Retinol Metabolism in the Mollusk Osilinus lineatus Indicates an Ancient Origin for Retinyl Ester Storage Capacity

Although retinoids have been reported to be present and active in vertebrates and invertebrates, the presence of mechanisms for retinoid storage in the form of retinyl esters, a key feature to maintain whole-organism retinoid homeostasis, have been considered to date a vertebrate innovation. Here we demonstrate for the first time the presence of retinol and retinyl esters in an invertebrate lophotrochozoan species, the gastropod mollusk Osilinus lineatus. Furthermore, through a pharmacological approach consisting of intramuscular injections of different retinoid precursors, we also demonstrate that the retinol esterification pathway is active in vivo in this species. Interestingly, retinol and retinyl esters were only detected in males, suggesting a gender-specific role for these compounds in the testis. Females, although lacking detectable levels of retinol or retinyl esters, also have the biochemical capacity to esterify retinol, but at a lower rate than males. The occurrence of retinyl ester storage capacity, together with the presence in males and females of active retinoids, i.e., retinoic acid isomers, indicates that O. lineatus has a well developed retinoid system. Hence, the present data strongly suggest that the capacity to maintain retinoid homeostasis has arisen earlier in Bilateria evolution than previously thought

Molecular Evolution of Ultraspiracle Protein (USP/RXR) in Insects

Ultraspiracle protein/retinoid X receptor (USP/RXR) is a nuclear receptor and transcription factor which is an essential component of a heterodimeric receptor complex with the ecdysone receptor (EcR). In insects this complex binds ecdysteroids and plays an important role in the regulation of growth, development, metamorphosis and reproduction. In some holometabolous insects, including Lepidoptera and Diptera, USP/RXR is thought to have experienced several important shifts in function. These include the acquisition of novel ligand-binding properties and an expanded dimerization interface with EcR. In light of these recent hypotheses, we implemented codon-based likelihood methods to investigate if the proposed shifts in function are reflected in changes in site-specific evolutionary rates across functional and structural motifs in insect USP/RXR sequences, and if there is any evidence for positive selection at functionally important sites. Our results reveal evidence of positive selection acting on sites within the loop connecting helices H1 and H3, the ligand-binding pocket, and the dimer interface in the holometabolous lineage leading to the Lepidoptera/Diptera/Trichoptera. Similar analyses conducted using EcR sequences did not indicate positive selection. However, analyses allowing for variation across sites demonstrated elevated non-synonymous/synonymous rate ratios (dN/dS), suggesting relaxed constraint, within the dimerization interface of both USP/RXR and EcR as well as within the coactivator binding groove and helix H12 of USP/RXR. Since the above methods are based on the assumption that dS is constant among sites, we also used more recent models which relax this assumption and obtained results consistent with traditional random-sites models. Overall our findings support the evolution of novel function in USP/RXR of more derived holometabolous insects, and are consistent with shifts in structure and function which may have increased USP/RXR reliance on EcR for cofactor recruitment. Moreover, these findings raise important questions regarding hypotheses which suggest the independent activation of USP/RXR by its own ligand

Supplemental Information 1: Images of control and 9- cis

Trichoplax adhaerens, the only known species of Placozoa is likely to be closely related to an early metazoan that preceded branching of Cnidaria and Bilateria. This animal species is surprisingly well adapted to free life in the World Ocean inhabiting tidal costal zones of oceans and seas with warm to moderate temperatures and shallow waters. The genome of T. adhaerens (sp. Grell) includes four nuclear receptors, namely orthologue of RXR (NR2B), HNF4 (NR2A), COUP-TF (NR2F) and ERR (NR3B) that show a high degree of similarity with human orthologues. In the case of RXR, the sequence identity to human RXR alpha reaches 81% in the DNA binding domain and 70% in the ligand binding domain. We show that T. adhaerens RXR (TaRXR) binds 9-cis retinoic acid (9-cis-RA) with high affinity, as well as high specificity and that exposure of T. adhaerens to 9-cis-RA regulates the expression of the putative T. adhaerens orthologue of vertebrate L-malate-NADP+ oxidoreductase (EC 1.1.1.40) which in vertebrates is regulated by a heterodimer of RXR and thyroid hormone receptor. Treatment by 9-cis-RA alters the relative expression profile of T. adhaerens nuclear receptors, suggesting the existence of natural ligands. Keeping with this, algal food composition has a profound effect on T. adhaerens growth and appearance. We show that nanomolar concentrations of 9-cis-RA interfere with T. adhaerens growth response to specific algal food and causes growth arrest. Our results uncover an endocrine-like network of nuclear receptors sensitive to 9-cis-RA in T. adhaerens and support the existence of a ligand-sensitive network of nuclear receptors at the base of metazoan evolution

Locust retinoid X receptors: 9-Cis-retinoic acid in embryos from a primitive insect

The retinoid X receptor (RXR) is activated by its often elusive cognate ligand, 9-cis-retinoic acid (9-cis-RA). In flies and moths, molting is mediated by a heterodimer ecdysone receptor consisting of the ecdysone monomer (EcR) and an RXR homolog, ultraspiracle (USP); the latter is believed to have diverged from its RXR origin. In the more primitive insect, Locusta migratoria (Lm), RXR is more similar to human RXRs than to USPs. LmRXR was detected in early embryos when EcR transcripts were absent, suggesting another role apart from ecdysone signaling. Recombinant LmRXRs bound 9-cis-RA and all-trans-RA with high affinity (IC50 = 61.2–107.7 nM; Kd = 3 nM), similar to human RXR. To determine whether specific binding had functional significance, the presence of endogenous retinoids was assessed. Embryos were extracted by using modified Bligh and Dyer and solid-phase protocols to avoid the oily precipitate that makes this material unsuitable for assay. These extracts contained retinoids (5.4 nM) as assessed by RA-inducible Cyp26A1-promoter luciferase reporter cell lines. Furthermore, the use of HPLC and MS confirmed the presence of retinoids and identified in any embryo, 9-cis-RA, in addition to all-trans-RA. We estimate that whole embryos contain 3 nM RA, including 9-cis-RA at a concentration of 1.6 nM. These findings strongly argue for a functional role for retinoids in primitive insects and favor a model where signaling through the binding of 9-cis-RA to its RXR is established relatively early in evolution and embryonic development