The Echinodermata PPAR: Functional characterization and exploitation by the model lipid homeostasis regulator tributyltin☆
Graphical abstract
Introduction
Sea urchins, members of the Echinodermata phylum, play an important ecological role in ecosystem functioning through their grazing activity that controls the algae biomass (González-Irusta et al., 2010, Ribeiro et al., 2015, Romero et al., 2016). From an economic standpoint, species such as the herbivorous Paracentrotus lividus, widely distributed in the Atlantic and Mediterranean coasts (Arafa et al., 2012, Carboni et al., 2012, Kabeya et al., 2017), also hold a high commercial value, as their gonads are considered a gastronomic delicacy (Arafa et al., 2012, Guidetti, 2004, Guidetti et al., 2004, Shpigel et al., 2005a).
The sea urchin gonads are rich in lipids, carbohydrates and proteins (Archana and Babu, 2016). Among lipids, fatty acids display essential roles in gonad maturation and larvae development (Carboni et al., 2013). Before reaching the feeding stage, sea urchin embryos use nutrients provided by the egg. This makes maternal provisions, including essential fatty acids, crucial for embryo development and offspring success (Carboni et al., 2013). In agreement, previous studies found that total lipid levels were maintained constant prior to hatching; yet, they decreased after the digestion of the envelope, between free-swimming blastula and the first feeding stages (Sewell, 2005, Smith et al., 2008). Among fatty acids, several long-chain are known to play an important role in larvae development: docosahexaenoic acid (C22:6 n-3, DHA), eicosapentaenoic acid (C20:5 n-3, EPA), and arachidonic acid (20:4n-6, ARA) (Carboni et al., 2013). The sea urchin P. lividus is able to synthetize ARA and EPA from the precursors linoleic acid (18: 2n-6, LA) and α-linolenic acid (18: 3n-3, ALA) (Kabeya et al., 2017). Moreover, the equilibrium between n-6 and n-3 fatty acids is important from the economic standpoint since the imbalance between n-6 and n-3 consumption in humans can lead to health disorders, such as cardiovascular diseases (González-Mañán et al., 2012, Rincón-Cervera et al., 2016).
Animal lipid composition is not static and can be altered by diet, life-cycle stage or external stimuli (Arafa et al., 2012). Recently, several environmental chemicals were shown to modulate lipid homeostasis (Castro and Santos, 2014, De Cock and Van de Bor, 2014, Diamanti-Kandarakis et al., 2009, Grün and Blumberg, 2009, Jordão et al., 2016b, Lyssimachou et al., 2015, Ouadah-Boussouf and Babin, 2016, Santos et al., 2012). Those compounds, able to interfere with lipid homeostasis in favour of lipid storage are commonly known as obesogens and were primarily found to modulate lipid homeostasis in mammals; yet, recent studies suggested that their scope of action transcends mammals, or even vertebrates (Capitão et al., 2017, Janer et al., 2007, Jordão et al., 2015, Lyssimachou et al., 2009). Tributyltin (TBT), an endocrine disrupting chemical (EDC), is a well-recognised model of lipid homeostasis modulator. Although TBT is no longer used as biocide in anti-fouling paint for boats, its levels are still high in some areas: reaching 241,8 μg Sn/Kg, as reported in biological samples from one of the largest harbour regions in China (Chen et al., 2017). TBT was found to disrupt mammalian lipid homeostasis through the interaction with the nuclear receptors peroxisome proliferator-activated receptor γ (PPARγ) and retinoid X receptor (RXR) (Capitão et al., 2018, Harada et al., 2015, Hiromori et al., 2009, le Maire et al., 2009). PPARγ and RXR cooperate in the form of a permissive heterodimer (PPARγ/RXR) that is considered a master regulator of lipid homeostasis in vertebrates (Ahmadian et al., 2013, Berkenstam and Gustafsson, 2005, Hiromori et al., 2015, Janesick and Blumberg, 2011, Ouadah-Boussouf and Babin, 2016, Santos et al., 2012). PPARγ is member of a nuclear receptor superfamily that in vertebrates include also peroxisome proliferator-activated receptor α and β (PPARα and PPARβ). PPARs act as ligand-activated transcription factors by the binding of specific ligands, such as fatty acids, inducing a conformational change that cause the replacement of corepressors with coactivators triggering the transcription of specific genes from pathways involved in lipid homeostasis (Echeverría et al., 2016, Lodhi and Semenkovich, 2014, Tyagi and Gupta, 2011).
TBT was identified and shown to modulate RXR transactivation in several invertebrate groups, including annelids (André et al., 2017), gastropods (Nishikawa et al., 2004), crustaceans (Wang and LeBlanc, 2009) and rotifers (Lee et al., 2019). In vertebrates, TBT, and the related organotin, triphenyltin (TPT), were also shown to interact with PPARγ exhibiting a similar binding mode as that described for RXR: through the interaction of the tin atom and a cysteine residue (Harada et al., 2015, Hiromori et al., 2009, le Maire et al., 2009). Outside deuterostomes, PPAR was only reported in the mollusk Crassostrea gigas; yet, its characterization was limited to in silico analysis (Vogeler et al., 2014, Vogeler et al., 2017). Although nuclear receptors (NRs) are widespread in metazoans (Bridgham et al., 2010, Santos et al., 2018), the available functional data has limited taxonomic scope (Castro and Santos, 2014, Santos et al., 2018; Tan and Palli, 2008, Thornton, 2003, Vogeler et al., 2017). Since echinoderms are deuterostomes (Lavado et al., 2006), the characterization of their NRs involved in lipid homeostasis is essential to better understand the evolutionary response to lipid modulating chemicals.
Echinoderms are deuterostomes and, together with the sister group of hemichordates, share a closer ancestry with chordates than any other invertebrate phyla (Lavado et al., 2006, Rottinger and Lowe, 2012). In this work, we aimed to get additional insights into the adverse outcomes of a model lipid homeostasis modulator in early diverging deuterostomes. To achieve this aim, we isolated and functionally characterized in vitro, and for the first time, the PPAR orthologue of a non-chordate, the echinoderm P. lividus, using transactivation assays. We also evaluated the ability of the model lipid modulator, TBT, to alter lipid homeostasis following 3 weeks of exposure to environmentally relevant concentrations (100 and 250 ng Sn/L). In parallel, screening of the fatty acid profile and key gene expression levels was performed.
Section snippets
Gene isolation and cloning
The full sequence of the gene rxr and ppar, and the partial sequences of the genes acc (acetyl-CoA carboxylase) and acsl (long-chain acyl-CoA synthetase) were obtained using a combination of PCR-based approaches. Briefly, degenerate PCR primers were designed from conserved regions using CODEHOP (Rose et al., 2003) (COnsensus-DEgenerate Hybrid Oligonucleotide Primer) program to obtain the initial fragment or fragments. For rxr, 5′ and 3′ ends were further extended using the SMARTer™ RACE cDNA
Results and discussion
It is well established that different endocrine disrupting chemicals (EDCs) are able to interfere with the lipid metabolism of mammals; yet, data on the effect of those compounds in other lineages is still very limited. Nonetheless, recent research supports the hypothesis that lipid homeostasis and obesogenic outcomes transcend mammals (Capitão et al., 2017, Janer et al., 2007, Jordão et al., 2015, Jordão et al., 2016b, Lyssimachou et al., 2009, Lyssimachou et al., 2015, Maradonna et al., 2015
Conclusions
The present study is, to our knowledge, the first to isolate and functionally characterize the NRs PPAR and RXR from an echinoderm. Additionally, the in vivo observations support the hypothesis that TBT acts as a lipid homeostasis modulator in this group. The present work provides robust evidence for the ability of TBT to interfere with sea urchin lipid metabolism and reveal the modulation of PPAR/RXR as a potential mechanism. Future developmental and full life cycle studies should further
Funding
This work was supported by the project 031544 cofinanced by COMPETE 2020, Portugal 2020, and the European Union through the ERDF, and by Fundação para a Ciência e a Tecnologia through national funds and the support to A.M.F.C (SFRH/BD/90664/2012).
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
We acknowledge Ana André, Tiago Torres and Ricardo Capela for their help in the sampling process.
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