Transcriptional Activation of Estrogen Receptor-alpha and Estrogen Receptor-beta from Elephant Shark (Callorhynchus milii)

Humans and other vertebrates contain two estrogen receptors (ERs), ER and ER{beta}. Among cartilaginous fish (sharks, rays, skates), which are chondrichthyans that evolved about 425 million years ago, only activation by steroids of ER{beta} orthologs has been characterized. To remedy this gap in understanding estrogen signaling in chondrichthyans, we studied estrogen activation of orthologs of human ER and ER{beta} from elephant shark (Callorhynchus milii). Unexpectedly, we found that C. milii contained three estrogen-responsive ER genes: ER1 (596 amino acids), ER2 (600 amino acids), and ER3 (599 amino acids) with strong sequence similarity to each other. We also found an estrogen-unresponsive gene, ER4 (561 amino acids), with a 39 amino acid deletion in the DNA-binding domain. An estrogen-responsive ER{beta} ortholog (580 amino acids) also was present in C. milii. The three active C. milii ERs are of similar length to human ER (595 amino acids); however, C. milii ER{beta} is longer than human ER{beta} (530 amino acids). We studied transcriptional activation of ER and ER{beta} by estradiol (E2), the main reproductive estrogen in humans. We also studied estrone (E1), the main postmenopausal estrogen, and estriol (E3), which is synthesized during pregnancy. We determined the half-maximal response (EC50) and fold-activation to E2, E1, and E3 of C. milii ER1, ER2, ER3, and ER{beta}. Among these estrogens, E2 had the lowest EC50 for all four ERs. Fold-activation by E2 and E3 was similar for ER1, ER2, ER3, and ER{beta}. Overall, estrogen activation of C. milii ER and ER{beta} was similar to that for human ER and ER{beta}, indicating substantial conservation of the vertebrate ER during the 425 million years since the divergence of cartilaginous fish and humans from a common ancestor.