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

Abstract

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 alltrans-RA with high affinity (IC 50 ‫؍‬ 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.all-trans-retinoic acid ͉ Locusta migratoria ͉ ultraspiracle I nsect development and metamorphosis are directed by two principal lipophilic hormones: 20-hydroxyecdysone (20-OH-Ec), the active molting hormone, and juvenile hormone (JH), whose titer determines the nature of the molt (1, 2). As demonstrated in the fruitfly, Drosophila melanogaster (Dm), 20-OH-Ec binds to the ecdysone receptor (EcR), which in turn is bound to its obligate heterodimerization partner ultraspiracle (USP), a homologue of the vertebrate retinoid X receptor (RXR) (3-6). As members of the nuclear receptor superfamily, EcR and USP/RXR share a common modular structure (7) comprised of a N-terminal variable domain (A/B), a DNA binding domain (C), hinge (D), and C-terminal ligand-binding domain (LBD; or domain E/F).The vertebrate RXRs are known heterodimeric partners of several members of the nuclear receptor superfamily, including the retinoid, thyroid, and vitamin D receptors (8). As demonstrated in vivo, these RXRs can also form homodimers and conceivably mediate an independent retinoid signaling pathway (9, 10). Indeed, the vertebrate RXRs are known ligand-activated transcription factors that bind 9-cis-retinoic acid (9-cis-RA), a stereoisomer of the vitamin A derivative, all-trans-RA (11, 12). RA receptors (RARs), reported only in vertebrates, are distinct in that they bind both all-trans-RA and 9-cis-RA with high affinity (13, 6). In contrast to the vertebrate RXRs, crystal structures reveal that DmUSP and t...

Detection of Endogenous Retinoids in the Molluscan CNS and Characterization of the Trophic and Tropic Actions of 9-cisRetinoic Acid on Isolated Neurons

“…We report here that both atRA and 9-cis-RA are present in the molluscan CNS (and hemolymph) in nM concentrations, although the concentration of atRA was estimated to be slightly higher than 9-cis-RA. The estimated concentrations reported in this study are in accordance with previous detection levels of RA in vertebrate limb tissue (Thaller and Eichele, 1987;Scadding and Maden, 1994) as well as locust embryo (Nowickyj, 2008). Interestingly, in the locust embryo, 9-cis-RA was slightly more abundant than atRA (Nowickyj et al, 2008).…”

“…Our samples were immediately frozen in liquid nitrogen and protected from heat, air and light, thus minimizing the potential effects of endogenous isomerases. In addition, neither Nowickyj et al (2008), or Biesalski et al (1992 detected any significant trans to cis-isomerization, while Brodeur et al (2007) also failed to demonstrate endogenous RA isomerase activity in cultured HeLa cells. Indeed, the existence of cis-retinol isomers and the enzymes capable of oxidizing them to their characteristic RAs (Labrecque et al, 1995, Driessen et al, 1998, Romert et al, 1998, Paik et al, 2005, strongly suggests that 9-cis-RA (and other cis-RA isomers) may be synthesized independently from atRA.…”

“…However, in contrast to vertebrates, where 9-cis-RA has been difficult to detect, both atRA and 9-cis-RA isomers have been directly detected in the prevertebrate amphioxus (Dalfó et al, 2002), fiddler crab limb blastemas (Hopkins, 2001) and recently, the locust embryo (Nowickyj et al, 2008). Although RA has been shown to alter eye formation in the mollusc Lymnaea (Créton et al, 1993) and vitamin A was detected in the mollusc Helix pomatia (Eakin and Brandenburger, 1968), little is known about endogenous retinoids in molluscs.…”

“…This suggests that the molluscan RXR has the capability to bind 9-cis-RA, activate transcription and may play a dominant role in invertebrate retinoid signaling. Interestingly, Nowickyj et al (2008) showed that the locust RXR binds both 9-cis and atRA isomers with similar affinity, and at a level 2 magnitudes higher than DHA (docosahexaenoic acid, a putative nonretinoid RXR agonist), suggesting it is a retinoid-specific receptor. atRA has also been shown to upregulate RXR expression in the marine sponge in addition to exerting morphogenetic effects (Wiens et al, 2003).…”

Ecdysteroid hormone action

“…In heterometabolous insects like Locusta migratoria, this transcription factor is more similar to human RXR. LmRXR was detected in early embryos, when EcR transcripts were absent, suggesting an additional role apart from ecdysone signalling [32]. Phylogenetic analysis suggests that RXR-USPs have undergone remarkable functional shifts during evolution especially in the ligand binding domain of Usp-RXR [33].…”