RAR/RXR heterodimer (Germain et al., 2002). The peculiar behaviour of P. vulgata RAR also supports this hypothesis. Unlike other tested molluscs, P. vulgata RAR/ RXR yielded no response when exposed to HX630. While shown to activate P. vulgata, and all other tested RXRs, this RXR agonist also induced activity of P. vulgata RAR. Thus, this ability to bind RAR might modulate corepressor release in P. vulgata, preventing the repressive behaviour observed in the remaining mollusc species. Nonetheless, further studies are needed to address these issues. In fact, a spectrum of regulatory modes, including constitutive gene repression, was attributed to human RAR (le Maire et al., 2010).

heterodimers can negatively regulate transcription, even in the presence of RAR ligands, through corepressor interaction, in a DNA response element-dependent manner (DiRenzo et al., 1997; Kurokawa et al., 1995). Regarding the gastropods T. clavigera and N. lapillus, it was shown that RAR is able of form heterodimers with RXR (Gutierrez-Mazariegos et al., 2014a; Urushitani et al., 2013); yet, it is still unclear if this protein complex induces gene transcriptional activation upon ligand exposure. Since mollusc RARs seem unresponsive to the ligands tested so far, while mollusc RXRs conserved the ability to bind and be activated by 9-cis-RA (Castro et al., 2007; Gutierrez-Mazariegos et al., 2014a; Urushitani et al., 2013), we aimed to understand the functional behaviour of this protein complex in molluscs. Thus, in addition to the retinoid ligands, we selected two specific RXR agonists, tributyltin (TBT) and the synthetic ligand HX360, and examined the transcriptional behaviour of RAR and RXR (as positive control) alone, and in heterodimer (Fig. 3). Besides specificity, the choice of these ligands also related to their ability to produce adverse effects in molluscs, thus allowing to gain insight into the molecular mechanisms of signalling interference. Regarding TBT, the best known adverse health effect on aquatic wildlife is imposex development in gastropods (Lima et al., 2011); yet, adverse effects have been also reported in other mollusc classes. For instance, TBT also affects C. gigas, inducing changes in shell thickening and leads to perturbation at the level of reproduction mainly fertility, offspring mortality, decrease in larval and juveniles growth (Heral et al., 1989; Vogeler et al., 2017). The selective RXR agonist HX630 was also reported to induce imposex in gastropods (Stange et al., 2012; Umemiya et al., 1997). Using a two-hybrid protein-protein interaction strategy, we first confirmed the ability of the RAR and RXR pairs to form heterodimeric complexes. Indeed, for the selected mollusc species, co-transfection of RAR and RXR fusion proteins, LBD-GAL4 and LBD-VP16, respectively, significantly enhanced luciferase expression in COS-1 cells (see Supplementary Fig. 3). Next, we exposed COS-1 cells transfected with human and mollusc RAR/RXR fusion constructs to single concentrations of all-trans-RA, 4-oxo-RA, TBT and HX630 (Fig. 3C). As expected, the human RARγ in complex with RXRα yielded significant activations in the presence of all-trans-RA and 4-oxo-RA (Idres et al., 2002). Regarding mollusc heterodimeric complexes, transcriptional activation activities were not observed in the presence of the selected ligands; yet, significant transcription repression activities were detected. In detail, for N. lapillus all tested compounds induced a significant depression of luciferase induction, for A. crinita significant repressions were obtained for all-trans-RA, TBT and HX630, C. gigas was only significantly responsive to TBT and HX630 and P. vulgata heterodimers elicited no significant transcriptional repressions. Regarding human RARγ/RXRα, TBT also yielded a significant repression. To further explore the roles of the individual receptors within RAR/ RXR heterodimer in molluscs, we measured the transactivation abilities of RXR alone in the presence of the test compounds, and RAR in the presence of TBT and HX630, considered as RXR-specific ligands (Fig. 3A and B). Regarding HX630 and TBT exposures, RAR constructs were unable to mediate reporter gene transcription for the tested species, with the exception of P. vulgata RAR which yielded a significant activation when exposed to HX630 (Fig. 3A). On the other hand, human RXRα, T. clavigera and N. lapillus RXRs transactivation activities were significantly induced in the presence of TBT, known to be a high affinity ligand (Hiromori et al., 2015; Kanayama et al., 2005; Nishikawa et al., 2004; Urushitani et al., 2018). Fig. 3B shows that, similarly to T. clavigera and N. lapillus (Urushitani et al., 2018), A. crinita and P. vulgata RXRs yielded significant reporter gene transactivation upon TBT exposure. C. gigas RXR elicited a non-significant transactivation signal. Additionally, significant transactivation were measured with RXRs from all tested species when exposed to HX630, all-trans-RA, or, for the human construct, 9-cis-RA (Fig. 3B). All-trans-RA-mediated RXR activation across tested species was unexpected. Previous studies reported

3.4. Transcriptional activity of molluscs NRs mutation for gain and loss-offunction To further understand the roles of the individual receptors in the observed transcriptional signals obtained in the presence of all-trans-RA we first mutated two key residues in the binding pocket of RAR (Ala230Ser and Val353Ile), using N. lapillus RAR/RXR and according to previous gain-of-function studies (see Supplementary Fig. 4) (GutierrezMazariegos et al., 2014a). In agreement with previous works, the double mutant alone was able to recapitulate RAR-dependent transactivation in the presence of 10 μM all-trans-RA, similarly to the human RARγ (Fig. 4A). Yet, the heterodimer RAR/RXR was unresponsive to alltrans-RA (Fig. 4A). The ability of the mutant RAR construct to form dimers with RXR was not affected (Supplementary Fig. 5). In agreement with the results observed with P. vulgata, the accommodation of a RAR ligand impaired RAR/RXR-mediated repression. However, the N. lapillus gain-of-function mutant was not sufficient to recapitulate the response observed with the human RAR/RXR couple. Thus, other yet unidentified structural changes in molluscan RARs might contribute to this response. Next, we performed a loss-of-function mutations in order to abolish TBT interaction with the LBDs of human and N. lapillus RXRs (Cys432Ala and Cys409Ala, respectively). In human RXRα mutation of the key cysteine residue was previously shown to be crucial for binding to TBT (Supplementary Fig. 6) (le Maire et al., 2009). In agreement, RXR mutants alone, for both human and N. lapillus, were not responsive to TBT (Fig. 4B). A similar result was observed for mutant RXRs in complex with RARs, in both species (Fig. 4B), thus abolishing the repressions previously observed with the intact LBDs. The cysteine mutation did not impair heterodimerization between RXR mutants and respective RAR partners (see supplementary Fig. 7). These results further support the modulatory role of RXR within the RAR/RXR heterodimer. For both human and N. lapillus, RAR/RXR exposure to TBT 85

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Fig. 5. RAR-mediated luciferase activity in response to 1 μM synthetic RAR agonist and antagonists (LE135, CD2665, TTNPB). Results are expressed as average fold induction with respect to vehicle control DMSO (means ± SEM); * denotes significant differences from vehicle control DMSO, calculated using one-way ANOVA followed by FISHER LSD Test.

and RARα/β/γ agonist TTNPB. A recent study revealed that Lymnaea stagnalis embryos when exposed to the vertebrate RARβ selective antagonist LE135, develop defects during eyes and shell formation (Carter et al., 2015), similar to defects reported by RA application (Créton et al., 1993). As depicted in Fig. 5 none of the tested synthetic compounds were able to induce gene transcriptional activity with gastropod RARs. For human RARγ, results are in agreement with previous findings reporting the ability of LE135, CD2665 and TTNPB to induce RARγ–dependent transcription (Bernard et al., 1992; Lemaire et al., 2005; Li et al., 1999). Thus, our results suggested that the adverse effects of LE135 exposure observed with the gastropod L. stagnalis might be mediated by alternative signalling pathways, or that the morphologic response might be due the compound’s toxicity. 3.6. Transcriptional activity of N. lapillus RAR in the presence of common EDCs Fig. 4. Transactivation activity of mutant constructs in response to 0.1 μM TBT and 10 μM all-tans-RA. (A) RXR loss-of-function mutants (RXRm): Human RXRα Cys432Ala and N. lapillus RXRa Cys409Ala; (B) RAR gain-of-function mutant (RARm): N. Lapillus RAR Ala230Ser-Val353Ile. Results are expressed as average fold induction with respect to vehicle control DMSO (means ± SEM); * denotes significant differences from vehicle control DMSO, calculated using one-way ANOVA followed by FISHER LSD Test.

In general for invertebrates, and in particular for mollusc species, little is known about the mechanisms of endocrine disruption of RA signalling pathways mostly because of the lack of knowledge concerning the retinoid system. As mentioned before, the only known example of retinoid signalling modulation by endocrine disruptive chemicals (ECDs) is related to the imposex development through RXRdependent signalling following organotin exposure. Given that mammalian RAR can be modulated by environmental pollutants, we tested if synthetic chemicals, known to interact with human RARs, were able to induce mollusc RAR gene transactivation. Based on previously studies (Kamata et al., 2008; Lemaire et al., 2005), we selected two organochlorine pesticides, Endrin and Dieldrin. None of the tested EDCs were capable to induce N. lapillus RAR-dependent transactivation responses (Fig. 6).

yielded a transcriptional repression. However, mutational silencing of a crucial TBT-binding residue produced an unresponsive heterodimer. Modulation of the equilibrium of corepressor and coactivator recruitment and release should be further explored to understand the graded signalling mechanisms observed with human and mollusc RAR/RXR heterodimers. 3.5. Transcriptional response of N. lapillus and P. vulgata RARs in the presence of synthetic agonists and antagonists

4. Conclusion

Since none of the putative natural active retinoids were capable of induce reporter gene transactivation mediated by molluscs RAR, we next addressed if synthetic agonists and antagonist were able to modulate RAR from the gastropods P. vulgata and N. lapillus, using the human RARγ construct as control. Test compounds included selective vertebrate RARβ antagonist LE135 and RARβ/γ antagonist CD2665,

In the present work we characterized the ability of RARs from molluscs belonging to different classes to induce gene expression in the presence of potential natural ligands, synthetic agonist and antagonist, and organochlorine pesticides. Our transactivation assays revealed that none of mollusc RARs were activated by the tested retinoids, consistent with the poorly conserved key residues on LBD known to be crucial to 86

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Comp. Endocrinol. 208, 134–145. André, A., Ruivo, R., Capitão, A., Froufe, E., Páscoa, I., Costa Castro, L.F., Santos, M.M., 2017. Cloning and functional characterization of a retinoid X receptor orthologue in Platynereis dumerilii: an evolutionary and toxicological perspective. Chemosphere 182, 753–761. Anisimova, M., Gil, M., Dufayard, J.F., Dessimoz, C., Gascuel, O., 2011. Survey of branch support methods demonstrates accuracy, power, and robustness of fast likelihoodbased approximation schemes. Syst. Biol. 60, 685–699. Aranda, A., Pascual, A., 2001. Nuclear hormone receptors and gene expression. Physiol. Rev. 81, 1269–1304. Benson, D.A., Cavanaugh, M., Clark, K., Karsch-Mizrachi, I., Lipman, D.J., Ostell, J., Sayers, E.W., 2013. GenBank. Nucleic Acids Res. 41, D36–42. Bernard, B.A., Bernardon, J.M., Delescluse, C., Martin, B., Lenoir, M.C., Maignan, J., Charpentier, B., Pilgrim, W.R., Reichert, U., Shroot, B., 1992. 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Fig. 6. RAR-mediated luciferase activity in response to 10 μM organochlorine pesticides (Endrin and Dieldrin). Results are expressed as average fold induction with respect to vehicle control DMSO (means ± SEM); * denotes significant differences from vehicle control DMSO, calculated using one-way ANOVA followed by FISHER LSD Test.

interact with the ligands. This data advocates that, during evolution, the mollusc RARs lost the ability to mediated signal transduction upon retinoid interaction, a feature that might be conserved across the phylum. Furthermore, mollusc RARs conserved the ability to form heterodimer with RXR and, like vertebrate RXRs, the mollusc RXRs, are responsive and able to mediated target gene transcription. Moreover, we provide evidences supporting a major modulatory role of mollusc RXRs within the RAR/RXR heterodimer, a role that might be ancient and conserved at least with mammals, leading to target gene transcription repression. Yet, further studies are needed to understand the mechanics underlying RXR modulation, notably regarding corepressor stabilization and release. Additionally, our findings also emphasise differences in RAR modulation by environmental pollutants, with mollusc RAR showing unresponsiveness toward organochloride pesticides. It would be pertinent, in future studies, to focus in RAR-mediated signalling pathway modulation by organochlorine pesticides, and other EDCs, in additional invertebrate species such as chordates and annelids where the receptor has the ability to respond to retinoids in a similar manner as vertebrates.

Author contributions Conceived and designed the experiments: A.A., R.R., L.F.C.C., M.S.; Performed the experiments: A.A., E.F.; Analysed the data: A.A., R.R., E.F. (1), E.F. (2), L.F.C.C., M.S.; Contributed reagents/ materials/ analysis tools: A.A., R.R., F.F. (2), L.F.C.C., M.S.; Wrote the paper: A.A., R.R., L.F.C.C., M.S.

Acknowledgments This research was supported by Norte 2020 and FEDER (Coral—Sustainable Ocean Exploitation—Norte-01-0145-FEDER000036) and by Project No. 031342 co-financed by COMPETE 2020, Portugal 2020 and the European Union through the ERDF, and by Fundação para a Ciência e a Tecnologia (FCT) through national funds; PhD grants were awarded to Ana André (SFRH/BD/81243/2011) and Elza Fonseca (SFRH/BD/100262/2014), and a Postdoctoral grant was awarded to Raquel Ruivo (SFRH/BPD/72519/2010) by FCT. 87

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