The Echinodermata PPAR: Functional characterization and exploitation by the model lipid homeostasis regulator tributyltinā
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 (GonzĆ”lez-Irusta etĀ al., 2010, Ribeiro etĀ al., 2015, Romero etĀ al., 2016). From an economic standpoint, species such as the herbivorous Paracentrotus lividus, widely distributed in the Atlantic and Mediterranean coasts (Arafa etĀ al., 2012, Carboni etĀ al., 2012, Kabeya etĀ al., 2017), also hold a high commercial value, as their gonads are considered a gastronomic delicacy (Arafa etĀ al., 2012, Guidetti, 2004, Guidetti etĀ al., 2004, Shpigel etĀ 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 etĀ 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 etĀ 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 etĀ 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 etĀ al., 2013). The sea urchin P.Ā lividus is able to synthetize ARA and EPA from the precursors linoleic acid (18: 2n-6, LA) and Ī±-linolenic acid (18: 3n-3, ALA) (Kabeya etĀ 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 (GonzĆ”lez-MaƱƔn etĀ al., 2012, RincĆ³n-Cervera etĀ al., 2016).
Animal lipid composition is not static and can be altered by diet, life-cycle stage or external stimuli (Arafa etĀ 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 etĀ al., 2009, GrĆ¼n and Blumberg, 2009, JordĆ£o etĀ al., 2016b, Lyssimachou etĀ al., 2015, Ouadah-Boussouf and Babin, 2016, Santos etĀ 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 (CapitĆ£o etĀ al., 2017, Janer etĀ al., 2007, JordĆ£o etĀ al., 2015, Lyssimachou etĀ 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 241,8Ā Ī¼g Sn/Kg, as reported in biological samples from one of the largest harbour regions in China (Chen etĀ al., 2017). TBT was found to disrupt mammalian lipid homeostasis through the interaction with the nuclear receptors peroxisome proliferator-activated receptor Ī³ (PPARĪ³) and retinoid X receptor (RXR) (CapitĆ£o etĀ al., 2018, Harada etĀ al., 2015, Hiromori etĀ al., 2009, le Maire etĀ al., 2009). PPARĪ³ and RXR cooperate in the form of a permissive heterodimer (PPARĪ³/RXR) that is considered a master regulator of lipid homeostasis in vertebrates (Ahmadian etĀ al., 2013, Berkenstam and Gustafsson, 2005, Hiromori etĀ al., 2015, Janesick and Blumberg, 2011, Ouadah-Boussouf and Babin, 2016, Santos etĀ al., 2012). PPARĪ³ is member of a nuclear receptor superfamily that in vertebrates include also peroxisome proliferator-activated receptor Ī± and Ī² (PPARĪ± and PPARĪ²). 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 (EcheverrĆa etĀ 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 (AndrĆ© etĀ al., 2017), gastropods (Nishikawa etĀ al., 2004), crustaceans (Wang and LeBlanc, 2009) and rotifers (Lee etĀ al., 2019). In vertebrates, TBT, and the related organotin, triphenyltin (TPT), were also shown to interact with PPARĪ³ exhibiting a similar binding mode as that described for RXR: through the interaction of the tin atom and a cysteine residue (Harada etĀ al., 2015, Hiromori etĀ al., 2009, le Maire etĀ al., 2009). Outside deuterostomes, PPAR was only reported in the mollusk Crassostrea gigas; yet, its characterization was limited to in silico analysis (Vogeler etĀ al., 2014, Vogeler etĀ al., 2017). Although nuclear receptors (NRs) are widespread in metazoans (Bridgham etĀ al., 2010, Santos etĀ al., 2018), the available functional data has limited taxonomic scope (Castro and Santos, 2014, Santos etĀ al., 2018; Tan and Palli, 2008, Thornton, 2003, Vogeler etĀ al., 2017). Since echinoderms are deuterostomes (Lavado etĀ 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 etĀ 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 inĀ vitro, and for the first time, the PPAR orthologue of a non-chordate, the echinoderm P.Ā 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 250Ā 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 etĀ al., 2003) (COnsensus-DEgenerate Hybrid Oligonucleotide Primer) program to obtain the initial fragment or fragments. For rxr, 5ā² and 3ā² ends were further extended using the SMARTerā¢ 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 (CapitĆ£o etĀ al., 2017, Janer etĀ al., 2007, JordĆ£o etĀ al., 2015, JordĆ£o etĀ al., 2016b, Lyssimachou etĀ al., 2009, Lyssimachou etĀ al., 2015, Maradonna etĀ 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 inĀ 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 FundaĆ§Ć£o para a CiĆŖncia 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 AndrƩ, Tiago Torres and Ricardo Capela for their help in the sampling process.
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This paper has been recommended for acceptance by Christian Sonne.