The Echinodermata PPAR: Functional characterization and exploitation by the model lipid homeostasis regulator tributyltin

Highlights

• TBT is a known modulator of lipid homeostasis in mammals.

• The previous studies in the field are mainly focus in vertebrates.

• First time an Echinodermata gene orthologue of PPAR characterized.

• TBT modulates PPAR/RXR and alter gene expression and lipid profile in P. lividus.

Abstract

The wide ecological relevance of lipid homeostasis modulators in the environment has been increasingly acknowledged. Tributyltin (TBT), for instance, was shown to cause lipid modulation, not only in mammals, but also in fish, molluscs, arthropods and rotifers. In vertebrates, TBT is known to interact with a nuclear receptor heterodimer module, formed by the retinoid X receptor (RXR) and the peroxisome proliferator-activated receptor (PPAR). These modulate the expression of genes involved in lipid homeostasis. In the present work, we isolated for the first time the complete coding region of the Echinodermata (Paracentrotus lividus) gene orthologues of PPAR and RXR and evaluated the ability of a model lipid homeostasis modulator, TBT, to interfere with the lipid metabolism in this species. Our results demonstrate that TBT alters the gonadal fatty acid composition and gene expression patterns: yielding sex-specific responses in fatty acid levels, including the decrease of eicosapentaenoic acid (C20:5 n-3, EPA) in males, and increase of arachidonic acid (20:4n-6, ARA) in females, and upregulation of long-chain acyl-CoA synthetase (acsl), ppar and rxr. Furthermore, an in vitro test using COS-1 cells as host and chimeric receptors with the ligand binding domain (LBD) of P. lividus PPAR and RXR shows that organotins (TBT and TPT (Triphenyltin)) suppressed activity of the heterodimer PPAR/RXR in a concentration-dependent manner. Together, these results suggest that TBT acts as a lipid homeostasis modulator at environmentally relevant concentrations in Echinodermata and highlight a possible conserved mode of action via the PPAR/RXR heterodimer.

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.