Nuclear Ecdysteroid Receptors Research Articles

20-Hydroxyecdysone activates the protective arm of the RAAS via the MAS receptor.

20-Hydroxyecdysone (20E) is a steroid hormone that plays a key role in insect development through nuclear ecdysteroid receptors (EcR/RXR complex) and at least one membrane GPCR receptor (DopEcR). It also displays numerous pharmacological effects in mammals, where its mechanism of action is still debated, involving either an unidentified GPCR or the estrogen ERβ receptor. The goal of this study was to better understand 20E mechanism of action in mammals. A mouse myoblast cell line (C2C12) and the gene expression of myostatin (a negative regulator of muscle growth) were used as a reporter system of anabolic activity. Experiments using protein-bound 20E established the involvement of a membrane receptor. 20E-like effects were also observed with angiotensin(1-7), the endogenous ligand of MAS. Additionally, the effect on myostatin gene expression was abolished by Mas receptor knock-down using siRNA or pharmacological inhibitors. 17β-Estradiol (E2) also inhibited myostatin gene expression, but protein-bound E2 was inactive, and E2 activity was not abolished by angiotensin(1-7) antagonists. A mechanism involving cooperation between the MAS receptor and a membrane-bound palmitoylated estrogen receptor is proposed. The possibility to activate the MAS receptor with a safe steroid molecule is consistent with the pleiotropic pharmacological effects of ecdysteroids in mammals and, indeed, the proposed mechanism may explain the close similarity between the effects of angiotensin(1-7) and 20E. Our findings open up many possible therapeutic developments involving stimulation of the protective arm of the renin-angiotensin-aldosterone system (RAAS) with 20E.

Steroid hormone signaling is involved in the age-dependent behavioral response to sex pheromone in the adult male moth Agrotis ipsilon

In most animals, including insects, male reproduction depends on the detection and processing of female-produced sex pheromones. In the male moth, Agrotis ipsilon, both behavioral response and neuronal sensitivity in the primary olfactory center, the antennal lobe (AL), to female sex pheromone are age- and hormone-dependent. In many animal species, steroids are known to act at the brain level to modulate the responsiveness to sexually relevant chemical cues. We aimed to address the hypothesis that the steroidal system and in particular 20-hydroxyecdysone (20E), the main insect steroid hormone, might also be involved in this olfactory plasticity. Therefore, we first cloned the nuclear ecdysteroid receptor EcR (AipsEcR) and its partner Ultraspiracle (AipsUSP) of A. ipsilon, the expression of which increased concomitantly with age in ALs. Injection of 20E into young sexually immature males led to an increase in both responsiveness to sex pheromone and amount of AipsEcR and AipsUSP in their ALs. Conversely, the behavioral response decreased in older, sexually mature males after injection of cucurbitacin B (CurB), an antagonist of the 20E/EcR/USP complex. Also, the amount of AipsEcR and AipsUSP significantly declined after treatment with CurB. These results suggest that 20E is involved in the expression of sexual behavior via the EcR/USP signaling pathway, probably acting on central pheromone processing in A. ipsilon.

Properties of nuclear and cytosolic ecdysteroid receptors from an epithelial cell line from Chironomus tentans

Recently ecdysteroid receptors have been identified in extracts of an epithelial cell line of the dipteran insect Chironomus tentans. These proteins have been shown to bind with high affinity to DNA and preferably to known ecdysteroid response elements. DNA-binding assays were performed under excess of DNA-binding sites in comparison to ecdysteroid receptors. Thereby, a proportion of about 20% of total ecdysteroid receptors in cytosolic extracts and an average of 50% of nuclear receptors could be demonstrated to bind to DNA. Ecdysteroid receptors from nuclear and cytosolic extracts displayed different S-value patterns. Cytosolic extract showed maximal ecdysteroid binding at S-values of about 4.4 S and greater than 8 S regardless of salt concentration in the buffer. Nucleic ecdysteroid receptors displayed a single peak of ecdysteroid binding at about 5 S. Furthermore, 0.4M NaCl could not completely elute nuclear ecdysteroid receptors from calf thymus-DNA cellulose columns whereas cytosolic receptors could almost be completely eluted with buffers of this ionic strength. This suggests a higher affinity to DNA for nuclear ecdysteroid receptors. These findings indicate that ecdysteroid receptors in this insect cell line may require activation step(s) similar to those discussed for vertebrate steroid receptors.

Co-localization of ecdysteroid receptors and c-fos-like protein in the brain of Manduca sexta larvae.

The presence of c-fos, a marker for cell activation, was investigated in cerebral neurons actively expressing ecdysteroid receptors during larval-pupal development in the tobacco hornworm, Manduca sexta. Colocalization was accomplished by ecdysteroid autoradiography using the tritiated high affinity 20-hydroxyecdysone agonist ponasterone A and immunocytochemistry with an antibody to a peptide sequence which is highly conserved in both human and murine c-fos. Immunoreactivity to a c-fos-like protein(s) was present in nuclei of many neurons of all the developmental stages examined. However, with the exception of the optic lobe, cells expressing nuclear ecdysteroid receptors were more immunoreactive than non-ecdysteroid-binding neurons. These data suggest that ecdysteroid-induced gene activation and translation may involve c-fos expression.

Uptake and Binding of Moulting Hormones in Crayfish

According to the “classical” scheme of steroid hormone action, steroid hormones are bound to transport proteins, the hormones are taken up by the target tissue and there are bound to the cytoplasmic receptor. This hormone-receptor complex then migrates to the nucleus and alters genome activity. We tried to determine whether this scheme is also valid for crustaceans. There is no evidence for a specific ecdysteroid binding protein in the hemolymph of 3 crustacean species studied so far. As concerns the next step in this cascade of events, the uptake, we could demonstrate that besides diffusion a carrier-mediated transport of ecdysteroids into crayfish hypodermis is involved. Both cytoplasmic and nuclear ecdysteroid receptors were demonstrated by us in 4 different species, the receptors of crayfish being best characterized. The receptors are proteins of high affinity and steroid specificity. Their size is about 70–80,000 da and their isoelectric point is in the range of 5.7 to 5.9. Free SH groups, pH and ionic strength are important factors for the binding. Non-derivatized tritium labelled ecdysteroids can be covalently attached to the receptor by photoaffinity labelling. The usefulness of this method is demonstrated in this paper through investigation of steroid specificity, tissue distribution, affinity and size of the receptor with both conventional binding assays and after photoactivation. Ecdysteroid antibodies recognized the photochemically crosslinked hormone-receptor complex and were used as immunoabsorbents.

Exogenous ecdysteroids elicit low‐threshold sensory responses in spiny lobsters

AbstractElectrophysiological recordings from chemoreceptors in the lateral antennule of Panulirus interruptus reveal low threshold detection of 10−3 M β‐ecdysone or 10−12 M α‐ecdysone with a latency of less than 1 second. Responses to either α‐ or β‐ecdysone were recorded from juvenile, female (with and without spermatophores or bearing eggs), and male lobsters in intermolt and premolt stages. Receptors responding to α‐ecdysone did not respond to 2.9 × 10−11 M β‐ecdysone. The behavioral function of this low threshold detection is not known, but ecdysteroids may be important in intra‐ or interspecific communication. The short latency of this neural response to ecdysteroids strongly implicates interaction with a membrane‐associated component. Thus, the chemoreceptors in the lateral antennule of P. interruptus may provide a valuable system for the study of the affinity and specificity of membrane‐bound ecdysteroid receptors for comparison to previously described cytoplasmic and nuclear ecdysteroid receptors and membrane‐associated receptors specific for other steroid compounds.

Characterization of nuclear ecdysteroid receptor from crayfish integument

The existence of nuclear ecdysteroid receptors in isolated nuclei from crayfish integument was demonstrated. Nuclei were prepared from crayfish hypodermis by use of a modified Chauveau-method and exposed to [ 3H]-ecdysteroids. The nuclear hormone receptor complex sedimented at about 5S and the available binding sites had a high affinity to [ 3H] ecdysteroids. Treatment of the nuclei with pronase and DNase completed or partially reduced nuclear bound ecdysone, whereas RNase was without any effect. Competition of nuclear bound ecdysone by various steroids confirmed ponasterone A and kaladasterone as the best competitors followed by 20-OH-ecdysone and ecdysone, whereas compounds like poststerone, “triol” or hydrocortisone did not compete. If [ 3H]-20-OH-ecdysone was used as the radio-ligand, 20-OH-ecdysone was a better competitor than ecdysone indicating that there may be two different binding sites for ecdysone and 20-OH-ecdysone; this was further substantiated by the finding of two different number of binding sites for these steroids.

Isolation of Nuclei from Crayfish Tissues and Demonstration of Nuclear Ecdysteroid Receptors

Nuclei from hypodermis and midgut gland of the crayfish, Orconectes limosus, have been isolated with a slightly modified Chauveau-method. The isolated nuclei are well preserved and free from cytoplasmic contamination. They are able to incorporate [3H]UTP into acid-insoluble material. This labelling is α-amanitin sensitive. The isolated nuclei are devoid of the mitochondrial/microsomal ecdysone-C-20-hydroxylase. The DNA: RNA: protein ratio is 1:0.8: 3.2 (using Triton X-100 during the isolation procedure the ratio is 1:0.4: 4.6) for nuclei from the hypodermis and 1:1: 3.8 for nuclei from the midgut gland. The absolute amount of DNA per cell is 1.6, resp. 3.5 pg. The final DNA-recovery is about 20%. The yield increases to about 60% if the final ultrazentrifugation step is omitted. The isolated nuclei bind 3H-ecdysone and 3H-20-OH-ecdysone specifically. As revealed by Scatchard analysis the affinity of the nuclear 20-OH-ecdysone receptor is 1-2 × 10-9 ᴍ