0013 7227 06 15 00 0 Printed in U S A Endocrinology 147 8 3861 3869 Copyright 2006 by The Endocrine Society doi 10 1210 en 2006 0363 The Octopus vulgaris Estrogen Receptor Is a Constitutive Transcriptional Activator Evolutionary and Functional Implications June Keay Jamie T Bridgham and Joseph W Thornton Center for Ecology and Evolutionary Biology University of Oregon Eugene Oregon 97403 Steroid hormones such as estrogens and androgens are important regulators of reproduction physiology and development in a variety of animal taxa including vertebrates and mollusks Steroid hormone receptors which mediate the classic cellular responses to these hormones were thought to be vertebrate specific which left the molecular mechanisms of steroid action in invertebrates unresolved Recently an estrogen receptor ER ortholog was isolated from the sea hare Aplysia californica but the functional significance of the receptor was unclear because estrogens and other steroids are not known to be important in that species Furthermore the Aplysia ER was found to be a constitutive transcriptional activator but it was unclear whether the estrogen independence of the ER was an Aplysia specific novelty or a more ancient character general to the mollusks Here we report on S TEROID HORMONE RECEPTORS SRs play crucial roles in regulating reproduction development and metabolism in vertebrates 1 They act as high affinity molecular mediators between steroid hormone ligands specific target sequences in genomic DNA and coregulator proteins that activate or repress transcription of nearby genes SRs have a modular structure in which two highly conserved domains the DNA binding and ligand binding domains DBD and LBD respectively are particularly important to ligand activated transcription In the classical mode of action of steroid hormone receptors the receptor is in an inactive conformation in the absence of a hormone ligand When the hormone is present it binds tightly and specifically to the pocket of the LBD changing its conformation and facilitating dimerization and nuclear transport 2 The receptor DBD binds to a specific response element a 15 bp sequence of genomic DNA in the control region of target genes 3 In this active conformation stabilized by ligand surfaces of the LBD are exposed for tight interactions with coactivator proteins that modify chromatin or otherwise affect transcription ExFirst Published Online May 11 2006 Abbreviations AF Activation function DBD DNA binding domain E2 17 estradiol ER estrogen receptor ERE estrogen response element ERR ER related receptor ERRE estrogen related response element GRE glucocorticoid response element HAP hydroxyapatite LBD ligand binding domain LRH liver receptor homolog RACE rapid amplification of cDNA ends SF steroidogenic factor SR steroid hormone receptor TEGDK buffer of Tris HCl EDTA glycerol dithiothreitol and KCl UAS upstream activating sequence Endocrinology is published monthly by The Endocrine Society http www endo society org the foremost professional society serving the endocrine community the isolation and functional characterization of the first ER ortholog from an invertebrate in which estrogens are produced and play an apparent role the cephalopod Octopus vulgaris We show that the Octopus ER is a strong constitutive transcriptional activator from canonical estrogen response elements The receptor does not bind estradiol and is unresponsive to estrogens and other vertebrate steroid hormones These characteristics are similar to those observed with the Aplysia ER and support the hypothesis that the evolving ER gained constitutive activity deep in the mollusk lineage The apparent reproductive role of estrogens in Octopus and other mollusks is unlikely to be mediated by the ER and may take place through an ancient non ER mediated pathway Endocrinology 147 3861 3869 2006 pression of the target gene is then selectively up or downregulated 4 In this way steroid hormones stimulate coordinated cascades of gene expression that underlie such functions as secondary sexual differentiation female reproductive cycling long term response to stress and adaptation to changing osmolarity throughout the vertebrates Steroid hormones are also found in invertebrates including mollusks in which there is evidence that they play an endocrine role 5 10 In the cephalopod Octopus vulgaris for example 17 estradiol E2 and progesterone are found in oviduct and ovarian tissues and the concentration of these hormones in females correlates with phases of the reproductive cycle 7 8 In addition specific high affinity estradiol binding and immunoreactivity to antihuman estrogen receptor ER antibodies have been detected in O vulgaris female reproductive tract 7 In the bivalve mollusk Mytilus edulis E2 has been detected in gonads pedal ganglia and hemolymph 11 12 In explants of hemocytes and pedal ganglia of this species very low doses of estradiol produce strong and rapid cell signaling effects 12 13 There is evidence that synthetic pollutants that act as estrogens in vertebrates cause endocrine disruption in gastropods 14 Numerous other steroids have also been found in a variety of mollusks and may have functional roles 15 16 The molecular mechanisms of steroid action in mollusks remain unknown however Steroid receptors were long thought to be vertebrate specific novelties based on their complete absence from the fully sequenced genomes of insects and nematodes but a recent study identified an ER ortholog in the mollusk Aplysia californica 17 Functional studies showed that the Aplysia ER does not bind E2 and is 3861 3862 Endocrinology August 2006 147 8 3861 3869 a powerful constitutive transcriptional activator even in the absence of E2 or any other added hormones 17 To understand the evolution of this feature the ancestor of all SRs was reconstructed synthesized and functionally characterized This receptor was found to specifically bind E2 and activate transcription only when estrogens were added a result consistent with the ancestor s high sequence similarity to vertebrate ERs particularly at critical sites in the ligand binding pocket 17 18 The constitutive activity of the Aplysia ER was therefore inferred to be a derived state Several other nuclear receptors are also constitutively active including the liver receptor homolog LRH 1 of rodents and the ER related receptors ERRs the receptors most closely related to the steroid receptors 19 Both of these receptor groups have evolved
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